Method for improving in-vitro maturation effect of vitrification bovine oocytes in GV stage

Abstract

The invention provides a method for improving the in-vitro maturation effect of vitrified bovine GV-stage oocytes, and in the cryopreservation process of the bovine GV-stage oocytes, the developmental ability of the bovine GV-stage oocytes after vitrification is improved by independently or jointly adding ergothioneine (EGT), isocitric acid (ICA), PLCzeta and Scriptaid. Experiments show that by adding EGT and ICA into a pretreatment solution, a freezing solution, an unfreezing solution and an oocyte in-vitro maturation solution and adding PLCzeta and Scriptaid into the unfreezing solution and the oocyte in-vitro maturation solution, damage of freezing to oocytes can be relieved to a great extent.

Description

Technical Field

[0001] This invention relates to the field of animal embryo engineering technology, and more specifically, to a method for improving the in vitro maturation effect of vitrified bovine GV stage oocytes. Background Technology

[0002] Cryopreservation of GV-stage oocytes provides a stable source of oocytes, enabling the establishment of oocyte banks and improving oocyte utilization, which is crucial for seasonally breeding animals (Kamoshita et al., 2024). Simultaneously, GV-stage oocyte cryopreservation can be used for the preservation of genetic material in endangered species, a vital means of maintaining the diversity of livestock genetic resources (Bhatand Sofi, 2021). Cryopreservation of GV-stage oocytes can meet the needs of infertile patients seeking to have children. In human assisted reproduction, GV-stage oocyte cryopreservation has several clinical advantages. Vitrification of GV-stage oocytes can reduce the ovarian stimulation caused by drugs, lowering the risk of ovarian hyperstimulation syndrome and hormone-sensitive malignancies (Khalili et al., 2017). Furthermore, patients undergoing chemotherapy or radiotherapy cannot obtain normal oocytes; cryopreservation of GV-stage oocytes before chemotherapy or radiotherapy effectively solves this problem, thus gaining widespread application in assisted reproductive medicine (Kato, 2016).

[0003] Although most GV oocytes can be successfully fertilized and develop into fetuses after freezing, the negative effects of freezing and thawing cannot be ignored, such as mechanical damage caused by ice crystal formation, microenvironmental imbalance caused by abnormally high levels of reactive oxygen species (ROS), and ATP level disturbances. Vitrification technology uses high-concentration osmotic protectants to replace most of the water inside the oocyte to reduce ice crystal formation. However, Anzar et al., using accelerator X-ray diffraction (SXRD), revealed that hexagonal ice crystals formed in bovine cumulus-oocyte complexes (COCs) during vitrification, which can cause mechanical damage to the cytoskeleton, organelles, etc. (Anzar et al., 2014). Furthermore, cryopreserved GV exhibits abnormally elevated ROS activity levels upon thawing, leading to redox imbalance and inhibited oocyte development. This phenomenon is commonly observed in mice (Qin et al., 2021), cattle (Gutierrez-Castillo et al., 2023), sheep (Zhang et al., 2024), and humans (Nohales-Córcoles et al., 2016). Additionally, cryopreserved GV oocytes show impaired mitochondrial structure and function, resulting in decreased mitochondrial membrane potential (ΔΨm) and ATP levels, thus inhibiting oocyte development (Mogas et al., 2024). Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the in vitro maturation effect of vitrified bovine GV stage oocytes.

[0005] To achieve the objective of this invention, in a first aspect, this invention provides a method for improving the in vitro maturation effect of vitrified bovine GV stage oocytes. During the cryopreservation of bovine GV stage oocytes, EGT and ICA are added to the pretreatment solution, freezing solution, thawing solution, and oocyte in vitro maturation solution, while PLCzeta and Scriptaid are added to the thawing solution and oocyte in vitro maturation solution, thereby improving the developmental capacity of bovine GV stage oocytes after vitrification.

[0006] ICA is isocitrate, EGT is ergothioneine, PLCzeta is bovine phosphatidylinositol-4,5-bisphosphate phosphodiesterase ζ (NCBI code NC_037332.1), and Scriptaid is a histone deacetylase inhibitor. Scriptaid is available from Sigma-Aldrich, catalog number S7817.

