An oocyte in vitro maturation culture medium additive and application thereof

Abstract

The application provides an oocyte in-vitro maturation culture solution additive and application thereof, and belongs to the technical field of cell culture. The additive takes dimethyl succinate as an active ingredient, and the final concentration is 1-4 mM. In application, the sheep cumulus-oocyte complex is placed in TCM-199 culture solution containing the additive for culture. Experiments show that the additive can significantly improve the cumulus diffusion index and the first polar body discharge rate, up-regulate the expression of genes such as HAS2 and PTX3, reduce the active oxygen level, improve the mitochondrial membrane potential and ATP content, and enhance the antioxidant capacity. Transcriptome analysis confirms that the additive improves the oocyte quality by regulating the mitochondrial metabolism, oxidative phosphorylation and apoptosis pathway. The subsequent cleavage rate, blastocyst rate and blastocyst cell number of the treated oocyte are significantly improved. The application solves the problems of poor oocyte maturation quality and low embryo development potential in the existing system, is simple to operate, low in cost, and suitable for large-scale sheep embryo production.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to an additive for oocyte in vitro maturation culture medium and its application. Background Technology

[0002] In vitro maturation (IVM) of sheep oocytes is a crucial step in in vitro embryo production (IVP) technology, directly impacting the efficiency of subsequent in vitro fertilization (IVF) and in vitro embryo culture (IVC), as well as embryo quality. However, existing sheep oocyte IVM culture systems still have many shortcomings, primarily manifested in: low oocyte maturation rates, asynchronous cytoplasmic and nuclear maturation, high levels of oxidative stress, impaired mitochondrial function, and weak developmental potential in subsequent embryos. These factors severely restrict the propagation efficiency of superior sheep germplasm resources and the development of transgenic technology.

[0003] Therefore, it is necessary to develop an additive and culture medium for oocyte in vitro maturation to improve oocyte maturation rate and quality. Summary of the Invention

[0004] The purpose of this invention is to provide an additive for oocyte in vitro maturation culture medium, which can improve the maturation rate of sheep oocytes when added to the oocyte in vitro maturation culture medium.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, the use of dimethyl succinate in the preparation of an additive for oocyte in vitro maturation culture medium is provided.

[0006] In some embodiments, the concentration of the oocyte in vitro maturation culture medium additive is 1-4 mM. Oocytes cultured in vitro with a concentration of 1-4 mM exhibit the highest maturation rate and the best quality.

[0007] Dimethyl succinate (DMS), CAS number 106-65-0, also known as dimethyl succinate or dimethyl succinate, is an important organic chemical intermediate widely used in pharmaceuticals, pesticides, dyes, food additives, and basic research. Its molecular formula is C6H2O. 10 O4, with a molecular weight of 146.14. This compound is miscible with diethyl ether, soluble in acetone, and slightly soluble in water and ethanol. It is mainly used in gas chromatography analysis standards, organic synthesis, and fragrance preparation. Furthermore, the oocyte is a non-human mammalian / livestock oocyte.

[0008] The structural formula of dimethyl succinate is as follows: .

[0009] In a second aspect of the invention, an additive for oocyte in vitro maturation culture medium is provided, the additive comprising dimethyl succinate.

[0010] In a third aspect, the present invention provides an in vitro maturation culture medium for oocytes, wherein the in vitro maturation culture medium for oocytes contains the aforementioned in vitro maturation culture medium additive.

[0011] Furthermore, the basal culture medium includes TCM-199 medium, serum, sodium pyruvate, L-glutamine, cysteine, estradiol, epidermal growth factor, follicle-stimulating hormone, and luteinizing hormone.

[0012] In some embodiments, the content of each component per liter of culture medium is as follows: 80-120 mL fetal bovine serum, 0.2-0.4 mmol sodium pyruvate, 1-3 mmol L-glutamine, 80-120 μmol cysteine, 0.5-1.5 mg estradiol, 8-12 μg epidermal growth factor, 8000-12000 U follicle-stimulating hormone, 8000-12000 U luteinizing hormone, and 1-4 mmol dimethyl succinate.

