Pharmaceutical composition for delaying oocyte senescence and application thereof

Abstract

The invention discloses a pharmaceutical composition for delaying oocyte senescence and application thereof, and relates to the technical field of biological medicine, and the pharmaceutical composition comprises alpha-ketoglutaric acid, a bicarbonate buffer solution, an energy metabolism substrate, polyvinyl alcohol, hormone, a growth factor and insulin-transferrin-selenium. The pharmaceutical composition for delaying oocyte aging disclosed by the invention is beneficial to improving the maturation rate of oocytes, reducing the cytoplasm fragmentation rate and the cleavage fragmentation rate, and improving the cleavage rate and the blastocyst rate; according to the invention, the content of beta-galactosidase is reduced, the function of mitochondria in the aging process after ovulation of the mature oocyte is improved, the membrane potential of the mitochondria in the aging process after ovulation of the mature oocyte is stabilized, the activity of the mitochondria is improved, and the normalization of RNA methylation modification in the aging process after ovulation of the mature oocyte is promoted.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a pharmaceutical composition for delaying oocyte aging and its application. Background Art

[0002] As biological age increases, the quality of mature oocytes irreversibly declines with age. If the mature oocytes after ovulation are not fertilized in time, they will initiate programmed aging in the fallopian tube microenvironment. Its characteristics include abnormal spindle arrangement, reduced chromosome integrity, disordered cortical granule excretion, sclerosis of the zona pellucida, reduced mitochondrial activity and dysfunction, explosive accumulation of reactive oxygen species, and disordered DNA methylation modification, which seriously affect the fertilization and embryonic development ability of mature oocytes, leading to embryonic malformations and pregnancy failure.

[0003] Currently, there are efforts to improve ovarian aging and oocyte quality by using black phosphorus nanosheets, which have strong reactive oxygen species scavenging capabilities, as drug carriers. Furthermore, there are efforts to improve early embryonic development by using mevalonate to prepare drugs, thereby improving oocyte fertilization rates, two-cell formation rates, and high-quality blastocyst formation rates. However, oocytes still suffer from reduced mitochondrial activity and disrupted RNA methylation modifications. Summary of the Invention

[0004] In view of the deficiencies of the above-mentioned related technologies, the present application provides a pharmaceutical composition for delaying oocyte aging and its application. The pharmaceutical composition for delaying oocyte aging in the present application is beneficial for improving the maturation rate of oocytes, reducing the cytoplasmic fragmentation rate and the cleavage fragmentation rate, and improving the cleavage rate and blastocyst rate; reducing the content of β-galactosidase, improving the function of mitochondria in mature oocytes during postovulatory aging, stabilizing the mitochondrial membrane potential in mature oocytes during postovulatory aging, increasing mitochondrial activity, and promoting the normalization of RNA methylation modification in mature oocytes during postovulatory aging.

[0005] On the one hand, the present application provides a pharmaceutical composition for delaying oocyte aging using the following technical solution: A pharmaceutical composition for delaying oocyte aging comprises alpha-ketoglutaric acid, bicarbonate buffer, energy metabolism substrate, polyvinyl alcohol, hormone, growth factor and insulin-transferrin-selenium.

[0006] Preferably, the concentration of α-ketoglutarate is 10-80 μM.

[0007] Preferably, the concentration of α-ketoglutarate is 10-40 μM.

[0008] Preferably, the concentration of α-ketoglutarate is 20 μM.

[0009] Preferably, the energy metabolism substrate comprises 0.9-0.95 mM sodium pyruvate, 3.0-3.1 mM glucose and 0.55-0.6 mM L-cysteine.

[0010] Preferably, the energy metabolism substrate comprises 0.91 mM sodium pyruvate, 3.05 mM glucose and 0.57 mM L-cysteine.

[0011] Preferably, the hormones include 8-12 IU / mL of equine chorionic gonadotropin, 8-12 IU / mL of human chorionic gonadotropin, and 2-3 IU / mL of follicle-stimulating hormone.

