WO2023100925A1 - Asymmetric cell division inhibitor, chromosomal aneuploidy inhibitor, medium, in vitro fertilization medium, cell treatment kit, asymmetric cell division inhibition method, in vitro fertilization method, transplantation method, infertility treatment method, and offspring acquisition method - Google Patents
Asymmetric cell division inhibitor, chromosomal aneuploidy inhibitor, medium, in vitro fertilization medium, cell treatment kit, asymmetric cell division inhibition method, in vitro fertilization method, transplantation method, infertility treatment method, and offspring acquisition method Download PDFInfo
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- WO2023100925A1 WO2023100925A1 PCT/JP2022/044127 JP2022044127W WO2023100925A1 WO 2023100925 A1 WO2023100925 A1 WO 2023100925A1 JP 2022044127 W JP2022044127 W JP 2022044127W WO 2023100925 A1 WO2023100925 A1 WO 2023100925A1
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- asymmetric division
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- vitro fertilization
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention provides mammalian cell asymmetric cell division inhibitors, chromosomal aneuploidy inhibitors, media, in vitro fertilization media, cell treatment kits, asymmetric division inhibition methods, in vitro fertilization methods, transplantation methods, infertility treatment methods, and a method for obtaining offspring.
- Patent Document 1 a method for promoting egg maturation in assisted reproductive medicine is described. This technology reduces the rate of ovarian hyperstimulation syndrome (OHSS), achieves equal or improved pregnancy rates, shortens time to conception, and prevents premature ovulation.
- OHSS ovarian hyperstimulation syndrome
- the present invention has been made in view of such circumstances, and aims to solve the above-mentioned problems.
- the asymmetric division inhibitor of the present invention is characterized by containing a spontaneous activation inhibitor that inhibits asymmetric division caused by spontaneous activation of pluripotent cells.
- the asymmetric division inhibitor of the present invention is characterized in that the spontaneous activation is accompanied by chromosomal dispersion.
- the agent for suppressing asymmetric division of the present invention is characterized in that the suppression of the asymmetric division induces suppression of the spontaneous activation and/or reassortment of the dispersed chromosomes.
- the asymmetric division inhibitor of the present invention is characterized in that the cells are pluripotent stem cells, germ cells, eggs, or embryos.
- the asymmetric division inhibitor of the present invention is characterized in that, when the cell is the germ cell or the egg, the spontaneous activation is due to exposure to carbon dioxide before fertilization or aging. .
- the asymmetric division inhibitor of the present invention is characterized in that the spontaneous activation inhibitor is a proteasome inhibitor.
- the asymmetric division inhibitor of the present invention is characterized in that the proteasome inhibitor contains MG132.
- the asymmetric division inhibitor of the present invention is characterized in that the proteasome inhibitor contains ALLN.
- the chromosomal aneuploidy inhibitor of the present invention is characterized by containing a spontaneous activation inhibitor that suppresses asymmetric division due to spontaneous activation of pluripotent cells.
- the medium of the present invention is characterized by containing the asymmetric division inhibitor.
- the in vitro fertilization medium of the present invention is characterized by containing the asymmetric division inhibitor.
- the cell treatment kit of the present invention is characterized by containing the asymmetric division inhibitor.
- the method for suppressing asymmetric division of the present invention is characterized by suppressing spontaneous activation of pluripotent cells and suppressing asymmetric division of the cells.
- the in vitro fertilization method of the present invention is a method of in vitro fertilization of collected germ cells or eggs, wherein the asymmetric division inhibitor is added and treated to prevent asymmetric division of the germ cells, eggs, or embryos. It is characterized by suppressing.
- the transplantation method of the present invention is characterized by transplanting fertilized eggs or embryos derived from germ cells or eggs treated by the in vitro fertilization method.
- the infertility treatment method of the present invention is a mammalian infertility treatment method, wherein the collected germ cells or eggs are treated by adding the asymmetric division inhibitor, and the germ cells or the fertilized eggs derived from the eggs or It is characterized by suppressing asymmetric division of embryos.
- the method for obtaining offspring of the present invention is characterized by obtaining offspring of mammals by the transplantation method or the infertility treatment method.
- asymmetric division inhibitor capable of suppressing a phenomenon in which chromosomes are dispersed due to fertilization and abortion of embryos despite fertilization, thereby increasing the birth rate.
- Fig. 1 is a graph showing the birth rate by natural mating (vivo) and fertilized eggs exposed to CO 2 according to Example 1 of the present invention.
- 4 is a graph showing the relationship between CO 2 exposure time and fertilization rate when collecting unfertilized ova according to Example 1 of the present invention.
- 4 is a graph showing the relationship between CO 2 exposure time and birth rate when unfertilized eggs are collected according to Example 1 of the present invention.
- Fig. 2 is a photograph showing the results of IVF and transplantation of the anesthetized harvested ovum according to Example 1 of the present invention and the ovum exposed to CO2 for 150 seconds into the fallopian tube.
- FIG. 1 is a photograph showing the results of whole-mount staining of CO 2 -exposed ova according to Example 1 of the present invention.
- 1 is a graph showing the results of whole-mount staining of CO 2 -exposed eggs according to Example 1 of the present invention.
- 4 is a graph showing results of spontaneous activation under acidic conditions according to Example 1 of the present invention.
- FIG. 2 is a photograph showing the results of microinsemination of the anesthetized oocyte and the oocyte exposed to CO 2 according to Example 1 of the present invention.
- FIG. 4 is a graph showing the results of microinsemination of the anesthetized harvested oocyte and the CO 2 -exposed oocyte according to Example 1 of the present invention.
- FIG. 2 is a photograph showing the presence or absence of fertilization in the anesthetized collected oocytes and the CO 2 -exposed oocytes according to Example 1 of the present invention, examined by the expression of Nanog.
- 2 is a graph showing the relationship between the CO 2 exposure time and the rate of 2-cell formation in mice according to Example 1 of the present invention.
- 2 is a graph showing the relationship between CO 2 exposure time and birth rate of mice according to Example 1 of the present invention.
- 1 is a photograph showing the results of IVF and transplantation into oviducts of anesthetized mouse oocytes according to Example 1 of the present invention and oocytes exposed to CO 2 for 330 seconds.
- FIG. 2 is a conceptual diagram and photographs of immunostaining for examining the state of ovum after IVF according to Example 1 of the present invention.
- 1 is a graph showing the results of immunostaining with DAPI and H3K9me3 by IVF of anesthetized oocytes according to Example 1 of the present invention.
- FIG. 2 is a graph showing the results of immunostaining with DAPI and H3K9me3 by IVF of CO 2 -exposed eggs according to Example 1 of the present invention.
- FIG. FIG. 2 is a photograph of immunostaining for examining the post-IVF state of anesthetized collected oocytes and CO 2 -exposed oocytes according to Example 1 of the present invention.
- Figure 2 is a graph (6.5 hours) of the number of female pronuclei after IVF in anesthetized retrieved oocytes and CO2 - exposed oocytes according to Example 1 of the present invention
- Figure 2 is a graph (20-21 hours) of the number of female pronuclei after IVF in anesthetized retrieved oocytes and CO2 exposed oocytes according to Example 1 of the present invention
- FIG. 2 is a photograph of immunostaining of the anesthetized retrieved egg and the CO 2 -exposed egg 24 hours after IVF according to Example 1 of the present invention.
- FIG. FIG. 2 is a photograph of karyotype analysis when the control oocytes according to Example 1 of the present invention were developed to 2-cells after IVF.
- FIG. FIG. 2 is a photograph of karyotype analysis when 2-cells were developed after IVF of CO 2 -exposed ova according to Example 1 of the present invention.
- FIG. FIG. 2 is a photograph of eggs exposed to CO 2 according to Example 1 of the present invention, which were grown to 2-cells after IVF.
- FIG. 2 is a graph showing the unequal division rate of chromosomes when the ovum exposed to CO 2 according to Example 1 of the present invention is developed to 2-cell after IVF.
- 2 is a graph showing the state of chromosomes and the fertilization rate when MG132 was added to eggs exposed to CO 2 according to Example 1 of the present invention.
- FIG. 4 is a graph showing the state of chromosomes in fertilized ova when MG132 was added to the ova exposed to CO 2 according to Example 1 of the present invention.
- FIG. 2 is a photograph of immunostaining with DAPI and H3K9me3 when MG132 was added to the eggs exposed to CO 2 according to Example 1 of the present invention.
- FIG. 1 is a graph of the number of female pronuclei when MG132 is added to eggs exposed to CO 2 according to Example 1 of the present invention.
- Fig. 10 is a photograph showing the development of 2-cells in the eggs exposed to CO 2 according to Example 1 of the present invention to which MG132 was added.
- FIG. 2 is a graph showing the rate of suppression of chromosome dispersion when MG132 was added to CO 2 -exposed eggs according to Example 1 of the present invention.
- 1 is a photograph of offspring produced when MG132 was added to eggs exposed to CO 2 according to Example 1 of the present invention.
- 1 is a graph showing the birth rate when MG132 was added to eggs exposed to CO 2 according to Example 1 of the present invention.
- FIG. 2 is a photograph of immunostaining with DAPI and H3K9me3 when ALLN was added to the eggs exposed to CO 2 according to Example 1 of the present invention.
- FIG. 1 is a graph of the number of female pronuclei when ALLN was added to CO2 - exposed eggs according to Example 1 of the present invention.
- FIG. 2 is a photograph of offspring when ALLN was added to eggs exposed to CO2 according to Example 1 of the present invention.
- 1 is a graph showing the birth rate when ALLN was added to eggs exposed to CO 2 according to Example 1 of the present invention.
- 4 is a graph showing the relationship between the age of rats and the ratio of MII and spontaneous activation of ova according to Example 1 of the present invention. 4 is a graph showing the relationship between the age and fertility of rats according to Example 1 of the present invention.
- Fig. 10 is a photograph of immunostaining with DAPI and H3K9me3 when MG132 was added to the aged infertile ovum according to Example 1 of the present invention.
- FIG. 4 is a graph showing the number of female pronuclei when MG132 was added to the aged infertile ovum according to Example 1 of the present invention.
- 1 is a photograph of a litter when MG132 is added to the aged infertile ovum according to Example 1 of the present invention.
- 2 is a graph showing the birth rate when MG132 was added to the ovum of elderly infertility according to Example 1 of the present invention.
- FIG. 10 is a graph showing the chromosomal composition when MG132 was added to the aged infertile ovum according to Example 2 of the present invention.
- FIG. 10 is a pie chart showing the chromosomal composition of a 2-cell stage embryo (comparative example) when MG132 was not added to an aged infertile egg in Example 2 of the present invention.
- FIG. FIG. 10 is a pie chart showing the chromosomal composition of a 2-cell stage embryo (Example) when MG132 was added to an aged infertile ovum in Example 2 of the present invention.
- FIG. 9 is a table showing details of the pie chart shown in FIG. 8;
- CO 2 carbon dioxide
- rice field Normal unfertilized rat oocytes lose their developmental potential after fertilization when the mother is exposed to CO 2 for only about 2 minutes. This phenomenon was an infertility that resulted in developmental failure through dispersion of female chromosomes due to spontaneous activation of eggs while maintaining fertilization ability with sperm.
- proteasome inhibitor a reversible ubiquitin proteasome inhibitor
- the inventors have found that treating ova with proteasome inhibitors according to the present embodiments to treat infertility, such as those associated with spontaneous activation and dispersal of the female pronucleus, reduces spontaneous activation.
- the present inventors have found that fertilized eggs can develop normally and offspring can be obtained as a result, leading to the completion of the present invention. Specifically, by in vitro fertilizing eggs collected from mothers exposed to CO 2 using a medium containing two types of proteasome inhibitors MG132 or ALLN, the birth rate of 1 to 2% was reduced to 20%. It has been found that it can be increased to ⁇ 30%.
- the asymmetric division inhibitor according to the present embodiment can be expected to improve the birth rate associated with childbirth at an advanced age.
- BEST MODE FOR CARRYING OUT THE INVENTION will be described in more detail by way of embodiments.
- An asymmetric division inhibitor according to an embodiment of the present invention is characterized by containing a spontaneous activation inhibitor that inhibits asymmetric division caused by spontaneous activation of pluripotent cells.
- the chromosomal aneuploidy suppressing agent according to the embodiment of the present invention is characterized by containing a spontaneous activation suppressing agent that suppresses asymmetric division due to spontaneous activation of pluripotent cells. This spontaneous activation is caused by prefertilization CO2 exposure or aging if the cell is an egg. Due to this spontaneous activation, the ovum enters a state in which the cell cycle has progressed from the MII stage in which nuclear division has stopped.
- the female pronucleus disperses, that is, it is accompanied by chromosomal dispersal. If the cell divides up to 2-cells without resolving this chromosomal dispersion, the chromosomal asymmetry will occur, causing abnormalities in subsequent cleavage, embryogenesis, etc., and development will stop. In other words, no offspring can be obtained, resulting in infertility. Therefore, in the present embodiment, by including this spontaneous activation suppressor, it functions as a suppressor that suppresses asymmetric division of chromosomes. In addition, in this embodiment, the spontaneous activation suppressing agent can also function as a chromosome aneuploidy suppressing agent when asymmetric division occurs by the same mechanism as spontaneous activation.
- the spontaneous activation inhibitor may be a proteasome inhibitor.
- a proteasome inhibitor By using a proteasome inhibitor in this way, it is possible to suppress asymmetric division by suppressing spontaneous activation and/or inducing reassortment of dispersed chromosomes.
- Proteasome inhibitors when activated through fertilization, inhibit the degradation of proteins that are required for the normal transport and alignment of the endoskeleton, spindle fibers, and other chromosomes through the steps that occur as the cell cycle progresses. It is conceivable that asymmetric division may be suppressed by suppressing progression.
- the proteasome inhibitor may contain MG132 or ALLN.
- MG132 CAS 133407-82-6), ALLN (CAS 110044-82-1), etc.
- ALLN CAS 110044-82-1
- the proteasome inhibitor may contain MG132 or ALLN.
- MG132 CAS 133407-82-6), ALLN (CAS 110044-82-1), etc.
- Concerning the dose it is possible to use such a concentration as to produce a normal proteasome inhibitory effect.
- MG132 can be used at a final concentration of 1 to 20 ⁇ M
- ALLN can be used at a final concentration of 10 to 50 ⁇ M, etc., so that the optimum concentration can be set and used. be.
- proteasome inhibitors other than MG132 and ALLN can be used as proteasome inhibitors.
- proteasome inhibitors include low-molecular-weight compounds that bind to ubiquitin or proteasomes, or that inactivate or degrade ubiquitin or proteasomes themselves; synthetic molecules such as proteins, peptides, nucleic acids, and PNAs (peptide nucleic acids); including.
- these proteins and peptides include ubiquitin or proteasome neutralizing antibodies that inhibit binding to ubiquitin or proteasome, peptides similar or similar to ubiquitin or proteasome binding sites, ubiquitin-specific degrading enzymes, cleaving enzymes, etc. including.
- the antibody may be an antibody-like peptide, PNA, etc. that has been truncated (shortened) to change the molecular weight, added with various tag molecules, or modified.
- this proteasome activity inhibitor may be a peptide composition such as a synthetic inactive ubiquitin-like peptide, a peptide containing a proteasome binding site, and the like.
- the spontaneous activation inhibitor of the present embodiment is not particularly limited, and includes, for example, low-molecular-weight compounds, nucleic acids, amino acids, proteins, and various other organic compounds.
- spontaneous activation suppressing agent it may also be possible to use temperature adjustment, addition of a magnetic field, an electric field, radiation, or other methods for physically inhibiting protein degradation. .
- asymmetric division inhibitor according to the embodiment of the present invention can be used in combination with other compositions and the like.
- the cells with pluripotency are pluripotent stem cells, germ cells, ova, or embryos.
- the present inventors have made preliminary observations that even during the generation of pluripotent stem cells, unequal division occurs and the number and length of chromosomes in the nucleus, the karyotype, etc. are likely to differ from those of normal cells.
- the asymmetric division inhibitor according to the present embodiment makes it possible to induce suppression of spontaneous activation and/or reassortment of dispersed chromosomes in pluripotent stem cells, germ cells, eggs, or embryos. , it becomes possible to proceed with division, generation, development, etc. as normal cells.
- the pluripotent stem cells of the present embodiment include multipotent stem cells that can differentiate into various cells in organisms such as mammals including humans and other vertebrates.
- the pluripotent stem cells of the present embodiment can be passaged, maintain a state in which differentiation does not progress even after passage, and are difficult to change in karyotype or the like, or are difficult to change in epigenetic phenotype. It is preferred to have the properties Also, in relation to this, the pluripotent stem cells of the present embodiment preferably have sufficient proliferative ability in vitro or in vivo.
- pluripotent stem cells of the present embodiment include embryonic stem cells (hereinafter referred to as "ES cells”), induced pluripotent stem cells (hereinafter referred to as “iPS cells”). cells”), other artificially generated or selected stem cells with pluripotency, and the like.
- ES cells embryonic stem cells
- iPS cells induced pluripotent stem cells
- cells other artificially generated or selected stem cells with pluripotency, and the like.
- pluripotent stem cells of this embodiment are stem cells created by reprogramming somatic cells with various vectors such as retroviruses, adenoviruses, and plasmids containing specific genes, RNA, low-molecular-weight compounds, etc. good too.
- the pluripotent stem cells of this embodiment may be cells used for processing prior to being reprogrammed and generated.
- the pluripotent stem cells according to the present embodiment are preferably totipotent, they do not necessarily have totipotent pluripotency. Furthermore, as the pluripotent stem cells according to the present embodiment, it is also possible to use naive cells with higher pluripotency than usual.
- the pluripotent stem cells of the present embodiment can be generated as individuals by chimerizing with germ cells described below, or can be differentiated into various cells of tissues and organs without being differentiated to that extent. It is preferable to have differentiation capacity.
- the pluripotent stem cells of the present embodiment are obtained or cryopreserved after being cultured on feeder cells or on a cell culture plate or the like coated with a basement membrane matrix such as collagen and maintained. It is possible to obtain
- the pluripotent stem cells of the present embodiment include cells prepared from cells obtained from disease patients, cells that serve as models for other diseases, cells incorporating a reporter gene (reporter cells), and conditioned cells. Cells capable of null knockout or knockin, other genetically modified cells, and the like may also be used.
- This genetic recombination includes addition, modification and deletion of genes in chromosomes, addition of genes by various vectors and artificial chromosomes, changes in epigenetic control, addition of artificial genetic materials such as PNA, and other genetic recombination.
- Germ cells of this embodiment include primordial germ cells, spermatogonia, oocytes, pre-meiotic germ cells, other germ cell-derived cells, egg cells, sperm, cells capable of parthenogenesis, Cells with teratoma-forming ability that are different from pluripotent stem cells, and other cells that may be manipulated into ontogeny are included.
- the ovum of the present embodiment includes matured ovum in the MII stage, ovum obtained in the previous stage in a harvestable state, ovum generated from primordial germ cells, oocytes, etc., and pluripotent stem cells. Including generated egg-like cells.
- the ovum of the present embodiment also includes cells that can be fertilized or parthenogenetic and that can develop into embryos, embryoid bodies, and individuals (offspring) in some form.
- the fertilized egg of this embodiment is a fertilized egg obtained by fertilizing an animal egg and a sperm, a parthenogenetic egg, and other developable oocyte.
- This fertilized egg may be the egg according to the present embodiment fertilized by in vitro fertilization or microinsemination, and is a cell obtained at the time when cleavage is initiated and has totipotency (fertilized egg clone cell) etc., or the one that has been frozen and thawed may be used.
- other types of fertilized eggs commonly used by those skilled in the art can be used.
- the mammalian cells of the present embodiment are mammalian embryos whose genetic information has been processed by techniques such as transgenics, gene knockouts, conditional knockouts, etc., in which genes are introduced by genetic recombination techniques using various vectors and the like. There may be.
- This processing of genetic information includes the introduction or removal of genes into the genome by genome editing, etc., or the introduction of genes into extrachromosomal sites such as plasmids and artificial chromosomes. It may be epigenetic regulation such as regulation or histone modification.
- RNA, PNA (Peptide Nucleic Acid), addition of other artificial bases, triple helix formation, etc. may be used, and other various genetic information processing techniques may be used.
- the cells of the present embodiment may be those selected in the form of colonies or the like using various markers or visual observation.
- the cells of this embodiment may contain a mixture of cells in various states of differentiation and development. That is, each cell may be in a stage of development and not sufficiently differentiated or immature.
- Animals to which cells according to this embodiment belong are not particularly limited, and include a wide range of vertebrates and invertebrates. Vertebrates include fish, amphibians, reptiles, birds, and mammals. Mammals to be treated for embryos of this embodiment are, for example, Primates, Rodentia, Lagomorpha, Cetartiodactyla, Perissodactyla, or carnivorous It is derived from the order Carnivora and can be treated, for example, with the in vitro fertilization medium of the present embodiment prepared for different embryos for each order and species. Embryos of placental mammals of Eutheria, including rare mammals other than these orders, are all applicable to the embryo treatment of the present embodiment.
- Animals to which the cells according to the present embodiment belong may be described from a different point of view than the above-mentioned order unit, for example, humans (Homo sapiens), experimental animals, domestic animals, companion animals, and the like.
- experimental animals include mice (Mus musculus), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), guinea pigs (Cavia porcellus) and the like as rodents.
- Animals of the order Lagomorpha include rabbits (Leporinae Trouessart) and the like.
- Livestock includes, for example, cetacean artiodactyl animals such as pigs (Sus scrofa domesticus), cattle (Bos taurus), and sheep (Ovis aries).
- Perissodactyla animals include horses (Equus caballus) and the like.
- Companion animals include cats (Felis silvestris catus), dogs (Canis lupus familiaris), ferrets (Mustela putorius), etc., as carnivorous animals.
- non-human primate animals include apes such as Gorilla, Pan troglodytes, Macaca mulatta, other Simiiformes, and other primates.
- heterologous animals of different species are also included.
- the animal according to the present embodiment may be a hybrid, an inbred strain that is a system of animals and plants obtained by continuous inbreeding for 20 or more generations, or the like. Further, it may be a mutant (genetic mutation) animal such as a nude mouse, a predetermined disease model animal, a heterologous animal that has changed as a species, a hybrid animal between species, or the like. It also includes artificially improved breeds, animals fixed as species, and subspecies. Furthermore, the classification of laboratory animals, domestic animals, companion animals, and the like described above is for convenience, and is used for different purposes, such as breeding purposes and medical purposes. In addition, the animal of the present embodiment includes a wide range of animals such as chordates, mollusks, annelids, arthropods, etc., which undergo egg cleavage and development, even if they are invertebrates.
- the cells of this embodiment may be subjected to various manipulations necessary for developmental engineering and the like.
- This manipulation includes not only the manipulation of the cell itself, but also manipulation of the maternal body, reproductive cells, etc. associated with the acquisition of the cell.
- the manipulation of the cells of the present embodiment includes, for example, superovulation treatment to the mother for easy supply of embryos, freezing or thawing of embryos accompanying the transfer of frozen embryos, dissociation of cells, nuclear transfer (Nuclear Transplantation, NT), microinsemination (intracytoplasmic sperm injection, in vitro fertilization, intracytoplasmic sperm injection, ICSI), microinjection (MI), electroporation (electroporation, EP), and the like.
- operations such as cell fusion are also included in the operations of this embodiment.
- Cell fusion and the like also include preparation of polyploid cells such as aneuploids and tetraploids.
- manipulations to cells of the present embodiment include various treatments such as osmotic pressure change, perforation with other chemical substances, perforation with forceps or the like, In Vitro Fertilization (IVF), and the like.
- treatments include, as described above, those performed to introduce nuclei, chromosomes, DNA, RNA, etc. into cells for purposes such as developmental engineering.
- This introduction may be performed using a variety of media.
- a DDS Drug Delivery System
- viral vectors may be constructed using viruses commonly used by those skilled in the art, such as adenovirus, adeno-associated virus, and retrovirus.
- the manipulation of the cells of this embodiment also includes, for example, the generation of genetically modified animals.
- This genetically-mutated animal includes, for example, techniques for producing gene-knockout, knock-in, conditional knock-in animals, and the like.
- the above-described operations can be performed to produce the above-described pluripotent stem cells, primordial germ cells, and the like.
- Various treatments related to immature ovo maturation in vitro maturation, IVM), embryo transfer (Blastocyst Transfer, BT), etc. may also be included in the operations of this embodiment.
- the medium according to the present embodiment is characterized by containing the asymmetric division inhibitor described above.
- This medium may be the medium for treating the cells described above. Specifically, it may be a medium for generating pluripotent stem cells, a medium for processing collected oocytes, or the like.
- the medium contains an asymmetric division inhibitor at a concentration appropriate for the purpose.
- the medium according to this embodiment may be an in vitro fertilization medium characterized by containing the asymmetric division inhibitor described above. That is, the in vitro fertilization medium according to the present embodiment is an in vitro fertilization medium for reducing spontaneous activation, and is characterized by containing a spontaneous activation inhibitor.
- the medium according to the present embodiment contains components according to the type, state, and density of cells, the type and content of manipulation, other conditions, and the like.
- the components may include, for example, components and water for composing the medium necessary for culturing the in vitro fertilization medium.
- the in vitro fertilization medium of the present embodiment may be used by adding pH buffer compounds, amino acids, vitamins, antioxidants, antibiotics, collagen precursors, trace metal ions and complexes, various salts, and the like. More specifically, the in vitro fertilization medium of the present embodiment may contain, for example, components of media commonly used by those skilled in the art to culture cells.
- a medium such as a general HTF medium, a modified HTF medium, and the like as an in vitro fertilization medium, and a general medium such as DMEM (Dulbecco's Modified Eagle Medium).
- DMEM Dulbecco's Modified Eagle Medium
- the medium may contain serum or various serum substitutes. The medium containing these various serum substitutes may be used in xeno-free (Xeno-Free, XF, or Animal Component-Free, ACF) culture systems.
- the medium according to this embodiment may also contain various RNAs, peptides, proteins, etc. for promoting development, differentiation, growth, and the like. These include various differentiation-inducing factors, growth factors, and the like.
- a low-molecular-weight compound for inducing differentiation such as retinoic acid, which is necessary depending on the type of operation, may be included.
- the in vitro fertilization medium of this embodiment may contain only components such as PBS that prevent the cells from dying in a short period of time.
- concentration of each agent contained in the in vitro fertilization medium of this embodiment can be appropriately set by a person skilled in the art according to the type, state, density of the culture, type and content of operation, and other conditions.
- concentration of the inhibitor of spontaneous activation of the present embodiment may be constant during each period during treatment, may be varied during each period, or may be varied stepwise during each period.
- cytochalasins and the like may be added in order to prevent physical damage during operations such as cell fusion, nuclear transfer, and injection.
- the treatment kit of this embodiment is characterized by containing the asymmetric division inhibitor described above.
- the processing kit of the present embodiment may contain solutions and culture media suitable for various operations, and reagents necessary for other operations.
- reagents include, for example, the probes and primers of the present embodiment, various enzymes, buffer solutions, washing solutions, dissolving solutions, test reagents, and the like.
- cells, containers, other materials, equipment, tools, etc. necessary for the operation of this embodiment may be added to provide a processing kit of this embodiment.
- the treatment kit of the present embodiments may include reagents, food, cages, drinking water, etc. for maintaining the developmental engineering products described below.
- a treatment kit comprising carriers and other reagents necessary for cell collection, treatment, transplantation, and the like. Furthermore, it is also possible to provide a treatment kit to which a spontaneous activation inhibitor, which can be added to a normal medium to complete an in vitro fertilization medium, and a manual describing concentrations and treatment methods, etc. are attached.
- the method for suppressing asymmetric division of the present embodiment is a method for suppressing spontaneous activation of pluripotent cells and suppressing asymmetric division of cells. Specifically, the method for inhibiting asymmetric division of the present embodiment is characterized by treating with an in vitro fertilization medium containing a spontaneous activation inhibitor at a specific concentration for a specific period of time.
- in vitro fertilization is performed with the in vitro fertilization medium containing the asymmetric division suppression according to the present embodiment, and the eggs and sperm are subjected to in vitro fertilization for about 1 to 5 hours. After this, in vitro fertilization is continued for 1 to 5 hours in a medium containing no asymmetric division suppression according to the present embodiment. Furthermore, 3 to 9 hours after insemination, the oocytes are washed and cultured continuously in a medium containing no asymmetric division inhibition. Then, by culturing overnight, it becomes possible to perform transplantation or the like into animals on the following day.
- the asymmetric division inhibitor according to the present embodiment is immersed in the cells, suppresses spontaneous activation, regulates the cell cycle, and disperses accompanying spontaneous activation.
- Spontaneous activation and chromosomal aneuploidy can be suppressed by inducing spontaneous chromosomal reassortment and suppressing pathways such as developmental arrest due to asymmetric division that would otherwise be triggered by spontaneous activation. It is speculated to recover from sterilization due to causative. Based on this, it is presumed that embryonic development arrest is suppressed by restoring the normal cell cycle and allowing the cells to repair themselves.
- the cells of this embodiment are pluripotent stem cells, they may be produced after being treated with an asymmetrical division inhibitor, or may be treated with an asymmetrical division inhibitor after production.
- the obtained pluripotent stem cells or the like may be induced to differentiate, cultured for a specific period of time, and obtained at any stage as cells, cell clusters, tissues, organs, or the like. These obtained embryos, tissues, and cells may be dissociated and processed. Alternatively, it may be used for treatment such as injection into a diseased site of a patient, or transplantation as at least part of a sheet, tissue or organ.
- the embryos, tissues, and cells of the present embodiment may be prepared into a single-layered or multi-layered sheet using culture equipment used by those skilled in the art, and then transplanted to the patient.
- the cells of the present embodiment can be cultured using an appropriate carrier or layered using a 3D printer or the like to transplant a more organized culture. That is, the transplantation method of this embodiment also functions as a treatment method of this embodiment.
- embryos or embryoid bodies generated from the cells subjected to the above-described treatment are obtained as the medicament (medical composition) of the present embodiment and used for treatment.
- the treatment method of this embodiment can be used as regenerative medicine to treat diseases of animals including humans.
- the in vitro fertilization method according to this embodiment is a method for in vitro fertilization of collected germ cells or eggs, which is treated by adding the asymmetric division inhibitor according to this embodiment, and fertilized eggs derived from germ cells or eggs Alternatively, it is characterized by suppressing asymmetric division of the embryo.
- the above-described method for suppressing asymmetric division can function as an in vitro fertilization method by treating germ cells, eggs, or embryos according to the present embodiment in the same manner as the method for suppressing asymmetric division described above. is.
- the infertility treatment method according to the present embodiment is a mammalian infertility treatment method, wherein the collected germ cells or eggs are treated by adding the asymmetric division inhibitor according to the present embodiment, and the germ cells or eggs It is characterized by suppressing asymmetric division of the derived fertilized egg or embryo. Furthermore, with this infertility treatment method, it is possible to obtain mammalian offspring with a high probability, even with CO 2 -exposed oocytes and aged oocytes.
- the method for obtaining offspring according to this embodiment is not particularly limited.
- the embryo of this embodiment can be obtained by internal fertilization, external fertilization, or nuclear transfer.
- the optimum values can be adjusted by those skilled in the art according to the animal species, strain, type of operation, etc.
- the cell division, development, and development by treating the cells of the present embodiment include any or any combination of individuals (offspring), organs, tissues, and cells (hereinafter referred to as "developmental engineering product” ).
- individuals include chimera individuals, model organism individuals, other experimental animal individuals, individuals required for reproductive engineering, individuals required for medical treatment, and the like.
- Organs and tissues do not necessarily have to be matured to the level of organs, as long as specific differentiated cells have a specific structure as a cell cluster.
- cells include dissociated cell aggregates without specific structures.
- the in vitro fertilization method, transplantation method, infertility treatment method, and offspring acquisition method of this embodiment can be used in reproductive medicine. As a result, for example, it can be applied to mothers in which it is difficult for embryos to settle due to genetic background, old age, various diseases, or the like. In this case, by transplanting the fertilized egg, embryo, or embryoid body according to this embodiment into the mother's womb, the transplantation method of this embodiment also functions as a therapeutic method.
- the in vitro fertilization method, the transplantation method, and the infertility treatment method of the present embodiment are applied to humans among mammals, they should be implemented within the necessary scope and limit in accordance with the ethics of various reproductive medicines. In other words, genetic modification, etc. should normally not be done, and even if genetic modification, etc. is necessary for the purpose, it should be done to a minimum extent according to specific criteria, such as the prevention of genetic diseases and infectious diseases. . Further, when the present invention is implemented in Japan, oocyte collection, implantation after in vitro fertilization, and treatment are performed by a doctor. Therefore, in Japan, the term "animal” in the therapeutic method of the present invention does not include Homo sapiens. On the other hand, in other countries, the definition of "animal” and “therapeutic method” is not limited.
- the in vitro fertilization method, transplantation method, infertility treatment method, and offspring acquisition method can also be used for animals other than humans for treatment.
- This animal is not particularly limited and includes a wide range of vertebrates and invertebrates. Vertebrates include fish, amphibians, reptiles, birds, and mammals. Specifically, the mammal is, for example, of the order Primates, Rodentia, Lagomorpha, Cetartiodactyla, or Perissodactyla, Carnivora, as described above. ) may be various animals.
- mice mice, rats, hamsters, guinea pigs, rabbits, sheep, pigs, cows, horses, dogs, cats, ferrets, non-human transgenic primates, and the like may be used.
- wild animals include fish, birds including poultry, and reptiles. It also broadly includes crustaceans including shrimps and insects, and other invertebrates such as squid. That is, in addition to humans, it can also be used for methods such as breeding of various animals.
- the developmental engineering product according to the embodiment of the present invention can be used as a therapeutic target for a part of the body of an animal, or an organ or tissue excised or excreted from an animal.
- this treatment is a treatment in a broad sense, and can be applied to bioreactors, culture in model animals, culture of human transplant-like cultured organs, and the like.
- the developmental engineering product of the present embodiment can be used for various therapeutic applications other than regenerative medicine, such as bioreactors, production of artificial organs, and production of individual clones.
- the processing method is appropriately selected according to various conditions such as the condition of the mother's womb and the condition of the subject. It can be selected and changed.
- MG132 manufactured by Merck millipore, MG132 (1 mg) in solution. 474791-1MGCN
- HTF medium manufactured by Arc Resources
- sperm and in vitro fertilization described below were incubated in this medium for 3 hours. gone.
- in vitro fertilization was continued for 3 hours in HTF medium without MG132.
- the ova were washed and continued to be cultured in HTF medium.
- immunostaining was performed at 6.5 hours, 10 hours, and 20 to 22 hours after insemination, and the cells were cultured overnight and transplanted to pseudopregnant rats the next day. This transplantation was carried out in accordance with a standard method, and a specific number was transplanted into each of the right and left fallopian tubes of pseudopregnant rats.
- ALLN (Merck millipore, 208719-5MGCN) was used at a final concentration of 25 ⁇ M. Other usage was the same as for MG132.