[0007] Furthermore, bovine GV stage oocytes were treated in a pretreatment solution containing EGT and ICA, followed by vitrification and thawing. The pretreatment solution was M199 medium containing 10% (v / v) ethylene glycol (EG) and 10% (v / v) dimethyl sulfoxide (DMSO).

[0008] Preferably, the concentrations of EGT and ICA added to the pretreatment solution are 25 µM and 0.5 M, respectively.

[0009] Furthermore, vitrification freezing is performed using a freezing solution containing EGT and ICA.

[0010] The cryosol was M199 medium containing 20% ​​(v / v) ethylene glycol, 20% (v / v) dimethyl sulfoxide, 0.5 mol / L sucrose, 20% (v / v) fetal bovine serum (FBS) and 300 g / L Ficoll (polysucrose).

[0011] Preferably, the concentrations of EGT and ICA added to the cryosol are 25 µM and 0.5 M, respectively.

[0012] Furthermore, the thawing solution consists of thawing solution II and thawing solution II, which are M199 medium containing 0.25 M sucrose and M199 medium containing 0.15 M sucrose, respectively. The thawing method includes: removing the OPS tube from liquid nitrogen, blowing the oocytes in the tube into thawing solution I containing EGT, ICA, PLCzeta, and Scriptaid, incubating for 1 min, then transferring to thawing solution II containing EGT, ICA, PLCzeta, and Scriptaid, incubating for 5 min, then transferring to oocyte in vitro maturation solution containing EGT, ICA, PLCzeta, and Scriptaid for 30 min of recovery, and finally selecting oocytes with uniform cytoplasm and good membrane integrity to undergo in vitro maturation in oocyte in vitro maturation solution containing EGT, ICA, PLCzeta, and Scriptaid.

[0013] The oocyte in vitro maturation medium was M199 medium containing 10% (v / v) fetal bovine serum, 10 μg / mL follicle-stimulating hormone (FSH), 10 μg / mL luteinizing hormone (LH), 10 μg / mL heparin, 40 ng / mL insulin-like growth factor (IGF), 1 μg / mL estradiol and 50 ng / mL epidermal growth factor (EGF).

[0014] Preferably, the concentrations of EGT, ICA, PLCzeta, and Scriptaid added to the oocyte in vitro maturation medium are 25 µM, 0.5 M, 0.5 μg / mL, and 10 nM, respectively.

[0015] Preferably, the culture conditions used for in vitro maturation are: 38.5℃, 5% CO2, maturation for 22-24 h.

[0016] Secondly, this invention provides the application of combined treatment with EGT, ICA, PLCzeta and Scriptaid in promoting in vitro maturation of vitrified bovine GV stage oocytes; The application aims to improve the developmental capacity of bovine GV-stage oocytes after vitrification.

[0017] In this invention, EGT is a natural amino acid derived from plants and capable of accumulating in animals. Studies have shown that it has antioxidant properties, effectively scavenging -OH groups and chelating ferrous and copper ions to prevent the formation of -OH groups from H2O2 under the influence of ferrous or copper ions. EGTEGT is absorbed by cells via a specific transporter (OCTN1) and highly concentrated in the most protected sites, such as mitochondria (the main source of ROS) and the cell nucleus (which protects DNA). This targeting makes its protective efficiency extremely high. Selenocysteine ​​is a special selenium-containing amino acid, known as the "21st amino acid." It is a component of the active center of selenium-containing enzymes such as glutathione peroxidase (GPX) and plays a crucial role in antioxidation, detoxification, immune regulation, and metabolism. Furthermore, it participates in protein synthesis through specific gene encoding.

