Composition and method for regulating in-vitro embryo DNA methylation modification and application of composition and method in improvement of embryo development efficiency and quality

Abstract

The invention discloses a composition and a method for regulating in-vitro embryo DNA methylation modification and application of the composition and the method in improvement of embryo development efficiency and quality. Aiming at the problems of low embryo development rate, low subsequent development potential and the like caused by abnormal apparent modification of an in-vitro embryo, the invention provides a composition comprising more than two regulators selected from vitamin C or a precursor thereof, RELN and FGF2, DNA methylation can be effectively corrected, the development rate and quality of the in-vitro fertilized embryo are further greatly improved, and the development period of the in-vitro fertilized embryo is shortened. And an effective strategy is provided for upgrading the livestock industry.

Description

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

[0002] The invention belongs to the field of biotechnology, and in particular relates to the combined administration of vitamin C, RELN and FGF2 to improve the development efficiency and quality of embryos obtained by in vitro fertilization. Background Art

[0003] Mammalian in vitro embryo production technology has been widely used in the fields of livestock genetic improvement, breeding and human assisted reproduction. However, in vitro embryo production still has technical bottlenecks such as low development efficiency and poor embryo quality, which have been difficult to break through. It is generally believed that the in vitro culture environment is different from the in vivo development environment, especially the imperfect composition of the in vitro embryo development culture medium, which causes abnormal epigenetic modifications of in vitro embryos, is an important reason for the low development potential and poor quality of in vitro embryos, greatly limiting the development of in vitro embryo production technology. Therefore, optimizing the composition of in vitro embryo culture medium to correct abnormal epigenetic modifications of in vitro embryos is currently the most direct and effective strategy to improve the efficiency and quality of in vitro embryo development and improve in vitro embryo production technology.

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

[0005] However, due to the complexity of methylation modification, current correction technologies for in vitro embryonic development targeting DNA methylation modification still have problems of low developmental efficiency and poor quality.

[0006] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0007] In order to solve at least some of the technical problems in the prior art, the present invention provides a composition and application for regulating embryonic DNA methylation level in vitro. Specifically, the present invention includes the following contents.

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

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

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

[0011] (b) vitamin C or its precursor and FGF2, wherein the weight ratio of the vitamin C or its precursor to the 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, and the weight ratio of the vitamin C or its precursor, the RELN and the FGF2 is (800-1500):1:(1-10).

[0014] In certain embodiments, in the composition for regulating embryonic DNA methylation levels in vitro according to the present invention, the amount of vitamin C or its precursor can make the working concentration of vitamin C reach 0.5-5000 μg / ml; the amount of FGF2 can make the working concentration of FGF2 reach 0.001-5 μg / ml; or the amount of vitamin C or its precursor can make the working concentration of vitamin C reach 0.001-5 μg / ml.

[0015] In certain embodiments, the composition for regulating embryonic DNA methylation level in vitro according to the present invention further comprises a basal culture medium.

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

[0017] In certain 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, reducing the amount of 5mC and / or increasing the ratio of 5hmC / 5mC; or making the ratio of 5hmC / 5mC close to the level of the developing embryo in vivo.

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

[0019] In certain embodiments, according to the method for improving in vitro embryo development of the present invention, the improvement comprises improving embryo development efficiency and / or quality.

[0020] In certain embodiments, according to the method for improving in vitro embryo development described in the present invention, the improvement includes increasing the cleavage rate in vitro, increasing the number of blastocysts or the blastocyst rate obtained in vitro and / or the implantation rate of embryos after transplantation and the number of fetuses produced.

[0021] The fourth aspect of the present 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 the data released by the International Embryo Tissue Society (IETS), the number of in vitro embryos of cattle has exceeded the number of in vivo embryos since 2016, and it has shown a trend of increasing year by year. Therefore, in vitro embryo production technology has become an effective way to efficiently utilize improved resources. The present invention provides effective correction pathways and methods for DNA methylation for problems such as low embryo development rate and low subsequent development potential caused by abnormal epigenetic modification of in vitro embryos, greatly improves the development rate and quality of in vitro fertilized embryos, and provides an effective strategy for the upgrading of the animal husbandry industry. At the same time, it has important reference value in the field of assisted reproduction, especially in the acquisition of high-quality embryos. Therefore, the present invention will produce huge economic value and social value for my country to promote the production of in vitro embryos of livestock. DETAILED DESCRIPTION

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

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

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

[0027] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (eg, an animal, plant, or microorganism).

