Preparation for in-vitro embryo production and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation for in-vitro embryo production and application of the preparation, and the preparation contains at least one of the following components: SEMA4 protein; an Fbxo48 inhibitor; the invention discloses a flavone C-glycoside compound. According to the preparation for in-vitro embryo production, at least one of SEMA4 type protein, Fbxo48 inhibitor and flavone C-glycoside compound is added, so that mitochondrial division and biosynthesis are promoted, mitochondrial functions (including membrane potential, respiratory intensity, ATP level and the like) are enhanced, active oxygen is effectively removed, lipid peroxidation is inhibited and the like; therefore, the maturation quality of in-vitro oocytes and the development potential of in-vitro embryos are remarkably improved in a plurality of stages such as in-vitro maturation of the oocytes, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to formulations for in vitro embryo production and their applications. Background Technology

[0002] In the field of in vitro embryo culture, the production process of in vitro embryos begins with the collection of oocytes and mainly includes three stages: in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), and in vitro culture of fertilized eggs (IVC). Among them, in vitro maturation of oocytes is the starting point and foundation of the entire production process. Its success or failure directly determines the potential for subsequent fertilization and embryo development, and thus becomes the rate-limiting step that affects the overall efficiency and quality of in vitro embryo production.

[0003] At present, the existing IVM system still has significant limitations, specifically: (1) Only 30%-50% of immature oocytes in the conventional culture system can complete meiosis, and a large number of oocytes cannot meet the fertilization standard due to problems such as meiosis arrest and abnormal spindle assembly; (2) Even if the oocytes are morphologically mature, their cytoplasmic maturity is often insufficient, and there are problems such as mitochondrial functional defects and weak antioxidant capacity, which leads to their development potential after fertilization being far lower than that of mature oocytes in vivo. Their embryo cleavage rate and blastocyst formation rate are 30%-50% lower than those of mature systems in vivo, and the risk of embryonic chromosomal abnormalities is significantly increased, which seriously affects the clinical pregnancy outcome.

[0004] The underlying reasons for the above problems mainly stem from the differences between the in vitro culture environment and the in vivo physiological environment. First, mitochondria, as the "energy factory" of oocytes, directly determine the quality of maturation due to their functional state. However, in the in vitro culture environment, oocyte mitochondria often exhibit problems such as insufficient quantity, structural abnormalities (e.g., cristae morphology destruction), and decreased membrane potential. This leads to a decrease in respiratory chain complex activity and severely insufficient ATP production (50%-80% lower than in vivo mature oocytes). Energy deficiency directly affects highly energy-consuming meiotic processes such as spindle assembly and chromosome separation, thereby increasing the aneuploidy rate. Second, fluctuations in oxygen concentration and imbalances in nutrient metabolism in the in vitro culture environment lead to a large accumulation of reactive oxygen species (ROS) in the cells, causing lipid peroxidation (significantly increased MDA levels), protein and DNA damage. At the same time, the endogenous antioxidant defense system of in vitro mature oocytes (such as the activity of enzymes like SOD and GPx) is often insufficiently activated, failing to effectively clear excess ROS, forming a vicious cycle of oxidative damage, and ultimately triggering oocyte apoptosis.

[0005] Although some optimization strategies have addressed the aforementioned problems to some extent by adding single antioxidants or growth factors to in vitro culture systems, they lack a systematic approach and are unable to fully simulate the complex regulatory networks in vivo, nor can they synergistically improve multi-dimensional functions such as mitochondrial function, redox homeostasis, and epigenetic regulation.

[0006] Therefore, there is an urgent need to develop an innovative in vitro culture system that can target multiple points and systematically intervene to simultaneously improve the maturation quality of oocyte nuclei and cytoplasm (synergistically enhance mitochondrial function, antioxidant capacity, etc.), which is of vital importance for comprehensively improving the efficiency and quality of in vitro embryo production. Summary of the Invention

[0007] This invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, this invention provides a formulation for in vitro embryo production and its application. The formulation for in vitro embryo production of this invention, by adding at least one of SEMA4-like proteins, Fbxo48 inhibitors, and flavonoid C-glycosides, promotes mitochondrial division and biosynthesis, enhances mitochondrial function (including membrane potential, respiratory intensity, and ATP levels), effectively scavenges reactive oxygen species, and inhibits lipid peroxidation, thereby significantly improving the maturation quality of in vitro oocytes and the developmental potential of in vitro embryos at multiple stages, including in vitro oocyte maturation, and has broad application prospects.

[0008] This invention is based on the inventor's discoveries and understanding of the following problems:

[0009] The inventors of this invention have long been engaged in the research and development of animal gamete and embryo engineering technology. They first conducted extensive screening of protein factors, metabolites, natural products, and small chemical molecules that might affect the in vitro maturation of oocytes. Ultimately, experiments revealed that the single addition of SEMA4 proteins, Fbxo48 inhibitors, and flavonoid C-glycosides could promote in vitro oocyte maturation. Furthermore, the combined use of SEMA4D, BC1618, and Isoschaftoside could promote in vitro oocyte maturation. It was found that Isoschaftoside could enhance the effects of SEMA4D and BC1618. By combining protein factors, signal-regulating small molecules, and natural antioxidants, a multifunctional composition integrating "mitochondrial fission promoter + biogenic inducer + reactive oxygen species scavenger + lipid peroxidation inhibitor + antioxidant gene expression enhancer" was developed. This composition significantly improved the microenvironment for in vitro oocyte maturation, effectively increasing the quality of in vitro maturation and the development rate after in vitro fertilization.

[0010] In a first aspect, the present invention provides a formulation for in vitro embryo production. According to embodiments of the invention, the formulation contains at least one of the following components: a SEMA4 protein; an Fbxo48 inhibitor; and a flavonoid glycoside. The formulation according to embodiments of the invention, by adding at least one of a SEMA4 protein, an Fbxo48 inhibitor, and a flavonoid glycoside, promotes in vitro oocyte maturation, improves the maturation quality of in vitro oocytes, and enhances the developmental potential of in vitro embryos.

[0011] According to embodiments of the present invention, the above-described formulation for in vitro embryo production may further have the following additional technical features:

[0012] According to embodiments of the present invention, the SEMA4 proteins include one or more of SEMA4A, SEMA4B, SEMA4C, SEMA4D, SEMA4E, SEMA4F, and SEMA4G proteins.

[0013] According to a preferred embodiment of the present invention, the SEMA4 protein is the SEMA4D protein.

[0014] According to an embodiment of the present invention, the Fbxo48 inhibitor is BC1618 and / or BC1583.

[0015] According to embodiments of the present invention, the flavonoid glycosides include one or more of isochaftoside, orientin, homoorientin, vitexin, isovitexin, and puerarin.

[0016] According to an embodiment of the present invention, the concentration of the SEMA4 protein is 5–200 ng / mL.

[0017] According to an embodiment of the present invention, the concentration of the Fbxo48 inhibitor is 1–10 μmol / L.

[0018] According to an embodiment of the present invention, the concentration of the flavonoid C-glycoside compound is 1–10 μmol / L.

[0019] According to an embodiment of the present invention, the formulation contains a SEMA4 protein and an Fbxo48 inhibitor, wherein the concentration of the SEMA4 protein is 5–200 ng / mL and the concentration of the Fbxo48 inhibitor is 1–10 μmol / L.

[0020] According to an embodiment of the present invention, the formulation contains SEMA4 protein, Fbxo48 inhibitor and isoxavorin, wherein the concentration of SEMA4 protein is 5-200 ng / mL, the concentration of Fbxo48 inhibitor is 1-10 μmol / L, and the concentration of isoxavorin is 1-10 μmol / L.

[0021] According to an embodiment of the present invention, the formulation contains at least one of the following components: SEMA4D protein; BC1618; isoxazoline.

[0022] According to an embodiment of the present invention, the concentration of the SEMA4D protein is 5–200 ng / mL.

[0023] According to an embodiment of the present invention, the concentration of BC1618 is 1–10 μmol / L.

[0024] According to an embodiment of the present invention, the isosulfanol is 1–10 μmol / L.

[0025] According to an embodiment of the present invention, the formulation contains SEMA4D protein and BC1618, wherein the concentration of SEMA4D protein is 5-200 ng / mL and the concentration of BC1618 is 1-10 μmol / L.

[0026] According to an embodiment of the present invention, the formulation contains SEMA4D protein, BC1618 and isoxavorin, wherein the concentration of SEMA4D protein is 5-200 ng / mL, the concentration of BC1618 is 1-10 μmol / L, and the concentration of isoxavorin is 1-10 μmol / L.

[0027] According to an embodiment of the present invention, the formulation is used in the in vitro maturation stage of oocytes.

[0028] According to embodiments of the present invention, the formulation further includes one or more of inorganic salts, amino acids, vitamins, sugars, follicle-stimulating hormone, luteinizing hormone, estradiol, fetal bovine serum, pH indicators, and antibiotics.

[0029] According to embodiments of the present invention, the formulation further comprises 80–95% v / v TCM199 culture medium, 5–10 IU / mL pregnant mare serum gonadotropin, 5–10 IU / mL luteinizing hormone, 1–10 μg / mL estrogen, 100–200 IU / mL penicillin, 100–200 μg / mL streptomycin, 5–20% follicular fluid, 0.4–0.8 mg / mL glucose, 1.5–2.5 mg / mL sodium bicarbonate, 0.05–0.08 mg / mL sodium pyruvate, and 0.3–0.6 mg / mL cysteine.

[0030] According to an embodiment of the present invention, the oocyte is derived from a non-human mammal.

[0031] According to embodiments of the present invention, the mammals include one or more of non-human primates, cattle, sheep, pigs, horses, and mice.

[0032] In a second aspect of the invention, the invention proposes the use of SEMA4 proteins, Fbxo48 inhibitors, flavonoid C-glycosides, or the formulations for in vitro embryo production described in the first aspect in in vitro embryo production.

