Composition and method for regulating in vitro embryonic DNA methylation modification, and use thereof in improving embryonic development efficiency and quality

Abstract

Provided are a composition and method for regulating in vitro embryonic DNA methylation modification, and use thereof in improving embryonic development efficiency and quality. Aiming at the problems of low embryonic development rate and low subsequent development potential caused by abnormal epigenetic modification of in vitro embryos, provided is a composition comprising two or more regulators selected from vitamin C or a precursor thereof, RELN, and FGF2, which can effectively correct DNA methylation, thereby greatly improving the development rate and quality of in vitro fertilized embryos, and providing an effective strategy for upgrading the livestock industry.

Description

Compositions, methods of modulating DNA methylation modification of in vitro embryos and applications in improving efficiency and quality of embryo development

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the application with the filing date of June 27, 2024, application number CN202410845339.5, titled “Compositions, methods of modulating DNA methylation modification of in vitro embryos and applications in improving efficiency and quality of embryo development”, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of biotechnology, and particularly 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

[0004] Mammalian in vitro embryo production technology has been widely used in livestock genetic improvement, elite breed propagation and human assisted reproduction fields. However, the technical bottleneck of low development efficiency and poor quality of in vitro embryos has been difficult to break through. It is currently generally believed that the abnormal epigenetic modification of in vitro embryos caused by the imperfect composition of in vitro embryo development culture medium is an important reason for the low development potential and poor quality of in vitro embryos, which greatly limits the development of in vitro embryo production technology. Therefore, optimizing the composition of in vitro embryo culture medium to correct the abnormal epigenetic modification of in vitro embryos is the most direct and effective strategy to improve the development efficiency and quality of in vitro embryos and perfect the in vitro embryo production technology.

[0005] During early embryonic development, there are dramatic changes in DNA methylation epigenetic modification, including DNA methylation erasure (active demethylation) and establishment (de novo methylation). Current gene knockout evidence clearly shows that abnormalities in DNA demethylation and de novo methylation in early embryos can cause embryonic development abnormalities or low development potential in later stages. However, a large amount of research evidence shows that there is a common problem of abnormal DNA methylation modification in in vitro fertilized embryos of different species, including insufficient active demethylation of DNA (incomplete erasure) and imperfect establishment of de novo methylation of DNA. Therefore, correcting abnormal DNA methylation modification in in vitro fertilized embryos is one of the key means to improve the development efficiency and development potential of in vitro embryos.

[0006] However, due to the complexity of methylation modification, the current correction technology for DNA methylation modification of in vitro embryo development still has the problems of low development efficiency and poor quality.

[0007] The information in the background section is only for the purpose of promoting an understanding of the general background of the application. It should not be taken as an acknowledgement or any form of suggestion that it forms the general knowledge of those skilled in the art before the present application was conceived.

[0008] SUMMARY

[0009] To solve at least part of the technical problems in the prior art, the present application provides a composition for in vitro regulating DNA methylation level of embryo and application. Specifically, the present application comprises the following contents.

[0010] In a first aspect, the present application provides a composition for in vitro regulating DNA methylation level of embryo, comprising two or more modulators selected from vitamin C or its precursor, RELN and FGF2.

[0011] In some embodiments, the composition for in vitro regulating DNA methylation level of embryo according to the present application comprises any combination of (a)-(d):

[0012] (a) vitamin C or its precursor and RELN, and the weight ratio of the vitamin C or its precursor to the RELN is 800-1500:1;

[0013] (b) vitamin C or its precursor and FGF2, and the weight ratio of the vitamin C or its precursor to the FGF2 is 50-500:1;

[0014] (c) RELN and FGF2, and the weight ratio of the RELN to the FGF2 is 1:1-10;

[0015] (d) vitamin C or its precursor, RELN and FGF2, and the weight ratio of the vitamin C or its precursor, the RELN to the FGF2 is (800-1500):1:(1-10).

[0016] In some embodiments, the composition for in vitro regulating DNA methylation level of embryo according to the present application, the amount of vitamin C or its precursor can achieve a working concentration of 0.5-5000 μg / ml; the amount of FGF2 can achieve a working concentration of 0.001-5 μg / ml; or the amount of vitamin C or its precursor can achieve a working concentration of 0.001-5 μg / ml.

[0017] In some embodiments, the composition for in vitro regulating DNA methylation level of embryo according to the present application further comprises a basal medium.

[0018] In a second aspect, the present application provides a method for in vitro regulating DNA methylation level of embryo, comprising the step of contacting the embryo with the composition of the first aspect in vitro.

[0019] In some embodiments, the method for modulating the level of DNA methylation of an embryo in vitro according to the present application, the modulating comprises increasing the amount of 5hmC, decreasing the amount of 5mC and / or increasing the ratio of 5hmC / 5mC in the DNA of the embryo; or bringing the ratio of 5hmC / 5mC close to the level of in vivo developing embryos.