[0013] In a fourth aspect of the invention, the use of the aforementioned oocyte in vitro maturation culture medium additive in improving the maturation rate and / or quality of oocytes is provided.

[0014] In a fifth aspect of the invention, the use of the oocyte in vitro maturation culture medium in improving the maturation rate and / or quality of oocytes, and / or in improving the developmental potential of embryos after in vitro fertilization is provided.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The sheep oocyte in vitro maturation culture medium containing dimethyl succinate (DMS) and its application method provided by this invention have the following significant improvements and beneficial effects compared with the prior art: 1. Significantly improves the synchronicity and quality of oocyte nuclear-cytoplasmic maturation. Existing culture systems often suffer from asynchronous nucleoplasmic maturation. This invention addresses this issue by adding a specific concentration of DMS to the basal culture medium, which significantly upregulates the expression of cumulus cell expansion-related genes (HAS2, PTX3, TNFAIP6, GREM1) and increases the cumulus diffusion index (CEI). Simultaneously, it significantly increases the first polar body expulsion rate (PBE rate) in a concentration-dependent manner, with the 2 mM DMS treatment group showing the best results. This indicates that DMS effectively promotes the resumption of meiosis and arrest in the MII phase of oocyte maturation, solving the problem of delayed cytoplasmic maturation during in vitro maturation and ensuring the quality of oocyte maturation at both the morphological and molecular levels.

[0016] 2. Effectively alleviates oxidative stress and reconstructs cellular redox homeostasis. The in vitro culture environment readily induces excessive reactive oxygen species (ROS) production in oocytes, leading to developmental arrest. This invention demonstrates that DMS treatment significantly reduces ROS levels in MII-stage oocytes. Mechanistic studies show that DMS upregulates the expression of nuclear factor E2-related factor 2 (NRF2) and its downstream antioxidant enzyme genes (SOD2, CAT, GPX4). This activation of the antioxidant pathway enhances the oocyte's ability to scavenge free radicals under external stress, thereby protecting organelles from oxidative damage and maintaining normal physiological functions.

[0017] 3. Optimize mitochondrial function and enhance cellular energy metabolism. Mitochondria are the energy supply center for oocyte maturation and early embryonic development. This invention found that DMS treatment significantly increases mitochondrial mass (enhanced MitoTracker fluorescence intensity) and improves mitochondrial membrane potential (increased JC-1 red / green fluorescence ratio), and upregulates the expression of key genes in mitochondrial biosynthesis (TFAM). More importantly, the relative ATP content in oocytes treated with DMS was significantly increased. This indicates that DMS provides sufficient energy support for oocyte meiosis and subsequent fertilization by promoting mitochondrial biosynthesis and oxidative phosphorylation, which is the material basis for enhancing embryonic developmental potential.

[0018] 4. Inhibits apoptosis and improves oocyte survival rate. High levels of ROS and energy deprivation typically trigger apoptosis. This invention, through DMS intervention, significantly downregulated the expression of pro-apoptotic genes (BAX, CASPASE3, CASPASE9) while upregulating the expression of anti-apoptotic gene (BCL2). This effective inhibition of the apoptosis signaling pathway reduces cell loss during culture, ensuring a sufficient number of high-quality oocytes for subsequent fertilization, thereby improving the efficiency of the entire in vitro embryo production process.

[0019] 5. Significantly enhances the developmental potential of subsequent embryos and increases the yield of high-quality embryos. The core advantage of this invention lies not only in improving the condition of the oocytes themselves, but also in directly translating into enhanced subsequent developmental capabilities. Experimental data show that mature oocytes treated with DMS exhibited significantly higher cleavage rates, 8-cell embryo rates, and blastocyst rates after in vitro fertilization compared to the control group. In particular, DMS treatment significantly increased the total number of cells in the blastocysts (confirmed by Hoechst 33342 staining), meaning that the obtained blastocysts have a higher inner cell mass cell count, potentially leading to a substantial increase in pregnancy and litter sizes after embryo transfer.

