KR20200145804A - Composition and Method for enhancing developmental rate of embryos using melatonin - Google Patents

Abstract

The present invention relates to a composition for embryonic development comprising melatonin, and a method for improving the embryonic development rate using the same. The composition prevents the stopping of the development of the 2-cell stage of an embryo, thereby increasing the efficiency of developing into a blastocyst, and thus a blastocyst that is excellent in quality or quantity may be acquired.

Description

Composition and method for enhancing developmental rate of embryos using melatonin, including melatonin, and composition for embryonic development using the same

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

Somatic cell nuclear transfer (SCNT)-based reprogramming and derivation of embryonic stem cells (ESCs) can produce patient-specific stem cells for regenerative medicine. Has been proposed. Recently, three separate research groups have successfully derived several somatic nuclear transfer-embryonic stem cell (SCNT-ESC) lineages using high-quality human oocytes and fibroblasts from several sources. As much of the developmental arrest of human SCNT embryos is partially overcome by the regulation of histone methylation, it has become a technology applicable to cell therapy at present. In addition, the establishment of human leukocyte antigen (HLA)-matched homozygous pluripotent stem cells (PSCs) may increase the potential effectiveness of SCNT-ESCs, but many for research and clinical applications. It requires a number of donated human oocytes.

Recently, the technique of vitrification of oocytes has been used as a practical technique for a widely used cryopreservation method as well as a human assisted reproduction technique. In fact, it has been reported that the cell development and developmental arrest (cell bolck) of offspring born from vitrified (cold-frozen) oocytes did not increase when compared with conventional in vitro fertilization (IVF). This technology provides a valuable opportunity to maintain the fertility potential of infertile women and to treat women of childbearing age at risk of reduced age-induced fertility and the risk of fertility from cancer treatment. In addition, vitrification freezing of human excess oocytes can provide sustained eggs for research, such as SCNTs, and their use can also reduce ethical concerns. However, until recently, the production of cloned eggs using cryopreserved oocytes and induction of the SCNT-ESC line have not been achieved. Even with a survival rate of 90% or more, the clinical results of vitrified frozen oocytes were lower than those of fresh oocytes from the human reproductive technology program. This is thought to be due to cytoskeletal damage, changes in spindle structure, microtubules, cortical granule distribution, and oocyte hardening. In addition, cryopreserved oocytes are particularly susceptible to oxidative stress due to their high levels of lipids and produce large amounts of free radicals that affect the balance between oxidation and reduction reactions and the intracellular antioxidant system.

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 of increasing the efficiency of embryo development or increasing embryo 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, it provides a composition comprising melatonin for increasing the formation of embryos and/or increasing the efficiency of embryo development.

In the present specification, the term "formation of an embryo" means that a zygote becomes several cells through cell division, and these cells form an embryo through cell division and differentiation, or to develop into an embryo. .

In the present specification, the term "increased efficiency" means an increase in the development of blastocyst or blastocyst development of an egg. The blastocyst is developed so that it can be divided into an inner cell mass to differentiate into a fetus and a trophectoderm to differentiate into a placenta by forming a blastocyst after a densified morula during the process of growing the embryo. It means a fertilized egg in a state of being. Therefore, the increase of the efficiency is that the embryonic development efficiency of the egg is increased compared to the egg cultured in the absence of an agent that reduces H3K9me3 methylation, the development of the embryo to the blastocyst stage is increased, and the occurrence of the blastocyst stage. This increases, the production efficiency of blastocysts increases, the acquisition efficiency of blastocysts increases, or the developmental formation rate of blastocysts increases.

In one embodiment, the composition may be one containing melatonin in a concentration of 0.1 μM to 50 μM in a basic medium. Melatonin is a molecule involved in the cycle of biological reactions, also known as N-acetyl-5-methoxytryptamine. In mammals, melatonin regulates the cell cycle and shows strong resistance to oxidative stress and apoptosis. That is, the method according to one aspect is to reduce free radicals and apoptosis of the somatic cell nuclear transfer oocyte by culturing the nucleus of somatic cells and the oocyte from which the nucleus has been removed in a medium containing melatonin. There is an advantage that an improved blastocyst can be obtained. In addition, it is possible to improve the implantation rate of fertilized eggs artificially fertilized with somatic cell nuclear transfer oocytes, and promote embryonic stem cell induction. Specifically, melatonin in the basic medium is 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 basic medium is less than the above range, active oxygen cannot be effectively removed, and thus the efficiency of the development of blastocysts of somatic cell nuclear transfer oocytes is impaired, and there is a problem that a good quality oocyte cannot be obtained. In addition, when it exceeds the above range, there is a problem in that melatonin acts on the somatic cell nucleus transplanted egg for a long time, preventing the egg from maturation. Specifically, the basic medium may be a basic medium used for culturing mammalian eggs. The basic medium varies 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 medium known to those skilled in the art. As the inorganic salts, NaCl, KCl, and NaHCO ₃ may be used, and as a carbon source, glucose, sodium, pyruvate and calcium lactate salts may be used, and as amino acids, essential amino acids including glutamine and non-essential amino acids, Other trace elements and buffers can be used as cofactors. 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 may be selected from the group consisting of. In addition, the medium is a neutral buffer (e.g., phosphate and/or high concentration bicarbonate) and protein nutrients (e.g. serum, e.g. FBS, FCS (fetal calf serum), horse serum, serum substitute, albumin, or essential) in isotonic solution. Amino acids and non-essential amino acids such as glutamine, L-glutamine). Furthermore, lipids (fatty acids, cholesterol, serum HDL or LDL extracts) and other components found in most preservative media of this kind (e.g., transferrin, nucleoside or nucleotide, 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, antibiotics may be additionally included in the basic medium. The melatonin may be included in the basic medium at 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 basic medium is less than the above range, active oxygen cannot be effectively removed, and thus the efficiency of blastocyst development of somatic cell nuclear transfer oocytes is impaired. In addition, when exceeding the above range, there is a problem in that melatonin acts on the somatic cell nucleus transplanted egg for a long time to hinder the maturation of the egg.

