KR102306054B1 - Composition and Method for enhancing developmental rate of embryos using melatonin - Google Patents

Abstract

It relates to a composition for embryonic development comprising melatonin and a method for improving the embryonic development rate using the same, wherein the composition increases the efficiency of development into blastocysts by preventing the two-cell phase developmental arrest of the embryo, qualitative or Quantitatively superior blastocysts can be obtained.

Description

A composition for embryo development, including melatonin, and a method for improving the efficiency of embryonic development using the same

It relates to a composition for embryo development, including melatonin, and a method for improving the efficiency of embryo development using the same.

Somatic cell nuclear transfer (SCNT)-based reprogramming and derivation of embryonic stem cells (ESCs) could produce patient-specific stem cells for regenerative medicine. has been proposed Recently, three separate research groups have successfully derived several somatic cell nuclear transfer-embryonic stem cell (SCNT-ESC) lineages using high-quality human oocytes and fibroblasts from multiple sources. As many of the developmental arrests of human SCNT embryos are partially overcome by modulation of histone methylation, it is now an applicable technique for cell therapy. In addition, the establishment of human leukocyte antigen (HLA)-matched homozygous pluripotent stem cells (PSCs) could increase the potential efficiency of SCNT-ESCs, but there are many Requires a number of donated human oocytes.

Recently, the oocyte vitrification technique has been used as a practical technique for a widely used cryopreservation method as well as a human assisted reproduction technique. In fact, it was reported that cell development and cell bolck of progeny born from vitrified-frozen (low-temperature frozen) oocytes did not increase when compared with conventional in vitro fertilization (IVF). This technology provides a valuable opportunity to maintain fertility in infertile women and treat fertility risks from cancer treatment and women of childbearing potential at risk for age-induced fertility decline. In addition, vitrification-freezing of human excess oocytes may provide persistent oocytes for studies such as SCNTs, and their use may also reduce ethical concerns. However, until recently, production of cloned eggs and induction of SCNT-ESC lines using cryopreserved oocytes were not achieved. Clinical outcomes of vitrified-frozen oocytes were lower than fresh oocytes from human reproductive technology programs, even when survival rates were greater than 90%. This is thought to be due to cytoskeletal damage, changes in spindle structure, microtubules, cortical granular distribution, and hardening of oocytes. In addition, cryopreserved oocytes are particularly vulnerable to oxidative stress due to their high levels of lipids, and produce large amounts of free radicals that affect the balance between oxidative and reduction reactions and intracellular antioxidant systems.

One aspect is to provide a composition for increasing embryonic development efficiency or forming an embryo, including melatonin.

Another aspect is to provide a method for increasing the efficiency of embryonic development or increasing embryonic formation, comprising culturing an egg in a medium containing melatonin.

One aspect provides a composition for increasing the formation of an embryo comprising melatonin. Another aspect provides a composition for increasing the efficiency of embryonic development comprising melatonin. That is, there is provided a composition comprising melatonin for increasing the formation of an embryo and/or for increasing the efficiency of embryo development.

As used herein, the term "formation of an embryo" means that a zygote becomes several cells through cell division, and these cells form an embryo or develop into an embryo through cell division and differentiation. .

As used herein, the term "increase in efficiency" means an increase in blastocyst development or blastocyst development of an egg. In the process of the blastocyst and fertilized egg growing repeatedly, the blastocyst forms a blastocyst after the densified morula, and develops to the extent that it is divided into an inner cell mass that will differentiate into a fetus and a trophectoderm that will differentiate into a placenta. It means a fertilized egg. Therefore, the increase in efficiency is related to the increase in the embryonic development efficiency of the oocyte compared to the oocyte cultured in the absence of an agent that reduces H3K9me3 methylation, the increase in the development of the embryo to the blastocyst stage, and the development to the blastocyst stage This increase means that the production efficiency of blastocysts is increased, that the yield efficiency of blastocysts is increased, or that the rate of developmental formation of blastocysts is increased.

In one embodiment, the composition may contain melatonin in a concentration of 0.1 μM to 50 μM in the basal medium. Melatonin is a molecule involved in the cycle of biological reactions, also known as N-acetyl-5-methoxytryptamine. In mammals, melatonin modulates the cell cycle and exhibits strong resistance to oxidative stress and apoptosis. That is, the method according to one aspect reduces reactive oxygen species and apoptosis of the somatic cell nuclear-transferred oocyte by culturing the nucleus and nuclei of somatic cells in a medium containing melatonin, thereby preventing cell damage and improving quality. There is an advantage that an improved blastocyst can be obtained. In addition, it is possible to enhance the implantation rate of an artificially fertilized fertilized egg with a somatic cell nuclear transfer oocyte, and promote embryonic stem cell induction. Specifically, 0.1 μM to 50 μM, 0.1 μM to 40 μM, 0.1 μM to 30 μM, 1 μM to 50 μM, 1 μM to 30 μM, 5 μM to 30 μM, 5 μM to 25 μM, 5 μM to 20 μM, 10 μM to 25 μM or 5 μM to 15 μM. At this time, when the concentration of melatonin contained in the basal medium is less than the above range, active oxygen cannot be effectively removed, thereby inhibiting the blastocyst development efficiency of somatic cell nuclear transfer oocytes. In addition, when the above range is exceeded, there is a problem in that melatonin acts on the somatic cell nuclear transfer oocyte for a long time and prevents the maturation of the ovum. Specifically, the basal medium may be a basal medium used for culturing mammalian eggs. The basic medium is different depending on the species of the mammal, but includes any one or more selected from the group consisting of inorganic salts, carbon sources, amino acids, bovine serum albumin, and cofactors, and may include all common mediums known to those skilled in the art. As the inorganic salt, NaCl, KCl and NaHCO ₃ and the like can be used, and as a carbon source, glucose, sodium, pyruvate and calcium lactate salt can be used, and the amino acid includes essential amino acids and non-essential amino acids including glutamine, As the cofactor, other trace elements and buffers may be used. The medium is, for example, MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium), K-SFM (Keratinocyte Serum Free Medium), IMDM (Iscove's Modified Dulbecco's Medium), F12 and DMEM/F12. The medium may also contain a neutral buffer (eg phosphate and/or high bicarbonate concentration) and protein nutrients (eg serum, eg FBS, fetal calf serum (FCS), horse serum, serum replacement, albumin, or essential amino acids and non-essential amino acids such as glutamine, L-glutamine). Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most stock media of this kind (eg transferrin, nucleosides or nucleotides, pyruvate, any ionized form or salt) sugar sources such as glucose, glucocorticoids such as hydrocortisone and/or reducing agents such as β-mercaptoethanol). In addition, the basal medium may further include an antibiotic. The melatonin may be included in the basal medium in an amount of 0.1 µM to 50 µM, 0.1 µM to 40 µM, 0.1 µM to 30 µM, 1 µM to 50 µM, 1 µM to 30 µM, 5 µM to 30 µM or 5 µM to 25 µM. . At this time, when the concentration of melatonin contained in the basal medium is less than the above range, active oxygen cannot be effectively removed, thereby inhibiting the blastocyst development efficiency of somatic cell nuclear transfer oocytes. In addition, when it exceeds the above range, there is a problem in that melatonin acts on the somatic cell nuclear-transferred oocyte for a long time and prevents the maturation of the ovum.

In another embodiment, the composition may further include an agent that reduces H3K9me3 methylation. The agent that reduces the methylation may be an agent that increases the expression of a member of the KDM4 family of histone demethylases. For example, the agent may increase the expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D), or a combination thereof.

When the embryo is in the two-cell stage , Amd1 , Fam46C , Oxt , Ppt2 , Ctss , Dffa , Eif3f , Hacl1 , Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 and The expression of any one or more genes selected from the group consisting of Wbscr22 may be increased. Also, Atp6v0c, Cd52 , Dapk2 , Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 And Rnd3 may be one in which the expression of any one or more genes selected from the group consisting of is reduced.

When the embryo develops into a blastocyst , the expression of any one or more genes selected from the group consisting of Amd1 , Fam46C , Oxt , Ppt2 , Gulo and Txnip may be increased. Also, Adh1, Car2, Gsta3, Gstm2 , Mb, Phlda2, S100a1, Akr1c13, Amd2, Clu, Hist1h3a, Hspb1, Il1rn, Nat8, Efemp1, Glipr1, Lgals4, Plac1, Snora15, Snora 21, Snora 34, Anxa1, Clec2f, the Hrsp12, and any one or more genes selected from the group consisting of Plat may be reduced. The embryo may be an embryo formed through artificial insemination in vitro, or the embryo may be formed by transplanting a nucleus of a somatic cell into an egg from which the nucleus has been removed. The embryo may be formed through artificial insemination in vitro, or the embryo may be formed by transferring the nucleus of a somatic cell to an egg from which the nucleus has been removed.

