Composition for promoting DNA demethylation in vitro, method for producing embryo, embryo, and embryo-receiving livestock animal

Abstract

To provide a composition and the like capable of promoting DNA demethylation in embryos.SOLUTION: Provided is a composition for promoting DNA demethylation in embryos in vitro, the composition comprising miRNA.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a composition for promoting DNA demethylation in vitro, a method for producing an embryo, an embryo, and an embryo recipient livestock animal. [Background technology]

[0002] Embryo transfer is widely used in Wagyu cattle production and human reproductive medicine. Non-Patent Document 1 reports that embryos developed outside the body (ex vivo embryos) have highly methylated DNA in early cleavage embryos compared to embryos developed inside the body (in vivo embryos). High DNA methylation in ex vivo embryos has also been confirmed in cattle. In cattle, epigenetic abnormalities in offspring derived from ex vivo embryos (oversized offspring due to methylation abnormalities) are a major problem with embryo transfer, and health risks to offspring derived from ex vivo embryos have also been reported in humans (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Epigenetics 2011;6:177-87. [Non-Patent Document 2] Hum Reprod. 2010 Mar;25(3):605-12. Summary of the Invention [Problem to be solved by the invention]

[0004] DNA in sperm and eggs is highly methylated at the time of fertilization, and undergoes rapid demethylation after fertilization. This process is called reprogramming, and is an important step in subsequent embryonic development. This process is thought to be slower in in vitro embryos. Therefore, creating a culture environment that promotes DNA demethylation after fertilization may be able to intervene not only in the development of fertilized eggs and embryos, but also in the lifespan and prognosis of offspring.

[0005] An object of the present invention is to provide a composition capable of promoting DNA demethylation in an embryo. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and as a result have found that a composition containing miRNA can promote DNA demethylation in an embryo, thereby completing the present invention.

[0007] That is, the present invention is as follows. [1] A composition for promoting DNA demethylation in vitro in an embryo, comprising miRNA. [2] The composition described in [1], wherein the miRNA is at least one selected from the group consisting of miR-199a, miR-29b, and miR-148a, and mimics thereof. [3] The composition described in [2], wherein the miR-199a is miR-199a-3p. [4] The composition according to any one of [1] to [3], further comprising TGFβ1. [5] The composition according to any one of [1] to [4], wherein the composition contains follicular fluid or extracellular vesicles derived from follicular fluid. [6] The composition according to any one of [1] to [5], for culturing a fertilized egg or embryo within 48 hours after fertilization treatment. [7] The composition according to any one of [1] to [6], which suppresses expression of DNA methyltransferase in the embryo. [8] The composition described in [7], wherein the DNA methyltransferase is at least one of DNMT1 and DNMT3. [9] The composition according to any one of [1] to [8], which promotes development of the embryo.

[10] The composition according to any one of [1] to [9], wherein the embryo is a bovine embryo.

[11] [1] to

[10] , comprising culturing a fertilized egg or embryo in vitro in the presence of the composition. A method for producing embryos with enhanced DNA demethylation.

[12]

[11] An embryo produced by the method described in

[11] .

[13]

[12] A recipient livestock animal having an embryo according to the above. Effect of the Invention

[0008] According to the present invention, it is possible to provide a composition capable of promoting DNA demethylation in an embryo. [Brief description of the drawings]