[0020] 6. The optimal concentration and mechanism of action have been determined, possessing extremely high clinical translational value. This invention, through systematic concentration gradient experiments (1 mM, 2 mM, 4 mM), clarified that 2 mM is the optimal addition concentration, avoiding the problems of ineffectiveness due to excessively low concentrations or toxicity due to excessively high concentrations. Furthermore, combined with transcriptome sequencing analysis, it elucidated that DMS primarily functions through the core network of "mitochondrial function-oxidative phosphorylation-redox balance-cell cycle / apoptosis." This provides a solid theoretical basis for the expanded application of DMS in assisted reproduction in cattle, sheep, and other livestock, and even in humans. The method is simple to operate, requiring only the addition of trace amounts of DMS to existing commercial culture media, making it low-cost and easily applicable in large-scale embryo production. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Figures show the in vitro maturation of sheep oocytes and the expression of related genes after treatment with different concentrations of dimethyl succinate (DMS). In the figures, A shows the morphology of COCs before and after in vitro maturation, and low-power and high-power fields of MII-stage oocytes; B shows the cumulus oophorus diffusion index (CEI); C-F show the relative expression levels of HAS2, PTX3, TNFAIP6, and GREM1, respectively; G shows the first polar body expulsion rate (PBE rate); and H-K show the relative expression levels of BCL2, BAX, CASPASE3, and CASPASE9.

[0023] Figure 2Figure 1 shows the results of oxidative stress, mitochondrial function, and related gene expression in sheep MII oocytes after treatment with different concentrations of DMS. In the figure, A-B represent DCFH-DA staining and relative fluorescence intensity; C-D represent MitoTracker staining and relative fluorescence intensity; E-F represent JC-1 staining and red / green fluorescence ratio; G represents relative ATP content; and H-L represent the relative expression levels of TFAM, NRF2, SOD2, CAT, and GPX4, respectively.

[0024] Figure 3 This image shows the transcriptome analysis results of sheep oocytes at stage MII after DMS treatment. A represents principal component analysis; B is a volcano plot of differentially expressed genes; C is a heatmap of differentially expressed gene clusters; DE represents GO enrichment analysis of differentially expressed genes; FG represents KEGG pathway enrichment analysis; and HI represents the GSEA enrichment curve.

[0025] Figure 4 Figures show the results of in vitro fertilization and early embryonic development of sheep oocytes after treatment with different concentrations of DMS. AB represents cleavage embryos and cleavage rate; CD represents 8-cell embryos and 8-cell rate; EF represents blastocysts and blastocyst rate; and GH represents Hoechst 33342 staining and total blastocyst cell count. Detailed Implementation

[0026] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0027] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0028] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0029] The following will provide a detailed description of an oocyte in vitro maturation culture medium additive and its application, in conjunction with embodiments and experimental data.

[0030] Example 1: In vitro maturation culture medium for oocytes Example 1 provides a formulation for an in vitro maturation culture medium for oocytes: basal culture medium + 1 mM DMS (dimethyl succinate, CAS: 106-65-0, purity ≥98%). The basal culture medium consists of TCM-199 (Gibco), 10% fetal bovine serum (FBS), 0.3 mM sodium pyruvate, 2 mM L-glutamine, 100 µM cysteine, 1 µg / mL estradiol, 10 ng / mL epidermal growth factor, 10 IU / mL follicle-stimulating hormone (FSH), and 10 IU / mL luteinizing hormone (LH). The DMS is first dissolved with a small amount of DMSO, then diluted with PBS to the required concentration, with the final DMSO concentration ≤0.1%.

[0031] Example 2: In vitro maturation culture medium for oocytes Example 2 provides a formulation for an in vitro maturation culture medium for oocytes: basal culture medium + 2 mM DMS. The basal culture medium is the same as in Example 1.