Another aspect provides a method of increasing embryonic formation or efficiency of embryonic development, comprising culturing the egg in a medium containing melatonin. Specific details of the medium containing melatonin are the same as described above.

In one embodiment, it may include contacting the egg with an agent that reduces H3K9me3 methylation. Specific details of the agent for reducing the H3K9me3 methylation are the same as described above. In addition, the egg may be thawed after freezing. In addition, the egg may be a somatic cell nuclear transfer egg.

The method may include culturing the egg in a medium containing melatonin for 1 to 10 days. Specifically, it may be cultured for 1 to 10 days, 1 to 8 days, 1 to 7 days, 2 to 8 days, 2 to 6 days, or 3 to 6 days. At this time, when the culture period is less than the above range, there is a problem that the embryo is not sufficient to develop into a blastocyst.

In addition, the method may further comprise the step of developing the embryo obtained in the culturing step as an individual. The subject may be a morula, a blastula and/or a gastrula.

Another aspect provides an embryo, blastocyst and/or embryonic stem cell produced by the method. Another aspect provides a composition for transplantation comprising the embryo and / or blastocyst prepared by the above method as an active ingredient.

Another aspect comprises the steps of preparing an egg from which the nucleus and nucleus of a somatic cell have been removed; And it provides a method of increasing the efficiency of somatic-cell nuclear transfer (SCNT) comprising; culturing the nucleus and the nucleus of the somatic cells in a medium containing melatonin in the removed egg. The efficiency may be a success rate of somatic cell nuclear transfer and an embryonic development rate of cells generated by the somatic cell nuclear transfer. The embryonic development rate may refer to the development of an embryo into a blastocyst through the 2-cell stage, 4-cell stage, and 8-cell stage.

The present inventors confirmed that KDM4A overexpression was induced by injection of KDM4A mRNA to prevent developmental arrest at the time of zygotic gene activation (ZGA), that is, the 2-cell stage, and significantly improve the development of somatic cell nuclear transfer embryos. did. Thus, it expands the usefulness of oocyte donors for somatic cell nuclear transfer and promotes the production of therapeutic cloning and nuclear-transplanted embryonic stem cells (NT-ESCs), particularly for therapeutic use and nuclear-transplanted ESCs for both research and disease modeling. It can be used in a method to enhance somatic cell nuclear transfer. The present invention is not intended for human reproductive cloning.

The method comprises the steps of removing the nucleus of the oocyte, transplanting the nucleus (donor nucleus) of one or more somatic cells, activating the reconstructed nuclear-transferred oocyte (embryo), and further culturing the blastocyst. may further include. These steps for somatic-cell nuclear transfer (SCNT) may be performed by those skilled in the art with appropriate modifications according to methods disclosed in literature (Nature 419, 583-587, 10 October 2002). The nuclear-transferred oocytes (embryos) are generated from injecting donor somatic cell-derived nuclei (eg, nuclear genetic material) into denucleated recipient oocytes to form nuclear-transferred oocytes, which are activated or fusion to form a nuclear-transferred embryo.

As used herein, the term “somatic cell” refers to a plant or animal cell that is not a germ cell or a germ cell precursor. The somatic cells include cumulus cells, epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, red blood cells, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells. It may be one selected from the group consisting of cells, fetal cells and adult cells.

As used herein, the term "somatic cell nuclear transfer" or "somatic cell nuclear replacement" refers to a technology for transplanting a nucleus collected from a donor cell into a recipient cell from which the nucleus has been removed, and refers to a technology for generating cells that are genetically identical to the donor cell. .

The oocytes from which the nucleus has been removed may be cumulus-oocyte complexes collected from an individual's follicle, or may be commercially obtained. The subject may be a mammal including a human. For example, the mammal may include a rat, a pig, a sheep, a dog, a cow, a horse, a goat, and the like. In one embodiment, somatic cell nuclear transfer oocytes (SCNT-FOC) using fresh oocyte cytoplasm (FOC) and cytoplasm of vitrified-frozen/thawed (cold-preserved/thawed) oocytes ( As a result of injecting Kdm4a mRNA into SCNT-CROC), it was confirmed that the blastocyst development rate was about 17% lower in SCNT-CROC ( FIG. 2c ). Thereafter, the SCNT-CROC and SCNT-FOC were further treated with melatonin to confirm the blastocyst development rate. As a result, melatonin improved the blastocyst development rate of SCNT-CROC independently of Kdm4a mRNA. It was confirmed that the blastocyst development efficiency, which could not be overcome, was improved by about 16%, indicating a blastocyst development rate similar to that of SCNT-FOC (Table 2). Accordingly, the egg from which the nucleus has been removed may be thawed or thawed after being frozen and/or cryopreserved. As the freezing method, slow-freezing or vitrification, which is an ultra-rapid freezing method, may be used. As SCNT-CROC treated with melatonin exhibits a blastocyst development rate similar to that of SCNT-FOC, it can be used for nuclear transfer directly without separate cryo-thaw treatment of donor eggs during somatic cell replication, thus shortening the process of somatic cell nuclear transfer. And it is possible to improve the replication efficiency.

The method according to one embodiment may include forming a blastocyst by culturing the nucleus of a somatic cell and an egg from which the nucleus has been removed in vitro.

The method according to one embodiment may include contacting with an agent that reduces H3K9me3 methylation. The method according to another embodiment may include injecting an agent that reduces H3K9me3 methylation. The agent for reducing H3K9me3 methylation may be an agent for increasing the expression of a member of the KDM4 family of histone demethylases. The agent may, for example, increase the expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D) or a combination thereof. The step of contacting or injecting may be performed on a nuclear-transferred embryo after fusion of the nucleus of the somatic cell and the egg from which the nucleus has been removed. Specifically, the embryo may be an embryo before the activation of the somatic cell nuclear transfer oocyte gene starts. For example, 5 hours post activation (5 hpa) or between 10-12 hpa (ie, 1-cell phase), or about 20 hpa (ie, early 2-cell phase) after activation of the embryo or between 20 and 28 hpa (ie, two-cell phase) with one or more histone demethylase KDM4 family.

In addition, in the step of contacting or injecting, one or more histone demethylase KDM4 family is contacted or may be injected. The contacting or infusion may be contact with a donor somatic cell for at least 1 hour, or for at least 2 hours, wherein the contacting is at least 1 day (24 hours) prior to denucleation from the donor somatic cell to the denucleated oocyte, 2 It may be carried out for more than one day, more than three days, or more than three days.

Therefore, by contacting the nucleus of the somatic cell and the nucleus-removed egg with an agent that reduces H3K9me3 methylation, the activity of histone methylase, which inhibited replication in somatic cell nuclear transfer embryos, is reduced, and the expression of embryogenesis-related genes is enhanced. Bar, it is possible to enhance the generation and development efficiency of blastocysts. For example, the developmental efficiency may be to increase the percentage development of somatic cell nuclear transfer embryos that develop to 2-cell, 4-cell and 8-cell or blastocyst stage, and 8-cell or blastocyst stage of somatic cell nuclear transfer embryo. It is possible to increase the developmental efficiency prior to transplantation. That is, the agent that reduces H3K9me3 methylation can overcome the phenomenon of two-cell phase arrest in somatic cell nuclear transfer embryos and maintain continuous embryonic development.

The method of one embodiment may include culturing the nucleus and nuclei of somatic cells in the medium containing melatonin for 1 to 10 days. Specifically, it may be cultured for 1 to 10 days, 1 to 8 days, 1 to 7 days, 2 to 8 days, 2 to 6 days, or 3 to 6 days. At this time, when the culture period is less than the above range, there is a problem that the somatic cell nuclear transfer oocytes are not sufficient to develop into blastocysts. There is a problem with this difficulty.

In one embodiment, the method comprises about 5% or more, about 10% or more, about 13% or more, about 15% or more, the efficiency of nuclear transfer compared to somatic cell nuclear transfer performed in the absence of an agent that reduces H3K9me3 methylation; It may be an increase of about 30% or more, about 50% or more, 50 to 80% or more than 80%. That is, to increase the efficiency of the pre-transplant development of the somatic cell nuclear transfer embryo, or increase the development of the embryo to the blastocyst stage, or the development of the embryo to the expanded blastocyst stage by about 5%, about 7%, about 10 %, more than about 12, or more than 12% expanded blastocyst stage. In another embodiment, the method comprises at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, and at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, It may be increased by 7 times or more, 8 times or more, or more than 8 times. The increase in somatic cell nuclear transfer efficiency means an increase in the production or yield of blastocysts. The increase in the production or yield of blastocysts indicates that the generation or yield of blastocysts is at least about 110%, at least about 120%, at least about 130%, at least about 140 compared to somatic cell nuclear transfer performed in the absence of an agent that reduces H3K9me3 methylation. % or more, about 150% or more, or more than about 150%.