[0009] [Figure 1] The experimental schedule is as follows: Follicular fluid-derived components were added to the culture medium from the pronuclear stage embryos 18 hours after fertilization until 48 hours later, which is the time of genome activation. After that, the embryos were allowed to develop into blastocyst stage embryos in a normal medium. [Diagram 2] The DNA methylation levels in 8-cell embryos are shown. A: Condition 1 (follicular fluid (FF) added), B: Condition 2 (EV added), C: Condition 3 (miRNA added), D: Condition 4 (TGFβ1 added). * indicates significant difference. P<0.05 [Diagram 3] The expression level of methyltransferase after addition of follicular fluid and the DNA methylation level in blastocyst-stage embryos are shown. AB: Effect of follicular fluid on the expression level of methyltransferase in 8-cell stage embryos. C: DNA methylation status of blastocyst-stage embryos obtained under condition 1 (addition of follicular fluid). * indicates a significant difference. P<0.05 [Figure 4] The effect of miRNA is shown. A: DNMT3A expression level after addition of miRNA, B: cleavage rate up to 8-cell stage embryos, C: development rate up to blastocyst stage embryos. * indicates significant difference. P<0.05 [Diagram 5]The effect of miRNA (miR-199a-3p and miR-29b) concentrations in follicular fluid on DNA methylation levels in 8-cell embryos is shown. A: Summary of the measurement method, B: Measurement results. * indicates significant difference. P<0.05 [Figure 6] The effect of TGFβ1 concentration in follicular fluid on DNA methylation levels in 8-cell embryos and on the development rate to blastocysts is shown. A: Summary of the measurement method, B: Measurement results, C: Development rate at the time of addition. * indicates a significant difference. P<0.05 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Below, we will explain in detail the form for implementing the present invention (hereinafter referred to as the "present embodiment"). However, the present invention is not limited to this, and various modifications are possible without departing from the gist of the present invention.

[0011] In vivo, a large amount of follicular fluid flows into the fallopian tube, which is the environment for egg fertilization and embryo development, at the time of ovulation. The present inventors hypothesized that a small amount of remaining follicular fluid causes important changes in early embryos, and considered that factors that support such changes exist in the components of follicular fluid. As shown in the examples below, the present inventors found that follicular fluid and the components contained in follicular fluid promote DNA demethylation in embryos. This led to the discovery that a composition containing miRNA can promote DNA demethylation in embryos in vitro.

[0012] Thus, one aspect of the present invention relates to a composition for promoting DNA demethylation in an embryo in vitro. One aspect of the invention relates to a method for promoting DNA demethylation in an embryo. One aspect of the present invention relates to a method for producing an embryo with enhanced DNA demethylation. One aspect of the present invention relates to embryos in which DNA demethylation has been enhanced. One aspect of the present invention relates to an embryo-recipient livestock animal having an embryo in which DNA demethylation has been promoted. One aspect of the invention relates to the use of miRNAs to promote DNA demethylation in embryos in vitro. One aspect of the invention relates to the use of miRNA for use in the preparation of a composition for promoting DNA demethylation in vitro in an embryo.

[0013] [Composition] The composition of the present embodiment contains miRNA and is used to promote DNA demethylation in embryos in vitro. The composition of the present embodiment may be used to produce "Nodai fertilized eggs".

[0014] miRNA is a microRNA, which is a single-stranded or double-stranded RNA having the function of suppressing gene expression. In the present invention, miRNA may be a precursor miRNA or a mature miRNA, but is preferably a mature miRNA.

[0015] The length (base length) of the base sequence in mature miRNA is not particularly limited, but is preferably 30 bases or less, more preferably 17 to 27 bases, and even more preferably 21 to 25 bases. The length (base length) of the base sequence in the precursor miRNA is not particularly limited, but is preferably 120 bases or less, more preferably 50 to 100 bases, and even more preferably 60 to 80 bases. The length of the base sequence in miRNA (base length) is not particularly limited, but is preferably 120 bases or less, more preferably 17 to 100 bases, and even more preferably 21 to 80 bases.

[0016] In the present specification, "miRNA" includes not only natural miRNA but also miRNA mimics artificially synthesized or modified to mimic natural miRNA. A miRNA mimic is a molecule having similar or identical activity to a miRNA molecule. Similar activity means a level acceptable for realizing the use of the present embodiment. Examples of miRNA mimics include synthetic oligonucleotides containing one or more nucleotide analogs, such as locked nucleic acid monomers, and nucleotides containing scaffold modifications and / or nucleotides containing base modifications. The miRNA mimic is preferably a double-stranded mimic.

[0017] The miRNA mimic may be, for example, a double-stranded oligonucleotide comprising a passenger strand and a guide strand.Since natural miRNA is complementary to the sense sequence of the target, the miRNA mimic may have a guide strand with the same sequence as natural miRNA or a fragment thereof.The miRNA mimic may have a passenger strand that is at least partially reverse-complementary to the guide strand and can form a double-stranded mimic.The passenger strand may have the function of stabilizing the guide strand, preventing the degradation of the double strand, and / or facilitating the delivery of the double strand.

