Application of Scirpusin in Improving Cryopreservation and in Vitro Maturation of Oocytes

Abstract

This invention relates to the field of reproductive engineering technology, specifically to the application of rutabagastone in improving the cryopreservation and in vitro maturation of oocytes. This invention is the first to clearly demonstrate that rutabagastone can improve the quality of cryopreservation and in vitro maturation of frozen oocytes. By adding rutabagastone to the cryopreservation solution, thawing solution, and in vitro maturation solution, this invention reduces cryopreservation damage to oocytes, resulting in increased survival rate after thawing, improved mitochondrial function, reduced damage to the oocyte spindle and zona pellucida, reduced oxidative stress levels, and ultimately improved subsequent developmental capacity of frozen oocytes.

Description

Technical Field

[0001] This invention relates to the field of reproductive engineering technology, specifically to the application of triscutellarin in improving the cryopreservation and in vitro maturation of oocytes. Background Technology

[0002] Oocyte cryopreservation technology is of great significance for the preservation of genetic resources in endangered animals, rapid breeding of superior breeds, and the treatment of human infertility. However, due to the inherent fragility of cells, achieving a high development rate after thawing remains challenging. Currently, effective strategies for cell and tissue cryopreservation include slow freezing and vitrification. Compared to slow freezing, vitrification offers a faster freezing rate and less cell damage.

[0003] DMSO is the most commonly used cell cryoprotectant (CPA), inhibiting ice crystal formation by disrupting hydrogen bonds in water. However, high concentrations of DMSO often exhibit cytotoxicity, potentially leading to cell death. These limitations highlight the urgent need for alternative cryoprotectants that can mitigate the physical and biochemical damage during vitrification. Therefore, there is an urgent need to find novel cryoprotectants to improve upon traditional cryoprotectants and reduce their cytotoxicity.

[0004] Currently, with advancements in oocyte cryopreservation technology, the survival rate of cryopreserved immature oocytes (GV stage) is very high. However, the damage caused by freezing inflicts varying degrees of damage on oocyte organelles, such as mitochondria, spindle fibers, and zona pellucida, ultimately leading to a decline in oocyte developmental capacity. Finding agents to improve the maturation efficiency of cryopreserved oocytes has become a crucial breakthrough in this technology. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of scutellarin in improving the cryopreservation and in vitro maturation of oocytes.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides the application of salicylic acid ketone in improving the cryopreservation and in vitro maturation of oocytes, wherein the application is: adding salicylic acid ketone to the balancing solution, freezing solution, thawing solution and in vitro maturation solution used in the two processes of vitrification and in vitro maturation culture of oocytes.

[0007] In a second aspect, the present invention provides an oocyte cryopreservation agent, the working solution of which contains 1-25 μM of trisalicylic acid.

[0008] A third aspect of the present invention provides a method for improving the maturation quality of cryopreserved oocytes, characterized in that, in both the vitrification and in vitro maturation processes of oocytes, leucotrienol is added to the balancing solution, freezing solution, thawing solution, and in vitro maturation solution.

[0009] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention is the first to clearly demonstrate that rutin can improve the maturation quality of frozen oocytes. By adding rutin to the cryopreservation solution, thawing solution, and in vitro maturation solution, this invention reduces cryopreservation damage to oocytes, resulting in improved post-thawing survival rate, improved mitochondrial function and spindle assembly, reduced zona pellucida damage, and decreased oxidative stress levels, ultimately enhancing the subsequent developmental capacity of frozen oocytes.

[0010] This invention provides a new use for scutellarin, which can be used to prepare novel oocyte vitrification cryoprotectants and in vitro maturation culture media. The high-quality oocytes obtained by this method can not only support embryo biotechnology research such as in vitro fertilization and somatic cell nuclear transfer, but also be applied to the field of human assisted reproductive technology. Attached Figure Description

[0011] Figure 1 The effects of triscutellarin on oocyte cryopreservation survival rate and blastocyst development rate are shown in the figure. A represents the effect of different concentrations of triscutellarin on oocyte cryopreservation survival rate; B represents the effect of different concentrations of triscutellarin on oocyte cleavage rate after cryopreservation; C represents the effect of different concentrations of triscutellarin on oocyte blastocyst rate after cryopreservation; D represents the effect of adding 10 µM triscutellarin during oocyte cryopreservation and / or in vitro maturation on oocyte cleavage rate; and E represents the effect of adding 10 µM triscutellarin during oocyte cryopreservation and / or in vitro maturation on oocyte blastocyst rate. In the figure, different letters indicate significant differences between groups, while the same letter indicates no significant difference.