[0033] Those skilled in the art will understand that the features and advantages described above for formulations used in in vitro embryo production also apply to this application, and will not be repeated here.

[0034] According to embodiments of the present invention, the above application may also have the following additional technical features:

[0035] According to embodiments of the present invention, the SEMA4 protein, Fbxo48 inhibitor, flavonoid C-glycoside compound, or the preparation for in vitro embryo production described in the first aspect has at least one of the following uses: improving the maturation quality of in vitro oocytes; improving the developmental potential of in vitro embryos; wherein improving the developmental potential of in vitro embryos includes improving the developmental efficiency and / or developmental quality of in vitro embryos.

[0036] According to embodiments of the present invention, the SEMA4 protein, Fbxo48 inhibitor, flavonoid C-glycoside compound, or the preparation for in vitro embryo production described in the first aspect has at least one of the following uses: promoting mitochondrial division in oocytes; increasing the number of mitochondria in oocytes; increasing the mitochondrial membrane potential of oocytes; increasing the mitochondrial respiration intensity of oocytes; increasing the ATP level in oocytes; reducing the lipid peroxidation level in oocytes; and increasing the expression level of antioxidant genes in oocytes; wherein the antioxidant genes include one or more of SOD1, SOD2, SOD3, CATALASE, GCLM, and GCLC.

[0037] In a third aspect, the present invention provides a method for in vitro embryo production. According to embodiments of the invention, the method includes: co-culturing oocytes with SEMA4-like proteins, Fbxo48 inhibitors, flavonoid C-glycosides, or the preparations for in vitro embryo production described in the first aspect; wherein the oocytes are derived from non-human mammals. The method according to embodiments of the invention, by co-culturing oocytes with SEMA4-like proteins, Fbxo48 inhibitors, flavonoid C-glycosides, or the aforementioned preparations, can provide precise regulatory support throughout the entire process of in vitro embryo production, thereby comprehensively improving the efficiency and quality of in vitro embryo production.

[0038] According to embodiments of the present invention, the above method may further have the following additional technical features:

[0039] According to embodiments of the present invention, the non-human mammals include one or more of non-human primates, cattle, sheep, pigs, horses, and mice.

[0040] According to an embodiment of the present invention, the concentration of the SEMA4 protein is 5–200 ng / mL.

[0041] According to an embodiment of the present invention, the concentration of the Fbxo48 inhibitor is 1–10 μmol / L.

[0042] According to an embodiment of the present invention, the concentration of the flavonoid C-glycoside compound is 1–10 μmol / L.

[0043] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0045] Figure 1 The images show the mitochondrial staining results of mature oocytes in each group in Example 8 of the present invention. In this image, A is the MitoTracker Green staining image of mitochondria in mature oocytes of the control group / treatment group, and B is the statistical result of the fluorescence intensity of mitochondria in mature oocytes of the control group / treatment group. ** indicates P < 0.01.

[0046] Figure 2 The images show the results of mitochondrial membrane potential detection in mature oocytes of each group in Example 8 of the present invention. In this image, A is the staining image of mitochondrial membrane potential of mature oocytes in the control group / treatment group, and B is the statistical result of fluorescence intensity of mitochondrial membrane potential staining in mature oocytes in the control group / treatment group. ** indicates P < 0.01.

[0047] Figure 3 The images show the results of mitochondrial respiration intensity detection in mature oocytes of each group in Example 8 of the present invention. In this image, A is a staining image of mitochondrial respiration intensity in mature oocytes of the control group / treatment group, and B is a statistical result of mitochondrial respiration intensity staining fluorescence intensity in mature oocytes of the control group / treatment group. ** indicates P < 0.01.

[0048] Figure 4The figures show the results of mitochondrial ATP content detection in mature oocytes of each group in Example 8 of the present invention. In this figure, A is the ATP fluorescence staining image of mature oocytes in the control group / treatment group, B is the statistical graph of ATP staining fluorescence intensity of mature oocytes in the control group / treatment group, and **** indicates P < 0.0001.

[0049] Figure 5 The images show the results of lipid peroxidation level detection in mature oocytes of each group in Example 8 of the present invention. In the image, A is the fluorescence staining image of lipid peroxidation in mature oocytes of the control group / treatment group, B is the statistical graph of fluorescence intensity of lipid peroxidation staining in mature oocytes of the control group / treatment group, and **** indicates P < 0.0001.

[0050] Figure 6 The figures shown are the results of detecting the expression levels of antioxidant genes in mature oocytes of each group in Example 8 of the present invention. Among them, A is a statistical graph of the detection results of the expression level of SOD1 gene in mature oocytes of the control group / treatment group, B is a statistical graph of the expression level of SOD2 gene in mature oocytes of the control group / treatment group, C is a statistical graph of the expression level of SOD3 gene in mature oocytes of the control group / treatment group, D is a statistical graph of the expression level of Catalase gene in mature oocytes of the control group / treatment group, E is a statistical graph of the expression level of GCLM gene in mature oocytes of the control group / treatment group, and F is a statistical graph of the expression level of GCLC gene in mature oocytes of the control group / treatment group. * indicates P < 0.05, ** indicates P < 0.01. Detailed Implementation

[0051] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0052] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0053] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0054] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0055] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0056] Terms and Definitions

[0057] In this article, the term "cleavage rate" refers to the percentage of fertilized eggs that have undergone cleavage, reflecting the proportion of fertilized eggs that have successfully begun to divide.

[0058] In this article, the term "blastocyst rate" refers to the percentage of embryos that have developed to the blastocyst stage out of the total number of fertilized eggs, representing the efficiency of in vitro embryo development to the blastocyst stage.

[0059] In this document, the term "Mito-Tracker Green concentrated stock solution" refers to the high-concentration fluorescent probe stock solution used for oocyte mitochondrial staining in the examples, which needs to be diluted before use to detect the fluorescence signal intensity of mitochondria.

[0060] In this paper, the term "Mito-Tracker Green fluorescence intensity" refers to the number of mitochondria quantitatively assessed by fluorescence intensity after staining with the Mito-Tracker Green probe. Higher fluorescence intensity indicates a greater number of mitochondria.

[0061] In this paper, the term "JC-1 staining buffer" refers to a buffer system used to optimize the staining effect of JC-1, where JC-1 is a fluorescent probe for detecting mitochondrial membrane potential, including 5×JC-1 staining buffer, 200×JC-1 staining buffer, etc.

[0062] In this article, the term "mitochondrial membrane potential" refers to the level of mitochondrial membrane potential detected by the JC-1 probe. This parameter is an important indicator for measuring the functional status of mitochondria, and a higher membrane potential indicates better mitochondrial function.

[0063] In this paper, the term "mitochondrial respiratory intensity" refers to the level of reactive oxygen species (ROS) generation in mitochondria as detected by the MitoSOX probe. This parameter reflects the respiratory function of mitochondria, and a higher respiratory intensity indicates more active mitochondrial energy metabolism.

[0064] In this paper, the term "ATP content" refers to the ATP concentration level in oocytes detected by the pCMV-Mito-AT1.03 probe. This parameter reflects the energy metabolism status of the cell, and the higher the ATP content, the more active its energy metabolism.

[0065] In this paper, the term "lipid peroxidation level" refers to the degree of lipid peroxidation detected by the C11-BODIPY fluorescent probe. This parameter is an important indicator for measuring cellular oxidative damage. The higher the fluorescence intensity, the higher the degree of lipid peroxidation and the more severe the oxidative damage.

[0066] In this paper, the term "antioxidant gene expression level" refers to the expression level of antioxidant genes (such as SOD1, CATALASE, etc.) detected by real-time quantitative PCR, which reflects the antioxidant capacity of oocytes.

[0067] Preparations for in vitro embryo production

[0068] This invention provides a formulation for in vitro embryo production. According to embodiments of the invention, the formulation contains at least one of the following components: SEMA4-like protein; Fbxo48 inhibitor; and flavonoid C-glycoside compound. The formulation according to embodiments of the invention, by adding at least one of SEMA4-like protein, Fbxo48 inhibitor, and flavonoid C-glycoside compound, promotes in vitro oocyte maturation, improves the maturation quality of in vitro oocytes, and enhances the developmental potential of in vitro embryos.

[0069] It should be noted that the formulation contains at least one of SEMA4 protein, Fbxo48 inhibitor, and flavonoid C-glycosides. This mainly includes several situations: adding one of the three components (SEMA4 protein, Fbxo48 inhibitor, and flavonoid C-glycosides), adding any two of the three components (SEMA4 protein and Fbxo48 inhibitor, SEMA4 protein and flavonoid C-glycosides, Fbxo48 inhibitor and flavonoid C-glycosides), and adding all three components simultaneously (SEMA4 protein, Fbxo48 inhibitor, and flavonoid C-glycosides). All of these fall within the scope of protection of this invention.

[0070] In this article, the term "SEMA4 proteins" refers to the fourth group of members in the semaphorin family. These are transmembrane regulatory molecules involved in various physiological and pathological processes, including nervous system development, immune responses, cytoskeleton organization, angiogenesis, and cell-cell interactions. SEMA4 proteins comprise seven distinct subfamilies: SEMA4A, SEMA4B, SEMA4C, SEMA4D, SEMA4E, SEMA4F, and SEMA4G proteins.

[0071] It should be noted that, although the functional characteristics of SEMA4D protein were only used for verification in the embodiments of the present invention, SEMA4D and other SEMA4 proteins are a class of proteins with similar structural features and metabolic functions. When added to the formulation for in vitro embryo production, they will have similar technical effects. Therefore, all SEMA4 proteins (including SEMA4A, SEMA4B, SEMA4C, SEMA4E, SEMA4F and SEMA4G proteins) should be within the scope of protection of the present invention.