[0018] As described above, the present invention was made by finding that follicular fluid and the components contained in follicular fluid promote DNA demethylation in embryos.Therefore, the miRNA is preferably the miRNA contained in follicular fluid or the miRNA extracted from follicular fluid, and more preferably the miRNA contained in extracellular vesicles contained in follicular fluid or the miRNA extracted from extracellular vesicles contained in follicular fluid.The miRNA and follicular fluid may be derived from the same animal as the embryo to which the composition of the present embodiment is applied.The miRNA and follicular fluid may be derived from humans, cows, pigs, rats, mice, rabbits, sheep, cats, dogs, monkeys, etc.

[0019] The miRNA is preferably at least one selected from the group consisting of, for example, miR-199a, miR-29b, and miR-148a, and mimics thereof. That is, the composition of the present embodiment preferably contains miR-199a, miR-29b, miR-148a, or mimics thereof. Here, miR-199a is preferably miR-199a-3p. miR-199a, miR-199a-3p, miR-148a, and miR-29b may be derived from humans, cows, pigs, rats, mice, rabbits, sheep, cats, dogs, monkeys, etc. miR-199a-3p may have, for example, the amino acid sequence of SEQ ID NO: 1. miR-29b may have, for example, the amino acid sequence of SEQ ID NO: 2. miR-148a may have, for example, the amino acid sequence of SEQ ID NO: 3.

[0020] [Table 1]

[0021] The miR-199a mimic may have (1) a guide strand having the same nucleotide sequence as the guide strand of miR-199a, (2) a guide strand having a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the guide strand of miR-199a, or (3) a guide strand having a nucleotide sequence in which 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides are added, deleted, and / or substituted relative to the guide strand of miR-199a. The miR-199a mimic may have a passenger strand that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% complementary to any of the above guide strands.

[0022] The miR-199a-3p mimic may have (1) a guide strand having the same nucleotide sequence as the guide strand of miR-199a-3p (SEQ ID NO: 1), (2) a guide strand having a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the guide strand of miR-199a-3p, or (3) a guide strand having a nucleotide sequence in which 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides are added, deleted, and / or substituted relative to the guide strand of miR-199a-3p. The miR-199a-3p mimic may have a passenger strand that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% complementary to any of the above guide strands.

[0023] The miR-29b mimic may have (1) a guide strand having the same nucleotide sequence as the guide strand of miR-29b (SEQ ID NO: 2), (2) a guide strand having a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the guide strand of miR-29b, or (3) a guide strand having a nucleotide sequence in which 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides are added, deleted, and / or substituted relative to the guide strand of miR-29b. The miR-29b mimic may have a passenger strand that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% complementary to any of the above guide strands.

[0024] The miR-148a mimic may have (1) a guide strand having the same nucleotide sequence as the miR-148a guide strand (SEQ ID NO: 3), (2) a guide strand having a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the miR-148a guide strand, or (3) a guide strand having a nucleotide sequence in which 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides are added, deleted, and / or substituted relative to the miR-148a guide strand. The miR-148a mimic may have a passenger strand that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% complementary to any of the above guide strands.

[0025] As the miR-199a, miR-199a-3p, miR-148a, and miR-29b mimics, commercially available miR-199a, miR-199a-3p, miR-148a, and miR-29b mimics may be used, respectively.

[0026] In the composition of this embodiment, miRNA (particularly, miR-199a (including miR-199a-3p), miR-148a, and miR-29b, and mimics thereof) may suppress the expression of DNA methyltransferase in an embryo. That is, miRNA (particularly, miR-199a (including miR-199a-3p), miR-148a, and miR-29b, and mimics thereof) may suppress the expression of DNA methyltransferase-related genes in an embryo. The DNA methyltransferase may be at least one of DNMT1 and DNMT3. The suppression of the expression may be suppressed so that the expression level of DNA methyltransferase is 95%, 90%, 85%, 80%, or 75% or less compared to the case without application of miRNA.

