Compositions, methods for regulating DNA methylation modification in vitro and their application in improving embryonic development efficiency and quality.

Abstract

This invention discloses compositions, methods, and applications for regulating DNA methylation modification in in vitro embryos, and their use in improving embryo development efficiency and quality. Addressing the problems of low embryo development rate and low subsequent developmental potential caused by abnormal epigenetic modifications in in vitro embryos, this invention provides compositions comprising two or more regulators selected from vitamin C or its precursors, RELN, and FGF2. These compositions can effectively correct DNA methylation, thereby significantly improving the development rate and quality of in vitro fertilized embryos, providing an effective strategy for upgrading the livestock industry.

Description

[0001] This application is a divisional application filed on June 27, 2024, with application number CN202410845339.5, entitled "Compositions, methods for regulating in vitro embryonic DNA methylation modification and their application in improving embryonic development efficiency and quality", which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention belongs to the field of biotechnology, and in particular relates to the combined application of vitamin C, RELN and FGF2 to improve the developmental efficiency and quality of embryos obtained by in vitro fertilization. Background Technology

[0003] In vitro embryo production (IVF) technology for mammals has been widely applied in livestock genetic improvement, breeding, and human assisted reproduction. However, low developmental efficiency and poor embryo quality remain significant technical bottlenecks that have been difficult to overcome. It is generally believed that the difference between the in vitro culture environment and the in vivo developmental environment, particularly the imperfect composition of the in vitro embryo culture medium leading to abnormal epigenetic modifications, is a major reason for the low developmental potential and poor quality of in vitro embryos, severely limiting the development of IVF technology. Therefore, optimizing the composition of the in vitro embryo culture medium to correct abnormal epigenetic modifications is currently the most direct and effective strategy to improve the developmental efficiency and quality of in vitro embryos and perfect IVF technology.

[0004] Early embryonic development involves dramatic changes in DNA methylation epigenetic modifications, including the erasure (active demethylation) and establishment (de novo methylation) of DNA methylation. Current evidence from gene knockout clearly indicates that abnormalities in both DNA demethylation and de novo methylation in early embryos can lead to developmental abnormalities or low developmental potential later in life. However, extensive research evidence shows that abnormal DNA methylation modifications are prevalent in in vitro fertilized embryos from different species, including insufficient active demethylation (incomplete erasure) and incomplete establishment of de novo methylation. Therefore, correcting abnormal DNA methylation modifications in in vitro fertilized embryos is one of the key means to improve in vitro embryonic developmental efficiency and developmental potential.

[0005] However, due to the complexity of methylation modification, current techniques for correcting DNA methylation modifications in in vitro embryonic development still suffer from low developmental efficiency and poor quality.

[0006] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address at least some of the technical problems in the prior art, the present invention provides compositions and applications for in vitro regulation of embryonic DNA methylation levels. Specifically, the present invention includes the following.

[0008] In a first aspect, the present invention provides a composition for in vitro regulation of embryonic DNA methylation levels, comprising two or more regulators selected from vitamin C or its precursors, RELN, and FGF2.

[0009] In some embodiments, the composition for in vitro regulation of embryonic DNA methylation levels according to the present invention comprises any combination of (a)-(d):

[0010] (a) Vitamin C or its precursor and RELN, wherein the weight ratio of said vitamin C or its precursor to said RELN is 800-1500:1;

[0011] (b) Vitamin C or its precursor and FGF2, wherein the weight ratio of said vitamin C or its precursor to said FGF2 is 50-500:1;

[0012] (c) RELN and FGF2, wherein the weight ratio of RELN to FGF2 is 1:1-10;

[0013] (d) Vitamin C or its precursor, RELN and FGF2, wherein the weight ratio of said vitamin C or its precursor, said RELN and said FGF2 is (800-1500):1:(1-10).

[0014] In some embodiments, in the composition for in vitro regulation of embryonic DNA methylation levels according to the present invention, the amount of vitamin C or its precursor is sufficient to achieve a working concentration of vitamin C of 0.5-5000 μg / ml; the amount of FGF2 is sufficient to achieve a working concentration of FGF2 of 0.001-5 μg / ml; or the amount of vitamin C or its precursor is sufficient to achieve a working concentration of vitamin C of 0.001-5 μg / ml.

[0015] In some embodiments, the composition for regulating embryonic DNA methylation levels according to the present invention further includes a basal culture medium.

[0016] In a second aspect, the present invention provides a method for in vitro regulation of embryonic DNA methylation levels, comprising the step of contacting an embryo with the composition described in the first aspect in vitro.

[0017] In some embodiments, in the method for regulating embryonic DNA methylation levels in vitro according to the present invention, the regulation includes increasing the amount of 5hmC in embryonic DNA, decreasing the amount of 5mC and / or increasing the 5hmC / 5mC ratio; or making the 5hmC / 5mC ratio close to the level of an in vivo developing embryo.

[0018] A third aspect of the invention provides a method for improving in vitro embryonic development, comprising the step of contacting an embryo with the composition described in the first aspect in vitro.

[0019] In some embodiments, the method for improving in vitro embryonic development according to the present invention includes improving embryonic development efficiency and / or quality.

[0020] In some embodiments, the method for improving in vitro embryonic development according to the present invention includes improving the in vitro cleavage rate, increasing the number or rate of blastocysts obtained in vitro, and / or the implantation rate of embryos after transfer and the number of fetuses produced.

[0021] A fourth aspect of the invention provides the use of the composition described in the first aspect in animal genetic improvement, breeding, in vitro embryo production or culture.