[0032] Example 3: In vitro maturation culture medium for oocytes Example 3 provides a formulation for an in vitro maturation culture medium for oocytes: basal culture medium + 4 mM DMS. The basal culture medium is the same as in Example 1.

[0033] Comparative Example 1, Control Group Comparative Example 1 was the PBS control group, consisting of basal culture medium plus an equal volume of PBS (containing the same amount of DMSO as in Example 1, so that the final DMSO volume fraction was ≤0.1%). The basal culture medium was the same as in Example 1.

[0034] Experimental Example 1: Effects of DMS on in vitro maturation of sheep oocytes I. Experimental Objective To investigate the effects of different concentrations of dimethyl succinate (DMS) on the in vitro maturation efficiency and apoptosis-related gene expression of sheep cumulus-oocyte complexes (COCs), and to screen for the optimal DMS concentration to promote oocyte maturation.

[0035] II. Experimental Methods 1. Obtaining sheep oocytes After obtaining sheep ovarian tissue from the slaughterhouse, it was placed in 0.9% physiological saline containing 200 IU / mL penicillin and 200 IU / mL streptomycin and transported back to the laboratory at approximately 28°C. Upon arrival at the laboratory, the ovarian tissue was washed five times with 0.9% physiological saline containing 200 IU / mL penicillin and 200 IU / mL streptomycin, and excess surface tissue was removed with surgical scissors. It was then placed in 0.9% physiological saline for later use.

[0036] Ovarian follicles were mechanically dissected using forceps and a No. 4 scalpel to release cumulus-oocyte complexes (COCs). COCs were released into an oocyte collection solution containing 10 mg / mL heparin, 10 mM HEPES, 1× penicillin-streptomycin antibiotics, and 10% fetal bovine serum (FBS) in TCM-199 (Gibco). After settling, COCs were collected, and those with homogeneous cytoplasm, intact morphology, and encapsulated by multiple layers of cumulus cells were preferred for subsequent in vitro maturation culture.

[0037] 2. Grouping: The sheep COCs collected in vitro were randomly divided into 4 groups: Comparative Example 1 (PBS control group, basal culture medium + equal volume of PBS), Example 1 (basal culture medium + 1 mM DMS), Example 2 (basal culture medium + 2 mM DMS), and Example 3 (basal culture medium + 4 mM DMS).

[0038] 3. In vitro maturation culture: Prepare and equilibrate four-well plates for in vitro maturation culture in advance. Add 650 µL of oocyte in vitro maturation culture medium to each well and cover with 250 µL of assisted reproductive mineral oil. Incubate at 38.5℃, 5% CO2, and saturated humidity for at least 30 min to equilibrate. Wash the collected sheep COCs three times in the in vitro processing medium and transfer them into the four-well plates, placing approximately 50 COCs in each well. Incubate at 38.5℃, 5% CO2, and saturated humidity for 22–24 h.

[0039] 3. Indicator Testing: Morphological observation: Record the cumulus diffusion index (CEI) and the expulsion of the first polar body (PB1).

[0040] Gene expression: The relative expression levels of maturation-related genes (HAS2, PTX3, TNFAIP6, GREM1) and apoptosis-related genes (BCL2, BAX, CASPASE3, CASPASE9) were detected by qRT-PCR.

[0041] III. Experimental Results 1. Maturity rate: Figure 1 A shows the typical morphology of COCs before and after in vitro maturation and the low- and high-power fields of MII stage oocytes. It can be seen that the cumulus diffusion degree of Examples 1-3 is significantly better than that of Comparative Example 1. Figure 1 The quantitative results of the cumulus diffusion index (CEI) of B showed that the CEI of Examples 1 to 3 were significantly improved compared with Comparative Example 1 (P<0.05), with Example 2 (2 mM DMS) showing the best effect. Figure 1The results of the first polar body expulsion rate (PBE rate) of G showed that the PBE rates of Examples 1 to 3 were significantly higher than those of Comparative Example 1 (P<0.05), and were concentration-dependent, with the highest PBE rate in Example 2.