[0018] PLCzeta: Phosphatidylinositol-4,5-bisphosphate phosphodiesterase ζ (PLCzeta) is a phospholipase C (PLC) isoenzyme specifically expressed primarily in sperm (Heytens and Parrington et al., 2009; Gonzalez-Castro and Carnevale, 2023). In mammalian fertilization, PLCzeta is considered a "sperm-borne oocyte activating factor" (SOAF) released after sperm enters the oocyte, and its core physiological function is to initiate a series of cascade reactions that activate the oocyte. After sperm and oocyte fuse, PLCzeta is released into the oocyte's cytoplasm. Subsequently, it hydrolyzes phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) on the cell membrane with extremely high calcium ion sensitivity, producing two key second messengers: inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 diffuses into the cytoplasm and binds to the IP3 receptor (IP3R) on the endoplasmic reticulum membrane, leading to the release of calcium ions (Ca) stored in the endoplasmic reticulum. 2+ A large amount of Ca is released into the cytoplasm. 2+A sudden increase in concentration activates a series of downstream signaling pathways, and simultaneously, Ca... 2+ When the concentration increases, it will be pumped back to the endoplasmic reticulum or expelled from the cell through a feedback mechanism, leading to a decrease in concentration. The continuous action of PLCzeta causes this process to repeat cyclically at a specific frequency and amplitude, forming the so-called "calcium ion oscillation".

[0019] Due to the central role of PLCzeta in oocyte activation, researchers have developed a technique to artificially activate oocytes using exogenous PLCzeta. By microinjecting cRNA or recombinant protein encoding bovine PLCzeta into bovine oocytes, the physiological fertilization process can be effectively simulated, inducing calcium oscillations similar to sperm induction patterns. This method has been shown to successfully activate oocytes and support subsequent parthenogenesis or clonal embryo development (Ross and Beyhan et al., 2008). Studies have shown that activating SCNT embryos by injecting bovine PLCzeta cRNA can achieve developmental rates comparable to or even higher than those of in vitro fertilization (IVF) embryos. A detailed comparative experiment showed that among 332 SCNT embryos activated with PLCzeta cRNA, the cleavage rate reached 78.9% (262 / 332), and the blastocyst formation rate was 27.7% (92 / 332). This result was not statistically significantly different from the blastocyst rate of the Iono / DMAP chemical activation group (29.2%) and the IVF control group (36.5%), but significantly better than the Iono / CHX group (19.4%). Furthermore, cloned embryos activated by PLCzeta exhibited a lower rate of chromosomal abnormalities, and their gene expression profiles and epigenetic modifications were closer to those of IVF embryos, suggesting that this activation method may be more beneficial for nuclear reprogramming and healthy embryonic development. PLCzeta function exhibits significant species specificity. Studies in bovine oocytes have found that bovine PLCzeta (b PLCzeta) induces calcium oscillations more effectively in the homologous system (i.e., bovine oocytes) than heterologous mouse PLCzeta (m PLCzeta). For example, low concentrations of bPLCzeta cRNA can induce robust calcium oscillations in bovine oocytes, while mPLCzeta cRNA requires higher concentrations to achieve a similar effect. The calcium oscillation pattern is considered a physiological signal necessary to activate oocytes and ensure normal embryonic development.

[0020] Scriptaid, a histone deacetylase inhibitor, functions by inhibiting HDAC activity, increasing histone acetylation levels, thereby relaxing chromatin structure and promoting gene expression reprogramming (SUN, H, et al., 2015). In reproductive medicine, this compound is widely used to improve the quality and developmental potential of gametes and embryos. Studies have shown that adding specific concentrations of Scriptaid during sperm processing in patients with asthenospermia can effectively enhance sperm motility, ATP concentration, mitochondrial membrane potential, and hyaluronic acid binding activity, and significantly improve the fertilization rate of intracytoplasmic sperm injection (ICSI). In embryo engineering, Scriptaid is particularly effective in treating somatic cell nuclear transfer embryos. For example, in sheep cloning experiments, treating donor cells or reconstructed embryos with 0.2–0.29 μmol / L Scriptaid significantly improves blastocyst development and pregnancy rates. This mechanism is closely related to increasing the acetylation levels of histone sites such as H3K9 and H4K12 in early embryos. Bovine blastocysts treated with 5 nM showed the best quality. Furthermore, the study also found that Scriptaid helps improve the developmental potential of embryos generated from round sperm injection and better maintains the methylation status of imprinted genes. Notably, the effects of Scriptaid exhibit a significant concentration- and time-dependent effect; excessively high concentrations (e.g., 0.89 μmol / L) or simultaneous treatment of donor cells and embryos may, however, reduce embryonic developmental capacity due to cytotoxicity. Therefore, precisely optimizing the concentration and window of action is crucial to maximizing its positive effects.