[0012] Preferably, the hormones include 10 IU / mL of equine chorionic gonadotropin, 10 IU / mL of human chorionic gonadotropin, and 2.5 IU / mL of follicle-stimulating hormone.

[0013] Preferably, the growth factors include 35-45 ng / mL of fibroblast growth factor 2, 15-25 ng / mL of insulin-like growth factor 1, 15-25 ng / mL of leukemia inhibitory factor, and 8-12 ng / mL of epidermal growth factor.

[0014] Preferably, the growth factors include 40 ng / mL of fibroblast growth factor 2, 20 ng / mL of insulin-like growth factor 1, 20 ng / mL of leukemia inhibitory factor, and 10 ng / mL of epidermal growth factor.

[0015] Preferably, the concentration of the polyvinyl alcohol is 0.9-1.1 mg / mL.

[0016] Preferably, the concentration of the polyvinyl alcohol is 1 mg / mL.

[0017] Preferably, the volume fraction of the insulin-transferrin-selenium is 0.9%-1.1%.

[0018] Preferably, the volume fraction of the insulin-transferrin-selenium is 1%.

[0019] On the other hand, the present application provides a pharmaceutical composition for delaying oocyte aging for use in delaying post-ovulation aging of mature oocytes.

[0020] Preferably, the mature oocyte is obtained by the following steps: stripping the connective tissue attached to the surface of the ovary, disinfecting and then rinsing with double-antibiotic saline; aspirating the follicular fluid in the follicle, and screening out the mature oocyte-cumulus complex that is coated with at least three layers of dense cumulus cells and has uniform cytoplasm and no dark granules or vacuoles; The mature oocyte-cumulus complex is cultured until the first polar body is expelled, and then the cumulus cells are removed to obtain the mature oocyte.

[0021] Preferably, the pharmaceutical composition is beneficial for improving mitochondrial function in mature oocytes during post-ovulatory aging.

[0022] Preferably, the pharmaceutical composition is beneficial for stabilizing the mitochondrial membrane potential during the post-ovulatory aging process of mature oocytes.

[0023] Preferably, the pharmaceutical composition is beneficial for promoting the normalization of RNA methylation modification during the post-ovulatory aging process of mature oocytes.