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

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

When the embryo is developed as a blastocyst, expression of any one or more genes selected from the group consisting of Amd1 , Fam46C , Oxt , Ppt2 , Gulo, and Txnip may be increased. In addition, Adh1 , Car2 , Gsta3 , Gstm2 , Mb , Phlda2 , S100a1 , Akr1c13 , Amd2 , Clu , Hist1h3a , Hspb1 , Il1rn , Nat8 , Efemp1 , Glipr1 , Lgals4 , Plac1 , Snora15 , Snora1 , Snora2 34, Anxa1 , Snora2 34, Anxa It may be that the expression of any one or more genes selected from the group consisting of Hrsp12 , and Plat is reduced. The embryo may be an embryo formed in vitro through artificial insemination, or the embryo may be formed by implanting a nucleus of a somatic cell into an egg from which the nucleus has been removed. The embryo may be formed by artificial insemination outside the body, or the embryo may be formed by implanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.

Another aspect provides a method of increasing the efficiency of embryo development or embryo formation, comprising culturing an egg in a medium containing melatonin. Details of the medium containing melatonin are as described above.

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

The method may include culturing the eggs for 1 to 10 days in a medium containing melatonin. 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, if the culture period is less than the above range, there is a problem that the embryo is not sufficient to develop into a blastocyst, and if it exceeds the above range, there is a problem in that it is difficult to obtain a qualitatively improved blastocyst due to excessive maturation of the egg.

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

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

Another aspect is the step of preparing a nucleus of somatic cells and an egg from which the nucleus has been removed; And culturing the nucleus of the somatic cells and the oocyte from which the nucleus has been removed in a medium containing melatonin. It provides a method of increasing the efficiency of somatic-cell nuclear transfer (SCNT). The efficiency may be a success rate of somatic cell nuclear transplantation and an embryonic development rate of cells generated by the somatic cell nuclear transplantation. The embryonic development rate may mean the development of a blastocyst through the 2-cell stage, 4-cell stage, and 8-cell stage of the embryo.

The present inventors confirmed that by injecting KDM4A mRNA, the zygotic gene activation (ZGA) of the egg that induced KDM4A overexpression prevents developmental arrest at the 2-cell phase, and significantly improves the embryonic development of somatic cell nuclear transfer. I did. Thus, it expands the utility 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 uses and for both research and disease modeling. It can be used in a method to improve nuclear transfer of somatic cells. The present invention is not intended for reproduction of human reproduction.

The method includes the steps of removing the nucleus of the oocyte, transplanting the nucleus (donor nucleus) of one or more somatic cells, activating the reconstructed nuclear-transplanted oocyte (embryo), and additionally culturing it with a blastocyst. It may further include. The steps for such somatic-cell nuclear transfer (SCNT) can be performed by appropriate modifications by a person skilled in the art according to a method disclosed in the literature (Nature 419, 583-587, 10 October 2002). The nuclear-transplanted oocytes (embryos) are generated by injecting a donor somatic cell-derived nucleus (eg, nuclear genetic material) into a recipient oocyte from which the nucleus has been removed to form a nuclear-transplanted oocyte, which is activated or It may be fused to form a nuclear-transplanted 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 are oocytes, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, red blood cells, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryo reproduction It may be 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 technique in which a nucleus collected from a donor cell is transplanted into a recipient cell from which the nucleus has been removed, and refers to a technique that generates cells that are genetically identical to the donor cell. .

The oocyte from which the nucleus has been removed may be cumulus-oocyte complexs collected from an individual follicle, and may be obtained commercially. The individual may be a mammal including a human. For example, the mammal may include a mouse, a pig, a sheep, a dog, a cow, a horse, a goat, and the like. In one embodiment, an egg transplanted with somatic cell nuclei using the cytoplasm of a somatic cloned egg (SCNT-FOC) using fresh oocyte cytoplasm (FOC) and a vitrified frozen/thawed (cold preserved/thawed) oocyte ( As a result of injecting Kdm4a mRNA into SCNT-CROC), it was confirmed that the blastocyst development rate was low by about 17% in SCNT-CROC (FIG. 2c ). Thereafter, the SCNT-CROC and SCNT-FOC were additionally 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, and previously, only injection of Kdm4a mRNA It was confirmed that the blastocyst development efficiency, which could not be overcome, was improved by about 16%, indicating a similar blastocyst development rate to that of SCNT-FOC (Table 2). Thus, the egg from which the nucleus has been removed may be frozen and/or frozen and then thawed or melted. As the freezing method, a slow-freezing method or a vitrification method, which is an ultra-fast freezing method, may be used. Since SCNT-CROC treated with melatonin exhibits a similar blastocyst development rate to SCNT-FOC, it can be used for nuclear transplantation without separate freezing and thawing of donor oocytes during somatic cell cloning, so the process of somatic cell nuclear transplantation can be shortened. And it can improve the replication efficiency.