[0072] In this paper, the term "Fbxo48 inhibitor" refers to a class of compounds that can inhibit the function of F-box protein 48 (FBXO48). As an orphan ubiquitin E3 ligase subunit protein, it can target active phosphorylated AMPKα (pAMPKα), induce its multiple ubiquitination, and then be degraded by the proteasome. Fbxo48 inhibitors enhance the activity of the AMPK signaling pathway by preventing the degradation of pAMPKα. In the embodiments of this invention, BC1618 is used as an example to verify that this class of Fbxo48 inhibitors, whether added alone or in combination with components such as SEMA4D protein, have the ability to promote in vitro maturation of oocytes. All Fbxo48 inhibitors (discovered or undiscovered) should fall within the scope of protection of this invention.

[0073] In this paper, the term "flavonoid C-glycosides" refers to a class of natural products with antioxidant functions, in which glycosyl groups are directly linked to carbon atoms of the flavonoid skeleton through carbon-carbon bonds (CC bonds). In the embodiments of this invention, isosulfanol is used as an example to verify that this class of flavonoid C-glycosides, when added alone, has the ability to promote the in vitro maturation of oocytes. All flavonoid C-glycosides should fall within the protection scope of this invention.

[0074] According to embodiments of the present invention, the SEMA4 proteins include one or more of SEMA4A, SEMA4B, SEMA4C, SEMA4D, SEMA4E, SEMA4F, and SEMA4G proteins. This further increases the flexibility and adaptability of the formulations of the present invention for in vitro embryo production, allowing the selection of the most suitable SEMA4 proteins according to specific needs to achieve optimal in vitro embryo production results.

[0075] According to a preferred embodiment of the present invention, the SEMA4 protein is SEMA4D protein. Therefore, SEMA4D protein is further preferred among SEMA4 proteins, as it has excellent effects in promoting in vitro oocyte maturation and embryo development, and can significantly improve the success rate and quality of in vitro embryo production.

[0076] According to an embodiment of the present invention, the Fbxo48 inhibitor is BC1618 and / or BC1583.

[0077] According to embodiments of the present invention, the flavonoid C-glycosides include one or more of isoxastatin, styracil, isoxastatin, vitexin, isovitellin, and puerarin. Thus, by providing flavonoid C-glycosides such as isoxastatin, the diversity of formulation components is increased. Specifically, when the formulation of the present invention for in vitro embryo production adds flavonoid C-glycosides without adding Fbxo48 inhibitors or SEMA4 proteins, suitable flavonoid C-glycosides can be selected according to different needs, thereby further improving the success rate and quality of in vitro embryo production when using the formulation of the present invention for in vitro embryo production.

[0078] According to embodiments of the present invention, the concentration of the SEMA4 protein is 5–200 ng / mL. Thus, by optimizing the control of the SEMA4 protein concentration, it is further ensured that the SEMA4 protein exerts its best effect in regulating oocyte maturation culture. This concentration range effectively allows the SEMA4 protein to perform its biological functions without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of the SEMA4 protein is 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL, preferably 20–200 ng / mL, and more preferably 50–200 ng / mL.

[0079] According to an embodiment of the present invention, the concentration of the Fbxo48 inhibitor is 1–10 μmol / L. Thus, by optimizing the control of the Fbxo48 inhibitor concentration, the optimal effect of the Fbxo48 inhibitor in regulating oocyte maturation culture is further ensured. This concentration range effectively exerts the biological function of the Fbxo48 inhibitor without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of the Fbxo48 inhibitor is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–5 μmol / L, and more preferably 1–3 μmol / L.

[0080] According to embodiments of the present invention, the concentration of the flavonoid C-glycoside compound is 1–10 μmol / L. Thus, by optimizing the control of the concentration of the flavonoid C-glycoside compound, the optimal effect of the flavonoid C-glycoside compound in regulating oocyte maturation culture is further ensured. This concentration range effectively exerts the biological function of the flavonoid C-glycoside compound without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of the flavonoid C-glycoside compound is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–5 μmol / L, and more preferably 1–3 μmol / L.

[0081] According to embodiments of the present invention, the formulation contains SEMA4 protein and Fbxo48 inhibitor, wherein the concentration of SEMA4 protein is 5–200 ng / mL and the concentration of Fbxo48 inhibitor is 1–10 μmol / L. Thus, when the formulation contains SEMA4 protein and Fbxo48 inhibitor, the concentrations of the two components are further optimized and controlled to achieve synergistic effects, thereby exerting the best effect in regulating oocyte maturation culture. For example, when the formulation contains SEMA4 protein and Fbxo48 inhibitor, the concentrations of SEMA4 protein are 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, etc. The concentrations of the Fbxo48 inhibitor are 1 μmol / L, 120 ng / L, 150 ng / L, 170 ng / L, 200 ng / L, preferably 20–200 ng / L, more preferably 50–100 ng / L; the concentrations of the Fbxo48 inhibitor are 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, preferably 1–3 μmol / L, more preferably 1–2 μmol / L.

[0082] It should be noted that, in the embodiments of the present invention, the combination of SEMA4D protein from the SEMA4 class of proteins and BC1618 from the Fbxo48 inhibitor showed that this combination also had a good ability to promote oocyte in vitro maturation and embryo development. Therefore, when all SEMA4 class proteins and all Fbxo48 inhibitors (discovered or undiscovered) are combined and added to the formulation of the present invention for in vitro embryo production, they all fall within the scope of protection of the present invention.

[0083] According to an embodiment of the present invention, the formulation contains SEMA4 protein, Fbxo48 inhibitor and isoxavorin, wherein the concentration of SEMA4 protein is 5-200 ng / mL, the concentration of Fbxo48 inhibitor is 1-10 μmol / L, and the concentration of isoxavorin is 1-10 μmol / L. Therefore, when the formulation contains SEMA4 protein, Fbxo48 inhibitor, and isosulfanilidine, the concentration of the three components when added synergistically is further optimized to ensure their optimal effect in regulating oocyte maturation culture. For example, when the formulation contains SEMA4 protein, Fbxo48 inhibitor, and isosulfanilidine, the concentration of the SEMA4 protein is 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, or 200 ng / mL, preferably 20–200 ng / mL. More preferably, the concentration is 50–100 ng / mL; the concentration of the Fbxo48 inhibitor is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–3 μmol / L, more preferably 1–2 μmol / L; the concentration of isosulfanilidine is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–10 μmol / L, more preferably 1–3 μmol / L.

[0084] It should be noted that, in the embodiments of the present invention, after combining SEMA4D protein (a SEMA4 protein), BC1618 (an Fbxo48 inhibitor), and isoxavorin, it was found that the addition of isoxavorin has the ability to further enhance the combined effect of SEMA4 protein and Fbxo48 inhibitor. That is, the combination has a better ability to promote oocyte in vitro maturation and embryo development. Therefore, when all SEMA4 proteins, all Fbxo48 inhibitors (discovered or undiscovered), and isoxavorin are combined and added to the formulation of the present invention for in vitro embryo production, they all fall within the scope of protection of the present invention.

[0085] According to embodiments of the present invention, the formulation contains at least one of the following components: SEMA4D protein; BC1618; and isoxastatin. Therefore, by further preferentially selecting SEMA4D protein from the SEMA4 protein family, BC1618 from the Fbxo48 inhibitor, and isoxastatin from the flavonoid C-glycosides, the efficacy of the formulation of the present invention for in vitro embryo production in promoting oocyte maturation and embryo development is further optimized.

[0086] It should be noted that the formulation contains at least one of SEMA4D protein, BC1618, and isosulfanilidine, which mainly includes several situations: adding one of the three components (SEMA4D protein, BC1618, and isosulfanilidine), adding any two of the three components (SEMA4D protein and BC1618, SEMA4D protein and isosulfanilidine, BC1618 and isosulfanilidine), and adding all three components simultaneously (SEMA4D protein, BC1618, and isosulfanilidine), all of which fall within the scope of protection of this invention.

[0087] According to an embodiment of the present invention, the concentration of the SEMA4D protein is 5–200 ng / mL. Therefore, by optimizing and controlling the concentration of SEMA4D protein, it is possible to further ensure that SEMA4D protein exerts its best effect in regulating oocyte maturation culture. This concentration range can effectively exert the biological function of SEMA4D protein without negatively impacting embryonic development due to excessive concentration. For example, the concentration of SEMA4D protein is 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, or 200 ng / mL, preferably 20–200 ng / mL, and more preferably 50–100 ng / mL.

[0088] According to an embodiment of the present invention, the concentration of BC1618 is 1–10 μmol / L. Thus, by optimizing the control of the BC1618 concentration, it is further ensured that BC1618 exerts its best effect in regulating oocyte maturation culture. This concentration range effectively allows BC1618 to perform its biological functions without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of BC1618 is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–5 μmol / L, and more preferably 1–3 μmol / L.

[0089] According to embodiments of the present invention, the isosulfanilidine is 1–10 μmol / L. Thus, by optimizing the control of the isosulfanilidine concentration, it is further ensured that isosulfanilidine exerts its optimal effect in regulating oocyte maturation culture. This concentration range effectively exerts the biological function of isosulfanilidine without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of isosulfanilidine is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–5 μmol / L, and more preferably 1–3 μmol / L.

[0090] According to embodiments of the present invention, the formulation contains SEMA4D protein and BC1618, wherein the concentration of SEMA4D protein is 5–200 ng / mL and the concentration of BC1618 is 1–10 μmol / L. Therefore, when the formulation contains SEMA4D protein and BC1618, the concentrations of the two components are further optimized and controlled to achieve a synergistic effect, thereby exerting the best effect in regulating oocyte maturation culture. For example, when the formulation contains SEMA4D protein and BC1618, the concentrations of SEMA4D protein are 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, etc. The concentrations of BC1618 are 1 μmol / L, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, and 200 ng / mL, preferably 50–200 ng / mL, and more preferably 50–100 ng / mL; the concentrations of BC1618 are 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, and 10 μmol / L, preferably 1–3 μmol / L, and more preferably 1–2 μmol / L.