As described above, in the method according to an aspect, cell damage can be prevented by culturing somatic cell nuclear transfer cells in a medium containing melatonin, thereby reducing active oxygen in the somatic cell nuclear transfer cells. can be improved In addition, since apoptosis of somatic cell nuclear transfer oocytes frozen and thawed by melatonin is reduced, blastocyst formation and production efficiency can be improved. In addition, as the implantation rate of somatic cell nuclear-transferred oocytes is improved, endangered animals can be produced through in vitro fertilization, and as embryonic stem cell induction is enhanced, embryonic stem cell lines can be efficiently produced.

Another aspect provides a somatic cell nuclear transfer embryo produced according to the method. Another aspect provides a somatic cell nuclear transfer blastocyst prepared according to the method. The embryo is genetically modified, e.g., one or more transgenes in the genetic material of the donor nucleus have been modified prior to the somatic cell nuclear transfer step (i.e., prior to collecting the donor nucleus and fusing it with the cytoplasm of the recipient oocyte). it could be In one embodiment, the embryo may include nuclear DNA derived from a donor somatic cell, cytoplasm derived from a recipient oocyte, and mitochondrial DNA derived from a third donor individual. The embryo or blastocyst has up- or down-regulated expression of genes related to cell survival, tissue regeneration, antioxidant function, inflammation or apoptosis, etc. compared to somatic cell nuclear transfer embryos or blastocysts performed in the absence of an agent that reduces H3K9me3 methylation it could be

In one embodiment, the expression of genes involved in cell survival and tissue regeneration in the embryo may be up-regulated. Specifically, the gene may be Amd1 , Fam46C , Oxt, and/or Ppt2 . In addition, the expression of genes involved in antioxidant function, inflammation or apoptosis may be up-regulated. Specifically, the genes are Ctss , Dffa , Eif3f , Hacl1 , Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 . and/or It may be Wbscr22. In addition, cell death or degeneration-related genes, such as Atp6v0c, Cd52 , Dapk2 , Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 and/or Rnd3 expression may be down-regulated.

In another embodiment, the blastocyst may be one in which the expression of genes related to cell survival or tissue regeneration, for example, Amd1 , Fam46C , Oxt , and/or Ppt2 is up-regulated. In particular, it is a heredity associated with antioxidant function, inflammation or apoptosis, e.g. Gulo and/or the expression of Txnip may be up-regulated. On the other hand, genes associated with oxidative stress, such as Adh1 , Car2 , Gsta3 , Gstm2 , Mb , Phlda2 , and/or S100a1 ; It may be that the expression of cell death or degeneration-related genes, for example, Akr1c13 , Amd2 , Clu , Hist1h3a , Hspb1 , Il1rn , and/or Nat8 is down-regulated. In addition, genes associated with cell and tumor proliferation, such as Efemp1 , Glipr1 , Lgals4 , Plac1 , Snora15 , Snora 21 , Snora 34 , and/or Anxa1 ; It may be that the expression of a gene related to an immune response, for example, Clec2f , Hrsp12, and/or Plat is down-regulated.

Another aspect provides a somatic cell nuclear transfer embryonic stem cell prepared according to the above method. Separating the embryonic stem cells from the inner cell mass in the blastocyst prepared according to the method; And it may be formed by culturing the undifferentiated cells derived from the inner cell mass. In addition, the embryonic stem cells may be pluripotent stem cells or totipotent stem cells.

Another aspect comprises the steps of preparing an egg from which the nucleus and nucleus of a somatic cell have been removed; culturing the nucleus of the somatic cell and an egg from which the nucleus has been removed with a candidate material; and evaluating the H3K9me3 methylation activity of the somatic cell nuclear transfer cells generated after the culturing.

The cells derived from the somatic cell nuclear transfer embryo or blastocyst, for example, embryonic stem cells, etc. can be used in a test to determine whether the agent affects differentiation or cell proliferation. For example, as the ability of the cell to differentiate or proliferate is assessed from the presence or absence of the agent, it can be used for screening to select agents that affect cells derived from the somatic cell nuclear transfer embryo or blastocyst. . The test compound can be any compound of interest, including chemical compounds, small molecules, polypeptides, or other biological agents (eg, antibodies or cytokines, etc.).

In the method according to one embodiment, the step of evaluating the H3K9me3 methylation activity is to confirm the expression level of a gene or protein that reduces H3K9me3 methylation, using a technique well known in the art, such as RT-PCR or immunostaining. can be done by In addition, the step of evaluating the H3K9me3 methylation activity may be performed together with the step of evaluating the somatic cell replication efficiency. If the number of somatic cell nuclear-transferred oocytes, 2-cell stage cells, 4-cell stage cells or blastocyst stage cells in the culture is changed after addition compared to before addition of the candidate substance, the candidate substance is the H3K9me3 methylation It can be determined to be a substance that reduces the activity and further increases the efficiency of somatic cell replication by somatic cell nuclear transfer.

Another aspect comprises the steps of preparing an egg from which the nucleus and nucleus of a somatic cell have been removed; obtaining somatic-cell nuclear transfer (SCNT) cells by culturing the nucleus and nuclei of the somatic cells in a medium containing melatonin; and in vitro fertilization of the somatic cell nuclear-transferred cells and liquid semen. Specific details of the method of culturing the nucleus of the somatic cell and the nucleus-removed egg in a medium containing melatonin are the same as described above. The somatic cell nuclear transfer cell may be a somatic cell nuclear transfer egg, specifically, a 2-cell stage, a 4-cell stage, and an 8-cell stage embryo, and may be a cell that has developed and has reached the blastocyst stage.

The method according to one embodiment includes in vitro fertilization of the somatic cell nuclear transfer cells and liquid semen. The semen may be semen collected from the vas deferens of an individual. The subject may be a mammal including a human. For example, the mammal may include a rat, a pig, a sheep, a dog, a cow, a horse, a goat, and the like. The somatic cell nuclear transfer cells and liquid semen may be fertilized in vitro for 1 to 7 days in a medium composition for in vitro fertilization. In this case, when the in vitro fertilization period is less than the above range, there is a problem that fertilization cannot be performed, and when the in vitro fertilization period exceeds the above range, there is a problem that the embryo is degenerate. In addition, in the above step, the step of contacting or injecting with an agent that reduces H3K9me3 methylation may be further included. Specific details of the agent for reducing the methylation are the same as described above. The agent that reduces the methylation is in contact with a somatic cell transplanted fertilized egg after the injection of semen into the somatic cell nuclear transfer cells, before pronuclear formation (within 18 hours after semen injection), specifically after the injection of semen, and before the 2-cell phase may be injected. Therefore, the somatic cell transplanted fertilized egg is a somatic cell nuclear transfer cell (embryo) that proceeds efficiently without developmental arrest and successfully develops into a blastocyst through the 2-, 4- and 8-cell stages without developmental defects or loss of viability. can increase

It relates to a composition for embryo development comprising melatonin and a method for improving embryonic development rate using the same, wherein the cells efficiently develop into blastocysts without cell damage and/or developmental arrest, in vitro fertilization procedure It can be used to produce somatically cloned fertilized eggs.

1a is a photograph showing the results of observing the demethylation of H3K9me3 by injecting Kdm4a mRNA into the SCNT-FOC and SCNT-CROC groups.
1b is a graph showing the results of observing the demethylation of H3K9me3 by injecting Kdm4a mRNA into the SCNT-FOC and SCNT-CROC groups.
Figure 2a is a photograph confirming the change in the number of inner cells and total cells of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected.
2b is a photograph confirming the degree of embryonic development of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected.
Figure 2c is a graph showing the blastocyst quality ratio of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected.
3a shows genes with different expression patterns in 2-cell embryos of SCNT-FOC and SCNT-CROC groups depending on whether Kdm4a mRNA is injected.
3b shows genes whose expression is regulated (up/down) in 2-cell embryos of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected or not.
Figure 4a is a photograph confirming the level of DNA fragmentation in blastocysts of SCNT-CROC and SCNT-CROC + K groups with or without melatonin treatment by TUNEL staining.
Figure 4b is a graph confirming the number of TUNEL-positive cells in the blastocysts of the SCNT-CROC and SCNT-CROC + K groups with or without melatonin treatment.
Figure 5a is a photograph confirming the fluorescence staining (apoptosis; green, nuclear; blue staining) of apoptosis in the SCNT-CROC + K group with or without melatonin treatment.
Figure 5b is a graph showing the level of reactive oxygen species in the morula of the SCNT-CROC + K group with or without melatonin treatment.
Figure 6a is a photograph of visualizing whether or not implantation in the uterus of a mouse of the SCNT-CROC + K group according to the presence or absence of melatonin treatment.
Figure 6b is a photograph visualizing the results of repeated experiments whether or not implanted in the uterus of the SCNT-CROC + K group according to the presence or absence of melatonin treatment.
Figure 6c is a graph confirming the embryo transfer rate of the SCNT-CROC + K group according to the presence or absence of melatonin treatment.
Figure 7a is the result of confirming the mESC induction rate of the SCNT-CROC + K group according to the presence or absence of melatonin treatment with an optical microscope.
Figure 7b is a graph showing the mESC induction rate of the SCNT-CROC + K group with or without melatonin treatment.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Preparation example]

To collect recipient oocytes and SCNT donor oocytes, 8-10 week old female B6D2F1 mice (Orient-bio, Gyeonggi-do, Korea) were used. Female ICR mice aged 8-10 weeks were used as poster mothers for embryo transfer. To induce pseudopragnancy, the mice were crossed with male mice of the same strain. The protocol for the use of animals in this study was approved by the Animal Experimentation and Use Committee (IACUC) (Project No. IACUC-170119) of CHA University, and all experiments were performed according to the approved protocol.