[0027] The composition of the present embodiment may contain components other than miRNA, so long as they do not inhibit the effects of the present invention. Examples of such components include components that can be contained in culture fluids such as synthetic oviductal fluid (SOF) that can be used in the culture of fertilized eggs or embryos. The components other than miRNA may be added in the form of synthetic oviductal fluid. In addition, the composition of the present embodiment may contain components contained in follicular fluid as components other than miRNA.

[0028] The composition of the present embodiment preferably contains TGFβ1 as a component contained in the follicular fluid other than miRNA. TGFβ1 is transforming growth factor-β1, a cytokine belonging to the TGF-β family. TGFβ1 may be an active form that binds to a TGF-β receptor, or a latent form in which active TGFβ1 is bound to LTBP (latent TGF-β binding proteins) and LAP (latency-associated peptide), but the active form is preferred. TGFβ1 may be derived from the same animal as the embryo to which the composition of the present embodiment is applied. TGFβ1 may be derived from humans, cows, pigs, rats, mice, rabbits, sheep, cats, dogs, monkeys, or the like. TGFβ1 may have, for example, the amino acid sequence of SEQ ID NO: 4 of human-derived TGFβ1, or an amino acid sequence having 80% or more, 85% or more, 90% or more, or 94% or more sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0029] [ka]

[0030] The composition of the present embodiment may contain follicular fluid. The composition of the present embodiment may be follicular fluid collected from an animal. The composition of the present embodiment may be a composition in which follicular fluid collected from an animal is added to synthetic oviductal fluid. The composition of the present embodiment may be a composition in which a specific component is removed from follicular fluid collected from an animal. When the composition of the present embodiment contains follicular fluid, the follicular fluid may contain miRNA, or the composition may contain miRNA separately from the follicular fluid. The animal from which the follicular fluid is collected is preferably an animal from which a fertilized egg or an embryo is derived, but follicular fluid collected from a different animal species may be used.

[0031] The composition of the present embodiment may contain extracellular vesicles contained in follicular fluid, i.e., extracellular vesicles derived from follicular fluid. The composition of the present embodiment may be a composition in which extracellular vesicles extracted from follicular fluid collected from an animal are added to synthetic oviductal fluid. In the present invention, the extracellular vesicles may be exosomes. Extraction of extracellular vesicles may be performed using a commercially available kit for extracting extracellular vesicles. An example of such a kit is ExoQuick manufactured by System Biosciences (SBI).

[0032] Components that can be contained in a culture medium that can be used in culturing a fertilized egg or an embryo include, for example, inorganic salts, lactate, pyruvate, amino acids (e.g., glutamine, glycine, taurine, etc.), glucose, streptomycin, penicillin, glucose, phenol red, etc. Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium hydrogen phosphate, sodium bicarbonate, etc.

[0033] The concentration of miRNA in the composition of this embodiment may be, for example, 5.0 nM or more, and is preferably 10 nM or more, 30 nM or more, 40 nM or more, 50 nM or more, or 60 nM or more. In the above range, the concentration of miRNA is preferably 200 nM or less, 150 nM or less, 100 nM or less, 90 nM or less, or 80 nM or less.

[0034] The concentration of TGFβ1 in the composition of this embodiment may be, for example, 0.01 ng / mL or more, 0.1 ng / mL or more, or 1 ng / mL or more, preferably 3 ng / mL or more, 5 ng / mL or more, 8 ng / mL or more, or 10 ng / mL or more. In the above range, the concentration of TGFβ1 is preferably 50 ng / mL or less, 40 ng / mL or less, 30 ng / mL or less, 25 ng / mL or less, or 20 ng / mL or less.

[0035] The concentrations of components other than miRNA and TGFβ1 in the composition of this embodiment are not particularly limited and may be similar to those of synthetic oviductal fluid or follicular fluid.