[0021] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention improves the developmental capacity of bovine GV-stage oocytes after vitrification by adding EGT, ICA, PLCzeta, and Scriptaid, either alone or in combination, during the cryopreservation of bovine GV-stage oocytes. Experiments show that adding EGT and ICA to the pretreatment solution, freezing solution, thawing solution, and oocyte in vitro maturation solution, and adding PLCzeta and Scriptaid to the thawing solution and oocyte in vitro maturation solution, can significantly mitigate the damage to GV oocytes caused by freezing, and improve the efficiency of in vitro maturation after freezing and thawing of GV-stage oocytes and the embryonic developmental capacity after in vitro fertilization. Attached Figure Description

[0022] Figure 1 In a preferred embodiment of the present invention, the combined addition of EGT, ICA, PLCzeta, and Scriptaid affects gene expression in vitrified GV-stage oocytes after maturation.

[0023] Figure 2The effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on gene expression in IVF blastocysts after vitrification are described in a preferred embodiment of the present invention. Detailed Implementation

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0025] The PLCzeta used in the following examples was derived from bovine follicles, and Scriptaid was purchased from Sigma, catalog number S7817. Other ingredients included EGT (EGT, Sigma E7521), isocitrate (ICA, Sigma I16005), PLCzeta (Sigma P5542), follicle-stimulating hormone (FSH, Sigma F4021), luteinizing hormone (LH, Sigma L9773), heparin (Sigma H4784), insulin-like growth factor (IGF, Sigma I8779), estradiol (Sigma E2257), epidermal growth factor (EGF, Sigma E4127), hyaluronidase (Sigma H3884), and M199 1× (Gibco 12340-030).

[0026] The pretreatment solution was M199 medium containing 10% (v / v) ethylene glycol (EG) and 10% (v / v) dimethyl sulfoxide (DMSO).

[0027] The cryosol was M199 medium (M199 1×) containing 20% ​​(v / v) ethylene glycol, 20% (v / v) dimethyl sulfoxide, 0.5 mol / L sucrose, 20% (v / v) fetal bovine serum (FBS) and 300 g / L Ficoll.

[0028] The thawing solution consists of two thawing solutions: one is M199 medium containing 0.25 M sucrose, and the other is M199 medium containing 0.15 M sucrose.

[0029] The in vitro maturation medium for oocytes (IVM medium) was M199 medium containing 10% (v / v) fetal bovine serum, 10 μg / mL follicle-stimulating hormone (FSH), 10 μg / mL luteinizing hormone (LH), 10 μg / mL heparin, 40 ng / mL insulin-like growth factor (IGF), 1 μg / mL estradiol and 50 ng / mL epidermal growth factor (EGF).

[0030] Example GV stage oocyte collection, freezing, IVM, experimental grouping, etc.

[0031] 2.1 Oocyte Collection Ovaries were obtained from slaughterhouses (Holstein dairy cows) and placed in 37°C physiological saline containing 1% penicillin-streptomycin. The ovaries were then transported to the laboratory within 2 hours. Cumulus-oocyte complexes (COCs) were extracted from follicles with a diameter of 2–8 mm. The COCs were then treated with 0.1% (w / v) hyaluronidase to remove cumulus cells, yielding cumulus-depleted GV-stage oocytes. Viable GV-stage oocytes were selected for subsequent experiments.

[0032] 2.2 Vitrification and thawing GV stage oocytes were vitrified using the OPS method, with the OPS being drawn from a 0.25 mL thin straw.