[Example]

Example 1. Collection of oocytes

5 IU of pregnant mare serum gonadotropin (PMSG; Sigma-Aldrich, St. Louis, MO) was injected into the mice of the preparation example, and after 48 hours, 5 IU of human chorionic gonadotropin (chorionic) was injected. gonadotropin, hCG; Sigma-Aldrich) was injected to induce superovulation. After injecting hCG into mice, oocytes were collected in M2 (Sigma-Aldrich) medium 14 hours later, and cumulus cells were removed with a medium containing 0.1% hyaluronidase (Sigma-Aldrich). Denuded. Then, for the experiment, cumulus-free oocytes were cultured in potassium simplex optimized medium (KSOM, Millipore, Darmstadt, Germany) The dispersed cumulus cells were treated with hyaluronidase. Then, the pellet was resuspended in a small amount of 3% (v/v) of polyvinylpyrrolidone (PVP) in M2 and stored at 4°C until use.

Example 2. Oocyte vitrification and thawing (warming)

Quinn' with Knockout Serum Replacement (KSR, Gibco, Grand Island, NY) 20% (v/v) and HEPES (Sage, Malov, Denmark) as basal media for the preparation of vitrified freezing and warming solutions of advantage medium was used. A combination of ethylene glycol (EG, Sigma-Aldrich) and dimethylsulfoxide (DMSO, Sigma-Aldrich) was subjected to vitrification and freezing with two cryoprotectant agents. MII oocytes were pre-equilibrated with HEPES medium containing 7.5% ethylene glycol and 7.5% DMSO for 2 minutes and 30 seconds. Pre-equilibrated oocytes were equilibrated for 20 seconds in an equal volume of HEPES medium supplemented with 15% ethylene glycol, 15% DMSO, and 0.5M sucrose (Sigma-Aldrich). Equilibrated oocytes were loaded onto electron microscopic copper grids (EM Grid, PELCO, Redding, CA) and slush nitrogen (SN2) using a Vit-master (IMT, Ness Ziona, Israel). were cryopreserved in Vitrified-frozen oocytes were stored in LN2 tanks. For warming, vitrified-frozen oocytes were warmed in a four-step method. The EM grids were sequentially transferred to 0.5, 0.25, 0.125 and 0 M sucrose at an interval of 2 minutes and 30 seconds at 37°C. Thereafter, the oocytes were washed three times with modified HTF (Millipore) medium and cultured in HTF medium until the experiment was started.

Example 3. Somatic cell nuclear transfer (SCNT)

Fresh or vitrification/warmed oocytes prepared in Example 2 were incubated at 37° C., and then the oocytes were removed. Fresh cumulus cells were used as nuclear donor eggs. Nuclei of fresh, vitrified, frozen/thawed cumulus cells were removed in M2 medium containing 5 μg/ml of cytochalasin B. For nuclear transfer, cumulus cells were injected into the enucleated oocytes in M2 medium using a piezo-driven micromanipulator (Primetech LTD, Tsuchiura-shi, Japan). _{Thereafter, embryos regenerated with 10 mM SrCl 2} , 2 mM EGTA, and 5 μg/ml cytochalasin B in M16 (Millipore) medium were activated for 6 hours, and then at 37° C., 5% CO _{2 in} a humidified atmosphere. was cultured in KSOM. The group of somatic cloned oocytes using fresh oocyte cytoplasm (FOC) was named SCNT-FOC, and the group using the cytoplasm of vitrified/frozen/thawed (cryopreserved/thawed) oocytes was named SCNT-CROC. did.

Example 4. Kdm4a mRNA Confirmation of embryonic development and transcriptional differences in somatic cell nuclear transfer cells following injection

4-1. Kdm4a Preparation of mRNA

Full-length mouse Kdm4a / Jhdm3a cDNA was cloned into pcDNA3.1 plasmid containing poly(A)83 at the 3' end of the cloning site using In-Fusion Kit (Clonetech # 638909). mRNA was synthesized from the linearized template plasmid by in vitro transcription using the mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies # AM1345). The synthesized mRNA was dissolved in nuclease-free water. The concentration of mRNA was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies); Aliquots of mRNA were stored at -80°C until use.

4-2. Confirmation of embryonic development of somatic cell nuclear transfer cells

mRNA injection of Kdm4a, encoding H3K9me3 demethylase, is known to enhance egg development in somatic cell nuclear-transferred mammals. First, in order to analyze the effect of Kdm4a mRNA on the development of cloned oocytes (SCNT-CROC) using the cytoplasm of vitrified/frozen/thawed oocytes, embryonic development was evaluated whether or not Kdm4a mRNA was injected. Next, ~10 pl of water (control) or 2 μg/μl Kdm4a mRNA was injected into the two groups of cloned eggs (SCNT-FOC group and SCNT-CROC group) prepared in Example 3 using a piezo-driven micromanipulator. did. In addition, embryo development was confirmed by immunofluorescence. Specifically, after washing the cloned embryos with phosphate-buffered saline (PBS) containing 0.1% polyvinyl alcohol (PVA), 4% w/v paraformaldehyde at room temperature was fixed for 30 min. After washing the oocytes with PBS/PVA, they were cultured overnight in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 at 4°C. Thereafter, the oocytes were washed 3 times in PBS-0.1% BSA and incubated with 1:200 dilutions of H3K9me3 antibody and purified mouse anti-OCT-3/4 (BD) at the 1 and 2 cell stage for 2 hours. The cloned embryos were washed 3 times with PBS-0.1% BSA and incubated for 1 hour at room temperature with a 1:200 dilution of goat anti-mouse antibody. Then, it was washed three more times in PBS-0.1% BSA. DNA was visualized by staining the oocytes with 4',6-diamidino-2-phenylindole (DAPI). Embryos were mounted on glass slides with fluorescent mounting medium and observed using a fluorescence microscope (Zeiss LSM880, Zeiss, Jena, Germany).

group mRNA (μg/μl) Number of nuclear-transferred oocytes 2-cell stage embryo count (%) ¶ 4-cell stage embryo count (%) # Number of 2-cell block embryos (%±SEM) # Number of blastocysts (%±SEM) # SCNT-FOC - 112 105 (94) 70 (67) 35 (33±2.9) ^a 27 (26±3.4) ^a SCNT-FOC+K 2 109 103 (95) 102 (99) 1 (1±1) ^b 85 (83±3.5) ^b SCNT-CROC - 139 132 (95) 93 (70) 39 (30±1.8) ^a 30 (23±3.1) ^a SCNT-CROC+K 2 130 125 (96) 123 (98) 2 (2±1.2) ^b 82 (66±2.4) ^c

a,b,c: different superscripts within the same column, indicating a significant difference in values (P<0.05; n=5) SCNT-FOC: somatic cell nuclear transfer cloned egg using fresh oocyte cytoplasm

SCNT-CROC: Somatic cell nuclear transfer cloned egg using cytoplasm of vitrified/frozen/thawed oocytes

K: injection of lysine (K)-specific demethylase 4A (Kdm4a) mRNA

¶: Based on the number of SCNT oocytes

#: based on the number of 2-cell embryos

1 is the result of observing the demethylation of H3K9me3 by injecting Kdm4a mRNA into the SCNT-FOC and SCNT-CROC groups, and Table 1 shows the effect of the injection of Kdm4a mRNA on the development of the SCNT-FOC and SCNT-CROC groups. am. It was confirmed that demethylation of H3K9me3 was normally performed in the nuclei of the first and second cell stages of the SCNT-FOC group and the SCNT-CROC group (FIG. 1a). As a result of quantifying the methylation level, it was confirmed that when Kdm4a mRNA was injected into the SCNT-FOC group and SCNT-CROC, the level of H3K9me3 was significantly reduced ( FIG. 1b ). In addition, when Kdm4a mRNA was injected into the SCNT-FOC group and the SCNT-CROC group, it was confirmed that the blastocyst efficiency of both groups was significantly increased. That is, it was confirmed that down-regulation of H3K9me3 activity improved both the blastocyst formation rate of the SCNT-FOC group and the SCNT-CROC group embryo. Therefore, it can be confirmed that injection of Kdm4a mRNA inhibits H3K9me3 activity and overcomes the 2-cell block in cloned eggs (Table 1).