- in vitro fertilization (in vitro fertilization (IVF)) Caudal epididymal spermatozoa were collected in HTF medium and allowed to acquire fertilization capacity for 1 hour. Anesthetized (An.), after a predetermined time (90 seconds, 120 seconds, 150 seconds, etc.) after inhaling 99.9% CO 2 , cervical dislocation treatment (CO 2 (c.d.)) or nothing Cervical dislocation (c.d.) was performed. In each case, the ovum-cumulus cell complex was collected from the oviduct ampulla of the treated female and mixed with capacitation-treated sperm to initiate in vitro fertilization. After insemination to combine eggs and sperm, the eggs were fixed at each time and subjected to immunostaining and the like.
- Whole mount staining was performed according to a standard method. Oocytes were sandwiched between a slide glass and a cover glass, fixed with glutaraldehyde, dehydrated with ethanol, and then stained with orcein acetate for nuclei.
- FIG. 1A is a graph showing the birth rate (Birth rate (%)) of rats when fertilized eggs that were naturally mated (vivo) and unfertilized eggs were exposed to CO 2 for 3 minutes (vitro). . Although these rats die due to cessation of spontaneous respiration 90 seconds after the start of CO 2 exposure, they are completely euthanized by cervical dislocation immediately thereafter.
- FIG. 1B shows the results, the vertical axis is fertilization rate (%), and the horizontal axis is anesthesia mixed with three kinds of anesthesia (An.), CO 2 exposure (CO 2 ), euthanasia treatment with cervical dislocation. (c.d.). In anesthetized (An.), acquisition when alive (live) and acquisition after cervical dislocation (cd) were compared.
- the fertilization rate was examined by changing the CO 2 exposure time to 90 seconds, (90 s), 120 seconds (120 s), 150 seconds (150 s), 210 seconds (210 s), and 300 seconds (300 s).
- FIG. 1C shows the results of examining the relationship between CO 2 exposure time and birth rate.
- the vertical axis in FIG. 1C is the birth rate, and the horizontal axis is the same as in FIG. 1B.
- FIG. 1D shows 2-cells that were anesthetized (An.) and could undergo IVF, and 2-cells that could undergo IVF with oocytes collected after 150 seconds of CO 2 exposure (CO 2 150 s). 13.5-day embryos transplanted into the left and right oviducts are shown. As a result, although the eggs collected under anesthesia developed normally, the 2-cells derived from the eggs exposed to CO 2 did not even leave an implantation scar.
- FIG . 1E shows the results of culturing anesthetized and CO 2 -exposed oocytes for 3 hours after IVF and subjecting them to whole-mount staining to see what happens to oocytes upon exposure to CO 2 .
- the photograph of "An.” shows the staining results of eggs collected under anesthesia and subjected to IVF.
- the " CO2 " picture shows the staining results after 150 seconds of CO2 exposure.
- the arrow in the photograph of "An.” is a feature indicating that the ovum is in the MII stage, in which the first polar body is released during nuclear fission and the nuclear fission is stopped.
- spontaneous activation spontaneous activation, hereinafter also referred to as “s.a." occurs in the egg, and the second polar body (PB2) is released. It was confirmed that
- FIG. 1F is a graph showing the results of whole-mount staining of the anesthetized oocytes in FIG. 1E and the oocytes exposed to CO 2 for varying seconds after IVF, cultured for 3 hours.
- the vertical axis is the MII stage and the rate of spontaneous activation (s.a.), and the horizontal axis is the control cervical dislocation under anesthesia (An. (c.d.)), 90 seconds, 120 seconds. , respectively show the results with 150 seconds of CO 2 exposure. Numbers within each bar indicate the experimental rank. As a result, it was found that 60% of the eggs were spontaneously activated when exposed to CO 2 for 90 seconds, and most of them were spontaneously activated when exposed to CO 2 for 120 seconds or longer.
- cont (c.d.) is the control for cervical dislocation
- An (c.d.) is the cervical dislocation under anesthesia, 90 s after exposure to CO 2 (CO 2 90 s) for 3 cases.
- pCO2 is carbon dioxide partial pressure
- pO2 oxygen partial pressure
- cNa sodium concentration (mmol/L)
- cK is potassium concentration (mmol/L)
- Hct is electrical conductivity
- Temp temperature
- cHCO3 the bicarbonate index.
- a blood gas analysis revealed that the pH of the patient exposed to CO 2 was 6.6, indicating that acidosis had occurred.
- the oxygen partial pressure was normal, it was not in an oxygen-deficient state.
- the spontaneous activation was examined by whole-mount staining after exposing the ova to pH 6.5 medium for 10 minutes.
- the results are shown in FIG. 1G.
- the vertical axis indicates the ratio of stage MII (MII) and spontaneous activation (s.a.). Numbers within each bar indicate the number of experiments. As a result, no spontaneous activation occurred due to the lowered pH. Therefore, it was found that acidosis was not the cause of the spontaneous activation.
- FIG. 1H A photograph of this result is shown in FIG. 1H.
- FIG. 1I is a graph showing this result. The vertical axis indicates the rate of growth into embryos (developmental rate (%)). Numbers in each bar indicate the number of normal embryos/experimental number. As a result, the incidence of micro-insemination embryos derived from CO2 - exposed eggs was significantly lower despite the injection of a single sperm.
- FIG. 1J shows the results of IVF using sperm from Nanog-EGFP Tg rats (F344.W-Tg(Nanog-GFP,-PuroR)Kyo). Nanog is known to be expressed in morula stage embryos after fertilization. Conversely, Nanog is not expressed in parthenogenesis.
- the ovum collected under anesthesia (An.) and the ovum collected after CO 2 exposure for 120 seconds (CO 2 120 s) are micrographs (Phase) of the morula stage embryos in each embryo. , and photographs showing the expression of EGFP (EGFP), respectively.
- EGFP values indicate the number of embryos that captured fluorescence/the number of embryos.
- about half of the blastocysts of the CO2 - exposed eggs and the eggs collected under anesthesia were GFP-positive and Nanog was expressed. From this, it was found that embryos produced by CO 2 exposure were produced by fertilization with sperm rather than by parthenogenesis.
- mice in vitro fertilization and CO2 exposure Next, by exposing mouse eggs to CO 2 during harvesting, and using CO 2 during euthanasia, the eggs spontaneously activate, resulting in impaired post-fertilization developmental potential. was examined to see if it was rat-specific. Mice were also exposed to CO 2 for 90 seconds, 210 seconds, and 330 seconds by the same procedure as rats, and then eggs were collected and subjected to IVF. All mice, like rats, stopped spontaneous respiration and died 90 seconds after the start of CO 2 exposure. Egg collection was performed at each subsequent hour. As a control, an egg collection was also performed 240 seconds after death by cervical dislocation under anesthesia (An. (c.d.)) (section).
- FIG. 1K shows the 2-cell rate (%).
- FIG. 1L shows the rate of development into embryos after implantation (developmental rate (%)). Numbers within each bar indicate the respective number/number of experiments. As a result, in mice, exposure to CO 2 had a profound effect not only on developmental but also on fertility.
- FIG. 1M shows 2-cells transplanted into the left and right fallopian tubes as in FIG. 1D. Compared with the control (An.), the 2-cells (CO 2 330s) obtained with exposure for 330 seconds did not produce any scars upon implantation.
- FIG. 2A is a conceptual diagram of immunostaining with DAPI (4′,6-diamidino-2-phenylindole), which stains the nucleus, and H3K9me3, a histone protein that attaches to female nucleic acids, to examine the state of eggs. , shows an actual dyed photograph.
- the conceptual diagram on the left shows the concept of the state of staining according to the state of the ovum.
- MII Metaphase II
- AII Anaphase II
- TII Telophase II
- Sc chromosome dispersal. Scattered state and "sa” indicate examples of spontaneous activation states, respectively.
- the above abbreviations have the same meanings.
- Fig. 2B shows the eggs collected under control anesthesia
- Fig. 2C shows the ratio of the conditions after IVF and confirmed by immunostaining with DAPI and H3K9me3 for the eggs exposed to CO 2 for 120 seconds (120s). , respectively.
- the horizontal axis indicates the time after insemination
- the vertical axis indicates the confirmed chromosomal state of the egg (chromosome status (%)).
- the letters in each bar indicate the same state as in FIG. 2A described above, and "Fz" indicates the state of fertilization.
- the ova collected under control anesthesia stopped at the MII stage for about 4.5 hours after insemination, and then entered the fertilized (Fz) state.
- oocytes exposed to CO2 did not remain arrested at MII, spontaneous activation occurred from 0.5-1.5 hours after insemination, and the chromosomes dispersed over time. It was gone. That is, rat oocytes undergo spontaneous activation to chromosomal dispersal upon exposure to CO 2 . After 3 hours after egg retrieval, the chromosomes were dispersed in all the eggs. However, 5.5 hours after insemination, the egg with dispersed chromosomes was found to be fertilized with the sperm (Fz). That is, even when spontaneous activation occurred, fertilization was possible even when the chromosomes were dispersed.
- FIG. 2D shows eggs harvested under control anesthesia (An.) and eggs exposed to CO 2 for 120 seconds (CO 2 ) at 6.5 hours (6.5 h), 10 hours (10 h), The state of immunostaining by DAPI, H3K9me3, and co-staining (Merge) in each state at 20 to 21 hours (20-21 h) is shown.
- Arrowheads indicate female pronuclei.
- Arrows indicate the male nucleus or male pronucleus.
- CO2 - exposed oocytes had dispersed female pronuclei when stained 6.5 hours after insemination.
- the chromosomes were dispersed to form dispersed female pronuclei.
- FIG. 2E shows the number of female pronuclei at 6.5 hours (6.5 h) after insemination in the retrieved oocytes (An.) and CO2 - exposed oocytes ( CO2 ) collected under anesthesia in Figure 2D above. is plotted.
- FIG. 2F plots the number of female pronuclei at 20-21 hours (20-21 h) as in FIG. 2E.
- the vertical axis indicates the number of female pronuclei (No. of female pronuclei)
- each circle indicates the number
- the thick line indicates the median value
- the thin square line indicates the standard deviation (1 ⁇ ), which is rounded down to less than 1. Show each thing.
- most of the oocytes collected under anesthesia had a single female pronucleus, but most of the CO 2 oocytes had 2 to 7 dispersed female pronuclei.
- FIG. 2G is the result of immunostaining 24 hours after insemination. Twenty-four hours after insemination, both the embryos derived from the eggs collected under anesthesia (An.) and the embryos derived from the eggs exposed to CO 2 (CO 2 ) crossed male and female pronuclei and entered the M phase.
- PB2 indicates a second polar body.
- it pauses as the MII phase in the middle of the second division of meiosis, is activated with the entry of sperm, and the cell cycle restarts. At this time, the second polar body is confirmed in the fertilized egg.
- HDV Hidden Degradation of Viable Oocytes
- FIG. 3A is a photograph of karyotype analysis when eggs collected from control cervical dislocation under anesthesia (cd) were subjected to IVF and developed into 2-cells. When the ovum collected under anesthesia is fertilized and becomes 2-cell, most of the blastomeres have 42 chromosomes.
- FIG. 3B is a photograph of karyotype analysis when eggs exposed to CO 2 for 120 seconds were subjected to IVF and developed into 2-cells. In embryos that are fertilized into 2-cells after being exposed to CO 2 in this way, the number of chromosomes is heterogeneous for each blastomere. That is, in one 2-cell, each blastomere had a different number of chromosomes.
- FIG. 3C is a time-series photograph of ova exposed to CO 2 for 120 seconds and then subjected to IVF to develop into 2-cells, followed by real-time observation by live imaging with an H2B-mCherry probe. It can be seen that eggs exposed to CO 2 intersect with male nuclei and are fertilized while multiple female pronuclei remain scattered.
- arrowheads indicate dispersed chromosomes and arrows indicate female pronuclei. After that, it divides and reaches the 2-cell stage, but at that time, it can be seen that the microchromosome is incorporated into only one blastomere.
- FIG. 3D counts such chromosome segregation errors.
- the ovaries collected under control anesthesia (An.) and the ova exposed to CO 2 for 120 seconds (CO 2 120s) the vertical axis represents the chromosome segmentation error rate (chromosome segmentation error (%)).
- Gray areas indicate 2-cells with distribution errors. Unfilled areas indicate 2-cells with no distribution error. Numbers in bars indicate respective counts. In this way, counting chromosome segregation errors, chromosomal asymmetry was confirmed in about 45% of the eggs exposed to CO 2 . That is, in the CO 2 -exposed oocytes, chromosome asymmetry occurred at the stage of division into 2-cells after IVF.
- 4A shows the state of chromosomes in the state of MG132 addition, the vertical axis indicates the state of chromosomes (chromosome status (%)), and the horizontal axis indicates the time from insemination. Each number in the bar indicates the number counted. Spontaneous egg activation (s.a.) was significantly reduced and more eggs remained in the MII stage. However, it was found that 3 hours after insemination, when MG132 was removed, spontaneous activation occurred, and although chromosome dispersion occurred, fertilization occurred.
- FIG. 4B shows temporal changes in the state of chromosomes when MG132 was added.
- the vertical axis indicates the chromosome status (chromosome status (%)). Each number in the bar indicates the number counted. Even with the addition of MG132, about half of the fertilized eggs were fertilized with scattered chromosomes.
- FIG. 4C shows DAPI in each state 6.5 hours (6.5 h), 10 hours (10 h), 20-22 hours (20-22 h), 24 hours (24 h) after insemination for those with MG132 addition, The state of immunostaining by H3K9me3, co-staining (Merge) is shown. Arrowheads indicate the second polar body (PB2).
- Figure 4D shows the number of female pronuclei (No. of female pronuclei) to which MG132 was added in the same format as Figure 2E.
- FIG. 4E is a photograph observed in real time by live imaging after MG132-added 2-cells were generated.
- FIG. 4F shows the rate of suppression of chromosome dispersion in 2-cell stage embryos by the addition of MG132 to CO 2 -exposed embryos.
- Distribution error rates are shown, and 2-cells with distribution errors are shown in gray areas. Unfilled areas indicate 2-cells with no distribution error. Numbers in bars indicate respective counts. "p” indicates the p-value of the statistical test. From these results, it was found that MG132 improved chromosomal asymmetry in 2-cells and ameliorated chromosomal aneuploidy.
- FIG. 4G shows a comparison between the addition of MG132 and no MG132.
- the collected eggs, the collected eggs exposed to CO 2 for 120 seconds (CO 2 120s), and the eggs exposed to CO 2 for 150 seconds (CO 2 150s) were treated with MG132 (+) and not (-). shows the results for Thus, even the CO 2 -exposed eggs were able to develop and produce offspring when treated with the proteasome inhibitor MG132.
- the fertility rate also increased with the addition of MG132 to the ova of the control cervical dislocation under anesthesia. This indicates that MG132 may be used for promoting pregnancy (fertility treatment).
- FIG. 4I shows the results of immunostaining of CO 2 only (CO 2 ( 120 s)) and ALLN-added (CO 2 ( 120 s) + ALLN) co-stained with DAPI, H3K9me3 (Merge). .
- the arrowheads in the figure show the appearance of the female pronucleus, and if there are more than one, they are dispersed.
- FIG. 4J shows the number of female pronuclei counted in 2-cells (No. of female pronuclei) in a format similar to that of FIG. 2E. As a result, similar to the MG132-added group, it was clearly observed that the female pronuclei were organized 20 to 22 hours after insemination.
- FIG. 4K is a photograph of the offspring actually obtained, and FIG. 4L shows the birth rate with (+) and without (-) addition of ALLN.
- FIG. 4K is a photograph of the offspring actually obtained
- FIG. 4L shows the birth rate with (+) and without (-) addition of ALLN.
- FIG. 5A shows the age of the rat mother, 1 month old (1: juvenile), 6 months old (6: before 40 years old in terms of human reproductive age), 8 to 10 months old (8-10: 40 years old or later in terms of human reproductive age), the results of investigating the spontaneous activation state of eggs collected under anesthesia are shown.
- the vertical axis indicates the percentage of MII stage (MII) and spontaneous activation (sa) (Percentage of MII and sa (%)).
- the numbers in each bar indicate the number of counts.
- Figure 5B shows fertility rates at these ages. As described above, it was found that spontaneous activation occurred in about half of the eggs at 6 months of age and more than 70% at 8 months of age even though the eggs were collected under anesthesia. Also, when in vitro fertilization was performed with these eggs, the fertilization rate itself did not change much, as with the CO 2 -exposed eggs described above.
- FIG. 5C is a photograph of immunostaining with DAPI and H3K9me3 when ova were obtained under anesthesia from a 9-month-old infertile rat and MG132 was added.
- the treatment for adding MG132 at this time was the same as the treatment for the eggs exposed to CO 2 for 120 seconds as described above.
- the figures show the conditions 20 to 22 hours after insemination with (+) and without (-) the addition of MG132.
- Arrowheads indicate female pronuclei
- arrows indicate male pronuclei.
- PB2 indicates the second polar body.
- FIG. 5D shows the number of female pronuclei in 2-cells (No.
- FIG. 5E shows a photograph of the offspring actually obtained.
- FIG. 5F shows birth rates (Birth rate (%)) at 6 months old (6), 8-10 months old (8-10), with (+) and without (-) addition of MG132.
- the litter rate of 8- to 10-month-old rats decreased to 10% in the absence of MG132 treatment.
- MG132 can restore (rescue) the litter rate to the same level as that at young age. This made it possible to ameliorate the low litter rate of older births.
- HDV ⁇ Even if eggs are collected after inhaling CO 2 , or just adding (immersing) the proteasome inhibitor ALLN and performing IVF, HDV can be recovered and offspring will be born.
- HDV was also treatable with proteasome inhibitors other than MG132.
- ⁇ HDV also occurs in aging, which is the biggest factor in infertility.
- treatment with HTF supplemented with proteasome inhibitors could completely cure HDV. As a result, it is possible to realize a safe and reliable elderly childbirth with a high birth rate even at an old age.
- Fig. 6 shows the single cell whole genome sequence ( It is a graph which shows the result of having performed scWGS). That is, we decoded the whole genome of each blastomere and elucidated the chromosomal organization.
- MG132(-) on the left side of FIG. 6 is a graph showing the results of a comparative example in which MG132 processing is not performed.
- scWGS was performed on 7 2-cell stage embryos, ie 14 blastomeres.
- Ap indicates aneuploidy
- Ep indicates normal chromosome number (Euploid).
- aneuploidy occurred in 10 of 14 (71.4%) blastomeres.
- MG132(+) on the right side of FIG. 6 is a graph showing the results of the example in which the MG132 treatment was performed. In this example, MG132 treatment reduced aneuploidy to 6 (27.3%) of 22 blastomeres.
- the graph in FIG. 7 shows in detail the configuration of MG132 untreated (comparative example) in FIG.
- Each pie chart represents 2-cell stage embryos
- 7 pie charts represent 7 2-cell stage embryos untreated with MG132
- the inner circle in each graph is one blastomere
- the outer circle is Another blastomere is shown.
- the chromosomes the number of 20 autosomes+X chromosomes is indicated by the difference in density. Black indicates that the chromosome was duplicated (normal), gray indicates that it was not duplicated.
- the letters in the center of the circle are in lower case, for example, in the case of "a, b", they indicate embryos that have not been treated with MG132.
- the graph of FIG. 8 details the configuration of the MG132 process (example) of FIG.
- Each pie chart represents 12 two-cell stage embryos treated with MG132 in the same manner as in FIG.
- the letters in the center of the pie chart are capitalized like "A, B", and similarly, males are indicated as “male” and females as “female”.
- males are indicated as “male” and females as “female”.
- only two in the lower right are all gray blastomeres, but this blastomere could not be analyzed.
- FIG. 9 shows the drawing of FIG. 8 in more detail regarding the number of chromosomes in each blastomere.
- each column with a gray frame indicates that there were other than two chromosomes.
- an asymmetric division inhibitor that improves the birth rate due to childbirth at an advanced age, and is industrially applicable.
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Abstract
Provided is an asymmetric cell division inhibitor with which infertility due to aging or the like can be ameliorated. The asymmetric cell division inhibitor includes a spontaneous activation inhibitor that inhibits asymmetric cell division caused by the spontaneous activation of cells having pluripotency. When the cells are reproductive cells or ova, spontaneous activation occurs due to aging or exposure to carbon dioxide before fertilization. The spontaneous activation is accompanied by the dispersion of chromosomes. Therefore, the asymmetric cell division inhibitor inhibits asymmetric cell division by inducing the inhibition of spontaneous activation and/or the reaggregation of dispersed chromosomes. Specifically, the spontaneous activation inhibitor may be a proteasome inhibitor, and includes MG132 or ALLN. The treatment in an in vitro fertilization medium containing the collected asymmetric cell division inhibitor can inhibit the asymmetric cell division of reproductive cells or fertilized ova.
Description
本発明は、特に、哺乳類の細胞の不等分裂抑制剤、染色体異数性抑制剤、培地、体外受精培地、細胞処理キット、不等分裂抑制方法、体外受精方法、移植方法、不妊治療方法、及び産仔取得方法に関する。
In particular, the present invention provides mammalian cell asymmetric cell division inhibitors, chromosomal aneuploidy inhibitors, media, in vitro fertilization media, cell treatment kits, asymmetric division inhibition methods, in vitro fertilization methods, transplantation methods, infertility treatment methods, and a method for obtaining offspring.
近年、出生率の低下は女性の社会進出が進む先進諸国において大きな社会問題となっている。日本産科婦人科学会による最新の統計では、晩婚化等で不妊に悩む夫婦が増えており、２０１９年には４５万件以上もの不妊治療が行われている。我が国では全出生児の１４人に１人（６０５９８人）が体外受精由来である。このように不妊治療は少子化を回復する術として有効であるため、我が国の政府は、不妊治療に保険適用をすることになってきている。
ここで、ヒトの女性では、４０歳以上になると体外受精後の妊娠率が１３．４％、出生率はわずかに６．７％となり、多くの胚が移植後に流産している。 In recent years, the declining birth rate has become a major social problem in developed countries where women's social advancement is progressing. According to the latest statistics from the Japan Society of Obstetrics and Gynecology, the number of couples suffering from infertility is increasing due to factors such as late marriage, and more than 450,000 cases of infertility treatment were performed in 2019. In Japan, 1 out of 14 (60,598) out of all births are derived from in vitro fertilization. In this way, fertility treatment is effective as a means of recovering from the declining birthrate, so the Japanese government has decided to cover fertility treatment with insurance.
Here, in human females over the age of 40, the pregnancy rate after in vitro fertilization is 13.4%, and the live birth rate is only 6.7%, and many embryos miscarry after transplantation.
ここで、ヒトの女性では、４０歳以上になると体外受精後の妊娠率が１３．４％、出生率はわずかに６．７％となり、多くの胚が移植後に流産している。 In recent years, the declining birth rate has become a major social problem in developed countries where women's social advancement is progressing. According to the latest statistics from the Japan Society of Obstetrics and Gynecology, the number of couples suffering from infertility is increasing due to factors such as late marriage, and more than 450,000 cases of infertility treatment were performed in 2019. In Japan, 1 out of 14 (60,598) out of all births are derived from in vitro fertilization. In this way, fertility treatment is effective as a means of recovering from the declining birthrate, so the Japanese government has decided to cover fertility treatment with insurance.
Here, in human females over the age of 40, the pregnancy rate after in vitro fertilization is 13.4%, and the live birth rate is only 6.7%, and many embryos miscarry after transplantation.
これについて、特許文献１を参照すると、生殖補助医療において、卵子の成熟を促す方法が記載されている。この技術は、卵巣過剰刺激症候群（ＯＨＳＳ）の比率を低下させたり、同等の妊娠率を実現するかまたは妊娠率を向上させたり、妊娠までの期間を短縮したり、及び早発排卵を阻止したりするために、医薬成分の２－（Ｎ－アセチル－Ｄ－チロシル－トランス－４－ヒドロキシ－Ｌ－プロリル－Ｌ－アスパラギニル－Ｌ－スレオニル－Ｌ－フェニルアラニル）ヒドラジノカルボニル－Ｌ－ロイシル－Ｎω－メチル－Ｌ－アルギニル－Ｌ－トリプトファンアミド又はその製薬学的に許容可能な塩を治療上有効な量投与する工程を含む方法である。
Regarding this, referring to Patent Document 1, a method for promoting egg maturation in assisted reproductive medicine is described. This technology reduces the rate of ovarian hyperstimulation syndrome (OHSS), achieves equal or improved pregnancy rates, shortens time to conception, and prevents premature ovulation. the pharmaceutical ingredient 2-(N-acetyl-D-tyrosyl-trans-4-hydroxy-L-prolyl-L-asparaginyl-L-threonyl-L-phenylalanyl)hydrazinocarbonyl-L-leucyl -Nω-methyl-L-arginyl-L-tryptophanamide or a pharmaceutically acceptable salt thereof in a therapeutically effective amount.
しかしながら、特許文献１に記載されたような技術にもかかわらず、そもそも、何故、高齢化により胚が流産するのかの原因が判明していなかった。原因が判明していないため、出生率を劇的に改善させるような技術も開発されていなかった。
However, despite the technology described in Patent Document 1, the cause of embryo miscarriage due to aging has not been clarified in the first place. Because the cause is unknown, no technology has been developed to dramatically improve the birth rate.
本発明は、このような状況に鑑みてなされたものであり、上述の課題を解消することを課題とする。
The present invention has been made in view of such circumstances, and aims to solve the above-mentioned problems.
本発明の不等分裂抑制剤は、多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含むことを特徴とする。
本発明の不等分裂抑制剤は、前記自発的活性化は、染色体の分散を伴うものであることを特徴とする。
本発明の不等分裂抑制剤は、前記不等分裂の抑制は、前記自発的活性化の抑制及び／又は分散した前記染色体の再集合を誘導することを特徴とする。
本発明の不等分裂抑制剤は、前記細胞は、多能性幹細胞、生殖細胞、卵子、又は胚であることを特徴とする。
本発明の不等分裂抑制剤は、前記細胞が前記生殖細胞又は前記卵子である場合、前記自発的活性化は、受精前の二酸化炭素への暴露又は加齢によるものであることを特徴とする。
本発明の不等分裂抑制剤は、前記自発的活性化抑制剤は、プロテアソーム阻害剤であることを特徴とする。
本発明の不等分裂抑制剤は、前記プロテアソーム阻害剤は、ＭＧ１３２を含むことを特徴とする。
本発明の不等分裂抑制剤は、前記プロテアソーム阻害剤は、ＡＬＬＮを含むことを特徴とする。
本発明の染色体異数性抑制剤は、多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含むことを特徴とする。
本発明の培地は、前記不等分裂抑制剤を含むことを特徴とする。
本発明の体外受精培地は、前記不等分裂抑制剤を含むことを特徴とする。
本発明の細胞処理キットは、前記不等分裂抑制剤を含むことを特徴とする。
本発明の不等分裂抑制方法は、多能性を備える細胞の自発的活性化を抑制し、前記細胞の不等分裂を抑制することを特徴とする。
本発明の体外受精方法は、採取された生殖細胞又は卵子の体外受精方法であって、前記不等分裂抑制剤を添加して処理し、前記生殖細胞、前記卵子、又は胚の不等分裂を抑制することを特徴とする。
本発明の移植方法は、前記体外受精方法により処理された生殖細胞又は卵子由来の受精卵又は胚を移植することを特徴とする。
本発明の不妊治療方法は、哺乳類の不妊治療方法であって、採取された生殖細胞又は卵子を、前記不等分裂抑制剤を添加して処理し、前記生殖細胞又は前記卵子由来の受精卵又は胚の不等分裂を抑制することを特徴とする。
本発明の産仔取得方法は、前記移植方法、又は前記不妊治療方法により哺乳類の産仔を取得することを特徴とする。 The asymmetric division inhibitor of the present invention is characterized by containing a spontaneous activation inhibitor that inhibits asymmetric division caused by spontaneous activation of pluripotent cells.
The asymmetric division inhibitor of the present invention is characterized in that the spontaneous activation is accompanied by chromosomal dispersion.
The agent for suppressing asymmetric division of the present invention is characterized in that the suppression of the asymmetric division induces suppression of the spontaneous activation and/or reassortment of the dispersed chromosomes.
The asymmetric division inhibitor of the present invention is characterized in that the cells are pluripotent stem cells, germ cells, eggs, or embryos.
The asymmetric division inhibitor of the present invention is characterized in that, when the cell is the germ cell or the egg, the spontaneous activation is due to exposure to carbon dioxide before fertilization or aging. .
The asymmetric division inhibitor of the present invention is characterized in that the spontaneous activation inhibitor is a proteasome inhibitor.
The asymmetric division inhibitor of the present invention is characterized in that the proteasome inhibitor contains MG132.
The asymmetric division inhibitor of the present invention is characterized in that the proteasome inhibitor contains ALLN.
The chromosomal aneuploidy inhibitor of the present invention is characterized by containing a spontaneous activation inhibitor that suppresses asymmetric division due to spontaneous activation of pluripotent cells.
The medium of the present invention is characterized by containing the asymmetric division inhibitor.
The in vitro fertilization medium of the present invention is characterized by containing the asymmetric division inhibitor.
The cell treatment kit of the present invention is characterized by containing the asymmetric division inhibitor.
The method for suppressing asymmetric division of the present invention is characterized by suppressing spontaneous activation of pluripotent cells and suppressing asymmetric division of the cells.
The in vitro fertilization method of the present invention is a method of in vitro fertilization of collected germ cells or eggs, wherein the asymmetric division inhibitor is added and treated to prevent asymmetric division of the germ cells, eggs, or embryos. It is characterized by suppressing.
The transplantation method of the present invention is characterized by transplanting fertilized eggs or embryos derived from germ cells or eggs treated by the in vitro fertilization method.
The infertility treatment method of the present invention is a mammalian infertility treatment method, wherein the collected germ cells or eggs are treated by adding the asymmetric division inhibitor, and the germ cells or the fertilized eggs derived from the eggs or It is characterized by suppressing asymmetric division of embryos.
The method for obtaining offspring of the present invention is characterized by obtaining offspring of mammals by the transplantation method or the infertility treatment method.
本発明の不等分裂抑制剤は、前記自発的活性化は、染色体の分散を伴うものであることを特徴とする。
本発明の不等分裂抑制剤は、前記不等分裂の抑制は、前記自発的活性化の抑制及び／又は分散した前記染色体の再集合を誘導することを特徴とする。
本発明の不等分裂抑制剤は、前記細胞は、多能性幹細胞、生殖細胞、卵子、又は胚であることを特徴とする。
本発明の不等分裂抑制剤は、前記細胞が前記生殖細胞又は前記卵子である場合、前記自発的活性化は、受精前の二酸化炭素への暴露又は加齢によるものであることを特徴とする。
本発明の不等分裂抑制剤は、前記自発的活性化抑制剤は、プロテアソーム阻害剤であることを特徴とする。
本発明の不等分裂抑制剤は、前記プロテアソーム阻害剤は、ＭＧ１３２を含むことを特徴とする。
本発明の不等分裂抑制剤は、前記プロテアソーム阻害剤は、ＡＬＬＮを含むことを特徴とする。
本発明の染色体異数性抑制剤は、多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含むことを特徴とする。
本発明の培地は、前記不等分裂抑制剤を含むことを特徴とする。
本発明の体外受精培地は、前記不等分裂抑制剤を含むことを特徴とする。
本発明の細胞処理キットは、前記不等分裂抑制剤を含むことを特徴とする。
本発明の不等分裂抑制方法は、多能性を備える細胞の自発的活性化を抑制し、前記細胞の不等分裂を抑制することを特徴とする。
本発明の体外受精方法は、採取された生殖細胞又は卵子の体外受精方法であって、前記不等分裂抑制剤を添加して処理し、前記生殖細胞、前記卵子、又は胚の不等分裂を抑制することを特徴とする。
本発明の移植方法は、前記体外受精方法により処理された生殖細胞又は卵子由来の受精卵又は胚を移植することを特徴とする。
本発明の不妊治療方法は、哺乳類の不妊治療方法であって、採取された生殖細胞又は卵子を、前記不等分裂抑制剤を添加して処理し、前記生殖細胞又は前記卵子由来の受精卵又は胚の不等分裂を抑制することを特徴とする。
本発明の産仔取得方法は、前記移植方法、又は前記不妊治療方法により哺乳類の産仔を取得することを特徴とする。 The asymmetric division inhibitor of the present invention is characterized by containing a spontaneous activation inhibitor that inhibits asymmetric division caused by spontaneous activation of pluripotent cells.
The asymmetric division inhibitor of the present invention is characterized in that the spontaneous activation is accompanied by chromosomal dispersion.
The agent for suppressing asymmetric division of the present invention is characterized in that the suppression of the asymmetric division induces suppression of the spontaneous activation and/or reassortment of the dispersed chromosomes.
The asymmetric division inhibitor of the present invention is characterized in that the cells are pluripotent stem cells, germ cells, eggs, or embryos.
The asymmetric division inhibitor of the present invention is characterized in that, when the cell is the germ cell or the egg, the spontaneous activation is due to exposure to carbon dioxide before fertilization or aging. .
The asymmetric division inhibitor of the present invention is characterized in that the spontaneous activation inhibitor is a proteasome inhibitor.
The asymmetric division inhibitor of the present invention is characterized in that the proteasome inhibitor contains MG132.
The asymmetric division inhibitor of the present invention is characterized in that the proteasome inhibitor contains ALLN.
The chromosomal aneuploidy inhibitor of the present invention is characterized by containing a spontaneous activation inhibitor that suppresses asymmetric division due to spontaneous activation of pluripotent cells.
The medium of the present invention is characterized by containing the asymmetric division inhibitor.
The in vitro fertilization medium of the present invention is characterized by containing the asymmetric division inhibitor.
The cell treatment kit of the present invention is characterized by containing the asymmetric division inhibitor.
The method for suppressing asymmetric division of the present invention is characterized by suppressing spontaneous activation of pluripotent cells and suppressing asymmetric division of the cells.
The in vitro fertilization method of the present invention is a method of in vitro fertilization of collected germ cells or eggs, wherein the asymmetric division inhibitor is added and treated to prevent asymmetric division of the germ cells, eggs, or embryos. It is characterized by suppressing.
The transplantation method of the present invention is characterized by transplanting fertilized eggs or embryos derived from germ cells or eggs treated by the in vitro fertilization method.
The infertility treatment method of the present invention is a mammalian infertility treatment method, wherein the collected germ cells or eggs are treated by adding the asymmetric division inhibitor, and the germ cells or the fertilized eggs derived from the eggs or It is characterized by suppressing asymmetric division of embryos.
The method for obtaining offspring of the present invention is characterized by obtaining offspring of mammals by the transplantation method or the infertility treatment method.