[0022] According to data released by the International Embryo Tissue Society (IETS), since 2016, the number of in vitro embryos in cattle has exceeded the number of in vivo embryos, and this number is increasing year by year. Therefore, in vitro embryo production technology has become an effective way to efficiently utilize superior breeding resources. This invention addresses the problems of low embryonic development rate and low subsequent developmental potential caused by abnormal epigenetic modifications in in vitro embryos by providing an effective DNA methylation correction pathway and method. This significantly improves the development rate and quality of in vitro fertilized embryos, providing an effective strategy for upgrading the livestock industry. It also has important reference value in the field of assisted reproduction, especially in obtaining high-quality embryos. Therefore, this invention will generate significant economic and social value for promoting in vitro embryo production in livestock in my country. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] In this article, the term "embryo" refers to the early stage of development from a fertilized egg. Since development is a continuous process, the term "embryo" in this article includes different forms of embryos generated at any developmental process or stage, generally referring to pre-implantation embryos, such as 2-cell embryos, 4-cell embryos, 8-cell embryos, morula, blastocysts, etc.

[0027] In this article, the term "in vitro" refers to events that occur in artificial environments, such as in test tubes or reaction vessels, in cell cultures, in petri dishes, etc., rather than in living organisms (e.g., animals, plants, or microorganisms).

[0028] In this article, the term "DNA methylation level" refers to the degree of methylation of DNA, such as the genome or its regions or genes, obtained from an embryo; that is, the extent to which methyl groups are added to specific bases. DNA methylation or demethylation is an important mechanism for gene expression regulation, capable of altering genetic expression or developmental progression without changing the DNA sequence.

[0029] In this article, the term "regulation," sometimes also referred to as "modulation," refers to any behavior that alters methylation levels, including upregulating or downregulating the methylation levels of specific bases, as well as optimizing methylation levels to approach or reach a certain standard. These standards can be artificially set or natural standards, such as the methylation levels of an embryo during in vivo development.

[0030] In this article, the term "vitamin C" or "ascorbic acid," abbreviated as "VitC," is associated with a variety of diseases and has been identified by the World Health Organization as an essential medicine in its list of essential medicines.

[0031] In this document, the term "precursor" refers to any substance, molecule, or entity that, upon chemical or physical alteration, can function as or produce vitamin C. Precursors can be covalently or chelated in some manner and are released or converted into the active ingredient, particularly vitamin C, before, during, or after administration to a subject or target cells or tissues. Precursors can be prepared by modifying functional groups present in a compound in a manner that allows the modified compound to cleave into the parent compound during routine procedures or in vivo. Precursors include compounds in which a hydroxyl, amino, thiol, or carboxyl group is bound to any group that, upon administration to a mammalian subject, undergoes cleavage to form a free hydroxyl, amino, thiol, or carboxyl group, respectively.

[0032] In this article, the term "RELN" refers to filoprotein or its active fragments or nucleic acids. Filoprotein refers to the full-length form of filoprotein or filoprotein having or containing the complete sequence, including proteins or isoforms homologous and / or orthologous to human filoprotein. An active fragment refers to a portion of the full-length filoprotein, i.e., an active peptide, polypeptide, etc. Filoprotein can be naturally isolated or artificially synthesized, including through genetic engineering or biotechnology, and chemical synthesis methods. In the case of isolation, the source of filoprotein is not limited; it can be from the same source as the subject or their tissues or cells, or from a different source.

[0033] In this article, the terms "FGF2" or "bFGF" refer to fibroblast growth factor II (sometimes also called "basic fibroblast growth factor"), which refers to fibroblast growth factor II or its active fragments or nucleic acids. Fibroblast growth factor II refers to the full-length form of fibroblast growth factor II or fibroblast growth factor II with or containing the complete sequence, and also includes proteins or isoforms that are homologous and / or orthologous to human fibroblast growth factor II. The fibroblast growth factor II described here can be naturally isolated or artificially synthesized, including through genetic engineering or biotechnology, and chemical synthesis methods. In the case of isolation, the source of fibroblast growth factor II is not limited; it can be from the same source as the subject or their tissues or cells, or from a different source.

[0034] In this document, the term "nucleic acid" includes DNA and RNA or their modified forms, including polynucleotides and oligonucleotides. Preferably, it is mRNA capable of producing active proteins such as RELN and FGF2. Here, "modified form" refers to a component of the nucleic acid, i.e., one or more components of the sugar, base, and phosphate ester structure, that differs from the natural component, preferably from the natural component produced by the human body. Nucleoside substitutes are molecules in which the ribose phosphate backbone is replaced by a non-ribose phosphate structure that places the bases in the correct spatial relation so that hybridization is substantially similar to hybridization observed with the ribose phosphate backbone, for example, analogues of an uncharged ribose phosphate backbone.

[0035] In this article, the term "working concentration," also known as "use concentration," refers to the initial concentration at which the regulator is brought into contact with the embryo for effective treatment. The unit of concentration is not limited and can be, for example, ng / ml, μg / ml, mg / ml, etc.

[0036] [Composition]

[0037] A first aspect of this invention provides a composition for in vitro regulation of embryonic DNA methylation levels, sometimes simply referred to herein as "the composition of this invention," comprising a combination of different regulators, wherein the regulators are selected from two or more of vitamin C or its precursors, RELN, and FGF2. The regulators in the combination of this invention interact with each other to produce a synergistic effect, significantly enhancing the regulatory effect compared to a single regulator. The form of the composition of this invention is not limited; it can be a solid, such as a dry powder, or a liquid, such as a solution.