[0020] In a third aspect, the present application provides a method for improving the development of an embryo in vitro, comprising the step of contacting the embryo in vitro with the composition of the first aspect.

[0021] In some embodiments, the method for improving the development of an embryo in vitro according to the present application, the improving comprises increasing the efficiency and / or quality of embryo development.

[0022] In some embodiments, the method for improving the development of an embryo in vitro according to the present application, the improving comprises increasing the cleavage rate in vitro, increasing the number or rate of blastocysts obtained in vitro and / or the implantation rate of embryos after implantation and the number of fetuses produced.

[0023] In a fourth aspect, the present application provides the use of the composition of the first aspect in animal genetic improvement, elite breed propagation, in vitro embryo production or culture.

[0024] According to the data released by the International Embryo Transfer Society (IETS), the number of in vitro embryos of cattle has exceeded that of in vivo embryos since 2016, and shows a trend of increasing year by year, so the in vitro embryo production technology has become an effective way to efficiently utilize elite breed resources. The present application provides an effective correction approach and method for DNA methylation in view of the problems of 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 upgrading the livestock 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 application will have great economic and social value for promoting in vitro embryo production of domestic livestock in China. DETAILED DESCRIPTION

[0025] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is intended to be illustrative, but not to be limiting, of the present application, and is understood that it is provided in the interest of promoting the understanding of the concepts of the present application. The details are grouped by functional or structural similarity, and, when possible, described with overlapping reference numerals.

[0026] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range as well as each smaller range between any stated value or intervening value within the stated range is also specifically disclosed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the limits are also included.

[0027] Unless defined otherwise, 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 application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are described in them. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0028] As used herein, the term "embryo" refers to an early stage of a developing organism that is derived from a fertilized egg. Since development is a continuous process, the embryo herein includes different morphological stages of the developing organism, and generally refers to pre-attachment or pre-implantation embryos, such as 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae, blastulae, etc.

[0029] As used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a culture dish, etc., rather than in a living organism (e.g., an animal, a plant, or a microorganism).

[0030] As used herein, the term "DNA methylation level" refers to the degree of methylation of DNA, such as genome or a region thereof or a gene, obtained from an embryo, i.e., the degree of methylation groups added to a particular base. DNA methylation or demethylation is an important mechanism for regulating gene expression, which can change genetic performance or developmental progress without changing the DNA sequence.

[0031] As used herein, the term "modulate", which can also be referred to as "regulate", refers to any change in the methylation level, including up-regulating the methylation level of a particular base, or down-regulating the methylation level of a particular base, and also includes optimizing the methylation level to approach or reach a certain standard. The standard can be a man-made standard or a natural state standard, such as the methylation level of an embryo during in vivo development.

[0032] As used herein, the term "vitamin C", also known as "ascorbic acid", abbreviated as "VitC", is associated with a variety of diseases and is identified by the World Health Organization as an essential drug in the Essential Medicine List.

[0033] Herein, the term "precursor" refers to any substance, molecule or entity that, upon chemical or physical alteration, is capable of acting as or producing Vitamin C. The precursor can be covalently bound or chelated in some manner and released or converted into the active ingredient, in particular Vitamin C, before, simultaneously with or after administration to a subject or target cell or tissue. The precursor can be prepared by modifying a functional group present in the compound in such a way that the modification can be cleaved in a routine operation or in vivo to the parent compound. Precursors include compounds in which a hydroxyl, amino, thiol or carboxyl group is bound to any group that is subject to cleavage to form a free hydroxyl, amino, thiol or carboxyl group, respectively, when administered to a mammalian subject.

[0034] Herein, the term "RELN" means reelin or an active fragment, nucleic acid thereof. Herein, reelin means reelin in full-length form or having or containing the complete sequence, and also includes proteins or isoforms that are homologous and / or orthologous to human reelin. An active fragment means a portion of reelin in full-length form, i.e. a peptide, polypeptide, etc. that is active. Here, reelin can be isolated naturally or can be obtained by artificial synthesis, including genetic engineering or biotechnology, chemical synthesis methods. In the case of isolation, the source of reelin is not limited, and can be the same source as the subject or its tissue, cell, or can be a different source, which is not limited.

[0035] Herein, the term "FGF2" or "bFGF" means fibroblast growth factor II (sometimes also referred to as "basic fibroblast growth factor"), and means fibroblast growth factor II or an active fragment, nucleic acid thereof. Herein, fibroblast growth factor II means fibroblast growth factor II in full-length form or having or containing the complete sequence, and also includes proteins or isoforms that are homologous and / or orthologous to human fibroblast growth factor II. Here, fibroblast growth factor II can be isolated naturally or can be obtained by artificial synthesis, including genetic engineering or biotechnology, 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 tissue, cell, or can be a different source, which is not limited.