[0028] Herein, the term "DNA methylation level" refers to the degree of methylation of DNA, such as a genome or a region or gene thereof, of a sample obtained from an embryo, i.e., the degree to which methyl groups are added to specific bases. DNA methylation or demethylation is an important mechanism for regulating gene expression, which can change genetic expression or developmental progression without changing the DNA sequence.

[0029] Herein, the term "regulation", sometimes also referred to as "regulation", refers to any behavior that changes the methylation level, including increasing the methylation level of a specific base, or decreasing the methylation level of a specific base, and also includes optimizing the methylation level to make it close to or reach a certain standard. These standards can be artificially set standards or standards of natural states, such as the methylation level of an embryo during in vivo development.

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

[0031] Herein, the term "precursor" refers to any substance, molecule or entity that can act as or produce vitamin C after chemical or physical changes. The precursor can be covalently bound or chelated in some manner and released or converted into an active ingredient, particularly vitamin C, before, simultaneously or after administration to a subject or target cell or tissue. Precursors can be prepared by modifying the functional groups present in the compound in a manner that allows the modification to be cleaved into the parent compound in routine manipulation or in vivo. Precursors include compounds in which hydroxyl, amino, sulfhydryl or carboxyl groups are bound to any group that is cleaved to form free hydroxyl, amino, sulfhydryl or carboxyl groups, respectively, when administered to a mammalian subject.

[0032] Herein, the term "RELN" means reelin or its active fragment or nucleic acid. Among them, reelin refers to the full-length form of reelin or reelin with or containing the complete sequence, and also includes proteins or isoforms that are homologous and / or orthologous to human reelin. Active fragment refers to a part of the full-length form of reelin, that is, active peptides, polypeptides, etc. Here, reelin can be isolated from nature or obtained by artificial synthesis, including genetic engineering or biotechnology, and chemical synthesis methods. In the case of separation, the source of reelin is not limited, and it can be the same source as the subject or its tissues and cells, or it can be a different source, and there is no limitation on this.

[0033] As used herein, the term "FGF2" or "bFGF" refers to fibroblast growth factor II (sometimes also referred to as "basic fibroblast growth factor"), and 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 having 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 herein can be isolated from nature or obtained by artificial synthesis, including genetic engineering or biotechnology, or chemical synthesis methods. In the case of isolation, the source of fibroblast growth factor II is not limited, and can be the same source as the subject or its tissues or cells, or a different source, and there is no limitation on this.

[0034] Herein, the term "nucleic acid" includes DNA and RNA or modified forms thereof, including polynucleotides, oligonucleotides. Preferably, the mRNA capable of producing active proteins such as RELN and FGF2 is used. Here, the modified form refers to the components of nucleic acid, i.e. one or more components of sugar, base and phosphate structure, which are different from natural components, preferably different from natural components produced from human body. Nucleoside substitutes are molecules in which the ribose phosphate backbone is replaced by a non-ribose phosphate structure, which places the base in the correct spatial relationship, so that hybridization is substantially similar to that seen with the ribose phosphate backbone, for example, an analog of a non-charged ribose phosphate backbone.

[0035] Herein, the term "working concentration" is also referred to as "use concentration", which refers to the initial concentration when the regulator is brought into contact with the embryo and effectively treated. The concentration unit is not limited, and can be, for example, ng / ml, μg / ml, mg / ml, etc.

[0036] [Composition]

[0037] The first aspect of the present invention provides a composition for regulating embryonic DNA methylation levels in vitro, sometimes referred to herein as the "composition of the present invention", which includes a combination of different regulators, wherein the regulators are selected from two or more of vitamin C or its precursor, RELN and FGF2. The regulators in the regulator combination of the present invention interact with each other to produce a synergistic effect, and the combination greatly improves the regulatory effect compared to a single regulator. The form of the composition of the present invention is not limited, and can be a solid such as a dry powder, or a liquid such as a solution.