[0036] The composition of the present embodiment is a composition for promoting DNA demethylation in an embryo in vitro, and is distinguished from a composition for promoting DNA demethylation in an embryo in vivo. The composition of the present embodiment may be an artificially prepared composition. The artificially prepared composition also includes a composition produced in vivo to which a specific component has been added, and a composition produced in vivo to which a specific component has been removed.

[0037] The composition of the present embodiment is used to promote DNA demethylation in an embryo, in other words, the composition of the present embodiment is used to obtain an embryo in which DNA demethylation is promoted compared to an embryo obtained without application of the composition of the present embodiment. The composition of the present embodiment is used to produce an embryo in which DNA demethylation is promoted in vitro. The embryo may have promoted DNA demethylation in a state ranging from an 8-cell stage embryo to a blastocyst stage embryo.

[0038] In the present specification, a fertilized egg refers to a cell in which a sperm has entered the egg and fusion of a male pronucleus and a female pronucleus has been completed. In this embodiment, the fertilized egg may be obtained by in vitro fertilization, and the fertilized egg obtained by in vitro fertilization may be frozen and stored, and then thawed for use.

[0039] As used herein, an embryo refers to a fertilized egg that has begun cell division after fertilization. In the present invention, an embryo may be an embryo up to the blastocyst stage, for example, a cleavage stage embryo, a morula, or a blastocyst stage embryo.

[0040] The composition of the present embodiment promotes DNA demethylation in an embryo in vitro, but it is not necessarily required to apply the composition in the state of an embryo. The composition may be applied in the state of a fertilized egg, and DNA demethylation in the resulting embryo may be promoted. That is, the composition of the present embodiment may be applied to a fertilized egg or an embryo, and DNA demethylation in the resulting embryo is promoted. The composition of the present embodiment may be applied, for example, until the fertilized egg becomes a blastocyst stage embryo, or until the fertilized egg leaves the blastocyst stage embryo. The composition of the present embodiment may be applied, for example, until the fertilized egg becomes an 8-cell stage embryo. The composition of the present embodiment may be applied, for example, to a fertilized egg or an embryo within 48 hours (for example, 1 to 48 hours, 3 to 40 hours, 5 to 30 hours, 9 to 28 hours, or 12 to 25 hours) after fertilization treatment.

[0041] The composition of the present embodiment may be a culture medium for culturing a fertilized egg or an embryo. When a fertilized egg is cultured using the composition of the present embodiment as a culture medium, demethylation of DNA is promoted in an embryo developed from the fertilized egg. When an embryo is cultured using the composition of the present embodiment as a culture medium, demethylation of DNA is promoted in the embryo. The composition of the present embodiment may be a culture medium for culturing a fertilized egg or an embryo until the fertilized egg becomes a blastocyst stage embryo or until the fertilized egg leaves the blastocyst stage embryo. The composition of the present embodiment may be a culture medium for culturing a fertilized egg or an embryo until the fertilized egg becomes an 8-cell stage embryo, for example. The composition of the present embodiment may be used as a culture medium for culturing within 48 hours (for example, 1 hour to 48 hours, 3 hours to 40 hours, 5 hours to 30 hours, 9 hours to 28 hours, or 12 hours to 25 hours) after fertilization treatment.

[0042] The embryos in which demethylation is promoted by the composition of the present embodiment may be human embryos or non-human embryos, such as cows, pigs, rats, mice, rabbits, sheep, cats, dogs, monkeys, etc. The embryos may be livestock animals, particularly cows. The promotion of demethylation by the composition of this embodiment does not include demethylation that occurs in the human body. In addition, biological components that promote DNA demethylation in embryos in vivo, including miRNA, are distinguished from the composition of this embodiment.

[0043] The composition of the present embodiment is used to promote DNA demethylation in an embryo, and may further increase the cell number of a blastocyst stage embryo during the process of embryo development, promote embryo development, and / or suppress the expression of DNA methyltransferases (e.g., at least one of DNMT1 and DNMT3) in the embryo.