[0036] The term "nucleic acid" herein includes DNA and RNA or modified forms thereof, including polynucleotides, oligonucleotides. Preferred is mRNA capable of producing an active protein such as RELN and FGF2. Herein, modified forms refer to one or more components of the nucleic acid, i.e. sugar, base and phosphate structure, which are different from the natural components, preferably different from the natural components produced in the human body. Nucleotide substitutes are molecules in which the ribose phosphate backbone is replaced by a non-ribose phosphate backbone, which places the bases in the correct spatial relationship for hybridization substantially similar to that seen with ribose phosphate backbones, for example, analogs of uncharged ribose phosphate backbones.

[0037] The term "working concentration" also referred to as "use concentration" herein refers to the initial concentration at which the modulators are contacted with the embryos and processed for effective processing. The unit of concentration is not limited and can be, for example, ng / ml, μg / ml, mg / ml, etc.

[0038] [Composition]

[0039] In a first aspect of the present application, there is provided a composition for in vitro regulation of DNA methylation level of an embryo, sometimes simply referred to herein as "the composition of the present application", which comprises a combination of different modulators, wherein the modulators are selected from two or more of vitamin C or a precursor thereof, RELN and FGF2. The modulators in the combination of the present application interact with each other to produce a synergistic effect, greatly enhancing the regulation effect compared with a single modulator. The form of the composition of the present application is not limited and can be a solid such as a dry powder or a liquid such as a solution.

[0040] In certain embodiments, the composition of the present application can be a combination of the above-mentioned modulators, i.e. contains no other components than the modulators and inevitable impurities. The modulators in the composition of the present application can be present in a mixed form or in a form in which two or more modulators are present separately. In use, the modulators present separately can be mixed in advance before use or used simultaneously or sequentially.

[0041] In certain embodiments, the composition of the present application comprises the above-mentioned combination of modulators and further comprises other components in addition thereto. The composition or type of the other components is not limited and can be freely selected as desired. Such other components can be selected from any known components, particularly reagents, compositions related to embryo culture, such as culture medium, culture solution or additives thereof, etc.

[0042] In certain embodiments, the modulator combination of the application is a combination of Vitamin C or a precursor thereof 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 of both Vitamin C and RELN can be achieved, greatly enhancing the regulatory effect.

[0043] In certain embodiments, the modulator combination of the application is a combination of Vitamin C or a precursor thereof and FGF2. Preferably, the weight ratio of Vitamin C to FGF2 is 50-500: 1, such as 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 of both Vitamin C and FGF2 can be achieved, greatly enhancing the regulatory effect.

[0044] In certain embodiments, the modulator combination of the application 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 of both RELN and FGF2 can be achieved, greatly enhancing the regulatory effect.

[0045] In certain embodiments, the amount of vitamin C or its precursor of the present application is capable of achieving a working concentration of vitamin C in use of 0.5 to 5000 μg / ml. The concentration or amount in the composition is not limited as long as it is capable of achieving the above range in use. In the case where the composition is a solution and is added as an ingredient, the concentration of vitamin C or its precursor in the composition can be the working concentration, so that it can be diluted to the above working concentration when the composition is added to the embryo culture medium. In the case where the composition is a solution and is used directly as the 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 to 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, 9.5 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 120 μg / ml, 140 μg / ml, 160 μg / ml, 180 μg / ml, 200 μg / ml, 220 μg / ml, 240 μg / ml, 260 μg / ml, 280 μg / ml, 300 μg / ml, 350 μg / ml, 400 μg / ml, 450 μg / ml, 500 μg / ml, 550 μg / ml, 600 μg / ml, 650 μg / ml, 700 μg / ml, 750 μg / ml, 800 μg / ml, 900 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml, 2500 μg / ml, 3000 μg / ml, 3500 μg / ml, 4000 μg / ml, 4500 μg / ml.

[0046] In certain embodiments, the amount of FGF2 of the present application is capable of achieving a working concentration of FGF2 of 0.001 to 5 μg / ml. The concentration or amount of FGF2 in the composition is not limited as long as it is capable of achieving the above range at the time of use. In the case where the composition is a solution and is used as an additive, the concentration of FGF2 in the composition can be a working concentration, so that it is capable of being diluted to achieve the above working concentration when the composition is added to an embryo culture solution. In the case where the composition is a solution and is directly used as a culture solution, the concentration of FGF2 in the composition is usually substantially equal to the working concentration. The working concentration of FGF2 is preferably 0.05 to 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, 0.55 μg / ml, 0.6 μg / ml, 0.65 μg / ml, 0.7 μg / ml, 0.75 μg / ml, 0.8 μg / ml, 0.85 μg / ml, 0.9 μg / ml, 0.95 μg / ml, 1 μg / ml, 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.