[0038] In certain embodiments, the composition of the present invention may be a combination of the above-mentioned conditioning agents, i.e., it does not contain any component other than the conditioning agents except for the inevitable impurities. In the composition of the present invention, each conditioning agent may exist in a mixed form, or two or more conditioning agents may exist in a form of being present alone. When used, the conditioning agents present alone may be pre-mixed and then used, or each conditioning agent may be used alone simultaneously or separately in sequence.

[0039] In certain embodiments, the composition of the present invention comprises the above-mentioned combination of regulators, and further comprises other ingredients in addition. The composition or type of other ingredients is not limited and can be freely selected as needed. Such other ingredients can select any known ingredients, particularly reagents and compositions related to embryo culture, such as culture medium, culture fluid or its additives, etc.

[0040] In certain 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, such as 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 improving the regulatory effect.

[0041] In certain embodiments, the modulator 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 , 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, 480:1, etc. Within the above range, effective synergy between vitamin C and FGF2 can be achieved, greatly enhancing the regulatory effect.

[0042] In certain embodiments, the modulator combination of the present invention is a combination of RELN and FGF2. Preferably, the weight ratio of RELN to FGF2 is 1:1-10, such as 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 certain embodiments, the amount of vitamin C or its precursor of the present invention can make the working concentration of vitamin C reach 0.5-5000 μg / ml when used. As long as vitamin C can reach the above range when used, the concentration or amount in the composition is not limited. In the case where the composition is a solution and is used as an added component, the concentration of vitamin C or its precursor in the composition can be a working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to reach the above working concentration. In the case where the composition is a solution and is directly used as a culture medium, the concentration of vitamin C or its precursor in the composition is usually substantially equal to the working concentration. The working concentration of vitamin C is preferably 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 certain embodiments, the amount of FGF2 of the present invention can make the working concentration of FGF2 reach 0.001-5 μg / ml. As long as FGF2 can reach the above range when used, the concentration or amount of FGF2 in the composition is not limited. When the composition is a solution and is used as an added component, the concentration of FGF2 in the composition can be a working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to reach the above working concentration. When the composition is a solution and is directly used as a culture medium, the concentration of FGF2 in the composition is usually substantially equal to the working concentration. The working concentration of FGF2 is preferably 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, 0.5 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 certain embodiments, the amount of RELN of the present invention can make the working concentration of RELN reach 0.001-5 μg / ml. The working concentration of RELN is preferably 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, 0.5 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] The second aspect of the present invention provides a method for regulating embryonic DNA methylation levels in vitro, comprising the step of contacting the embryo with the composition of the first aspect in vitro.

[0048] In certain embodiments, the contacting of the present invention is performed in liquid, ie, the embryos are treated in a culture medium containing a combination of modulators.

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

[0050] In certain embodiments, the regulation of the present invention includes reducing the amount of 5mC, wherein 5mC refers to 5-methylcytosine, and reducing the amount of 5mC means that the relative amount (especially relative to the untreated group or the group treated with a single regulator) or the absolute amount of 5-methylcytosine in the DNA base is increased.

[0051] In certain embodiments, the regulation of the present invention refers to increasing the ratio of 5hmC / 5mC. The increase or increase of 5hmC relative to 5mC includes the decrease or reduction of 5mC when the amount of 5hmC remains unchanged; the increase or increase of 5hmC when the amount of 5mC remains unchanged; and the increase or increase of 5hmC while the amount of 5mC decreases or decreases.

[0052] In certain embodiments, the regulation of the present invention refers to making the ratio of 5hmC / 5mC close to the level of the embryo developed in vivo. Compared with the embryo developed in vivo, the DNA methylation in the embryo cultured in vitro is abnormal, especially the 5hmC / 5mC ratio is reduced. The composition of the present invention can make the 5hmC / 5mC ratio close to the corresponding level of the embryo developed in vivo, or substantially consistent with the corresponding level of the embryo developed in vivo. Here, "close" means that the difference with the DNA methylation level in the embryo developed in vivo is within 10%, preferably within 6%, more preferably within 5%, such as within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.1%, and even within 0.05%. Here, "substantially" means that the corresponding level of the embryo developed in vivo is consistent with 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%, and more than 99.9%.