[0038] In some embodiments, the composition of the present invention may be a combination of the above-mentioned regulators, i.e., containing no components other than unavoidable impurities besides the regulators. The regulators in the composition of the present invention may exist in a mixed form or in the form of two or more regulators existing individually. When used, the individually existing regulators may be pre-mixed before use, or each regulator may be used individually, simultaneously, or sequentially.

[0039] In some embodiments, the compositions of the present invention comprise the above-described combination of regulators, and further comprise other components. The composition or type of these other components is not limited and can be freely selected as needed. Such other components can be any known ingredients, particularly embryo culture-related reagents and compositions, such as culture media, culture solutions, or additives thereof.

[0040] In some embodiments, the regulator combination of the present invention is a combination of vitamin C or its precursor and RELN. Preferably, the weight ratio of vitamin C to RELN is 800-1500:1, for example, 850:1, 900:1, 950:1, 1000:1, 1050:1, 1100:1, 1150:1, 1200:1, 1250:1, 1300:1, 1350:1, 1400:1, 1450:1, etc. Within the above range, effective synergy between vitamin C and RELN can be achieved, greatly enhancing the regulatory effect.

[0041] In some embodiments, the regulator combination of the present invention is a combination of vitamin C or its precursor and FGF2. Preferably, the weight ratio of vitamin C to FGF2 is 50-500:1, for example 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1. The ratios of vitamin C and FGF2 within the specified ratios are 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, 250:1, 260:1, 270:1, 280:1, 290:1, 300:1, 320:1, 340:1, 360:1, 380:1, 400:1, 420:1, 440:1, 460:1, and 480:1. Within these ratios, vitamin C and FGF2 can achieve effective synergy, significantly enhancing their regulatory effect.

[0042] In some embodiments, the regulator combination of the present invention is a combination of RELN and FGF2. Preferably, the weight ratio of RELN to FGF2 is 1:1-10, for example, 1:1.1, 1:1.2, 1:1.5, 1:1.8, 1:2.0, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, etc. Within the above range, effective synergy between RELN and FGF2 can be achieved, greatly enhancing the regulatory effect.

[0043] In some embodiments, the amount of vitamin C or its precursor of the present invention is sufficient to achieve a working concentration of vitamin C of 0.5-5000 μg / ml during use. The concentration or amount in the composition is not limited as long as the vitamin C can achieve the above range during use. When the composition is a solution and is used as an additive, the concentration of vitamin C or its precursor in the composition can be the working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to achieve the above working concentration. When the composition is a solution and is used directly as a culture medium, the concentration of vitamin C or its precursor in the composition is generally substantially equal to the working concentration. The preferred working concentration of vitamin C is 1-4000 μg / ml, such as 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3 μg / ml, 3.5 μg / ml, 4 μg / ml, 4.5 μg / ml, 5 μg / ml, 5.5 μg / ml, 6 μg / ml, 6.5 μg / ml, 7 μg / ml, 7.5 μg / ml, 8 μg / ml, 8.5 μg / ml, 9 μg / ml, 9 .5μg / ml, 10μg / ml, 11μg / ml, 12μg / ml, 13μg / ml, 14μg / ml, 15μg / ml, 16μg / ml, 17μg / ml, 18μg / ml, 19μg / ml, 20μg / ml, 21μg / ml, 22μg / ml, 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 50μg / ml, 60 μg / ml, 70μg / ml, 80μg / ml, 90μg / ml, 100μg / ml, 120μg / ml, 140μg / ml, 160μg / ml, 180μg / ml, 20 0μg / ml, 220μg / ml, 240μg / ml, 260μg / ml, 280μg / ml, 300μg / ml, 350μg / ml, 400μg / ml, 450μg / m l, 500μg / ml, 550μg / ml, 600μg / ml, 650μg / ml, 700μg / ml, 750μg / ml, 800μg / ml, 900μg / ml, 100 0μg / ml, 1500μg / ml, 2000μg / ml, 2500μg / ml, 3000μg / ml, 3500μg / ml, 4000μg / ml, 4500μg / ml.

[0044] In some embodiments, the amount of FGF2 in this invention enables a working concentration of 0.001-5 μg / ml. The concentration or amount of FGF2 in the composition is not limited as long as it allows FGF2 to reach the above range during use. When the composition is a solution and is used as an additive, the concentration of FGF2 in the composition can be the working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to achieve the above working concentration. When the composition is a solution and is used directly as a culture medium, the concentration of FGF2 in the composition is generally substantially equal to the working concentration. The preferred working concentration of FGF2 is 0.05-5 μg / ml, such as 0.06 μg / ml, 0.07 μg / ml, 0.08 μg / ml, 0.09 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.20 μg / ml, 0.25 μg / ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, etc. 5μg / ml, 0.6μg / ml, 0.65μg / ml, 0.7μg / ml, 0.75μg / ml, 0.8μg / ml, 0.85μg / ml, 0.9μg / ml, 0.95 μg / ml, 1μg / ml, 1.5μg / ml, 2μg / ml, 2.5μg / ml, 3μg / ml, 3.5μg / ml, 4μg / ml, 4.5μg / ml, 5μg / ml.