[0024] In summary, this application includes at least one of the following beneficial technical effects: 1. The mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of the present application, which simulates the post-ovulatory aging of mature oocytes, have an increased maturation rate, a decreased percentage of fragmented oocytes, an increased cleavage rate, a decreased embryo fragmentation rate, and an increased blastocyst rate compared to the oocytes treated with the pharmaceutical composition of Comparative Example 1. In particular, the cleavage rate and blastocyst rate are increased to levels close to those of the normal mature oocyte control group that does not simulate the post-ovulatory aging of mature oocytes. In addition, the β-galactosidase content of the mature oocytes treated with the pharmaceutical composition for delaying mature oocyte aging of the present application, which simulates the post-ovulatory aging of mature oocytes, is much lower than that of the mature oocytes treated with the pharmaceutical composition of Comparative Example 1, indicating that the pharmaceutical composition for delaying mature oocyte aging of the present application is beneficial for delaying the post-ovulatory aging of mature oocytes. 2. The mitochondrial fluorescence intensity and mitochondrial membrane potential of mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of the present application simulated the post-ovulation aging of mature oocytes compared to the mature oocytes treated with the pharmaceutical composition of Comparative Example 1 were both increased, indicating that the pharmaceutical composition for delaying mature oocyte aging of the present application is beneficial for improving the function of mitochondria in the post-ovulation aging process of mature oocytes and stabilizing the mitochondrial membrane potential; 3. The pharmaceutical composition for delaying oocyte aging of the present application simulates the post-ovulation aging of mature oocytes. The expression level of 5-methylcytosine in mature oocytes treated with the pharmaceutical composition of Comparative Example 1 is significantly reduced, and is reduced to a level close to that of normal mature oocytes that do not simulate the post-ovulation aging of mature oocytes. This shows that the pharmaceutical composition for delaying mature oocyte aging of the present application is beneficial to promoting the normalization of RNA methylation modification during the post-ovulation aging process of mature oocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 1 is a comparison chart of the maturation rates of mature oocytes treated with the pharmaceutical compositions for delaying oocyte aging according to Examples 1-4, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 2 1 is a comparison chart of the cytoplasmic fragmentation rates of mature oocytes treated with the pharmaceutical compositions for delaying oocyte aging of Examples 1-4, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 3 3. It is a comparison chart of the cleavage rates of mature oocytes treated with the pharmaceutical compositions for delaying oocyte aging of Examples 1-4, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1 at the early embryonic stage; Figure 4 1 is a comparison chart of the cleavage fragmentation rates of mature oocytes treated with the pharmaceutical compositions for delaying oocyte aging of Examples 1-4, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1 at the early embryonic stage; Figure 5 This is a comparison chart of the blastocyst rates of mature oocytes treated with the pharmaceutical compositions for delaying oocyte aging of Examples 1-4, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1 at the early embryonic stage; Figure 6 This is a comparison of the fluorescence staining of β-galactosidase in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 7 This is a comparison of the fluorescence intensity of β-galactosidase in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 8 This is a comparison of fluorescent staining of mitochondria in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 9 This is a comparison of the fluorescence intensity of mitochondria in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging according to Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 10This is a fluorescence staining comparison of mitochondrial membrane potential in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 11 This is a comparison of the fluorescence intensity of mitochondrial membrane potential in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 12 This is a comparison chart of the expression levels of Am, Cm, Gm, m5C, and m6A in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1; Figure 13 This is a comparison chart of the expression levels of DNMT3B, Mettl3, Mettl14, NSUN2, FTO and ALKBH5 in mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Example 2, normal mature oocytes, and mature oocytes treated with the pharmaceutical composition of Comparative Example 1. DETAILED DESCRIPTION

[0026] The present application is described in further detail below in conjunction with Examples. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The methods used are conventional methods well known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise indicated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0027] The raw materials used in the examples and comparative examples can all be obtained commercially.

[0028] Example 1 Example 1 of the present application discloses a pharmaceutical composition for delaying oocyte aging, and the preparation steps are as follows: TCM-199 bicarbonate buffer (Gibco, product number 11150059) is used as the base solution, and 10 μM α-ketoglutaric acid (Sigma-Aldrich), 1 mg / mL polyvinyl alcohol (PVA, Sigma-Aldrich, USA, product number P8136), 1% (100×) insulin-transferrin-selenium (ITS, Gibco, product number 2953825), 0.91 mM sodium pyruvate (Sigma-Aldrich, product number SLCJ1780), 3.05 mM glucose (Sigma-Aldrich, product number G6152), 0.57 mM L-cysteine ​​(Sigma-Aldrich, Catalog No. 168149), 10 IU / mL equine chorionic gonadotropin (eCG, Ningbo Second Hormone Factory, Cixi, China), 10 IU / mL human chorionic gonadotropin (hCG, Ningbo Second Hormone Factory), 2.5 IU / mL follicle-stimulating hormone (FSH, Ningbo Second Hormone Factory), 40 ng / mL fibroblast growth factor 2 (FGF-2, Sino Biological, Beijing, China, Catalog No. 10014-HNAE), 20 ng / mL insulin-like growth factor 1 (IGF-1, Sino Biological, Catalog No. 10598-HNAE), 20 ng / mL leukemia inhibitory factor (LIF, Sino Biological, Catalog No. 14890-HNAH), and 10 ng / mL epidermal growth factor (EGF, Gibco, Catalog No. PHG0311).

[0029] Example 2 Example 2 of the present application discloses a pharmaceutical composition for delaying oocyte aging. The difference between Example 2 and Example 1 is that the concentration of α-ketoglutaric acid in Example 2 is 20 μM.