[0091] According to an embodiment of the present invention, the formulation contains SEMA4D protein, BC1618 and isoxavorin, wherein the concentration of SEMA4D protein is 5-200 ng / mL, the concentration of BC1618 is 1-10 μmol / L, and the concentration of isoxavorin is 1-10 μmol / L. Therefore, when the formulation contains SEMA4D protein, BC1618, and isoxastatin, the concentrations of the three components are further optimized and controlled to ensure synergistic effects and optimal efficacy in regulating oocyte maturation culture. For example, when the formulation contains SEMA4D protein, BC1618, and isoxastatin, the concentration of SEMA4D protein is 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, etc. The concentrations of the BC1618 are 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, and 10 μmol / L, preferably 1 to 3 μmol / L, more preferably 1 to 2 μmol / L; the concentrations of the isosulfanol are 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, and 10 μmol / L, preferably 1 to 5 μmol / L, more preferably 1 to 3 μmol / L.

[0092] According to an embodiment of the present invention, the formulation is used in the in vitro maturation stage of oocytes. Thus, oocyte maturation and development can be promoted by adding the formulation during the in vitro maturation stage of oocytes.

[0093] According to embodiments of the present invention, the formulation further includes one or more of the following: inorganic salts, amino acids, vitamins, sugars, follicle-stimulating hormone, luteinizing hormone, estradiol, fetal bovine serum, pH indicators, and antibiotics. Thus, the presence of these substances provides the basic conditions for the growth and development of oocytes, and together with the functional components in the formulation (at least one of SEMA4 proteins, Fbxo48 inhibitors, and flavonoid C-glycosides), constitutes a complete nutritional system. This system provides an ideal growth environment for the in vitro maturation of oocytes, further improving the maturation efficiency and quality of oocytes, and ensuring that they exhibit better developmental potential and efficiency during subsequent embryonic development.

[0094] According to embodiments of the present invention, the formulation further includes 80-95% v / v TCM199 culture medium, 5-10 IU / mL pregnant mare serum gonadotropin, 5-10 IU / mL luteinizing hormone, 1-10 μg / mL estrogen, 100-200 IU / mL penicillin, 100-200 μg / mL streptomycin, 5-20% follicular fluid, 0.4-0.8 mg / mL glucose, 1.5-2.5 mg / mL sodium bicarbonate, 0.05-0.08 mg / mL sodium pyruvate, and 0.3-0.6 mg / mL cysteine. Thus, this component provides the basic conditions for the growth and development of oocytes.

[0095] It should be noted that the specific types, concentrations, and combinations of the other components in the formulation, except for at least one of SEMA4 proteins, Fbxo48 inhibitors, and flavonoid C-glycosides (this part provides basic metabolic conditions for oocytes), can be appropriately adjusted according to the source of oocytes and experimental requirements. Any schemes that reasonably optimize or equivalently replace the formulation components of the present invention for in vitro embryo production should fall within the protection scope of the present invention.

[0096] According to embodiments of the present invention, the oocytes are derived from non-human mammals. Therefore, the formulations of the present invention for in vitro embryo production are applicable to oocytes from a variety of non-human mammalian sources.

[0097] According to embodiments of the present invention, the mammals include one or more of non-human primates, cattle, sheep, pigs, horses, and mice.

[0098] It should be noted that the scope of application of the formulation for in vitro embryo production of the present invention is not limited to the aforementioned non-human mammals, but also applies to all other mammals, such as buffalo, sheep, goats, donkeys, camels, rats, rabbits, guinea pigs, hamsters, ferrets, cats, dogs, etc., all of which should fall within the protection scope of the present invention.

[0099] Applications in in vitro embryo production

[0100] This invention proposes the application of SEMA4 proteins, Fbxo48 inhibitors, flavonoid C-glycosides, or the aforementioned formulations for in vitro embryo production in in vitro embryo production.

[0101] Those skilled in the art will understand that the features and advantages described above for formulations used in in vitro embryo production also apply to this application, and will not be repeated here.

[0102] It should be noted that the scope of application of this invention includes many non-human mammals, such as non-human primates, cattle, sheep, pigs, horses, mice, buffalo, sheep, goats, donkeys, camels, rats, rabbits, guinea pigs, hamsters, ferrets, cats, dogs, etc., all of which should fall within the protection scope of this invention.

[0103] According to embodiments of the present invention, the SEMA4 protein, Fbxo48 inhibitor, flavonoid C-glycoside compound, or the aforementioned preparations for in vitro embryo production have at least one of the following uses: improving the maturation quality of in vitro oocytes; improving the developmental potential of in vitro embryos; wherein improving the developmental potential of in vitro embryos includes improving the developmental efficiency and / or developmental quality of in vitro embryos.

[0104] According to embodiments of the present invention, the SEMA4 protein, Fbxo48 inhibitor, flavonoid C-glycoside compound, or the aforementioned preparations for in vitro embryo production have at least one of the following uses: promoting mitochondrial division in oocytes; increasing the number of mitochondria in oocytes; increasing the mitochondrial membrane potential of oocytes; increasing the mitochondrial respiration intensity of oocytes; increasing the ATP level in oocytes; reducing the lipid peroxidation level in oocytes; and increasing the expression level of antioxidant genes in oocytes; wherein the antioxidant genes include one or more of SOD1, SOD2, SOD3, CATALASE, GCLM, and GCLC.

[0105] It should be noted that the antioxidant genes that can be enhanced after the application of the preparation are not limited to the SOD1, SOD2 and other genes specifically listed above. The above list is only an example and does not exhaust all possibilities. All genes that can respond to oxidative stress, scavenge reactive oxygen species (ROS) and maintain cellular redox homeostasis, such as PRDX1 (peroxidoreductase 1), PRDX3 (peroxidoreductase 3), NQO1 (quinone oxidoreductase 1), and HMOX1 (heme oxygenase 1), are within the scope of protection of this invention.

[0106] Methods of in vitro embryo production

[0107] This invention proposes a method for in vitro embryo production. According to embodiments of the invention, the method includes: co-culturing oocytes with SEMA4-like proteins, Fbxo48 inhibitors, flavonoid C-glycosides, or the aforementioned preparations for in vitro embryo production; wherein the oocytes are all derived from non-human mammals. According to the method of this invention, by co-culturing oocytes with SEMA4-like proteins, Fbxo48 inhibitors, flavonoid C-glycosides, or the aforementioned preparations, precise regulatory support can be provided throughout the entire process of in vitro embryo production, thereby comprehensively improving the efficiency and quality of in vitro embryo production.

[0108] According to embodiments of the present invention, the flavonoid glycosides include one or more of isochaftoside, orientin, homoorientin, vitexin, isovitexin, and puerarin.

[0109] According to embodiments of the present invention, the non-human mammals include one or more of non-human primates, cattle, sheep, pigs, horses, and mice. Therefore, the in vitro embryo production method of the present invention is applicable to oocytes from a variety of non-human mammalian sources.

[0110] It should be noted that the scope of application of the method of the present invention includes many non-human mammals, such as non-human primates, cattle, sheep, pigs, horses, mice, buffalo, sheep, goats, donkeys, camels, rats, rabbits, guinea pigs, hamsters, ferrets, cats, dogs, etc., all of which should fall within the protection scope of the present invention.

[0111] According to embodiments of the present invention, the concentration of the SEMA4 protein is 5–200 ng / mL. Thus, by optimizing the control of the SEMA4 protein concentration, it is further ensured that the SEMA4 protein exerts its best effect in regulating oocyte maturation culture. This concentration range effectively allows the SEMA4 protein to perform its biological functions without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of the SEMA4 protein is 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL, preferably 50–200 ng / mL, and more preferably 50–100 ng / mL.

[0112] According to an embodiment of the present invention, the concentration of the Fbxo48 inhibitor is 1–10 μmol / L. Thus, by optimizing the control of the Fbxo48 inhibitor concentration, the optimal effect of the Fbxo48 inhibitor in regulating oocyte maturation culture is further ensured. This concentration range effectively exerts the biological function of the Fbxo48 inhibitor without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of the Fbxo48 inhibitor is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–5 μmol / L, and more preferably 1–3 μmol / L.

[0113] According to embodiments of the present invention, the concentration of the flavonoid C-glycoside compound is 1–10 μmol / L. Thus, by optimizing the control of the concentration of the flavonoid C-glycoside compound, the optimal effect of the flavonoid C-glycoside compound in regulating oocyte maturation culture is further ensured. This concentration range effectively exerts the biological function of the flavonoid C-glycoside compound without negatively impacting embryonic development due to excessively high concentrations. Exemplarily, the concentration of the flavonoid C-glycoside compound is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, or 10 μmol / L, preferably 1–5 μmol / L, and more preferably 1–3 μmol / L.

[0114] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0115] Example 1: In vitro maturation, in vitro fertilization, and embryo culture of sheep oocytes

[0116] 1. In vitro maturation of sheep oocytes

[0117] Sheep ovaries were collected from the slaughterhouse and placed in physiological saline at 30-35℃. They were transported back to the laboratory within 2 hours and washed three times with physiological saline. Then, the follicles were punctured using a disposable syringe with a 20G needle containing 5mL of oocyte aspiration fluid to release the cumulus-oocyte complex. Sheep cumulus-oocyte complexes with uniform cytoplasm and more than 3 layers of cumulus cells were collected under a stereomicroscope. The selected sheep cumulus-oocyte complexes were washed three times with oocyte aspiration fluid and then three times with oocyte in vitro maturation basal culture medium pre-equilibrated in an incubator for 3 hours. They were then transferred to four-well plates containing 600μL of oocyte in vitro maturation basal culture medium and 300μL of mineral oil per well for culture. Each well contained 30 sheep cumulus-oocyte complexes. The four-well plates were then placed in a saturated humidity incubator at 38.5℃ with 5% CO2 for 22 hours.