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

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

[0055] The improvements of the present invention include improving the efficiency and / or quality of embryo development, including improving the in vitro cleavage rate, the number of blastocysts obtained or the blastocyst rate, the implantation rate after transplantation, and the number of fetuses produced.

[0056] [use]

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

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

[0059] In certain embodiments, the genetic improvement and enrichment of the present invention include genetic improvement or enrichment relying on in vitro embryo production technology.

[0060] Example 1

[0061] This example is the application of the combined administration of VitC, RELN and FGF2 in improving DNA methylation modification of mouse in vitro fertilization embryos and improving the development efficiency and quality of mouse in vitro fertilization embryos.

[0062] 1. Experimental animals and reagents:

[0063] If not otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The culture medium KSOM+AA in the examples was purchased from Millipore, USA, VitC, RELN and FGF2 were purchased from Sigma-Aldrich, USA, ICR mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., and M2 culture medium was purchased from Zhongke Maichen (Beijing) Technology Co., Ltd.

[0064] 2. Experimental methods

[0065] (1) Experimental groups

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

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

[0068] Treatment group 1: VitC alone administration group;

[0069] Treatment group 2: FGF2 alone administration group;

[0070] Treatment group 3: RELN alone administration group;

[0071] Treatment group 4: VitC and FGF2 combined administration group;

[0072] Treatment group 5: VitC and RELN combined administration group;

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

[0074] Treatment group 7: VitC, RELN, and FGF2 combined administration group.

[0075] (2) Collection of fertilized mouse embryos

[0076] Eight-week-old female mice in good health were selected, and 5 IU of pregnant mare serum gonadotropin (PMSG) was intraperitoneally injected for estrus synchronization. 48 hours later, 5 IU of human chorionic gonadotropin (hCG) was intraperitoneally injected for superovulation. The female mice were then caged with 10-week-old male mice, and the female mice with vaginal plugs were placed alone in the mouse cage the next morning. The default fertilization time was 24:00 at night, and the embryos at this time were recorded as E0.5. When collecting blastocysts, 96 hours after the injection of hCG, the abdominal cavity of the female mice was opened surgically, the uterus was cut and placed in M2 culture medium, and the blastocysts were flushed out using an egg flushing needle.

[0077] (3) Mouse in vitro fertilization embryo collection

[0078] 8-week-old young female mice were used as experimental materials. Each female mouse was intraperitoneally injected with 5 IU pregnant mare serum gonadotropin (PMSG). After 48 hours, each female mouse was intraperitoneally injected with 5 IU human chorionic gonadotropin (hCG). After 12 hours, the female mice were killed by neck dislocation, the oviducts were removed, and placed in preheated 37°C M2. The COCs (cumulus oocyte complex) in the ampulla of the oviduct were drawn into the fertilization drop with a 1 ml syringe, and the COCs were placed in a CO2 incubator for 30 minutes. Then, the 8-week-old young male mice were killed by neck dislocation, the epididymis was cut, and the sperm was gently squeezed from the epididymis into the capacitation drop with forceps, and placed in a CO2 incubator for capacitation for 1 hour. After sperm capacitation, an appropriate amount of semen was drawn with a pipette and added to the fertilization drop containing COCs, and the sperm and COCs were incubated for 4 hours. After that, the fertilized eggs were selected for in vitro culture according to the discharge of polar bodies, and the blastocysts were collected 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, USA), and then the embryos were transferred to 4% paraformaldehyde fixative for fixation at room temperature for 1 hour, and then permeabilized with DPBS (Dulbecco's phosphate buffer) containing 0.5% Triton X-100 at room temperature for 1 hour, and then washed once with 0.1% PBS-PVA, and then treated with 4M HCl for 20 minutes, and then treated with 100mM Tris-HCl for 10 minutes, and then blocked with DPBS solution containing 1% BSA at 4°C overnight. The next day, the embryos were incubated with primary antibodies (5-hydroxymethylcytosine and 5-methylcytosine antibodies) at room temperature for 1 hour, and then 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-diamidino-2-phenylindole) for 15 min to stain the cell nucleus, and the fluorescence signals were observed and photographed under a BX51 microscope. The fluorescence intensities of 5-hydroxymethylcytosine and 5-methylcytosine were counted using Image J software, and the ratio was calculated.