[0030] Example 3 Example 3 of the present application discloses a pharmaceutical composition for delaying oocyte aging. The difference between Example 3 and Example 1 is that the concentration of α-ketoglutaric acid in Example 3 is 40 μM.

[0031] Example 4 Example 4 of the present application discloses a pharmaceutical composition for delaying oocyte aging. The difference between Example 4 and Example 1 is that the concentration of α-ketoglutaric acid in Example 4 is 80 μM.

[0032] Comparative Example 1 Comparative Example 1 discloses a pharmaceutical composition for delaying oocyte aging. The difference between Comparative Example 1 and Example 1 is that α-ketoglutaric acid is not added in Comparative Example 1.

[0033] Test and Inspection (1) Obtaining normal mature oocytes: After obtaining pig ovaries from the slaughterhouse, they were immediately placed in 37°C preheated double-antibiotic saline (containing penicillin-streptomycin) and transported to the laboratory within 2-4 hours. The connective tissue attached to the ovarian surface was stripped off and the surface was disinfected with 75% ethanol for 30 seconds. Then, the ovaries were rinsed 2-3 times with 37°C double-antibiotic saline to remove residual impurities. Follicular fluid from follicles with a diameter of 3-8 mm was punctured and aspirated using an 18-20G needle syringe. Mature oocyte-cumulus complexes (COCs) were screened under a stereomicroscope (40× magnification) that were coated with at least three layers of dense cumulus cells and had uniformly clear cytoplasm without dark granules or vacuoles. The mature oocyte-cumulus complex was placed on culture medium and cultured in an incubator containing 5% CO2, 38.5℃ and saturated humidity for 44-46 hours. After the first polar body was excluded, the mature oocyte-cumulus complex was transferred into 0.1% hyaluronidase. The cumulus cells were removed by repeated blowing to obtain normal mature oocytes. The maturation rate and cytoplasmic fragmentation rate of normal mature oocytes were tested. The normal mature oocytes were fertilized and cultured in embryo culture medium for 7 days to enter the early embryo stage. The cleavage rate, cleavage fragmentation rate and blastocyst rate were tested.

[0034] The pharmaceutical compositions for delaying the aging of mature oocytes of Examples 1-4 and the pharmaceutical composition of Comparative Example 1 were used to culture normal mature oocytes for 24 hours to simulate the post-ovulation aging of mature oocytes, and then the maturation rate and cytoplasmic fragmentation rate were detected respectively, and the comparison diagrams between the groups were obtained as follows: Figure 1-2 The oocytes obtained above were fertilized and cultured in embryo culture medium for 7 days to enter the early embryo stage. The cleavage rate, cleavage fragment rate and blastocyst rate were detected, and the comparison charts between the groups were obtained as shown in the following order. Figure 3-5 The results of the mature oocytes treated with the pharmaceutical composition for delaying the aging of mature oocytes in Example 1 are marked as Aging+10μMα-KG in the figure, the results of Example 2 are marked as Aging+20μMα-KG in the figure, the results of Example 3 are marked as Aging+40μMα-KG in the figure, the results of Example 4 are marked as Aging+80μMα-KG in the figure, the results of the normal mature oocyte control group are marked as Control in the figure, and the results of Comparative Example 1 are marked as Aging in the figure.

[0035] (2) Fluorescence staining of aging-specific markers (β-galactosidase) and Cell Event™ Senescence Green (a fluorescent probe based on β-galactosidase activity) was performed on normal mature oocytes, mature oocytes treated with the pharmaceutical composition for delaying mature oocyte aging in Example 2, and mature oocytes treated with the pharmaceutical composition in Comparative Example 1, respectively, to obtain fluorescence staining comparison images as shown in FIG. Figure 6 As shown in the fluorescence intensity comparison diagram Figure 7 shown.