[0118] The preparation method for the egg-absorption fluid is as follows:

[0119] 49 mL TCM199 culture medium (purchased from Thermo Fisher, catalog number 11150059) + 1 mL FBS (purchased from Gibco, catalog number A5256701).

[0120] The preparation method for the basal culture medium for in vitro maturation of oocytes is as follows:

[0121] 85% TCM199 culture medium, 8 IU / mL pregnant mare serum gonadotropin, 8 IU / mL luteinizing hormone, 6 ng / mL estrogen, 150 IU / mL penicillin, 150 μg / mL streptomycin, 12% follicular fluid, 0.65 mg / mL glucose, 1.85 mg / mL sodium bicarbonate, 0.06 mg / mL sodium pyruvate, 0.45 mg / mL cysteine.

[0122] (2) In vitro fertilization

[0123] The sheep cumulus-oocyte complexes that matured in vitro for 22 hours obtained in step (1) were placed in 0.5% hyaluronidase and gently and repeatedly pipetted to remove most of the cumulus cells. The complexes were then washed three times with fertilization fluid to obtain mature oocytes. The oocytes were then transferred into fertilization fluid that had been equilibrated in an incubator for more than 2 hours and cultured in four-well plates. Each well contained 400 μL of fertilization fluid and 300 μL of mineral oil (30 mature oocytes / well) for in vitro fertilization.

[0124] Frozen sperm were removed from liquid nitrogen and rapidly thawed in a 38°C water bath. The thawed sperm were then transferred to 600 μL of sperm surfacing medium (purchased from IVF Bioscience, catalog number 73671004) and incubated in an incubator for 30 min to allow the sperm to float fully. 100 μL of the supernatant was then added to a four-well plate containing mature oocytes. The sperm and eggs were incubated for 19 h under fertilization conditions of saturated humidity, 38.5°C, 5% CO2, and 95% air.

[0125] The preparation method for the fertilized fluid is as follows:

[0126] mSOFaa culture medium + 2% v / v bovine serum + 100 μg / mL streptomycin + 100 IU / mL penicillin.

[0127] The preparation method for mSOFaa culture medium (all reagents used in its preparation were purchased from Sigma-Aldrich) is as follows:

[0128] Each 1L contains 6.34g NaCl, 0.54g KCl, 0.23g CaCl2·2H2O, 0.01g MgSO4·7H2O, 0.17g KH2PO4, 0.24g NaHCO3, 0.30g glucose, 0.04g sodium pyruvate, 5.8mL sodium lactate, 2% v / v BME amino acid solution, and 1% v / v MEM non-essential amino acid solution.

[0129] (3) Embryo culture

[0130] After co-incubating the sperm and eggs for 19 hours in step (2), the oocytes were aspirated, and the remaining cumulus cells and sperm were gently removed by blowing. Then, the oocytes were washed three times with mSOFaa culture medium and transferred to mSOFaa culture medium that had been equilibrated in an incubator for more than 2 hours. The oocytes were placed in different wells of a four-well plate and cultured in a saturated humidity incubator at 38.5℃, 5% O2, 5% CO2, and 90% N2. After 48 hours of culture, the cleavage rate was calculated. Culture continued until day 7, and the blastocyst rate was calculated. The calculation method for the cleavage rate is shown in Formula 1, and the calculation method for the blastocyst rate is shown in Formula 2.

[0131] Cleavage rate = (Number of cleaved eggs / Total number of fertilized eggs) * 100% Formula 1

[0132] Blastocyst rate = (Number of embryos that developed to the blastocyst stage / Total number of fertilized eggs) * 100% Formula 2

[0133] Example 2: Effects of adding different concentrations of SEMA4D on embryonic development

[0134] Referring to step 1 of Example 1, after the cleaned sheep cumulus-oocyte complex was cultured in vitro for 22 hours using in vitro maturation basal culture medium (600 μL), it was divided into a control group and treatment groups 1-9. The treatment conditions for each group were as follows:

[0135] (1) Control group: Oocytes were cultured in vitro for 22 hours in basal culture medium for in vitro maturation (without the addition of other components);

[0136] (2) Treatment groups 1-9: oocytes were cultured for 22 h in basal culture medium containing SEMA4D (purchased from R&D Systems, catalog number 7470-S4-050) at concentrations of 1 ng / mL (treatment group 1), 5 ng / mL (treatment group 2), 10 ng / mL (treatment group 3), 20 ng / mL (treatment group 4), 50 ng / mL (treatment group 5), 100 ng / mL (treatment group 6), 200 ng / mL (treatment group 7), 500 ng / mL (treatment group 8), and 1000 ng / mL (treatment group 9).

[0137] Sheep cumulus-oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation basal culture medium for the aforementioned groups. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were statistically analyzed.

[0138] The effects of adding SEMA4D to the basal culture medium for in vitro maturation of oocytes on embryonic development after fertilization are shown in Table 1.

[0139] Table 1. Effects of adding SEMA4D to the basal culture medium for in vitro maturation of oocytes on post-fertilization embryonic development.

[0140] Grouping Number of eggs Cleavage rate (%) Blastocyst rate (%) control group 205 <![CDATA[60.98±4.27 b ]]> <![CDATA[18.05±1.37 c ]]> Processing Group 1 218 <![CDATA[56.88±5.83 b ]]> <![CDATA[16.97±2.14 c ]]> Processing Group 2 209 <![CDATA[62.20±6.12 b ]]> <![CDATA[20.10±1.92 b ]]> Processing Group 3 213 <![CDATA[67.14±5.05 a ]]> <![CDATA[22.07±2.65 b ]]> Processing Group 4 201 <![CDATA[68.16±4.96 a ]]> <![CDATA[23.88±1.78 b ]]> Processing Group 5 215 <![CDATA[71.16±6.45 a ]]> <![CDATA[26.98±2.03 a ]]> Processing Group 6 207 <![CDATA[72.95±5.44 a ]]> <![CDATA[30.92±2.29 a ]]> Processing Group 7 211 <![CDATA[69.19±4.68 a ]]> <![CDATA[28.91±1.55 a ]]> Processing Group 8 219 <![CDATA[63.01±5.71 b ]]> <![CDATA[19.18±2.51 c ]]> Processing Group 9 203 <![CDATA[56.16±5.19 b ]]> <![CDATA[12.81±2.08 d ]]>

[0141] In Table 1, different superscript letters (a, b, c) in the same column indicate significant differences between groups (P < 0.05).

[0142] The results showed that compared with the control group (60.98±4.27%), the cleavage rates of treatment groups 3, 4, 5, 6, and 7 (i.e., supplemented with 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL LSM 4D, respectively) were significantly higher (67.14±5.05%, 68.16±4.96%, 71.16±6.45%, 72.95±5.44%, and 69.19±4.68%, respectively) (P<0.05). Furthermore, compared with the control group, the blastocyst rate was significantly higher. Compared with the control group (18.05±1.37%), the blastocyst rates of treatment groups 2, 3, 4, 5, 6, and 7 (i.e., 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL LSM EMA 4D added, respectively) were significantly higher (P<0.05).

[0143] The above results indicate that adding 5–200 ng / mL SEMA4D to the basal culture medium for in vitro maturation of oocytes has a significant promoting effect on embryonic development after fertilization (the effect is not significant when 500 ng / mL SEMA4D is added, but there is a partial improvement).

[0144] Example 3: Effects of adding different concentrations of BC1618 on embryonic development

[0145] Referring to step 1 of Example 1, after the cleaned sheep cumulus-oocyte complex was cultured in vitro for 22 hours using in vitro maturation basal culture medium (600 μL), it was divided into a control group and treatment groups 1-6. The treatment conditions for each group were as follows:

[0146] (1) Control group: Oocytes were cultured in vitro for 22 hours in basal culture medium for in vitro maturation (without the addition of other components);

[0147] (2) Treatment groups 1–6: oocytes were cultured for 22 h in basal culture medium containing BC1618 (purchased from MedChemExpress, catalog number HY-134656) at concentrations of 0.1 μmol / L (treatment group 1), 0.3 μmol / L (treatment group 2), 1 μmol / L (treatment group 3), 3 μmol / L (treatment group 4), 10 μmol / L (treatment group 5), and 30 μmol / L (treatment group 6), respectively.

[0148] Sheep cumulus-oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation basal culture medium for the aforementioned groups. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were statistically analyzed.

[0149] The effects of adding BC1618 to the basal culture medium for oocyte in vitro maturation on embryonic development after fertilization are shown in Table 2.

[0150] Table 2. Effects of adding BC1618 to the basal culture medium for oocyte in vitro maturation on post-fertilization embryo development.

[0151] Grouping Number of eggs Cleavage rate (%) Blastocyst rate (%) control group 203 <![CDATA[56.16±5.67 b ]]> <![CDATA[18.23±1.56 b ]]> Processing Group 1 198 <![CDATA[58.08±4.91 b ]]> <![CDATA[17.17±1.98 b ]]> Processing Group 2 209 <![CDATA[58.85±6.03 b ]]> <![CDATA[18.18±1.72 b ]]> Processing Group 3 195 <![CDATA[68.21±5.12 a ]]> <![CDATA[26.15±2.09 a ]]> Processing Group 4 207 <![CDATA[71.98±4.78 a ]]> <![CDATA[28.02±1.47 a ]]> Processing Group 5 201 <![CDATA[61.19±5.45 b ]]> <![CDATA[20.90±1.83 b ]]> Processing Group 6 210 <![CDATA[52.86±6.24 c ]]> <![CDATA[13.81±2.02 c ]]>

[0152] In Table 2, different superscript letters (a, b, c) in the same column indicate significant differences between groups (P < 0.05).