[0047] In certain embodiments, the amount of RELN of the present application is capable of achieving a working concentration of RELN of 0.001 to 5 μg / ml. The working concentration of RELN is preferably 0.05 to 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, 0.55 μg / ml, 0.6 μg / ml, 0.65 μg / ml, 0.7 μg / ml, 0.75 μg / ml, 0.8 μg / ml, 0.85 μg / ml, 0.9 μg / ml, 0.95 μg / ml, 1 μg / ml, 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.

[0048] [Method for controlling DNA methylation level of embryo in vitro]

[0049] In a second aspect, the present application provides a method for modulating the level of DNA methylation in an embryo in vitro, comprising the step of contacting the embryo in vitro with the composition of the first aspect.

[0050] In certain embodiments, the contacting of the present application is performed in a liquid, i.e. the embryo is treated in a culture medium containing the combination of modulators.

[0051] In certain embodiments, the modulation of the present application comprises increasing the amount of 5hmC in the DNA of the embryo. Herein, 5hmC refers to 5-hydroxymethylcytosine, and increasing the amount of 5hmC in the DNA of the embryo refers to the relative amount (especially relative to the untreated group or the group treated with a single modulator) or the absolute amount of 5-hydroxymethylcytosine in the DNA bases being increased.

[0052] In certain embodiments, the modulation of the present application comprises decreasing the amount of 5mC. Herein, 5mC refers to 5-methylcytosine, and decreasing the amount of 5mC refers to the relative amount (especially relative to the untreated group or the group treated with a single modulator) or the absolute amount of 5-methylcytosine in the DNA bases being decreased.

[0053] In certain embodiments, the modulation of the present application refers to increasing the ratio of 5hmC / 5mC. Herein, the increase or improvement of 5hmC relative to 5mC includes the case where the amount of 5hmC is unchanged, and the amount of 5mC is decreased; includes the case where the amount of 5mC is unchanged, and the amount of 5hmC is increased; and also includes the case where the amount of 5hmC is increased, and the amount of 5mC is decreased.

[0054] In certain embodiments, the modulation of the present application refers to bringing the ratio of 5hmC / 5mC close to the level of in vivo developing embryos. Compared with in vivo developing embryos, the DNA methylation in embryos cultured in vitro is abnormal, especially the ratio of 5hmC / 5mC is decreased. The composition of the present application can bring the ratio of 5hmC / 5mC close to or substantially consistent with the corresponding level of in vivo developing embryos. Herein, "close to" refers to the difference 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%, or even within 0.05% of the level of DNA methylation in in vivo developing embryos. Herein, "substantially consistent with" refers to the ratio being consistent with the corresponding level of in vivo developing embryos by 90% or more, preferably by 92% or more, more preferably by 95% or more, such as by 96% or more, by 97% or more, by 98% or more, by 99% or more, by 99.5% or more, by 99.8% or more, or by 99.9% or more.

[0055] [Method for improving the development of embryos in vitro]

[0056] In a third aspect of the present application, a method for improving in vitro embryo development is provided, which comprises the step of contacting an embryo with the composition of the first aspect in vitro.

[0057] The improvements of the present application include improving embryo development efficiency and / or quality, including improving cleavage rate, number of blastocysts or blastocyst rate in vitro, and implantation rate and number of fetuses produced after implantation.

[0058] [Uses]

[0059] In a fourth aspect of the present application, the composition of the first aspect is used in animal genetic improvement, elite breeding, and in vitro embryo production. The composition and its modulators have been described in detail in the first aspect, and will not be repeated here.

[0060] In certain embodiments, the animal of the present application 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, 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 certain embodiments, the animal of the present application is a livestock, including a pig, a cow, a sheep.

[0061] In certain embodiments, the genetic improvement and elite breeding of the present application include genetic improvement or breeding relying on in vitro embryo production technology.

[0062] Example 1

[0063] This example is the application of combined administration of VitC, RELN, and FGF2 in improving DNA methylation modification of mouse in vitro fertilized embryos and improving the efficiency and quality of mouse in vitro fertilized embryo development.

[0064] 1. Experimental animals and reagents:

[0065] If not specifically indicated, the technical means used in the examples are conventional means well known to those skilled in the art. In the examples, the culture medium KSOM+AA was purchased from Millipore Corporation of the United States, VitC, RELN, and FGF2 were purchased from Sigma-Aldrich Corporation of the United States, ICR mice were purchased from Spafas (Beijing) Biotechnology Co., Ltd., and M2 culture medium was purchased from Zhongke Meichen (Beijing) Technology Co., Ltd.