Figure 2 confirms the change in the number of internal cells and total cells of the SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected. In this case, the inner cell population means the number of cells that can be developed into embryonic stem cells in the future, type 1 indicates less than 10 inner cells, and type 2 indicates more than 10 inner cells.

As shown in Figures 2a and 2b, the embryonic developmental defects in the SCNT-FOC group and the SCNT-CROC group were almost overcome after the injection of mRNA, but the embryogenesis and the degree of embryonic development in the SCNT-CROC group were the SCNT-FOC group. was significantly lower than that of embryonic development and development in In particular, as shown in Figure 2c, the number of cloned embryos with a high ratio of the number of ICMs (over 10 ICMs per blastocyst) in the SCNT-CROC+K group was significantly lower than that in the SCNT-FOC+K group (p<0.05). . That is, injection of Kdm4a mRNA can improve blastocyst quality by significantly increasing the number of inner cell populations.

4-3. Confirmation of Transcription Differences in 2-cell Embryos of Somatic Cell Nuclear Transplantation Cells

To determine the transcriptional differences in the earliest embryos between the SCNT-FOC group and the SCNT-CROC group, 24 h after oocyte activation and Kdm4a mRNA injection, two groups of pooled 2-cell embryos (100 embryos per sample; RNA sequencing was performed twice with 3 replicates). In round 1, gene expression was assessed in 2-cell embryos between SCNT-FOC+K and SCNT-CROC+K (100 embryos per sample, replicates 3). In round 2, gene expression was assessed in blastocysts between SCNT-CROC+K and SCNT-CROC+K+M (50 blastocysts per sample, replicates 2 times). Complementary DNA (cDNA) was amplified using the SMARTer Ultra Low Input RNA cDNA preparation kit (Takara, 634890) according to the manufacturer's instructions. The cDNA was fragmented into approximately 200 bp fragments using an M220 sonicator (Covaris). The fragmented cDNA was end-repaired and adapter-ligated. Sequencing libraries were prepared using the ScriptSeq v2 kit (Illumina) according to the manufacturer's instructions. Single-ended sequencing was performed on a HiSeq2500 (Illumina) and mapped to the mm9 mouse genome using STAR (v2.5.2b, https://github.com/alexdobin/STAR). Then, fragments per kilobase per million read (FPKM) were calculated with Cufflinks (v2.2.1) using the default options. Genes were considered to be differentially expressed in fold change>3 and FPKM>5. Statistical analysis and scatter plot generation were performed using the R (v3.3.2) package. Expression of most genes was similar in both groups, and only a few genes (159) showed different expression patterns (FIG. 3a). GO terms and KEGG analysis confirmed that several pathways including apoptosis and p53 signaling pathways were remarkably activated. When DEG was analyzed by GO terms-database, only 16 genes (0.92%) out of 1,730 apoptosis-related genes showed a more than 3-fold difference between the SCNT-FOC group and the SCNT-CROC group. In the SCNT-CROC group, eight pro-apoptotic genes (Cyt, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7, Pmaip) (50%) and three anti-apoptotic genes (Adrb2, Scin, Six1) were It was confirmed that the expression was up-regulated compared to the SCNT-FOC group. In addition, it was confirmed that only two apoptosis-related genes (Ifng and Siah1a) (18.2%) and three anti-apoptosis-related genes (Syce3, Tsc22d3 and Vegfa) (27.2%) were down-regulated ( FIG. 3b ).

In addition, in the SCNT-CROC group, only 6 genes (0.41%) out of 1,449 cell cycle-related genes showed a difference in expression level, and it was confirmed that one cell arrest-related gene (Gadd45g) was up-regulated (Fig. 3b). On the other hand, in the SCNT-FOC group and the SCNT-CROC group, it can be seen that the expression patterns of 30 gene expression-related genes (0.62%) out of 4,841 genes and 61 genes (0.60%) out of 10,234 metabolic process-related genes are different from each other. there was. However, it can be seen that most of the genes do not have a negative function on gene expression or metabolic processes ( FIG. 3b ).

Example 5. Embryonic development of somatic cloned eggs with or without melatonin

5-1. Confirmation of embryonic development of somatic cell nuclear transfer cells with and without melatonin

Kdm4a (SCNT-CROC+K) was first injected into cloned eggs of the SCNT-CROC group, and then cultured in KSOM medium containing 10 μM melatonin (Sigma) and KSOM medium not containing it. Embryonic development of cloned eggs was assessed 5 days (120 hours) after activation. The melatonin concentration was selected through a preliminary experiment using a conventional fertilized mouse embryo as a concentration showing the best quality and high blastocyst formation rate. The results for each group are shown in Table 2 below.

group Melatonin (μM) Number of nuclear-transferred oocytes 2-cell stage embryo count (%) ¶ 4-cell stage embryo count (%) # Number of 2-cell block embryos (%±SEM) # Number of blastocysts (%±SEM) # SCNT-CROC - 125 112 (90) 66 (59) 46 (42±3.7) ^a 23 (20±1.5) ^a SCNT-CROC +M 10 125 114 (91) 81 (71) 33 (31±3.7) ^a 44 (39±1.2) ^b SCNT-CROC + K - 138 128 (98) 124 (94) 4 (3±1.5) ^a 76 (59±3.3) ^a SCNT-CROC + K + M 10 138 123 (96) 119 (96) 4 (4±1.8) ^a 91 (75±3.3) ^b

a,b,c: different superscripts within the same column, indicating a significant difference in values (P<0.05; n=5) SCNT-FOC: somatic cell nuclear transfer cloned egg using fresh oocyte cytoplasm

SCNT-CROC: Somatic cell nuclear transfer cloned egg using cytoplasm of vitrified/frozen/thawed oocytes

M: Melatonin treatment

K: injection of lysine (K)-specific demethylase 4A (Kdm4a) mRNA

¶: Based on the number of SCNT oocytes

#: based on the number of 2-cell embryos

As a result, melatonin played a positive role in the embryonic development of the SCNT-CROC group regardless of the injection of Kdm4a mRNA (Table 2). That is, it can be seen that the treatment of melatonin improves the blastocyst development rate of the SCNT-CROC group independently of Kdm4a mRNA injection. In addition, it was confirmed that the SCNT-CROC group exhibited a blastocyst development rate similar to that of the SCNT-FOC group by increasing the blastocyst development efficiency, which could not be overcome only by injection of Kdm4a mRNA into the SCNT-CROC group.

5-2. Confirmation of DNA fragmentation following melatonin treatment

In the group of Example 5-1, the level of DNA fragmentation in the blastocyst according to whether or not melatonin was treated was detected using the TUNEL staining method (in situ Cell Death Detection Kit, Roche, Indianapolis, IN). SCNT-derived blastocysts were washed three times in DPBS 0.1% PVA. Embryos were incubated in TUNEL reaction medium at 37° C. under dark conditions for 1 hour. DNA stained with 1 μg/ml Hoechst 33342 (Bis-benzimide, Sigma) was used for nuclear counter-staining. The signals of the embryos were observed with a confocal microscope (Zeiss, LSM880).

As a result, it was confirmed that the number of TUNEL-positive cells in the blastocyst of the SCNT-CROC group decreased when melatonin was treated (FIG. 4a). The overall proportion of TUNEL-positive embryos was significantly reduced (7.8% vs. 54.3% and 24.1) in the melatonin-treated group (SCNT-CROC+K+M) compared to the non-melatonin-treated group (SCNT-CROC and SCNT-CROC+K). %) (p<0.05; Figure 4b).

5-3. Effects of melatonin treatment on the production of free radicals in embryos

In the group of Example 5-1, the effect of melatonin treatment on active oxygen in blastocysts was confirmed. Specifically, in the SCNT-CROC+K group, embryos at morula or blastocyst stage were treated with or without melatonin, and culture medium containing 5 μM CellROX Oxidative Stress Reagent (Molecular Probes, Eugene, OR) for 30 min. After incubation in 0.1% polyvinyl alcohol (PVA)-D-PBS was treated. Embryos were observed under a fluorescence confocal microscope (Zeiss, LSM880). The recorded fluorescence intensity was analyzed using ImageJ software. Fluorescent pixel values of embryos were measured within a certain region from the cytoplasmic region of other embryos. Before analyzing statistically significant differences between groups, background fluorescence values were excluded from the final values. Experiments were performed with 3 replicates of 5 to 10 embryos in each replicate.

As a result, it was confirmed that the treatment of melatonin significantly reduced the level of active oxygen in the morula. In addition, as a result of examining the effect of melatonin treatment on the qualitative aspect of blastocysts through fluorescent staining (cell death; green, nuclear; blue staining) of the part where apoptosis occurred, the number of apoptosis stained with purple was significantly reduced. , it was confirmed that the apoptosis rate was also significantly reduced (FIG. 5a). In addition, it was confirmed that the level of reactive oxygen species in the morula was significantly lower in the SCNT-CROC group compared to the SCNT-CROC + K group (FIG. 5b). That is, the treatment of melatonin improves apoptosis pathways such as apoptosis, peroxisomal and oxidative phosphorylation, and thus a qualitatively improved blastocyst can be obtained.