[0012] Figure 2 The effect of triscutellarin on the antioxidant capacity of cryopreserved oocytes was investigated. In the figure, A represents the ROS level of in vitro matured oocytes after cryopreservation, and B represents the GSH level of in vitro matured oocytes after cryopreservation. In the figure, different letters in the intergroup comparisons indicate significant differences, while the same letter indicates no significant differences.

[0013] Figure 3The effect of triscutellarin on mitochondrial distribution in cryopreserved oocytes is shown in the figure. A represents normal mitochondrial distribution, B represents abnormal mitochondrial distribution, and C represents the mitochondrial distribution in in vitro matured oocytes after cryopreservation. In the figure, different letters in the intergroup comparisons indicate significant differences, while the same letter indicates no significant differences.

[0014] Figure 4 The effect of triscutellarin on apoptosis in cryopreserved oocytes is shown in the figure. A represents normal oocytes, B represents apoptotic oocytes, and C represents apoptosis in mature oocytes after cryopreservation. In the figure, different letters in the intergroup comparisons indicate significant differences, while the same letter indicates no significant difference.

[0015] Figure 5 The effect of triscutellarin on ATP levels in cryopreserved oocytes is shown in the figure. Different letters in the intergroup comparison indicate significant differences, while the same letter indicates no significant differences.

[0016] Figure 6 The effect of triscutellarin on antioxidant genes in cryopreserved oocytes.

[0017] Figure 7 The effect of triscutellarin on spindle damage in cryopreserved oocytes is shown in the figure. In the figure, A represents spindle assembly in normal oocytes, B represents spindle assembly in abnormal oocytes, and C represents the comparison of spindle damage in different treatment groups. In the figure, different letters in the inter-group comparison indicate significant differences, and the same letter indicates no significant differences.

[0018] Figure 8 The effect of triscutellarin on zona pellucida damage in cryopreserved oocytes; in the figure, different letters in the intergroup comparisons indicate significant differences, while the same letter indicates no significant differences. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0021] In a typical embodiment of the present invention, the application of triscutellarin in improving the cryopreservation and in vitro maturation of oocytes is provided. The application is as follows: triscutellarin is added to the balancing solution, freezing solution, thawing solution and in vitro maturation solution used in the two processes of vitrification and in vitro maturation culture of oocytes.

[0022] Sauchinone is a diastereomer of lignan obtained from *Saururus chinensis*. Its molecular formula is C2. 20 H 20 O6, CAS number 177931-17-8, has the following structure: .

[0023] Currently, there are no research reports on the application of triscutellarin in the field of reproductive technology to improve the efficiency of oocyte cryopreservation and enhance the quality of in vitro cultured oocytes or embryos.

[0024] This invention discovers that scutellarin improves the maturation quality of frozen oocytes by enhancing mitochondrial function and spindle assembly after in vitro maturation of frozen oocytes, and reducing zona pellucida hardening. Specifically, it improves the survival rate of frozen oocytes, increases the rate of parthenogenetic cleavage activation, increases the blastocyst development rate, and enhances the subsequent developmental capacity of frozen oocytes.

[0025] In some embodiments, the product contains 1-25 μM of trisalicylic acid.

[0026] Furthermore, the product contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 25 or 25 μM, or a range of any two values ​​between them.

[0027] In some embodiments, the equilibration solution is a basal culture medium supplemented with 10% (v / v) fetal bovine serum, 7.5% (v / v) ethylene glycol, 7.5% (v / v) DMSO, 0.25 M trehalose, and 10 µM triscutellarin.

[0028] In some embodiments, the cryosol is a basal culture medium supplemented with 10% (v / v) fetal bovine serum, 7.5% (v / v) ethylene glycol, 7.5% (v / v) DMSO, 0.25 M trehalose, and 10 µM triscutellarin.