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

[0082] (5) Mouse blastocyst uterine transplantation

[0083] On the day of in vitro fertilization, young female mice in natural estrus were selected to mate with young male mice with vasectomy and ligation. The next morning, female mice with vaginal plugs were regarded as pseudopregnant recipients and regarded as day 0.5. The 4.5-day blastocysts obtained in step (2) were used for embryo transfer, and 6 well-developed blastocysts were selected and transplanted into the uterine horns on both sides of the pseudopregnant recipients. On embryonic day 19.5 (14 days after embryo transfer), fetuses were obtained to evaluate the developmental capacity of each embryo.

[0084] (6) Combined administration of VitC, RELN, and FGF2 regulates DNA methylation of in vitro embryos to improve the quality of in vitro fertilized embryo development

[0085] Specific experimental groups:

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

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

[0088] Treatment group 1: VitC (100 μg / ml) alone administration group;

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

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

[0091] Treatment group 4: VitC (50 μg / ml) and FGF2 (250 ng / ml) combined administration group;

[0092] Treatment group 5: VitC (50 μg / ml) and RELN (50 ng / ml) combined administration group;

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

[0094] Treatment group 7: VitC (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml) combined administration group.

[0095] After 96 hours of in vitro culture, the blastocyst rate of each group was counted and calculated. At the same time, 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 of the in vivo group and the blastocysts of each group after 96 hours of in vitro culture were used for embryo transfer. On embryonic day 19.5 (14 days of embryo transfer), the recipient mothers were killed, the number of implantation sites was counted, and then the fetuses and placentas were taken out respectively, and the number of live fetuses was recorded.

[0097] 3. Experimental results

[0098] Table 1 Effect of combined administration of VitC, RELN and FGF2 on the blastocyst rate and 5hmC / 5mC ratio of mice in vitro fertilization

[0099]

[0100]

[0101] Note: The superscripts a, b, and c indicate significant differences, and the significance was tested using one-way analysis of variance. 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 group 1 (41.1±3.0%), treatment group 2 (40.9±2.2%), treatment group 4 (50.7±3.2%), treatment group 6 (49.1±2.2%) or treatment group 7 (53.1±2.0%) can significantly improve the efficiency of in vitro blastocyst development. In addition, VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combined treatment produced a synergistic effect, which was significantly higher than the blastocyst development efficiency of VitC, FGF2 and RELN added alone; and the combination effect of VitC+FGF2+RELN was closest to the in vivo group. It can be seen that the combined use of VitC, FGF2 and RELN helps to 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 can significantly increase the ratio of 5hmC / 5mC in in vitro blastocysts. Moreover, the ratio of 5hmC / 5mC after treatment with VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN was not significantly different from that in the in vivo group, indicating that the combination of VitC, RELN, and FGF2 or the combination of the three has an effective correction effect on DNA methylation modification in IVF embryos.

[0104] Table 2 Effect of combined administration of VitC, RELN and FGF2 on the implantation rate and number of offspring after in vitro fertilization embryo transfer in mice

[0105]

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

[0107] As can be seen from Table 2, for the implantation rate, the treatment groups 1 / 2 / 4 / 5 / 6 / 7 were significantly higher than the control group (in vitro fertilization group) (50.0±4.3%), among which, the combination treatments of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN produced a synergistic effect, which were significantly higher than the embryo implantation rates of VitC, FGF2, and RELN added alone; and the combination effect of VitC+FGF2+RELN was closest to the in vivo group.

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

[0109] Example 2

[0110] This example is the application of combined administration of VitC, RELN and FGF2 in improving the development efficiency and quality of sheep in vitro fertilization embryos.