[0042] 2. Gene expression: Figure 1 C-F showed that the expression levels of oocyte maturation-related genes HAS2, PTX3, TNFAIP6 and GREM1 were significantly upregulated in Examples 1-3 (P<0.05), with the largest upregulation in Example 2. Figure 1 H~K showed that the expression of the anti-apoptotic gene BCL2 was significantly upregulated (P<0.05), while the expression of the pro-apoptotic genes BAX, CASPASE3 and CASPASE9 was significantly downregulated (P<0.05), and the changes were most significant in the second example group.

[0043] IV. Experimental Conclusions DMS (1~4 mM) can effectively promote the synchronicity of nucleocytoplasmic maturation in sheep oocytes, improve cumulus diffusion and first polar body expulsion, and improve oocyte maturation quality by upregulating maturation-related genes and inhibiting apoptosis pathways, with 2 mM being the optimal concentration.

[0044] Experimental Example 2: Effects of DMS on Oxidative Stress and Mitochondrial Function in MII Oocytes I. Experimental Objective To verify whether DMS enhances the antioxidant capacity and energy metabolism of mature oocytes by reducing reactive oxygen species (ROS) levels and improving mitochondrial function.

[0045] II. Experimental Methods 1. Sample collection: M2 oocytes were collected from each group (Examples 1-3 and Comparative Example 1) after treatment. 2. ROS Detection: The levels of reactive oxygen species (ROS) in MII stage oocytes of each group were detected using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Approximately 30 MII stage oocytes were randomly collected from each group, washed once with in vitro manipulation solution, and then incubated in 10 µM DCFH-DA working solution at 38.5℃ in the dark for 30 min. After incubation, the oocytes were thoroughly washed with DPBS and slides were prepared for fluorescence microscopy observation. Fluorescence images of all samples were acquired under the same microscopic imaging parameters, and the DCFH-DA fluorescence intensity was measured using ImageJ software.

[0046] 3. Mitochondrial function testing: Mitochondrial Distribution: The distribution and functional status of mitochondria in MII-stage oocytes were detected using the MitoTracker fluorescent probe. Approximately 30 MII-stage oocytes were randomly collected from each group, washed once with in vitro manipulation solution, and then incubated in MitoTracker working solution at 38.5℃ in the dark for 30 min. After incubation, the oocytes were thoroughly washed with DPBS and slides were prepared for fluorescence microscopy observation. Images of all samples were acquired under the same microscopic imaging parameters, and the MitoTracker fluorescence intensity was measured using ImageJ software.

[0047] Membrane potential detection: The mitochondrial membrane potential (ΔΨm) of MII stage oocytes in each group was further detected using the JC-1 detection kit. MII stage oocytes from each group were placed in JC-1 working solution and incubated at 38.5℃ in the dark for 30 min. After incubation, the oocytes were thoroughly washed with DPBS and slides were prepared for fluorescence microscopy observation. JC-1 fluorescence signals from each group were acquired under the same imaging parameters, and the ratio of red fluorescence of JC-1 aggregates to green fluorescence of monomers was analyzed using ImageJ software to evaluate the mitochondrial membrane potential level.

[0048] ATP content: To further evaluate the effect of DMS on mitochondrial energy metabolism in oocytes, ATP content was measured in MII stage oocytes from each group. After washing with DPBS, oocytes from each group were lysed, reacted, and detected according to the ATP assay kit instructions. The relative ATP content was calculated based on the standard curve or relative luminescence / fluorescence signal.

[0049] 4. Gene expression: qRT-PCR was used to detect the expression of genes related to mitochondrial biosynthesis (TFAM) and antioxidants (NRF2, SOD2, CAT, GPX4).

[0050] III. Experimental Results 1. Oxidative stress: Figure 2 A shows representative images of DCFH-DA fluorescence staining in MII stage oocytes from each group. Figure 2 B represents the quantitative results of relative fluorescence intensity. Compared with Comparative Example 1, the fluorescence intensity of DCFH-DA in Examples 1-3 was significantly reduced (P<0.05), indicating that DMS effectively reduced intracellular ROS levels, with the most significant decrease observed in Example 2.