[0066] 2. Experimental methods

[0067] (1) Experimental grouping

[0068] In vivo group: in vivo fertilization group;

[0069] Control group: normal in vitro fertilization group;

[0070] Treatment group 1: VitC alone administration group;

[0071] Treatment group 2: FGF2 alone administration group;

[0072] Treatment group 3: RELN alone administration group;

[0073] Treatment group 4: VitC and FGF2 combined administration group;

[0074] Treatment group 5: VitC and RELN combined administration group

[0075] Treatment group 6: RELN and FGF2 combined administration group;

[0076] Treatment group 7: VitC, RELN, FGF2 combined administration group.

[0077] (2) Collection of in vivo fertilized embryos in mice

[0078] Healthy 8-week-old female mice were selected, and 5 IU of pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally for synchronization of estrus, and 48 h later, 5 IU of human chorionic gonadotropin (hCG) was injected intraperitoneally for superovulation. Then, the female mice were caged with 10-week-old male mice, and the next morning, the female mice with vaginal plugs were placed in separate cages. The default fertilization time was 24 hours at night, and the embryos were recorded as E0.5. When collecting blastocysts, 96 h after hCG injection, the female mice were surgically opened, and the uteruses were cut and placed in M2 culture solution. The blastocysts were collected using an oocyte flushing needle.

[0079] (3) Collection of in vitro fertilized embryos in mice

[0080] 8-week-old young female mice were used as experimental materials, and each female mouse was injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin (PMSG), and 48 h later, each female mouse was injected intraperitoneally with 5 IU of human chorionic gonadotropin (hCG). Twelve hours later, the female mice were sacrificed by cervical dislocation, and the oviducts were removed and placed in preheated 37°C M2. The COCs (cumulus-oocyte complexes) in the ampulla of the oviducts were drawn into the fertilization drop using a 1 ml syringe, and the oviducts were placed in a CO2 incubator for 30 minutes. Then, 8-week-old young male mice were sacrificed by cervical dislocation, and the epididymides were cut and the sperm was gently squeezed from the epididymides into the capacitation drop using forceps. After capacitation, an appropriate amount of sperm was added to the COCs-containing fertilization drop, and the sperm was incubated with the COCs for 4 h. After that, the fertilized eggs were selected according to the presence of polar bodies for in vitro culture, and the blastocysts were collected 96 h after in vitro culture.

[0081] (4) Embryo 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) immunofluorescence staining

[0082] The embryo obtained in step (2) is washed three times with 0.1% PBS-PVA, the zona pellucida is removed with acid Tyrode solution (T1788, Sigma, USA), and then the embryo is transferred into 4% paraformaldehyde fixing solution for room temperature fixation for 1 h, followed by permeation with 0.5% Triton X-100 in DPBS (Dulbecco's phosphate buffered saline) at room temperature for 1 h, followed by washing once with 0.1% PBS-PVA, followed by treatment with 4M HCl for 20 min, followed by treatment with 100 mM Tris-HCl for 10 min, followed by blocking the embryo with 1% BSA in DPBS solution at 4°C overnight. The next day, the primary antibody (5-hydroxymethylcytosine and 5-methylcytosine antibody) is incubated at room temperature for 1 h, followed by washing three times with 0.5% Triton X-100 in DPBS at room temperature, followed by incubation with the secondary antibody at room temperature for 1 h. Finally, DAPI (4', 6-diamidino-2-phenylindole) is incubated for 15 min to stain the nucleus, the fluorescence signal is observed under a BX51 microscope, and the fluorescence intensity of 5-hydroxymethylcytosine and 5-methylcytosine is counted using ImageJ software, and the ratio is calculated.

[0083] The primary antibody used is 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 antibody used is as follows: Alexafluor594 goat anti-rabbit antibody (1:1000 dilution, Invitrogen, A-11034, USA), Alexafluor488 goat anti-mouse antibody (1:1000 dilution, Invitrogen, A-11030, USA).

[0084] (5) Mouse blastocyst uterine transplantation

[0085] On the day of in vitro fertilization, a young female mouse in natural estrus is mated with a vasectomized and ligated young male mouse. The next morning, the female mouse with a vaginal plug is considered a pseudopregnant recipient, and is considered day 0.5. The 4.5-day blastocysts obtained in step (2) are subjected to embryo transplantation, and 6 well-developed blastocysts are selected for transplantation into the two uterine horn positions of the pseudopregnant recipient. On day 19.5 of the embryo (14 days after embryo transplantation), the fetuses are obtained to evaluate the developmental capacity of each embryo.

[0086] (6) Combined administration of VitC, RELN, and FGF2 regulates DNA methylation of in vitro embryos to improve the developmental quality of in vitro fertilized embryos

[0087] Specific experimental grouping:

[0088] In vivo group: in vivo fertilization group;

[0089] Control group: normal in vitro fertilization group;

[0090] Treatment group 1: VitC (100 μg / ml) alone administration group;

[0091] Treatment group 2: FGF2 (500 ng / ml) alone administration group;

[0092] Treatment group 3: RELN (100 ng / ml) alone administration group;

[0093] Treatment group 4: VitC (50 μg / ml) and FGF2 (250 ng / ml) combined administration group;

[0094] Treatment group 5: VitC (50 μg / ml) and RELN (50 ng / ml) combined administration group;

[0095] Treatment group 6: RELN (50 ng / ml) and FGF2 (250 ng / ml) combined administration group;

[0096] Treatment group 7: VitC (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml) combined administration group.