[0153] The results showed that compared with the control group (56.16±5.67%) and blastocyst rate (18.23±1.56%), the treatment groups 3 and 4 (i.e., 1 μmol / L and 3 μmol / L BC1618 added, respectively) had significantly higher cleavage rates (68.21±5.12% and 71.98±4.78%) and blastocyst rates (26.15±2.09% and 28.02±1.47%) (P<0.05). In addition, the cleavage rate and blastocyst rate of the treatment group (10 μmol / L BC1618 added, respectively) were also higher than those of the control group.

[0154] The above results indicate that adding 1–10 μmol / L of BC1618 to the basal culture medium for in vitro maturation of oocytes can improve the efficiency of embryonic development after fertilization.

[0155] Example 4: Effects of adding different concentrations of Isoschaftoside on embryonic development

[0156] Referring to step 1 of Example 1, after the cleaned sheep cumulus-oocyte complex was cultured in vitro for 22 hours using in vitro maturation basal culture medium (600 μL), it was divided into a control group and treatment groups 1-6. The treatment conditions for each group were as follows:

[0157] (1) Control group: Oocytes were cultured in vitro for 22 hours in basal culture medium for in vitro maturation (without the addition of other components);

[0158] (2) Treatment groups 1–6: oocytes were cultured for 22 h in basal culture medium containing Isoschaftoside (purchased from MedChemExpress, catalog number HY-N0703) at concentrations of 0.1 μmol / L (treatment group 1), 0.3 μmol / L (treatment group 2), 1 μmol / L (treatment group 3), 3 μmol / L (treatment group 4), 10 μmol / L (treatment group 5), and 30 μmol / L (treatment group 6), respectively.

[0159] Sheep cumulus-oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation basal culture medium for the aforementioned groups. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were statistically analyzed.

[0160] The effects of adding Isoschaftoside to the basal culture medium for in vitro maturation of oocytes on post-fertilization embryo development are shown in Table 3.

[0161] Table 3. Effects of adding Isoschaftoside to the basal culture medium for oocyte in vitro maturation on post-fertilization embryo development.

[0162] Grouping Number of eggs Cleavage rate (%) Blastocyst rate (%) control group 211 <![CDATA[56.87±3.12 b ]]> 19.91±1.23 Processing Group 1 217 <![CDATA[58.06±4.83 b ]]> 18.89±1.87 Processing Group 2 205 <![CDATA[57.07±4.35 b ]]> 19.02±1.45 Processing Group 3 220 <![CDATA[61.82±5.17 b ]]> 20.91±2.61 Processing Group 4 214 <![CDATA[71.03±5.28 a ]]> 21.03±2.12 Processing Group 5 209 <![CDATA[67.94±4.26 a ]]> 21.05±2.28 Processing Group 6 222 <![CDATA[59.01±5.41 b ]]> 17.12±1.39

[0163] In Table 3, different superscript letters (a, b, c) in the same column indicate significant differences between groups (P < 0.05).

[0164] The results showed that compared with the control group (56.87±3.12%), the cleavage rates of treatment groups 4 and 5 (i.e., 3 μmol / L and 10 μmol / L Isoschaftoside added, respectively) were significantly higher (71.03±5.28% and 67.94±4.26%, respectively) (P<0.05). In addition, the cleavage rate of the 1 μmol / L Isoschaftoside added group (treatment group 3) was also higher than that of the control group. There was no difference in blastocyst rate between all Isoschaftoside added treatment groups and the control group (P>0.05).

[0165] The above results indicate that adding 1–10 μmol / L Soschaftoside to the basal culture medium for oocyte in vitro maturation can improve the cleavage rate after fertilization without affecting the blastocyst rate.

[0166] Example 5: Effects of combined addition of SEMA4D and BC1618 on embryonic development after in vitro fertilization

[0167] Referring to step 1 of Example 1, after the cleaned sheep cumulus-oocyte complex was cultured in vitro for 22 hours using in vitro maturation basal culture medium (600 μL), it was divided into a control group and treatment groups 1-11. The treatment conditions for each group were as follows:

[0168] (1) Control group: Oocytes were cultured in vitro for 22 hours in basal culture medium for in vitro maturation (without the addition of other components);

[0169] (2) Treatment group 1: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with an additional 50 ng / mL SEMA4D added);

[0170] (3) Treatment group 2: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with an additional 100 ng / mL SEMA4D added);

[0171] (4) Treatment group 3: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with an additional 200 ng / mL SEMA4D added);

[0172] (5) Treatment group 4: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with an additional 1 μmol / L BC1618 added);

[0173] (6) Treatment group 5: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with an additional 3 μmol / L BC1618 added);

[0174] (7) Treatment group 6: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 50 ng / mL SEMA4D and 1 μmol / LBC1618);

[0175] (8) Treatment group 7: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 50 ng / mL SEMA4D and 3 μmol / LBC1618);

[0176] (9) Treatment group 8: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D and 1 μmol / LBC1618);

[0177] (10) Treatment group 9: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D and 3 μmol / L BC1618);

[0178] (11) Treatment group 10: oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 200 ng / mL SEMA4D and 1 μmol / L BC1618);

[0179] (12) Treatment group 11: oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 200 ng / mL SEMA4D and 3 μmol / L BC1618).

[0180] Sheep cumulus-oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation basal culture medium for the aforementioned groups. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were statistically analyzed.

[0181] The effects of adding SEMA4D and BC1618 to the basal culture medium for oocyte in vitro maturation on embryonic development after fertilization are shown in Table 4.

[0182] Table 4. Effects of SEMA4D and BC1618 addition to basal culture medium for oocyte in vitro maturation on post-fertilization embryo development.

[0183] Grouping Number of eggs Cleavage rate (%) Blastocyst rate (%) control group 232 <![CDATA[56.03±3.72 d ]]> <![CDATA[16.81±1.56 d ]]> Processing Group 1 241 <![CDATA[65.15±4.36 c ]]> <![CDATA[24.07±2.89 c ]]> Processing Group 2 228 <![CDATA[65.79±4.98 c ]]> <![CDATA[25.88±1.92 c ]]> Processing Group 3 237 <![CDATA[67.09±5.81 c ]]> <![CDATA[23.21±2.37 c ]]> Processing Group 4 245 <![CDATA[68.16±4.35 c ]]> <![CDATA[22.04±3.05 c ]]> Processing Group 5 230 <![CDATA[69.13±5.12 c ]]> <![CDATA[23.91±1.31 c ]]> Processing Group 6 247 <![CDATA[74.09±6.07 b ]]> <![CDATA[29.15±2.18 b ]]> Processing Group 7 235 <![CDATA[74.04±4.59 b ]]> <![CDATA[31.06±2.64 b ]]> Processing Group 8 229 <![CDATA[79.04±5.63 a ]]> <![CDATA[35.81±1.78 a ]]> Processing Group 9 243 <![CDATA[71.19±4.84 b ]]> <![CDATA[32.10±2.53 b ]]> Processing Group 10 238 <![CDATA[63.03±5.42 c ]]> <![CDATA[28.15±1.45 b ]]> Processing Group 11 231 <![CDATA[52.81±4.95 d ]]> <![CDATA[15.15±1.91 d ]]>

[0184] In Table 4, different superscript letters (a, b, c) in the same column indicate significant differences between groups (P < 0.05).

[0185] The results showed that the cleavage rate and blastocyst rate of treatment group 8 (combined supplementation of 100 ng / mL SEMA4D and 1 μmol / L BC1618) were significantly higher than those of other groups (P<0.05).

[0186] The above results indicate that the combined addition of 100 ng / mL SEMA4D and 1 μmol / L BC1618 to the basal culture medium for oocyte in vitro maturation is most effective in improving embryo development efficiency after in vitro fertilization.

[0187] Example 6: Effects of adding SEMA4D, BC1618 and Isoschaftoside on embryonic development after in vitro fertilization

[0188] Referring to step 1 of Example 1, after the cleaned sheep cumulus-oocyte complex was cultured in vitro for 22 hours using in vitro maturation basal culture medium (600 μL), it was divided into a control group and treatment groups 1-5. The treatment conditions for each group were as follows:

[0189] (1) Control group: Oocytes were cultured in vitro for 22 hours in basal culture medium for in vitro maturation (without the addition of other components);

[0190] (2) Treatment group 1: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D and 1 μmol / LBC1618);

[0191] (3) Treatment group 2: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 0.3 μmol / L Isoschaftoside);

[0192] (4) Treatment group 3: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 1 μmol / L Isoschaftoside);

[0193] (5) Treatment group 4: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L Isoschaftoside);

[0194] (6) Treatment group 5: oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 10 μmol / L Isoschaftoside).

[0195] Sheep cumulus-oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation basal culture medium for the aforementioned groups. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were statistically analyzed.

[0196] The effects of combined addition of SEMA4D, BC1618 and Isoschaftoside to the basal culture medium for oocyte in vitro maturation on post-fertilization embryo development are shown in Table 5.

[0197] Table 5. Effects of combined addition of SEMA4D, BC1618, and Isoschaftoside to basal culture medium for oocyte in vitro maturation on post-fertilization embryo development.

[0198] Grouping Number of eggs Cleavage rate (%) Blastocyst rate (%) control group 251 <![CDATA[58.17±3.89 c ]]> <![CDATA[19.12±1.38 d ]]> Processing Group 1 258 <![CDATA[79.07±5.13 b ]]> <![CDATA[36.82±2.76 c ]]> Processing Group 2 249 <![CDATA[77.91±4.62 b ]]> <![CDATA[34.94±2.91 c ]]> Processing Group 3 261 <![CDATA[80.84±5.07 b ]]> <![CDATA[42.15±3.42 b ]]> Processing Group 4 255 <![CDATA[92.16±6.25 a ]]> <![CDATA[65.88±4.09 a ]]> Processing Group 5 253 <![CDATA[75.89±5.84 b ]]> <![CDATA[35.97±2.25 c ]]>

[0199] In Table 5, different superscript letters (a, b, c) in the same column indicate significant differences between groups (P < 0.05).