[0051] 2. Mitochondrial state: Figure 2 As shown in C~D, the MitoTracker fluorescence intensity of Examples 1~3 was significantly higher than that of Comparative Example 1 (P<0.05), indicating an increase in mitochondrial mass; Figure 2E~F showed that the red / green fluorescence ratio of JC-1 was significantly increased in all three groups of Examples 1 to 3 (P<0.05), indicating enhanced mitochondrial membrane potential stability, with the most significant increase in the second group of Examples.

[0052] 3. Energy metabolism: Figure 2 G shows that the relative ATP content of Examples 1-3 is significantly higher than that of Comparative Example 1 (P<0.05). Figure 2 H~L showed that the expression levels of TFAM, a key gene in mitochondrial biosynthesis, and antioxidant-related genes NRF2, SOD2, CAT, and GPX4 were significantly upregulated in groups 1-3 of the examples (P<0.05), with the largest upregulation in group 2 of the examples.

[0053] IV. Experimental Conclusions DMS enhances the expression of antioxidant enzymes and reduces ROS levels by activating the NRF2 signaling pathway; at the same time, it promotes mitochondrial biosynthesis, increases membrane potential and ATP production, thereby optimizing mitochondrial function and providing sufficient energy and a stable redox environment for oocyte maturation.

[0054] Experiment Example 3: Analysis of the transcriptome mechanism of DMS regulation of sheep oocyte maturation I. Experimental Objective Revealing the intrinsic molecular mechanism by which DMS improves the maturation quality of sheep oocytes at the transcriptomic level.

[0055] II. Experimental Methods 1. Sample grouping: Comparative Example 1 was the NC group, and Example 2 (treated with 2 mM DMS) was the DMS group.

[0056] 2. Sequencing and Analysis: Total RNA was extracted from MII stage oocytes of each group and subjected to transcriptome sequencing (RNA-seq). Principal component analysis (PCA), differentially expressed gene (DEG) screening, GO and KEGG enrichment analysis, and GSEA enrichment analysis were performed on the data. To further illustrate the molecular basis of DMS in improving the maturation quality of sheep oocytes, MII stage oocytes obtained in Comparative Example 1 were used as the NC group, and MII stage oocytes obtained in Example 2 were used as the DMS group. Samples were collected from each group for transcriptome sequencing analysis. After quality control, sequence alignment, and expression quantification of the sequencing data, differentially expressed genes were screened, and principal component analysis, differentially expressed gene clustering analysis, GO enrichment analysis, KEGG pathway enrichment analysis, and gene set enrichment analysis were performed.

[0057] III. Experimental Results 1. Overall differences: Figure 3Principal component analysis (PCA) of sample A showed that the NC group (Comparative Example 1) and the DMS group (Example 2) were significantly separated in the principal component space, indicating that DMS caused a significant change in the overall transcriptional profile of oocytes. Figure 3 The differentially expressed gene volcano plot of B showed that a large number of significantly differentially expressed genes were screened (|log2FC|≥1, padj<0.05), among which the number of upregulated and downregulated genes was clearly defined. Figure 3 The cluster heatmap of C visually demonstrates the significant differences in gene expression patterns between the DMS group and the NC group.

[0058] 2. Functional enrichment: Figure 3 GO enrichment analysis of D~E showed that differentially expressed genes were mainly enriched in the mitochondrial electron transport chain, oxidative phosphorylation, ATP synthesis, oocyte meiosis, cell cycle regulation, and p53 apoptosis signaling pathway. Figure 3 KEGG pathway enrichment analysis of F~G further confirmed that differentially expressed genes were significantly enriched in the TCA cycle (succinate node), oxidative phosphorylation, cell cycle and p53 signaling pathways, and signaling pathways related to granulosa cell-oocyte dialogue (such as SUCNR1 downstream signaling) also showed a significant enrichment trend.