[0044] [method] The method for producing an embryo with promoted DNA demethylation according to this embodiment (hereinafter also simply referred to as the "method for producing an embryo according to this embodiment") comprises culturing a fertilized egg or embryo in vitro in the presence of the composition according to this embodiment (hereinafter also simply referred to as the "culture step"). The composition according to this embodiment may be in any of the forms described above. The method for producing an embryo according to this embodiment may also be referred to as the "Nature University Embryo Production Method", and the embryo obtained thereby may be referred to as the "Nature University Fertilized Egg".

[0045] The above culture may be culture of a fertilized egg or embryo until the fertilized egg becomes a blastocyst stage embryo or until the fertilized egg leaves the blastocyst stage embryo, and is preferably culture of a fertilized egg or embryo until the fertilized egg becomes an 8-cell stage embryo. The above culture may be culture of a fertilized egg or embryo within 48 hours after fertilization (e.g., 1 to 48 hours, 3 to 40 hours, 5 to 30 hours, 9 to 28 hours, or 12 to 25 hours). The above culture may be started from an unfertilized egg after fertilization, or may be performed by selecting a fertilized egg that has been confirmed to be fertilized. The above culture may be started within 30 hours after fertilization (e.g., 0 to 30 hours, 1 to 28 hours, 5 to 25 hours, 9 to 23 hours, or 12 to 20 hours).

[0046] The culture time in the above culture step may be, for example, until the embryo becomes a blastocyst stage embryo or until the embryo leaves the blastocyst stage, but is preferably until the fertilized egg becomes an 8-cell stage embryo. The culture time may be, for example, from 10 hours to 60 hours, from 15 hours to 50 hours, from 20 hours to 40 hours, or from 25 hours to 35 hours.

[0047] The method for producing an embryo of this embodiment may include another step before or after the above-mentioned culture step. For example, the method for producing an embryo of this embodiment may include a fertilization treatment step of contacting an egg with sperm in vitro prior to the above-mentioned culture step. The fertilization treatment step may be a step of contacting an egg with sperm in vitro to produce a fertilized egg, or a step of producing a fertilized egg by in vitro fertilization using sperm and an egg. The fertilization treatment step may be performed in the presence of the composition of this embodiment, or in the absence of the composition of this embodiment.

[0048] The method for producing an embryo according to the present embodiment may include a selection step of selecting fertilized eggs that have been confirmed to be fertilized after the fertilization treatment step. The selection step may be a step of selecting fertilized eggs by, for example, confirming fertilized eggs using a microscope or the like. The fertilized eggs selected by the selection step may be subjected to the above-mentioned culture step.

[0049] The method for producing an embryo of this embodiment may include a step of further culturing in the absence of the composition of this embodiment after the above-mentioned culturing step. When culturing in vitro, the culture medium in such culture may be a culture medium usually used for embryo development. Alternatively, the embryo may be cultured for a certain period of time in the oviduct of an animal. Such culture may be until the embryo becomes a blastocyst stage embryo, or until the embryo leaves the blastocyst stage embryo.

[0050] The method for promoting DNA demethylation in an embryo of the present embodiment includes culturing a fertilized egg or embryo in vitro in the presence of the composition of the present embodiment. The composition of the present embodiment may be in any of the above-mentioned forms. The culturing in such a method may be the same as the culturing step in the method for producing an embryo of the present embodiment.

[0051] [Embryos and livestock recipients] The embryo of this embodiment is produced by the above-mentioned embryo production method of this embodiment, and has promoted DNA demethylation. The embryo of this embodiment has promoted DNA demethylation compared to embryos produced by methods other than the embryo production method of this embodiment. The embryo of this embodiment has promoted DNA demethylation compared to embryos produced by in vitro fertilization (excluding embryos produced by the embryo production method of this embodiment). The embryo of this embodiment may be in a frozen state. The embryo of this embodiment may be an embryo from the 8-cell stage to the blastocyst stage. The embryo of this embodiment can be said to be an "Agricultural University fertilized egg".

[0052] By implanting the embryo of this embodiment into a recipient animal, an animal derived from the embryo can be produced. The produced animal is derived from an embryo in which DNA demethylation has been promoted, and therefore may be an animal with a reduced probability of developing epigenetic abnormalities (e.g., oversized offspring due to methylation abnormalities). The recipient animal may be a livestock animal, such as a cow or a pig, or may be a cow.