[0045] In some embodiments, the amount of RELN in this invention enables a working concentration of 0.001-5 μg / ml. The preferred working concentration of RELN is 0.05-5 μg / ml, such as 0.06 μg / ml, 0.07 μg / ml, 0.08 μg / ml, 0.09 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.20 μg / ml, 0.25 μg / ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, etc. 5μg / ml, 0.6μg / ml, 0.65μg / ml, 0.7μg / ml, 0.75μg / ml, 0.8μg / ml, 0.85μg / ml, 0.9μg / ml, 0.95 μg / ml, 1μg / ml, 1.5μg / ml, 2μg / ml, 2.5μg / ml, 3μg / ml, 3.5μg / ml, 4μg / ml, 4.5μg / ml, 5μg / ml.

[0046] [Methods for regulating embryonic DNA methylation levels in vitro]

[0047] A second aspect of the invention provides a method for in vitro regulation of embryonic DNA methylation levels, comprising the step of contacting an embryo in vitro with the composition described in the first aspect.

[0048] In some embodiments, the contact of the present invention is carried out in a liquid, even if the embryo is treated in a culture medium containing a combination of regulators.

[0049] In some embodiments, the regulation of the present invention includes increasing the amount of 5hmC in embryonic DNA. Here, 5hmC refers to 5-hydroxymethylcytosine, and increasing the amount of 5hmC in embryonic DNA means that the relative amount (particularly relative to the untreated group or the treatment group with a single regulator) or absolute amount of 5-hydroxymethylcytosine in the DNA bases is increased.

[0050] In some embodiments, the regulation of the present invention includes reducing the amount of 5mC. Here, 5mC refers to 5-methylcytosine, and reducing the amount of 5mC means increasing the relative amount (particularly relative to the untreated group or the treatment group with a single regulator) or the absolute amount of 5-methylcytosine in the DNA bases.

[0051] In some embodiments, the regulation of the present invention refers to increasing the ratio of 5hmC / 5mC. This increase or improvement of 5hmC relative to 5mC includes, but is not limited to, a decrease in the amount of 5mC; an increase in the amount of 5hmC while keeping the amount of 5hmC constant; and an increase in the amount of 5hmC while simultaneously decreasing the amount of 5mC.

[0052] In some embodiments, the regulation of the present invention refers to bringing the 5hmC / 5mC ratio close to the level of in vivo developing embryos. Compared with in vivo developing embryos, DNA methylation is abnormal in in vitro cultured embryos, particularly with a reduced 5hmC / 5mC ratio. The compositions of the present invention can bring the 5hmC / 5mC ratio close to, or substantially consistent with, the corresponding level of in vivo developing embryos. Here, "close to" means within 10%, preferably within 6%, more preferably within 5%, such as within 4%, 3%, 2%, 1%, 0.5%, 0.1%, or even within 0.05%, of the DNA methylation level in in vivo developing embryos. Here, "substantially" means consistent with the corresponding level of in vivo developing embryos by more than 90%, preferably more than 92%, more preferably more than 95%, such as more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.8%, or more than 99.9%.

[0053] [Methods for improving in vitro embryonic development]

[0054] A third aspect of the invention provides a method for improving in vitro embryonic development, comprising the step of contacting an embryo in vitro with the composition described in the first aspect.

[0055] The improvements of this invention include increased embryo development efficiency and / or quality, including increased in vitro cleavage rate, number of blastocysts or blastocyst rate, and implantation rate and number of fetuses produced after transfer.

[0056] [use]

[0057] A fourth aspect of the invention provides the use of the composition described in the first aspect in animal genetic improvement, breeding, and in vitro embryo production. The composition and its modifiers have been described in detail in the first aspect and will not be repeated here.

[0058] In some embodiments, the animal of the present invention refers to a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, or pig). In some embodiments, the animal includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone. In some embodiments, the animal of the present invention is livestock, including pigs, cattle, and sheep.

[0059] In some embodiments, the genetic improvement and propagation of improved varieties of the present invention include genetic improvement or propagation relying on in vitro embryo production technology.

[0060] Example 1

[0061] This embodiment describes the application of combined administration of Vitamin C, RELN, and FGF2 in improving DNA methylation modification in mouse in vitro fertilized embryos and enhancing the developmental efficiency and quality of mouse in vitro fertilized embryos.

[0062] 1. Laboratory animals and reagents:

[0063] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art. In the examples, the culture medium KSOM+AA was purchased from Millipore, USA; Vitamin C, RELN, and FGF2 were purchased from Sigma-Aldrich, USA; ICR mice were purchased from Spiefol (Beijing) Biotechnology Co., Ltd.; and M2 culture medium was purchased from Zhongke Maichen (Beijing) Technology Co., Ltd.

[0064] 2. Experimental Methods

[0065] (1) Experimental Grouping

[0066] In vivo group: In vivo fertilization group;

[0067] Control group: Normal in vitro fertilization group;

[0068] Treatment group 1: Vitamin C administered alone;

[0069] Treatment group 2: FGF2 alone;

[0070] Treatment group 3: RELN alone;

[0071] Treatment group 4: Combined administration of Vitamin C and FGF2;

[0072] Treatment group 5: Combined administration of Vitamin C and RELN;

[0073] Treatment group 6: Combined administration of RELN and FGF2;

[0074] Treatment group 7: Combined administration of Vitamin C, RELN, and FGF2.

[0075] (2) Collection of in vivo fertilized embryos in mice

[0076] Healthy 8-week-old female mice were selected and intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG) for estrus synchronization. 48 hours later, 5 IU of human chorionic gonadotropin (hCG) was injected intraperitoneally for superovulation. The female mice were then paired with 10-week-old male mice. The following morning, the female mice with vaginal plugs were placed separately in their cages. Fertilization was assumed to occur at midnight (24:00), and the embryos at this time were designated as E0.5. For blastocyst collection, 96 hours after hCG injection, the peritoneum of the female mice was surgically opened, the uterus was removed and placed in M2 culture medium, and the blastocysts were flushed using a flushing needle.