本発明によれば、多能性を備える細胞の自発的活性化による不等分裂を自発的活性化抑制剤により抑制することで、母体等の環境変化に応じて卵子等の細胞が自発的活性化による染色体の分散が生じ、受精はするのにその胚が流産するような現象を抑えることができ、出生率を高めることが可能な不等分裂抑制剤を提供することができる。
According to the present invention, by suppressing asymmetric division due to spontaneous activation of cells with pluripotency with a spontaneous activation inhibitor, cells such as eggs spontaneously activate in response to environmental changes such as the mother's body. It is possible to provide an asymmetric division inhibitor capable of suppressing a phenomenon in which chromosomes are dispersed due to fertilization and abortion of embryos despite fertilization, thereby increasing the birth rate.
＜実施の形態＞
ラットの体外受精（Ｉｎ Ｖｉｔｒｏ Ｆｅｒｔｉｌｉｚａｔｉｏｎ、以下、「ＩＶＦ」とも省略する。）は、再現が困難とされており、マウスのゲノム編集では体外受精卵子が使われている一方で、ラットは長い間自然交配卵子が使われてきた。
このような状況下で、本発明者らは、麻酔下で採卵することにより効率よく体外受精できることを見いだした。このため、本発明者らは、ラットで体外受精ができなかった原因は、従来のラットの卵子採取のためのＣＯ2による安楽殺が原因ではないかと推測した。この安楽殺において、ラットは、ＣＯ2暴露後９０秒で自発呼吸が停止して死に至る。自然交配（ｖｉｖｏ）の受精卵を採取する際にＣＯ2に３分暴露しても産仔は得られるが、未受精卵子を採取する際に３分暴露した場合は、その卵子を体外受精してもまったく産仔は得られなかった。 <Embodiment>
In vitro fertilization (hereinafter also abbreviated as “IVF”) in rats is considered difficult to reproduce. Crossed eggs have been used.
Under such circumstances, the present inventors found that in vitro fertilization can be efficiently performed by collecting eggs under anesthesia. Therefore, the present inventors speculated that the reason why rats could not undergo in vitro fertilization was the conventional euthanasia with CO 2 for oocyte collection. In this euthanasia, rats die by cessation of spontaneous respiration 90 seconds after CO2 exposure. Even if exposed to CO 2 for 3 minutes when collecting fertilized eggs of natural mating (vivo), offspring can be obtained, but if exposed for 3 minutes when collecting unfertilized eggs, the eggs can be fertilized in vitro. However, no offspring were obtained at all.
ラットの体外受精（Ｉｎ Ｖｉｔｒｏ Ｆｅｒｔｉｌｉｚａｔｉｏｎ、以下、「ＩＶＦ」とも省略する。）は、再現が困難とされており、マウスのゲノム編集では体外受精卵子が使われている一方で、ラットは長い間自然交配卵子が使われてきた。
このような状況下で、本発明者らは、麻酔下で採卵することにより効率よく体外受精できることを見いだした。このため、本発明者らは、ラットで体外受精ができなかった原因は、従来のラットの卵子採取のためのＣＯ2による安楽殺が原因ではないかと推測した。この安楽殺において、ラットは、ＣＯ2暴露後９０秒で自発呼吸が停止して死に至る。自然交配（ｖｉｖｏ）の受精卵を採取する際にＣＯ2に３分暴露しても産仔は得られるが、未受精卵子を採取する際に３分暴露した場合は、その卵子を体外受精してもまったく産仔は得られなかった。 <Embodiment>
In vitro fertilization (hereinafter also abbreviated as “IVF”) in rats is considered difficult to reproduce. Crossed eggs have been used.
Under such circumstances, the present inventors found that in vitro fertilization can be efficiently performed by collecting eggs under anesthesia. Therefore, the present inventors speculated that the reason why rats could not undergo in vitro fertilization was the conventional euthanasia with CO 2 for oocyte collection. In this euthanasia, rats die by cessation of spontaneous respiration 90 seconds after CO2 exposure. Even if exposed to CO 2 for 3 minutes when collecting fertilized eggs of natural mating (vivo), offspring can be obtained, but if exposed for 3 minutes when collecting unfertilized eggs, the eggs can be fertilized in vitro. However, no offspring were obtained at all.
このため、本発明者らは、鋭意実験を繰り返し、ラットの体外受精が困難である原因が、安楽殺の際に用いる二酸化炭素（以下「ＣＯ2」という。）への暴露であることを突き止めた。
正常なラット未受精卵子は母体がわずか２分程度ＣＯ2に暴露されるだけで、受精後の発生能を消失する。この現象は、精子との受精能を保持したまま卵子が自発的活性化による雌性染色体の分散を経て発生不全に至る不妊症であった。
この不妊症を治療するための薬剤を探したところ、体外受精の場において卵子を、可逆的ユビキチンプロテアソーム阻害剤（以下、単に「プロテアソーム阻害剤」という。）で処理することにより、分散した雌性前核をふたたび集合させることが可能となり、その結果、出生率を有意に改善できることが判明した。 For this reason, the present inventors have conducted extensive experiments and found that the cause of the difficulty of in vitro fertilization in rats is exposure to carbon dioxide (hereinafter referred to as "CO 2 ") used during euthanasia. rice field.
Normal unfertilized rat oocytes lose their developmental potential after fertilization when the mother is exposed to CO 2 for only about 2 minutes. This phenomenon was an infertility that resulted in developmental failure through dispersion of female chromosomes due to spontaneous activation of eggs while maintaining fertilization ability with sperm.
When I searched for a drug to treat this infertility, I found that by treating eggs with a reversible ubiquitin proteasome inhibitor (hereinafter simply referred to as "proteasome inhibitor") in the field of in vitro fertilization, dispersed female progenitors It was found that the nuclei could be reassembled, resulting in a significant improvement in fertility.
正常なラット未受精卵子は母体がわずか２分程度ＣＯ2に暴露されるだけで、受精後の発生能を消失する。この現象は、精子との受精能を保持したまま卵子が自発的活性化による雌性染色体の分散を経て発生不全に至る不妊症であった。
この不妊症を治療するための薬剤を探したところ、体外受精の場において卵子を、可逆的ユビキチンプロテアソーム阻害剤（以下、単に「プロテアソーム阻害剤」という。）で処理することにより、分散した雌性前核をふたたび集合させることが可能となり、その結果、出生率を有意に改善できることが判明した。 For this reason, the present inventors have conducted extensive experiments and found that the cause of the difficulty of in vitro fertilization in rats is exposure to carbon dioxide (hereinafter referred to as "CO 2 ") used during euthanasia. rice field.
Normal unfertilized rat oocytes lose their developmental potential after fertilization when the mother is exposed to CO 2 for only about 2 minutes. This phenomenon was an infertility that resulted in developmental failure through dispersion of female chromosomes due to spontaneous activation of eggs while maintaining fertilization ability with sperm.
When I searched for a drug to treat this infertility, I found that by treating eggs with a reversible ubiquitin proteasome inhibitor (hereinafter simply referred to as "proteasome inhibitor") in the field of in vitro fertilization, dispersed female progenitors It was found that the nuclei could be reassembled, resulting in a significant improvement in fertility.
すなわち、本発明者らは、自発的活性化や雌性前核の分散を伴うような不妊症を治療するために、本実施形態に係るプロテアソーム阻害剤で卵子を処理すると、自発的活性化を減少させ、分散した雌性前核の再集合を誘導し、その結果、受精卵が正常に発生して産仔が得られることが分かり、本発明を完成させるに至った。
具体的には、ＣＯ2暴露された母体から採卵した卵子を２種類のプロテアソーム阻害剤ＭＧ１３２又はＡＬＬＮ存在化の培地を用いて体外受精することにより、未処理では１～２％の出生率を、２０～３０％にまで高めることができることを解明した。 Thus, the inventors have found that treating ova with proteasome inhibitors according to the present embodiments to treat infertility, such as those associated with spontaneous activation and dispersal of the female pronucleus, reduces spontaneous activation. The present inventors have found that fertilized eggs can develop normally and offspring can be obtained as a result, leading to the completion of the present invention.
Specifically, by in vitro fertilizing eggs collected from mothers exposed to CO 2 using a medium containing two types of proteasome inhibitors MG132 or ALLN, the birth rate of 1 to 2% was reduced to 20%. It has been found that it can be increased to ~30%.
具体的には、ＣＯ2暴露された母体から採卵した卵子を２種類のプロテアソーム阻害剤ＭＧ１３２又はＡＬＬＮ存在化の培地を用いて体外受精することにより、未処理では１～２％の出生率を、２０～３０％にまで高めることができることを解明した。 Thus, the inventors have found that treating ova with proteasome inhibitors according to the present embodiments to treat infertility, such as those associated with spontaneous activation and dispersal of the female pronucleus, reduces spontaneous activation. The present inventors have found that fertilized eggs can develop normally and offspring can be obtained as a result, leading to the completion of the present invention.
Specifically, by in vitro fertilizing eggs collected from mothers exposed to CO 2 using a medium containing two types of proteasome inhibitors MG132 or ALLN, the birth rate of 1 to 2% was reduced to 20%. It has been found that it can be increased to ~30%.
ここで、女性では、なぜ高齢化により胚が流産するのか原因が判明していないため、その出生率を劇的に改善させるような技術開発にも至っていなかった。つまり、高齢出産が増加している現状で、その低い出生率の原因解明と改善が強く求められていた。
これについて、本発明者らは、上述のＣＯ2暴露によるものと同様の現象による不妊症が加齢ラットの卵子でも発生しており、加齢個体における低出生率の原因となっていることを突き止めた。
加齢させたラットの個体から採卵した卵子も同様にＭＧ１３２で処理すると、分散した雌性前核を再集合させることが可能となり、その結果、胚移植数に対して３０％越える効率で産仔が得られた。
すなわち、本実施形態に係る不等分裂抑制剤は、高齢出産に係る出生率を改善することが期待できる。
以下、本発明を実施形態により、さらに詳しく説明する。 Here, since the cause of embryo miscarriage due to aging has not been clarified in women, no technology has been developed to dramatically improve the birth rate. In other words, in the current situation where the number of children giving birth at an advanced age is increasing, there is a strong need to clarify the causes of the low birth rate and improve it.
In this regard, the present inventors have found that infertility due to a phenomenon similar to that due to CO 2 exposure described above also occurs in oocytes of aged rats, and is responsible for the low birth rate in aged individuals. rice field.
Similarly, MG132 treatment of oocytes retrieved from aged rats allowed the dispersed female pronuclei to reassemble, resulting in a litter efficiency of over 30% relative to the number of embryos transferred. Got.
That is, the asymmetric division inhibitor according to the present embodiment can be expected to improve the birth rate associated with childbirth at an advanced age.
BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail by way of embodiments.
これについて、本発明者らは、上述のＣＯ2暴露によるものと同様の現象による不妊症が加齢ラットの卵子でも発生しており、加齢個体における低出生率の原因となっていることを突き止めた。
加齢させたラットの個体から採卵した卵子も同様にＭＧ１３２で処理すると、分散した雌性前核を再集合させることが可能となり、その結果、胚移植数に対して３０％越える効率で産仔が得られた。
すなわち、本実施形態に係る不等分裂抑制剤は、高齢出産に係る出生率を改善することが期待できる。
以下、本発明を実施形態により、さらに詳しく説明する。 Here, since the cause of embryo miscarriage due to aging has not been clarified in women, no technology has been developed to dramatically improve the birth rate. In other words, in the current situation where the number of children giving birth at an advanced age is increasing, there is a strong need to clarify the causes of the low birth rate and improve it.
In this regard, the present inventors have found that infertility due to a phenomenon similar to that due to CO 2 exposure described above also occurs in oocytes of aged rats, and is responsible for the low birth rate in aged individuals. rice field.
Similarly, MG132 treatment of oocytes retrieved from aged rats allowed the dispersed female pronuclei to reassemble, resulting in a litter efficiency of over 30% relative to the number of embryos transferred. Got.
That is, the asymmetric division inhibitor according to the present embodiment can be expected to improve the birth rate associated with childbirth at an advanced age.
BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail by way of embodiments.
〔不等分裂抑制剤、染色体異数性抑制剤〕
本発明の実施の形態に係る不等分裂抑制剤は、多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含むことを特徴とする。
また、本発明の実施の形態に係る染色体異数性抑制剤は、多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含むことを特徴とする。
この自発的活性化は、細胞が卵子であれば、受精前のＣＯ2暴露又は加齢により生じる。この自発活性化により、卵子については、核分裂が停止したＭＩＩ期から細胞周期が進行した状態となる。この際、雌性前核が分散する、すなわち、染色体の分散を伴うものである。この染色体の分散が解消しないままで細胞が２－ｃｅｌｌまで分裂すると、染色体の不等分裂となり、その後の卵割や胚形成等に異常をきたし、発生が停止する。すなわち、産仔も得られず、不妊症となる。
このため、本実施形態においては、この自発的活性化抑制剤を含ませることで、染色体の不等分裂を抑制する抑制剤として機能させる。
また、本実施形態においては、この自発的活性化抑制剤は、自発的活性化と同様の機構で不当分裂が生じる際の染色体異数性抑制剤として機能させることも可能である。 [Anisotropic agent, Chromosome aneuploidy inhibitor]
An asymmetric division inhibitor according to an embodiment of the present invention is characterized by containing a spontaneous activation inhibitor that inhibits asymmetric division caused by spontaneous activation of pluripotent cells.
In addition, the chromosomal aneuploidy suppressing agent according to the embodiment of the present invention is characterized by containing a spontaneous activation suppressing agent that suppresses asymmetric division due to spontaneous activation of pluripotent cells.
This spontaneous activation is caused by prefertilization CO2 exposure or aging if the cell is an egg. Due to this spontaneous activation, the ovum enters a state in which the cell cycle has progressed from the MII stage in which nuclear division has stopped. At this time, the female pronucleus disperses, that is, it is accompanied by chromosomal dispersal. If the cell divides up to 2-cells without resolving this chromosomal dispersion, the chromosomal asymmetry will occur, causing abnormalities in subsequent cleavage, embryogenesis, etc., and development will stop. In other words, no offspring can be obtained, resulting in infertility.
Therefore, in the present embodiment, by including this spontaneous activation suppressor, it functions as a suppressor that suppresses asymmetric division of chromosomes.
In addition, in this embodiment, the spontaneous activation suppressing agent can also function as a chromosome aneuploidy suppressing agent when asymmetric division occurs by the same mechanism as spontaneous activation.
本発明の実施の形態に係る不等分裂抑制剤は、多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含むことを特徴とする。
また、本発明の実施の形態に係る染色体異数性抑制剤は、多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含むことを特徴とする。
この自発的活性化は、細胞が卵子であれば、受精前のＣＯ2暴露又は加齢により生じる。この自発活性化により、卵子については、核分裂が停止したＭＩＩ期から細胞周期が進行した状態となる。この際、雌性前核が分散する、すなわち、染色体の分散を伴うものである。この染色体の分散が解消しないままで細胞が２－ｃｅｌｌまで分裂すると、染色体の不等分裂となり、その後の卵割や胚形成等に異常をきたし、発生が停止する。すなわち、産仔も得られず、不妊症となる。
このため、本実施形態においては、この自発的活性化抑制剤を含ませることで、染色体の不等分裂を抑制する抑制剤として機能させる。
また、本実施形態においては、この自発的活性化抑制剤は、自発的活性化と同様の機構で不当分裂が生じる際の染色体異数性抑制剤として機能させることも可能である。 [Anisotropic agent, Chromosome aneuploidy inhibitor]
An asymmetric division inhibitor according to an embodiment of the present invention is characterized by containing a spontaneous activation inhibitor that inhibits asymmetric division caused by spontaneous activation of pluripotent cells.
In addition, the chromosomal aneuploidy suppressing agent according to the embodiment of the present invention is characterized by containing a spontaneous activation suppressing agent that suppresses asymmetric division due to spontaneous activation of pluripotent cells.
This spontaneous activation is caused by prefertilization CO2 exposure or aging if the cell is an egg. Due to this spontaneous activation, the ovum enters a state in which the cell cycle has progressed from the MII stage in which nuclear division has stopped. At this time, the female pronucleus disperses, that is, it is accompanied by chromosomal dispersal. If the cell divides up to 2-cells without resolving this chromosomal dispersion, the chromosomal asymmetry will occur, causing abnormalities in subsequent cleavage, embryogenesis, etc., and development will stop. In other words, no offspring can be obtained, resulting in infertility.
Therefore, in the present embodiment, by including this spontaneous activation suppressor, it functions as a suppressor that suppresses asymmetric division of chromosomes.
In addition, in this embodiment, the spontaneous activation suppressing agent can also function as a chromosome aneuploidy suppressing agent when asymmetric division occurs by the same mechanism as spontaneous activation.
しかしながら、本発明の実施の形態に係る不等分裂抑制剤において、自発的活性化抑制剤は、プロテアソーム阻害剤であってもよい。
このようにプロテアソーム阻害剤を用いることで、自発的活性化の抑制及び／又は分散した染色体の再集合を誘導することで、不等分裂の抑制を行うことが可能となる。プロテアソーム阻害剤は、本来は受精を経て活性化された際に細胞サイクルの進行に伴い段階を経て行われる細胞内骨格、紡錘糸、その他の染色体の正常な輸送及び整列に必要なタンパクの分解が進行するのを抑えることで、不等分裂を抑制する可能性が考えられる。さらに、この受精に寄らない意図しない自発的活性化による細胞サイクルの進行を抑え、必要なタンパクの分解を抑えることで、受精進行のある種のセーフティー機構により、分散した染色体の再集合を誘導することが可能性の一つとして考えられ得る。 However, in the asymmetric division inhibitor according to the embodiment of the present invention, the spontaneous activation inhibitor may be a proteasome inhibitor.
By using a proteasome inhibitor in this way, it is possible to suppress asymmetric division by suppressing spontaneous activation and/or inducing reassortment of dispersed chromosomes. Proteasome inhibitors, when activated through fertilization, inhibit the degradation of proteins that are required for the normal transport and alignment of the endoskeleton, spindle fibers, and other chromosomes through the steps that occur as the cell cycle progresses. It is conceivable that asymmetric division may be suppressed by suppressing progression. Furthermore, by suppressing cell cycle progression due to this unintentional spontaneous activation unrelated to fertilization and suppressing the degradation of necessary proteins, a kind of safety mechanism during the fertilization process induces the reassortment of dispersed chromosomes. can be considered as one of the possibilities.
このようにプロテアソーム阻害剤を用いることで、自発的活性化の抑制及び／又は分散した染色体の再集合を誘導することで、不等分裂の抑制を行うことが可能となる。プロテアソーム阻害剤は、本来は受精を経て活性化された際に細胞サイクルの進行に伴い段階を経て行われる細胞内骨格、紡錘糸、その他の染色体の正常な輸送及び整列に必要なタンパクの分解が進行するのを抑えることで、不等分裂を抑制する可能性が考えられる。さらに、この受精に寄らない意図しない自発的活性化による細胞サイクルの進行を抑え、必要なタンパクの分解を抑えることで、受精進行のある種のセーフティー機構により、分散した染色体の再集合を誘導することが可能性の一つとして考えられ得る。 However, in the asymmetric division inhibitor according to the embodiment of the present invention, the spontaneous activation inhibitor may be a proteasome inhibitor.
By using a proteasome inhibitor in this way, it is possible to suppress asymmetric division by suppressing spontaneous activation and/or inducing reassortment of dispersed chromosomes. Proteasome inhibitors, when activated through fertilization, inhibit the degradation of proteins that are required for the normal transport and alignment of the endoskeleton, spindle fibers, and other chromosomes through the steps that occur as the cell cycle progresses. It is conceivable that asymmetric division may be suppressed by suppressing progression. Furthermore, by suppressing cell cycle progression due to this unintentional spontaneous activation unrelated to fertilization and suppressing the degradation of necessary proteins, a kind of safety mechanism during the fertilization process induces the reassortment of dispersed chromosomes. can be considered as one of the possibilities.
本発明の実施の形態に係る不等分裂抑制剤において、プロテアソーム阻害剤は、ＭＧ１３２又はＡＬＬＮを含んでもよい。
ここで、本実施形態係るプロテアソーム阻害剤は、ＭＧ１３２（ＣＡＳ １３３４０７－８２－６）、ＡＬＬＮ（ＣＡＳ １１００４４－８２－１）等を用いることが可能である。用量については、通常のプロテアソームの阻害効果が生じるような濃度で用いることが可能である。たとえば、ＭＧ１３２では後述の実施例１、２に示したように、最終濃度１～２０μＭ、ＡＬＬＮについては最終濃度１０～５０μＭ等の濃度で、適宜、最適濃度となるように設定して使用可能である。 In the asymmetric division inhibitor according to the embodiment of the present invention, the proteasome inhibitor may contain MG132 or ALLN.
Here, MG132 (CAS 133407-82-6), ALLN (CAS 110044-82-1), etc. can be used as the proteasome inhibitor according to this embodiment. Concerning the dose, it is possible to use such a concentration as to produce a normal proteasome inhibitory effect. For example, as shown in Examples 1 and 2 below, MG132 can be used at a final concentration of 1 to 20 μM, and ALLN can be used at a final concentration of 10 to 50 μM, etc., so that the optimum concentration can be set and used. be.
ここで、本実施形態係るプロテアソーム阻害剤は、ＭＧ１３２（ＣＡＳ １３３４０７－８２－６）、ＡＬＬＮ（ＣＡＳ １１００４４－８２－１）等を用いることが可能である。用量については、通常のプロテアソームの阻害効果が生じるような濃度で用いることが可能である。たとえば、ＭＧ１３２では後述の実施例１、２に示したように、最終濃度１～２０μＭ、ＡＬＬＮについては最終濃度１０～５０μＭ等の濃度で、適宜、最適濃度となるように設定して使用可能である。 In the asymmetric division inhibitor according to the embodiment of the present invention, the proteasome inhibitor may contain MG132 or ALLN.
Here, MG132 (CAS 133407-82-6), ALLN (CAS 110044-82-1), etc. can be used as the proteasome inhibitor according to this embodiment. Concerning the dose, it is possible to use such a concentration as to produce a normal proteasome inhibitory effect. For example, as shown in Examples 1 and 2 below, MG132 can be used at a final concentration of 1 to 20 μM, and ALLN can be used at a final concentration of 10 to 50 μM, etc., so that the optimum concentration can be set and used. be.
なお、プロテアソーム阻害剤としては、ＭＧ１３２やＡＬＬＮ以外の各種のプロテアソーム阻害剤を用いることが可能である。このプロテアソーム阻害剤としては、例えば、ユビキチンやプロテアソームと結合したり、ユビキチンやプロテアソーム自体を不活性化や分解等したりする低分子化合物、タンパク質やペプチド、核酸、ＰＮＡ（ペプチド核酸）等の合成分子を含む。具体的には、このタンパク質やペプチドは、ユビキチンやプロテアソームとの結合を阻害するユビキチン又はプロテアソームの中和抗体、ユビキチンやプロテアソーム結合部位と同様又は類似するペプチド、ユビキチン特異的な分解酵素、切断酵素等を含む。加えて、この抗体は、トランケート（短縮）されて分子量を変更されたり、各種タグ分子が加えられたり、修飾されたりした抗体様ペプチドやＰＮＡ等であってもよい。さらに、このプロテアソーム活性阻害剤は、合成された活性のないユビキチン類似のペプチド、プロテアソーム結合部位を含むペプチド等のペプチド組成物であってもよい。
Various proteasome inhibitors other than MG132 and ALLN can be used as proteasome inhibitors. Examples of proteasome inhibitors include low-molecular-weight compounds that bind to ubiquitin or proteasomes, or that inactivate or degrade ubiquitin or proteasomes themselves; synthetic molecules such as proteins, peptides, nucleic acids, and PNAs (peptide nucleic acids); including. Specifically, these proteins and peptides include ubiquitin or proteasome neutralizing antibodies that inhibit binding to ubiquitin or proteasome, peptides similar or similar to ubiquitin or proteasome binding sites, ubiquitin-specific degrading enzymes, cleaving enzymes, etc. including. In addition, the antibody may be an antibody-like peptide, PNA, etc. that has been truncated (shortened) to change the molecular weight, added with various tag molecules, or modified. Furthermore, this proteasome activity inhibitor may be a peptide composition such as a synthetic inactive ubiquitin-like peptide, a peptide containing a proteasome binding site, and the like.
なお、本実施形態の自発的活性化抑制剤は、他にも、例えば、低分子化合物、核酸、アミノ酸、タンパク質、その他の各種有機化合物等であり、特に限定されない。
In addition, the spontaneous activation inhibitor of the present embodiment is not particularly limited, and includes, for example, low-molecular-weight compounds, nucleic acids, amino acids, proteins, and various other organic compounds.
さらに、本実施形態に係る自発的活性化抑制剤として、物理的にタンパク質の分解を阻害するための温度調整、磁場や電場や放射線の付加、その他の手法を用いることも可能であってもよい。
Furthermore, as the spontaneous activation suppressing agent according to the present embodiment, it may also be possible to use temperature adjustment, addition of a magnetic field, an electric field, radiation, or other methods for physically inhibiting protein degradation. .
また、本発明の実施の形態に係る不等分裂抑制剤は、他の組成物等と併用することも可能である。
In addition, the asymmetric division inhibitor according to the embodiment of the present invention can be used in combination with other compositions and the like.
〔細胞〕
本発明の実施の形態に係る不等分裂抑制剤において、多能性を備える細胞は、多能性幹細胞、生殖細胞、卵子、又は胚であることを特徴とする。 〔cell〕
In the asymmetric division inhibitor according to the embodiment of the present invention, the cells with pluripotency are pluripotent stem cells, germ cells, ova, or embryos.
本発明の実施の形態に係る不等分裂抑制剤において、多能性を備える細胞は、多能性幹細胞、生殖細胞、卵子、又は胚であることを特徴とする。 〔cell〕
In the asymmetric division inhibitor according to the embodiment of the present invention, the cells with pluripotency are pluripotent stem cells, germ cells, ova, or embryos.
ここで、多能性幹細胞の生成の際においても、不平等分裂が生じて、核内の染色体の数や長さ、核型等が正常細胞と異なりやすいことが本発明者らの予備的な実験により分かっている。すなわち、多能性幹細胞の生成の際にも、下記の実施例１、２で示す卵子と同様の機構による不平等分裂が生じているため、本実施形態に係る自発的活性化抑制剤によりこれを抑制可能となる。
さらに、卵子以外の生殖細胞のいくつかについても、同様の機構による不平等分裂が生じていると考えられるため、これについても本実施形態に係る自発的活性化抑制剤により抑制可能となる。加えて、胚の発生段階においても、いくつかの症例や状況において、自発的活性化と同様の機構で不平等分裂が生じていると考えられ、これも抑制することが期待できる。
すなわち、本実施形態に係る不等分裂抑制剤により、多能性幹細胞、生殖細胞、卵子、又は胚で、自発的活性化の抑制及び／又は分散した染色体の再集合を誘導することが可能となり、正常な細胞として分裂、発生、発育等を進行させることが可能となる。 Here, the present inventors have made preliminary observations that even during the generation of pluripotent stem cells, unequal division occurs and the number and length of chromosomes in the nucleus, the karyotype, etc. are likely to differ from those of normal cells. We know from experiment. That is, even when pluripotent stem cells are generated, unequal division occurs by a mechanism similar to that of eggs shown in Examples 1 and 2 below. can be suppressed.
Furthermore, since unequal division is thought to occur in some reproductive cells other than ovum due to a similar mechanism, this can also be suppressed by the inhibitor of spontaneous activation according to the present embodiment. In addition, in some cases and situations during embryonic development, unequal division is thought to occur by a mechanism similar to that of spontaneous activation, and it is expected that this can also be suppressed.
That is, the asymmetric division inhibitor according to the present embodiment makes it possible to induce suppression of spontaneous activation and/or reassortment of dispersed chromosomes in pluripotent stem cells, germ cells, eggs, or embryos. , it becomes possible to proceed with division, generation, development, etc. as normal cells.
さらに、卵子以外の生殖細胞のいくつかについても、同様の機構による不平等分裂が生じていると考えられるため、これについても本実施形態に係る自発的活性化抑制剤により抑制可能となる。加えて、胚の発生段階においても、いくつかの症例や状況において、自発的活性化と同様の機構で不平等分裂が生じていると考えられ、これも抑制することが期待できる。
すなわち、本実施形態に係る不等分裂抑制剤により、多能性幹細胞、生殖細胞、卵子、又は胚で、自発的活性化の抑制及び／又は分散した染色体の再集合を誘導することが可能となり、正常な細胞として分裂、発生、発育等を進行させることが可能となる。 Here, the present inventors have made preliminary observations that even during the generation of pluripotent stem cells, unequal division occurs and the number and length of chromosomes in the nucleus, the karyotype, etc. are likely to differ from those of normal cells. We know from experiment. That is, even when pluripotent stem cells are generated, unequal division occurs by a mechanism similar to that of eggs shown in Examples 1 and 2 below. can be suppressed.
Furthermore, since unequal division is thought to occur in some reproductive cells other than ovum due to a similar mechanism, this can also be suppressed by the inhibitor of spontaneous activation according to the present embodiment. In addition, in some cases and situations during embryonic development, unequal division is thought to occur by a mechanism similar to that of spontaneous activation, and it is expected that this can also be suppressed.
That is, the asymmetric division inhibitor according to the present embodiment makes it possible to induce suppression of spontaneous activation and/or reassortment of dispersed chromosomes in pluripotent stem cells, germ cells, eggs, or embryos. , it becomes possible to proceed with division, generation, development, etc. as normal cells.
具体的には、本実施形態の多能性幹細胞は、例えば、ヒトを含む哺乳類、その他の脊椎動物等の生物で各種細胞に分化可能な、多分化能を備える幹細胞（Ｓｔｅｍ Ｃｅｌｌ）を含む。ここで、本実施形態の多能性幹細胞は、継代可能であり、継代しても分化が進まない状態を保ち、核型等が変化しにくく、又はエピジェネティックな表現型が変化しにくい性質を有することが好適である。また、本実施形態の多能性幹細胞は、これに関連して、生体外（ｉｎ ｖｉｔｒｏ）又は生体内（ｉｎ ｖｉｖｏ）で十分な増殖能力を備えていることが好適である。このような本実施形態の多能性幹細胞の具体例としては、胚性幹細胞（Ｅｍｂｒｙｏｎｉｃ Ｓｔｅｍ Ｃｅｌｌ、以下、「ＥＳ細胞」という。）、人工多能性幹細胞（Ｉｎｄｕｃｅｄ Ｐｌｕｒｉｐｏｔｅｎｔ Ｓｔｅｍ Ｃｅｌｌ、以下、「ｉＰＳ細胞」という。）、その他の人工的に生成され若しくは選択された多能性を備える幹細胞等が挙げられる。これら本実施形態の多能性幹細胞は、特定の遺伝子を含むレトロウイルスやアデノウイルスやプラスミド等の各種ベクター、ＲＮＡ、低分子化合物等により、体細胞を再プログラミングして作成された幹細胞であってもよい。
加えて、本実施形態の多能性幹細胞は、再プログラミングされて生成される前の段階の処理に用いられる細胞であってもよい。 Specifically, the pluripotent stem cells of the present embodiment include multipotent stem cells that can differentiate into various cells in organisms such as mammals including humans and other vertebrates. Here, the pluripotent stem cells of the present embodiment can be passaged, maintain a state in which differentiation does not progress even after passage, and are difficult to change in karyotype or the like, or are difficult to change in epigenetic phenotype. It is preferred to have the properties Also, in relation to this, the pluripotent stem cells of the present embodiment preferably have sufficient proliferative ability in vitro or in vivo. Specific examples of such pluripotent stem cells of the present embodiment include embryonic stem cells (hereinafter referred to as "ES cells"), induced pluripotent stem cells (hereinafter referred to as "iPS cells"). cells”), other artificially generated or selected stem cells with pluripotency, and the like. These pluripotent stem cells of this embodiment are stem cells created by reprogramming somatic cells with various vectors such as retroviruses, adenoviruses, and plasmids containing specific genes, RNA, low-molecular-weight compounds, etc. good too.
In addition, the pluripotent stem cells of this embodiment may be cells used for processing prior to being reprogrammed and generated.
加えて、本実施形態の多能性幹細胞は、再プログラミングされて生成される前の段階の処理に用いられる細胞であってもよい。 Specifically, the pluripotent stem cells of the present embodiment include multipotent stem cells that can differentiate into various cells in organisms such as mammals including humans and other vertebrates. Here, the pluripotent stem cells of the present embodiment can be passaged, maintain a state in which differentiation does not progress even after passage, and are difficult to change in karyotype or the like, or are difficult to change in epigenetic phenotype. It is preferred to have the properties Also, in relation to this, the pluripotent stem cells of the present embodiment preferably have sufficient proliferative ability in vitro or in vivo. Specific examples of such pluripotent stem cells of the present embodiment include embryonic stem cells (hereinafter referred to as "ES cells"), induced pluripotent stem cells (hereinafter referred to as "iPS cells"). cells”), other artificially generated or selected stem cells with pluripotency, and the like. These pluripotent stem cells of this embodiment are stem cells created by reprogramming somatic cells with various vectors such as retroviruses, adenoviruses, and plasmids containing specific genes, RNA, low-molecular-weight compounds, etc. good too.
In addition, the pluripotent stem cells of this embodiment may be cells used for processing prior to being reprogrammed and generated.
加えて、本実施形態に係る多能性幹細胞としては、全能性を備えている方が好適であるものの、必ずしも全能性に近い多分化能を備えている細胞である必要はない。さらに、本実施形態に係る多能性幹細胞としては、通常より多分化能が高いナイーブ（Ｎａｉｖｅ）な細胞を用いることも可能である。また、本実施形態の多能性幹細胞は、下記で説明する生殖細胞とキメラ化することで個体として発生する等、又はそこまで分化しなくても組織や臓器の様々な細胞に分化する等の分化能力を備えることが好適である。
また、本実施形態の多能性幹細胞は、フィーダー細胞上又はコラーゲン等の基底膜マトリックスをコーティングした細胞培養用プレート等で培養され、維持された後、これを取得したり、凍結保存したものを取得したりすることが可能である。 In addition, although the pluripotent stem cells according to the present embodiment are preferably totipotent, they do not necessarily have totipotent pluripotency. Furthermore, as the pluripotent stem cells according to the present embodiment, it is also possible to use naive cells with higher pluripotency than usual. In addition, the pluripotent stem cells of the present embodiment can be generated as individuals by chimerizing with germ cells described below, or can be differentiated into various cells of tissues and organs without being differentiated to that extent. It is preferable to have differentiation capacity.
In addition, the pluripotent stem cells of the present embodiment are obtained or cryopreserved after being cultured on feeder cells or on a cell culture plate or the like coated with a basement membrane matrix such as collagen and maintained. It is possible to obtain
また、本実施形態の多能性幹細胞は、フィーダー細胞上又はコラーゲン等の基底膜マトリックスをコーティングした細胞培養用プレート等で培養され、維持された後、これを取得したり、凍結保存したものを取得したりすることが可能である。 In addition, although the pluripotent stem cells according to the present embodiment are preferably totipotent, they do not necessarily have totipotent pluripotency. Furthermore, as the pluripotent stem cells according to the present embodiment, it is also possible to use naive cells with higher pluripotency than usual. In addition, the pluripotent stem cells of the present embodiment can be generated as individuals by chimerizing with germ cells described below, or can be differentiated into various cells of tissues and organs without being differentiated to that extent. It is preferable to have differentiation capacity.