[0053] The embryo-recipient livestock animal of this embodiment is produced by the above-mentioned method for producing an embryo of this embodiment, and has an embryo in which DNA demethylation has been promoted. The embryo-recipient livestock animal of this embodiment can be produced by implanting an embryo produced as described above into an embryo-recipient animal. An animal in which DNA demethylation has been promoted can be produced by using the embryo-recipient livestock animal of this embodiment. The implantation of the embryo may be performed when the embryo is at the blastocyst stage. The embryo-recipient livestock animal may be a cow or a pig, and may be a cow. EXAMPLES

[0054] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to the following examples.

[0055] First, as previously reported (T. Noguchi et al., J Reprod Dev. 2020;66:35-39), RNA-seq analysis was performed on bovine in vivo and in vitro embryos to examine differentially expressed genes. Using Ingenuity Pathway Analysis, upstream factors of in vivo development were identified from differentially expressed genes. Among the identified miRNAs, miR-199a-3p, miR-29b, and miR-148a were selected as miRNAs present at high concentrations in oviductal and follicular fluids.

[0056] Next, as shown in Figure 1, in vitro fertilized bovine pronuclear embryos (18 hours after fertilization) were transferred to culture media under any of the following conditions 1 to 4, and cultured for 30 hours until they reached the 8-cell stage, which is the time of genome activation. After that, they were cultured using a conventional culture method (for 5 days in synthetic oviductal fluid without the addition of any of the following follicular fluid-derived components) until they reached the blastocyst stage.

[0057] Condition 1: Synthetic oviductal fluid supplemented with 1% follicular fluid Condition 2: Synthetic oviductal fluid to which extracellular vesicles (EVs) extracted from follicular fluid were added so that the amount of EVs was the same as when 1% follicular fluid was added. Condition 3: Synthetic oviductal fluid supplemented with miRNA (miR-199a-3p or miR-29b) (60 nM) extracted from EVs Condition 4: Synthetic oviductal fluid supplemented with a selected growth factor (TGFβ1) (10 ng / mL) as follows: The composition of the synthetic oviductal fluid was that described in Theriogenology. 1992 May;37(5):963-78. Human-derived TGFβ1 (Fujifilm Wako Pure Chemical Industries, Ltd.) having the amino acid sequence of SEQ ID NO: 4 was used as TGFβ1, and double-stranded miRNA mimics (ThermoFisher, MC11779 and MC10103) containing guide strands having the base sequences of SEQ ID NO: 1 and 2, respectively, were used as miR-199a-3p and miR-29b. The miRNA was added to pronuclear embryos from which the zona pellucida had been removed using Lipofectamine 2000 (ThermoFisher, Inc.).

[0058] The follicular fluid was collected from bovine antral follicles with a diameter of 3-6 mm. EVs were collected from follicular fluid using ExoQuick (System Biosciences (SBI)). The selection of growth factors was carried out as follows. RNA-seq analysis was performed on 8-cell embryos produced under condition 1 above and 8-cell embryos produced without the addition of follicular fluid to examine genes with altered expression. TGF-β1 was selected as a growth factor that was presumed to be an upstream factor on the follicular fluid-added side and was present in high concentrations in follicular fluid.

[0059] As a result, the DNA methylation status of 8-cell embryos was significantly decreased under all conditions, including the addition of follicular fluid, EVs, miRNAs, and TGFβ1 (Fig. 2A-D). In addition, detailed analysis of embryos in condition 1, in which follicular fluid was added, revealed that the expression levels of DNMT1 and DNMT3 were significantly decreased (Fig. 3A-B). Furthermore, the DNA methylation status of blastocysts obtained by culturing 8-cell embryos obtained under condition 1, in which follicular fluid was added, was also significantly decreased (Fig. 3C). miR-199a-3p and miR-29b significantly decreased the expression of DNMT3A (Fig. 4A), and were also found to promote embryo development (Fig. 4B-C).