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

A method according to an embodiment may include the step of contacting an agent that reduces H3K9me3 methylation. The method according to another embodiment may include the step of 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 demethylase. The agent may be, for example, increasing 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-transplanted embryo after the nucleus of a somatic cell and an egg from which the nucleus has been removed are fused. Specifically, the embryo may be an embryo before the activation of the somatic cell nuclear transfer oocyte gene starts. For example, within 5 hours (5 hours post activation, 5 hpa) or between 10 and 12 hpa (i.e., 1-cell phase), or about 20 hpa (i.e., initial 2-cell phase) after activation of the embryo. Alternatively, it may be to contact one or more histone demethylase KDM4 families between 20 and 28 hpa (ie, 2-cell phase).

In addition, in the step of contacting or injecting, the donor cell, for example, the final differentiated somatic cell nucleus or cytoplasm, is contacted with at least one histone demethylase KDM4 family before injecting the nucleus of the donor cell into the nucleated oocyte. It may be injected. The contact or injection may be in contact with a donor somatic cell for 1 hour or more, or for 2 hours or more, and the contact is 1 day (24 hours) or more before nucleus removal from the donor somatic cell to the nucleated oocyte, 2 It may be performed 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 oocyte from which the nucleus has been removed with an agent that reduces H3K9me3 methylation, the activity of histone methyl enzyme that inhibited replication in somatic cell nuclear transfer embryos is reduced, and the expression of genes related to embryogenesis is enhanced. Bar, it is possible to improve the generation and development efficiency of blastocysts. For example, the developmental efficiency may be to increase the% of development of a somatic cell nuclear transfer embryo that develops in a 2-cell, 4-cell and 8-cell or blastocyst stage, and an 8-cell or blastocyst stage of a somatic cell nuclear transfer embryo It can increase the developmental efficiency before transplantation into the furnace. That is, the agent that reduces H3K9me3 methylation can overcome the 2-cell phase arrest phenomenon in somatic cell nuclear transfer embryos and maintain continuous embryonic development.

The method of one embodiment may include culturing the nucleus of somatic cells and the oocyte from which the nucleus has been removed 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, if the culture period is less than the above range, there is a problem that the somatic cell nuclear transfer egg is not sufficient to develop into a blastocyst, and if it exceeds the above range, the somatic cell nuclear transfer egg is excessively mature to obtain a qualitatively improved blastocyst. There is a problem that this is difficult.

In one embodiment, the method has an efficiency of nuclear transplantation of about 5% or more, about 10% or more, about 13% or more, about 15% or more, compared to somatic cell nuclear transplantation performed in the absence of an agent that reduces H3K9me3 methylation, It may be about 30% or more, about 50% or more, 50-80% or more than 80%. That is, to increase the efficiency of development before transplantation of somatic cell nuclear transfer embryos, or to increase the development of the embryo to the blastocyst stage, or to increase the development of the embryo to the expanded blastocyst stage by about 5%, about 7%, or about 10. %, about 12 or more, or more than 12% dilated blastocyst stage. In another embodiment, the method is 3 times or more, 4 times or more, 5 times or more, 6 times or more, compared to somatic cell nuclear transplantation performed in the absence of an agent that reduces H3K9me3 methylation, It may be increased by more than 7 times, more than 8 times or more than 8 times. The increase in the somatic cell nuclear transplantation efficiency means an increase in the production or production of blastocysts. The increase in the production or yield of blastocysts is about 110% or more, about 120% or more, about 130% or more, about 140, compared to somatic cell nuclear transplantation 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, the method according to one aspect can prevent cell damage by culturing somatic cell nuclear transfer cells in a medium containing melatonin, thereby reducing free radicals of somatic cell nuclear transfer cells, thereby preventing cell damage. Can be improved. In addition, apoptosis of somatic cell nuclear transfer oocytes frozen and thawed by melatonin is reduced, and blastocyst formation and production efficiency can be improved. In addition, as the implantation rate of somatic cell nucleus-transplanted oocytes is improved, an endangered animal can be produced through in vitro fertilization, and as embryonic stem cell induction is enhanced, an embryonic stem cell line can be efficiently produced.

Another aspect provides a somatic cell nuclear transfer embryo (embryo) prepared according to the above method. Another aspect provides a somatic cell nuclear transfer blastocyst (blastocyst) prepared according to the above method. The embryo is genetically modified and, for example, prior to the somatic cell nucleus transplantation step (i.e., prior to collecting the donor nucleus and fusion with the cytoplasm of the recipient oocyte), one or more transgenes in the genetic material of the donor nucleus have been modified. Can be. In one embodiment, the embryo may include a donor somatic cell-derived nuclear DNA, a recipient oocyte-derived cytoplasm, and a third donor individual-derived mitochondrial DNA. 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. Can be.