In addition, the pluripotent stem cells of the present embodiment are obtained or cryopreserved after being cultured on feeder cells or on a cell culture plate or the like coated with a basement membrane matrix such as collagen and maintained. It is possible to obtain
さらに加えて、本実施形態の多能性幹細胞は、疾患の患者から得られた細胞から作成された細胞、その他の疾患のモデルとなる細胞、レポーター遺伝子が組み込まれた細胞（レポーター細胞）、コンディショナルノックアウトやノックインが可能な細胞、その他の遺伝子組み換えされた細胞等であってもよい。この遺伝子組み換えは、染色体内の遺伝子の追加や修飾や削除、各種ベクターや人工染色体による遺伝子等の付加、エピジェネティック制御の変更、ＰＮＡ等の人工遺伝物質の付加、その他の遺伝子組み換えを含む。
In addition, the pluripotent stem cells of the present embodiment include cells prepared from cells obtained from disease patients, cells that serve as models for other diseases, cells incorporating a reporter gene (reporter cells), and conditioned cells. Cells capable of null knockout or knockin, other genetically modified cells, and the like may also be used. This genetic recombination includes addition, modification and deletion of genes in chromosomes, addition of genes by various vectors and artificial chromosomes, changes in epigenetic control, addition of artificial genetic materials such as PNA, and other genetic recombination.
本実施形態の生殖細胞は、始原生殖細胞、精原細胞、卵祖細胞、減数分裂をする前の生殖細胞、生殖細胞由来のその他の細胞、卵細胞、精子、単為発生可能な細胞、上述の多能性幹細胞とは異なる奇形腫形成能力のある細胞、その他、何らかの操作で個体発生する可能性のある細胞を含む。
Germ cells of this embodiment include primordial germ cells, spermatogonia, oocytes, pre-meiotic germ cells, other germ cell-derived cells, egg cells, sperm, cells capable of parthenogenesis, Cells with teratoma-forming ability that are different from pluripotent stem cells, and other cells that may be manipulated into ontogeny are included.
本実施形態の卵子は、成熟してＭＩＩ期にある卵子、その前の段階の採取可能な状態で取得された卵子、始原生殖細胞や卵祖細胞等から生成された卵子、多能性幹細胞から生成された卵子様の細胞等を含む。本実施形態の卵子は、その他にも、受精又は単為発生可能であり、何らかの形で胚、胚様体、個体（産仔）に発生する可能性のある細胞を含む。
The ovum of the present embodiment includes matured ovum in the MII stage, ovum obtained in the previous stage in a harvestable state, ovum generated from primordial germ cells, oocytes, etc., and pluripotent stem cells. Including generated egg-like cells. The ovum of the present embodiment also includes cells that can be fertilized or parthenogenetic and that can develop into embryos, embryoid bodies, and individuals (offspring) in some form.
本実施形態の受精卵は、動物の卵子と精子とが受精された受精卵、単為発生卵、その他の発生可能な卵様細胞である。この受精卵は、本実施形態に係る卵子が体外受精や顕微授精で受精されたものであってもよく、卵割が開始され全能性がある時点で取得された細胞（受精卵クローンの細胞）等であってもよく、凍結保存されたものが解凍されたものが用いられてもよい。本実施形態では、その他、当業者に一般的に用いられる形式の受精卵を用いることが可能である。
The fertilized egg of this embodiment is a fertilized egg obtained by fertilizing an animal egg and a sperm, a parthenogenetic egg, and other developable oocyte. This fertilized egg may be the egg according to the present embodiment fertilized by in vitro fertilization or microinsemination, and is a cell obtained at the time when cleavage is initiated and has totipotency (fertilized egg clone cell) etc., or the one that has been frozen and thawed may be used. In this embodiment, other types of fertilized eggs commonly used by those skilled in the art can be used.
加えて、本実施形態の哺乳類の細胞は、各種ベクター等により、遺伝子組み換えの手法で遺伝子を導入されたトランスジェニック、遺伝子ノックアウト、コンディショナルノックアウト等の手法で遺伝情報を加工された哺乳類の胚であってもよい。この遺伝情報の加工は、ゲノム編集等によるゲノム中への遺伝子導入若しくは除去であっても、プラスミドや人工染色体のような染色体外への遺伝子導入であっても、染色体の特定部位のメチル化の制御やヒストンの修飾等のエピジェネティック制御であってもよい。または、ＲＮＡ、ＰＮＡ（Ｐｅｐｔｉｄｅ Ｎｕｃｌｅｉｃ Ａｃｉｄ）、その他の人工的な塩基の付加、三重螺旋化等であってもよく、その他の各種遺伝情報の加工手法が使用可能である。
In addition, the mammalian cells of the present embodiment are mammalian embryos whose genetic information has been processed by techniques such as transgenics, gene knockouts, conditional knockouts, etc., in which genes are introduced by genetic recombination techniques using various vectors and the like. There may be. This processing of genetic information includes the introduction or removal of genes into the genome by genome editing, etc., or the introduction of genes into extrachromosomal sites such as plasmids and artificial chromosomes. It may be epigenetic regulation such as regulation or histone modification. Alternatively, RNA, PNA (Peptide Nucleic Acid), addition of other artificial bases, triple helix formation, etc. may be used, and other various genetic information processing techniques may be used.
ここで、本実施形態の細胞は、各種マーカーや目視等によりコロニー等の形式で選択したものであってもよい。
また、本実施形態の細胞は、様々な分化や発生の状態のものを混合して含んでいてもよい。すなわち、各細胞は、発生の段階にあって十分、分化していなかったり、未成熟であったりしてもよい。 Here, the cells of the present embodiment may be those selected in the form of colonies or the like using various markers or visual observation.
In addition, the cells of this embodiment may contain a mixture of cells in various states of differentiation and development. That is, each cell may be in a stage of development and not sufficiently differentiated or immature.
また、本実施形態の細胞は、様々な分化や発生の状態のものを混合して含んでいてもよい。すなわち、各細胞は、発生の段階にあって十分、分化していなかったり、未成熟であったりしてもよい。 Here, the cells of the present embodiment may be those selected in the form of colonies or the like using various markers or visual observation.
In addition, the cells of this embodiment may contain a mixture of cells in various states of differentiation and development. That is, each cell may be in a stage of development and not sufficiently differentiated or immature.
本実施形態に係る細胞が属する動物は、特に限定されるものではなく、脊椎動物及び無脊椎動物を広く含む。脊椎動物としては、魚類、両生類、は虫類、鳥類、及び哺乳類を含む。
本実施形態の胚処理の対象となる哺乳類は、例えば、霊長目（Ｐｒｉｍａｔｅｓ）、齧歯目（Ｒｏｄｅｎｔｉａ）、ウサギ目（Ｌａｇｏｍｏｒｐｈａ）、鯨偶蹄目（Ｃｅｔａｒｔｉｏｄａｃｔｙｌａ）、奇蹄目（Ｐｅｒｉｓｓｏｄａｃｔｙｌａ）、又は食肉目（Ｃａｒｎｉｖｏｒａ）由来であり、例えば、それぞれの目や種毎に異なる胚用に用意した、本実施形態の体外受精培地で処理可能である。なお、これらの目以外の稀少な哺乳類を含む、真獣下綱（Ｅｕｔｈｅｒｉａ）の有胎盤哺乳類の胚について、本実施形態の胚処理の対象としてすべて適用可能である。 Animals to which cells according to this embodiment belong are not particularly limited, and include a wide range of vertebrates and invertebrates. Vertebrates include fish, amphibians, reptiles, birds, and mammals.
Mammals to be treated for embryos of this embodiment are, for example, Primates, Rodentia, Lagomorpha, Cetartiodactyla, Perissodactyla, or carnivorous It is derived from the order Carnivora and can be treated, for example, with the in vitro fertilization medium of the present embodiment prepared for different embryos for each order and species. Embryos of placental mammals of Eutheria, including rare mammals other than these orders, are all applicable to the embryo treatment of the present embodiment.
本実施形態の胚処理の対象となる哺乳類は、例えば、霊長目（Ｐｒｉｍａｔｅｓ）、齧歯目（Ｒｏｄｅｎｔｉａ）、ウサギ目（Ｌａｇｏｍｏｒｐｈａ）、鯨偶蹄目（Ｃｅｔａｒｔｉｏｄａｃｔｙｌａ）、奇蹄目（Ｐｅｒｉｓｓｏｄａｃｔｙｌａ）、又は食肉目（Ｃａｒｎｉｖｏｒａ）由来であり、例えば、それぞれの目や種毎に異なる胚用に用意した、本実施形態の体外受精培地で処理可能である。なお、これらの目以外の稀少な哺乳類を含む、真獣下綱（Ｅｕｔｈｅｒｉａ）の有胎盤哺乳類の胚について、本実施形態の胚処理の対象としてすべて適用可能である。 Animals to which cells according to this embodiment belong are not particularly limited, and include a wide range of vertebrates and invertebrates. Vertebrates include fish, amphibians, reptiles, birds, and mammals.
Mammals to be treated for embryos of this embodiment are, for example, Primates, Rodentia, Lagomorpha, Cetartiodactyla, Perissodactyla, or carnivorous It is derived from the order Carnivora and can be treated, for example, with the in vitro fertilization medium of the present embodiment prepared for different embryos for each order and species. Embryos of placental mammals of Eutheria, including rare mammals other than these orders, are all applicable to the embryo treatment of the present embodiment.
本実施形態に係る細胞が属する動物について、上述の目単位とは異なる観点で説明すると、例えば、ヒト（Ｈｏｍｏ ｓａｐｉｅｎｓ）、実験動物、家畜、伴侶動物等が挙げられる。このうち、実験動物としては、齧歯目の動物として、マウス（Ｍｕｓ ｍｕｓｃｕｌｕｓ）、ラット（Ｒａｔｔｕｓ ｎｏｒｖｅｇｉｃｕｓ）、ハムスター（Ｍｅｓｏｃｒｉｃｅｔｕｓ ａｕｒａｔｕｓ）、モルモット（Ｃａｖｉａ ｐｏｒｃｅｌｌｕｓ）等が挙げられる。ウサギ目の動物としては、ウサギ（Ｌｅｐｏｒｉｎａｅ Ｔｒｏｕｅｓｓａｒｔ）等が挙げられる。家畜としては、例えば、鯨偶蹄目の動物として、ブタ（Ｓｕｓ ｓｃｒｏｆａ ｄｏｍｅｓｔｉｃｕｓ）、ウシ（Ｂｏｓ ｔａｕｒｕｓ）、ヒツジ（Ｏｖｉｓ ａｒｉｅｓ）等が挙げられる。奇蹄目の動物としては、ウマ（Ｅｑｕｕｓ ｃａｂａｌｌｕｓ）等が挙げられる。また、伴侶動物としては、食肉目の動物として、ネコ（Ｆｅｌｉｓ ｓｉｌｖｅｓｔｒｉｓ ｃａｔｕｓ）、イヌ（Ｃａｎｉｓ ｌｕｐｕｓ ｆａｍｉｌｉａｒｉｓ）、フェレット（Ｍｕｓｔｅｌａ ｐｕｔｏｒｉｕｓ）等が挙げられる。また、ヒト以外の霊長目の動物としては、類人猿であるゴリラ（Ｇｏｒｉｌｌａ）やチンパンジー（Ｐａｎ ｔｒｏｇｌｏｄｙｔｅｓ）、アカゲサル（Ｍａｃａｃａ ｍｕｌａｔｔａ）、その他の真猿類（Ｓｉｍｉｉｆｏｒｍｅｓ）、それ以外の霊長類等が挙げられる。この他にも、種が異なる異種の動物等も挙げられる。これに加え、上述の他の目の異種についても含まれてもよい。また、本実施形態に係る動物は、交雑系（ｈｙｂｒｉｄ）、近親交配を２０世代以上継続して得られた動植物の系統である近交系（Ｉｎｂｒｅｄ ｓｔｒａｉｎ）等であってもよい。さらに、ヌードマウス等のミュータント（遺伝子変異）系の動物、所定の疾患モデル動物、又は、種として変化した異種の動物、種同士の交雑（Ｈｙｂｒｉｄ）動物等であってもよい。さらに、人工的に品種改良、種として固定された動物、亜種等も含む。
さらに、上述の実験動物、家畜、伴侶動物等の分類は、便宜上のものであり、それぞれ別の目的、更に、繁殖目的や医療目的等でも用いられる。
また、本実施形態の動物は、無脊椎動物等であっても、脊索動物、軟体動物、環形動物、節足動物等の卵割を行い発生する動物も広く含む。 Animals to which the cells according to the present embodiment belong may be described from a different point of view than the above-mentioned order unit, for example, humans (Homo sapiens), experimental animals, domestic animals, companion animals, and the like. Among them, experimental animals include mice (Mus musculus), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), guinea pigs (Cavia porcellus) and the like as rodents. Animals of the order Lagomorpha include rabbits (Leporinae Trouessart) and the like. Livestock includes, for example, cetacean artiodactyl animals such as pigs (Sus scrofa domesticus), cattle (Bos taurus), and sheep (Ovis aries). Perissodactyla animals include horses (Equus caballus) and the like. Companion animals include cats (Felis silvestris catus), dogs (Canis lupus familiaris), ferrets (Mustela putorius), etc., as carnivorous animals. Examples of non-human primate animals include apes such as Gorilla, Pan troglodytes, Macaca mulatta, other Simiiformes, and other primates. In addition, heterologous animals of different species are also included. In addition, the other eye species described above may also be included. In addition, the animal according to the present embodiment may be a hybrid, an inbred strain that is a system of animals and plants obtained by continuous inbreeding for 20 or more generations, or the like. Further, it may be a mutant (genetic mutation) animal such as a nude mouse, a predetermined disease model animal, a heterologous animal that has changed as a species, a hybrid animal between species, or the like. It also includes artificially improved breeds, animals fixed as species, and subspecies.
Furthermore, the classification of laboratory animals, domestic animals, companion animals, and the like described above is for convenience, and is used for different purposes, such as breeding purposes and medical purposes.
In addition, the animal of the present embodiment includes a wide range of animals such as chordates, mollusks, annelids, arthropods, etc., which undergo egg cleavage and development, even if they are invertebrates.
さらに、上述の実験動物、家畜、伴侶動物等の分類は、便宜上のものであり、それぞれ別の目的、更に、繁殖目的や医療目的等でも用いられる。
また、本実施形態の動物は、無脊椎動物等であっても、脊索動物、軟体動物、環形動物、節足動物等の卵割を行い発生する動物も広く含む。 Animals to which the cells according to the present embodiment belong may be described from a different point of view than the above-mentioned order unit, for example, humans (Homo sapiens), experimental animals, domestic animals, companion animals, and the like. Among them, experimental animals include mice (Mus musculus), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), guinea pigs (Cavia porcellus) and the like as rodents. Animals of the order Lagomorpha include rabbits (Leporinae Trouessart) and the like. Livestock includes, for example, cetacean artiodactyl animals such as pigs (Sus scrofa domesticus), cattle (Bos taurus), and sheep (Ovis aries). Perissodactyla animals include horses (Equus caballus) and the like. Companion animals include cats (Felis silvestris catus), dogs (Canis lupus familiaris), ferrets (Mustela putorius), etc., as carnivorous animals. Examples of non-human primate animals include apes such as Gorilla, Pan troglodytes, Macaca mulatta, other Simiiformes, and other primates. In addition, heterologous animals of different species are also included. In addition, the other eye species described above may also be included. In addition, the animal according to the present embodiment may be a hybrid, an inbred strain that is a system of animals and plants obtained by continuous inbreeding for 20 or more generations, or the like. Further, it may be a mutant (genetic mutation) animal such as a nude mouse, a predetermined disease model animal, a heterologous animal that has changed as a species, a hybrid animal between species, or the like. It also includes artificially improved breeds, animals fixed as species, and subspecies.
Furthermore, the classification of laboratory animals, domestic animals, companion animals, and the like described above is for convenience, and is used for different purposes, such as breeding purposes and medical purposes.
In addition, the animal of the present embodiment includes a wide range of animals such as chordates, mollusks, annelids, arthropods, etc., which undergo egg cleavage and development, even if they are invertebrates.
ここで、本実施形態の細胞は、発生工学等に必要な各種操作を行われていてもよい。この操作は、細胞自体への操作に加え、細胞の取得に伴う母体や生殖細胞等への操作も含む。
具体的には、本実施形態の細胞への操作は、例えば、胚の易供給性に関する母体への過排卵処置、凍結胚の移植に伴う胚の凍結又は解凍、細胞の解離、核移植（Ｎｕｃｌｅａｒ Ｔｒａｎｓｐｌａｎｔａｔｉｏｎ、ＮＴ）、顕微授精（卵細胞質内精子注入法、体外受精、ｉｎｔｒａｃｙｔｏｐｌａｓｍｉｃ ｓｐｅｒｍ ｉｎｊｅｃｔｉｏｎ、ＩＣＳＩ）、マイクロインジェクション（Ｍｉｃｒｏｉｎｊｅｃｔｉｏｎ、ＭＩ）、エレクトロポレーション（電気穿孔法、Ｅｌｅｃｔｒｏｐｏｒａｔｉｏｎ、ＥＰ）等を含む。さらに、細胞融合等の操作も、本実施形態の操作に含む。細胞融合等においては、異数体、４倍体等の倍数体の細胞の作成も含まれる。 Here, the cells of this embodiment may be subjected to various manipulations necessary for developmental engineering and the like. This manipulation includes not only the manipulation of the cell itself, but also manipulation of the maternal body, reproductive cells, etc. associated with the acquisition of the cell.
Specifically, the manipulation of the cells of the present embodiment includes, for example, superovulation treatment to the mother for easy supply of embryos, freezing or thawing of embryos accompanying the transfer of frozen embryos, dissociation of cells, nuclear transfer (Nuclear Transplantation, NT), microinsemination (intracytoplasmic sperm injection, in vitro fertilization, intracytoplasmic sperm injection, ICSI), microinjection (MI), electroporation (electroporation, EP), and the like. Furthermore, operations such as cell fusion are also included in the operations of this embodiment. Cell fusion and the like also include preparation of polyploid cells such as aneuploids and tetraploids.
具体的には、本実施形態の細胞への操作は、例えば、胚の易供給性に関する母体への過排卵処置、凍結胚の移植に伴う胚の凍結又は解凍、細胞の解離、核移植（Ｎｕｃｌｅａｒ Ｔｒａｎｓｐｌａｎｔａｔｉｏｎ、ＮＴ）、顕微授精（卵細胞質内精子注入法、体外受精、ｉｎｔｒａｃｙｔｏｐｌａｓｍｉｃ ｓｐｅｒｍ ｉｎｊｅｃｔｉｏｎ、ＩＣＳＩ）、マイクロインジェクション（Ｍｉｃｒｏｉｎｊｅｃｔｉｏｎ、ＭＩ）、エレクトロポレーション（電気穿孔法、Ｅｌｅｃｔｒｏｐｏｒａｔｉｏｎ、ＥＰ）等を含む。さらに、細胞融合等の操作も、本実施形態の操作に含む。細胞融合等においては、異数体、４倍体等の倍数体の細胞の作成も含まれる。 Here, the cells of this embodiment may be subjected to various manipulations necessary for developmental engineering and the like. This manipulation includes not only the manipulation of the cell itself, but also manipulation of the maternal body, reproductive cells, etc. associated with the acquisition of the cell.
Specifically, the manipulation of the cells of the present embodiment includes, for example, superovulation treatment to the mother for easy supply of embryos, freezing or thawing of embryos accompanying the transfer of frozen embryos, dissociation of cells, nuclear transfer (Nuclear Transplantation, NT), microinsemination (intracytoplasmic sperm injection, in vitro fertilization, intracytoplasmic sperm injection, ICSI), microinjection (MI), electroporation (electroporation, EP), and the like. Furthermore, operations such as cell fusion are also included in the operations of this embodiment. Cell fusion and the like also include preparation of polyploid cells such as aneuploids and tetraploids.
これらに加えて、本実施形態の細胞への操作は、浸透圧変化、その他の化学物質による穿孔、鉗子等による穿孔、体外受精（Ｉｎ Ｖｉｔｒｏ Ｆｅｒｔｉｌｉｚａｔｉｏｎ、ＩＶＦ）、等の各種処理を含む。これらの処理は、上述のように、発生工学等の目的で、核、染色体、ＤＮＡやＲＮＡ等を細胞へ導入するために行われるものを含む。この導入は、各種媒体を用いて行われてもよい。この媒体としては、例えば、プラスミドやウイルスベクター、リポソーム等のＤＤＳ（Ｄｒｕｇ Ｄｅｌｉｖｅｒｙ Ｓｙｓｔｅｍ）、その他の高分子を細胞内に導入する各種手法を用いてもよい。このうち、ウイルスベクターは、アデノウイルス、アデノ随伴ウイルス、レトロウイルス等の当業者に一般的なウイルスを用いて構成されてもよい。加えて、本実施形態の細胞への操作は、例えば、遺伝子変異動物の作製も含む。この遺伝子変異動物は、例えば、遺伝子ノックアウト、ノックイン、コンディショナルノックイン動物等を作製する手法も含む。
または、上述の多能性幹細胞や始原生殖細胞等を作製するために、上述の操作を行うことも可能である。なお、未熟卵体外成熟（ｉｎ ｖｉｔｒｏ ｍａｔｕｒａｔｉｏｎ、ＩＶＭ）、胚移植（Ｂｌａｓｔｏｃｙｓｔ Ｔｒａｎｓｆｅｒ、ＢＴ）等に関連する各種の処理も、本実施形態の操作に含まれてもよい。 In addition to these, manipulations to cells of the present embodiment include various treatments such as osmotic pressure change, perforation with other chemical substances, perforation with forceps or the like, In Vitro Fertilization (IVF), and the like. These treatments include, as described above, those performed to introduce nuclei, chromosomes, DNA, RNA, etc. into cells for purposes such as developmental engineering. This introduction may be performed using a variety of media. As this medium, for example, a DDS (Drug Delivery System) such as a plasmid, a viral vector, a liposome, or other various techniques for introducing macromolecules into cells may be used. Among these, viral vectors may be constructed using viruses commonly used by those skilled in the art, such as adenovirus, adeno-associated virus, and retrovirus. In addition, the manipulation of the cells of this embodiment also includes, for example, the generation of genetically modified animals. This genetically-mutated animal includes, for example, techniques for producing gene-knockout, knock-in, conditional knock-in animals, and the like.
Alternatively, the above-described operations can be performed to produce the above-described pluripotent stem cells, primordial germ cells, and the like. Various treatments related to immature ovo maturation (in vitro maturation, IVM), embryo transfer (Blastocyst Transfer, BT), etc. may also be included in the operations of this embodiment.
または、上述の多能性幹細胞や始原生殖細胞等を作製するために、上述の操作を行うことも可能である。なお、未熟卵体外成熟（ｉｎ ｖｉｔｒｏ ｍａｔｕｒａｔｉｏｎ、ＩＶＭ）、胚移植（Ｂｌａｓｔｏｃｙｓｔ Ｔｒａｎｓｆｅｒ、ＢＴ）等に関連する各種の処理も、本実施形態の操作に含まれてもよい。 In addition to these, manipulations to cells of the present embodiment include various treatments such as osmotic pressure change, perforation with other chemical substances, perforation with forceps or the like, In Vitro Fertilization (IVF), and the like. These treatments include, as described above, those performed to introduce nuclei, chromosomes, DNA, RNA, etc. into cells for purposes such as developmental engineering. This introduction may be performed using a variety of media. As this medium, for example, a DDS (Drug Delivery System) such as a plasmid, a viral vector, a liposome, or other various techniques for introducing macromolecules into cells may be used. Among these, viral vectors may be constructed using viruses commonly used by those skilled in the art, such as adenovirus, adeno-associated virus, and retrovirus. In addition, the manipulation of the cells of this embodiment also includes, for example, the generation of genetically modified animals. This genetically-mutated animal includes, for example, techniques for producing gene-knockout, knock-in, conditional knock-in animals, and the like.
Alternatively, the above-described operations can be performed to produce the above-described pluripotent stem cells, primordial germ cells, and the like. Various treatments related to immature ovo maturation (in vitro maturation, IVM), embryo transfer (Blastocyst Transfer, BT), etc. may also be included in the operations of this embodiment.
〔細胞用培地及び体外受精培地〕
本実施形態に係る培地は、上述の不等分裂抑制剤を含むことを特徴とする。
この培地は、上述の細胞を処理するための培地であってもよい。具体的には、多能性幹細胞を生成するための培地、採取された卵子を処理するための培地等であってもよい。本実施形態において、この培地には、目的に応じた濃度の不等分裂抑制剤が含まれている。 [Cell medium and in vitro fertilization medium]
The medium according to the present embodiment is characterized by containing the asymmetric division inhibitor described above.
This medium may be the medium for treating the cells described above. Specifically, it may be a medium for generating pluripotent stem cells, a medium for processing collected oocytes, or the like. In this embodiment, the medium contains an asymmetric division inhibitor at a concentration appropriate for the purpose.
本実施形態に係る培地は、上述の不等分裂抑制剤を含むことを特徴とする。
この培地は、上述の細胞を処理するための培地であってもよい。具体的には、多能性幹細胞を生成するための培地、採取された卵子を処理するための培地等であってもよい。本実施形態において、この培地には、目的に応じた濃度の不等分裂抑制剤が含まれている。 [Cell medium and in vitro fertilization medium]
The medium according to the present embodiment is characterized by containing the asymmetric division inhibitor described above.
This medium may be the medium for treating the cells described above. Specifically, it may be a medium for generating pluripotent stem cells, a medium for processing collected oocytes, or the like. In this embodiment, the medium contains an asymmetric division inhibitor at a concentration appropriate for the purpose.
より具体的には、本実施形態に係る培地は、上述の不等分裂抑制剤を含むことを特徴とする体外受精培地であってもよい。
すなわち、本実施形態に係る体外受精培地は、自発的活性化を減少させるための体外受精培地であって、自発的活性化抑制剤を含むことを特徴とする。 More specifically, the medium according to this embodiment may be an in vitro fertilization medium characterized by containing the asymmetric division inhibitor described above.
That is, the in vitro fertilization medium according to the present embodiment is an in vitro fertilization medium for reducing spontaneous activation, and is characterized by containing a spontaneous activation inhibitor.
すなわち、本実施形態に係る体外受精培地は、自発的活性化を減少させるための体外受精培地であって、自発的活性化抑制剤を含むことを特徴とする。 More specifically, the medium according to this embodiment may be an in vitro fertilization medium characterized by containing the asymmetric division inhibitor described above.
That is, the in vitro fertilization medium according to the present embodiment is an in vitro fertilization medium for reducing spontaneous activation, and is characterized by containing a spontaneous activation inhibitor.
その他、本実施形態に係る培地は、細胞の種類、状態、密度、操作の種類や内容、その他の条件等に応じた成分を含む。
この成分としては、例えば、体外受精培地を培養するのに必要な培地を構成するための成分及び水を含んでいてもよい。たとえば、本実施形態の体外受精培地は、ｐＨバッファー化合物、アミノ酸、ビタミン類、抗酸化剤、抗生物質、コラーゲン前駆体、微量金属イオンや錯体、各種塩等が加えられて使用されてもよい。
より具体的には、本実施形態の体外受精培地は、例えば、当業者が細胞の培養に一般的に使用する培地の成分を含んでいてもよい。この培地としては、例えば、体外受精培地として一般的なＨＴＦ培地、改変ＨＴＦ培地等と同様の成分、一般的なＤＭＥＭ（Ｄｕｌｂｅｃｃｏ'ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ）等の培地を用いることが可能である。または、多能性幹細胞、生殖細胞、卵子、受精卵、胚等に特化した特定成分を含む培地等を用いることが可能である。加えて、培地には、血清や各種血清代替物が含まれていてもよい。この各種血清代替物が含まれた培地は、異種由来成分不含有（Ｘｅｎｏ－Ｆｒｅｅ、ＸＦ、又はＡｎｉｍａｌ Ｃｏｍｐｏｎｅｎｔ－Ｆｒｅｅ、ＡＣＦ）の培養系で使用されてもよい。 In addition, the medium according to the present embodiment contains components according to the type, state, and density of cells, the type and content of manipulation, other conditions, and the like.
The components may include, for example, components and water for composing the medium necessary for culturing the in vitro fertilization medium. For example, the in vitro fertilization medium of the present embodiment may be used by adding pH buffer compounds, amino acids, vitamins, antioxidants, antibiotics, collagen precursors, trace metal ions and complexes, various salts, and the like.
More specifically, the in vitro fertilization medium of the present embodiment may contain, for example, components of media commonly used by those skilled in the art to culture cells. As this medium, it is possible to use, for example, a medium such as a general HTF medium, a modified HTF medium, and the like as an in vitro fertilization medium, and a general medium such as DMEM (Dulbecco's Modified Eagle Medium). Alternatively, it is possible to use a medium or the like containing specific components specialized for pluripotent stem cells, germ cells, eggs, fertilized eggs, embryos, and the like. In addition, the medium may contain serum or various serum substitutes. The medium containing these various serum substitutes may be used in xeno-free (Xeno-Free, XF, or Animal Component-Free, ACF) culture systems.
この成分としては、例えば、体外受精培地を培養するのに必要な培地を構成するための成分及び水を含んでいてもよい。たとえば、本実施形態の体外受精培地は、ｐＨバッファー化合物、アミノ酸、ビタミン類、抗酸化剤、抗生物質、コラーゲン前駆体、微量金属イオンや錯体、各種塩等が加えられて使用されてもよい。
より具体的には、本実施形態の体外受精培地は、例えば、当業者が細胞の培養に一般的に使用する培地の成分を含んでいてもよい。この培地としては、例えば、体外受精培地として一般的なＨＴＦ培地、改変ＨＴＦ培地等と同様の成分、一般的なＤＭＥＭ（Ｄｕｌｂｅｃｃｏ'ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ）等の培地を用いることが可能である。または、多能性幹細胞、生殖細胞、卵子、受精卵、胚等に特化した特定成分を含む培地等を用いることが可能である。加えて、培地には、血清や各種血清代替物が含まれていてもよい。この各種血清代替物が含まれた培地は、異種由来成分不含有（Ｘｅｎｏ－Ｆｒｅｅ、ＸＦ、又はＡｎｉｍａｌ Ｃｏｍｐｏｎｅｎｔ－Ｆｒｅｅ、ＡＣＦ）の培養系で使用されてもよい。 In addition, the medium according to the present embodiment contains components according to the type, state, and density of cells, the type and content of manipulation, other conditions, and the like.
The components may include, for example, components and water for composing the medium necessary for culturing the in vitro fertilization medium. For example, the in vitro fertilization medium of the present embodiment may be used by adding pH buffer compounds, amino acids, vitamins, antioxidants, antibiotics, collagen precursors, trace metal ions and complexes, various salts, and the like.
More specifically, the in vitro fertilization medium of the present embodiment may contain, for example, components of media commonly used by those skilled in the art to culture cells. As this medium, it is possible to use, for example, a medium such as a general HTF medium, a modified HTF medium, and the like as an in vitro fertilization medium, and a general medium such as DMEM (Dulbecco's Modified Eagle Medium). Alternatively, it is possible to use a medium or the like containing specific components specialized for pluripotent stem cells, germ cells, eggs, fertilized eggs, embryos, and the like. In addition, the medium may contain serum or various serum substitutes. The medium containing these various serum substitutes may be used in xeno-free (Xeno-Free, XF, or Animal Component-Free, ACF) culture systems.
さらに、本実施形態に係る培地には、発生、分化、成長等を促進するための各種ＲＮＡ、ペプチド、タンパク質等も含んでいてもよい。これらは、各種分化誘導因子、成長因子等を含む。また、操作の種類によって必要なレチノイン酸のような分化誘導用の低分子化合物等を含んでいてもよい。
または、本実施形態の体外受精培地は、ＰＢＳのように、細胞が短い期間で死滅しないようにした成分のみが含まれていてもよい。 Furthermore, the medium according to this embodiment may also contain various RNAs, peptides, proteins, etc. for promoting development, differentiation, growth, and the like. These include various differentiation-inducing factors, growth factors, and the like. In addition, a low-molecular-weight compound for inducing differentiation such as retinoic acid, which is necessary depending on the type of operation, may be included.
Alternatively, the in vitro fertilization medium of this embodiment may contain only components such as PBS that prevent the cells from dying in a short period of time.
または、本実施形態の体外受精培地は、ＰＢＳのように、細胞が短い期間で死滅しないようにした成分のみが含まれていてもよい。 Furthermore, the medium according to this embodiment may also contain various RNAs, peptides, proteins, etc. for promoting development, differentiation, growth, and the like. These include various differentiation-inducing factors, growth factors, and the like. In addition, a low-molecular-weight compound for inducing differentiation such as retinoic acid, which is necessary depending on the type of operation, may be included.
Alternatively, the in vitro fertilization medium of this embodiment may contain only components such as PBS that prevent the cells from dying in a short period of time.
本実施形態の体外受精培地に含まれる各剤の濃度は、培養物の種類、状態、密度、操作の種類や内容、その他の条件等に応じて、当業者により適宜設定可能である。
The concentration of each agent contained in the in vitro fertilization medium of this embodiment can be appropriately set by a person skilled in the art according to the type, state, density of the culture, type and content of operation, and other conditions.
その他の体外受精培地に含まれる各剤は、上述の濃度の範囲から設定できる。加えて、本実施形態の自発的活性化抑制剤の濃度は、処理中の各期間において、一定でもよく、各期間において変化させてもよく、それぞれの期間で段階的に変化させてもよい。
Each agent contained in other in vitro fertilization media can be set from the above concentration range. In addition, the concentration of the inhibitor of spontaneous activation of the present embodiment may be constant during each period during treatment, may be varied during each period, or may be varied stepwise during each period.
なお、上述の不等分裂抑制剤に加え、細胞融合、核移植、インジェクション等の操作時の物理的破損を防止するために、サイトカラシン類等が添加されていてもよい。
In addition to the asymmetric division inhibitors described above, cytochalasins and the like may be added in order to prevent physical damage during operations such as cell fusion, nuclear transfer, and injection.
〔処理キット〕
本実施形態の処理キットは、上述の不等分裂抑制剤を含むことを特徴とする。
具体的には、本実施形態の処理キットは、各種の操作に合わせた溶液、培養液、その他の操作に必要な試薬を含んでいてもよい。このような試薬としては、例えば、本実施形態のプローブやプライマー、各種酵素、緩衝液、洗浄液、溶解液、検査用の試薬等も含まれる。
加えて、本実施形態の操作に必要な細胞、容器、その他の資材、器材、道具類等を加えて、本実施形態の処理キットとして提供されてもよい。さらに、本実施形態の処理キットは、後述する発生工学産物を維持するための試薬、食餌、ケージ、飲料水、その他のものが含まれていてもよい。
加えて、細胞の採取、処理、移植等に必要な担体その他の試薬を含んだ構成の処理キットを提供することも可能である。
さらに、通常培地に付加して体外受精培地として完成するような自発的活性化抑制剤と、濃度や処理方法を記載したマニュアル等を添付した処理キットとして提供することも可能である。 [Processing kit]
The treatment kit of this embodiment is characterized by containing the asymmetric division inhibitor described above.