[0060] The DNA methylation status was measured by immunostaining. Anti-5-methylcytosine (5-mC) antibody (D3S2Z, Cell Signaling, Danvers, MA, USA) was used. DNMT1 and DNMT3A were also measured by immunostaining. Anti-DNMT1 antibody (60B1220.1, NOVUS, USA) and anti-DNMT3A antibody (AI12955, ABCEPTA, USA) were used. The following secondary antibody was used: anti-rabbit IgG (H+L), 1:500, F(ab')2 fragment (Alexa Fluor 488 conjugate), Cat# 4412. Then, the sections were observed under a fluorescent microscope (DMI 6000 B; Leica, Wetzlar, Germany), and the images were quantified and analyzed using ImageJ software.

[0061] It was also confirmed that follicular fluid had a similar demethylation effect in pig embryos.Furthermore, a significant decrease in DNA methylation was confirmed when a miR-148a mimic (a double-stranded miRNA mimic containing a guide strand having the base sequence of SEQ ID NO:3 (manufactured by ThermoFisher)) was added at 60 nM as miRNA.

[0062] Next, follicular fluid was collected from multiple individuals (30 cattle) and extracellular vesicles (EVs) were extracted using ExoQuick (System Biosciences (SBI)). miRNA was extracted from the extracellular vesicles and the concentrations of miR-199a-3p and miR-29b were measured using Mir-X miRNA RT-PCR Kit (Takara). After the measurement, as shown in Figure 5A, synthetic oviductal fluid containing 1% follicular fluid collected from individuals with relatively high concentrations of miR-199a-3p and miR-29b, or follicular fluid collected from individuals with relatively low concentrations of miR-199a-3p and miR-29b, was used to culture in vitro fertilized cattle pronuclear embryos (18 hours after fertilization) for 30 hours until they reached the 8-cell stage, which is the time of genome activation.

[0063] As a result, when follicular fluid containing high concentrations of miRNAs (miR-199a-3p and miR-29b) was used, the DNA methylation status in 8-cell embryos was significantly reduced (Figure 5B).

[0064] Next, follicular fluid was collected from multiple cows (10 cows) in the same manner, and the concentration of TGFβ1 was measured using the Legend Max Elisa Kit (BioLegend). After the measurement, as shown in Figure 6A, follicular fluid collected from three cows with relatively high TGFβ1 concentrations (average concentration 33 ng / mL) or synthetic oviductal fluid containing 1% follicular fluid collected from three cows with relatively low TGFβ1 concentrations (average concentration 9 ng / mL) was used to culture in vitro fertilized cow pronuclear embryos (18 hours after fertilization) for 30 hours until they reached the 8-cell stage, which is the time of genome activation.

[0065] As a result, when follicular fluid with a high concentration of TGFβ1 was used, the DNA methylation status of 8-cell embryos was significantly decreased (Figure 6B), and the development rate of blastocysts was also significantly improved when TGFβ1 was added (Figure 6C).

Claims

1. A composition for promoting DNA demethylation in an embryo in vitro, comprising miRNA.

2. 2. The composition of claim 1, wherein the miRNA is at least one selected from the group consisting of miR-199a, miR-29b, and miR-148a, and mimics thereof.

3. The composition of claim 2, wherein the miR-199a is miR-199a-3p.

4. The composition of claim 1 further comprising TGFβ1.

5. The composition of claim 1 , wherein the composition comprises follicular fluid or extracellular vesicles derived from follicular fluid.

6. The composition according to claim 1 for culturing a fertilized egg or embryo within 48 hours after fertilization.

7. The composition of claim 1 , which suppresses expression of DNA methyltransferase in the embryo.

8. The composition according to claim 7 , wherein the DNA methyltransferase is at least one of DNMT1 and DNMT3.

9. The composition of claim 1 , which promotes development of the embryo.

10. The composition of claim 1 , wherein the embryo is a bovine embryo.

11. The method comprises culturing a fertilized egg or embryo in vitro in the presence of a composition according to any one of claims 1 to 10. A method for producing embryos with enhanced DNA demethylation.

12. An embryo produced by the method of claim 11.

13. A recipient livestock animal having an embryo according to claim 12.

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