[0029] In some embodiments, the thawing solution includes thawing solution I and thawing solution II, wherein thawing solution I is a basal culture medium supplemented with 10% (v / v) fetal bovine serum, 10 µM trisalicylic acid, and 1 M trehalose; and thawing solution II is a basal culture medium supplemented with 10% (v / v) fetal bovine serum, 10 µM trisalicylic acid, and 1 M trehalose.

[0030] In some embodiments, the in vitro maturation solution is a basal culture medium supplemented with 10% (v / v) fetal bovine serum, 500 IU / mL penicillin, 500 μg / mL streptomycin, 50 ng / mL epidermal growth factor (EGF), 0.05 IU / mL follicle-stimulating hormone (FSH), 0.05 IU / mL luteinizing hormone (LH), 1 μg / mL 17β-estradiol, 0.0242 mg / mL sodium pyruvate, and 10 µM trisperazine.

[0031] In some embodiments, the basal culture medium is selected from any one of DMEM culture medium, TCM199 culture medium, M16, or MEM culture medium. As an example, the present invention uses TCM199 culture medium.

[0032] In another typical embodiment of the present invention, an oocyte cryopreservation agent is provided, the working solution of which contains 1~25μM of triscutellarin.

[0033] Furthermore, the working fluid contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 25 or 25 μM, or a range of any two values ​​between them.

[0034] In some embodiments, the oocyte cryopreservation agent further includes at least one additional cryoprotectant.

[0035] The additional cryoprotectant is selected from acetamide, agarose, alginate or ester, isomalt oligosaccharide, 1-aniline, albumin, amino acids, ammonium acetate, butylene glycol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethylacetamide, dimethylformamide, dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, α-glycerol phosphate or ester, glycerol monoacetate, hyaluronic acid, glycine, hydroxyethyl starch, inositol, lactose, and magnesium chloride. Magnesium sulfate, maltose, mannitol, mannose, methanol, methylacetamide, methylformamide, methylurea, hydrogel, dextrin, dextran, phenol, prolinic polyol, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, serine, sodium bromide, sodium chloride, sodium iodide, pyridine N-oxide, ribose, sodium nitrate, sucrose, trehalose, sodium sulfate, sorbitol, triethylene glycol, trimethylamine acetate, urea, valine, and xylose, or any group thereof.

[0036] In some embodiments, the oocyte cryopreservation agent further includes a cryopreservation-compatible pH buffer capable of maintaining a neutral pH value at low temperatures.

[0037] The terms "low temperature" or "freezing temperature" as used in this article generally refer to temperatures ranging from below zero to... 196℃, if from 50℃ to 196℃, if from 80℃ to 196℃, for example, below 55℃, for example, below 60℃, for example, below 65℃, for example, below 70℃, for example, below 75℃, for example, below 80℃, for example, below 85℃, for example, below 90℃, for example, below 95℃, for example, below 100℃, for example, below 105℃, for example, below 110℃, for example, below 115℃, for example, below 120℃, for example, below 125℃, for example, below 130℃, for example, below 135℃, for example, below 140℃, for example, below 145℃, for example, below 150℃, for example, below 155℃, for example, below 160℃, for example, below 165℃, for example, below 170℃, for example, below 175℃, for example, below 180℃, for example, below 185℃, for example, below The temperature is 190℃.

[0038] In a typical embodiment of the present invention, a method for cryopreserving oocytes is provided, comprising the following steps: contacting the oocytes to be cryopreserved with the above-mentioned oocyte cryopreservation agent to obtain a cryopreservation composition, and then lowering the temperature of the cryopreservation composition to the cryopreservation temperature.

[0039] In another typical embodiment of the present invention, a method for improving the quality of oocyte maturation is provided, wherein scutellarin is added to the balancing solution, freezing solution, thawing solution and in vitro maturation solution used in the two processes of oocyte vitrification and in vitro maturation culture.

[0040] In some embodiments, the final concentration of salicylic acid added to the equilibration solution, freezing solution, thawing solution or in vitro maturation solution is 1~25 μM.