[0033] Vitrification: Pretreatment solutions were prepared as follows: M199 medium + 10% (v / v) ethylene glycol (EG) + 10% (v / v) dimethyl sulfoxide (DMSO); vitrification solution was prepared as follows: M199 medium + 20% (v / v) EG + 20% (v / v) DMSO, 0.5 mol / L sucrose, 20% fetal bovine serum (FBS), and 300 g / L Ficoll. Oocytes were equilibrated in the pretreatment solution at room temperature for 3 min, transferred to the vitrification solution, loaded into OPS tubes within 25 seconds, and immediately immersed in liquid nitrogen (LN2).

[0034] Thawing: Prepare two separate glass thawing solutions: Solution I: M199 medium + 0.25 M sucrose solution; Solution II: M199 medium + 0.15 M sucrose solution. Remove the OPS tubes from liquid nitrogen, shake them to remove residual liquid nitrogen, immerse the OPS tubes in Glass thawing solution I, expel the oocytes, incubate at room temperature for 1 minute, then transfer to Glass thawing solution II and incubate at room temperature for 5 minutes. After thawing, allow the oocytes to recover at room temperature for 30 minutes in IVM medium. Only select oocytes with homogeneous cytoplasm and good membrane integrity for the next step.

[0035] 2.3 In vitro maturation of oocytes Select 50 COCs as a group and place them in an in vitro maturation solution ( in vitroIn four-well plates of IVM (Intracytoplasmic Variation) culture, the culture conditions were set at 38.5℃, 5% CO2, and maturation time was 22–24 h. The maturation rate of oocytes (including the first polar body) in each group was counted. The IVM medium consisted of M199 (Gibco BRL; Grand Island, New York, USA), 10% (v / v) fetal bovine serum (FBS; Gibco BRL Division), 10 μg / mL follicle-stimulating hormone (FSH), 10 μg / mL luteinizing hormone (LH), 10 μg / mL heparin, 40 ng / mL insulin-like growth factor (IGF), 1 μg / mL estradiol, and 50 ng / mL epidermal growth factor (EGF).

[0036] 2.4 Detection of ROS levels in oocytes The ROS level of oocytes was detected using a reactive oxygen species (ROS) assay kit (Beyotime, S0033S) according to the manufacturer's instructions: oocytes were aspirated from the culture medium, washed three times with PBS, and placed in PBS solution containing 10 μM DCFH-DA (dye 2,7-dichlorofluorescein diacetate) and incubated at 37°C for 20 minutes; subsequently, oocytes were aspirated, washed three times with PBS, placed on a glass slide, covered with a coverslip, and fluorescence was observed under a laser confocal microscope with excitation wavelength of 488 nm and emission wavelength of 525 nm.

[0037] 2.5 Detection of ATP levels in oocytes The ATP levels of oocytes were detected using the Mitochondrial Membrane Potential Assay Kit (JC-1, Beyotime, C2006) according to the manufacturer's instructions: oocytes were aspirated from the culture medium and transferred to 200 μL of JC-1 staining working solution, and incubated at 37°C for 20 minutes in a cell culture incubator; subsequently, the oocytes were aspirated, washed twice with JC-1 staining buffer (1×), transferred to the culture medium, and fluorescence was observed under a laser confocal microscope with the parameters set to 485 nm excitation wavelength and 590 nm emission wavelength.

[0038] 2.6 Quantitative Real-Time PCR Blastocysts were placed in streptomycin solution (5 mg / mL), pipetted to remove the zona pellucida, and then washed three times with DPBS. The blastocysts were then placed in 1.5 mL nuclease-free centrifuge tubes and stored at -80°C for later use. cDNA was synthesized using the Cells-to-cDNA II Kit (Invitrogen, USA), and gene expression levels were detected using the PowerUp™ SYBR™ Green Master Mix Kit (Applied Biosystems, USA). The specific steps are as follows: (1) Sample processing Add 100 μL of pre-chilled Cell Lysis II Buffer to the collected sample, mix well, incubate at 75°C for 10 min, and then place on ice for later use. Then, add 2 μL of DNase I, mix gently, centrifuge briefly, incubate at 37°C for 15 min, and then incubate at 75°C for 5 min.