[0200] The results showed that the cleavage rate and blastocyst rate of treatment group 4 (i.e., combined addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L LIsoschaftoside) were significantly higher than those of other groups (P<0.05); at the same time, the cleavage rate and blastocyst rate of other treatment groups were also significantly higher than those of the control group (P<0.05).

[0201] The above results indicate that the combined addition of SEMA4D, BC1618, and Isoschaftoside to the basal culture medium for oocyte in vitro maturation can effectively improve the developmental efficiency of embryos after in vitro fertilization. Among them, the combined addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618, and 3 μmol / L Isoschaftoside has the best effect on improving the developmental efficiency of embryos after in vitro fertilization.

[0202] Example 7: Effects of combined addition of SEMA4D, BC1618 and other flavonoid C-glycosides on embryonic development after in vitro fertilization

[0203] To investigate whether other flavonoid C-glycosides also have the ability to enhance the combined effect of SEMA4D and BC1618, the inventors added SEMA4D, BC1618 and other flavonoid C-glycosides to the in vitro maturation basal culture medium of oocytes and examined their effects on embryo development rate after in vitro fertilization.

[0204] Referring to step 1 of Example 1, after the cleaned sheep cumulus-oocyte complex was cultured in vitro for 22 hours using in vitro maturation basal culture medium (600 μL), it was divided into a control group and treatment groups 1-7. The treatment conditions for each group were as follows:

[0205] (1) Control group: Oocytes were cultured in vitro for 22 hours in basal culture medium for in vitro maturation (without the addition of other components);

[0206] (2) Treatment group 1: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D and 1 μmol / LBC1618);

[0207] (3) Treatment group 2: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L Isoschaftoside);

[0208] (4) Treatment group 3: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L Orientin (purchased from MedChemExpress, catalog number HY-N0405);

[0209] (5) Treatment group 4: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L Homoorientin (purchased from MedChemExpress, catalog number HY-N0767);

[0210] (6) Treatment group 5: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L Vitexin (purchased from MedChemExpress, catalog number HY-N0013);

[0211] (7) Treatment group 6: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L Isovitexin (purchased from MedChemExpress, catalog number HY-N0773);

[0212] (8) Treatment group 7: oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional addition of 100 ng / mL SEMA4D, 1 μmol / LBC1618 and 3 μmol / L Puerarin (purchased from MedChemExpress, catalog number HY-N0145).

[0213] Sheep cumulus-oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation basal culture medium for the aforementioned groups. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were statistically analyzed.

[0214] The effects of combined addition of SEMA4D, BC1618 and different flavonoid C-glycosides to the basal culture medium for oocyte in vitro maturation on embryonic development are shown in Table 6.

[0215] Table 6. Effects of combined addition of SEMA4D, BC1618, and different flavonoid glycosides to the basal culture medium for oocyte in vitro maturation on embryonic development.

[0216] Grouping Number of eggs Cleavage rate (%) Blastocyst rate (%) control group 241 <![CDATA[61.00±4.25 c ]]> <![CDATA[21.16±1.15 c ]]> Processing Group 1 237 <![CDATA[81.01±5.89 b ]]> <![CDATA[35.86±1.89 b ]]> Processing Group 2 252 <![CDATA[92.86±7.12 a ]]> <![CDATA[67.86±4.16 a ]]> Processing Group 3 239 <![CDATA[77.82±6.03 b ]]> <![CDATA[31.80±3.02 b ]]> Processing Group 4 248 <![CDATA[77.02±5.56 b ]]> <![CDATA[33.87±2.23 b ]]> Processing Group 5 244 <![CDATA[65.16±5.48 c ]]> <![CDATA[22.13±1.67 c ]]> Processing Group 6 235 <![CDATA[82.13±6.01 b ]]> <![CDATA[37.02±2.31 b ]]> Processing Group 7 250 <![CDATA[75.20±4.69 b ]]> <![CDATA[19.20±1.34 c ]]>

[0217] In Table 5, different superscript letters (a, b, c) in the same column indicate significant differences between groups (P < 0.05).

[0218] The results showed that only treatment group 2 (i.e., combined addition of SEMA4D, BC1618 and Isoschaftoside) had significantly higher cleavage and blastocyst rates (92.86±7.12% and 67.86±4.16%, respectively) than the control group (i.e., combined addition of SEMA4D and BC1618) (81.01±5.89% and 35.86±1.89%, respectively, P<0.05); while other flavonoid C-glycosides (treatment groups 3-7) did not further improve the combined effect of SEMA4D and BC1618.

[0219] The above results indicate that among flavonoid C-glycosides, only Isoschaftoside has the ability to enhance the combined effect of SEMA4D and BC1618 (improving embryonic development efficiency after in vitro fertilization).

[0220] Example 8: Effects of combined addition of SEMA4D, BC1618 and Isoschaftoside on oocyte maturation quality

[0221] (1) Conditions for the control group / treatment group

[0222] Referring to step 1 of Example 1, after the cleaned sheep cumulus-oocyte complex was cultured in vitro for 22 hours using in vitro maturation basal culture medium (600 μL), it was divided into a control group and a treatment group. The treatment conditions for each group were as follows:

[0223] Control group: Oocytes were cultured in vitro for 22 hours in basal culture medium for in vitro maturation (without the addition of other components);

[0224] Treatment group: Oocytes were cultured in vitro for 22 h in basal culture medium for in vitro maturation (with additional additions of 100 ng / mL SEMA4D, 1 μmol / L BC1618 and 3 μmol / L Isoschaftoside).

[0225] (2) In vitro maturation of oocytes, in vitro fertilization and embryo culture

[0226] The sheep cumulus-oocyte complex was collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation basal culture medium for the aforementioned groups to obtain mature oocytes. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1.

[0227] (3) Oocyte mitochondrial staining

[0228] Mito-Tracker Green concentrated stock solution (1 mM, purchased from MedChemExpress, catalog number HY-135056) was prepared using DMSO. Then, Mito-Tracker Green working solution was prepared using oocyte manipulation solution M2 (purchased from Merck KGaA, catalog number M7167) at a volume ratio of 1:20000 (Mito-Tracker Green concentrated stock solution: oocyte manipulation solution M2). The mature oocytes obtained in step (2) were digested in 0.1% hyaluronidase for 2 min, and then washed twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). They were then placed in Mito-Tracker Green working solution pre-equilibrated at 37℃, with 30 oocytes per 100 μL of Mito-Tracker Green working solution, and incubated in a CO2 incubator at 37℃ for 30 min. After incubation, the oocytes were pressed onto glass slides and observed using a laser confocal microscope. The fluorescence intensity of Mito-Tracker Green was analyzed using ImageJ software.

[0229] The results of mitochondrial staining in mature oocytes of each group are shown below. Figure 1 .

[0230] The results showed that the mitochondrial MitoTracker staining fluorescence intensity of mature oocytes in the treatment group (114.34±7.82%) was significantly higher than that in the control group (76.37%±5.32%) (P<0.05).

[0231] The above results indicate that the combined addition of SEMA4D, BC1618 and Isoschaftoside can significantly increase the number of mitochondria in oocytes.

[0232] (4) Detection of mitochondrial membrane potential of oocytes

[0233] First, 800 μL of oocyte in vitro manipulation solution M2 and 200 μL of 5×JC-1 staining buffer (purchased from Beyotime Biotechnology Co., Ltd., catalog number C2006) were vigorously mixed to obtain a mixture. Then, 5 μL of 200×JC-1 was added to the mixture to obtain JC-1 working solution (Note: JC-1 working solution should be prepared and used immediately). Then, the mature oocytes obtained in step (2) were placed in 0.1% hyaluronidase for 2 min for digestion, and the oocytes were aspirated with a pipette and placed in manipulation solution M2. Then, they were washed twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). Then, they were placed in JC-1 working solution and incubated in a 37℃ incubator for 15 min. After incubation, they were washed 3 times, then pressed with a glass slide, and the oocyte mitochondrial membrane potential was observed by taking pictures with a laser confocal microscope and analyzed by ImageJ software.

[0234] The results of mitochondrial membrane potential detection in mature oocytes of each group are shown below. Figure 2 .

[0235] The results showed that the mitochondrial membrane potential of mature oocytes in the control group (0.46±0.04%) was significantly lower than that in the treatment group (0.73±0.08%) (P<0.05).

[0236] The above results indicate that the combined addition of SEMA4D, BC1618 and Isoschaftoside can significantly increase the mitochondrial membrane potential of oocytes.

[0237] (5) Detection of mitochondrial respiration intensity in oocytes

[0238] The mature oocytes obtained in step (2) were incubated with 5 μmol / L MitoSOX (purchased from Invitrogen) for 30 min, then washed three times with 0.1% PVA-PBS, and then photographed with a laser confocal microscope to examine and analyze the fluorescence intensity of each oocyte. The fluorescence intensity of mitochondrial respiration was analyzed using ImageJ software.

[0239] The results of the mitochondrial respiration intensity detection of mature oocytes in each group are shown below. Figure 3 .

[0240] The results showed that the mitochondrial respiration intensity of mature oocytes in the treatment group (74.53±5.10%) was significantly higher than that in the control group (67.22±3.41%) (P<0.05).

[0241] The above results indicate that the combined addition of SEMA4D, BC1618 and Isoschaftoside can significantly enhance the mitochondrial respiratory function of oocytes.