[0111] 1. Experimental animals and reagents:

[0112] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. VitC, RELN, FGF2 and the components in the basal culture medium in the examples were purchased from Sigma-Aldrich, USA, and the sheep breed was Black-headed Suffolk, which was obtained from Inner Mongolia Sino Sheep Breeding Technology Co., Ltd.

[0113] 2. Experimental methods

[0114] (1) Experimental groups

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

[0116] Treatment group 1: VitC (100 μg / ml) alone administration group;

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

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

[0119] Treatment group 4: VitC (50 μg / ml) and FGF2 (250 ng / ml) combined administration group;

[0120] Treatment group 5: VitC (50 μg / ml) and RELN (50 ng / ml) combined administration group;

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

[0122] Treatment group 7: VitC (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml) combined administration group.

[0123] (2) Sheep oocyte in vitro maturation and embryonic development

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

[0125] Follicular fluid was extracted from the ovaries of living sheep using live egg collection technology, spread on a 60 mm culture dish, and placed on a 38.5°C warming table. COCs containing oocytes with uniform cytoplasm were selected under a stereomicroscope, washed three times with egg extraction fluid, and then washed three times with in vitro maturation fluid pre-equilibrated for 3 hours, transferred to in vitro maturation culture medium, and placed in a 5% CO2, 38.5°C saturated humidity incubator for 24 hours.

[0126] After 24 hours, first thaw the sperm in a 39°C water bath for 1 minute, then transfer it to the sperm upstream solution and place it in an incubator for culture. After 30 minutes, aspirate the supernatant and transfer it to a 1.5mL centrifuge tube, centrifuge it at 1500rpm for 5 minutes, and remove the supernatant. At the same time, place the COCs that have been matured in vitro for 24 hours in a hyaluronidase solution, repeatedly blow and remove the cumulus cells on the surface of the mature oocytes, and after washing, transfer the oocytes to the fertilization solution and add an appropriate amount of semen to incubate for 10-12 hours.

[0127] ② Embryo in vitro culture

[0128] The oocytes after in vitro fertilization were washed three times with embryo culture medium, further transferred to embryo culture medium, and cultured in a saturated humidity incubator with 5% CO2. After 48 hours of culture, the cleavage rate was counted, and culture was continued until day 7, and the D6 and D7 blastocyst rates were counted.

[0129] ③Embryo uterus transplantation and pregnancy testing.

[0130] Select blastocysts with better morphology on D6 or D7. Tie the recipient sheep to the operating stand, fix the tip of the uterine horn on the side of the ovary with the corpus luteum or the side with good corpus luteum, use a paper clip to pierce the wall of the uterine horn at the 1 / 3 of the blood vessel-free area, and then insert the transplant gun tip into the uterine cavity through the needle hole, swing the gun tip, and when it is confirmed that the gun tip is in the uterine cavity, push the piston of the syringe connected to the embryo transfer tube to inject the embryo, then withdraw the embryo transfer tube, and finally return the uterine horn to the abdominal cavity and disinfect it. Use B-ultrasound to detect pregnancy on the 45th day after transplantation, and count the pregnancy rate and the number of offspring born. Among them,

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

[0132] 3. Experimental results

[0133] As can be seen from Table 3, compared with the control group (37.7±2.4%), treatment groups 1 / 2 / 4 / 5 / 6 / 7 all significantly improved the blastocyst development rate of sheep in vitro fertilization embryos, and did not affect the 48h cleavage rate of in vitro fertilization embryos; among them, the blastocyst development rate of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN combination treatments were significantly higher than the blastocyst development rate of VitC, FGF2, and RELN added alone.

[0134] As can be seen from Table 4, compared with the control group (58.1%), the administration of VitC, RELN, and FGF2 alone could not improve the pregnancy rate after embryo transplantation, but the combination treatments of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN produced a synergistic effect, which were significantly higher than the pregnancy rate and the number of offspring born after embryo transplantation when VitC, FGF2, and RELN were treated alone.