[0077] (3) Collection of mouse in vitro fertilization embryos

[0078] Eight-week-old young female mice were used as experimental materials. Each female mouse was intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG). 48 hours later, each female mouse was intraperitoneally injected with 5 IU of human chorionic gonadotropin (hCG). Twelve hours later, the female mice were euthanized by cervical dislocation, and the oviducts were removed and placed in a preheated 37℃ M2 incubator. Using a 1 ml syringe, COCs (cumulus-oocyte complexes) from the ampulla of the oviduct were transferred into a fertilization drop. The incubator was placed in a CO2 incubator for equilibration for 30 minutes. Then, eight-week-old young male mice were euthanized by cervical dislocation, and the epididymis was removed. Sperm were gently squeezed from the epididymis into a capacitation drop using forceps and placed in a CO2 incubator for capacitation for 1 hour. After sperm capacitation, an appropriate amount of semen was added to the fertilization drop containing COCs using a pipette, and the sperm were incubated with COCs for 4 hours. Afterward, fertilized eggs were selected for in vitro culture based on polar body expulsion. Blastocysts were harvested 96 hours after in vitro culture.

[0079] (4) Immunofluorescence staining of embryonic 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC)

[0080] The embryos obtained in step (2) were washed three times with 0.1% PBS-PVA, and the zona pellucida was removed with acidic Tyrode solution (T1788, Sigma-Aldrich, USA). The embryos were then transferred to 4% paraformaldehyde fixative and fixed at room temperature for 1 hour. Afterward, they were permeabilized with DPBS (Durbeco phosphate buffer) containing 0.5% Triton X-100 at room temperature for 1 hour. They were then washed once with 0.1% PBS-PVA, treated with 4M HCl for 20 minutes, and then treated with 100mM Tris-HCl for 10 minutes. Finally, the embryos were blocked overnight at 4°C with DPBS containing 1% BSA. The next day, the embryos were incubated with primary antibodies (5-hydroxymethylcytosine and 5-methylcytosine antibodies) at room temperature for 1 hour, washed three times with DPBS containing 0.5% Triton X-100 at room temperature, and then incubated with secondary antibodies at room temperature for 1 hour. Finally, the cells were incubated with DAPI (4',6-diamidinyl-2-phenylindole) for 15 minutes to stain the nuclei. The fluorescence signal was observed and photographed under a BX51 microscope. The fluorescence intensity of 5-hydroxymethylcytosine and 5-methylcytosine was counted and the ratio was calculated using ImageJ software.

[0081] The primary antibodies used were: 5-hydroxymethylcytosine antibody (1:500 dilution, 36769, ActiviteMotif, USA) and 5-methylcytosine antibody (1:250 dilution, 36649, ActiviteMotif, USA). The secondary antibodies used were: Alexafluor 594 goat anti-rabbit antibody (1:1000 dilution, Invitrogen, USA, A-11034) and Alexafluor 488 goat anti-mouse antibody (1:1000 dilution, Invitrogen, USA, A-11030).

[0082] (5) Mouse blastocyst uterine transfer

[0083] On the day of in vitro fertilization, young female mice in natural estrus were mated with vasectomized young male mice. The next morning, female mice with vaginal plugs were considered pseudopregnant recipients, designated as day 0.5. The day 4.5 blastocysts obtained in step (2) were transferred, and six well-developed blastocysts were transferred to the uterine horns on both sides of the pseudopregnant recipient. On day 19.5 (day 14 after embryo transfer), fetuses were retrieved to assess the developmental capacity of each embryo.

[0084] (6) Combined administration of Vitamin C, RELN, and FGF2 regulates DNA methylation in in vitro embryos and improves the developmental quality of in vitro fertilized embryos.

[0085] Specific experimental groups:

[0086] In vivo group: In vivo fertilization group;

[0087] Control group: Normal in vitro fertilization group;

[0088] Treatment group 1: Vitamin C (100 μg / ml) administered alone;

[0089] Treatment group 2: FGF2 (500 ng / ml) administered alone;

[0090] Treatment group 3: RELN (100 ng / ml) administered alone;

[0091] Treatment group 4: Combined administration of Vitamin C (50 μg / ml) and FGF2 (250 ng / ml);

[0092] Treatment group 5: Combined administration of Vitamin C (50 μg / ml) and RELN (50 ng / ml);

[0093] Treatment group 6: Combined administration of RELN (50 ng / ml) and FGF2 (250 ng / ml);

[0094] Treatment group 7: Combined administration of Vitamin C (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml).

[0095] After 96 hours of in vitro culture, the blastocyst rate of each group was statistically analyzed and calculated. Simultaneously, the 5-hmC / 5-mC immunofluorescence intensity ratios of blastocysts 1 / 2 / 3 / 4 / 5 / 6 / 7 in the in vivo group, control group, and treatment group were compared.

[0096] The blastocysts from the in vivo group and the blastocysts from each group after 96 hours of in vitro culture were transferred. On day 19.5 (day 14 after embryo transfer), the recipient female mice were euthanized, the number of implantation sites was counted, and then the fetuses and placentas were removed separately, and the number of live fetuses was recorded.

[0097] 3. Experimental Results

[0098] Table 1. Effects of combined administration of Vitamin C, RELN, and FGF2 on the rate of in vitro fertilized blastocysts and the 5hmC / 5mC ratio in mice.