5-4. Confirmation of effect on transcription of blastocyst according to melatonin treatment

To determine which genes are regulated by melatonin, transcripts of 2-cell embryos and blastocysts (50 embryos or blastocysts per sample, replicates twice) in both the melatonin-treated and untreated groups of Example 5-1 was analyzed, and the results are shown in Table 3 below.

Related features genetic symbol gene ID gene name control cell survival and
tissue regeneration Deaf1 NM_001282072.1 DEAF1, transcription factor top Fxn NM_008044.2 frataxin lift Ppan NM_145610.2 peter pan homolog lift Rab10os NR_015551.1 RAB10, member RAS oncogene family, opposite strand lift Sprr2d NM_011470.2 small proline-rich protein 2D lift Stag3 NM_016964.2 stromal antigen 3 lift Tsen15 NM_025677.3 tRNA splicing endonuclease subunit 15 lift Zfp335 NM_199027.2 zinc finger protein 335 lift Adm NM_009627.2 adrenomedullin downward Zfp54 NM_011760.2 zinc finger protein 54 downward Zscan4f NM_001110316.2 zinc finger and SCAN domain containing 4F downward antioxidant, inflammation
and apoptosis ctss NM_001267695.2 cathepsin S lift dffa NM_001025296.2 DNA fragmentation factor, alpha subunit lift oxidative stress Eif3f NM_025344.2 eukaryotic translation initiation factor 3, subunit F lift Hacl1 NM_019975.3 2-hydroxyacyl-CoA lyase 1 lift Hspbp1 NM_001360629.1 HSPA (heat shock 70kDa) binding protein, cytoplasmic cochaperone 1 lift Mob2 NM_001347560.1 MOB kinase activator 2 lift Mrpl23 NM_011288.1 mitochondrial ribosomal protein L23 lift Mrpl33 NM_025796.3 mitochondrial ribosomal protein L33 lift pmvk NM_001310640.1 phosphomevalonate kinase lift Slc4a11 NM_001081162.1 solute carrier family 4, sodium bicarbonate transporter-like, member 11 lift Wbscr22 NM_001202560.2 BUD23, rRNA methyltransferase and ribosome maturation factor lift cell death /play ctsd NM_009983.3 cathepsin D lift Ifi27 NM_001364173.1 interferon, alpha-inducible protein 27 lift Mettl13 NM_144877.1 methyltransferase like 13 lift Nck1 NM_001324530.1 non-catalytic region of tyrosine kinase adapter protein 1 lift Nmi NM_001141948.1 N-myc (and STAT) interactor lift ATP6v0c NM_001361531.1 ATPase, H+ transporting, lysosomal V0 subunit C downward CD52 NM_013706.2 CD52 antigen downward Dapk2 NM_010019.3 death-associated protein kinase 2 downward Ddit4l NM_030143.4 DNA-damage-inducible transcript 4-like downward Duoxa2 NM_025777.2 dual oxidase maturation factor 2 downward Nfkbia NM_010907.2 nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha downward ptgds NM_008963.2 prostaglandin D2 synthase (brain) downward Rdh12 NM_001313971.1 retinol dehydrogenase 12 downward Rnd3 NM_028810.2 Rho family GTPase 3 downward Cell and tumor proliferation AY074887 NM_145229.2 cDNA sequence AY074887 lift Cdca2 NM_001110162.1 cell division cycle associated 2 lift Dnph1 NM_207161.3 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 lift Eif3d NM_018749.2 eukaryotic translation initiation factor 3, subunit D lift Eif3e NM_008388.2 eukaryotic translation initiation factor 3, subunit E lift gtf2h2 NM_001360706.1 general transcription factor II H, polypeptide 2 lift Nefl NM_010910.2 neurofilament, light polypeptide lift Recql NM_001204906.1 RecQ protein-like lift Snhg7 NR_024068.2 small nucleolar RNA host gene 7 lift Ccne2 NM_001037134.2 cyclin E2 downward Ly6a NM_001271416.1 lymphocyte antigen 6 complex, locus A downward immune response ccdc86 NM_023731.3 coiled-coil domain containing 86 lift Commd4 NM_025417.2 COMM domain containing 4 lift Il17ra NM_008359.2 interleukin 17 receptor A lift Lat2 NM_020044.3 linker for activation of T cells family, member 2 lift cstb NM_007793.3 cystatin B downward Qpct NM_001316729.1 glutaminyl-peptide cyclotransferase (glutaminyl cyclase) downward etc Arfgap2 NM_001166024.1 ADP-ribosylation factor GTPase activating protein 2 lift bcdin3d NM_029236.2 BCDIN3 domain containing lift Cenpl NM_001127181.2 centromere protein L lift Cep63 NM_001081122.1 centrosomal protein 63 lift Clpp NM_017393.2 caseinolytic mitochondrial matrix peptidase proteolytic subunit lift cmtm7 NM_001252479.1 CKLF-like MARVEL transmembrane domain containing 7 lift Cox17 NM_001017429.2 cytochrome c oxidase assembly protein 17, copper chaperone lift Dbnl NM_001146308.1 drebrin-like lift Defb22 NM_001002791.2 defensin beta 22 lift DNAf1 NM_026648.4 dynein, axonemal assembly factor 1 lift Dph2 NM_026344.3 DPH2 homolog lift Elmod3 NM_001253692.1 ELMO/CED-12 domain containing 3 lift gtsf1l NM_026630.2 gametocyte specific factor 1-like lift Hint3 NM_025798.3 histidine triad nucleotide binding protein 3 lift ltc4s NM_001313968.1 leukotriene C4 synthase lift Ltn1 NM_001081068.1 listerin E3 ubiquitin protein ligase 1 lift Mars2 NM_175439.3 methionine-tRNA synthetase 2 (mitochondrial) lift Meig1 NM_001355205.1 meiosis expressed gene 1 lift mppe1 NM_001357518.1 metallophosphoesterase 1 lift Mvb12a NM_028617.2 multivesicular body subunit 12A lift mvd NM_138656.2 mevalonate (diphospho) decarboxylase lift Ndufs7 NM_001364694.1 NADH:ubiquinone oxidoreductase core subunit S7 lift nt5c NM_015807.1 5',3'-nucleotidase, cytosolic lift Oxa1l NM_026936.3 oxidase assembly 1-like lift Pet100 NM_001195244.1 PET100 homolog lift psmd13 NM_011875.4 proteasome (prosome, macropain) 26S subunit, non-ATPase, 13 lift Psmg3 NM_001356963.1 proteasome (prosome, macropain) assembly chaperone 3 lift ptpn18 NM_011206.2 protein tyrosine phosphatase, non-receptor type 18 lift Pxmp4 NM_021534.3 peroxisomal membrane protein 4 lift Slc35f5 NM_001356294.1 solute carrier family 35, member F5 lift Sln NM_025540.2 sarcolipin lift Srp68 NM_146032.3 signal recognition particle 68 lift Surf2 NM_013678.2 surfeit gene 2 lift Tagap1 NM_147155.2 T cell activation GTPase activating protein 1 lift Tbccd1 NM_001081368.2 TBCC domain containing 1 lift Tead4 NM_001080979.1 TEA domain family member 4 lift Tmem126b NM_026734.1 transmembrane protein 126B lift Tmem205 NM_001253867.1 transmembrane protein 205 lift Tor1b NM_133673.3 torsin family 1, member B lift Trmt61a NM_001099792.1 tRNA methyltransferase 61A lift Wrb NM_207301.3 tryptophan rich basic protein lift Ythdf1 NM_173761.3 YTH domain family 1 lift Zfp42 NM_009556.3 zinc finger protein 42 lift Lhfpl1 NM_178358.3 lipoma HMGIC fusion partner-like 1 downward Lin7b NM_011698.1 lin-7 homolog B (C. elegans) downward Mid1ip1 NM_001166635.1 Mid1 interacting protein 1 (gastrulation specific G12-like (zebrafish))