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

In another embodiment, the blastocyst may be one whose expression of genes related to cell survival or tissue regeneration, for example, Amd1 , Fam46C , Oxt , and/or Ppt2 is upregulated. In particular, it is inherited related to antioxidant function, inflammation or cell death, for example Gulo And/or the expression of Txnip may be upregulated. On the other hand, genes associated with oxidative stress, such as Adh1 , Car2 , Gsta3 , Gstm2 , Mb , Phlda2 , and/or S100a1 ; The expression of apoptosis or regression-related genes, for example, Akr1c13 , Amd2 , Clu , Hist1h3a , Hspb1 , Il1rn, and/or Nat8 may be 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 ; The expression of genes related to the immune response, for example, Clec2f , Hrsp12, and/or Plat may be 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 cells derived from the undifferentiated inner cell mass. In addition, the embryonic stem cells may be pluripotent stem cells or pluripotent stem cells.

Another aspect is the step of preparing a nucleus of somatic cells and an egg from which the nucleus has been removed; Culturing the nucleus of the somatic cell and the egg from which the nucleus is removed together with a candidate material; And evaluating the H3K9me3 methylation activity of the somatic cell nucleus transplanted cells generated after the culture. It provides a method of screening for an agent that increases the efficiency of somatic cell replication.

Cells derived from the somatic cell nuclear transfer embryos or blastocysts, for example embryonic stem cells, etc., can be used in tests to determine whether the agent affects differentiation or cell proliferation. For example, since the ability of the cells to differentiate or proliferate is evaluated from the presence or absence of an agent, it can be used for screening to select an agent that affects cells derived from the somatic cell nuclear transfer embryo or blastocyst. . The test compound can be any compound of interest, including a chemical compound, small molecule, polypeptide or other biological agent (eg, antibody or cytokine, etc.).

In the method according to an 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 techniques well known in the art such as RT-PCR or immunostaining. You can do it. In addition, the step of evaluating the H3K9me3 methylation activity may be performed together with the step of evaluating the somatic cell replication efficiency. Compared with before the addition of the candidate substance, if the number of somatic cell nucleus transplanted oocytes, 2-cell stage cells, 4-cell stage cells or blastocyst stage cells in the culture after addition is changed, the candidate substance is of the H3K9me3 methylation. It can be determined to be a substance that decreases activity and further increases somatic cell replication efficiency by somatic cell nuclear transfer.

Another aspect is the step of preparing a nucleus of somatic cells and an egg from which the nucleus has been removed; Obtaining a somatic-cell nuclear transfer (SCNT) cell by culturing the nucleus of the somatic cell and the egg from which the nucleus is removed in a medium containing melatonin; And it provides a method for producing a somatic cell transplant fertilized egg comprising the step of in vitro fertilization of the somatic cell nuclear transfer cells and liquid semen. Details of the method for culturing the nucleus of the somatic cell and the egg from which the nucleus has been removed in a medium containing melatonin are 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 in which the embryo has developed and reached the blastocyst stage.

A method according to an embodiment includes in vitro fertilization of the somatic cell nuclear transfer cells and liquid semen. The semen may be semen collected from the individual's vas deferens. The individual may be a mammal including a human. For example, the mammal may include a mouse, a pig, a sheep, a dog, a cow, a horse, a goat, and the like. In vitro fertilization of the somatic cell nuclear transfer cells and liquid semen in an in vitro fertilization medium composition for 1 to 7 days. In this case, when the in vitro fertilization period is less than the above range, there is a problem that fertilization is not possible, and when the period exceeds the above range, there is a problem that the embryo is degraded. In addition, in the above step, it may further include a step of contacting or injecting with an agent for reducing H3K9me3 methylation. Details of the agent for reducing methylation are as described above. The methylation-reducing agent is, after injecting semen into somatic cell nucleus transplanted cells, before pronucleus formation (within 18 hours after semen injection), specifically after semen injection, in contact with somatic cell-transplanted fertilized eggs before the 2-cell stage, or It may be injected. Therefore, the somatic cell-transferred fertilized egg proceeds efficiently without stopping the development of somatic cell nuclear transfer cells (embryos) and successfully develops into blastocysts through the 2-, 4-, and 8-cell phase without developmental defects or loss of viability. Can increase

It relates to a composition for embryonic development comprising melatonin and a method for improving the embryonic development rate using the same, wherein the cells are efficiently developed into blastocysts without cell damage and/or stoppage of development, and in vitro fertilization procedures are performed. Can be used to produce somatically replicated fertilized eggs.