Specifically, the processing kit of the present embodiment may contain solutions and culture media suitable for various operations, and reagents necessary for other operations. Such reagents include, for example, the probes and primers of the present embodiment, various enzymes, buffer solutions, washing solutions, dissolving solutions, test reagents, and the like.
In addition, cells, containers, other materials, equipment, tools, etc. necessary for the operation of this embodiment may be added to provide a processing kit of this embodiment. Additionally, the treatment kit of the present embodiments may include reagents, food, cages, drinking water, etc. for maintaining the developmental engineering products described below.
In addition, it is possible to provide a treatment kit comprising carriers and other reagents necessary for cell collection, treatment, transplantation, and the like.
Furthermore, it is also possible to provide a treatment kit to which a spontaneous activation inhibitor, which can be added to a normal medium to complete an in vitro fertilization medium, and a manual describing concentrations and treatment methods, etc. are attached.
本実施形態の処理キットは、上述の不等分裂抑制剤を含むことを特徴とする。
具体的には、本実施形態の処理キットは、各種の操作に合わせた溶液、培養液、その他の操作に必要な試薬を含んでいてもよい。このような試薬としては、例えば、本実施形態のプローブやプライマー、各種酵素、緩衝液、洗浄液、溶解液、検査用の試薬等も含まれる。
加えて、本実施形態の操作に必要な細胞、容器、その他の資材、器材、道具類等を加えて、本実施形態の処理キットとして提供されてもよい。さらに、本実施形態の処理キットは、後述する発生工学産物を維持するための試薬、食餌、ケージ、飲料水、その他のものが含まれていてもよい。
加えて、細胞の採取、処理、移植等に必要な担体その他の試薬を含んだ構成の処理キットを提供することも可能である。
さらに、通常培地に付加して体外受精培地として完成するような自発的活性化抑制剤と、濃度や処理方法を記載したマニュアル等を添付した処理キットとして提供することも可能である。 [Processing kit]
The treatment kit of this embodiment is characterized by containing the asymmetric division inhibitor described above.
Specifically, the processing kit of the present embodiment may contain solutions and culture media suitable for various operations, and reagents necessary for other operations. Such reagents include, for example, the probes and primers of the present embodiment, various enzymes, buffer solutions, washing solutions, dissolving solutions, test reagents, and the like.
In addition, cells, containers, other materials, equipment, tools, etc. necessary for the operation of this embodiment may be added to provide a processing kit of this embodiment. Additionally, the treatment kit of the present embodiments may include reagents, food, cages, drinking water, etc. for maintaining the developmental engineering products described below.
In addition, it is possible to provide a treatment kit comprising carriers and other reagents necessary for cell collection, treatment, transplantation, and the like.
Furthermore, it is also possible to provide a treatment kit to which a spontaneous activation inhibitor, which can be added to a normal medium to complete an in vitro fertilization medium, and a manual describing concentrations and treatment methods, etc. are attached.
〔不等分裂抑制方法〕
本実施形態の不等分裂抑制方法は、多能性を備える細胞の自発的活性化を抑制し、細胞の不等分裂を抑制する方法である。
具体的には、本実施形態の不等分裂抑制方法は、特定期間、特定濃度で、自発的活性化抑制剤を含む体外受精培地で処理することを特徴とする。 [Asymmetric division suppression method]
The method for suppressing asymmetric division of the present embodiment is a method for suppressing spontaneous activation of pluripotent cells and suppressing asymmetric division of cells.
Specifically, the method for inhibiting asymmetric division of the present embodiment is characterized by treating with an in vitro fertilization medium containing a spontaneous activation inhibitor at a specific concentration for a specific period of time.
本実施形態の不等分裂抑制方法は、多能性を備える細胞の自発的活性化を抑制し、細胞の不等分裂を抑制する方法である。
具体的には、本実施形態の不等分裂抑制方法は、特定期間、特定濃度で、自発的活性化抑制剤を含む体外受精培地で処理することを特徴とする。 [Asymmetric division suppression method]
The method for suppressing asymmetric division of the present embodiment is a method for suppressing spontaneous activation of pluripotent cells and suppressing asymmetric division of cells.
Specifically, the method for inhibiting asymmetric division of the present embodiment is characterized by treating with an in vitro fertilization medium containing a spontaneous activation inhibitor at a specific concentration for a specific period of time.
より具体的には、例えば、本実施形態に係る不等分裂抑制を含む体外受精培地にて、媒精し、卵子と精子とを１～５時間程度、体外受精する。この上で、本実施形態に係る不等分裂抑制を含まない培地で１～５時間体外受精を継続する。さらに、媒精後、３～９時間で、卵子を洗って不等分裂抑制を含まない培地で培養継続する。この上で、一晩培養することで、翌日に、動物に移植等を行うことが可能となる。
More specifically, for example, in vitro fertilization is performed with the in vitro fertilization medium containing the asymmetric division suppression according to the present embodiment, and the eggs and sperm are subjected to in vitro fertilization for about 1 to 5 hours. After this, in vitro fertilization is continued for 1 to 5 hours in a medium containing no asymmetric division suppression according to the present embodiment. Furthermore, 3 to 9 hours after insemination, the oocytes are washed and cultured continuously in a medium containing no asymmetric division inhibition. Then, by culturing overnight, it becomes possible to perform transplantation or the like into animals on the following day.
これらの培地による処理の間に、細胞内に本実施形態に係る不等分裂抑制剤が浸漬され、自発的活性化を抑制したり、細胞サイクルを調整したり、自発的活性化に伴う分散された染色体の再集合を誘導したりして、本来なら自発的活性化により不等分裂による発生停止等の経路が惹起されるところを抑制することで、自発的活性化や染色体の異数性を原因とするによる不妊化から回復すると推測される。この上で、正常な細胞サイクルへ復帰させ、細胞が自ら修復されることで胚発生停止が抑制されると推測される。
During the treatment with these media, the asymmetric division inhibitor according to the present embodiment is immersed in the cells, suppresses spontaneous activation, regulates the cell cycle, and disperses accompanying spontaneous activation. Spontaneous activation and chromosomal aneuploidy can be suppressed by inducing spontaneous chromosomal reassortment and suppressing pathways such as developmental arrest due to asymmetric division that would otherwise be triggered by spontaneous activation. It is speculated to recover from sterilization due to causative. Based on this, it is presumed that embryonic development arrest is suppressed by restoring the normal cell cycle and allowing the cells to repair themselves.
ここで、本実施形態の細胞が多能性幹細胞の場合、不等分裂抑制剤による処理を行った上で製造されてもよく、製造の後に、不等分裂抑制剤で処理されてもよい。取得された多能性幹細胞等は、分化誘導され、その後、特定期間培養され、細胞、細胞塊、組織、又は器官等のいずれかの段階で取得されてもよい。
これらの取得された胚、組織、細胞は、解離等されて加工されてもよい。または、疾患等の患者の疾病の部位に注入、シートや組織や臓器の少なくとも一部として移植等の治療に用いてもよい。この際、本実施形態の胚、組織、細胞を、当業者に用いられる培養器材を用いて単層又は多層のシートを作成し、患患に移植してもよい。さらに、本実施形態の細胞を、適切な担体を用いて培養したり、３Ｄプリンター等を用いて積層したりして、より組織化された培養物を移植することも可能である。
すなわち、本実施形態の移植方法は、本実施形態の治療方法としても機能する。 Here, when the cells of this embodiment are pluripotent stem cells, they may be produced after being treated with an asymmetrical division inhibitor, or may be treated with an asymmetrical division inhibitor after production. The obtained pluripotent stem cells or the like may be induced to differentiate, cultured for a specific period of time, and obtained at any stage as cells, cell clusters, tissues, organs, or the like.
These obtained embryos, tissues, and cells may be dissociated and processed. Alternatively, it may be used for treatment such as injection into a diseased site of a patient, or transplantation as at least part of a sheet, tissue or organ. In this case, the embryos, tissues, and cells of the present embodiment may be prepared into a single-layered or multi-layered sheet using culture equipment used by those skilled in the art, and then transplanted to the patient. Furthermore, the cells of the present embodiment can be cultured using an appropriate carrier or layered using a 3D printer or the like to transplant a more organized culture.
That is, the transplantation method of this embodiment also functions as a treatment method of this embodiment.
これらの取得された胚、組織、細胞は、解離等されて加工されてもよい。または、疾患等の患者の疾病の部位に注入、シートや組織や臓器の少なくとも一部として移植等の治療に用いてもよい。この際、本実施形態の胚、組織、細胞を、当業者に用いられる培養器材を用いて単層又は多層のシートを作成し、患患に移植してもよい。さらに、本実施形態の細胞を、適切な担体を用いて培養したり、３Ｄプリンター等を用いて積層したりして、より組織化された培養物を移植することも可能である。
すなわち、本実施形態の移植方法は、本実施形態の治療方法としても機能する。 Here, when the cells of this embodiment are pluripotent stem cells, they may be produced after being treated with an asymmetrical division inhibitor, or may be treated with an asymmetrical division inhibitor after production. The obtained pluripotent stem cells or the like may be induced to differentiate, cultured for a specific period of time, and obtained at any stage as cells, cell clusters, tissues, organs, or the like.
These obtained embryos, tissues, and cells may be dissociated and processed. Alternatively, it may be used for treatment such as injection into a diseased site of a patient, or transplantation as at least part of a sheet, tissue or organ. In this case, the embryos, tissues, and cells of the present embodiment may be prepared into a single-layered or multi-layered sheet using culture equipment used by those skilled in the art, and then transplanted to the patient. Furthermore, the cells of the present embodiment can be cultured using an appropriate carrier or layered using a 3D printer or the like to transplant a more organized culture.
That is, the transplantation method of this embodiment also functions as a treatment method of this embodiment.
または、本実施形態に係る治療方法として、上述の処理を行った細胞から生成された胚や胚様体のようなものを本実施形態の医薬（医療用組成物）として取得し、治療に用いてもよい。すなわち、本実施形態の治療方法においては、再生医療として、ヒトを含む動物の疾患を治療するために用いることが可能である。
さらに、上述の処理を行った細胞から生成された胚や胚様体のようなものを子宮に移植さいて、産仔を取得することも可能であってもよい。 Alternatively, as a therapeutic method according to the present embodiment, embryos or embryoid bodies generated from the cells subjected to the above-described treatment are obtained as the medicament (medical composition) of the present embodiment and used for treatment. may In other words, the treatment method of this embodiment can be used as regenerative medicine to treat diseases of animals including humans.
Furthermore, it may also be possible to implant embryos or embryoid bodies produced from cells treated as described above into the uterus to obtain offspring.
さらに、上述の処理を行った細胞から生成された胚や胚様体のようなものを子宮に移植さいて、産仔を取得することも可能であってもよい。 Alternatively, as a therapeutic method according to the present embodiment, embryos or embryoid bodies generated from the cells subjected to the above-described treatment are obtained as the medicament (medical composition) of the present embodiment and used for treatment. may In other words, the treatment method of this embodiment can be used as regenerative medicine to treat diseases of animals including humans.
Furthermore, it may also be possible to implant embryos or embryoid bodies produced from cells treated as described above into the uterus to obtain offspring.
〔体外受精方法、移植方法、不妊治療方法、産仔取得方法〕
本実施形態に係る体外受精方法は、採取された生殖細胞又は卵子の体外受精方法であって、本実施形態に係る不等分裂抑制剤を添加して処理し、生殖細胞又は卵子由来の受精卵又は胚の不等分裂を抑制することを特徴とする。
ここで、上述の不等分裂抑制方法は、本実施形態に係る生殖細胞、卵子、又は胚を、上述の不等分裂抑制方法と同様に処理することにより、体外受精方法として機能させることが可能である。 [In vitro fertilization method, transplantation method, infertility treatment method, method of obtaining offspring]
The in vitro fertilization method according to this embodiment is a method for in vitro fertilization of collected germ cells or eggs, which is treated by adding the asymmetric division inhibitor according to this embodiment, and fertilized eggs derived from germ cells or eggs Alternatively, it is characterized by suppressing asymmetric division of the embryo.
Here, the above-described method for suppressing asymmetric division can function as an in vitro fertilization method by treating germ cells, eggs, or embryos according to the present embodiment in the same manner as the method for suppressing asymmetric division described above. is.
本実施形態に係る体外受精方法は、採取された生殖細胞又は卵子の体外受精方法であって、本実施形態に係る不等分裂抑制剤を添加して処理し、生殖細胞又は卵子由来の受精卵又は胚の不等分裂を抑制することを特徴とする。
ここで、上述の不等分裂抑制方法は、本実施形態に係る生殖細胞、卵子、又は胚を、上述の不等分裂抑制方法と同様に処理することにより、体外受精方法として機能させることが可能である。 [In vitro fertilization method, transplantation method, infertility treatment method, method of obtaining offspring]
The in vitro fertilization method according to this embodiment is a method for in vitro fertilization of collected germ cells or eggs, which is treated by adding the asymmetric division inhibitor according to this embodiment, and fertilized eggs derived from germ cells or eggs Alternatively, it is characterized by suppressing asymmetric division of the embryo.
Here, the above-described method for suppressing asymmetric division can function as an in vitro fertilization method by treating germ cells, eggs, or embryos according to the present embodiment in the same manner as the method for suppressing asymmetric division described above. is.
また、体外受精方法により処理された生殖細胞又は受精卵又はこれにより作出された胚を移植する移植方法を用いて、不妊治療とすることも可能である。
すなわち、本実施形態に係る不妊治療方法は、哺乳類の不妊治療方法であって、採取された生殖細胞又は卵子を本実施形態に係る不等分裂抑制剤を添加して処理し、生殖細胞又は卵子由来の受精卵又は胚の不等分裂を抑制することを特徴とする。
さらに、この不妊治療方法により、高い確率で、ＣＯ2暴露された卵子、高齢の卵子であっても、哺乳類の産仔を取得することが可能となる。 It is also possible to treat infertility using a transplantation method in which germ cells or fertilized eggs treated by in vitro fertilization methods or embryos produced thereby are transplanted.
That is, the infertility treatment method according to the present embodiment is a mammalian infertility treatment method, wherein the collected germ cells or eggs are treated by adding the asymmetric division inhibitor according to the present embodiment, and the germ cells or eggs It is characterized by suppressing asymmetric division of the derived fertilized egg or embryo.
Furthermore, with this infertility treatment method, it is possible to obtain mammalian offspring with a high probability, even with CO 2 -exposed oocytes and aged oocytes.
すなわち、本実施形態に係る不妊治療方法は、哺乳類の不妊治療方法であって、採取された生殖細胞又は卵子を本実施形態に係る不等分裂抑制剤を添加して処理し、生殖細胞又は卵子由来の受精卵又は胚の不等分裂を抑制することを特徴とする。
さらに、この不妊治療方法により、高い確率で、ＣＯ2暴露された卵子、高齢の卵子であっても、哺乳類の産仔を取得することが可能となる。 It is also possible to treat infertility using a transplantation method in which germ cells or fertilized eggs treated by in vitro fertilization methods or embryos produced thereby are transplanted.
That is, the infertility treatment method according to the present embodiment is a mammalian infertility treatment method, wherein the collected germ cells or eggs are treated by adding the asymmetric division inhibitor according to the present embodiment, and the germ cells or eggs It is characterized by suppressing asymmetric division of the derived fertilized egg or embryo.
Furthermore, with this infertility treatment method, it is possible to obtain mammalian offspring with a high probability, even with CO 2 -exposed oocytes and aged oocytes.
本実施形態に係る産仔を得るための方法は特に限定されない。たとえば、本実施形態の胚は、体内受精、体外受精、核移植によって得ることができる。
The method for obtaining offspring according to this embodiment is not particularly limited. For example, the embryo of this embodiment can be obtained by internal fertilization, external fertilization, or nuclear transfer.
上述の処理に必要な期間や処理する容量や手順等については、動物種、系統、操作の種類等により、最適な値を当業者により調整可能である。
With regard to the period required for the above-mentioned treatment, volume of treatment, procedure, etc., the optimum values can be adjusted by those skilled in the art according to the animal species, strain, type of operation, etc.
ここで、本実施形態の細胞を処理して細胞分裂、発生、発達させたものは、個体（産仔）、器官、組織、及び細胞のいずれか又は任意の組み合わせ（以下、「発生工学産物」という。）を含む。
このうち、個体としては、キメラ個体、モデル生物の個体、その他の実験用動物の個体、生殖工学上必要な個体、医療上に必要な個体等を含む。器官や組織は、必ずしも臓器のレベルまで成熟したものでなくてもよく、特定の分化した細胞が細胞塊として特定の構造を備えていればよい。さらに、細胞は、特定の構造がない細胞塊が解離されたものを含む。 Here, the cell division, development, and development by treating the cells of the present embodiment include any or any combination of individuals (offspring), organs, tissues, and cells (hereinafter referred to as "developmental engineering product" ).
Among these, individuals include chimera individuals, model organism individuals, other experimental animal individuals, individuals required for reproductive engineering, individuals required for medical treatment, and the like. Organs and tissues do not necessarily have to be matured to the level of organs, as long as specific differentiated cells have a specific structure as a cell cluster. Furthermore, cells include dissociated cell aggregates without specific structures.
このうち、個体としては、キメラ個体、モデル生物の個体、その他の実験用動物の個体、生殖工学上必要な個体、医療上に必要な個体等を含む。器官や組織は、必ずしも臓器のレベルまで成熟したものでなくてもよく、特定の分化した細胞が細胞塊として特定の構造を備えていればよい。さらに、細胞は、特定の構造がない細胞塊が解離されたものを含む。 Here, the cell division, development, and development by treating the cells of the present embodiment include any or any combination of individuals (offspring), organs, tissues, and cells (hereinafter referred to as "developmental engineering product" ).
Among these, individuals include chimera individuals, model organism individuals, other experimental animal individuals, individuals required for reproductive engineering, individuals required for medical treatment, and the like. Organs and tissues do not necessarily have to be matured to the level of organs, as long as specific differentiated cells have a specific structure as a cell cluster. Furthermore, cells include dissociated cell aggregates without specific structures.
本実施形態の体外受精方法、移植方法、不妊治療方法、及び産仔取得方法は、生殖医療に用いることが可能である。これにより、例えば、遺伝的背景、高齢、各種疾病等で胚の定着が難しい母体についても、適用可能となる。
この際、本実施形態に係る受精卵、胚、又は胚様体を母胎に移植することにより、本実施形態の移植方法は、治療方法としても機能する。 The in vitro fertilization method, transplantation method, infertility treatment method, and offspring acquisition method of this embodiment can be used in reproductive medicine. As a result, for example, it can be applied to mothers in which it is difficult for embryos to settle due to genetic background, old age, various diseases, or the like.
In this case, by transplanting the fertilized egg, embryo, or embryoid body according to this embodiment into the mother's womb, the transplantation method of this embodiment also functions as a therapeutic method.
この際、本実施形態に係る受精卵、胚、又は胚様体を母胎に移植することにより、本実施形態の移植方法は、治療方法としても機能する。 The in vitro fertilization method, transplantation method, infertility treatment method, and offspring acquisition method of this embodiment can be used in reproductive medicine. As a result, for example, it can be applied to mothers in which it is difficult for embryos to settle due to genetic background, old age, various diseases, or the like.
In this case, by transplanting the fertilized egg, embryo, or embryoid body according to this embodiment into the mother's womb, the transplantation method of this embodiment also functions as a therapeutic method.
なお、本実施形態の体外受精方法、移植方法、不妊治療方法を哺乳類の内、ヒトに適用する場合、各種生殖医療の倫理に則って、必要な範囲、限度での実施を行うようにする。すなわち、遺伝子改変等は通常はしないようにし、目的上、遺伝子改変等が必要であっても、例えば、遺伝疾患や感染症予防等の特定の基準に従って、最低限の程度で実施する必要がある。
また、本発明を日本国で実施する場合、卵子の採取、体外受精より後の移植、治療は医師により行われる。このため、日本国では、本発明の治療方法の「動物」は、ヒト（Ｈｏｍｏ ｓａｐｉｅｎｓ）を含まないものとする。一方、それ以外の国においては、「動物」「治療法」の定義は、限定されない。 When the in vitro fertilization method, the transplantation method, and the infertility treatment method of the present embodiment are applied to humans among mammals, they should be implemented within the necessary scope and limit in accordance with the ethics of various reproductive medicines. In other words, genetic modification, etc. should normally not be done, and even if genetic modification, etc. is necessary for the purpose, it should be done to a minimum extent according to specific criteria, such as the prevention of genetic diseases and infectious diseases. .
Further, when the present invention is implemented in Japan, oocyte collection, implantation after in vitro fertilization, and treatment are performed by a doctor. Therefore, in Japan, the term "animal" in the therapeutic method of the present invention does not include Homo sapiens. On the other hand, in other countries, the definition of "animal" and "therapeutic method" is not limited.
また、本発明を日本国で実施する場合、卵子の採取、体外受精より後の移植、治療は医師により行われる。このため、日本国では、本発明の治療方法の「動物」は、ヒト（Ｈｏｍｏ ｓａｐｉｅｎｓ）を含まないものとする。一方、それ以外の国においては、「動物」「治療法」の定義は、限定されない。 When the in vitro fertilization method, the transplantation method, and the infertility treatment method of the present embodiment are applied to humans among mammals, they should be implemented within the necessary scope and limit in accordance with the ethics of various reproductive medicines. In other words, genetic modification, etc. should normally not be done, and even if genetic modification, etc. is necessary for the purpose, it should be done to a minimum extent according to specific criteria, such as the prevention of genetic diseases and infectious diseases. .
Further, when the present invention is implemented in Japan, oocyte collection, implantation after in vitro fertilization, and treatment are performed by a doctor. Therefore, in Japan, the term "animal" in the therapeutic method of the present invention does not include Homo sapiens. On the other hand, in other countries, the definition of "animal" and "therapeutic method" is not limited.
一方、本発明の実施の形態に係る体外受精方法、移植方法、不妊治療方法、及び産仔取得方法は、ヒト以外の動物の治療を行う動物にも用いることが可能である。この動物は、特に限定されるものではなく、脊椎動物及び無脊椎動物を広く含む。脊椎動物としては、魚類、両生類、は虫類、鳥類、及び哺乳類を含む。具体的には、哺乳類は、例えば、上述の霊長目（Ｐｒｉｍａｔｅｓ）、齧歯目（Ｒｏｄｅｎｔｉａ）、ウサギ目（Ｌａｇｏｍｏｒｐｈａ）、鯨偶蹄目（Ｃｅｔａｒｔｉｏｄａｃｔｙｌａ）、又は奇蹄目（Ｐｅｒｉｓｓｏｄａｃｔｙｌａ）、食肉目（Ｃａｒｎｉｖｏｒａ）の各種の動物であってもよい。具体的には、マウス、ラット、ハムスター、モルモット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、イヌ、ネコ、フェレット、又は非ヒューマンのトランスジェニック霊長類等であってもよい。また、野生動物としては、哺乳類の他にも、魚類、家禽を含む鳥類、爬虫類等を含む。また、エビや昆虫等を含む甲殻類、その他のイカ等の無脊椎動物等も広く含む。すなわち、ヒトの他に、各種動物の増殖等の方法にも用いることができる。
On the other hand, the in vitro fertilization method, transplantation method, infertility treatment method, and offspring acquisition method according to the embodiments of the present invention can also be used for animals other than humans for treatment. This animal is not particularly limited and includes a wide range of vertebrates and invertebrates. Vertebrates include fish, amphibians, reptiles, birds, and mammals. Specifically, the mammal is, for example, of the order Primates, Rodentia, Lagomorpha, Cetartiodactyla, or Perissodactyla, Carnivora, as described above. ) may be various animals. Specifically, mice, rats, hamsters, guinea pigs, rabbits, sheep, pigs, cows, horses, dogs, cats, ferrets, non-human transgenic primates, and the like may be used. In addition to mammals, wild animals include fish, birds including poultry, and reptiles. It also broadly includes crustaceans including shrimps and insects, and other invertebrates such as squid. That is, in addition to humans, it can also be used for methods such as breeding of various animals.
加えて、本発明の実施の形態に係る発生工学産物は、動物の体内の一部分、又は動物から摘出又は排出された臓器や組織等についても、治療用の対象とすることができる。さらに、この治療は広義の治療であり、バイオリアクター、モデル動物での培養、人体移植様の培養臓器の培養等にも適用可能である。
加えて、本実施形態の発生工学産物は、再生医療以外の治療用途、例えば、バイオリアクター、人工臓器の製造、クローン個体の作成等、各種用途に使用可能である。 In addition, the developmental engineering product according to the embodiment of the present invention can be used as a therapeutic target for a part of the body of an animal, or an organ or tissue excised or excreted from an animal. Furthermore, this treatment is a treatment in a broad sense, and can be applied to bioreactors, culture in model animals, culture of human transplant-like cultured organs, and the like.
In addition, the developmental engineering product of the present embodiment can be used for various therapeutic applications other than regenerative medicine, such as bioreactors, production of artificial organs, and production of individual clones.
加えて、本実施形態の発生工学産物は、再生医療以外の治療用途、例えば、バイオリアクター、人工臓器の製造、クローン個体の作成等、各種用途に使用可能である。 In addition, the developmental engineering product according to the embodiment of the present invention can be used as a therapeutic target for a part of the body of an animal, or an organ or tissue excised or excreted from an animal. Furthermore, this treatment is a treatment in a broad sense, and can be applied to bioreactors, culture in model animals, culture of human transplant-like cultured organs, and the like.
In addition, the developmental engineering product of the present embodiment can be used for various therapeutic applications other than regenerative medicine, such as bioreactors, production of artificial organs, and production of individual clones.
本発明の実施の形態に係る体外受精方法、移植方法、不妊治療方法、及び産仔取得方法を行う際に、処理方法は、母胎の状況、さらに対象の状態等の種々の条件に応じて適宜選択及び変更することが可能である。
When performing the in vitro fertilization method, the transplantation method, the infertility treatment method, and the offspring acquisition method according to the embodiment of the present invention, the processing method is appropriately selected according to various conditions such as the condition of the mother's womb and the condition of the subject. It can be selected and changed.
以下で、本発明の実施の形態に係る不等分裂抑制剤及び不等分裂抑制方法を、実験を基にして、実施例としてさらに具体的に説明する。しかしながら、この実施例は一例にすぎず、これに限定されるものではない。
Below, the unequal fission suppressing agent and the unequal fission suppressing method according to the embodiment of the present invention will be described more specifically as an example based on experiments. However, this embodiment is only an example and is not intended to be limiting.
〔材料と方法〕
（動物）
用いたラットは全てウィスター系統のラット（クレアジャパン、及びチャールズリバー社製）である。１２時間／１２時間の昼夜周期で一般的な飼育環境で飼育した。過排卵は、非特許文献１に記載の手法と同様に行った。 [Materials and methods]
(animal)
All rats used were Wistar strain rats (Clea Japan and Charles River). They were reared in a general rearing environment with a 12 hour/12 hour day/night cycle. Superovulation was performed in the same manner as the method described inNon-Patent Document 1.
（動物）
用いたラットは全てウィスター系統のラット（クレアジャパン、及びチャールズリバー社製）である。１２時間／１２時間の昼夜周期で一般的な飼育環境で飼育した。過排卵は、非特許文献１に記載の手法と同様に行った。 [Materials and methods]
(animal)
All rats used were Wistar strain rats (Clea Japan and Charles River). They were reared in a general rearing environment with a 12 hour/12 hour day/night cycle. Superovulation was performed in the same manner as the method described in
（ＭＧ１３２による処理）
ＭＧ１３２（Ｍｅｒｃｋ ｍｉｌｌｉｐｏｒｅ製、ＭＧ１３２（１ｍｇ）ｉｎ ｓｏｌｕｔｉｏｎ． ４７４７９１－１ＭＧＣＮ）を、ＨＴＦ培地（アークリソース社製）に最終濃度１０μＭで添加し、この培地で精子と、下記で説明する体外受精を３時間行った。その後、ＭＧ１３２の含まれていないＨＴＦ培地で３時間体外受精を継続した。媒精後６時間で、卵子を洗浄し、ＨＴＦ培地で培養を継続させた。さらに、媒精後、６．５時間、１０時間、２０～２２時間で免疫染色をしたり、一晩培養して、翌日に偽妊娠ラットに移植したり等の処理をした。この移植は、定法に従い、偽妊娠ラットの左右卵管に、特定の個数ずつ移植した。 (Processing by MG132)
MG132 (manufactured by Merck millipore, MG132 (1 mg) in solution. 474791-1MGCN) was added to HTF medium (manufactured by Arc Resources) at a final concentration of 10 μM, and sperm and in vitro fertilization described below were incubated in this medium for 3 hours. gone. After that, in vitro fertilization was continued for 3 hours in HTF medium without MG132. Six hours after insemination, the ova were washed and continued to be cultured in HTF medium. In addition, immunostaining was performed at 6.5 hours, 10 hours, and 20 to 22 hours after insemination, and the cells were cultured overnight and transplanted to pseudopregnant rats the next day. This transplantation was carried out in accordance with a standard method, and a specific number was transplanted into each of the right and left fallopian tubes of pseudopregnant rats.
ＭＧ１３２（Ｍｅｒｃｋ ｍｉｌｌｉｐｏｒｅ製、ＭＧ１３２（１ｍｇ）ｉｎ ｓｏｌｕｔｉｏｎ． ４７４７９１－１ＭＧＣＮ）を、ＨＴＦ培地（アークリソース社製）に最終濃度１０μＭで添加し、この培地で精子と、下記で説明する体外受精を３時間行った。その後、ＭＧ１３２の含まれていないＨＴＦ培地で３時間体外受精を継続した。媒精後６時間で、卵子を洗浄し、ＨＴＦ培地で培養を継続させた。さらに、媒精後、６．５時間、１０時間、２０～２２時間で免疫染色をしたり、一晩培養して、翌日に偽妊娠ラットに移植したり等の処理をした。この移植は、定法に従い、偽妊娠ラットの左右卵管に、特定の個数ずつ移植した。 (Processing by MG132)
MG132 (manufactured by Merck millipore, MG132 (1 mg) in solution. 474791-1MGCN) was added to HTF medium (manufactured by Arc Resources) at a final concentration of 10 μM, and sperm and in vitro fertilization described below were incubated in this medium for 3 hours. gone. After that, in vitro fertilization was continued for 3 hours in HTF medium without MG132. Six hours after insemination, the ova were washed and continued to be cultured in HTF medium. In addition, immunostaining was performed at 6.5 hours, 10 hours, and 20 to 22 hours after insemination, and the cells were cultured overnight and transplanted to pseudopregnant rats the next day. This transplantation was carried out in accordance with a standard method, and a specific number was transplanted into each of the right and left fallopian tubes of pseudopregnant rats.
（ＡＬＬＮによる処理）
ＡＬＬＮ（Ｍｅｒｃｋ ｍｉｌｌｉｐｏｒｅ社製、２０８７１９－５ＭＧＣＮ）を、最終濃度を２５μＭで用いた。それ以外の用法は、ＭＧ１３２と同様に行った。 (Processing by ALLN)
ALLN (Merck millipore, 208719-5MGCN) was used at a final concentration of 25 μM. Other usage was the same as for MG132.
ＡＬＬＮ（Ｍｅｒｃｋ ｍｉｌｌｉｐｏｒｅ社製、２０８７１９－５ＭＧＣＮ）を、最終濃度を２５μＭで用いた。それ以外の用法は、ＭＧ１３２と同様に行った。 (Processing by ALLN)
ALLN (Merck millipore, 208719-5MGCN) was used at a final concentration of 25 μM. Other usage was the same as for MG132.
（体外受精（ＩＶＦ））
ＨＴＦ培地に精巣上体尾部精子を採取し、１時間受精能を獲得させた。麻酔した（Ａｎ．）、９９．９％ ＣＯ2を吸わせてから所定時間（９０秒、１２０秒、１５０秒等）経過後に頸椎脱臼処理（ＣＯ2 （ｃ．ｄ．））、又は何もせず頸椎脱臼処理（ｃ．ｄ．）した。
それぞれ、処理を行ったメスの卵管膨大部から卵子卵丘細胞複合体を採取して、受精能獲得処理を行った精子と混ぜ、体外受精を開始した。卵子と精子を合わせる媒精後、各時間で卵子を固定し免疫染色等を行った。 (in vitro fertilization (IVF))
Caudal epididymal spermatozoa were collected in HTF medium and allowed to acquire fertilization capacity for 1 hour. Anesthetized (An.), after a predetermined time (90 seconds, 120 seconds, 150 seconds, etc.) after inhaling 99.9% CO 2 , cervical dislocation treatment (CO 2 (c.d.)) or nothing Cervical dislocation (c.d.) was performed.
In each case, the ovum-cumulus cell complex was collected from the oviduct ampulla of the treated female and mixed with capacitation-treated sperm to initiate in vitro fertilization. After insemination to combine eggs and sperm, the eggs were fixed at each time and subjected to immunostaining and the like.
ＨＴＦ培地に精巣上体尾部精子を採取し、１時間受精能を獲得させた。麻酔した（Ａｎ．）、９９．９％ ＣＯ2を吸わせてから所定時間（９０秒、１２０秒、１５０秒等）経過後に頸椎脱臼処理（ＣＯ2 （ｃ．ｄ．））、又は何もせず頸椎脱臼処理（ｃ．ｄ．）した。
それぞれ、処理を行ったメスの卵管膨大部から卵子卵丘細胞複合体を採取して、受精能獲得処理を行った精子と混ぜ、体外受精を開始した。卵子と精子を合わせる媒精後、各時間で卵子を固定し免疫染色等を行った。 (in vitro fertilization (IVF))
Caudal epididymal spermatozoa were collected in HTF medium and allowed to acquire fertilization capacity for 1 hour. Anesthetized (An.), after a predetermined time (90 seconds, 120 seconds, 150 seconds, etc.) after inhaling 99.9% CO 2 , cervical dislocation treatment (CO 2 (c.d.)) or nothing Cervical dislocation (c.d.) was performed.
In each case, the ovum-cumulus cell complex was collected from the oviduct ampulla of the treated female and mixed with capacitation-treated sperm to initiate in vitro fertilization. After insemination to combine eggs and sperm, the eggs were fixed at each time and subjected to immunostaining and the like.
（ホールマウント染色）
定法に従い、ホールマウント（ｗｈｏｌｅ ｍｏｕｎｔ）染色を行った。卵子をスライドグラスとカバーガラスで挟み、グルタールアルデヒドで固定した後に、エタノールで脱水し、その後、酢酸オルセインで核を染色した。 (whole mount staining)
Whole mount staining was performed according to a standard method. Oocytes were sandwiched between a slide glass and a cover glass, fixed with glutaraldehyde, dehydrated with ethanol, and then stained with orcein acetate for nuclei.