[0041] In some embodiments, the final concentration of salicylic acid added to the equilibration solution, freezing solution, thawing solution or in vitro maturation solution is 10 μM.

[0042] In this invention, the oocyte is selected from mammalian cells or tissues. Further, the oocyte is a GV stage oocyte.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] The scutellarin used in this invention was purchased from MCE, catalog number HY-N0613, with the molecular formula C. 20 H 20 O6, CAS number 177931-17-8, has the following structure: .

[0045] Example 1 The basal culture medium used in the examples was TCM199 culture medium (GIBCO).

[0046] 1. Prepare the cryopreservation equilibration solution according to the following formula. The equilibration solution for oocyte cryopreservation consists of basal culture medium supplemented with fetal bovine serum, trisperidone, ethylene glycol, dimethyl sulfoxide (DMSO), and trehalose. The concentrations of fetal bovine serum in the equilibration solution are 10% (v / v), ethylene glycol 7.5% (v / v), DMSO 7.5% (v / v), and trehalose 0.25 M.

[0047] 2. Preparation of cryopreservation solution The cryoprotectant was prepared by adding 10% fetal bovine serum (v / v), 7.5% ethylene glycol (v / v), 7.5% DMSO (v / v), and 0.25 M trehalose to the basal culture medium.

[0048] 3. Preparation of oocyte thawing solution There are two types of thawing solutions for oocyte cryopreservation: thawing solution I and thawing solution II. Both thawing solution I and thawing solution II are composed of basal culture medium, fetal bovine serum, and trehalose. The concentration of fetal bovine serum in the thawing solution is 10% (v / v), the concentration of trehalose in thawing solution I is 1M, and the concentration of trehalose in thawing solution II is 0.5M.

[0049] 4. Preparation of in vitro maturation solution for oocytes Add 5 mL of fetal bovine serum, 5000 IU of penicillin, and 5000 μg of streptomycin to 45 mL of TCM199 culture medium (GIBCO). Gently stir to mix. Then add 2.5 IU of FSH, 2.5 IU of LH, 50 μg of 17β-estradiol, 1.21 mg of sodium pyruvate, and 500 ng of EGF. After standing for 2-3 hours, filter and sterilize using a 0.22 μm filter. Aliquot into 1.5 mL centrifuge tubes and store at 4°C for later use.

[0050] Example 2 1. Analyze the effects of trisqualene on the survival of cryopreserved oocytes and blastocyst development. Experimental group design: Fresh control group: The in vitro maturation solution used in the in vitro maturation culture of fresh oocytes does not contain trisperazine.

[0051] Frozen control group: The equilibration solution, freezing solution, thawing solution and in vitro maturation solution used in the vitrification and thawing of oocytes did not contain trisperazine, i.e. the equilibration solution, freezing solution, thawing solution and in vitro maturation solution prepared in Example 1.

[0052] The experimental group was divided into 4 groups: the equilibration solution, freezing solution, thawing solution and in vitro maturation culture of oocytes all contained 1 μM, 5 μM, 10 μM or 25 μM final concentration of triscutellarin, that is, the equilibration solution, freezing solution, thawing solution and in vitro maturation solution prepared in Example 1 were supplemented with the corresponding concentration of triscutellarin.

[0053] Preparation of the aforementioned scutellarin: Weigh 10 mg of scutellarin and dissolve it in 28.0607 mL of DMSO to prepare a 1 mM concentrated stock solution. After aliquoting, store at -80°C. When using, add the concentrated scutellarin stock solution to the above solutions and dilute to the desired final concentration.

[0054] The operation steps are as follows: (1) Oocyte collection GV phase oocyte acquisition: The experimental animals used in this experiment were all Kunming white mice. The mice were subjected to superovulation treatment, and 10 IU PMSG (pregnant mare serum gonadotropin) was injected at 17:00. 46-48 h later, the ovaries were collected and placed in a balanced manipulation solution. Under a stereomicroscope, the follicles on the ovaries were ruptured to release the oocytes. Round, plump oocytes with germinal follicles were selected with an oocyte retrieval needle.