[0036] (3) Fluorescence staining was performed on the mitochondria of normal mature oocytes, mature oocytes treated with the pharmaceutical composition for delaying the aging of mature oocytes in Example 2, and mature oocytes treated with the pharmaceutical composition in Comparative Example 1, and a fluorescence staining comparison diagram was obtained as shown in FIG. Figure 8 As shown in the fluorescence intensity comparison diagram Figure 9 shown.

[0037] (4) Detect the mitochondrial membrane potential of normal mature oocytes, mature oocytes treated with the pharmaceutical composition for delaying the aging of mature oocytes in Example 2, and mature oocytes treated with the pharmaceutical composition in Comparative Example 1, and obtain fluorescence staining comparison images as shown in FIG. Figure 10 As shown in the fluorescence intensity comparison diagram Figure 11 shown.

[0038] (5) The expression levels of Am, Cm, Gm, m5C and m6A in normal mature oocytes, mature oocytes treated with the pharmaceutical composition for delaying the aging of mature oocytes in Example 2, and mature oocytes treated with the pharmaceutical composition in Comparative Example 1 were detected, and a comparison chart of expression levels was obtained as shown in FIG. Figure 12 The results of Example 2 are marked as a-KG-treat in the figure, the results of the normal mature oocyte control group are marked as NC in the figure, and the results of Comparative Example 1 are marked as Aging in the figure.

[0039] (6) The expression levels of m5C-related modification enzymes (DNMT3B, Mettl3, Mettl14, NSUN2, FTO and ALKBH5) in normal mature oocytes, mature oocytes treated with the pharmaceutical composition for delaying the aging of mature oocytes in Example 2, and mature oocytes treated with the pharmaceutical composition in Comparative Example 1 were detected, and the expression level comparison diagram was obtained as shown in FIG. Figure 13 shown.

[0040] Result Analysis Reference Figure 1-5Compared with the mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Examples 1-4, the mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Examples 1-4 had lower maturation rates, lower percentage of fragmented oocyte, higher cleavage rate, lower embryo fragmentation rate, and higher blastocyst rate. In particular, the cleavage rate and blastocyst rate of the mature oocytes treated with the pharmaceutical composition for delaying oocyte aging of Examples 1-4 were increased to levels close to those of the normal mature oocyte control group. This indicates that the pharmaceutical composition for delaying oocyte aging of the present application can make the developmental phenotype of mature oocytes after simulated ovulation and aging approach that of normal mature oocytes, and can significantly save the developmental potential of mature oocytes.

[0041] Reference Figure 6-7 The fluorescence intensity of β-galactosidase of mature oocytes treated with the pharmaceutical composition for delaying oocyte aging in Example 2 was reduced (P<0.01), indicating that the lysosome-mediated aging pathway was inhibited, and the aging signal marked by Cell Event™ Senescence Green was weakened (P<0.01), indicating that the DNA damage response (DDR) and cell cycle arrest-related aging mechanisms were regulated, indicating that the pharmaceutical composition for delaying oocyte aging of the present application is beneficial for delaying the aging of mature oocytes after ovulation.

[0042] Reference Figure 8-11 Compared with the mature oocytes treated with the pharmaceutical composition for delaying oocyte aging in Example 2, the mitochondria of the mature oocytes treated with the pharmaceutical composition for delaying oocyte aging in Example 1 uniformly aggregated from a diffuse and disordered state to the cortical region and cytoplasm, approaching the polar distribution characteristics of normal mature oocytes. At the same time, the JC-1 probe red / green fluorescence ratio (P<0.05) of the mature oocytes treated with the pharmaceutical composition for delaying oocyte aging in Example 2 was increased compared with the mature oocytes treated with the pharmaceutical composition for delaying oocyte aging in Example 1, indicating that the mitochondrial energy metabolism activity was enhanced and the membrane potential depolarization phenomenon was reversed. This shows that the pharmaceutical composition for delaying oocyte aging in Example 2 of the present application is beneficial for improving the function of mitochondria in the aging process of mature oocytes after ovulation and stabilizing the mitochondrial membrane potential. Reference Figure 12The 5-methylcytosine (m5C) modification level of mature oocytes treated with the pharmaceutical composition for delaying oocyte aging in Example 2 was significantly reduced to a level close to that of the normal mature oocyte control group compared to the mature oocytes treated with the pharmaceutical composition of Comparative Example 1, indicating that the pharmaceutical composition for delaying oocyte aging in Example 2 of the present application is beneficial for reversing the epitranscriptome disorder of mature oocytes and promoting the normalization of RNA methylation modification during the aging process of mature oocytes after ovulation. Figure 13 The pharmaceutical composition for delaying oocyte aging in Example 2 is beneficial for increasing the expression of 5-methylcytosine-related modification enzymes, especially tRNA cytosine-5-methyltransferase (NSUN2), thereby restoring the methylation modification levels of mRNA and non-coding RNA in the aging process of mature oocytes after ovulation and improving the accuracy of post-transcriptional regulation.