[0097] After in vitro culture for 96 h, the blastocyst rates of each group were counted and calculated. At the same time, the 5-hmC / 5-mC immunofluorescence intensity ratio of blastocysts in the in vivo group, control group and treatment groups 1 / 2 / 3 / 4 / 5 / 6 / 7 were compared respectively.

[0098] The in vivo group blastocysts and the blastocysts of each group after in vitro culture for 96 h were subjected to embryo transfer. On the 19.5th day of the embryo (14 days after embryo transfer), after the recipient female mice were sacrificed, 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.

[0099] 3. Experimental results

[0100] Table 1 Effect of combined administration of VitC, RELN and FGF2 on blastocyst rate and 5hmC / 5mC ratio of mouse in vitro fertilization

[0101] Note: a, b, c superscript indicates different significant differences, and significance is tested by one-way ANOVA. Blastocyst rate = number of blastocysts / number of fertilized eggs.

[0102] As can be seen from Table 1, compared with the control group (in vitro fertilization group) (32.5±3.3%), the in vitro blastocyst development efficiency of the treatment group 1 (41.1±3.0%), the treatment group 2 (40.9±2.2%), the treatment group 4 (50.7±3.2%), the treatment group 6 (49.1±2.2%) or the treatment group 7 (53.1±2.0%) can be significantly improved, in addition, the VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combination treatment produces a synergistic effect, which is significantly higher than the VitC, FGF2 and RELN single addition blastocyst development efficiency; and the VitC+FGF2+RELN three combination effect is the closest to the in vivo group. It can be seen that the joint use of VitC, FGF2 and RELN helps to improve the development efficiency of mouse in vitro fertilization embryos.

[0103] Compared with the control group (in vitro fertilization group) (1.89±0.07), the treatment group 1 / 4 / 5 / 6 / 7 can significantly improve the ratio of 5hmC / 5mC in in vitro blastocyst. And the ratio of 5hmC / 5mC after VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combination treatment has no significant difference compared with the in vivo group, which shows that the joint use of two or three of VitC, RELN and FGF2 has an effective correction effect on the DNA methylation modification of in vitro fertilization embryos.

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

[0105] Note: a, b, c, superscript indicates different significant differences, and significant difference is tested by one-way ANOVA. Implantation rate = number of implantation sites / number of transferred embryos, average fetal number = total fetal number / pregnant recipient number.

[0106] As can be seen from Table 2, for the implantation rate, the treatment group 1 / 2 / 4 / 5 / 6 / 7 is significantly higher than the control group (in vitro fertilization group) (50.0±4.3%), among which, the VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combination treatment produces a synergistic effect, which is significantly higher than the VitC, FGF2 and RELN single addition embryo implantation rate; and the VitC+FGF2+RELN three combination effect is the closest to the in vivo group.

[0107] The above results show that the joint use of two or three of VitC, RELN and FGF2 can significantly improve the embryo implantation rate and the number of offspring after mouse in vitro fertilization embryo transfer.

[0108] Example 2

[0109] The application of combined administration of VitC, RELN and FGF2 in improving the efficiency and quality of embryo development in vitro fertilization of sheep.

[0110] 1. Experimental animals and reagents

[0111] If not specifically indicated, 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 basic culture medium in the examples are purchased from Sigma-Aldrich Company, USA, and the sheep breed is black head Suffolk, which is obtained from Inner Mongolia Saeno Sheep Breeding Technology Co., Ltd.

[0112] 2. Experimental methods

[0113] (1) Experimental grouping

[0114] Control group: normal in vitro fertilization group

[0115] Treatment group 1: VitC (100 μg / ml) alone administration group

[0116] Treatment group 2: FGF2 (500 ng / ml) alone administration group

[0117] Treatment group 3: RELN (100 ng / ml) alone administration group

[0118] Treatment group 4: VitC (50 μg / ml) and FGF2 (250 ng / ml) combined administration group

[0119] Treatment group 5: VitC (50 μg / ml) and RELN (50 ng / ml) combined administration group

[0120] Treatment group 6: RELN (50 ng / ml) and FGF2 (250 ng / ml) combined administration group

[0121] Treatment group 7: VitC (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml) combined administration group

[0122] (2) In vitro maturation of sheep oocytes and embryo development

[0123] ① Collection of oocytes, in vitro maturation and in vitro fertilization

[0124] 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.

[0125] 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.