[0055] (2) Vitrification cryopreservation of oocytes The vitrification cryopreservation method for oocytes is as follows: GV stage oocytes are equilibrated in cryopreservation equilibration solution for 3 minutes, then transferred to cryopreservation solution, immediately transferred to a cryopreservation loop, and quickly immersed in liquid nitrogen for freezing.

[0056] (3) Thawing of oocytes The frozen oocytes are removed from liquid nitrogen and immediately placed in thawing solution I for 3 minutes, then transferred to thawing solution II for 3 minutes to complete the thawing of the frozen oocytes.

[0057] (4) In vitro maturation of oocytes Thawed mouse GV stage oocytes were placed in an oocyte in vitro maturation culture medium preheated at 37°C for 2 hours, and then placed in an environment with a CO2 volume concentration of 5% for 14 hours for maturation culture.

[0058] (5) Parthenogenesis and embryo culture Parthenogenetic activation: Mouse oocytes were activated using SrCl2 (strontium chloride). Oocytes were first cultured in calcium-free CZB containing 10 mM SrCl2 and CB (cytochalasin) for 2.5 h, then in calcium-containing CZB containing CB for 3.5 h. Afterward, they were transferred to CZB for further culture, and after 48 hours at the 4-cell embryo stage, they were transferred to glycogen-containing CZB for blastocyst culture. Results are as follows... Figure 1 As shown in A, B, and C, 10 μM significantly improved the survival rate of frozen oocytes, the parthenogenetic activation cleavage rate, and the blastocyst rate (P<0.05).

[0059] 2. Effects of the addition of scutellarin during the cryopreservation stage on the survival of cryopreserved oocytes and blastocyst development 10 μM saurazol (SAU) was added during cryopreservation, in vitro maturation (IVM), and cryopreservation and in vitro maturation, respectively. The results are as follows: Figure 1 As shown in C and D, the addition of rutabagarin can improve the cleavage rate and blastocyst rate of frozen oocytes. Among them, the cleavage rate (P<0.05) and blastocyst rate (P<0.05) obtained by adding rutabagarin throughout the process are the highest.

[0060] Example 3: Effects of scutellarin on ROS and GSH levels in frozen oocytes after in vitro maturation Experimental group design: Fresh control group: The in vitro maturation solution used in the in vitro maturation culture of fresh oocytes does not contain trisperazine.

[0061] Frozen control group: The equilibration solution, freezing solution, thawing solution and in vitro maturation solution used in the vitrification and thawing of oocytes did not contain trisperazine, i.e. the equilibration solution, freezing solution, thawing solution and in vitro maturation solution prepared in Example 1.

[0062] The salicylic acid treatment group: The equilibration solution, freezing solution, thawing solution and in vitro maturation solution used in the vitrification and thawing of oocytes all contained a final concentration of 10 μM salicylic acid, that is, the equilibration solution, freezing solution, thawing solution and in vitro maturation solution prepared in Example 1 were supplemented with the corresponding concentration of salicylic acid.

[0063] After culturing COCs and removing cumulus cells as described in Example 2, naked oocytes were washed four times with 1% BSA-PBS and transferred to 96-well plates containing either DCFH-DA or CMF2HC dye. The plates were incubated at 37°C in the dark for 20 min, followed by three washes with 1% BSA-PBS to remove free dye. Finally, the oocytes were photographed and recorded under an inverted fluorescence microscope. The ROS and GSH levels of cryopreserved oocytes after in vitro maturation were measured in the fresh group, frozen control group, and the group treated with triglycerides. Results are as follows: Figure 2 As shown in A and B, the addition of 10 μM triyruvic acid during cryopreservation and in vitro maturation significantly reduced the ROS level of oocytes (P<0.05) and significantly increased the GSH level in frozen oocytes (P<0.05).

[0064] Example 4: Trichoderma can improve mitochondrial function in cryopreserved oocytes The experimental groups were the same as in Example 3.