定法に従い、ホールマウント（ｗｈｏｌｅ ｍｏｕｎｔ）染色を行った。卵子をスライドグラスとカバーガラスで挟み、グルタールアルデヒドで固定した後に、エタノールで脱水し、その後、酢酸オルセインで核を染色した。 (whole mount staining)
Whole mount staining was performed according to a standard method. Oocytes were sandwiched between a slide glass and a cover glass, fixed with glutaraldehyde, dehydrated with ethanol, and then stained with orcein acetate for nuclei.
〔結果〕
（ラットの体外受精とＣＯ2暴露）
まず、ラットを自然交配した受精卵と、未受精卵子を取得する際に、ＣＯ2暴露した際の影響について調べた。なお、本実施例１において、図１Ｉ、図１Ｊ、図１Ｋに係る結果以外は、ラットの卵子での実験結果である。
図１Ａは、自然交配（ｖｉｖｏ）した受精卵と、未受精卵子を採取する際に３分、ＣＯ2暴露した場合（ｖｉｔｒｏ）について、ラットの出生率（Ｂｉｒｔｈ ｒａｔｅ（％））を示すグラフである。これらのラットは、ＣＯ2暴露開始から９０秒で自発呼吸が停止して死に至るものの、その直後に頸椎脱臼にて完全に安楽殺する。その後、合計３分経過時点で採卵を行った。自然交配（ｖｉｖｏ）の場合、ＣＯ2に暴露３分経過した場合でも産仔は得られた。しかしながら、未受精卵子を採取する際に、ＣＯ2に暴露３分経過した卵子で体外受精しても、まったく産仔は得られなかった。 〔result〕
(In vitro fertilization and CO2 exposure in rats)
First, the effect of exposure to CO 2 was investigated when fertilized eggs obtained by natural mating of rats and unfertilized eggs were obtained. In addition, in Example 1, except for the results related to FIGS. 1I, 1J, and 1K, they are experimental results using rat ova.
FIG. 1A is a graph showing the birth rate (Birth rate (%)) of rats when fertilized eggs that were naturally mated (vivo) and unfertilized eggs were exposed to CO 2 for 3 minutes (vitro). . Although these rats die due to cessation of spontaneous respiration 90 seconds after the start of CO 2 exposure, they are completely euthanized by cervical dislocation immediately thereafter. After that, egg retrieval was performed after a total of 3 minutes had elapsed. In the case of natural mating (vivo), offspring were obtained even after exposure to CO 2 for 3 minutes. However, when unfertilized eggs were collected, even if in vitro fertilization was performed using eggs that had been exposed to CO 2 for 3 minutes, no offspring were produced.
（ラットの体外受精とＣＯ2暴露）
まず、ラットを自然交配した受精卵と、未受精卵子を取得する際に、ＣＯ2暴露した際の影響について調べた。なお、本実施例１において、図１Ｉ、図１Ｊ、図１Ｋに係る結果以外は、ラットの卵子での実験結果である。
図１Ａは、自然交配（ｖｉｖｏ）した受精卵と、未受精卵子を採取する際に３分、ＣＯ2暴露した場合（ｖｉｔｒｏ）について、ラットの出生率（Ｂｉｒｔｈ ｒａｔｅ（％））を示すグラフである。これらのラットは、ＣＯ2暴露開始から９０秒で自発呼吸が停止して死に至るものの、その直後に頸椎脱臼にて完全に安楽殺する。その後、合計３分経過時点で採卵を行った。自然交配（ｖｉｖｏ）の場合、ＣＯ2に暴露３分経過した場合でも産仔は得られた。しかしながら、未受精卵子を採取する際に、ＣＯ2に暴露３分経過した卵子で体外受精しても、まったく産仔は得られなかった。 〔result〕
(In vitro fertilization and CO2 exposure in rats)
First, the effect of exposure to CO 2 was investigated when fertilized eggs obtained by natural mating of rats and unfertilized eggs were obtained. In addition, in Example 1, except for the results related to FIGS. 1I, 1J, and 1K, they are experimental results using rat ova.
FIG. 1A is a graph showing the birth rate (Birth rate (%)) of rats when fertilized eggs that were naturally mated (vivo) and unfertilized eggs were exposed to CO 2 for 3 minutes (vitro). . Although these rats die due to cessation of spontaneous respiration 90 seconds after the start of CO 2 exposure, they are completely euthanized by cervical dislocation immediately thereafter. After that, egg retrieval was performed after a total of 3 minutes had elapsed. In the case of natural mating (vivo), offspring were obtained even after exposure to CO 2 for 3 minutes. However, when unfertilized eggs were collected, even if in vitro fertilization was performed using eggs that had been exposed to CO 2 for 3 minutes, no offspring were produced.
そこで、未受精卵子を採取する際に、ＣＯ2に暴露してから９０秒後、１２０秒後、１５０秒後で採卵し体外受精に供した場合の受精率の差異について調べた。
図１Ｂは、この結果を示し、縦軸は受精率（Ｆｅｒｔｉｌｉｚａｔｉｏｎ ｒａｔｅ（％））、横軸は三種混合の麻酔した（Ａｎ．）、ＣＯ2暴露（ＣＯ2）、頸椎脱臼での安楽殺処理（ｃ．ｄ．）について示す。麻酔した（Ａｎ．）においては、生存時に取得（ｌｉｖｅ）、頸椎脱臼（ｃ．ｄ．）後に取得したものを比較した。また、ＣＯ2暴露時間については、９０秒、（９０ｓ）、１２０秒（１２０ｓ）、１５０秒（１５０ｓ）、２１０秒（２１０ｓ）、３００秒（３００ｓ）と変化させて、受精率を調べた。また、頸椎脱臼（ｃ．ｄ．）後、１２０秒、ＣＯ2暴露したものについても調べた。各バー内の数字は、受精数／実験数の割合を示す。
１２０秒以内のＣＯ2暴露の場合、受精率に変化はなかった。その後は、段階的に低下するものの、３００秒でも受精率は５０％を超えているため、受精自体は行われることが多い。 Therefore, the difference in fertilization rate was examined when unfertilized oocytes were collected 90 seconds, 120 seconds, and 150 seconds after exposure to CO 2 and subjected to in vitro fertilization.
FIG. 1B shows the results, the vertical axis is fertilization rate (%), and the horizontal axis is anesthesia mixed with three kinds of anesthesia (An.), CO 2 exposure (CO 2 ), euthanasia treatment with cervical dislocation. (c.d.). In anesthetized (An.), acquisition when alive (live) and acquisition after cervical dislocation (cd) were compared. In addition, the fertilization rate was examined by changing the CO 2 exposure time to 90 seconds, (90 s), 120 seconds (120 s), 150 seconds (150 s), 210 seconds (210 s), and 300 seconds (300 s). We also examined those exposed to CO 2 for 120 seconds after cervical dislocation (c.d.). Numbers within each bar indicate the ratio of fertilizations/experiments.
There was no change in fertility within 120 seconds of CO2 exposure. After that, although it gradually decreases, the fertilization rate exceeds 50% even for 300 seconds, so fertilization itself is often performed.
図１Ｂは、この結果を示し、縦軸は受精率（Ｆｅｒｔｉｌｉｚａｔｉｏｎ ｒａｔｅ（％））、横軸は三種混合の麻酔した（Ａｎ．）、ＣＯ2暴露（ＣＯ2）、頸椎脱臼での安楽殺処理（ｃ．ｄ．）について示す。麻酔した（Ａｎ．）においては、生存時に取得（ｌｉｖｅ）、頸椎脱臼（ｃ．ｄ．）後に取得したものを比較した。また、ＣＯ2暴露時間については、９０秒、（９０ｓ）、１２０秒（１２０ｓ）、１５０秒（１５０ｓ）、２１０秒（２１０ｓ）、３００秒（３００ｓ）と変化させて、受精率を調べた。また、頸椎脱臼（ｃ．ｄ．）後、１２０秒、ＣＯ2暴露したものについても調べた。各バー内の数字は、受精数／実験数の割合を示す。
１２０秒以内のＣＯ2暴露の場合、受精率に変化はなかった。その後は、段階的に低下するものの、３００秒でも受精率は５０％を超えているため、受精自体は行われることが多い。 Therefore, the difference in fertilization rate was examined when unfertilized oocytes were collected 90 seconds, 120 seconds, and 150 seconds after exposure to CO 2 and subjected to in vitro fertilization.
FIG. 1B shows the results, the vertical axis is fertilization rate (%), and the horizontal axis is anesthesia mixed with three kinds of anesthesia (An.), CO 2 exposure (CO 2 ), euthanasia treatment with cervical dislocation. (c.d.). In anesthetized (An.), acquisition when alive (live) and acquisition after cervical dislocation (cd) were compared. In addition, the fertilization rate was examined by changing the CO 2 exposure time to 90 seconds, (90 s), 120 seconds (120 s), 150 seconds (150 s), 210 seconds (210 s), and 300 seconds (300 s). We also examined those exposed to CO 2 for 120 seconds after cervical dislocation (c.d.). Numbers within each bar indicate the ratio of fertilizations/experiments.
There was no change in fertility within 120 seconds of CO2 exposure. After that, although it gradually decreases, the fertilization rate exceeds 50% even for 300 seconds, so fertilization itself is often performed.
図１Ｃは、ＣＯ2の暴露時間と出生率との関係を調べた結果を示す。図１Ｃの縦軸は出生率、横軸は、図１Ｂと同様である。
結果として、二酸化炭素に暴露した後、１２０秒以上経過すると出生率（産仔率）に大きな相違が現れた。すなわち、ＣＯ2暴露開始後１２０秒で採卵しても産仔にはならなかった。一方、頸椎脱臼（ｃ．ｄ．）後にＣＯ2暴露しても、２分まで影響がなかった。
このため、生存時にＣＯ2に暴露されたか否かが発生率の変化の原因であることが推測された。 FIG. 1C shows the results of examining the relationship between CO 2 exposure time and birth rate. The vertical axis in FIG. 1C is the birth rate, and the horizontal axis is the same as in FIG. 1B.
As a result, there was a large difference in the birth rate (pup rate) after 120 seconds or more after exposure to carbon dioxide. In other words, even if eggs were collected 120 seconds after the start of CO 2 exposure, no offspring were produced. On the other hand, CO2 exposure after cervical dislocation (cd) had no effect up to 2 minutes.
Therefore, it was speculated that exposure to CO 2 during life was the cause of the change in incidence.
結果として、二酸化炭素に暴露した後、１２０秒以上経過すると出生率（産仔率）に大きな相違が現れた。すなわち、ＣＯ2暴露開始後１２０秒で採卵しても産仔にはならなかった。一方、頸椎脱臼（ｃ．ｄ．）後にＣＯ2暴露しても、２分まで影響がなかった。
このため、生存時にＣＯ2に暴露されたか否かが発生率の変化の原因であることが推測された。 FIG. 1C shows the results of examining the relationship between CO 2 exposure time and birth rate. The vertical axis in FIG. 1C is the birth rate, and the horizontal axis is the same as in FIG. 1B.
As a result, there was a large difference in the birth rate (pup rate) after 120 seconds or more after exposure to carbon dioxide. In other words, even if eggs were collected 120 seconds after the start of CO 2 exposure, no offspring were produced. On the other hand, CO2 exposure after cervical dislocation (cd) had no effect up to 2 minutes.
Therefore, it was speculated that exposure to CO 2 during life was the cause of the change in incidence.
図１Ｄは、麻酔した（Ａｎ．）で採卵しＩＶＦを行い得た２－ｃｅｌｌと、１５０秒のＣＯ2暴露（ＣＯ2 １５０ｓ）後に採卵した卵子でＩＶＦを行い得た２－ｃｅｌｌとをそれぞれ左右の卵管に移植した１３．５日胚の様子を示す。
結果として、麻酔下で採卵した卵は正常に発生するものの、ＣＯ2に暴露した卵子由来の２－ｃｅｌｌは着床痕さえ残らなかった。 FIG. 1D shows 2-cells that were anesthetized (An.) and could undergo IVF, and 2-cells that could undergo IVF with oocytes collected after 150 seconds of CO 2 exposure (CO 2 150 s). 13.5-day embryos transplanted into the left and right oviducts are shown.
As a result, although the eggs collected under anesthesia developed normally, the 2-cells derived from the eggs exposed to CO 2 did not even leave an implantation scar.
結果として、麻酔下で採卵した卵は正常に発生するものの、ＣＯ2に暴露した卵子由来の２－ｃｅｌｌは着床痕さえ残らなかった。 FIG. 1D shows 2-cells that were anesthetized (An.) and could undergo IVF, and 2-cells that could undergo IVF with oocytes collected after 150 seconds of CO 2 exposure (
As a result, although the eggs collected under anesthesia developed normally, the 2-cells derived from the eggs exposed to CO 2 did not even leave an implantation scar.
図１Ｅは、ＣＯ2に暴露することで卵子に何が起こるのかを確かめるために、麻酔した採卵とＣＯ2暴露した採卵した卵子をＩＶＦ後、３時間培養し、ホールマウント染色に供した結果の一例を示す。「Ａｎ．」の写真が、麻酔下で採卵しＩＶＦを行ったものの染色結果を示す。「ＣＯ2」の写真が、１５０秒のＣＯ2暴露をしたものの染色結果を示す。
「Ａｎ．」の写真中の矢印は、卵子核分裂の途中で第一極体が放出され、核分裂が停止したＭＩＩ期の卵子であることを示す特徴である。ところが、「ＣＯ2」の写真では、矢頭に示すように、卵子に自発的活性化（Ｓｐｏｎｔａｎｅｏｕｓ ａｃｔｉｖａｔｒｉｏｎ、以下「ｓ．ａ．」とも称する。）が生じており第二極体（ＰＢ２）が放出されている様子が確認された。 FIG . 1E shows the results of culturing anesthetized and CO 2 -exposed oocytes for 3 hours after IVF and subjecting them to whole-mount staining to see what happens to oocytes upon exposure toCO 2 . Here is an example. The photograph of "An." shows the staining results of eggs collected under anesthesia and subjected to IVF. The " CO2 " picture shows the staining results after 150 seconds of CO2 exposure.
The arrow in the photograph of "An." is a feature indicating that the ovum is in the MII stage, in which the first polar body is released during nuclear fission and the nuclear fission is stopped. However, in the photograph of "CO 2 ", as indicated by the arrowhead, spontaneous activation (spontaneous activation, hereinafter also referred to as "s.a.") occurs in the egg, and the second polar body (PB2) is released. It was confirmed that
「Ａｎ．」の写真中の矢印は、卵子核分裂の途中で第一極体が放出され、核分裂が停止したＭＩＩ期の卵子であることを示す特徴である。ところが、「ＣＯ2」の写真では、矢頭に示すように、卵子に自発的活性化（Ｓｐｏｎｔａｎｅｏｕｓ ａｃｔｉｖａｔｒｉｏｎ、以下「ｓ．ａ．」とも称する。）が生じており第二極体（ＰＢ２）が放出されている様子が確認された。 FIG . 1E shows the results of culturing anesthetized and CO 2 -exposed oocytes for 3 hours after IVF and subjecting them to whole-mount staining to see what happens to oocytes upon exposure to
The arrow in the photograph of "An." is a feature indicating that the ovum is in the MII stage, in which the first polar body is released during nuclear fission and the nuclear fission is stopped. However, in the photograph of "CO 2 ", as indicated by the arrowhead, spontaneous activation (spontaneous activation, hereinafter also referred to as "s.a.") occurs in the egg, and the second polar body (PB2) is released. It was confirmed that
図１Ｆは、図１Ｅの麻酔した採卵と、秒数を変化させてＣＯ2暴露した採卵した卵子をＩＶＦ後、３時間培養し、ホールマウント染色した結果を示すグラフである。縦軸は、ＭＩＩ期と、自発的活性化（ｓ．ａ．）が生じている割合、横軸は、コントロールの麻酔下頸椎脱臼（Ａｎ．（ｃ．ｄ．））、９０秒、１２０秒、１５０秒のＣＯ2暴露をした結果をそれぞれ示す。各バー内の数値は、実験階数を示す。
結果として、９０秒のＣＯ2暴露を行うことで６割の卵子が、自発的活性化を起こし、１２０ｓ以上の暴露では、ほとんどが自発的活性化を起こしていることが判明した。 FIG. 1F is a graph showing the results of whole-mount staining of the anesthetized oocytes in FIG. 1E and the oocytes exposed to CO 2 for varying seconds after IVF, cultured for 3 hours. The vertical axis is the MII stage and the rate of spontaneous activation (s.a.), and the horizontal axis is the control cervical dislocation under anesthesia (An. (c.d.)), 90 seconds, 120 seconds. , respectively show the results with 150 seconds of CO 2 exposure. Numbers within each bar indicate the experimental rank.
As a result, it was found that 60% of the eggs were spontaneously activated when exposed to CO 2 for 90 seconds, and most of them were spontaneously activated when exposed toCO 2 for 120 seconds or longer.
結果として、９０秒のＣＯ2暴露を行うことで６割の卵子が、自発的活性化を起こし、１２０ｓ以上の暴露では、ほとんどが自発的活性化を起こしていることが判明した。 FIG. 1F is a graph showing the results of whole-mount staining of the anesthetized oocytes in FIG. 1E and the oocytes exposed to CO 2 for varying seconds after IVF, cultured for 3 hours. The vertical axis is the MII stage and the rate of spontaneous activation (s.a.), and the horizontal axis is the control cervical dislocation under anesthesia (An. (c.d.)), 90 seconds, 120 seconds. , respectively show the results with 150 seconds of CO 2 exposure. Numbers within each bar indicate the experimental rank.
As a result, it was found that 60% of the eggs were spontaneously activated when exposed to CO 2 for 90 seconds, and most of them were spontaneously activated when exposed to
ここで、ＣＯ2に暴露されると血液は酸性に傾きアシドーシスが発生する。このアシドーシスが自発的活性化の原因であるのかについての検証を行った。
実際に、血液ガス分析を行い、ｐＨ他を測定した結果について、下記の表１に示す： Here, when exposed to CO2 , the blood becomes acidic and acidosis occurs. We examined whether this acidosis is the cause of spontaneous activation.
Blood gas analysis was actually performed, and the results of pH and other measurements are shown in Table 1 below:
実際に、血液ガス分析を行い、ｐＨ他を測定した結果について、下記の表１に示す： Here, when exposed to CO2 , the blood becomes acidic and acidosis occurs. We examined whether this acidosis is the cause of spontaneous activation.
Blood gas analysis was actually performed, and the results of pH and other measurements are shown in Table 1 below:
これらにおいて、ｃｏｎｔ（ｃ．ｄ．）は頸椎脱臼のコントロール、Ａｎ（ｃ．ｄ．）は麻酔下頸椎脱臼、ＣＯ2に暴露してから９０秒後（ＣＯ2 ９０ｓ）のものを３例について示す。表１の項目については、ｐＣＯ2は二酸化炭素分圧、ｐＯ2は酸素分圧、ｃＮａはナトリウム濃度（ｍｍｏｌ／Ｌ）、ｃＫはカリウム濃度（ｍｍｏｌ／Ｌ）、Ｈｃｔは電気伝導度、Ｔｅｍｐは温度、ｃｔＨｂは総ヘモグロビン濃度、ｃＨＣＯ3は、バイカーボネート指標を示す。
血液ガス分析より、ＣＯ2暴露したではｐＨが６．６となっており、アシドーシスが発生していた。一方、酸素分圧は正常であることから、酸欠状態ではなかった。 In these, cont (c.d.) is the control for cervical dislocation, An (c.d.) is the cervical dislocation under anesthesia, 90 s after exposure to CO 2 (CO 2 90 s) for 3 cases. show. For the items in Table 1, pCO2 is carbon dioxide partial pressure, pO2 is oxygen partial pressure, cNa is sodium concentration (mmol/L), cK is potassium concentration (mmol/L), Hct is electrical conductivity, and Temp is temperature, ctHb total hemoglobin concentration, cHCO3 the bicarbonate index.
A blood gas analysis revealed that the pH of the patient exposed to CO 2 was 6.6, indicating that acidosis had occurred. On the other hand, since the oxygen partial pressure was normal, it was not in an oxygen-deficient state.
血液ガス分析より、ＣＯ2暴露したではｐＨが６．６となっており、アシドーシスが発生していた。一方、酸素分圧は正常であることから、酸欠状態ではなかった。 In these, cont (c.d.) is the control for cervical dislocation, An (c.d.) is the cervical dislocation under anesthesia, 90 s after exposure to CO 2 (
A blood gas analysis revealed that the pH of the patient exposed to CO 2 was 6.6, indicating that acidosis had occurred. On the other hand, since the oxygen partial pressure was normal, it was not in an oxygen-deficient state.
次に、ｐＨの低下が自発的原因か否かを確かめるために、ｐＨ６．５の培地に卵子を１０分間暴露した後にホールマウント染色により自発的活性化を調べた。
図１Ｇに、この結果を示す。縦軸は、ＭＩＩ期（ＭＩＩ）と自発的活性化（ｓ．ａ．）の割合を示す。各バー内の数字は、実験数を示す。
結果として、ｐＨが低下したことによる自発的活性化は生じなかった。このため、アシドーシスが自発的活性化の原因ではないことが分かった。 Next, in order to confirm whether or not the decrease in pH is the spontaneous cause, the spontaneous activation was examined by whole-mount staining after exposing the ova to pH 6.5 medium for 10 minutes.
The results are shown in FIG. 1G. The vertical axis indicates the ratio of stage MII (MII) and spontaneous activation (s.a.). Numbers within each bar indicate the number of experiments.
As a result, no spontaneous activation occurred due to the lowered pH. Therefore, it was found that acidosis was not the cause of the spontaneous activation.
図１Ｇに、この結果を示す。縦軸は、ＭＩＩ期（ＭＩＩ）と自発的活性化（ｓ．ａ．）の割合を示す。各バー内の数字は、実験数を示す。
結果として、ｐＨが低下したことによる自発的活性化は生じなかった。このため、アシドーシスが自発的活性化の原因ではないことが分かった。 Next, in order to confirm whether or not the decrease in pH is the spontaneous cause, the spontaneous activation was examined by whole-mount staining after exposing the ova to pH 6.5 medium for 10 minutes.
The results are shown in FIG. 1G. The vertical axis indicates the ratio of stage MII (MII) and spontaneous activation (s.a.). Numbers within each bar indicate the number of experiments.
As a result, no spontaneous activation occurred due to the lowered pH. Therefore, it was found that acidosis was not the cause of the spontaneous activation.
また、酸性条件下では多精が起こると報告があるため、麻酔下頸椎脱臼で採卵した卵子（Ａｎ．（ｃ．ｄ．））と、１２０秒のＣＯ2暴露した卵子（ＣＯ2 １２０ｓ）に顕微授精を行った。すなわち、一つの卵子に一つの精子を入れた。
図１Ｈに、この結果の写真を示す。
図１Ｉは、この結果を示すグラフである。縦軸は、胚に成長した率（発生率、Ｄｅｖｅｌｏｐｍｅｎｔａｌ ｒａｔｅ（％））を示す。各バー内の数字は、正常胚数／実験数を示す。
結果として、一つの精子を注入したにもかかわらず、ＣＯ2暴露卵子由来の顕微授精胚の発生率は有意に低かった。 In addition, since it has been reported that polyspermia occurs under acidic conditions, eggs collected by cervical dislocation under anesthesia (An. (c.d.)) and eggs exposed to CO 2 for 120 seconds (CO 2 120s) performed microinsemination. That is, one sperm was put into one egg.
A photograph of this result is shown in FIG. 1H.
FIG. 1I is a graph showing this result. The vertical axis indicates the rate of growth into embryos (developmental rate (%)). Numbers in each bar indicate the number of normal embryos/experimental number.
As a result, the incidence of micro-insemination embryos derived from CO2 - exposed eggs was significantly lower despite the injection of a single sperm.
図１Ｈに、この結果の写真を示す。
図１Ｉは、この結果を示すグラフである。縦軸は、胚に成長した率（発生率、Ｄｅｖｅｌｏｐｍｅｎｔａｌ ｒａｔｅ（％））を示す。各バー内の数字は、正常胚数／実験数を示す。
結果として、一つの精子を注入したにもかかわらず、ＣＯ2暴露卵子由来の顕微授精胚の発生率は有意に低かった。 In addition, since it has been reported that polyspermia occurs under acidic conditions, eggs collected by cervical dislocation under anesthesia (An. (c.d.)) and eggs exposed to CO 2 for 120 seconds (
A photograph of this result is shown in FIG. 1H.
FIG. 1I is a graph showing this result. The vertical axis indicates the rate of growth into embryos (developmental rate (%)). Numbers in each bar indicate the number of normal embryos/experimental number.
As a result, the incidence of micro-insemination embryos derived from CO2 - exposed eggs was significantly lower despite the injection of a single sperm.
次に、自発的活性化でも単為発生により初期胚が生じることから、受精が起こるかどうかについて調べた。
図１Ｊに、Ｎａｎｏｇ－ＥＧＦＰ Ｔｇラット（Ｆ３４４．Ｗ－Ｔｇ（Ｎａｎｏｇ－ＧＦＰ，－ＰｕｒｏＲ）Ｋｙｏ）で精子を用いてＩＶＦを行った結果を示す。Ｎａｎｏｇは、受精後、桑実期胚から発現が確認されることが知られている。逆に、単為発生では、Ｎａｎｏｇは発現しない。
図１Ｊにおいて、麻酔で採卵した卵子（Ａｎ．）と、１２０秒のＣＯ2暴露後に採卵した卵子（ＣＯ2 １２０ｓ）の各胚において、桑実期胚になった状態の顕微鏡写真（Ｐｈａｓｅ）と、ＥＧＦＰの発現をみた写真（ＥＧＦＰ）をそれぞれ示す。ＥＧＦＰの数値は、蛍光を捉えた胚の数／胚の数を示す。
結果として、ＣＯ2暴露した卵子の胚盤胞も、麻酔で採卵した卵子も、約半分の胚盤胞がＧＦＰ陽性であり、Ｎａｎｏｇが発現していた。これにより、ＣＯ2暴露により生じる胚は、単為発生由来ではなく精子との受精により生じていることが分かった。 Next, we investigated whether fertilization occurs, since spontaneous activation also gives rise to early embryos through parthenogenesis.
FIG. 1J shows the results of IVF using sperm from Nanog-EGFP Tg rats (F344.W-Tg(Nanog-GFP,-PuroR)Kyo). Nanog is known to be expressed in morula stage embryos after fertilization. Conversely, Nanog is not expressed in parthenogenesis.
In FIG. 1J, the ovum collected under anesthesia (An.) and the ovum collected after CO 2 exposure for 120 seconds (CO 2 120 s) are micrographs (Phase) of the morula stage embryos in each embryo. , and photographs showing the expression of EGFP (EGFP), respectively. EGFP values indicate the number of embryos that captured fluorescence/the number of embryos.
As a result, about half of the blastocysts of the CO2 - exposed eggs and the eggs collected under anesthesia were GFP-positive and Nanog was expressed. From this, it was found that embryos produced by CO 2 exposure were produced by fertilization with sperm rather than by parthenogenesis.
図１Ｊに、Ｎａｎｏｇ－ＥＧＦＰ Ｔｇラット（Ｆ３４４．Ｗ－Ｔｇ（Ｎａｎｏｇ－ＧＦＰ，－ＰｕｒｏＲ）Ｋｙｏ）で精子を用いてＩＶＦを行った結果を示す。Ｎａｎｏｇは、受精後、桑実期胚から発現が確認されることが知られている。逆に、単為発生では、Ｎａｎｏｇは発現しない。
図１Ｊにおいて、麻酔で採卵した卵子（Ａｎ．）と、１２０秒のＣＯ2暴露後に採卵した卵子（ＣＯ2 １２０ｓ）の各胚において、桑実期胚になった状態の顕微鏡写真（Ｐｈａｓｅ）と、ＥＧＦＰの発現をみた写真（ＥＧＦＰ）をそれぞれ示す。ＥＧＦＰの数値は、蛍光を捉えた胚の数／胚の数を示す。
結果として、ＣＯ2暴露した卵子の胚盤胞も、麻酔で採卵した卵子も、約半分の胚盤胞がＧＦＰ陽性であり、Ｎａｎｏｇが発現していた。これにより、ＣＯ2暴露により生じる胚は、単為発生由来ではなく精子との受精により生じていることが分かった。 Next, we investigated whether fertilization occurs, since spontaneous activation also gives rise to early embryos through parthenogenesis.
FIG. 1J shows the results of IVF using sperm from Nanog-EGFP Tg rats (F344.W-Tg(Nanog-GFP,-PuroR)Kyo). Nanog is known to be expressed in morula stage embryos after fertilization. Conversely, Nanog is not expressed in parthenogenesis.
In FIG. 1J, the ovum collected under anesthesia (An.) and the ovum collected after CO 2 exposure for 120 seconds (
As a result, about half of the blastocysts of the CO2 - exposed eggs and the eggs collected under anesthesia were GFP-positive and Nanog was expressed. From this, it was found that embryos produced by CO 2 exposure were produced by fertilization with sperm rather than by parthenogenesis.
（マウスの体外受精とＣＯ2暴露）
次に、マウスの卵子を採取する際にもＣＯ2に暴露し、安楽殺の際にＣＯ2を用いることで卵子が自発的活性化を経由して受精後の発生能に障害が現れるこの現象が、ラット特異的か否かを調べた。
マウスについても、ラットの場合と同様の手法により、９０秒、２１０秒、３３０秒、ＣＯ2に暴露した後、卵子を採取してＩＶＦした。いずれのマウスも、ラットと同様にＣＯ2暴露開始から９０秒で自発呼吸が停止して死に至るものの、２１０秒、３３０秒のものについては、暴露後９０秒で頸椎脱臼により完全に安楽殺を行い、その後の各時間で採卵を行った。コントロールとして、麻酔下頸椎脱臼（Ａｎ．（ｃ．ｄ．））で死に至った後、２４０秒後に採卵を行ったもの（区）も用意した。 (Mouse in vitro fertilization and CO2 exposure)
Next, by exposing mouse eggs to CO 2 during harvesting, and using CO 2 during euthanasia, the eggs spontaneously activate, resulting in impaired post-fertilization developmental potential. was examined to see if it was rat-specific.
Mice were also exposed to CO 2 for 90 seconds, 210 seconds, and 330 seconds by the same procedure as rats, and then eggs were collected and subjected to IVF. All mice, like rats, stopped spontaneous respiration and died 90 seconds after the start of CO 2 exposure. Egg collection was performed at each subsequent hour. As a control, an egg collection was also performed 240 seconds after death by cervical dislocation under anesthesia (An. (c.d.)) (section).
次に、マウスの卵子を採取する際にもＣＯ2に暴露し、安楽殺の際にＣＯ2を用いることで卵子が自発的活性化を経由して受精後の発生能に障害が現れるこの現象が、ラット特異的か否かを調べた。
マウスについても、ラットの場合と同様の手法により、９０秒、２１０秒、３３０秒、ＣＯ2に暴露した後、卵子を採取してＩＶＦした。いずれのマウスも、ラットと同様にＣＯ2暴露開始から９０秒で自発呼吸が停止して死に至るものの、２１０秒、３３０秒のものについては、暴露後９０秒で頸椎脱臼により完全に安楽殺を行い、その後の各時間で採卵を行った。コントロールとして、麻酔下頸椎脱臼（Ａｎ．（ｃ．ｄ．））で死に至った後、２４０秒後に採卵を行ったもの（区）も用意した。 (Mouse in vitro fertilization and CO2 exposure)
Next, by exposing mouse eggs to CO 2 during harvesting, and using CO 2 during euthanasia, the eggs spontaneously activate, resulting in impaired post-fertilization developmental potential. was examined to see if it was rat-specific.
Mice were also exposed to CO 2 for 90 seconds, 210 seconds, and 330 seconds by the same procedure as rats, and then eggs were collected and subjected to IVF. All mice, like rats, stopped spontaneous respiration and died 90 seconds after the start of CO 2 exposure. Egg collection was performed at each subsequent hour. As a control, an egg collection was also performed 240 seconds after death by cervical dislocation under anesthesia (An. (c.d.)) (section).
図１Ｋは、２－ｃｅｌｌになった率（２－ｃｅｌｌ ｒａｔｅ（％））を示す。
図１Ｌは、移植後に胚に成長した率（発生率、Ｄｅｖｅｌｏｐｍｅｎｔａｌ ｒａｔｅ（％））を示す。各バー内の数字は、それぞれの数／実験数を示す。
結果として、マウスではＣＯ2に暴露することで発生能だけでなく受精能にも大きな影響があった。
図１Ｍは、図１Ｄと同様に２－ｃｅｌｌを左右の卵管に移植したものである。コントロール（Ａｎ．）と比べると、３３０秒の暴露で得られた２－ｃｅｌｌ（ＣＯ2 ３３０ｓ）は、移植しても着傷痕も得られなかった。 FIG. 1K shows the 2-cell rate (%).
FIG. 1L shows the rate of development into embryos after implantation (developmental rate (%)). Numbers within each bar indicate the respective number/number of experiments.
As a result, in mice, exposure to CO 2 had a profound effect not only on developmental but also on fertility.
FIG. 1M shows 2-cells transplanted into the left and right fallopian tubes as in FIG. 1D. Compared with the control (An.), the 2-cells (CO 2 330s) obtained with exposure for 330 seconds did not produce any scars upon implantation.
図１Ｌは、移植後に胚に成長した率（発生率、Ｄｅｖｅｌｏｐｍｅｎｔａｌ ｒａｔｅ（％））を示す。各バー内の数字は、それぞれの数／実験数を示す。
結果として、マウスではＣＯ2に暴露することで発生能だけでなく受精能にも大きな影響があった。
図１Ｍは、図１Ｄと同様に２－ｃｅｌｌを左右の卵管に移植したものである。コントロール（Ａｎ．）と比べると、３３０秒の暴露で得られた２－ｃｅｌｌ（ＣＯ2 ３３０ｓ）は、移植しても着傷痕も得られなかった。 FIG. 1K shows the 2-cell rate (%).
FIG. 1L shows the rate of development into embryos after implantation (developmental rate (%)). Numbers within each bar indicate the respective number/number of experiments.
As a result, in mice, exposure to CO 2 had a profound effect not only on developmental but also on fertility.