[0059] 3. Pathway validation: Figure 3 The GSEA enrichment curves of H~I showed that DMS treatment significantly activated the gene set associated with energy metabolism (such as oxidative phosphorylation) (NES>1, padj<0.05), while significantly inhibiting the gene set associated with apoptosis (NES<-1, padj<0.05).

[0060] IV. Experimental Conclusions Transcriptome analysis demonstrated at the whole-genome level that DMS improves oocyte quality by synergistically regulating the core networks of "TCA cycle-oxidative phosphorylation-energy metabolism" and "cell cycle-apoptosis". Its mechanism of action involves succinate metabolism nodes and granulosa cell-oocyte communication signaling pathways.

[0061] Experiment Example 4: Effects of DMS on the developmental potential of early sheep embryos I. Experimental Objective To assess the subsequent in vitro fertilization capacity and early embryonic developmental potential of mature oocytes pretreated with DMS.

[0062] II. Experimental Methods 1. In Vitro Fertilization (IVF): Mature oocytes from each group in Example 1 were co-incubated with sheep sperm selected by Percoll gradient centrifugation for 18–22 h. Specifically, mature oocytes obtained after treatment under different maturation culture conditions were used for in vitro fertilization. The oocytes in each group were derived from the in vitro maturation culture conditions corresponding to Comparative Example 1, Example 1, Example 2, and Example 3, respectively; the same conventional in vitro fertilization solution was used during the in vitro fertilization stage to evaluate the effect of DMS treatment during the maturation culture stage on subsequent fertilization and embryonic development.

[0063] The in vitro fertilization solution included SOF basal solution, 2% ESS (estrous sheep serum), 2 mM sodium pyruvate, 10 µg / mL heparin, 1 mM caffeine, and 3 mg / mL BSA. The PHE mixture included double-distilled water, 0.1 mg / mL taurine, 0.3 mg / mL penicillamine, and 0.045 mg / mL adrenaline. The Percoll gradient solution was constructed using 90% Percoll and 45% Percoll, wherein the 90% Percoll contained 0.3 mg / mL calcium chloride, 0.08 mg / mL magnesium chloride, 0.8 mg / mL sodium bicarbonate, and 184 µL of 60% sodium lactate.

[0064] Prepare and equilibrate four-well plates for in vitro fertilization culture in advance. Add 40 µL of PHE mixture to every 850 µL of in vitro fertilization fluid, mix well, add 500 µL to each well, and cover with 250 µL of mineral oil. Equilibrate for at least 30 min at 38.5℃, 5% CO2, 5% O2, 90% N2, and saturated humidity.

[0065] Mature oocytes with well-spread cumulus cells, clearly extruded first polar bodies, homogeneous cytoplasm, spherical shape, and intact zona pellucida were selected. These oocytes were transferred to a droplet of in vitro manipulation solution using a pipette with an approximate diameter of 120 µm, and the outer cumulus cells were removed with 0.1% (w / v) hyaluronidase. The oocytes were then washed five times in the in vitro manipulation solution and once in the in vitro fertilization solution before being transferred to a pre-equilibrated four-well plate for in vitro fertilization culture.

[0066] Frozen sperm were removed from the liquid nitrogen tank, thawed in a 38.5°C water bath for 30 s, and then added to Percoll gradient solution. The mixture was centrifuged horizontally at 2100 rpm for 10 min, and the sperm with higher motility at the bottom were collected. The selected sperm were then centrifuged at 1×10⁻⁶. 6 A final concentration of [value] mL was added to the in vitro fertilization culture wells and co-incubated with mature oocytes for 18–22 h. Excess sperm were then removed from the zona pellucida surface, and the fertilized eggs were transferred to the subsequent in vitro embryo culture system. Cleavage rate was calculated approximately 48 h post-fertilization, and the 8-cell rate was calculated on day 3.