1A is a photograph showing the results of observing demethylation of H3K9me3 by injecting Kdm4a mRNA into the SCNT-FOC and SCNT-CROC groups.
1B is a graph showing the results of observing demethylation of H3K9me3 by injecting Kdm4a mRNA into the SCNT-FOC and SCNT-CROC groups.
Figure 2a is a picture confirming the change in the number of inner cells and total cells of the SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected.
Figure 2b is a picture confirming the degree of embryonic development of the SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected.
Figure 2c is a graph showing the blastocyst quality ratio of the SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected.
Figure 3a shows genes having different expression patterns in the two-cell embryos of the SCNT-FOC and SCNT-CROC groups depending on whether Kdm4a mRNA is injected.
3B shows genes whose expression is regulated (upward/downward) in 2-cell embryos of the SCNT-FOC and SCNT-CROC groups according to whether or not Kdm4a mRNA is injected.
FIG. 4A is a photograph confirming the level of DNA fragmentation in blastocysts of the 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 blastocysts of the SCNT-CROC and SCNT-CROC+K groups according to the presence or absence of melatonin treatment.
Figure 5a is a photograph confirming the fluorescence staining (apoptosis; green, nucleus; blue staining) of a part where apoptosis occurred in the SCNT-CROC+K group according to the presence or absence of melatonin treatment.
5B is a graph showing the level of active oxygen in the morula of the SCNT-CROC+K group according to the presence or absence of melatonin treatment.
Figure 6a is a picture visualizing whether implantation in the uterus of the mouse SCNT-CROC + K group according to the presence or absence of melatonin treatment.
Figure 6b is a picture visualizing the results of the experiment by repeating whether or not implanted in the uterus of the mouse 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.
7A is a result of confirming the mESC induction rate of the SCNT-CROC+K group with or without melatonin treatment with an optical microscope.
7B is a graph showing the mESC induction rate of the SCNT-CROC+K group according to the presence or absence of melatonin treatment.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are 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, 8-10 weeks old, were used as poster mothers for embryo transfer. In order 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 Testing and Use Committee (IACUC) (Project No. IACUC-170119) of the University of Medical Sciences, and all experiments were performed according to the approved protocol.

[Example]

Example 1. Collection of oocytes

5 IU of pregnant male 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 gonadotropin) was injected. gonadotropin, hCG; Sigma-Aldrich) was injected to induce hyperovulation. 14 hours after injection of hCG into mice, oocytes were collected in M2 (Sigma-Aldrich) medium, and cumulus cells were removed with a medium containing 0.1% hyaluronidase (Sigma-Aldrich). Denuded. Thereafter, for the experiment, cumulus-free oocytes were cultured in a 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 (vitrification) and thawing (warming)

Quinn' with Knockout Serum Replacement (KSR, Gibco, Grand Island, NY) 20% (v/v) and HEPES (Sage, Malov, Denmark) as basic medium for the preparation of vitrified freezing and warming solutions Advantage medium of was used. It was subjected to vitrification freeze treatment with two cryoprotectant agents, a combination of ethylene glycol (EG, Sigma-Aldrich) and dimethylsulfoxide (dimethylsulfoxide, DMSO, Sigma-Aldrich). 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 on electron microscopic copper grids (EM Grid, PELCO, Redding, CA) and slush nitrogen (SN2) using Vit-master (IMT, Ness Ziona, Israel) To be cryopreserved. Freeze-frozen oocytes were stored in an LN2 tank. For warming, the vitrified frozen oocytes were heated in a four-step method. The EM grid was sequentially transferred to 0.5, 0.25, 0.125 and 0 M sucrose at intervals of 2 minutes and 30 seconds at 37°C. Thereafter, the oocytes were washed three times with a modified HTF (Millipore) medium, and cultured in HTF medium until the start of the experiment.

Example 3. Somatic cell nuclear transfer (SCNT)

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

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

4-1. Kdm4a mRNA preparation

The 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 template plasmid linearized 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

The injection of Kdm4a, which encodes H3K9me3 demethylase, is known to improve oocyte development in somatic cell nucleus-transferred mammals. First, in order to analyze the effect of Kdm4a mRNA on the development of cloned oocytes (SCNT-CROC) using the cytoplasm of freeze-frozen/thawed oocytes, embryo development was evaluated regardless of whether Kdm4a mRNA was injected. Next, injecting ~10 pl of water (control) or 2 µg/µl Kdm4a mRNA into the two groups of cloned eggs (SCNT-FOC group and SCNT-CROC group) prepared in Example 3 using a piezo-driven micromanipulator. I 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. It was fixed for 30 minutes. After washing the oocytes with PBS/PVA, they were incubated overnight in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 at 4°C. Thereafter, oocytes were washed three times in PBS-0.1% BSA, and a 1:200 dilution of H3K9me3 antibody and purified mouse anti-OCT-3/4 (BD) were incubated for 2 hours at the 1st and 2nd cell steps. The cloned embryo was 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 times in PBS-0.1% BSA. DNA was visualized by staining oocytes with 4',6-diamidino-2-phenylindole (DAPI). Embryos were placed on a glass slide with a fluorescent mounting medium and observed using a fluorescence microscope (Zeiss LSM880, Zeiss, Jena, Germany).

group mRNA (μg/μl) Nuclear transplant oocyte count Number of 2-cell embryos (%) ¶ Number of 4-cell embryos (%) # Number of 2-cell block embryos (%±SEM) # Blastocyst Count (%±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 row, indicating a significant difference in values (P<0.05; n=5) SCNT-FOC: Somatic cell nuclear transfer cloned egg using the cytoplasm of fresh oocytes

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

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 a 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. to be. It was confirmed that demethylation of H3K9me3 was normally performed in the nuclei of the 1st cell and 2nd cell groups of the SCNT-FOC group and the SCNT-CROC group (FIG. 1A). In the 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 improves the rate of blastocyst formation in both the SCNT-FOC group and the SCNT-CROC group. Therefore, it can be confirmed that the injection of Kdm4a mRNA inhibits H3K9me3 activity and overcomes the 2-cell block in the cloned egg (Table 1).