[0039] (2) Reverse transcription PCR reaction Add 5 μL of cell lysate, 4 μL of dNTP Mix, 2 μL of random primers, and 5 μL of nuclease-free water to the tube sequentially. After a brief centrifugation, incubate on ice for 1 min. Then, add 2 μL of 10 × RT Buffer, 1 μL of M-MLV Reverse Transcriptase, and 1 μL of RNase inhibitor, mix gently, and centrifuge briefly. Incubate at 42 °C for 1 h, followed by incubation at 95 °C for 10 min. Store the reaction product at -20 °C for later use.

[0040] (3) Real-time quantitative PCR reaction This experiment used bovine gene sequences from NCBI as a reference. Primers were designed using primer design software (https: / / bioinfo.ut.ee / primer3-0.4.0 / ) and synthesized by BGI Genomics. Reaction conditions: 95℃ pre-denaturation for 2 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles. β-actin was used as an internal control gene, and the relative expression level was calculated using 2-ΔΔCt. The primers used for quantitative real-time PCR are as follows:

[0041] Experimental Design: EGT treatment group: 25µM EGT was added to the pretreatment solution, freezing solution, thawing solution, and post-thawing IVM culture medium (in vitro maturation medium for oocytes).

[0042] ICA treatment group: 0.5 M ICA was added to the pretreatment solution, freezing solution, thawing solution, and post-thawing IVM culture medium.

[0043] PLCzeta treatment group: 0.5 μg / mL recombinant bovine PLCzeta was added to the thawing solution and the thawed IVM culture medium.

[0044] Scriptaid treatment group: 10 nM Scriptaid was added to the thawing solution and the thawed IVM culture medium.

[0045] Fresh group: Fresh GV stage oocytes were used for subsequent experiments without any treatment.

[0046] Vitrification group: GV stage oocytes were vitrified and frozen for subsequent experiments.

[0047] Experimental results: 1. Effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on maturation rate of vitrified GV oocytes and ATP content of MII oocytes. This study investigated the effects of combined EGT, ICA, PLCzeta, and Scriptaid on oocyte quality after vitrification. The results showed that the maturation rate in the Vitrification + EGT + ICA + PLCzeta + Scriptaid group (54.77 ± 6.35%) was significantly higher than that in the Vitrification group (24.49 ± 3.45%) and other treatment groups (26.06 ± 2.11% - 40.56 ± 3.26%), but not significantly different from the Fresh group (55.26 ± 6.45%). p > 0.05). This demonstrates that vitrification affects oocyte quality. The combined use of EGT, ICA, PLCzeta, and Scriptaid effectively restores the developmental capacity of vitrified oocytes; the optimal effect is achieved when all four are used together, significantly mitigating the damage caused by freezing to oocytes.

[0048] This experiment investigated the effects of combined EGT, ICA, PLCzeta, and Scriptaid on ATP content in oocytes after vitrification. The ATP content in oocytes of the Vitrification + EGT + ICA + PLCzeta + Scriptaid group (0.72 ± 0.07 pmol) was significantly higher than that of the Vitrification group (0.31 ± 0.02 pmol) and other treatment groups (0.31 ± 0.02 pmol - 0.67 ± 0.06 pmol), but there was no significant difference compared to the Fresh group (0.74 ± 0.06 pmol). p > 0.05). This indicates that vitrification affects the ATP content of oocytes, and the combined addition of EGT, ICA, PLCzeta, and Scriptaid can significantly mitigate the impact of freezing on oocyte ATP content.

[0049] The experimental results are shown in Tables 1 to 5.

[0050] Table 1. Effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on maturation rate of vitrified GV oocytes and ATP content of MII oocytes.

[0051] Note: Different superscripts indicate significant differences between groups. p < 0.05).

[0052] Table 2. Effects of EGT addition on maturation rate of vitrified GV oocytes and ATP content of MII oocytes.

[0053] Note: Different superscripts indicate significant differences between groups. p < 0.05).

[0054] Table 3. Effects of combined ICA addition on maturation rate of vitrified GV oocytes and ATP content of MII oocytes.