downward smco2 NM_027059.1 single-pass membrane protein with coiled-coil domains 2 downward Tmem263 NM_001013028.2 transmembrane protein 263 downward Tuba4a NM_001313723.1 tubulin, alpha 4A downward Zscan4c NM_001013765.2 zinc finger and SCAN domain containing 4C downward Zscan4d NM_001100186.1 zinc finger and SCAN domain containing 4D downward unknown 0610040B10Rik NR_027874.1 RIKEN cDNA 0610040B10 gene lift 1110007C09Rik NM_026738.2 caspase recruitment domain family, member 19 lift 1190002F15Rik NR_037955.1 lncRNA downstream of Cdkn1b lift 1700124L16Rik NR_105027.1 RIKEN cDNA 1700124L16 gene lift 1810063I02Rik NR_130986.1 RIKEN cDNA 1810063I02 gene lift 2010204K13Rik NR_027924.1 RIKEN cDNA 2010204K13 gene lift 2010320M18Rik NR_029440.1 RIKEN cDNA 2010320M18 gene lift 2900076A07Rik NR_045299.1 RIKEN cDNA 2900076A07 gene lift 4930444P10Rik NM_001243238.2 RIKEN cDNA 4930444P10 gene lift 5830454E08Rik NR_073359.1 RIKEN cDNA 5830454E08 gene lift Armc12 NM_026290.3 armadillo repeat containing 12 lift C130036L24Rik NR_015507.2 RIKEN cDNA C130036L24 gene lift ccdc59 NM_025602.3 coiled-coil domain containing 59 lift Commd8 NM_001356611.1 COMM domain containing 8 lift 2010204K13Rik NR_027924.1 RIKEN cDNA 2010204K13 gene lift GM12238 NR_028480.1 predicted gene 12238 lift GM15008 NR_045917.1 predicted gene 15008 lift GM16023 NR_040441.1 predicted gene 16023 lift H2-T9 NM_010399.2 histocompatibility 2, T region locus 9 lift I730030J21Rik NR_045781.1 RIKEN cDNA I730030J21 gene lift Kbtbd3 NM_001164574.1 kelch repeat and BTB (POZ) domain containing 3 lift LOC100504703 NR_040660.1 RIKEN cDNA A730063M14 gene lift Mir8112 NR_106192.1 microRNA 8112 lift Mrpl52 NM_026851.2 mitochondrial ribosomal protein L52 lift Ndufc1 NM_025523.1 NADH:ubiquinone oxidoreductase subunit C1 lift Olfr649 NM_147055.1 olfactory receptor 649 lift R3hcc1 NM_001146012.2 R3H domain and coiled-coil containing 1 lift Snora33 NR_037680.1 small nucleolar RNA, H/ACA box 33 lift Snora70 NR_002899.1 small nucleolar RNA, H/ACA box 70 lift Snord15a NR_002172.1 small nucleolar RNA, C/D box 15A lift Snord15b NR_002173.1 small nucleolar RNA, C/D box 14B lift Zfp868 XM_011242291.2 zinc finger protein 868 lift 1700061F12Rik NR_038180.1 RIKEN cDNA 1700061F12 gene downward 2700069I18Rik NR_045905.2 RIKEN cDNA 2700069I18 gene downward 4933430M04Rik NR_045857.2 RIKEN cDNA 4933430M04 gene downward BB287469 NM_001177573.1 expressed sequence BB287469 downward Eif3j1 NM_144545.4 eukaryotic translation initiation factor 3, subunit J1 downward GM11487 NM_001013393.1 predicted gene 11487 downward GM13078 NM_001085412.2 predicted gene 13078 downward GM13083 NM_001126324.1 predicted gene 13083 downward GM13152 NM_001039209.2 zinc finger protein 982 downward GM13242 NM_001103158.2 zinc finger protein 980 downward GM2016 NM_001122662.1 predicted gene 2016 downward GM2022 NM_001177574.1 predicted pseudogene 2022 downward Gm21319 NM_001270722.1 predicted gene, 21319 downward GM4027 NM_001177564.1 predicted gene 4027 downward Gm428 NM_001081644.1 predicted gene 428 downward GM5039 NR_003647.2 predicted gene 5039 downward GM5662 NM_001013824.3 predicted gene 5662 downward GM5868 NM_001024147.2 predicted gene 5868 downward GM8300 NM_001177565.1 predicted gene 8300 downward Gm8764 NM_001270900.1 predicted gene 8764 downward GM8994 NM_001142734.1 predicted gene 8994 downward Mir6340 NR_105758.1 microRNA 6340 downward

Nudt22 NM_026675.2 nudix (nucleoside diphosphate linked moiety X)-type motif 22 downward Olfr1463 NM_001011840.1 olfactory receptor 1463 downward Olfr376 NM_001172686.1 olfactory receptor 376 downward Olfr450 NM_146445.1 olfactory receptor 450 downward Olfr815 NM_146670.1 olfactory receptor 815 downward Pramef6 NM_001085414.2 PRAME family member 6 downward Rnase6 NM_001360117.1 ribonuclease, RNase A family, 6 downward Snora52 NR_034049.2 small nucleolar RNA, H/ACA box 52 downward Snora64 NR_002897.1 small nucleolar RNA, H/ACA box 64 downward Snora74a NR_002905.3 small nucleolar RNA, H/ACA box 74A downward Snord22 NR_004445.1 small nucleolar RNA, C/D box 22 downward Tdpoz3 NM_207271.2 TD and POZ domain containing 3 downward Tmem182 NM_001081198.1 transmembrane protein 182 downward Usp17la NM_007887.2 ubiquitin specific peptidase 17-like A downward usp17lb NM_001358922.1 ubiquitin specific peptidase 17-like B downward usp17ld NM_001001559.2 ubiquitin specific peptidase 17-like D downward Usp17le NM_001256973.1 ubiquitin specific peptidase 17-like E downward Zscan4a NR_033707.1 zinc finger and SCAN domain containing 4A downward

As a result, in the case of 2-cell embryos, 175 genes were differentially expressed in fold change>2 and FPKM>5 (Table 3). In particular, 111 genes were upregulated in the melatonin-treated group (SCNT-CROC+K+M) compared to the non-melatonin-treated group (SCNT-CROC+K). In particular, Amd1 , Fam46C , Oxt , and Ppt2 involved in cell survival and tissue regeneration were upregulated in 2-cell embryos of the melatonin-treated group (SCNT-CROC+K+M). In addition, Ctss , Dffa , Eif3f , Hacl1 , Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 have been implicated in antioxidant function, inflammation and apoptosis. and Wbscr22 was upregulated to high levels. On the other hand, cell death and degeneration-related genes ( Atp6v0c , Cd52 , Dapk2 , Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 ) and Rnd3 ) were down-regulated in 2-cell embryos of SCNT-CROC+K+M).

In the case of blastocysts, 81 genes were differentially expressed in fold change>2 and FPKM>5 (Table S2). In particular, 20 genes were upregulated in the melatonin-treated group (SCNT-CROC+K+M) compared to the non-melatonin-treated group (SCNT-CROC+K). Amd1 , Fam46C , Oxt , and Ppt2 , which are associated with cell survival and tissue regeneration, were upregulated in expression in blastocysts of the melatonin-treated group. In particular, Gulo is associated with antioxidant function, inflammation and apoptosis. and Txnip expression was upregulated. On the other hand, genes associated with oxidative stress ( Adh1 , Car2 , Gsta3 , Gstm2 , Mb , Phlda2 , and S100a1 ) and cell death and degeneration-related genes ( Akr1c13 , Amd2 , Clu , Hist1h3a , Hspb1 , Il1rn , and Nat8 ) are It was down-regulated in blastocysts of the treatment group (SCNT-CROC+K+M). In addition, the expression levels of cellular and tumor proliferation-related genes ( Efemp1 , Glipr1 , Lgals4 , Plac1 , Snora15 , Snora 21 , Snora 34 , and Anxa1 ) were decreased. In addition, some genes involved in the immune response ( Clec2f , Hrsp12 , and Plat ) were shown to be down-regulated in the melatonin-treated group (SCNT-CROC+K+M) (Table 4).