Figure 2 shows the change in the number of inner cells and total cells of the SCNT-FOC and SCNT-CROC groups depending on whether Kdm4a mRNA is injected. At this time, the inner cell population means the number of cells that can be generated as embryonic stem cells in the future, type 1 indicates less than 10 inner cells, type 2 indicates more than 10 inner cells.

As shown in FIGS. 2A and 2B, after the injection of mRNA, the embryonic developmental defect in the SCNT-FOC group and the SCNT-CROC group was almost overcome, but the degree of embryonic development and embryonic development in the SCNT-CROC group was determined by the SCNT-FOC group. Was significantly lower than that of embryonic development and development in In particular, as shown in Fig. 2c, the number of cloned embryos with a high ratio of the number of ICMs (more than 10 ICMs per blastocyst) in the SCNT-CROC+ K group was significantly less than in the SCNT-FOC + K group (p<0.05). . That is, the injection of Kdm4a mRNA significantly increases the number of inner cell populations, thereby improving blastocyst quality.

4-3. Identification of differences in transcription of somatic cell nuclear transfer cells in 2-cell embryos

To confirm the difference in transcription of the earliest embryos between the SCNT-FOC group and the SCNT-CROC group, after 24 hours of oocyte activation and Kdm4a mRNA injection, two groups of pooled 2-cell embryos (100 embryos per sample, RNA sequencing was performed twice). In round 1, gene expression was assessed in 2-cell embryos between SCNT-FOC+K and SCNT-CROC+K (100 embryos per sample, repeat 3 times). At the second round, gene expression was assessed in blastocysts between SCNT-CROC+K and SCNT-CROC+K+M (50 blastocysts per sample, repeat twice). Complementary DNA (cDNA) was amplified according to the manufacturer's instructions using the SMARTer Ultra Low Input RNA cDNA preparation kit (Takara, 634890). The cDNA was fragmented into about 200 bp fragments using an M220 sonicator (Covaris). Fragmented cDNA was end-repaired and adapter-ligated. The sequencing library was prepared using the ScriptSeq v2 kit (Illumina) according to the manufacturer's instructions. Single-ended sequencing was performed on HiSeq2500 (Illumina), and mapped to the mm9 mouse genome using STAR (v2.5.2b, https://github.com/alexdobin/STAR). Thereafter, fragments per kilobase per million read (FPKM) was calculated with Cufflinks (v2.2.1) using the default option. The gene was 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). In GO terms and KEGG analysis, several pathways including apoptosis and p53 signaling pathway were remarkably activated. When DEG was analyzed with the GO terms-database, only 16 genes (0.92%) of 1,730 apoptosis-related genes showed a 3-fold difference between the SCNT-FOC group and the SCNT-CROC group. In the SCNT-CROC group, 8 pre-apoptotic genes (Cyt, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7, Pmaip) (50%) and 3 anti-apoptotic genes (Adrb2, Scin, Six1) were Compared to the SCNT-FOC group, it was confirmed that the expression was upregulated. 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 upregulated (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 genes (0.62%) out of 4,841 genes and 61 genes (0.60%) out of 10,234 genes related to metabolic processes are different from each other. there was. However, it can be seen that most of the genes do not function negatively in gene expression or metabolic processes (FIG. 3B).

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

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

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

group Melatonin (μM) Nuclear transplant oocyte count Number of 2-cell embryos (%) ¶ Number of 4-cell embryos (%) # Number of 2-cell block embryos (%±SEM) # Blastocyst Count (%±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 row, indicating a significant difference in values (P<0.05; n=5) SCNT-FOC: Somatic cell nuclear transfer cloned egg using the cytoplasm of fresh oocytes

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

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 embryonic development of the SCNT-CROC group regardless of the injection of Kdm4a mRNA (Table 2). That is, it can be seen that treatment with melatonin improves the rate of blastocyst development of the SCNT-CROC group independently of Kdm4a mRNA injection. In addition, it was confirmed that the SCNT-CROC group showed a similar blastocyst development rate as the SCNT-FOC group by increasing the blastocyst development efficiency, which could not be overcome only by injection of Kdm4a mRNA.

5-2. Confirmation of DNA fragmentation according to melatonin treatment

In the group of Example 5-1, the level of DNA fragmentation in blastocysts depending on whether or not melatonin was treated was detected using TUNEL staining ( in situ Cell Death Detection Kit, Roche, Indianapolis, IN). SCNT-derived blastocysts were washed 3 times in DPBS 0.1% PVA. Embryos were incubated in TUNEL reaction medium at 37° C. for 1 hour in dark conditions. DNA stained with 1 μg/ml Hoechst 33342 (Bis-benzimide, Sigma) was used for nuclear counter-staining. The signal of the embryo was observed with a confocal microscope (Zeiss, LSM880).