[0126] ②In vitro embryo culture

[0127] 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 days 6 and 7 after continuing culture.

[0128] ③ Embryo transfer to the uterus and pregnancy testing.

[0129] 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.

[0130] Pregnancy rate = number of pregnant female animals / number of recipients of in vitro fertilization embryo transfer × 100%.

[0131] 3. Experimental Results

[0132] As shown in Table 3, compared with the control group (37.7±2.4%), the treatment groups 1 / 2 / 4 / 5 / 6 / 7 significantly improved the in vitro fertilization embryo blastocyst development rate of sheep, and did not affect the 48h cleavage rate of in vitro fertilization embryos; among them, the VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combined treatment was significantly higher than the VitC, FGF2 and RELN single addition blastocyst development rate.

[0133] As shown in Table 4, compared with the control group (58.1%), VitC, RELN, FGF2 alone cannot improve the pregnancy rate after embryo transfer, but VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combined treatment produces synergistic effect, which is significantly higher than the VitC, FGF2 and RELN single treatment embryo transfer pregnancy rate and offspring number.

[0134] Therefore, the combined use of VitC, RELN, FGF2 or the combined use of the three can significantly improve the development efficiency and quality of in vitro fertilization embryos of sheep.

[0135] Table 3 Effect of combined use of VitC, RELN, FGF2 on the development rate of in vitro fertilization embryos of sheep

[0136] Note: a, b superscript indicates significant difference, and significance is tested by one-way analysis of variance. Cleavage rate = two-cell number / egg number x 100%, blastocyst rate = blastocyst number / egg number x 100%.

[0137] Table 4 Effect of combined use of VitC, RELN, FGF2 on the pregnancy rate and offspring number of in vitro fertilization embryos of sheep

[0138] Note: a, b, c superscript indicates significant difference, and significance is analyzed by chi-square test. Pregnancy rate = pregnant ewe number / recipient ewe number x 100%.

[0139] Example 3

[0140] This example is the application of the combined use of VitC, RELN, FGF2 in improving the development efficiency and quality of in vitro fertilization embryos of cattle.

[0141] 1. The experimental animals and reagents involved in the experiments of the present application:

[0142] The technical means used in the examples are conventional means well known to those skilled in the art, unless otherwise specified. VitC, RELN, FGF2 and the components in the basal medium used in the examples were purchased from Sigma-Aldrich, USA, and the cows were from Adopt-a-Cow Biotech Co., Ltd.

[0143] 2. Experimental methods

[0144] (1) Experimental grouping

[0145] Control group: normal in vitro fertilization group;

[0146] Treatment group 1: VitC (100 μg / ml) alone administration group;

[0147] Treatment group 2: FGF2 (500 ng / ml) alone administration group;

[0148] Treatment group 3: RELN (100 ng / ml) alone administration group;

[0149] Treatment group 4: VitC (50 μg / ml) and FGF2 (250 ng / ml) combined administration group;

[0150] Treatment group 5: VitC (50 μg / ml) and RELN (50 ng / ml) combined administration group;

[0151] Treatment group 6: RELN (50 ng / ml) and FGF2 (250 ng / ml) combined administration group;

[0152] Treatment group 7: VitC (33.3 μg / ml), RELN (33.3 ng / ml) and FGF2 (166.7 ng / ml) combined administration group.

[0153] (2) In vitro maturation of bovine oocytes and embryonic development

[0154] ① Collection of oocytes, in vitro maturation and in vitro fertilization

[0155] The cow ovaries were kept at 32-35°C in physiological saline and sent to the laboratory. The visible follicles on the surface of the ovary were extracted with a 20 ml syringe. The cumulus-oocyte complex (COCs) containing 3 layers of cumulus cells were washed twice with oocyte washing solution, and then washed twice with oocyte maturation solution, and then placed in a four-well plate maturation medium (500 μl volume of maturation medium, about 50 oocytes, culture conditions: 5% CO2 in air, temperature 39°C, saturated humidity, culture time 24 h) pre-equilibrated in a CO2 incubator for more than 2 h.

[0156] Further, the fine tube semen is thawed in a 38℃ water bath, then placed in a 15 mL centrifuge tube containing washed semen, centrifuged at 1800 r / min for 5 min, the supernatant is discarded, and the operation is repeated twice. Then 50 μl of sperm suspension is added to 50 μl of fertilization drop which has been balanced for more than 2 h to form a 100 μl fertilization drop. The fertilization drop is balanced in the incubator for 1.5 h to allow the sperm to fully capacitate.