[0242] (6) Detection of ATP content in oocytes

[0243] The mature oocytes obtained in step (2) were incubated with 1 μmol / L pCMV-Mito-AT1.03 (purchased from Beyotime Biotechnology Co., Ltd.) for 30 min, and then the oocytes were washed three times with 0.1% PVA-PBS. The fluorescence intensity of each oocyte was examined and analyzed using a laser confocal microscope, and the fluorescence intensity of ATP staining was analyzed using ImageJ software.

[0244] The results of mitochondrial ATP content detection in mature oocytes of each group are shown below. Figure 4 .

[0245] The results showed that the ATP content in mature oocytes of the treatment group (69.37±4.36%) was significantly higher than that of the control group (55.18±3.42%) (P<0.05).

[0246] The above results indicate that the combined addition of SEMA4D, BC1618 and Isoschaftoside can significantly enhance the energy level of oocytes.

[0247] (7) Detection of lipid peroxidation level in oocytes

[0248] The mature oocytes obtained in step (2) were washed three times with PBS, and then freshly prepared C11-BODIPY fluorescent probe for lipid peroxidation detection (purchased from Invitrogen) was added and incubated at 37°C for 30 min. After incubation, the oocytes were gently washed three times with PBS to remove unbound excess reagents. The fluorescence intensity was observed under a fluorescence microscope at the appropriate excitation wavelength. Then, the fluorescence intensity of the lipid peroxidation staining was analyzed using a laser confocal microscope and ImageJ software.

[0249] The results of lipid peroxidation level detection in mature oocytes of each group are shown below. Figure 5 .

[0250] The results showed that the lipid peroxidation level in mature oocytes of the treatment group (0.57±0.03%) was significantly lower than that of the control group (0.66±0.02%) (P<0.05).

[0251] The above results indicate that the combined addition of SEMA4D, BC1618 and Isoschaftoside can significantly reduce oxidative damage to oocytes and protect oocyte viability.

[0252] (8) Detection of antioxidant gene expression levels in oocytes

[0253] First, RNA extraction, reverse transcription, and real-time quantitative PCR are performed. The specific steps are as follows:

[0254] The mature oocytes obtained in step (2) were collected by centrifugation and placed into 1.5 mL centrifuge tubes. Excess liquid was removed and the tubes were stored in an 80°C freezer. Total RNA was extracted using Trizol. The preparation of the reverse transcription system is shown in Table 6 and the reverse transcription reaction program is shown in Table 7.

[0255] Table 6. Preparation of the reverse transcription system

[0256] 5×Prime Script Buffer 4μL RNA ≤1μg RNase-free water Make up to 20 μL

[0257] Table 7 Reverse Transcription Reaction Program Settings

[0258]

[0259]

[0260] After reverse transcription, the cDNA sample was stored at 80°C for real-time quantitative PCR.

[0261] Following the real-time quantitative PCR reaction system and procedure settings, the cDNA template was mixed with the kit premix, and real-time quantitative PCR was performed in a BioRad CFX96 Touch RealTime PCR instrument, using GAPDH as an internal reference gene. -△Ct The relative expression levels of the target gene were calculated using the following methods. The preparation of the real-time quantitative PCR system is shown in Table 8, and the reaction program settings for the real-time quantitative PCR are shown in Table 9.

[0262] Table 8. Preparation of Real-Time Quantitative PCR System

[0263] Components Volume (μL) 2×TransStart Tip Green qPCR SuperMix 5.0 Primer-F 0.2 Primer-R 0.2 cDNA 1.0 Nuclease-free water 3.6 37℃ 15min 85℃ 5s

[0264] Table 9. Quantitative PCR Reaction Program Settings

[0265] temperature reaction time Cycle number 94℃ 30s 1 94℃ 5s 42 60℃ 30s 42

[0266] The results of the detection of antioxidant gene expression levels in mature oocytes of each group are shown below. Figure 6 .

[0267] The results showed that the expression levels of antioxidant genes SOD1, SOD2, SOD3, CATALASE, GCLM and GCLC in mature oocytes of the treatment group were significantly higher than those of the control group (P<0.05).

[0268] The above results indicate that the combined addition of SEMA4D, BC1618 and Isoschaftoside can increase the expression of antioxidant genes in oocytes and effectively enhance the antioxidant capacity of oocytes.

[0269] The above test results fully demonstrate that the multifunctional composition (SEMA4D, BC1618 and Isoschaftoside) of "mitochondrial division promoter + biogenesis inducer + reactive oxygen species scavenger + lipid peroxidation inhibitor + antioxidant gene expression enhancer" provided by this invention can effectively improve the in vitro maturation quality of oocytes and their developmental potential after fertilization.

[0270] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0271] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A formulation for in vitro embryo production, characterized in that, The formulation contains at least one of the following components: SEMA4 proteins; Fbxo48 inhibitors; Flavonoid C-glycosides.

2. The formulation according to claim 1, characterized in that, The SEMA4 proteins include one or more of the following: SEMA4A, SEMA4B, SEMA4C, SEMA4D, SEMA4E, SEMA4F, and SEMA4G. Optionally, the SEMA4 protein is a SEMA4D protein; Optionally, the Fbxo48 inhibitor is BC1618 and / or BC1583; Optionally, the flavonoid glycosides include one or more of isoxavorin, scutellarin, isoscutellarin, vitexin, isovitexin, and puerarin. Optionally, the concentration of the SEMA4 protein is 5–200 ng / mL; Optionally, the concentration of the Fbxo48 inhibitor is 1–10 μmol / L; Optionally, the concentration of the flavonoid C-glycoside compound is 1–10 μmol / L; Optionally, the formulation contains a SEMA4 protein and an Fbxo48 inhibitor, wherein the concentration of the SEMA4 protein is 5–200 ng / mL and the concentration of the Fbxo48 inhibitor is 1–10 μmol / L. Optionally, the formulation contains a SEMA4 protein, an Fbxo48 inhibitor, and isoxavorin, wherein the concentration of the SEMA4 protein is 5–200 ng / mL, the concentration of the Fbxo48 inhibitor is 1–10 μmol / L, and the concentration of isoxavorin is 1–10 μmol / L.

3. The formulation according to claim 1, characterized in that, The formulation contains at least one of the following components: SEMA4D protein; BC1618; Iso-Xia-Fo-Ta-Gan.

4. The formulation according to claim 3, characterized in that, The concentration of the SEMA4D protein is 5–200 ng / mL; Optionally, the concentration of BC1618 is 1–10 μmol / L; Optionally, the isoxavorin is 1–10 μmol / L; Optionally, the formulation contains SEMA4D protein and BC1618, wherein the concentration of SEMA4D protein is 5–200 ng / mL and the concentration of BC1618 is 1–10 μmol / L. Optionally, the formulation contains SEMA4D protein, BC1618 and isoxavorin, wherein the concentration of SEMA4D protein is 50–200 ng / mL, the concentration of BC1618 is 1–3 μmol / L, and the concentration of isoxavorin is 1–10 μmol / L.

5. The formulation according to any one of claims 1 to 4, characterized in that, The formulation is used in the in vitro maturation stage of oocytes; Optionally, the formulation further includes one or more of the following: inorganic salts, amino acids, vitamins, sugars, follicle-stimulating hormone, luteinizing hormone, estradiol, fetal bovine serum, pH indicators, and antibiotics. Optionally, the formulation further includes 80–95% v / v TCM199 medium, 5–10 IU / mL pregnant mare serum gonadotropin, 5–10 IU / mL luteinizing hormone, 1–10 μg / mL estrogen, 100–200 IU / mL penicillin, 100–200 μg / mL streptomycin, 5–20% follicular fluid, 0.4–0.8 mg / mL glucose, 1.5–2.5 mg / mL sodium bicarbonate, 0.05–0.08 mg / mL sodium pyruvate, and 0.3–0.6 mg / mL cysteine; Optionally, the oocyte is derived from a non-human mammal; Optionally, the mammals include one or more of non-human primates, cattle, sheep, pigs, horses, and mice.

6. The use of SEMA4 proteins, Fbxo48 inhibitors, flavonoid C-glycosides, or the formulations for in vitro embryo production as described in any one of claims 1 to 5 in in vitro embryo production.

7. The application according to claim 6, characterized in that, The SEMA4 protein, Fbxo48 inhibitor, flavonoid C-glycoside compound, or the formulation for in vitro embryo production according to any one of claims 1 to 5 has at least one of the following uses: Improve the quality of in vitro oocyte maturation; Enhance the developmental potential of in vitro embryos; in, Enhancing the developmental potential of in vitro embryos includes improving the developmental efficiency and / or quality of in vitro embryos.

8. The application according to claim 7, characterized in that, The SEMA4 protein, Fbxo48 inhibitor, flavonoid C-glycoside compound, or the formulation for in vitro embryo production according to any one of claims 1 to 5 has at least one of the following uses: Promotes mitochondrial division in oocytes; Increase the number of mitochondria in oocytes; Increase the mitochondrial membrane potential of oocytes; Increase the intensity of mitochondrial respiration in oocytes; Increase ATP levels in oocytes; Reduce lipid peroxidation levels in oocytes; Increase the expression level of antioxidant genes in oocytes; The antioxidant genes include one or more of SOD1, SOD2, SOD3, CATALASE, GCLM, and GCLC.

9. A method for in vitro embryo production, characterized in that, The method includes: Co-culture oocytes with SEMA4 protein, Fbxo48 inhibitor, flavonoid C-glycosides or the formulation for in vitro embryo production as described in any one of claims 1 to 5; The oocytes were derived from non-human mammals.

10. The method according to claim 9, characterized in that, The non-human mammals include one or more of the following: non-human primates, cattle, sheep, pigs, horses, and mice; Optionally, the concentration of the SEMA4 protein is 5–200 ng / mL; Optionally, the concentration of the Fbxo48 inhibitor is 1–10 μmol / L; Optionally, the concentration of the flavonoid C-glycoside compound is 1–10 μmol / L.

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