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

5-3. Effects of melatonin treatment on the production of reactive oxygen species in embryos

In the group of Example 5-1, the effect of melatonin treatment on free radicals in blastocysts was confirmed. Specifically, in the SCNT-CROC + K group, a morula or blastocyst stage embryo is treated with or without melatonin, and a culture medium containing 5 μM CellROX oxidative stress reagent (Molecular Probes, Eugene, OR) for 30 minutes After incubation in, it was treated with 0.1% polyvinyl alcohol (PVA)-D-PBS. Embryos were observed with a fluorescence confocal microscope (Zeiss, LSM880). The recorded fluorescence intensity was analyzed using ImageJ software. The fluorescence pixel values of the embryos were measured within a certain area from the cytoplasmic area of other embryos. Before analyzing the statistically significant difference between groups, the background fluorescence value was excluded from the final value. In each replicate, 5-10 embryos were replicated 3 times.

As a result, it was confirmed that treatment with melatonin significantly reduced the level of active oxygen in the morula. In addition, as a result of investigating the effect of melatonin treatment on the qualitative aspect of blastocysts through fluorescent staining (apoptosis; green, nucleus; blue staining) of the area where apoptosis occurred, the number of apoptosis stained in purple was significantly reduced. As shown, it was confirmed that the apoptosis rate was also significantly reduced (FIG. 5A). In addition, it was confirmed that the level of active oxygen 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, peroxisomes and oxidative phosphorylation, and thus qualitatively improved blastocysts can be obtained.

5-4. Confirmation of the effect of melatonin treatment on the transcription of blastocysts

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

group Melatonin (μM) Nuclear transplant oocyte count Number of 2-cell embryos (%) ¶ Number of 4-cell embryos (%) # Number of 2-cell block embryos (%±SEM) # Blastocyst Count (%±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 row, indicating a significant difference in values (P<0.05; n=5) SCNT-FOC: Somatic cell nuclear transfer cloned egg using the cytoplasm of fresh oocytes

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

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 embryonic development of the SCNT-CROC group regardless of the injection of Kdm4a mRNA (Table 2). That is, it can be seen that treatment with melatonin improves the rate of blastocyst development of the SCNT-CROC group independently of Kdm4a mRNA injection. In addition, it was confirmed that the SCNT-CROC group showed a similar blastocyst development rate as the SCNT-FOC group by increasing the blastocyst development efficiency, which could not be overcome only by injection of Kdm4a mRNA.

5-2. Confirmation of DNA fragmentation according to melatonin treatment

In the group of Example 5-1, the level of DNA fragmentation in blastocysts depending on whether or not melatonin was treated was detected using TUNEL staining ( in situ Cell Death Detection Kit, Roche, Indianapolis, IN). SCNT-derived blastocysts were washed 3 times in DPBS 0.1% PVA. Embryos were incubated in TUNEL reaction medium at 37° C. for 1 hour in dark conditions. DNA stained with 1 μg/ml Hoechst 33342 (Bis-benzimide, Sigma) was used for nuclear counter-staining. The signal of the embryo was observed with a confocal microscope (Zeiss, LSM880).

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

5-3. Effects of melatonin treatment on the production of reactive oxygen species in embryos

In the group of Example 5-1, the effect of melatonin treatment on free radicals in blastocysts was confirmed. Specifically, in the SCNT-CROC + K group, a morula or blastocyst stage embryo is treated with or without melatonin, and a culture medium containing 5 μM CellROX oxidative stress reagent (Molecular Probes, Eugene, OR) for 30 minutes After incubation in, it was treated with 0.1% polyvinyl alcohol (PVA)-D-PBS. Embryos were observed with a fluorescence confocal microscope (Zeiss, LSM880). The recorded fluorescence intensity was analyzed using ImageJ software. The fluorescence pixel values of the embryos were measured within a certain area from the cytoplasmic area of other embryos. Background fluorescence values were excluded from the final value before analyzing statistically significant differences between groups. In each replicate, 5-10 embryos were replicated 3 times.

As a result, it was confirmed that treatment with melatonin significantly reduced the level of active oxygen in the morula. In addition, as a result of investigating the effect of melatonin treatment on the qualitative aspect of blastocysts through fluorescent staining (apoptosis; green, nucleus; blue staining) of the area where apoptosis occurred, the number of apoptosis stained in purple was significantly reduced. As shown, it was confirmed that the apoptosis rate was also significantly reduced (FIG. 5A). In addition, it was confirmed that the level of active oxygen 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, peroxisomes and oxidative phosphorylation, and thus qualitatively improved blastocysts can be obtained.

5-4. Confirmation of the effect of melatonin treatment on the transcription of blastocysts

In order to determine which genes are regulated by melatonin, the transcripts of 2-cell embryos and blastocysts (50 embryos or blastocysts per sample, repeat 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 functions Genetic symbol Gene ID Gene name control Cell viability and
Tissue regeneration Deaf1 NM_001282072.1 DEAF1, transcription factor Condition 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 cell death 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 adaptor 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 Dnaaf1 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 a 2-cell embryo, it was found that 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 melatonin-treated group (SCNT-CROC+K). In particular, the Amd1, Fam46C, Oxt, and Ppt2 involved in cell survival and tissue was up-regulated at the 2-cell embryo of melatonin treatment group (SCNT-CROC + K + M ). In addition, Ctss , Dffa , Eif3f , Hacl1, Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 are associated with antioxidant function, inflammation and apoptosis. And Wbscr22 was upregulated to high levels. On the other hand, cell death and regression-related genes ( Atp6v0c , Cd52 , Dapk2, Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 And Rnd3 ) expression was down-regulated in the 2-cell embryo of SCNT-CROC+K+M).