[0135] Therefore, the combined administration of two or three of VitC, RELN and FGF2 can significantly improve the development efficiency and quality of sheep in vitro fertilization embryos.

[0136] Table 3 Effect of combined administration of VitC, RELN and FGF2 on the development rate of sheep IVF embryos

[0137]

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

[0139] Table 4 Effect of combined administration of VitC, RELN and FGF2 on pregnancy rate and number of offspring born in sheep IVF embryo transfer

[0140]

[0141]

[0142] Note: The superscripts a, b, and c indicate significant differences, and the significance was analyzed using the chi-square test. Pregnancy rate = number of pregnant females / number of recipient females × 100%.

[0143] Example 3

[0144] This example is the application of combined administration of VitC, RELN and FGF2 in improving the development efficiency and quality of bovine in vitro fertilization embryos.

[0145] 1. Experimental animals and reagents involved in the experiment of the present invention:

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

[0147] 2. Experimental methods

[0148] (1) Experimental groups

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

[0150] Treatment group 1: VitC (100 μg / ml) alone administration group;

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

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

[0153] Treatment group 4: VitC (50 μg / ml) and FGF2 (250 ng / ml) combined administration group;

[0154] Treatment group 5: VitC (50 μg / ml) and RELN (50 ng / ml) combined administration group;

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

[0156] Treatment group 7: VitC (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml) combined administration group.

[0157] (2) Bovine oocyte in vitro maturation and embryonic development

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

[0159] The ovaries of dairy cows were kept warm in 32℃-35℃ physiological saline and sent to the laboratory. The ovarian surface visible follicles were extracted with a 20ml syringe. The cumulus-oocyte complex (COCs) containing more than 3 layers of cumulus cells were washed twice with oocyte washing solution, washed twice with oocyte maturation solution, and then placed in a four-well plate maturation culture solution that was pre-balanced in a CO2 incubator for more than 2h (500μl volume maturation solution, about 50 oocytes, culture conditions are air containing 5% CO2, temperature 39℃, saturated humidity, and culture time is 24h.

[0160] Further, the semen in the capillary tube was thawed in a 38°C water bath, placed in a 15 mL centrifuge tube containing semen washing solution, centrifuged at 1800 r / min for 5 min, and the supernatant was discarded, and repeated twice. Then 50 μl of sperm suspension was added to the 50 μl fertilization drop that had been balanced for more than 2 h to form a 100 μl fertilization drop, which was balanced in an incubator for 1.5 h to allow the sperm to fully capacitate.

[0161] COCs cultured in vitro for 22-24 hours were digested with 0.1% hyaluronidase for about 1 minute, the outer layer of expanded granulosa cells was removed, and the oocytes were washed three times with pre-equilibrated fertilization solution, and the mature oocytes with uniform cytoplasm were aspirated and placed in fertilization drops (15 pieces / 100μl). After the sperm and egg were co-incubated for 8-10 hours in an incubator at 38.5°C and 5% CO2, the oocytes were placed in IVC for thorough washing, sperm adhering to the surrounding were removed, and the oocytes were cultured in mCR1a microdrops pre-equilibrated for more than 2 hours.

[0162] ② Embryo in vitro culture

[0163] The fertilized zygotes were transferred to four-well plates for culture, and 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 culture and continued to be cultured for 5 days. Thereafter, half of the medium was replaced every other day. After fertilization, the number of in vitro fertilized blastocysts was counted and the blastocyst rate was calculated after 7-8 days of in vitro culture.

[0164] ③Embryo uterus transplantation and pregnancy testing.

[0165] The blastocysts with good morphology on D7 or D8 were selected, and the recipient cows were anesthetized after being restrained according to the requirements of artificial insemination. After the vulva was cleaned and disinfected, the cervix was opened, the fallopian tube was flushed, and the embryo transfer tube was inserted into the deep part of one uterine horn by rectal grasping method. The syringe piston connected to the embryo transfer tube was pushed to inject the embryo, and then the embryo transfer tube was withdrawn. B-ultrasound was used to detect pregnancy on the 60th day after transplantation, and the pregnancy rate and number of offspring born were counted.