[0099]

[0100]

[0101] Note: The superscripts a, b, and c indicate different significant differences. The significance was tested using one-way ANOVA. Blastocyst rate = number of blastocysts / number of fertilized eggs.

[0102] As shown in Table 1, compared with the control group (IVF group) (32.5±3.3%), treatment groups 1 (41.1±3.0%), 2 (40.9±2.2%), 4 (50.7±3.2%), 6 (49.1±2.2%), and 7 (53.1±2.0%) all significantly improved the in vitro blastocyst development efficiency. Furthermore, the combined treatments of Vitamin C+FGF2, Vitamin C+RELN, RELN+FGF2, and Vitamin C+FGF2+RELN produced synergistic effects, all significantly higher than the blastocyst development efficiency of Vitamin C, FGF2, and RELN alone; and the effect of the Vitamin C+FGF2+RELN combination was closest to that of the in vivo group. Therefore, the combined use of Vitamin C, FGF2, and RELN helps improve the development efficiency of mouse IVF embryos.

[0103] Compared with the control group (IVF group) (1.89±0.07), treatment groups 1 / 4 / 5 / 6 / 7 significantly increased the 5hmC / 5mC ratio in in vitro blastocysts. Furthermore, the 5hmC / 5mC ratios after treatment with combinations of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN showed no significant difference compared to the in vivo group. This indicates that the combined application of two or all three of VitC, RELN, and FGF2 effectively corrects DNA methylation modification in IVF embryos.

[0104] Table 2. Effects of combined administration of Vitamin C, RELN, and FGF2 on implantation rate and number of offspring after in vitro fertilization embryo transfer in mice.

[0105]

[0106] Note: The superscripts a, b, and c indicate different significant differences. The significance was tested using one-way ANOVA. Implantation rate = number of implantation sites / number of embryos transferred; mean number of fetuses = total number of fetuses / number of pregnant recipients.

[0107] As shown in Table 2, the embryo implantation rate in treatment groups 1 / 2 / 4 / 5 / 6 / 7 was significantly higher than that in the control group (in vitro fertilization group) (50.0±4.3%). Among them, the combination treatments of Vitamin C+FGF2, Vitamin C+RELN, RELN+FGF2, and Vitamin C+FGF2+RELN produced a synergistic effect, all of which were significantly higher than the embryo implantation rates of Vitamin C, FGF2, and RELN added alone; and the effect of the combination of Vitamin C+FGF2+RELN was closest to that of the in vivo group.

[0108] The above results indicate that the combined administration of two or all three of Vitamin C, RELN, and FGF2 can significantly improve the embryo implantation rate and the number of offspring after in vitro fertilization embryo transfer in mice.

[0109] Example 2

[0110] This embodiment demonstrates the application of combined administration of Vitamin C, RELN, and FGF2 in improving the developmental efficiency and quality of sheep in vitro fertilized embryos.

[0111] 1. Laboratory animals and reagents:

[0112] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art. In the examples, VitC, RELN, FGF2, and components of the basal culture medium were all purchased from Sigma-Aldrich, USA, and the sheep breed was Blackhead Suffolk, sourced from Inner Mongolia Sainuo Sheep Breeding Technology Co., Ltd.

[0113] 2. Experimental Methods

[0114] (1) Experimental Grouping

[0115] Control group: Normal in vitro fertilization group;

[0116] Treatment group 1: Vitamin C (100 μg / ml) administered alone;

[0117] Treatment group 2: FGF2 (500 ng / ml) administered alone;

[0118] Treatment group 3: RELN (100 ng / ml) administered alone;

[0119] Treatment group 4: Combined administration of Vitamin C (50 μg / ml) and FGF2 (250 ng / ml);

[0120] Treatment group 5: Combined administration of Vitamin C (50 μg / ml) and RELN (50 ng / ml);

[0121] Treatment group 6: Combined administration of RELN (50 ng / ml) and FGF2 (250 ng / ml);

[0122] Treatment group 7: Combined administration of Vitamin C (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml).

[0123] (2) In vitro maturation of sheep oocytes and embryonic development

[0124] ①Oocyte collection, in vitro maturation, and in vitro fertilization

[0125] Follicular fluid was aspirated from the ovaries of live sheep using live oocyte retrieval technology. The follicular fluid was spread evenly in a 60 mm culture dish and placed on a 38.5°C incubator. Cocci containing homogeneous cytoplasm were selected under a stereomicroscope, washed three times with the oocyte extraction fluid, and then washed three times with pre-equilibrated in vitro maturation solution (3 hours prior). The resulting culture was transferred to in vitro maturation medium and incubated in a 5% CO2, 38.5°C, saturated humidity incubator for 24 hours.

[0126] After 24 hours, the sperm were thawed in a 39°C water bath for 1 minute and then transferred to sperm basal solution for incubation. After 30 minutes, the supernatant was aspirated and transferred to a 1.5 mL centrifuge tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Simultaneously, the cocci matured in vitro for 24 hours were placed in hyaluronidase solution, and the cumulus cells on the surface of the mature oocytes were repeatedly removed by pipetting. After washing, the oocytes were transferred to fertilization solution, and an appropriate amount of semen was added for co-incubation for 10-12 hours.

[0127] ②In vitro embryo culture

[0128] After in vitro fertilization, oocytes were washed three times with embryo culture medium and then transferred to embryo culture medium and cultured in a 5% CO2 saturated humidity incubator. Cleavage rate was calculated after 48 hours of culture, and blastocyst rate was calculated on day 6 and day 7 after continuing culture.

[0129] ③ Embryo transfer to the uterus and pregnancy testing.