[0065] After removing cumulus cells from in vitro matured cocci, oocytes were placed in a droplet containing 200 μM MitotrackerRed (Molecular Probes, Eugene, OR, USA) and incubated in a CO2 incubator for 30 minutes. The oocytes were washed three times with M2 solution, fixed in a droplet of 4% paraformaldehyde for 20 minutes, washed again, and incubated in 0.5% Triton X-100 for 20 minutes. The oocytes were then placed on a slide containing DAPI and observed under a laser confocal microscope. Figure 3 As shown in A and B, a normal distribution of mitochondria is characterized by their even distribution within the cytoplasm of the oocyte, while an abnormal distribution is characterized by mitochondria appearing in clusters or unevenly distributed within the cytoplasm. The distribution of mitochondria in cryopreserved oocytes from the fresh group, frozen control group, and triyruvic acid-treated group was examined, and the results are shown below. Figure 3 As shown in C, the addition of 10 μM triscutellarin during cryopreservation and in vitro maturation significantly reduced the proportion of abnormal mitochondrial distribution after oocyte maturation (P<0.05).

[0066] Example 5: Effect of Trichoderma on Early Apoptosis of Frozen Oocytes The experimental groups were the same as in Example 3.

[0067] Oocytes were incubated in M2 solution containing 20 μmol / L Annexin-V for 30 min, then washed three times in M2 solution for 5 min each time. Finally, the oocytes were transferred to a slide containing 10 μL DAPI. Fluorescence images were recorded under a fluorescence microscope. The fluorescence intensity in the images was statistically analyzed using ImageJ software and used as an assessment of early apoptosis expression levels. The proportion of early apoptosis in cryopreserved oocytes from the fresh group, frozen group, and triglyceride-treated group was determined. Figure 4 As shown in A, B, and C, the addition of 10 μM triscutellarin during cryopreservation and in vitro maturation significantly reduced the proportion of early apoptosis in oocytes after cryopreservation.

[0068] Example 6: Effect of Trichoderma on ATP Levels in Oocytes The experimental groups were the same as in Example 3.

[0069] The ATP content in oocytes was detected using an ATP assay kit under light-protected conditions. ATP standard solutions were serially diluted with lysis buffer. The ATP assay working solution was prepared by diluting the ATP assay solution at a 9:1 ratio with diluent. 20 μL of each concentration of standard solution was added to 100 μL of the ATP assay working solution, quickly mixed, and the relative light unit (RLU) value was measured using a multi-mode microplate reader, and a standard curve was plotted. Collected oocytes were lysed at 30 oocytes per group in 100 μL of lysis buffer. The lysed cells were centrifuged at 4°C and 12000 g for 5 min in a refrigerated centrifuge. 20 μL of the supernatant was added to 100 μL of the ATP assay working solution, quickly mixed, and the RLU value was measured using a multi-mode microplate reader. The average ATP content per cell was calculated based on the standard curve equation and the number of oocytes in each group. The ATP levels of cryopreserved oocytes in the fresh group, frozen group, and triglyceride-treated group were measured. Figure 5 As shown, the addition of 10 μM triscutellarin during cryopreservation and in vitro maturation significantly increased the ATP level of oocytes after maturation.

[0070] Example 7: Effects of scutellarin on the expression of antioxidant-related genes in frozen oocytes The experimental groups were the same as in Example 3.

[0071] Naked oocytes were washed three times with PBS and then lysed in 100 μL of lysis buffer. mRNA was extracted according to the mRNA extraction kit instructions, and then cDNA was synthesized by reverse transcription. The real-time quantitative polymerase chain reaction (PCR) system consisted of: 2 µL cDNA, 2 µL primers (sequences shown in Table 2), 10 µL SYBR Green, and 6 µL ddH2O. The reaction program was: 95 °C pre-denaturation for 30 s; 95 °C denaturation for 5 s, 60 °C annealing for 30 s, and 72 °C extension for 20 s, for a total of 40 cycles. 2 -ΔΔCT The relative expression level of mRNA was calculated using this method.

[0072] Table 2 Primer Sequences

[0073] The results are as follows Figure 6 As shown, the addition of 10 μM triyruvicone during cryopreservation and in vitro maturation significantly reduced the expression of the pro-apoptotic gene Bax and increased the expression of the anti-apoptotic gene Bcl-2 and antioxidant-related genes SOD, GPX, and CAT.

[0074] Example 8: Trichoderma alleviates spindle damage in cryopreserved oocytes The experimental groups were the same as in Example 3.