[0166] 3. Experimental results

[0167] As can be seen from Table 5, compared with the control group (28.9±2.2%), treatment groups 1 / 2 / 4 / 5 / 6 / 7 all significantly improved the blastocyst development rate of in vitro fertilized embryos in dairy cows, and did not affect 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 a synergistic effect, which was significantly higher than the blastocyst development rate of VitC, FGF2 and RELN added alone.

[0168] As can be seen from Table 6, compared with the control group (43.3%), the administration of VitC, RELN, and FGF2 alone could not improve the pregnancy rate after embryo transplantation, but the combination treatments of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN were significantly higher than the pregnancy rate and the number of offspring born after embryo transplantation when VitC, FGF2, and RELN were treated alone.

[0169] Therefore, the combined administration of VitC, RELN, and FGF2 or the combined administration of the three can significantly improve the development efficiency and quality of in vitro fertilization embryos in dairy cows.

[0170] Table 5 Effect of combined administration of VitC, RELN and FGF2 on the development 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 analysis of variance. Cleavage rate = number of two cells / number of eggs × 100%, blastocyst rate = number of blastocysts / number of eggs × 100%.

[0173] Table 6 Effect of combined administration of VitC, RELN and FGF2 on pregnancy rate of in vitro fertilization embryo transfer in dairy cows

[0174]

[0175]

[0176] Note: The superscripts a and b indicate significant differences, and the significance was analyzed using the chi-square test. Pregnancy rate = number of pregnant females / number of recipient females × 100%.

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

Claims

1. A composition for regulating embryonic DNA methylation levels in vitro, characterized in that: It comprises two or more regulators selected from vitamin C or its precursor, RELN and FGF2.

2. The composition for regulating embryonic DNA methylation level in vitro according to claim 1, characterized in that: The composition comprises any combination of (a)-(d): (a) vitamin C or its precursor and RELN, wherein the weight ratio of the vitamin C or its precursor to the RELN is 800-1500:1; (b) vitamin C or its precursor and FGF2, wherein the weight ratio of the vitamin C or its precursor to the FGF2 is 50-500:1; (c) RELN and FGF2, wherein the weight ratio of RELN to FGF2 is 1:1-10; (d) Vitamin C or its precursor, RELN and FGF2, and the weight ratio of the vitamin C or its precursor, the RELN and the FGF2 is (800-1500):1:(1-10).

3. The composition for regulating embryonic DNA methylation level in vitro according to claim 2, characterized in that: The amount of vitamin C or its precursor can make the working concentration of vitamin C reach 0.5-5000 μg / ml during in vitro culture; the amount of FGF2 can make the working concentration of FGF2 reach 0.001-5 μg / ml during in vitro culture; or the amount of RELN can make the working concentration of RELN reach 0.001-5 μg / ml during in vitro culture.

4. The composition for regulating embryonic DNA methylation level in vitro according to claim 1, characterized in that: Further comprising a basic culture medium.

5. A method for regulating embryonic DNA methylation levels in vitro, characterized in that: The method comprises the step of contacting the embryo in vitro with a composition according to any one of claims 1 to 4.

6. The method for regulating embryonic DNA methylation level in vitro according to claim 5, characterized in that: The regulation includes increasing the amount of 5hmC in embryonic DNA, decreasing the amount of 5mC and / or increasing the ratio of 5hmC / 5mC; or making the ratio of 5hmC / 5mC close to the level of developing embryos in vivo.

7. A method for improving in vitro embryo development, characterized in that: The method comprises the step of contacting the embryo in vitro with a composition according to any one of claims 1 to 4.

8. The method for improving in vitro embryo development according to claim 7, characterized in that: The improvement includes improving the efficiency and / or quality of embryo development.

9. The method for improving in vitro embryo development according to claim 7, characterized in that: The improvement includes increasing the cleavage rate in vitro, increasing the number of blastocysts or the blastocyst rate obtained in vitro and / or the implantation rate of embryos after transplantation and the number of fetuses produced.

10. Use of the composition according to any one of claims 1 to 4 in animal genetic improvement, breeding, in vitro embryo production or culture.

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