In the case of blastocysts, 81 genes were found to be 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 melatonin-treated group (SCNT-CROC+K). Amd1 , Fam46C , Oxt , and Ppt2 , which are associated with cell survival and tissue regeneration, were upregulated in the blastocyst of the melatonin-treated group. In particular, Gulo related to antioxidant function, inflammation and apoptosis And Txnip expression was always regulated. 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 melatonin. It was down-regulated in blastocysts of treatment group (SCNT-CROC+K+M). In addition, the expression levels of cells and tumor proliferation-related genes ( Efemp1 , Glipr1 , Lgals4 , Plac1 , Snora15 , Snora 21, Snora 34, and Anxa1 ) were found to be decreased. In addition, some genes associated with the immune response ( Clec2f , Hrsp12 , and Plat ) were shown to be downregulated in the melatonin-treated group (SCNT-CROC+K+M) (Table 4).

Related functions Genetic symbol Gene ID Gene name control Cell viability 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 cell death 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 Lgals4 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 transfer and implantation monitoring

Reconstituted embryos cultured in the blastocyst stage under melatonin-treated or non-treated conditions (SCNT-CROC+K and SCNT-CROC+K+M groups) were mated with male ICR mice undergoing vasectomy for 2.5 days pseudo-pregnancy (pseudo- pregnant) female ICR mice were implanted in the womb. At this time, the SCNT-CROC+K group and the SCNT-CROC+K+M group were transplanted to the left and right sides of the uterus of the same mouse, respectively. The rate of embryo transfer in female mice was confirmed on day 7.5 after intercourse of the mice. Thereafter, the mice were euthanized, and the normal uterus was washed with saline, then excised and the attached fat was removed. The tissue from which the fat was removed was photographed to visualize the implantation (FIG. 6A, 6B).

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

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

In order to obtain outgrowths, hatched blastocysts obtained from two groups (SCNT-CROC+K and SCNT-CROC+K+M) were used to inactivate mitosis in mouse embryonic stem cell (mESC) culture medium. MEF) placed on 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 the mES cell culture medium. First, the outgrowth was mechanically transferred to MEF feeder cells, and then passaged using trypsin-EDTA. All established mECS lines were monitored and characterized by morphology and alkaline phosphatase staining. Alkaline phosphatase activity was evaluated by histochemical staining. Colonies were fixed with 4% paraformaldehyde at room temperature for 1 minute and 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) It was treated at room temperature for 30 minutes. Alkaline phosphatase activity was detected colorimetrically with an optical microscope (Fig. 7A).

As a result, the induction rate of mESC was significantly higher in the melatonin-treated group compared to the non-treated 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. Embryo development was analyzed by one-way variance analysis (ANOVA) by DUNCAN's assay using SAS software and transplantation, and ESC-derivation rates were analyzed by Chi-square test, p. <0.05 was considered statistically significant.

Claims (18)

Contains an agent that reduces melatonin and H3K9me3 methylation, and when the embryo is in a two-cell phase, Amd1 , Fam46C , Oxt and Ppt2 A composition for increasing the efficiency of embryonic development or formation of embryos, wherein the expression of any one or more genes selected from the group consisting of is increased, and the eggs forming the embryos are 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 demethylase. 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 formation of a blastocyst. The composition of claim 1, wherein the embryo is formed in vitro through artificial insemination. 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. Contacting an agent that reduces H3K9me3 methylation to the egg isolated from the body; And
A method of increasing the efficiency of embryonic development or formation of an embryo, comprising the step of culturing the egg in a medium containing melatonin, wherein the embryo is in a 2- celled state, consisting of Amd1 , Fam46C , Oxt , and Ppt2 . Which is chosen from the county Wherein the expression of one or more genes is increased, and the egg forming the embryo is frozen or cryopreserved and then thawed or thawed. The method according to claim 10, wherein when the embryo is in a two-cell phase, expression of any one or more genes selected from the group consisting of Ctss, Dffa, Eif3f, Hacl1, Hspbp1, Mob2, Mrpl23, Mrpl33, Pmvk, Slc4a11 and Wbscr22 is increased. How to become. The method of claim 10, wherein when the embryo is in a two-cell state, expression of any one or more genes selected from the group consisting of Atp6v0c, Cd52, Dapk2, Ddit4l, Duoxa2, Nfkbia, Ptgds, Rdh12 and Rnd3 is reduced. . The method of claim 10, wherein when the embryo is developed into a blastocyst, expression of any one or more genes selected from the group consisting of Amd1 , Fam46C , Oxt, Ppt2, Gulo, and Txnip is increased. The method of claim 10, wherein when the embryo is developed as a blastocyst, 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 , Hrsp12, and the method that the expression of any one or more genes selected from the group consisting of Plat is reduced. The method of claim 10, further comprising developing an embryo obtained in the culturing step into an individual. An embryo prepared by the method of claim 10, wherein the embryo has increased expression of one or more genes selected from the group consisting of Ctss and Dffa . A blastocyst produced by the method of claim 10, wherein the blastocyst is a blastocyst in which the expression of any one or more genes selected from the group consisting of Clec2f , Hrsp12, and Plat is reduced. Embryonic stem cells produced by the method of claim 10.

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