[0055] Note: Different superscripts indicate significant differences between groups. p < 0.05).

[0056] Table 4. Effects of combined PLCzeta addition on maturation rate of vitrified GV oocytes and ATP content of MII oocytes.

[0057] Note: Different superscripts indicate significant differences between groups. p < 0.05).

[0058] Table 5. Effects of combined Scriptaid addition on maturation rate of vitrified GV oocytes and ATP content of MII oocytes.

[0059] Note: Different superscripts indicate significant differences between groups. p < 0.05).

[0060] 2. Effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on gene expression after maturation in vitrified GV-frozen oocytes. This experiment investigated the effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on gene expression in vitrified oocytes. Figure 1 As shown, the expression levels of CD9, JUNO, BMP15, and GDF9 genes in oocytes treated with vitrification + EGT + ICA + PLCzeta + Scriptaid were significantly higher than those in the vitrification group, vitrification + EGT + ICA group, and vitrification + EGT + ICA + PLCzeta group. However, there was no significant difference in CD9 and JUNO gene expression compared to the fresh group, and the expression levels of BMP15 and GDF9 genes were significantly higher in vitrification group than in the fresh group. This indicates that vitrification affects oocyte gene expression, and the expression levels significantly increase after treatment with the added substances. The combined effect of all four substances is optimal, and the combined addition of EGT, ICA, PLCzeta, and Scriptaid can significantly alter the effect of vitrification on oocyte gene expression.

[0061] 3. Effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on the maturation and developmental capacity and ATP content of vitrified GV-stage oocytes. This study investigated the effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on the developmental ability of IVF blastocysts after vitrification. The cleavage rate (58.06 ± 6.15%) and blastocyst rate (24.31 ± 3.46%) in the Vitrification + EGT + ICA + PLCzeta + Scriptaid group were significantly higher than those in the Vitrification group (32.99 ± 3.28%, 10.77 ± 1.65%, respectively). p < 0.05), but there was no significant difference compared to the Fresh group (60.26 ± 6.71%, 25.53 ± 3.42%). p > 0.05). This shows that vitrification freezing can affect the developmental capacity of oocytes and embryos. However, the combined addition of EGT, ICA, PLCzeta, and Scriptaid can effectively restore the developmental potential of blastocysts by simultaneously enhancing tricarboxylic acid cycle metabolism and reducing reactive oxygen species levels. The effect is best when all four work together, which can greatly alleviate the damage to blastocysts after freezing.

[0062] This experiment investigated the effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on the ATP content of IVF blastocysts after vitrification. As shown in Table 6, the ATP content of oocytes in the Vitrification + EGT + ICA + PLCzeta + Scriptaid group (0.72 ± 0.08 pmol) was significantly higher than that in the Vitrification group (0.57 ± 0.04 pmol). p < 0.05), but there was no significant difference compared to the Fresh group (0.74 ± 0.06 pmol). This indicates that vitrification freezing affects the ATP content of IVF blastocysts, with the best effect achieved when all four factors work together. The combined addition of EGT, ICA, PLCzeta, and Scriptaid can significantly mitigate the impact of freezing on the ATP content of IVF blastocysts.

[0063] Table 6. Effects of combined treatments of EGT, ICA, PLCzeta, and Scriptaid on in vitro fertilization efficiency and ATP content of blastocyst weight after maturation of bovine GV stage oocytes.

[0064] 4. Effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on gene expression in IVF blastocysts after maturation of vitrified GV-stage oocytes. This experiment investigated the effects of combined addition of EGT, ICA, PLCzeta, and Scriptaid on gene expression in IVF blastocysts after vitrification. Figure 2 As shown, the expression level of the pro-apoptotic molecule BAX in the Vitrification group was significantly higher than that in the Fresh group, while the expression levels of the anti-apoptotic molecule BCL2 and functional molecules IFN-tau and Oct4 were significantly lower in the Vitrification group than in the Fresh group. After the addition of substances, the expression level of BAX decreased, while the expression levels of BCL2, IFN-tau, and OCT4 increased. Among them, the BAX and BCL2 levels in the Vitrification + EGT + ICA + PLCzeta + Scriptaid group were similar to those in the Fresh group, while the IFN-tau and OCT4 levels were higher in the Vitrification group than in the Fresh group. This indicates that vitrification freezing affects gene expression in IVF blastocysts, but the expression levels of each gene gradually recover after treatment with the addition of substances. The effect is best when all four substances work together, and the combined addition of EGT, ICA, PLCzeta, and Scriptaid can significantly affect the gene expression levels of frozen IVF blastocysts.