FIG. 1M shows 2-cells transplanted into the left and right fallopian tubes as in FIG. 1D. Compared with the control (An.), the 2-cells (
（ラット卵子とＣＯ2暴露による染色体の分散）
次に、ＣＯ2に暴露した卵子には何が起こっているのかについて調べた。
図２Ａは、卵子の状態を調べるため、核を染色するＤＡＰＩ（４'，６－ｄｉａｍｉｄｉｎｏ－２－ｐｈｅｎｙｌｉｎｄｏｌｅ）と、雌性核酸に付着するヒストンタンパク質であるＨ３Ｋ９ｍｅ３との免疫染色で染色する概念図と、実際に染色した写真を示す。左図の概念図は、卵子の状態による染色の状態の概念を示す。ここでは、「ＭＩＩ」はＭｅｔａｐｈａｓｅ ＩＩ（ＭＩＩ期）、「ＡＩＩ」はＡｎａｐｈａｓｅ ＩＩ（ＡＩＩ期）、「ＴＩＩ」はＴｅｌｏｐｈａｓｅ ＩＩ（ＴＩＩ期、減数分裂再開（終期））、「Ｓｃ」は染色体が分散している（Ｓｃａｔｔｅｒｅｄ）状態、「ｓ．ａ．」は自発的活性化の状態の例を、それぞれ示す。以下、上述の略号は、同様の意味を示す。 (Dispersion of chromosomes by exposure to rat eggs and CO 2 )
Next, we investigated what happens to eggs exposed to CO2 .
FIG. 2A is a conceptual diagram of immunostaining with DAPI (4′,6-diamidino-2-phenylindole), which stains the nucleus, and H3K9me3, a histone protein that attaches to female nucleic acids, to examine the state of eggs. , shows an actual dyed photograph. The conceptual diagram on the left shows the concept of the state of staining according to the state of the ovum. Here, "MII" is Metaphase II (MII stage), "AII" is Anaphase II (AII stage), "TII" is Telophase II (TII stage, meiotic resumption (telophase)), and "Sc" is chromosome dispersal. Scattered state and "sa" indicate examples of spontaneous activation states, respectively. Hereinafter, the above abbreviations have the same meanings.
次に、ＣＯ2に暴露した卵子には何が起こっているのかについて調べた。
図２Ａは、卵子の状態を調べるため、核を染色するＤＡＰＩ（４'，６－ｄｉａｍｉｄｉｎｏ－２－ｐｈｅｎｙｌｉｎｄｏｌｅ）と、雌性核酸に付着するヒストンタンパク質であるＨ３Ｋ９ｍｅ３との免疫染色で染色する概念図と、実際に染色した写真を示す。左図の概念図は、卵子の状態による染色の状態の概念を示す。ここでは、「ＭＩＩ」はＭｅｔａｐｈａｓｅ ＩＩ（ＭＩＩ期）、「ＡＩＩ」はＡｎａｐｈａｓｅ ＩＩ（ＡＩＩ期）、「ＴＩＩ」はＴｅｌｏｐｈａｓｅ ＩＩ（ＴＩＩ期、減数分裂再開（終期））、「Ｓｃ」は染色体が分散している（Ｓｃａｔｔｅｒｅｄ）状態、「ｓ．ａ．」は自発的活性化の状態の例を、それぞれ示す。以下、上述の略号は、同様の意味を示す。 (Dispersion of chromosomes by exposure to rat eggs and CO 2 )
Next, we investigated what happens to eggs exposed to CO2 .
FIG. 2A is a conceptual diagram of immunostaining with DAPI (4′,6-diamidino-2-phenylindole), which stains the nucleus, and H3K9me3, a histone protein that attaches to female nucleic acids, to examine the state of eggs. , shows an actual dyed photograph. The conceptual diagram on the left shows the concept of the state of staining according to the state of the ovum. Here, "MII" is Metaphase II (MII stage), "AII" is Anaphase II (AII stage), "TII" is Telophase II (TII stage, meiotic resumption (telophase)), and "Sc" is chromosome dispersal. Scattered state and "sa" indicate examples of spontaneous activation states, respectively. Hereinafter, the above abbreviations have the same meanings.
図２Ｂはコントロールの麻酔下で採取した卵子、図２Ｃは１２０秒のＣＯ2暴露（１２０ｓ）卵子について、ＩＶＦし、媒精後の状態を、ＤＡＰＩ及びＨ３Ｋ９ｍｅ３で免疫染色して確認した状態の割合をそれぞれ示すグラフである。いずれも、横軸は媒精後の時間、縦軸は確認した卵子の染色体の状態（Ｃｈｒｏｍｏｓｏｍｅ ｓｔａｔｕｓ（％））を示す。各バー内の文字は、上述の図２Ａと同様の状態を示し、更に、「Ｆｚ」は受精（Ｆｅｒｔｉｌｉｚａｔｉｏｎ）の状態を示す。
この免疫染色の結果によれば、コントロールの麻酔下で採取した卵子は媒精後、４．５時間程度は、ＭＩＩ期で停止し、その後、受精（Ｆｚ）状態となった。しかしながら、ＣＯ2に暴露した卵子は、ＭＩＩで停止したままの状態とはならず、媒精後０．５～１．５時間から自発的活性化が生じ、時間の経過と共に、染色体が分散してしまっていた。
すなわち、ラット卵子は、ＣＯ2暴露により自発的活性化から染色体の分散に至っていた。採卵後３時間以降は、全ての卵子で、染色体が分散した。しかしながら、媒精５．５時間後にはその染色体の分散した状態の卵が精子と受精（Ｆｚ）することが分かった。すなわち、自発的活性化が起こっても、染色体が分散した状態でも、受精能はあった。 Fig. 2B shows the eggs collected under control anesthesia, and Fig. 2C shows the ratio of the conditions after IVF and confirmed by immunostaining with DAPI and H3K9me3 for the eggs exposed to CO 2 for 120 seconds (120s). , respectively. In each case, the horizontal axis indicates the time after insemination, and the vertical axis indicates the confirmed chromosomal state of the egg (chromosome status (%)). The letters in each bar indicate the same state as in FIG. 2A described above, and "Fz" indicates the state of fertilization.
According to the results of this immunostaining, the ova collected under control anesthesia stopped at the MII stage for about 4.5 hours after insemination, and then entered the fertilized (Fz) state. However, oocytes exposed to CO2 did not remain arrested at MII, spontaneous activation occurred from 0.5-1.5 hours after insemination, and the chromosomes dispersed over time. It was gone.
That is, rat oocytes undergo spontaneous activation to chromosomal dispersal upon exposure to CO 2 . After 3 hours after egg retrieval, the chromosomes were dispersed in all the eggs. However, 5.5 hours after insemination, the egg with dispersed chromosomes was found to be fertilized with the sperm (Fz). That is, even when spontaneous activation occurred, fertilization was possible even when the chromosomes were dispersed.
この免疫染色の結果によれば、コントロールの麻酔下で採取した卵子は媒精後、４．５時間程度は、ＭＩＩ期で停止し、その後、受精（Ｆｚ）状態となった。しかしながら、ＣＯ2に暴露した卵子は、ＭＩＩで停止したままの状態とはならず、媒精後０．５～１．５時間から自発的活性化が生じ、時間の経過と共に、染色体が分散してしまっていた。
すなわち、ラット卵子は、ＣＯ2暴露により自発的活性化から染色体の分散に至っていた。採卵後３時間以降は、全ての卵子で、染色体が分散した。しかしながら、媒精５．５時間後にはその染色体の分散した状態の卵が精子と受精（Ｆｚ）することが分かった。すなわち、自発的活性化が起こっても、染色体が分散した状態でも、受精能はあった。 Fig. 2B shows the eggs collected under control anesthesia, and Fig. 2C shows the ratio of the conditions after IVF and confirmed by immunostaining with DAPI and H3K9me3 for the eggs exposed to CO 2 for 120 seconds (120s). , respectively. In each case, the horizontal axis indicates the time after insemination, and the vertical axis indicates the confirmed chromosomal state of the egg (chromosome status (%)). The letters in each bar indicate the same state as in FIG. 2A described above, and "Fz" indicates the state of fertilization.
According to the results of this immunostaining, the ova collected under control anesthesia stopped at the MII stage for about 4.5 hours after insemination, and then entered the fertilized (Fz) state. However, oocytes exposed to CO2 did not remain arrested at MII, spontaneous activation occurred from 0.5-1.5 hours after insemination, and the chromosomes dispersed over time. It was gone.
That is, rat oocytes undergo spontaneous activation to chromosomal dispersal upon exposure to CO 2 . After 3 hours after egg retrieval, the chromosomes were dispersed in all the eggs. However, 5.5 hours after insemination, the egg with dispersed chromosomes was found to be fertilized with the sperm (Fz). That is, even when spontaneous activation occurred, fertilization was possible even when the chromosomes were dispersed.
図２Ｄは、コントロールの麻酔下で採取した卵子（Ａｎ．）、及び１２０秒のＣＯ2暴露卵子（ＣＯ2）について、媒精後６．５時間（６．５ｈ）、１０時間（１０ｈ）、２０～２１時間（２０－２１ｈ）の各状態のＤＡＰＩ、Ｈ３Ｋ９ｍｅ３、同時染色（Ｍｅｒｇｅ）による免疫染色の状態を示す。矢頭は、雌性前核を示す。矢印は、雄性核又は雄性前核を示す。
矢頭で示すように、媒精後６．５時間で染色すると、ＣＯ2暴露卵子は、雌性前核が分散していた。さらに、１０時間後、２０～２１時間後には、矢頭で示すように、染色体が分散し、分散した雌性前核となっていた。 FIG. 2D shows eggs harvested under control anesthesia (An.) and eggs exposed to CO 2 for 120 seconds (CO 2 ) at 6.5 hours (6.5 h), 10 hours (10 h), The state of immunostaining by DAPI, H3K9me3, and co-staining (Merge) in each state at 20 to 21 hours (20-21 h) is shown. Arrowheads indicate female pronuclei. Arrows indicate the male nucleus or male pronucleus.
As indicated by arrowheads, CO2 - exposed oocytes had dispersed female pronuclei when stained 6.5 hours after insemination. Furthermore, after 10 hours and 20 to 21 hours, as indicated by the arrowheads, the chromosomes were dispersed to form dispersed female pronuclei.
矢頭で示すように、媒精後６．５時間で染色すると、ＣＯ2暴露卵子は、雌性前核が分散していた。さらに、１０時間後、２０～２１時間後には、矢頭で示すように、染色体が分散し、分散した雌性前核となっていた。 FIG. 2D shows eggs harvested under control anesthesia (An.) and eggs exposed to CO 2 for 120 seconds (CO 2 ) at 6.5 hours (6.5 h), 10 hours (10 h), The state of immunostaining by DAPI, H3K9me3, and co-staining (Merge) in each state at 20 to 21 hours (20-21 h) is shown. Arrowheads indicate female pronuclei. Arrows indicate the male nucleus or male pronucleus.
As indicated by arrowheads, CO2 - exposed oocytes had dispersed female pronuclei when stained 6.5 hours after insemination. Furthermore, after 10 hours and 20 to 21 hours, as indicated by the arrowheads, the chromosomes were dispersed to form dispersed female pronuclei.
図２Ｅは、上述の図２Ｄの麻酔下で採取した採卵卵子（Ａｎ．）及びＣＯ2暴露した卵子（ＣＯ2）において、媒精後６．５時間（６．５ｈ）で雌性前核の数をプロットしたものである。図２Ｆは、図２Ｅと同様にして、２０～２１時間（２０－２１ｈ）で雌性前核の数をプロットしたものである。
各グラフについて、縦軸は雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）を示し、各○印はその数、太線は中央値、細い四角の線は標準偏差（１σ）の１未満を切り捨てしたものをそれぞれ示す。
結果として、麻酔下で採取した採卵卵子の雌性前核はほとんどが１個であるが、ＣＯ2卵子の雌性前核前核はその多くが２～７個程度に分散していた。 Figure 2E shows the number of female pronuclei at 6.5 hours (6.5 h) after insemination in the retrieved oocytes (An.) and CO2 - exposed oocytes ( CO2 ) collected under anesthesia in Figure 2D above. is plotted. FIG. 2F plots the number of female pronuclei at 20-21 hours (20-21 h) as in FIG. 2E.
For each graph, the vertical axis indicates the number of female pronuclei (No. of female pronuclei), each circle indicates the number, the thick line indicates the median value, and the thin square line indicates the standard deviation (1σ), which is rounded down to less than 1. Show each thing.
As a result, most of the oocytes collected under anesthesia had a single female pronucleus, but most of the CO 2 oocytes had 2 to 7 dispersed female pronuclei.
各グラフについて、縦軸は雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）を示し、各○印はその数、太線は中央値、細い四角の線は標準偏差（１σ）の１未満を切り捨てしたものをそれぞれ示す。
結果として、麻酔下で採取した採卵卵子の雌性前核はほとんどが１個であるが、ＣＯ2卵子の雌性前核前核はその多くが２～７個程度に分散していた。 Figure 2E shows the number of female pronuclei at 6.5 hours (6.5 h) after insemination in the retrieved oocytes (An.) and CO2 - exposed oocytes ( CO2 ) collected under anesthesia in Figure 2D above. is plotted. FIG. 2F plots the number of female pronuclei at 20-21 hours (20-21 h) as in FIG. 2E.
For each graph, the vertical axis indicates the number of female pronuclei (No. of female pronuclei), each circle indicates the number, the thick line indicates the median value, and the thin square line indicates the standard deviation (1σ), which is rounded down to less than 1. Show each thing.
As a result, most of the oocytes collected under anesthesia had a single female pronucleus, but most of the CO 2 oocytes had 2 to 7 dispersed female pronuclei.
図２Ｇは、媒精後２４時間の免疫染色の結果である。媒精して２４時間経過すると麻酔下で採卵した卵子由来の胚（Ａｎ．）も、ＣＯ2暴露した卵子由来の胚（ＣＯ2）も、雌雄前核が交わってＭ期に移行した。ＰＢ２は第二極体（Ｓｅｃｏｎｄ Ｐｏｌａｒ ｂｏｄｙ）を示す。ここで、正常な卵子では、減数分裂の第二分裂の途中でＭＩＩ期として休止し、精子の進入とともに活性化し、細胞サイクルが再開する。この際に、受精卵に確認されるのが、第二極体である。
すなわち、ＣＯ2暴露された状態の卵子は、正常な卵子では起こらない自発的活性化が生じて、染色体が分散し、分散したまま受精する。この上で、染色体が分散した雌性前核が、そのまま雄性前核と交わることが分かった。 FIG. 2G is the result ofimmunostaining 24 hours after insemination. Twenty-four hours after insemination, both the embryos derived from the eggs collected under anesthesia (An.) and the embryos derived from the eggs exposed to CO 2 (CO 2 ) crossed male and female pronuclei and entered the M phase. PB2 indicates a second polar body. Here, in a normal ovum, it pauses as the MII phase in the middle of the second division of meiosis, is activated with the entry of sperm, and the cell cycle restarts. At this time, the second polar body is confirmed in the fertilized egg.
That is, in the ova exposed to CO 2 , spontaneous activation that does not occur in normal ova occurs, the chromosomes are dispersed, and fertilization occurs while the chromosomes are dispersed. On top of this, it was found that the female pronucleus in which the chromosomes were dispersed intersects with the male pronucleus as it is.
すなわち、ＣＯ2暴露された状態の卵子は、正常な卵子では起こらない自発的活性化が生じて、染色体が分散し、分散したまま受精する。この上で、染色体が分散した雌性前核が、そのまま雄性前核と交わることが分かった。 FIG. 2G is the result of
That is, in the ova exposed to CO 2 , spontaneous activation that does not occur in normal ova occurs, the chromosomes are dispersed, and fertilization occurs while the chromosomes are dispersed. On top of this, it was found that the female pronucleus in which the chromosomes were dispersed intersects with the male pronucleus as it is.
このように、母体の環境変化に応じて卵子が自発的活性化と染色体の分散を経由し、その後、受精はするのにその胚が流産する現象を、「Ｈｉｄｄｅｎ Ｄｅｇｒａｄａｔｉｏｎ ｏｆ Ｖｉａｂｌｅ ｏｏｃｙｔｅｓ（ＨＤＶ）」と名付けた。ＨＤＶは、いってみれば、生きているように見える卵子が、実は死んでいるような状態である。
In this way, the ovum undergoes spontaneous activation and chromosome dispersal in response to environmental changes in the mother's body, and the phenomenon of fertilization but abortion of the embryo is called "Hidden Degradation of Viable Oocytes (HDV)". named. HDV is a state in which an egg that appears to be alive is actually dead.
（ＣＯ2暴露された卵子由来２細胞期胚の状態）
図３Ａは、コントロールの麻酔下頸椎脱臼（ｃ．ｄ）で採取した卵子をＩＶＦし、２－ｃｅｌｌに発生させた際の核型解析の写真である。このように麻酔で採卵した卵子が受精し、２－ｃｅｌｌになった場合は、多くの割球の染色体が４２本である。
図３Ｂは、１２０秒のＣＯ2暴露した卵子をＩＶＦし、２－ｃｅｌｌに発生させた際の核型解析の写真である。このようにＣＯ2暴露した後に受精して２－ｃｅｌｌになった胚では、割球ごとに染色体数が不均一になっている。すなわち、ひとつの２－ｃｅｌｌで、各割球の染色体数が異なっていた。 (State of 2-cell stage embryos derived from eggs exposed to CO 2 )
FIG. 3A is a photograph of karyotype analysis when eggs collected from control cervical dislocation under anesthesia (cd) were subjected to IVF and developed into 2-cells. When the ovum collected under anesthesia is fertilized and becomes 2-cell, most of the blastomeres have 42 chromosomes.
FIG. 3B is a photograph of karyotype analysis when eggs exposed to CO 2 for 120 seconds were subjected to IVF and developed into 2-cells. In embryos that are fertilized into 2-cells after being exposed to CO 2 in this way, the number of chromosomes is heterogeneous for each blastomere. That is, in one 2-cell, each blastomere had a different number of chromosomes.
図３Ａは、コントロールの麻酔下頸椎脱臼（ｃ．ｄ）で採取した卵子をＩＶＦし、２－ｃｅｌｌに発生させた際の核型解析の写真である。このように麻酔で採卵した卵子が受精し、２－ｃｅｌｌになった場合は、多くの割球の染色体が４２本である。
図３Ｂは、１２０秒のＣＯ2暴露した卵子をＩＶＦし、２－ｃｅｌｌに発生させた際の核型解析の写真である。このようにＣＯ2暴露した後に受精して２－ｃｅｌｌになった胚では、割球ごとに染色体数が不均一になっている。すなわち、ひとつの２－ｃｅｌｌで、各割球の染色体数が異なっていた。 (State of 2-cell stage embryos derived from eggs exposed to CO 2 )
FIG. 3A is a photograph of karyotype analysis when eggs collected from control cervical dislocation under anesthesia (cd) were subjected to IVF and developed into 2-cells. When the ovum collected under anesthesia is fertilized and becomes 2-cell, most of the blastomeres have 42 chromosomes.
FIG. 3B is a photograph of karyotype analysis when eggs exposed to CO 2 for 120 seconds were subjected to IVF and developed into 2-cells. In embryos that are fertilized into 2-cells after being exposed to CO 2 in this way, the number of chromosomes is heterogeneous for each blastomere. That is, in one 2-cell, each blastomere had a different number of chromosomes.
図３Ｃは、１２０秒のＣＯ2暴露した卵子をＩＶＦし、２－ｃｅｌｌに発生させた後で、Ｈ２Ｂ－ｍＣｈｅｒｒｙプローブによりライブイメージングでリアルタイムに観察したものを時間毎に取得した写真である。
ＣＯ2に暴露された卵子は、複数の雌性前核が散らばったまま、雄性核と交わり受精するのが分かる。ここで、矢頭は、分散した染色体を、矢印は雌性前核を示す。その後、分裂し２細胞期（２－ｃｅｌｌ）になるものの、その時に微小染色体が片方の割球にだけ取り込まれる様子が見える。 FIG. 3C is a time-series photograph of ova exposed to CO 2 for 120 seconds and then subjected to IVF to develop into 2-cells, followed by real-time observation by live imaging with an H2B-mCherry probe.
It can be seen that eggs exposed to CO 2 intersect with male nuclei and are fertilized while multiple female pronuclei remain scattered. Here, arrowheads indicate dispersed chromosomes and arrows indicate female pronuclei. After that, it divides and reaches the 2-cell stage, but at that time, it can be seen that the microchromosome is incorporated into only one blastomere.
ＣＯ2に暴露された卵子は、複数の雌性前核が散らばったまま、雄性核と交わり受精するのが分かる。ここで、矢頭は、分散した染色体を、矢印は雌性前核を示す。その後、分裂し２細胞期（２－ｃｅｌｌ）になるものの、その時に微小染色体が片方の割球にだけ取り込まれる様子が見える。 FIG. 3C is a time-series photograph of ova exposed to CO 2 for 120 seconds and then subjected to IVF to develop into 2-cells, followed by real-time observation by live imaging with an H2B-mCherry probe.
It can be seen that eggs exposed to CO 2 intersect with male nuclei and are fertilized while multiple female pronuclei remain scattered. Here, arrowheads indicate dispersed chromosomes and arrows indicate female pronuclei. After that, it divides and reaches the 2-cell stage, but at that time, it can be seen that the microchromosome is incorporated into only one blastomere.
図３Ｄは、このような染色体の分配エラーをカウントした。この図では、コントロールの麻酔下で採取した卵子（Ａｎ．）、１２０秒のＣＯ2に暴露した卵子（ＣＯ2 １２０ｓ）において、縦軸は染色体の分配エラー率（Ｃｈｒｏｍｏｓｏｍｅ ｓｅｇｍｅｎｔａｔｉｏｎ ｅｒｒｏｒ（％））を示す。灰色で塗りつぶした箇所が、分配エラーとなった２－ｃｅｌｌを示す。塗りつぶしていない箇所は、分配エラーとなっていない２－ｃｅｌｌを示す。バー内の数値は、それぞれのカウント数を示す。
このように、染色体の分配エラーをカウントすると、ＣＯ2に暴露した卵子では４５％程度で染色体の不等分裂が確認された。すなわち、ＣＯ2暴露した卵子では、ＩＶＦ後、２－ｃｅｌｌへ分裂する段階で、染色体の不等分裂が起こっていた。 FIG. 3D counts such chromosome segregation errors. In this figure, the ovaries collected under control anesthesia (An.) and the ova exposed to CO 2 for 120 seconds (CO 2 120s), the vertical axis represents the chromosome segmentation error rate (chromosome segmentation error (%)). show. Gray areas indicate 2-cells with distribution errors. Unfilled areas indicate 2-cells with no distribution error. Numbers in bars indicate respective counts.
In this way, counting chromosome segregation errors, chromosomal asymmetry was confirmed in about 45% of the eggs exposed to CO 2 . That is, in the CO 2 -exposed oocytes, chromosome asymmetry occurred at the stage of division into 2-cells after IVF.
このように、染色体の分配エラーをカウントすると、ＣＯ2に暴露した卵子では４５％程度で染色体の不等分裂が確認された。すなわち、ＣＯ2暴露した卵子では、ＩＶＦ後、２－ｃｅｌｌへ分裂する段階で、染色体の不等分裂が起こっていた。 FIG. 3D counts such chromosome segregation errors. In this figure, the ovaries collected under control anesthesia (An.) and the ova exposed to CO 2 for 120 seconds (
In this way, counting chromosome segregation errors, chromosomal asymmetry was confirmed in about 45% of the eggs exposed to CO 2 . That is, in the CO 2 -exposed oocytes, chromosome asymmetry occurred at the stage of division into 2-cells after IVF.
（プロテアソーム阻害剤ＭＧ１３２による効果）
この自発的活性化を抑えるため、様々な組成物を試したところ、プロテアソーム阻害剤を用いて、自発的活性化で分解されると不平等分裂をもたらすタンパクの分解を抑えることができた。
具体的には、１２０秒のＣＯ2に暴露した卵子を１０μＭのＭＧ１３２（可逆的プロテアソーム阻害剤）を含むＨＴＦ培地で３時間媒精し、その後ＭＧ１３２を含まない培地に卵子を移して媒精継続した（以下、「ＭＧ１３２添加」という。）。
図４Ａは、ＭＧ１３２添加の状態で染色体の状態を調べたものであり、縦軸は染色体の状態（Ｃｈｒｏｍｏｓｏｍｅ ｓｔａｔｕｓ（％））、横軸は媒精からの時間を示す。バー内の各数字は、カウントされた数を示す。
卵子の自発的活性化（ｓ．ａ．）は有意に低下し、ＭＩＩ期を維持する卵子が増えていた。ただし、媒精３時間後にＭＧ１３２を除去した時点から自発的活性化が発生し、染色体の分散が生じているものの、受精が起こっていることが分かった。 (Effect of proteasome inhibitor MG132)
Various compositions were tested to suppress this spontaneous activation, and proteasome inhibitors were used to suppress degradation of proteins that, when degraded by spontaneous activation, lead to unequal fission.
Specifically, eggs exposed to CO 2 for 120 seconds were inseminated in HTF medium containing 10 μM MG132 (reversible proteasome inhibitor) for 3 hours, and then transferred to a medium containing no MG132 to continue insemination. (hereinafter referred to as "MG132 addition").
FIG. 4A shows the state of chromosomes in the state of MG132 addition, the vertical axis indicates the state of chromosomes (chromosome status (%)), and the horizontal axis indicates the time from insemination. Each number in the bar indicates the number counted.
Spontaneous egg activation (s.a.) was significantly reduced and more eggs remained in the MII stage. However, it was found that 3 hours after insemination, when MG132 was removed, spontaneous activation occurred, and although chromosome dispersion occurred, fertilization occurred.
この自発的活性化を抑えるため、様々な組成物を試したところ、プロテアソーム阻害剤を用いて、自発的活性化で分解されると不平等分裂をもたらすタンパクの分解を抑えることができた。
具体的には、１２０秒のＣＯ2に暴露した卵子を１０μＭのＭＧ１３２（可逆的プロテアソーム阻害剤）を含むＨＴＦ培地で３時間媒精し、その後ＭＧ１３２を含まない培地に卵子を移して媒精継続した（以下、「ＭＧ１３２添加」という。）。
図４Ａは、ＭＧ１３２添加の状態で染色体の状態を調べたものであり、縦軸は染色体の状態（Ｃｈｒｏｍｏｓｏｍｅ ｓｔａｔｕｓ（％））、横軸は媒精からの時間を示す。バー内の各数字は、カウントされた数を示す。
卵子の自発的活性化（ｓ．ａ．）は有意に低下し、ＭＩＩ期を維持する卵子が増えていた。ただし、媒精３時間後にＭＧ１３２を除去した時点から自発的活性化が発生し、染色体の分散が生じているものの、受精が起こっていることが分かった。 (Effect of proteasome inhibitor MG132)
Various compositions were tested to suppress this spontaneous activation, and proteasome inhibitors were used to suppress degradation of proteins that, when degraded by spontaneous activation, lead to unequal fission.
Specifically, eggs exposed to CO 2 for 120 seconds were inseminated in HTF medium containing 10 μM MG132 (reversible proteasome inhibitor) for 3 hours, and then transferred to a medium containing no MG132 to continue insemination. (hereinafter referred to as "MG132 addition").
FIG. 4A shows the state of chromosomes in the state of MG132 addition, the vertical axis indicates the state of chromosomes (chromosome status (%)), and the horizontal axis indicates the time from insemination. Each number in the bar indicates the number counted.
Spontaneous egg activation (s.a.) was significantly reduced and more eggs remained in the MII stage. However, it was found that 3 hours after insemination, when MG132 was removed, spontaneous activation occurred, and although chromosome dispersion occurred, fertilization occurred.
図４Ｂは、ＭＧ１３２添加した場合における染色体の状態の時間変化を示す。縦軸は、染色体の状態（Ｃｈｒｏｍｏｓｏｍｅ ｓｔａｔｕｓ（％））を示す。バー内の各数字は、カウントされた数を示す。
ＭＧ１３２を添加しても、受精卵の約半分は染色体が散らばった状態で受精していた。 FIG. 4B shows temporal changes in the state of chromosomes when MG132 was added. The vertical axis indicates the chromosome status (chromosome status (%)). Each number in the bar indicates the number counted.
Even with the addition of MG132, about half of the fertilized eggs were fertilized with scattered chromosomes.
ＭＧ１３２を添加しても、受精卵の約半分は染色体が散らばった状態で受精していた。 FIG. 4B shows temporal changes in the state of chromosomes when MG132 was added. The vertical axis indicates the chromosome status (chromosome status (%)). Each number in the bar indicates the number counted.
Even with the addition of MG132, about half of the fertilized eggs were fertilized with scattered chromosomes.
図４Ｃは、ＭＧ１３２添加したものについて、媒精後６．５時間（６．５ｈ）、１０時間（１０ｈ）、２０～２２時間（２０－２２ｈ）、２４時間（２４ｈ）の各状態のＤＡＰＩ、Ｈ３Ｋ９ｍｅ３、同時染色（Ｍｅｒｇｅ）による免疫染色の状態を示す。矢頭は、第二極体（ＰＢ２）を示す。
図４Ｄは、図２Ｅと同様の形式で、ＭＧ１３２を添加したものの雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）をカウントしたものである。
図４Ｅは、ＭＧ１３２添加したものを２－ｃｅｌｌに発生させた後で、ライブイメージングでリアルタイムに観察した写真である。
結果として、媒精後６．５時間や１０時間では、雌性染色体又は雌性前核が分散しているものの、２０～２２時間で、まさに雄性前核と交わる直前には、分散していた雌性前核が一つにまとまっていた。すなわち、２－ｃｅｌｌにおける染色体の不等分裂が改善された。 FIG. 4C shows DAPI in each state 6.5 hours (6.5 h), 10 hours (10 h), 20-22 hours (20-22 h), 24 hours (24 h) after insemination for those with MG132 addition, The state of immunostaining by H3K9me3, co-staining (Merge) is shown. Arrowheads indicate the second polar body (PB2).
Figure 4D shows the number of female pronuclei (No. of female pronuclei) to which MG132 was added in the same format as Figure 2E.
FIG. 4E is a photograph observed in real time by live imaging after MG132-added 2-cells were generated.
As a result, although the female chromosomes or female pronuclei are dispersed at 6.5 and 10 hours after insemination, at 20 to 22 hours, just before intersecting with the male pronucleus, the dispersed female pronuclei The nucleus was united. That is, the asymmetric division of chromosomes in 2-cells was improved.
図４Ｄは、図２Ｅと同様の形式で、ＭＧ１３２を添加したものの雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）をカウントしたものである。
図４Ｅは、ＭＧ１３２添加したものを２－ｃｅｌｌに発生させた後で、ライブイメージングでリアルタイムに観察した写真である。
結果として、媒精後６．５時間や１０時間では、雌性染色体又は雌性前核が分散しているものの、２０～２２時間で、まさに雄性前核と交わる直前には、分散していた雌性前核が一つにまとまっていた。すなわち、２－ｃｅｌｌにおける染色体の不等分裂が改善された。 FIG. 4C shows DAPI in each state 6.5 hours (6.5 h), 10 hours (10 h), 20-22 hours (20-22 h), 24 hours (24 h) after insemination for those with MG132 addition, The state of immunostaining by H3K9me3, co-staining (Merge) is shown. Arrowheads indicate the second polar body (PB2).
Figure 4D shows the number of female pronuclei (No. of female pronuclei) to which MG132 was added in the same format as Figure 2E.
FIG. 4E is a photograph observed in real time by live imaging after MG132-added 2-cells were generated.
As a result, although the female chromosomes or female pronuclei are dispersed at 6.5 and 10 hours after insemination, at 20 to 22 hours, just before intersecting with the male pronucleus, the dispersed female pronuclei The nucleus was united. That is, the asymmetric division of chromosomes in 2-cells was improved.
図４Ｆは、ＣＯ2暴露胚へＭＧ１３２添加することにより、その２細胞期胚（２－ｃｅｌｌ）における染色体の分散が抑えられた率を表す。コントロールの麻酔下で採取した卵子（麻酔）、１２０秒のＣＯ2に暴露した卵子（ＣＯ2）、及びＭＧ１３２添加したもの（ＣＯ2に＋ＭＧ１３２）について、図３Ｄと同様に、縦軸は染色体の分配エラー率を示し、灰色で塗りつぶした箇所が、分配エラーとなった２－ｃｅｌｌを示す。塗りつぶしていない箇所は、分配エラーとなっていない２－ｃｅｌｌを示す。バー内の数値は、それぞれのカウント数を示す。「ｐ」は、統計検定のｐ値を示す。
この結果により、ＭＧ１３２により、２－ｃｅｌｌにおける染色体の不等分裂が改善され、染色体の異数性が改善されることが分かった。 FIG. 4F shows the rate of suppression of chromosome dispersion in 2-cell stage embryos by the addition of MG132 to CO 2 -exposed embryos. Eggs collected under control anesthesia (anesthesia), eggs exposed to CO 2 for 120 seconds (CO 2 ), and those with MG132 added (CO 2 +MG132). Distribution error rates are shown, and 2-cells with distribution errors are shown in gray areas. Unfilled areas indicate 2-cells with no distribution error. Numbers in bars indicate respective counts. "p" indicates the p-value of the statistical test.
From these results, it was found that MG132 improved chromosomal asymmetry in 2-cells and ameliorated chromosomal aneuploidy.
この結果により、ＭＧ１３２により、２－ｃｅｌｌにおける染色体の不等分裂が改善され、染色体の異数性が改善されることが分かった。 FIG. 4F shows the rate of suppression of chromosome dispersion in 2-cell stage embryos by the addition of MG132 to CO 2 -exposed embryos. Eggs collected under control anesthesia (anesthesia), eggs exposed to CO 2 for 120 seconds (CO 2 ), and those with MG132 added (CO 2 +MG132). Distribution error rates are shown, and 2-cells with distribution errors are shown in gray areas. Unfilled areas indicate 2-cells with no distribution error. Numbers in bars indicate respective counts. "p" indicates the p-value of the statistical test.
From these results, it was found that MG132 improved chromosomal asymmetry in 2-cells and ameliorated chromosomal aneuploidy.
図４Ｇに示すように、ＭＧ１３２を添加した卵子を偽妊娠雌ラットに移植したところ、ＣＯ2暴露した卵子からでも産仔が得られた。
図４Ｈは、このＭＧ１３２を添加した場合としない場合の比較として、縦軸はラットの出生率（Ｂｉｒｔｈ ｒａｔｅ（％））、横軸はコントロールの麻酔下頸椎脱臼（Ａｎ．（ｃ．ｄ．）で採取した卵子、１２０秒のＣＯ2暴露してから採取した卵子（ＣＯ2 １２０ｓ）、１５０秒のＣＯ2暴露した卵子（ＣＯ2 １５０ｓ）について、それぞれＭＧ１３２で処理の有（＋）無（－）についての結果を示す。
このように、ＣＯ2暴露された卵子であっても、プロテアソーム阻害剤ＭＧ１３２で処理することで発生の進行が可能となり、産仔が得られた。
なお、コントロールの麻酔下頸椎脱臼の卵子でも、ＭＧ１３２を添加したことにより出生率が上昇している。これは、ＭＧ１３２が妊娠促進（不妊治療）に用いられる可能性を示している。 As shown in FIG. 4G, when MG132-added ova were transplanted into pseudopregnant female rats, offspring were obtained even from CO 2 -exposed ova.