[0130] Select blastocysts with good morphology from day 6 or day 7. The recipient sheep is strapped to a surgical frame. The tip of the uterine horn on the side of the ovary with a corpus luteum or a well-developed corpus luteum is fixed. A paperclip is used to puncture the uterine horn wall at the avascular portion of the upper third. The embryo transfer syringe tip is then inserted into the uterine cavity through the puncture site. The tip is moved to confirm its position within the uterine cavity. The syringe plunger connected to the transfer tube is then pushed to inject the embryo. The transfer tube is then withdrawn. Finally, the uterine horn is returned to the abdominal cavity and disinfected. Pregnancy is assessed using ultrasound on day 45 post-transfer, and the pregnancy rate and number of offspring are recorded.

[0131] Pregnancy rate = number of pregnant female animals / number of recipients of in vitro fertilization embryo transfer × 100%.

[0132] 3. Experimental Results

[0133] As shown in Table 3, compared with the control group (37.7±2.4%), treatment groups 1 / 2 / 4 / 5 / 6 / 7 significantly improved the blastocyst development rate of sheep in vitro fertilized embryos without affecting the 48h cleavage rate of in vitro fertilized embryos. Among them, the treatments of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN combination were significantly higher than the blastocyst development rates of VitC, FGF2, and RELN alone.

[0134] As shown in Table 4, compared with the control group (58.1%), the administration of Vitamin C, RELN, and FGF2 alone did not improve the pregnancy rate after embryo transfer. However, the combined treatments of Vitamin C+FGF2, Vitamin C+RELN, RELN+FGF2, and Vitamin C+FGF2+RELN produced a synergistic effect, which was significantly higher than the pregnancy rate and number of offspring born after embryo transfer treated with Vitamin C, FGF2, and RELN alone.

[0135] Therefore, the combined administration of two or all three of Vitamin C, RELN, and FGF2 can significantly improve the developmental efficiency and quality of sheep in vitro fertilized embryos.

[0136] Table 3. Effects of combined administration of Vitamin C, RELN, and FGF2 on the developmental rate of sheep in vitro fertilized embryos.

[0137]

[0138] Note: The superscripts a and b indicate significant differences, and the significance was tested using one-way ANOVA. Cleavage rate = number of two-cell embryos / number of oocytes × 100%, blastocyst rate = number of blastocysts / number of oocytes × 100%.

[0139] Table 4. Effects of combined administration of Vitamin C, RELN, and FGF2 on pregnancy rate and number of offspring born in sheep in vitro fertilization embryo transfer.

[0140]

[0141]

[0142] Note: Superscripts for a, b, and c indicate significant differences; significance was analyzed using the chi-square test. Pregnancy rate = (Number of pregnant females / Number of recipient females) × 100%.

[0143] Example 3

[0144] This embodiment demonstrates the application of combined administration of Vitamin C, RELN, and FGF2 in improving the developmental efficiency and quality of bovine in vitro fertilization embryos.

[0145] 1. Experimental animals and reagents used in the experiments of this invention:

[0146] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art. In the examples, VitC, RELN, FGF2, and the components of the basal culture medium were all purchased from Sigma-Aldrich, USA, and the dairy cows were from Adopt a Cow Biotechnology Co., Ltd.

[0147] 2. Experimental Methods

[0148] (1) Experimental Grouping

[0149] Control group: Normal in vitro fertilization group;

[0150] Treatment group 1: Vitamin C (100 μg / ml) administered alone;

[0151] Treatment group 2: FGF2 (500 ng / ml) administered alone;

[0152] Treatment group 3: RELN (100 ng / ml) administered alone;

[0153] Treatment group 4: Combined administration of Vitamin C (50 μg / ml) and FGF2 (250 ng / ml);

[0154] Treatment group 5: Combined administration of Vitamin C (50 μg / ml) and RELN (50 ng / ml);

[0155] Treatment group 6: Combined administration of RELN (50 ng / ml) and FGF2 (250 ng / ml);

[0156] Treatment group 7: Combined administration of Vitamin C (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml).

[0157] (2) In vitro maturation of bovine oocytes and embryonic development

[0158] ①Oocyte collection, in vitro maturation, and in vitro fertilization

[0159] Bovine ovaries were incubated at 32℃-35℃ with physiological saline and transported to the laboratory. Follicles were observed on the surface of the ovary after extraction using a 20ml syringe. Cumulus-oocyte complexes (COCs) containing more than three layers of cumulus cells were washed twice with oocyte washing solution, followed by two washes with oocyte maturation solution. They were then placed in a four-well plate with maturation culture medium (500μl volume of maturation medium, approximately 50 oocytes, culture conditions: air containing 5% CO2, temperature 39℃, saturated humidity, culture time 24h) that had been pre-equilibrated in a CO2 incubator for more than 2 hours.

[0160] Further, after thawing the semen in a capillary tube at 38°C, it was placed in a 15mL centrifuge tube containing the washed semen solution and centrifuged at 1800 rpm for 5 min. The supernatant was discarded, and this process was repeated twice. Then, 50 μl of the sperm suspension was added to a 50 μl fertilization droplet that had been equilibrated for more than 2 hours to form a 100 μl fertilization droplet. The fertilization droplet was then incubated in an incubator for 1.5 hours to allow the sperm to fully capacimate.