[0075] (1) Fixed Wash oocytes three times with PBS containing 0.1% fetal bovine serum for 10 minutes each time. Then fix the oocytes in 4% paraformaldehyde at room temperature for 30 minutes. Finally, wash them three times with 0.1% BSA / PBS (fetal bovine serum / phosphate buffer) for 10 minutes each time.

[0076] (2) Penetration and sealing After transferring oocytes into Triton X-100 solution for 15 minutes to penetrate, wash three times with 0.1% BSA / PBS for 10 minutes each time, transfer to blocking solution with 1% BSA, and block at room temperature for 1 hour.

[0077] (3) Antibody incubation, staining and mounting observation Oocytes were transferred to α-Tubulin droplets and incubated overnight in a humidified chamber. After incubation, the oocytes were washed three times with 1% BSA for 10 minutes each time. The oocytes were then transferred to FITC-labeled secondary antibody and incubated for 1 hour. After incubation, the oocytes were washed three times with 1% BSA for 10 minutes each time. The oocytes were then transferred to DAPI staining solution and incubated for 10 minutes, followed by washing three times with 1% BSA for 5 minutes each time. Finally, the oocytes were placed on a glass slide, 3-5 µl of anti-quenching agent was added, and the slide was immediately sealed with a coverslip for observation under a laser confocal microscope.

[0078] The results are as follows Figure 7 As shown in A, B, and C, salicylic acid can alleviate spindle damage in cryopreserved oocytes.

[0079] Example 9: Triscutellarin alleviates zona pellucida damage in cryopreserved oocytes The experimental groups were the same as in Example 3.

[0080] Oocytes were transferred to 1 mg / mL α-chymotrypsin, coated with paraffin oil, and the zona pellucida dissolution was observed at 30°C. Observations were taken every 2 minutes for the first 30 minutes of treatment, and then every 5 minutes until the end of the test (3 hours). At the end of each time point, the oocytes adhering to the surface of the culture dish were considered to have undergone zona pellucida dissolution. The time when 75% of the oocytes underwent complete zona pellucida dissolution was defined as t0. 75 To assess the hardening of the zona pellucida.

[0081] The results are as follows Figure 8 As shown, the addition of triscutellarin to the cryopreservation solution, thawing solution, and in vitro maturation solution can reduce the proportion of zona pellucida damage.

[0082] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of scutellarin in improving oocyte cryopreservation and in vitro maturation, characterized in that, The application is as follows: adding triscutellarin to the balancing solution, freezing solution, thawing solution and in vitro maturation solution used in the two processes of oocyte vitrification and in vitro maturation culture.

2. The application as described in claim 1, characterized in that, By improving mitochondrial function and spindle assembly after frozen oocytes mature in vitro, and reducing zona pellucida hardening, the efficiency of frozen oocyte maturation in vitro can be improved.

3. The application as described in claim 1, characterized in that, The final concentration of salicylic acid added to the equilibration solution, freezing solution, thawing solution and in vitro maturation solution is 1~25 μM.

4. The application as described in claim 1, characterized in that, The oocytes are selected from mammalian cells or tissues.

5. The application as described in claim 1, characterized in that, The oocytes mentioned are GV stage oocytes.

6. A cryopreservation agent for oocytes, characterized in that, Its working solution contains 1~25 μM of triscutellarin.

7. The oocyte cryopreservation agent as described in claim 6, characterized in that, The oocyte cryopreservation agent also includes at least one additional cryoprotectant.

8. The oocyte cryopreservation agent as described in claim 6, characterized in that, The oocyte cryopreservation agent also includes a cryopreservation-compatible pH buffer that can maintain a neutral pH value at low temperatures.

9. A culture method for improving the quality of oocyte maturation, characterized in that, This includes adding trisqualene to the balancing solution, freezing solution, thawing solution, and in vitro maturation solution used in both oocyte vitrification and in vitro maturation culture processes.

10. The cultivation method as described in claim 9, characterized in that, The final concentration of salicylic acid added to the equilibration solution, freezing solution, thawing solution or in vitro maturation solution is 1~25 μM.

Images