[0067] 2. In vitro embryo culture and blastocyst cell count: After in vitro fertilization, fertilized eggs were placed in in vitro manipulation medium to wash and remove excess sperm from the zona pellucida surface. They were then transferred to in vitro embryo culture medium and cultured for 7–8 days, with 5% fetal bovine serum added on day 3. Four-well plates for in vitro embryo culture were prepared and equilibrated in advance. 500 µL of in vitro embryo culture medium was added to each well, and 250 µL of mineral oil was added to cover the plate. The plates were then equilibrated for at least 30 minutes at 38.5℃, 5% CO2, 5% O2, 90% N2, and saturated humidity. Embryos were transferred to droplets of in vitro manipulation medium using approximately 120 µm pipettes for washing, with approximately 50 embryos placed in each well. These were then cultured under the aforementioned hypoxic conditions for 7–8 days. Blastocyst formation was observed on day 7 or 8, and the blastocyst rate was recorded. On day 7, blastocysts from each group were randomly selected for Hoechst 33342 staining, and the total cell count was observed and recorded.

[0068] III. Experimental Results 1. Developmental efficiency: Figure 4 As shown in A~B, compared with Comparative Example 1, the cleavage rate of Examples 1~3 was significantly improved (P<0.05), with the highest rate in Example 2 group; Figure 4 As shown in C~D, the 8-cell rate on day 3 was significantly higher in all three groups of Example 1 to Example 3 than in Comparative Example 1 (P<0.05). Figure 4 E~F showed that the blastocyst rate on days 7-8 was significantly improved in all three groups (P<0.05), with the largest increase in group 2, where the blastocyst rate was about 13.98% higher than that in control group 1.

[0069] 2. Blastocyst quality: Figure 4 The Hoechst 33342 staining results of G~H showed that the total number of cells in the blastocysts obtained in Examples 1~3 was significantly higher than that in Comparative Example 1 (P<0.05), with the highest number of cells in the blastocyst group in Example 2, indicating that the blastocysts were of the best quality.

[0070] IV. Experimental Conclusions Mature oocytes pretreated with DMS showed significantly improved cleavage rate, 8-cell rate, and blastocyst rate after in vitro fertilization, and a significant increase in the total number of cells in the blastocysts. This indicates that DMS can not only accelerate embryo development but also improve blastocyst quality, thereby enhancing the embryo's developmental potential and post-implantation survival rate.

[0071] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0072] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of dimethyl succinate as an additive in the preparation of oocyte in vitro maturation culture medium.

2. The application according to claim 1, characterized in that, The final concentration of the dimethyl succinate in the culture medium is 1-4 mM.

3. An additive for oocyte in vitro maturation culture medium, characterized in that, Its active ingredient is dimethyl succinate, and the dimethyl succinate is at a concentration of 1~4 mM at the corresponding usage concentration of the additive.

4. An in vitro maturation culture medium for oocytes, characterized in that, It includes the basic culture medium and the additives described in claim 3.

5. The oocyte in vitro maturation culture medium according to claim 4, characterized in that, The basic culture medium includes TCM-199 medium, serum, sodium pyruvate, L-glutamine, cysteine, estradiol, epidermal growth factor, follicle-stimulating hormone, and luteinizing hormone.

6. The oocyte in vitro maturation culture medium according to claim 5, characterized in that, The components per liter of culture medium are: fetal bovine serum 80-120 mL, sodium pyruvate 0.2-0.4 mmol, L-glutamine 1-3 mmol, cysteine ​​80-120 μmol, estradiol 0.5-1.5 mg, epidermal growth factor 8-12 μg, follicle-stimulating hormone 8000-12000 U, luteinizing hormone 8000-12000 U, and dimethyl succinate 1-4 mmol.

7. The use of the additive according to claim 3, characterized in that, Used to improve the in vitro maturation rate and / or quality of oocytes.

8. The use of the culture medium according to any one of claims 4-6, characterized in that, Used to improve the in vitro maturation rate and / or quality of oocytes, and / or to improve the developmental potential of embryos after in vitro fertilization.

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