Related features genetic symbol gene ID gene name control cell survival and
tissue regeneration Amd1 NM_009665.5 S-adenosylmethionine decarboxylase 1 lift Fam46C NM_001142952.1 family with sequence similarity 46, member C lift Oxt NM_011025.4 oxytocin lift ppt2 NM_001302393.1 palmitoyl-protein thioesterase 2 lift antioxidant, inflammation
and apoptosis Gulo NM_178747.3 gulonolactone (L-) oxidase lift Txnip NM_001009935.2 thioredoxin interacting protein downward oxidative stress Adh1 NM_007409.2 alcohol dehydrogenase 1 downward Car2 NM_001357334.1 carbonic anhydrase 2 downward Gsta3 NM_001077353.2 glutathione S-transferase, alpha 3 downward gstm2 NM_008183.3 glutathione S-transferase, mu 2 downward Mb NM_001164047.1 myoglobin downward Phlda2 NM_009434.2 pleckstrin homology like domain, family A, member 2 downward S100a1 NM_011309.3 S100 calcium binding protein A1 downward cell death/regeneration Akr1c13 NM_013778.2 aldo-keto reductase family 1, member C13 downward Amd2 NM_007444.3 S-adenosylmethionine decarboxylase 2 downward Clu NM_013492.3 clusterin downward Hist1h3a NM_013550.5 histone cluster 1, H3a downward Hspb1 NM_013560.2 heat shock protein 1 downward Il1rn NM_001039701.3 interleukin 1 receptor antagonist downward Nat8 NM_023455.3 N-acetyltransferase 8 (GCN5-related) downward Cell and tumor proliferation Efemp1 NM_146015.2 epidermal growth factor-containing fibulin-like extracellular metrix protein downward Glipr1 NM_028608.3 GLI pathogenesis-related 1 (glioma) downward LG4 NM_010706.2 lectin, galactose binding, soluble 4 downward Plac1 NM_019538.4 placental specific protein 1 downward Snora15 NR_003681.1 small nucleolar RNA, H/ACA box 15 downward Snora21 NR_028078.1 small nucleolar RNA, H/ACA box 21 downward Snora34 NR_034051.1 small nucleolar RNA, H/ACA box 34 downward Anxa1 NM_010730.2 annexin A1 downward immune response Clec2f NM_001277202.1 C-type lectin domain family 2, member f downward Hrsp12 NM_008287.3 reactive intermediate imine deaminase A homolog downward Plat NM_008872.3 plasminogen activator, tissue downward Snora75 NR_028478.1 small nucleolar RNA, H/ACA box 75 lift etc Car4 NM_007607.2 carbonic anhydrase 4 downward Hotairm1 NR_131181.1 Hoxa transcript antisense RNA, myeloid-specific 1 downward Mt4 NM_008631.2 metallothionein 4 downward Rhox6 NM_008955.1 reproductive homeobox 6 downward Rmnd1 NM_025343.5 required for meiotic nuclear division 1 homolog downward S100a13 NM_009113.4 S100 calcium binding protein A13 downward S100a16 NM_001356605.1 S100 calcium binding protein A16 downward Serpinb6b NM_011454.1 serine (or cysteine) peptidase inhibitor, clade B, member 6b downward Zscan4a NR_033707.1 zinc finger and SCAN domain containing 4A downward Zscan4c NM_001013765.2 zinc finger and SCAN domain containing 4C downward Zscan4d NM_001100186.1 zinc finger and SCAN domain containing 4D downward unknown Eif3j1 NM_144545.4 eukaryotic translation initiation factor 3, subunit J1 lift GM19705 NR_045324.1 predicted gene, 19705 lift GM5512 NR_002891.1 predicted gene 5512 lift Lmo4 NM_001161769.1 LIM domain only 4 lift Mir6340 NR_105758.1 microRNA 6340 lift Mir6363 NR_105782.1 microRNA 6363 lift Mir8094 NR_106169.1 microRNA 8094 lift Pisd-ps3 NR_003518.2 phosphatidylserine decarboxylase, pseudogene 3 lift Snora26 NR_031758.1 small nucleolar RNA, H/ACA box 26 lift Snora28 NR_033168.1 small nucleolar RNA, H/ACA box 28 lift Snora30 NR_034045.1 small nucleolar RNA, H/ACA box 30 lift Snora31 NR_028481.1 small nucleolar RNA, H/ACA box 31 lift Snora70 NR_002899.1 small nucleolar RNA, H/ACA box 70 lift 4930461G14Rik NR_040736.1 RIKEN cDNA 4930461G14 gene downward ccdc160 NM_001034059.1 coiled-coil domain containing 160 downward ctsll3 NM_027344.3 cathepsin L-like 3 downward Eif3j2 NM_001256055.1 eukaryotic translation initiation factor 3, subunit J2 downward GM5547 NR_045845.1 predicted gene 5547 downward GM5662 NM_001013824.3 predicted gene 5662 downward GM7334 NR_002700.1 predicted gene 7334 downward GM9992 NM_001142539.1 predicted gene 9992 downward Hspb2 NM_001164708.1 heat shock protein 2 downward Klra22 NM_053152.2 killer cell lectin-like receptor subfamily A, member 22 downward LOC100504703 NR_040660.1 RIKEN cDNA A730063M14 gene downward Mir1191 NR_035422.1 microRNA 1191 downward unknown Mir6236 NR_105744.1 microRNA 6236 downward Mir6516 NR_105853.1 microRNA 6516 downward Ms4a10 NM_023529.2 membrane-spanning 4-domains, subfamily A, member 10 downward Pinlyp NM_001037143.2 phospholipase A2 inhibitor and LY6/PLAUR domain containing downward Pisd-ps1 NR_003517.1 phosphatidylserine decarboxylase, pseudogene 1 downward Platr21 NR_045455.1 pluripotency associated transcript 21 downward Pramef25 NM_001126315.2 PRAME family member 25 downward Snora47 NR_034043.1 small nucleolar RNA, H/ACA box 47 downward Snora64 NR_002897.1 small nucleolar RNA, H/ACA box 64 downward Snora81 NR_034048.1 small nucleolar RNA, H/ACA box 81 downward Tmem40 NM_001168256.1 transmembrane protein 40 downward usp17lb NM_201409.2 ubiquitin specific peptidase 17-like B downward

Example 6. Embryo metastasis and implantation monitoring

Reconstituted embryos cultured to blastocyst stage under melatonin-treated or untreated conditions (SCNT-CROC+K and SCNT-CROC+K+M groups) were pseudo-pregnant at 2.5 days mated with male ICR mice undergoing vasectomy. pregnant) were implanted in the uterus of female ICR mice. At this time, the SCNT-CROC+K group and the SCNT-CROC+K+M group were implanted in the left and right uterus of the same mouse, respectively. The embryo transfer rate in female mice was confirmed at 7.5 days after sexual intercourse of mice. Thereafter, the mice were euthanized and the normal uterus was washed with saline, excised, and the attached fat was removed. By imaging the tissue from which fat has been removed, implantation was visualized (FIGS. 6a and 6b).

As a result, the transplantation rate in the melatonin-treated group (SCNT-CROC+K+M) was significantly increased compared to that of the non-melatonin-treated group (SCNT-CROC+K) (66.2% (51/77) vs. 42.9% ( 33/77), p <0.05; FIG. 6c).

Example 7. Induction of mouse embryonic stem cells from SCNT blastocysts

To obtain outgrowths, the hatched blastocysts obtained from both groups (SCNT-CROC+K and SCNT-CROC+K+M) were transferred to mitotic-inactivated mouse embryonic fibroblasts (mESCs) in mouse embryonic stem cell (mESC) culture medium. MEF) feeder cells. 20% KRS, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco/Invitrogen, Grand Island, NY) and 1.5 x 10 ³ DMWM/F12 containing units/mL recombinant mLIF (Chemicon, Temecula, CA) was used as a culture medium for mES cell culture. The outgrowths were first mechanically transferred to MEF feeder cells and then passaged using trypsin-EDTA. All established mECS lineages were monitored and characterized by morphology and alkaline phosphatase staining. Alkaline phosphatase activity was assessed by histochemical staining. Colonies were fixed with 4% paraformaldehyde at room temperature for 1 min, washed twice with PBS, and alkaline phosphatase substrate solution (10 ml FRV-alkaline solution, 10 ml naphthol AS-BI alkaline solution; alkaline phosphatase kit, Sigma-Aldrich) was treated at room temperature for 30 minutes. Alkaline phosphatase activity was detected colorimetrically by light microscopy (Fig. 7a).

As a result, the melatonin-treated group had a significantly higher mESC induction rate than the untreated group ((21.3% (10/47) vs. 5.6% (2/36), p <0.05; FIG. 7b ).

Example 8. Statistical Analysis

All experiments were repeated at least 3 times. Results are expressed as the mean or standard error of the mean±mean. Embryonic development was analyzed by one-way analysis of variance (ANOVA) by DUNCAN's test using SAS software and transplantation, and ESC-derivation rates were analyzed by Chi-square test, p <0.05 was considered statistically significant.

Claims (18)

A composition for increasing the efficiency of embryonic development or the formation of an embryo, comprising an agent for reducing melatonin and H3K9me3 methylation, wherein, when the embryo is in a two-cell stage , Amd1 , Fam46C , Oxt and Ppt2 The composition wherein the expression of any one or more genes selected from the group consisting of is increased, and the egg forming the embryo is thawed or thawed after being frozen or cryopreserved. The composition of claim 1, wherein the melatonin is contained in a concentration of 5 μM to 20 μM. The composition of claim 1 , wherein the agent that reduces H3K9me3 methylation is an agent that increases the expression of a member of the KDM4 family of histone demethylases. The composition of claim 3, wherein the agent increases the expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D) or a combination thereof. The composition of claim 1, wherein the melatonin reduces free radicals and/or apoptosis of the embryo. The composition of claim 1, wherein the melatonin reduces apoptosis of the embryo. The composition of claim 1 , wherein the embryonic development is the development of an embryo into a blastocyst or the formation of a blastocyst. The composition of claim 1, wherein the embryo is formed through artificial insemination in vitro. The composition of claim 1, wherein the embryo is formed by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed. delete delete delete delete delete delete delete delete delete

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