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for improving the effect of in vitro maturation of vitrified bovine GV stage oocytes, characterized in that, During the cryopreservation of bovine GV stage oocytes, EGT and ICA were added to the pretreatment solution, freezing solution, thawing solution and oocyte in vitro maturation solution, while PLCzeta and Scriptaid were added to the thawing solution and oocyte in vitro maturation solution, thereby improving the developmental capacity of bovine GV stage oocytes after vitrification. Among them, ICA is isocitrate, EGT is ergothioneine, PLCzeta is bovine phosphatidylinositol-4,5-bisphosphate phosphodiesterase ζ, and Scriptaid is a histone deacetylase inhibitor.

2. The method according to claim 1, characterized in that, Bovine GV stage oocytes were treated in a pretreatment solution containing EGT and ICA, and then vitrified and thawed. The pretreatment solution was M199 medium containing 10% (v / v) ethylene glycol and 10% (v / v) dimethyl sulfoxide.

3. The method according to claim 2, characterized in that, The concentrations of EGT and ICA added to the pretreatment solution were 25 µM and 0.5 M, respectively.

4. The method according to claim 2, characterized in that, Vitrification freezing is performed using a freezing solution containing EGT and ICA; The cryosol is M199 medium containing 20% ​​(v / v) ethylene glycol, 20% (v / v) dimethyl sulfoxide, 0.5 mol / L sucrose, 20% (v / v) fetal bovine serum and 300 g / L Ficoll.

5. The method according to claim 4, characterized in that, The addition concentrations of EGT and ICA in the cryogenic fluid were 25 µM and 0.5 M, respectively.

6. The method according to claim 2, characterized in that, The thawing solution consists of two thawing solutions: one is M199 medium containing 0.25 M sucrose, and the other is M199 medium containing 0.15 M sucrose. The thawing method includes: removing the OPS tube from liquid nitrogen, blowing the oocytes in the tube into thawing solution I containing EGT, ICA, PLCzeta, and Scriptaid, incubating for 1 min, then transferring to thawing solution II containing EGT, ICA, PLCzeta, and Scriptaid, incubating for 5 min, then transferring to oocyte in vitro maturation solution containing EGT, ICA, PLCzeta, and Scriptaid for 30 min of recovery, and finally selecting oocytes with uniform cytoplasm and good membrane integrity to undergo in vitro maturation in oocyte in vitro maturation solution containing EGT, ICA, PLCzeta, and Scriptaid.

7. The method according to claim 6, characterized in that, The oocytes were matured in vitro in M199 medium containing 10% (v / v) fetal bovine serum, 10 μg / mL follicle-stimulating hormone, 10 μg / mL luteinizing hormone, 10 μg / mL heparin, 40 ng / mL insulin-like growth factor, 1 μg / mL estradiol and 50 ng / mL epidermal growth factor.

8. The method according to claim 6, characterized in that, The concentrations of EGT, ICA, PLCzeta, and Scriptaid added to the oocyte in vitro maturation medium were 25 µM, 0.5 M, 0.5 μg / mL, and 10 nM, respectively.

9. The method according to any one of claims 6-8, characterized in that, The culture conditions used for in vitro maturation were: 38.5℃, 5% CO2, maturation time 22–24 h.

10. Application of combined treatment with EGT, ICA, PLCzeta and Scriptaid in promoting in vitro maturation of vitrified bovine GV stage oocytes; The application aims to improve the developmental capacity of bovine GV-stage oocytes after vitrification.

Images