FIG. 4H shows a comparison between the addition of MG132 and no MG132. The collected eggs, the collected eggs exposed to CO 2 for 120 seconds (CO 2 120s), and the eggs exposed to CO 2 for 150 seconds (CO 2 150s) were treated with MG132 (+) and not (-). shows the results for
Thus, even the CO 2 -exposed eggs were able to develop and produce offspring when treated with the proteasome inhibitor MG132.
In addition, the fertility rate also increased with the addition of MG132 to the ova of the control cervical dislocation under anesthesia. This indicates that MG132 may be used for promoting pregnancy (fertility treatment).
図４Ｈは、このＭＧ１３２を添加した場合としない場合の比較として、縦軸はラットの出生率（Ｂｉｒｔｈ ｒａｔｅ（％））、横軸はコントロールの麻酔下頸椎脱臼（Ａｎ．（ｃ．ｄ．）で採取した卵子、１２０秒のＣＯ2暴露してから採取した卵子（ＣＯ2 １２０ｓ）、１５０秒のＣＯ2暴露した卵子（ＣＯ2 １５０ｓ）について、それぞれＭＧ１３２で処理の有（＋）無（－）についての結果を示す。
このように、ＣＯ2暴露された卵子であっても、プロテアソーム阻害剤ＭＧ１３２で処理することで発生の進行が可能となり、産仔が得られた。
なお、コントロールの麻酔下頸椎脱臼の卵子でも、ＭＧ１３２を添加したことにより出生率が上昇している。これは、ＭＧ１３２が妊娠促進（不妊治療）に用いられる可能性を示している。 As shown in FIG. 4G, when MG132-added ova were transplanted into pseudopregnant female rats, offspring were obtained even from CO 2 -exposed ova.
FIG. 4H shows a comparison between the addition of MG132 and no MG132. The collected eggs, the collected eggs exposed to CO 2 for 120 seconds (
Thus, even the CO 2 -exposed eggs were able to develop and produce offspring when treated with the proteasome inhibitor MG132.
In addition, the fertility rate also increased with the addition of MG132 to the ova of the control cervical dislocation under anesthesia. This indicates that MG132 may be used for promoting pregnancy (fertility treatment).
（プロテアソーム阻害剤ＡＬＬＮによる効果）
次に、ＭＧ１３２とは異なる可逆的プロテアソーム阻害剤ＡＬＬＮでも同様の効果があるか否かを確認した。このため、１２０秒のＣＯ2暴露を行った卵子に、２５μＭのＡＬＬＮをＭＧ１３２と同様に作用させた（以下、「ＡＬＬＥＮ添加」という。）。
図４Ｉは、ＣＯ2暴露を行ったのみ（ＣＯ2 (１２０ｓ））と、ＡＬＬＮ添加のもの（ＣＯ2 (１２０ｓ）＋ＡＬＬＮ）を、ＤＡＰＩ、Ｈ３Ｋ９ｍｅ３、同時染色（Ｍｅｒｇｅ）した免疫染色の結果を示す。図中の矢頭は、雌性前核の様子で、複数ある場合は分散していることを示す。矢印は、雄性前核を示す。「ＰＢ２」は、第二極体を示す。
図４Ｊは、図２Ｅと同様の形式で、２－ｃｅｌｌでの雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）をカウントした数を示す。
その結果、ＭＧ１３２添加群と同様に、明らかに媒精後２０～２２時間で、雌性前核がまとまっている様子がみられた。 (Effect of proteasome inhibitor ALLN)
Next, it was confirmed whether or not the reversible proteasome inhibitor ALLN, which is different from MG132, has the same effect. For this reason, 25 μM ALLN was allowed to act in the same manner as MG132 on eggs that had been exposed to CO 2 for 120 seconds (hereinafter referred to as “ALLEN addition”).
FIG. 4I shows the results of immunostaining of CO 2 only (CO 2 ( 120 s)) and ALLN-added (CO 2 ( 120 s) + ALLN) co-stained with DAPI, H3K9me3 (Merge). . The arrowheads in the figure show the appearance of the female pronucleus, and if there are more than one, they are dispersed. Arrows indicate male pronuclei. "PB2" indicates the second polar body.
FIG. 4J shows the number of female pronuclei counted in 2-cells (No. of female pronuclei) in a format similar to that of FIG. 2E.
As a result, similar to the MG132-added group, it was clearly observed that the female pronuclei were organized 20 to 22 hours after insemination.
次に、ＭＧ１３２とは異なる可逆的プロテアソーム阻害剤ＡＬＬＮでも同様の効果があるか否かを確認した。このため、１２０秒のＣＯ2暴露を行った卵子に、２５μＭのＡＬＬＮをＭＧ１３２と同様に作用させた（以下、「ＡＬＬＥＮ添加」という。）。
図４Ｉは、ＣＯ2暴露を行ったのみ（ＣＯ2 (１２０ｓ））と、ＡＬＬＮ添加のもの（ＣＯ2 (１２０ｓ）＋ＡＬＬＮ）を、ＤＡＰＩ、Ｈ３Ｋ９ｍｅ３、同時染色（Ｍｅｒｇｅ）した免疫染色の結果を示す。図中の矢頭は、雌性前核の様子で、複数ある場合は分散していることを示す。矢印は、雄性前核を示す。「ＰＢ２」は、第二極体を示す。
図４Ｊは、図２Ｅと同様の形式で、２－ｃｅｌｌでの雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）をカウントした数を示す。
その結果、ＭＧ１３２添加群と同様に、明らかに媒精後２０～２２時間で、雌性前核がまとまっている様子がみられた。 (Effect of proteasome inhibitor ALLN)
Next, it was confirmed whether or not the reversible proteasome inhibitor ALLN, which is different from MG132, has the same effect. For this reason, 25 μM ALLN was allowed to act in the same manner as MG132 on eggs that had been exposed to CO 2 for 120 seconds (hereinafter referred to as “ALLEN addition”).
FIG. 4I shows the results of immunostaining of CO 2 only (
FIG. 4J shows the number of female pronuclei counted in 2-cells (No. of female pronuclei) in a format similar to that of FIG. 2E.
As a result, similar to the MG132-added group, it was clearly observed that the female pronuclei were organized 20 to 22 hours after insemination.
図４Ｋは、実際に得られた産仔の写真、図４Ｌは、ＡＬＬＮ添加の有（＋）無（－）による出生率を示す。
実際に、ＡＬＬＮ入りの培地でＩＶＦを行うだけで、ＣＯ2暴露した卵子からでも産仔が３８％と高い割合で得られた。 FIG. 4K is a photograph of the offspring actually obtained, and FIG. 4L shows the birth rate with (+) and without (-) addition of ALLN.
In fact, just by performing IVF in a medium containing ALLN, offspring were produced at a high rate of 38% even from CO 2 -exposed oocytes.
実際に、ＡＬＬＮ入りの培地でＩＶＦを行うだけで、ＣＯ2暴露した卵子からでも産仔が３８％と高い割合で得られた。 FIG. 4K is a photograph of the offspring actually obtained, and FIG. 4L shows the birth rate with (+) and without (-) addition of ALLN.
In fact, just by performing IVF in a medium containing ALLN, offspring were produced at a high rate of 38% even from CO 2 -exposed oocytes.
（高齢不妊症）
上述のように、ＣＯ2暴露することによりラットの卵子が受精ではなく、その後の発生能を焼失する現象（ＨＤＶ）を見いだした。 それが他の不妊症でも発生しているのではないかと推測し、調べたところ、母体の加齢による不妊症と同じであることが判明した。 (elderly infertility)
As described above, a phenomenon (HDV) was found in which rat oocytes were not fertilized but lost their subsequent developmental potential due to CO 2 exposure. We speculated that it might have occurred in other infertility, and when we investigated it, it turned out that it was the same as infertility due to aging of the mother's body.
上述のように、ＣＯ2暴露することによりラットの卵子が受精ではなく、その後の発生能を焼失する現象（ＨＤＶ）を見いだした。 それが他の不妊症でも発生しているのではないかと推測し、調べたところ、母体の加齢による不妊症と同じであることが判明した。 (elderly infertility)
As described above, a phenomenon (HDV) was found in which rat oocytes were not fertilized but lost their subsequent developmental potential due to CO 2 exposure. We speculated that it might have occurred in other infertility, and when we investigated it, it turned out that it was the same as infertility due to aging of the mother's body.
図５Ａは、ラット母体の月齢（Ａｇｅ）で１ヶ月齢（１：幼弱）、６ヶ月齢（６：ヒトの生殖年齢で言うと４０歳前）、８～１０ヶ月齢（８－１０：ヒトの生殖年齢で言うと４０歳以降）、麻酔下で採卵した卵子の自発的活性化状態を調べた結果を示す。縦軸は、ＭＩＩ期（ＭＩＩ）と自発的活性化（ｓ．ａ．）の割合を示す（Ｐｅｒｃｅｎｔａｇｅ ｏｆ ＭＩＩ ａｎｄ ｓ．ａ．（％））。各バー内の数値は、カウント数を示す。
図５Ｂは、これらの月齢での受精率を示す。
このように、麻酔下で採卵したにもかかわらず、６ヶ月齢で約半分、８ヶ月齢では７割以上の卵子で自発的活性化が発生することが分かった。また、それら卵子で体外受精を行うと、受精率自体は、上述のＣＯ2に暴露された卵子と同様、さほど変わらなかった。 FIG. 5A shows the age of the rat mother, 1 month old (1: juvenile), 6 months old (6: before 40 years old in terms of human reproductive age), 8 to 10 months old (8-10: 40 years old or later in terms of human reproductive age), the results of investigating the spontaneous activation state of eggs collected under anesthesia are shown. The vertical axis indicates the percentage of MII stage (MII) and spontaneous activation (sa) (Percentage of MII and sa (%)). The numbers in each bar indicate the number of counts.
Figure 5B shows fertility rates at these ages.
As described above, it was found that spontaneous activation occurred in about half of the eggs at 6 months of age and more than 70% at 8 months of age even though the eggs were collected under anesthesia. Also, when in vitro fertilization was performed with these eggs, the fertilization rate itself did not change much, as with the CO 2 -exposed eggs described above.
図５Ｂは、これらの月齢での受精率を示す。
このように、麻酔下で採卵したにもかかわらず、６ヶ月齢で約半分、８ヶ月齢では７割以上の卵子で自発的活性化が発生することが分かった。また、それら卵子で体外受精を行うと、受精率自体は、上述のＣＯ2に暴露された卵子と同様、さほど変わらなかった。 FIG. 5A shows the age of the rat mother, 1 month old (1: juvenile), 6 months old (6: before 40 years old in terms of human reproductive age), 8 to 10 months old (8-10: 40 years old or later in terms of human reproductive age), the results of investigating the spontaneous activation state of eggs collected under anesthesia are shown. The vertical axis indicates the percentage of MII stage (MII) and spontaneous activation (sa) (Percentage of MII and sa (%)). The numbers in each bar indicate the number of counts.
Figure 5B shows fertility rates at these ages.
As described above, it was found that spontaneous activation occurred in about half of the eggs at 6 months of age and more than 70% at 8 months of age even though the eggs were collected under anesthesia. Also, when in vitro fertilization was performed with these eggs, the fertilization rate itself did not change much, as with the CO 2 -exposed eggs described above.
図５Ｃは、９ヶ月齢の高齢不妊症のラットから麻酔下で卵子を取得し、ＭＧ１３２を添加した場合のＤＡＰＩとＨ３Ｋ９ｍｅ３による免疫染色の写真である。この際のＭＧ１３２の添加の処理は、上述の１２０秒のＣＯ2に暴露した卵子に対する処理と同様に行った。図中、それぞれ、ＭＧ１３２添加の有（＋）無（－）における媒精後２０～２２時間の状態を示す。矢頭は雌性前核、矢印は雄性前核を示す。「ＰＢ２」は、第二極体を示す。
図５Ｄは、図２Ｅと同様の形式で、ＭＧ１３２添加の有（＋）無（－）において、２－ｃｅｌｌでの雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）をカウントした数を示す。
結果として、ＭＧ１３２の無添加の状態では雌性前核が複数見られ、高齢不妊症もＣＯ2暴露と同様のＨＤＶが起っていることを確認できた。これに対して、ＭＧ１３２の添加により、前核が全てまとまった。 FIG. 5C is a photograph of immunostaining with DAPI and H3K9me3 when ova were obtained under anesthesia from a 9-month-old infertile rat and MG132 was added. The treatment for adding MG132 at this time was the same as the treatment for the eggs exposed to CO 2 for 120 seconds as described above. The figures show theconditions 20 to 22 hours after insemination with (+) and without (-) the addition of MG132. Arrowheads indicate female pronuclei, arrows indicate male pronuclei. "PB2" indicates the second polar body.
FIG. 5D shows the number of female pronuclei in 2-cells (No. of female pronuclei) counted in the presence (+) and absence (−) of MG132 addition in the same format as in FIG. 2E.
As a result, multiple female pronuclei were observed in the MG132-free state, and it was confirmed that elderly infertility causes HDV similar to CO 2 exposure. In contrast, the addition of MG132 brought the pronuclei all together.
図５Ｄは、図２Ｅと同様の形式で、ＭＧ１３２添加の有（＋）無（－）において、２－ｃｅｌｌでの雌性前核の数（Ｎｏ． ｏｆ ｆｅｍａｌｅ ｐｒｏｎｕｃｌｅｉ）をカウントした数を示す。
結果として、ＭＧ１３２の無添加の状態では雌性前核が複数見られ、高齢不妊症もＣＯ2暴露と同様のＨＤＶが起っていることを確認できた。これに対して、ＭＧ１３２の添加により、前核が全てまとまった。 FIG. 5C is a photograph of immunostaining with DAPI and H3K9me3 when ova were obtained under anesthesia from a 9-month-old infertile rat and MG132 was added. The treatment for adding MG132 at this time was the same as the treatment for the eggs exposed to CO 2 for 120 seconds as described above. The figures show the
FIG. 5D shows the number of female pronuclei in 2-cells (No. of female pronuclei) counted in the presence (+) and absence (−) of MG132 addition in the same format as in FIG. 2E.
As a result, multiple female pronuclei were observed in the MG132-free state, and it was confirmed that elderly infertility causes HDV similar to CO 2 exposure. In contrast, the addition of MG132 brought the pronuclei all together.
図５Ｅは、実際に得られた産仔の写真を示す。
図５Ｆは、６ヶ月齢（６）、８～１０ヶ月齢（８－１０）、ＭＧ１３２添加の有（＋）無（－）による出生率（Ｂｉｒｔｈ ｒａｔｅ（％））を示す。
結果として、ＭＧ１３２処理を行わない場合、８～１０ヶ月齢のラットはその産仔率が１割と低下するが、ＭＧ１３２処理によりその産仔率は４割弱にまで向上させることができた。すなわち、ＭＧ１３２により、若齢と同じ産仔率に戻す（レスキュー）できる。これにより、高齢出産の低い産仔率を改善することが可能であった。 FIG. 5E shows a photograph of the offspring actually obtained.
FIG. 5F shows birth rates (Birth rate (%)) at 6 months old (6), 8-10 months old (8-10), with (+) and without (-) addition of MG132.
As a result, the litter rate of 8- to 10-month-old rats decreased to 10% in the absence of MG132 treatment. In other words, MG132 can restore (rescue) the litter rate to the same level as that at young age. This made it possible to ameliorate the low litter rate of older births.
図５Ｆは、６ヶ月齢（６）、８～１０ヶ月齢（８－１０）、ＭＧ１３２添加の有（＋）無（－）による出生率（Ｂｉｒｔｈ ｒａｔｅ（％））を示す。
結果として、ＭＧ１３２処理を行わない場合、８～１０ヶ月齢のラットはその産仔率が１割と低下するが、ＭＧ１３２処理によりその産仔率は４割弱にまで向上させることができた。すなわち、ＭＧ１３２により、若齢と同じ産仔率に戻す（レスキュー）できる。これにより、高齢出産の低い産仔率を改善することが可能であった。 FIG. 5E shows a photograph of the offspring actually obtained.
FIG. 5F shows birth rates (Birth rate (%)) at 6 months old (6), 8-10 months old (8-10), with (+) and without (-) addition of MG132.
As a result, the litter rate of 8- to 10-month-old rats decreased to 10% in the absence of MG132 treatment. In other words, MG132 can restore (rescue) the litter rate to the same level as that at young age. This made it possible to ameliorate the low litter rate of older births.
〔まとめ〕
・ラットは体外受精ができなかった。この原因は、安楽殺の際に吸わせるＣＯ2であった。
・ＣＯ2を吸わせると、卵子の核が分散し、染色体の不等分裂を介して受精後の発生能を失っていた。この不妊症の卵子の状態を「ＨＤＶ」と称した。
・ＣＯ2を吸わせてから採卵しても、プロテアソーム阻害剤ＭＧ１３２を添加（浸漬）してＩＶＦをするだけで、ＨＤＶを回復できて子供も生まれてくる。
すなわち、ＨＤＶは、プロテアソーム阻害剤で治療可能であった。
・ＣＯ2を吸わせてから採卵しても、プロテアソーム阻害剤ＡＬＬＮを添加（浸漬）してＩＶＦをするだけでも、ＨＤＶを回復できて子供も生まれてくる。
すなわち、ＨＤＶは、ＭＧ１３２以外のプロテアソーム阻害剤でも治療可能であった。
・不妊症の最大要因でもある「加齢」でもＨＤＶは発生していた。
さらに、プロテアソーム阻害剤を添加したＨＴＦでの処理によって、完全にＨＤＶを治癒できた。
これにより、高齢出産でも高い出生率で、安全で確実な高齢出産を実現することができる。 〔summary〕
• Rats could not be in vitro fertilized. The cause of this was the CO2 inhaled during euthanasia.
・When exposed to CO 2 , the nuclei of the oocytes dispersed and the developmental potential after fertilization was lost through chromosome asymmetry. This infertile oocyte condition was termed "HDV".
・Even if eggs are collected after inhaling CO 2 , HDV can be recovered and offspring can be born simply by adding (immersing) the proteasome inhibitor MG132 and performing IVF.
Thus, HDV was treatable with proteasome inhibitors.
・Even if eggs are collected after inhaling CO 2 , or just adding (immersing) the proteasome inhibitor ALLN and performing IVF, HDV can be recovered and offspring will be born.
Thus, HDV was also treatable with proteasome inhibitors other than MG132.
・HDV also occurs in aging, which is the biggest factor in infertility.
Furthermore, treatment with HTF supplemented with proteasome inhibitors could completely cure HDV.
As a result, it is possible to realize a safe and reliable elderly childbirth with a high birth rate even at an old age.
・ラットは体外受精ができなかった。この原因は、安楽殺の際に吸わせるＣＯ2であった。
・ＣＯ2を吸わせると、卵子の核が分散し、染色体の不等分裂を介して受精後の発生能を失っていた。この不妊症の卵子の状態を「ＨＤＶ」と称した。
・ＣＯ2を吸わせてから採卵しても、プロテアソーム阻害剤ＭＧ１３２を添加（浸漬）してＩＶＦをするだけで、ＨＤＶを回復できて子供も生まれてくる。
すなわち、ＨＤＶは、プロテアソーム阻害剤で治療可能であった。
・ＣＯ2を吸わせてから採卵しても、プロテアソーム阻害剤ＡＬＬＮを添加（浸漬）してＩＶＦをするだけでも、ＨＤＶを回復できて子供も生まれてくる。
すなわち、ＨＤＶは、ＭＧ１３２以外のプロテアソーム阻害剤でも治療可能であった。
・不妊症の最大要因でもある「加齢」でもＨＤＶは発生していた。
さらに、プロテアソーム阻害剤を添加したＨＴＦでの処理によって、完全にＨＤＶを治癒できた。
これにより、高齢出産でも高い出生率で、安全で確実な高齢出産を実現することができる。 〔summary〕
• Rats could not be in vitro fertilized. The cause of this was the CO2 inhaled during euthanasia.
・When exposed to CO 2 , the nuclei of the oocytes dispersed and the developmental potential after fertilization was lost through chromosome asymmetry. This infertile oocyte condition was termed "HDV".
・Even if eggs are collected after inhaling CO 2 , HDV can be recovered and offspring can be born simply by adding (immersing) the proteasome inhibitor MG132 and performing IVF.
Thus, HDV was treatable with proteasome inhibitors.
・Even if eggs are collected after inhaling CO 2 , or just adding (immersing) the proteasome inhibitor ALLN and performing IVF, HDV can be recovered and offspring will be born.
Thus, HDV was also treatable with proteasome inhibitors other than MG132.
・HDV also occurs in aging, which is the biggest factor in infertility.
Furthermore, treatment with HTF supplemented with proteasome inhibitors could completely cure HDV.
As a result, it is possible to realize a safe and reliable elderly childbirth with a high birth rate even at an old age.
なお、上記実施の形態の構成及び動作は例であって、本発明の趣旨を逸脱しない範囲で適宜変更して実行することができることは言うまでもない。
It should be noted that the configuration and operation of the above embodiment are examples, and it goes without saying that they can be modified and executed as appropriate without departing from the scope of the present invention.
次に、上述の実施例１のＭＧ１３２処理と同様の処理を行った際の実際の染色体構成を調べた結果を、実施例２として説明する。
図６は、加齢させたラット卵子（１１ヶ月～１２ヶ月齢由来）を麻酔下で採卵し、体外受精を行って得られた２細胞期胚について、割球毎にｓｉｎｇｌｅ ｃｅｌｌ ｗｈｏｌｅ ｇｅｎｏｍｅ ｓｅｑｕｅｎｃｅ（ｓｃＷＧＳ）を行った結果を示すグラフである。すなわち、割球毎の全ゲノムを解読し、染色体構成を解明した。 Next, as Example 2, the result of examining the actual chromosomal composition when the same treatment as the MG132 treatment of Example 1 was performed will be described.
Fig. 6 shows the single cell whole genome sequence ( It is a graph which shows the result of having performed scWGS). That is, we decoded the whole genome of each blastomere and elucidated the chromosomal organization.
図６は、加齢させたラット卵子（１１ヶ月～１２ヶ月齢由来）を麻酔下で採卵し、体外受精を行って得られた２細胞期胚について、割球毎にｓｉｎｇｌｅ ｃｅｌｌ ｗｈｏｌｅ ｇｅｎｏｍｅ ｓｅｑｕｅｎｃｅ（ｓｃＷＧＳ）を行った結果を示すグラフである。すなわち、割球毎の全ゲノムを解読し、染色体構成を解明した。 Next, as Example 2, the result of examining the actual chromosomal composition when the same treatment as the MG132 treatment of Example 1 was performed will be described.
Fig. 6 shows the single cell whole genome sequence ( It is a graph which shows the result of having performed scWGS). That is, we decoded the whole genome of each blastomere and elucidated the chromosomal organization.
図６の左側のＭＧ１３２（－）は、ＭＧ１３２処理を行わない比較例の結果を示すグラフである。この比較例では、７個の２細胞期胚、つまり１４個の割球についてｓｃＷＧＳを行った結果である。Ａｐは異数性（Ａｎｅｕｐｌｏｉｄ）、Ｅｐは正常染色体数（Ｅｕｐｌｏｉｄ）を示す。この比較例では、１４個中の１０個（７１．４％）の割球で異数性が生じた。
これに対して、図６の右側のＭＧ１３２（＋）は、ＭＧ１３２処理を行った実施例の結果を示すグラフである。この例では、ＭＧ１３２処理をすると、２２個の割球について、６個（２７．３％）にまで異数性が減少した。 MG132(-) on the left side of FIG. 6 is a graph showing the results of a comparative example in which MG132 processing is not performed. In this comparative example, scWGS was performed on 7 2-cell stage embryos,ie 14 blastomeres. Ap indicates aneuploidy, and Ep indicates normal chromosome number (Euploid). In this comparative example, aneuploidy occurred in 10 of 14 (71.4%) blastomeres.
On the other hand, MG132(+) on the right side of FIG. 6 is a graph showing the results of the example in which the MG132 treatment was performed. In this example, MG132 treatment reduced aneuploidy to 6 (27.3%) of 22 blastomeres.
これに対して、図６の右側のＭＧ１３２（＋）は、ＭＧ１３２処理を行った実施例の結果を示すグラフである。この例では、ＭＧ１３２処理をすると、２２個の割球について、６個（２７．３％）にまで異数性が減少した。 MG132(-) on the left side of FIG. 6 is a graph showing the results of a comparative example in which MG132 processing is not performed. In this comparative example, scWGS was performed on 7 2-cell stage embryos,
On the other hand, MG132(+) on the right side of FIG. 6 is a graph showing the results of the example in which the MG132 treatment was performed. In this example, MG132 treatment reduced aneuploidy to 6 (27.3%) of 22 blastomeres.
図７のグラフは、図６のＭＧ１３２未処理（比較例）の構成を詳細に示す。それぞれの円グラフは２細胞期胚を示し、７個の円グラフは、ＭＧ１３２未処理の２細胞期胚７個分を意味し、各グラフの内側の円が一つの割球、外側の円がもう一つの割球を示す。この上で、染色体のうち、常染色体２０本＋Ｘ染色体の数を濃度の差で示す。黒は、その染色体が２本（正常）であったことを示し、グレーは２本以外であったことを示す。なお、円の中心にある文字が小文字の場合、例えば「ａ，ｂ」の場合は、ＭＧ１３２未処理の胚を示す。つまり、１つの２細胞期胚のうち片方の割球が「ａ」、もう片方の割球が「ｂ」を意味する。各グラフの横に、オスであれば「ｍａｌｅ」、メスであれば「ｆｅｍａｌｅ」と示す。すなわち、オスにおいては、Ｘ染色体が１本であるためグレーとして表示している。より詳細な各割球の染色体数については図９に示す。
The graph in FIG. 7 shows in detail the configuration of MG132 untreated (comparative example) in FIG. Each pie chart represents 2-cell stage embryos, 7 pie charts represent 7 2-cell stage embryos untreated with MG132, the inner circle in each graph is one blastomere, and the outer circle is Another blastomere is shown. Of the chromosomes, the number of 20 autosomes+X chromosomes is indicated by the difference in density. Black indicates that the chromosome was duplicated (normal), gray indicates that it was not duplicated. In addition, when the letters in the center of the circle are in lower case, for example, in the case of "a, b", they indicate embryos that have not been treated with MG132. That is, one blastomere of one 2-cell stage embryo is "a" and the other blastomere is "b". Next to each graph is labeled "male" for males and "female" for females. That is, since males have one X chromosome, they are displayed in gray. More detailed chromosome number of each blastomere is shown in FIG.
図８のグラフは、図６のＭＧ１３２処理（実施例）の構成を詳細に示す。それぞれの円グラフは、図７と同様の記載方式で、ＭＧ１３２処理を施した２細胞期胚１２個を意味している。円グラフの中心の文字は、「Ａ，Ｂ」のように大文字表記としており、同様に、オスは「ｍａｌｅ」、メスは「ｆｅｍａｌｅ」と示している。なお、右下の二つだけ、全てグレーの割球があるものの、この割球については解析不能だったものを示す。
図９は、図８の図面をより詳細な各割球の染色体数について示したものである。図９において、各欄でグレーの枠がある欄は、染色体が２本以外であったことを示す。 The graph of FIG. 8 details the configuration of the MG132 process (example) of FIG. Each pie chart represents 12 two-cell stage embryos treated with MG132 in the same manner as in FIG. The letters in the center of the pie chart are capitalized like "A, B", and similarly, males are indicated as "male" and females as "female". In addition, only two in the lower right are all gray blastomeres, but this blastomere could not be analyzed.
FIG. 9 shows the drawing of FIG. 8 in more detail regarding the number of chromosomes in each blastomere. In FIG. 9, each column with a gray frame indicates that there were other than two chromosomes.
図９は、図８の図面をより詳細な各割球の染色体数について示したものである。図９において、各欄でグレーの枠がある欄は、染色体が２本以外であったことを示す。 The graph of FIG. 8 details the configuration of the MG132 process (example) of FIG. Each pie chart represents 12 two-cell stage embryos treated with MG132 in the same manner as in FIG. The letters in the center of the pie chart are capitalized like "A, B", and similarly, males are indicated as "male" and females as "female". In addition, only two in the lower right are all gray blastomeres, but this blastomere could not be analyzed.
FIG. 9 shows the drawing of FIG. 8 in more detail regarding the number of chromosomes in each blastomere. In FIG. 9, each column with a gray frame indicates that there were other than two chromosomes.
結果として、ＭＧ１３２処理することで、正常な染色体構成の割球が増えていることが分かった。このような結果から、実際に、加齢卵子の低出生率をＭＧ１３２で実際に改善することが可能であると考えられる。
As a result, it was found that MG132 treatment increased the number of blastomeres with normal chromosome composition. These results suggest that MG132 can actually improve the low fertility rate of aged oocytes.
本発明によれば、高齢出産による出生率等を改善させる不等分裂抑制剤を提供することができ、産業上利用可能である。
According to the present invention, it is possible to provide an asymmetric division inhibitor that improves the birth rate due to childbirth at an advanced age, and is industrially applicable.
Claims (17)
- 多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含む
ことを特徴とする不等分裂抑制剤。 An asymmetric division inhibitor comprising a spontaneous activation inhibitor that inhibits asymmetric division due to spontaneous activation of pluripotent cells. - 前記自発的活性化は、染色体の分散を伴うものである
ことを特徴とする請求項１に記載の不等分裂抑制剤。 2. The asymmetric division inhibitor according to claim 1, wherein the spontaneous activation is accompanied by chromosomal dispersal. - 前記不等分裂の抑制は、前記自発的活性化の抑制及び／又は分散した前記染色体の再集合を誘導する
ことを特徴とする請求項２に記載の不等分裂抑制剤。 3. The agent for suppressing asymmetric division according to claim 2, wherein the suppression of the asymmetric division induces suppression of the spontaneous activation and/or reassortment of the dispersed chromosomes. - 前記細胞は、
多能性幹細胞、生殖細胞、卵子、又は胚である
ことを特徴とする請求項１乃至３のいずれか１項に記載の不等分裂抑制剤。 The cells are
The asymmetric division inhibitor according to any one of claims 1 to 3, which is a pluripotent stem cell, germ cell, ovum, or embryo. - 前記細胞が前記生殖細胞又は前記卵子である場合、
前記自発的活性化は、受精前の二酸化炭素への暴露又は加齢によるものである
ことを特徴とする請求項４に記載の不等分裂抑制剤。 When the cell is the germ cell or the egg,
5. The asymmetric division inhibitor according to claim 4, wherein the spontaneous activation is due to exposure to carbon dioxide before fertilization or aging. - 前記自発的活性化抑制剤は、プロテアソーム阻害剤である
ことを特徴とする請求項１乃至５のいずれか１項に記載の不等分裂抑制剤。 The asymmetric division inhibitor according to any one of claims 1 to 5, wherein the spontaneous activation inhibitor is a proteasome inhibitor. - 前記プロテアソーム阻害剤は、
ＭＧ１３２を含む
ことを特徴とする請求項６に記載の不等分裂抑制剤。 The proteasome inhibitor is
7. The asymmetric division inhibitor according to claim 6, comprising MG132. - 前記プロテアソーム阻害剤は、
ＡＬＬＮを含む
ことを特徴とする請求項６に記載の不等分裂抑制剤。 The proteasome inhibitor is
7. The asymmetric division inhibitor according to claim 6, comprising ALLN. - 多能性を備える細胞の自発的活性化による不等分裂を抑制する自発的活性化抑制剤を含む
ことを特徴とする染色体異数性抑制剤。 A chromosomal aneuploidy suppressor comprising a spontaneous activation suppressor that suppresses asymmetric division due to spontaneous activation of pluripotent cells. - 請求項１乃至８のいずれか１項に記載の不等分裂抑制剤、又は請求項９に記載の染色体異数性抑制剤を含む
ことを特徴とする培地。 A culture medium comprising the asymmetric division inhibitor according to any one of claims 1 to 8 or the chromosomal aneuploidy inhibitor according to claim 9. - 請求項５乃至８のいずれか１項に記載の不等分裂抑制剤、又は請求項９に記載の染色体異数性抑制剤を含む
ことを特徴とする体外受精培地。 An in vitro fertilization medium comprising the asymmetric division inhibitor according to any one of claims 5 to 8 or the chromosomal aneuploidy inhibitor according to claim 9. - 請求項１乃至８のいずれか１項に記載の不等分裂抑制剤、又は請求項９に記載の染色体異数性抑制剤を含む
ことを特徴とする細胞処理キット。 A cell treatment kit comprising the asymmetric division inhibitor according to any one of claims 1 to 8 or the chromosomal aneuploidy inhibitor according to claim 9. - 多能性を備える細胞の自発的活性化を抑制し、
前記細胞の不等分裂を抑制する
ことを特徴とする不等分裂抑制方法。 suppressing the spontaneous activation of cells with pluripotency,
A method for suppressing asymmetric division, comprising suppressing asymmetric division of the cell. - 採取された生殖細胞又は卵子の体外受精方法であって、
請求項１１に記載の体外受精培地を添加して処理し、
前記生殖細胞、前記卵子、又は胚の不等分裂を抑制する
ことを特徴とする体外受精方法。 A method for in vitro fertilization of harvested germ cells or eggs, comprising:
treated by adding the in vitro fertilization medium according to claim 11,
An in vitro fertilization method characterized by suppressing asymmetric division of the germ cell, the egg, or the embryo. - 請求項１４に記載の体外受精方法により処理された生殖細胞又は卵子由来の受精卵又は胚を移植する
ことを特徴とする移植方法。 A transplantation method characterized by transplanting fertilized eggs or embryos derived from germ cells or eggs treated by the in vitro fertilization method according to claim 14 . - 哺乳類の不妊治療方法であって、
採取された生殖細胞又は卵子を、請求項１０に記載の培地を添加して処理し、
前記生殖細胞又は前記卵子由来の受精卵又は胚の不等分裂を抑制する
ことを特徴とする不妊治療方法。 A method of treating infertility in a mammal, comprising:
The collected germ cells or eggs are treated by adding the medium according to claim 10,
A method for treating infertility, comprising suppressing asymmetric division of the fertilized egg or embryo derived from the germ cell or the egg. - 請求項１５に記載の移植方法、又は請求項１６に記載の不妊治療方法により
哺乳類の産仔を取得する
ことを特徴とする産仔取得方法。 A method for obtaining offspring, comprising obtaining offspring from mammals by the transplantation method according to claim 15 or the method for treating infertility according to claim 16.
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| XIAO XIAO;XIANG-DONG ZI;HUI-RAN NIU;XIAN-RONG XIONG;JIN-CHENG ZHONG;JIAN LI;LI WANG;YONG WANG: "Effect of addition of FSH, LH and proteasome inhibitor MG132 to in vitro maturation medium on the developmental competence of yak (Bos grunniens) oocytes", REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY, BIOMED CENTRAL LTD., GB, vol. 12, no. 1, 22 April 2014 (2014-04-22), GB , pages 30, XP021182004, ISSN: 1477-7827, DOI: 10.1186/1477-7827-12-30 * |
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