[0157] The COCs cultured in vitro for 22-24 h are digested with 0.1% hyaluronidase for about 1 min to remove the outer layer of extended granulosa cells, washed with pre-equilibrated fertilization fluid for 3 times, and the mature oocytes with uniform cytoplasm are aspirated and placed in the fertilization drop (15 / 100 μl). After 8-10 h of co-incubation of the sperm and the oocyte in a 38.5℃, 5% CO2 incubator, the oocyte is washed in the IVC to remove the sperm adhered to the surrounding, and placed in the pre-equilibrated mCRlaa microdrop for more than 2 h for culture.

[0158] ②In vitro culture of embryos

[0159] The zygotes after fertilization are transferred to a four-well plate for culture, 500 μl of early development fluid is used for culture for 2 d, the cleavage embryos are counted, and then moved to 500 μl of late development fluid for continued culture for 5 d. The medium is replaced every other day in half amount. The number of in vitro fertilized blastocysts and the blastocyst rate are counted after 7-8 d of in vitro culture after fertilization.

[0160] ③Embryo uterine transplantation and pregnancy detection.

[0161] The good blastocysts of D7 or D8 morphology are selected, the recipient cow is fixed according to the requirements of artificial insemination, anesthetized, the vulva is washed and disinfected, the cervix is opened, the oviduct is flushed, the transfer tube is inserted into the deep part of the uterine horn by rectal control method, the syringe piston connected to the transfer tube is pushed to inject the embryo, and then the transfer tube is withdrawn. The pregnancy is detected by B-ultrasound on the 60th day after transplantation, and the pregnancy rate and the number of offspring are counted.

[0162] 3、Experimental results

[0163] As shown in Table 5, compared with the control group (28.9±2.2%), the treatment groups 1 / 2 / 4 / 5 / 6 / 7 significantly improved the in vitro fertilization embryo blastocyst development rate of the dairy cow, and did not affect the 48 h cleavage rate of the in vitro fertilized embryo; among them, the VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combination treatment produced a synergistic effect, and the blastocyst development rate was significantly higher than that of VitC, FGF2 and RELN alone.

[0164] From Table 6, it can be seen that compared with the control group (43.3%), VitC, RELN, FGF2 alone cannot improve the pregnancy rate after embryo transfer, but VitC+FGF2, VitC+RELN, RELN+FGF2, VitC+FGF2+RELN combination treatment are significantly higher than VitC, FGF2 and RELN alone in the pregnancy rate after embryo transfer and the number of offspring.

[0165] Therefore, the joint administration of VitC, RELN, FGF2 or the joint administration of all three can significantly improve the efficiency and quality of bovine in vitro fertilization embryo development.

[0166] Table 5 Effect of joint administration of VitC, RELN, FGF2 on bovine in vitro fertilization embryo development rate

[0167] Note: a, b superscript indicates significant difference, significance is tested using one-way ANOVA. Cleavage rate = 2-cell number / ovum number x 100%, blastocyst rate = blastocyst number / ovum number x 100%.

[0168] Table 6 Effect of joint administration of VitC, RELN, FGF2 on bovine in vitro fertilization embryo transfer pregnancy rate

[0169] Note: a, b superscript indicates significant difference, significance is analyzed using chi-square test. Pregnancy rate = pregnant female number / recipient female number x 100%.

[0170] Although the present application has been described with reference to the example embodiments, it will be appreciated that the application is not limited to the example embodiments disclosed. Various modifications or changes in the example embodiments of the application described herein can be made by those skilled in the art without departing from the scope or spirit of the application. The scope of the claims should be based on the broadest interpretation of the language used in the claims to encompass all modifications and equivalent structures and functions.

Claims

1. A composition for in vitro modulating the level of DNA methylation in an embryo, characterized in that, The composition comprises two or more modulators selected from the group consisting of Vitamin C or a precursor thereof, RELN and FGF2.

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

3. The composition for in vitro regulation of the methylation level of DNA of an embryo according to claim 2, characterized in that, The amount of the Vitamin C or a precursor thereof is capable of achieving a working concentration of Vitamin C in in vitro culture of 0.5-5000 μg / ml; the amount of the FGF2 is capable of achieving a working concentration of FGF2 in in vitro culture of 0.001-5 μg / ml; or the amount of the RELN is capable of achieving a working concentration of RELN in in vitro culture of 0.001-5 μg / ml.

4. The composition for regulating the DNA methylation level of an embryo in vitro according to claim 1, further comprising a basal medium.

5. A method of modulating the level of DNA methylation in an embryo in vitro, characterized in that, The method comprises the step of contacting the embryo with the composition according to any one of claims 1-4 in vitro.

6. The method of claim 5, wherein the method is in vitro. The regulation comprises increasing the amount of 5hmC, decreasing the amount of 5mC and / or increasing the ratio of 5hmC / 5mC in the DNA of the embryo; or bringing the ratio of 5hmC / 5mC close to the level of an in vivo developing embryo.

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

8. The method for improving in vitro embryo development according to claim 7, wherein, The improvement comprises increasing the efficiency and / or quality of embryo development.

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

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

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