[0043] Unless otherwise defined, the technical or scientific terms used in this application shall have the usual meanings understood by persons of ordinary skill in the field to which this application belongs. The words "first", "second", "third" and similar terms used in the specification and claims of this application do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "one" or "a" do not indicate a quantity limitation, but rather indicate the existence of at least one. Words such as "include" or "comprise" mean that the elements or objects appearing before "include" or "comprises" cover the elements or objects listed after "include" or "comprises" and their equivalents, and do not exclude other elements or objects. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0044] The above are all optional embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.

Claims

1. A pharmaceutical composition for delaying oocyte aging, characterized in that: These include α-ketoglutarate, bicarbonate buffer, energy metabolism substrates, polyvinyl alcohol, hormones, growth factors, and insulin-transferrin-selenium.

2. A pharmaceutical composition for delaying oocyte aging according to claim 1, characterized in that: The concentration of α-ketoglutarate is 10-80 μM.

3. A pharmaceutical composition for delaying oocyte aging according to claim 1, characterized in that: The energy metabolism substrates include 0.9-0.95 mM sodium pyruvate, 3.0-3.1 mM glucose and 0.55-0.6 mM L-cysteine.

4. A pharmaceutical composition for delaying oocyte aging according to claim 1, characterized in that: The hormones include 8-12 IU / mL of equine chorionic gonadotropin, 8-12 IU / mL of human chorionic gonadotropin, and 2-3 IU / mL of follicle-stimulating hormone.

5. A pharmaceutical composition for delaying oocyte aging according to claim 1, characterized in that: The growth factors include 35-45 ng / mL of fibroblast growth factor 2, 15-25 ng / mL of insulin-like growth factor 1, 15-25 ng / mL of leukemia inhibitory factor and 8-12 ng / mL of epidermal growth factor.

6. Use of the pharmaceutical composition for delaying oocyte aging according to any one of claims 1 to 5 in delaying post-ovulatory aging of mature oocytes.

7. The use according to claim 6, characterized in that: The mature oocytes are obtained by the following steps: stripping the connective tissue attached to the surface of the ovary, disinfecting and then rinsing with double-antibody saline; aspirating the follicular fluid in the follicles, and screening out mature oocyte-cumulus complexes that are coated with at least three layers of dense cumulus cells and have uniform cytoplasm and no dark granules or vacuoles; The mature oocyte-cumulus complex is cultured until the first polar body is expelled, and then the cumulus cells are removed to obtain the mature oocyte.

8. The use according to claim 6, characterized in that: The pharmaceutical composition is beneficial for improving the function of mitochondria in the aging process of mature oocytes after ovulation.

9. The use according to claim 6, characterized in that: The pharmaceutical composition is beneficial for stabilizing the mitochondrial membrane potential during the post-ovulation aging process of mature oocytes.

10. The use according to claim 6, characterized in that: The pharmaceutical composition is beneficial for promoting the normalization of RNA methylation modification during the post-ovulatory aging process of mature oocytes.

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