[0161] After 22-24 hours of in vitro culture, COCs were digested with 0.1% hyaluronidase for about 1 minute to remove the outer layer of expanded granulosa cells. The cells were then washed three times with pre-equilibrated fertilization solution, and mature oocytes with homogeneous cytoplasm were aspirated and placed in fertilization drops (15 oocytes / 100μl). After co-incubation with sperm and eggs at 38.5℃ and 5% CO2 for 8-10 hours, the oocytes were thoroughly washed in an IVC to remove adhering sperm, and then cultured in pre-equilibrated mCRlaa microdrops (equilibrated for at least 2 hours).

[0162] ②In vitro embryo culture

[0163] After fertilization, the zygotes were transferred to a four-well plate for culture. They were cultured in 500 μl of early development medium for 2 days. After counting the cleavage embryos, they were transferred to 500 μl of late development medium for 5 days. The medium was then changed in half every other day. After fertilization, the number of in vitro blastocysts was counted and the blastocyst rate was calculated after 7-8 days of in vitro culture.

[0164] ③ Embryo transfer to the uterus and pregnancy testing.

[0165] Blastocysts with good morphology (D7 or D8) are selected. The recipient cow is restrained and anesthetized according to artificial insemination requirements. After cleaning and disinfecting the vulva, the cervix is ​​opened, and the fallopian tubes are flushed. Using a rectal manipulation method, the embryo transfer tube is inserted deep into one uterine horn. The syringe plunger connected to the transfer tube is pushed to inject the embryo, and then the transfer tube is withdrawn. Pregnancy is assessed using ultrasound on day 60 post-transfer, and the pregnancy rate and number of offspring are recorded.

[0166] 3. Experimental Results

[0167] As shown in Table 5, compared with the control group (28.9±2.2%), treatment groups 1 / 2 / 4 / 5 / 6 / 7 significantly improved the blastocyst development rate of bovine in vitro fertilization embryos without affecting the 48h cleavage rate of in vitro fertilized embryos. Among them, the combination treatments of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN produced synergistic effects, and were significantly higher than the blastocyst development rate of VitC, FGF2, and RELN alone.

[0168] As shown in Table 6, compared with the control group (43.3%), the administration of Vitamin C, RELN, and FGF2 alone did not improve the pregnancy rate after embryo transfer. However, the combined treatments of Vitamin C+FGF2, Vitamin C+RELN, RELN+FGF2, and Vitamin C+FGF2+RELN were significantly higher than the combined treatments of Vitamin C, FGF2, and RELN alone in terms of pregnancy rate and number of offspring born after embryo transfer.

[0169] Therefore, the combined administration of two or all three of Vitamin C, RELN, and FGF2 can significantly improve the efficiency and quality of in vitro fertilization embryo development in dairy cows.

[0170] Table 5. Effects of combined administration of Vitamin C, RELN, and FGF2 on the developmental rate of in vitro fertilized embryos in dairy cows.

[0171]

[0172] Note: The superscripts a and b indicate significant differences, and the significance was tested using one-way ANOVA. Cleavage rate = number of two-cell embryos / number of oocytes × 100%, blastocyst rate = number of blastocysts / number of oocytes × 100%.

[0173] Table 6. Effects of combined administration of Vitamin C, RELN, and FGF2 on pregnancy rate of in vitro fertilization embryo transfer in dairy cows.

[0174]

[0175]

[0176] Note: Superscripts for a and b indicate significant differences; significance was determined using the chi-square test. Pregnancy rate = (Number of pregnant females / Number of recipient females) × 100%.

[0177] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A composition for regulating the level of DNA methylation in in vitro embryos, characterized in that, Includes at least one of the following: (a) 50 μg / ml of vitamin C and 50 ng / ml of RELN; (b) 50 ng / ml RELN and 250 ng / ml FGF2; (c) 33.3 μg / ml of vitamin C, 33.3 ng / ml of RELN and 166.7 ng / ml of FGF2.

2. The composition for regulating in vitro embryonic DNA methylation levels according to claim 1, characterized in that, This further includes the basic culture medium.

3. A method for in vitro regulation of DNA methylation levels in embryos from fertilized egg to blastocyst stage, characterized in that, The method involves contacting an embryo from fertilized egg to blastocyst stage in vitro with the composition according to claim 1 or 2, wherein, The embryo is a mammalian embryo; The regulation is to increase the amount of 5hmC in embryonic DNA from fertilized egg to blastocyst, decrease the amount of 5mC and / or increase the 5hmC / 5mC ratio; or to make the 5hmC / 5mC ratio close to the level of an in vivo developing embryo.

4. A method for improving the development of embryos from fertilized egg to blastocyst stage in vitro, characterized in that, The method involves contacting an embryo from the fertilized egg to the blastocyst stage in vitro with the composition according to claim 1 or 2.

5. The method for improving embryonic development from fertilized egg to blastocyst stage in vitro according to claim 4, characterized in that, The improvements include increased embryonic development efficiency and / or quality.

6. The method for improving in vitro embryonic development from fertilized egg to blastocyst stage according to claim 4, characterized in that, The improvements include increasing the in vitro cleavage rate, increasing the number or rate of blastocysts obtained in vitro, and / or the implantation rate of embryos after transfer and the number of fetuses produced.

7. The use of the composition according to claim 1 or 2 in animal genetic improvement, propagation of superior breeds, in vitro embryo production or culture, characterized in that, The genetic modification is to increase the amount of 5hmC in the embryonic DNA, decrease the amount of 5mC, and / or increase the 5hmC / 5mC ratio; or to make the 5hmC / 5mC ratio close to the level of an in vivo developing embryo, wherein the embryo is an embryo from the fertilized egg to the blastocyst stage, and the embryo is a mammalian embryo.

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