Composition, culture medium, product and method for alleviating aging of mature oocytes

Abstract

The present invention relates to the field of biotechnology, and the present invention provides a composition, a culture medium, a product and a method for alleviating the aging of mature eggs. The composition comprises SB265610, MTK458 and 8-Br-cGMP. The composition can reduce the level of inflammatory factors and the toxic effect of inflammatory reactions on eggs. At the same time, it can increase the number and activity of mitochondria to provide sufficient energy guarantee for eggs. In addition, the composition can also effectively reduce the level of reactive oxygen species and the damage of oxidative stress to eggs. Through a series of combined effects, the composition significantly reduces the incidence of egg apoptosis and the rate of spindle abnormalities. By using the composition of the present invention, the aging rate of mature eggs is significantly alleviated, and the development rate is increased by nearly 20 times. The present invention provides a new strategy for animal embryo engineering and human assisted reproductive technology and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition, a culture medium, a product and a method for alleviating the aging of mature eggs. Background Art

[0002] Post-ovulatory mature egg aging refers to the process in which the oocyte gradually becomes functionally impaired and its quality declines over time after ovulation. This process involves changes in many aspects of the oocyte's cell structure, metabolic function, and stability of genetic material. Mature egg aging is a common problem in human assisted reproductive technology, which can easily lead to pregnancy failure. The clinical pregnancy rate of human assisted reproduction is only 10%-30%, and has not increased in the past few decades. Mature egg aging seriously affects the efficiency of assisted reproduction and causes huge financial and psychological pressure on patients. Therefore, methods to delay the aging of mature eggs have important application value and social significance for improving egg quality and increasing pregnancy rates.

[0003] The aging of mature eggs after ovulation is caused by a complex mechanism of multiple factors. A single strategy cannot completely correct the aging of mature eggs. How to alleviate the aging of mature eggs is an urgent problem that technicians in this field need to solve. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. Therefore, the inventors found through a large number of screening experiments that the combined use of SB265610, MTK458 and 8-Br-cGMP can alleviate the aging of mature eggs after ovulation, reduce the level of reactive oxygen, inflammatory response, apoptosis rate and spindle assembly abnormality, and improve the development rate.

[0005] Therefore, in the first aspect of the present invention, the present invention proposes a composition. According to an embodiment of the present invention, the composition includes: SB265610, MTK458 and 8-Br-cGMP. After a large number of experiments, the inventors found that the use of the composition of SB265610, MTK458 and 8-Br-cGMP can alleviate the aging of mature eggs. The composition can reduce the level of inflammatory factors, reduce the toxic effects of inflammatory reactions on eggs, and provide a good culture environment for mature eggs. At the same time, it can increase the number and activity of mitochondria and provide sufficient energy guarantee for eggs. In addition, the composition can also effectively reduce the level of reactive oxygen and reduce the damage to eggs caused by oxidative stress. Through these synergistic effects, the composition significantly reduces the incidence of egg apoptosis and the rate of spindle abnormalities.

[0006] In the second aspect of the present invention, the present invention provides a culture medium. According to an embodiment of the present invention, the culture medium comprises the composition described in the first aspect. As described above, the composition of the present invention can reduce the level of inflammatory factors, reduce the toxic effect of the inflammatory response on the oocytes, and provide a good culture environment for mature oocytes. At the same time, it can increase the number and activity of mitochondria, providing sufficient energy guarantee for the oocytes. In addition, it can effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to the oocytes. Therefore, the culture medium of the present invention can alleviate the aging of mature oocytes. The experimental results show that culturing mature oocytes with the culture medium of this combination can increase the development rate of oocytes by nearly 20 times, which is much higher than the effect of the prior art.

[0007] In the third aspect of the present invention, the present invention provides a product. According to an embodiment of the present invention, the product comprises the composition described in the first aspect or the culture medium described in the second aspect. As described above, the composition of the present invention can reduce the level of inflammatory factors and the inflammatory response, providing a good culture environment for mature oocytes. At the same time, it can increase the number and activity of mitochondria, providing sufficient energy guarantee for the oocytes. It can also effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to the oocytes. Therefore, this product can alleviate the aging of mature oocytes and improve the development efficiency after fertilization. In addition, SB265610, MTK458 and 8-Br-cGMP are all safe and reliable components with clear action targets, ensuring the safety of this product during use. It can not only be applicable to the in vitro culture of mature oocytes after ovulation in various animals, but also provide a reference for human assisted reproductive technology. For example, it can accelerate the propagation of excellent varieties in the in vitro embryo production of animals such as cattle, pigs, horses, sheep, etc., or improve the success rate of test-tube babies in the field of human assisted reproduction.

[0008] In the fourth aspect of the present invention, the composition described in the first aspect, the culture medium described in the second aspect or the product described in the third aspect has at least one of the following uses: in vitro preservation of mature oocytes; delaying the in vitro aging of mature oocytes; increasing the development rate of in vitro cultured oocytes; increasing the number or activity of mitochondria of in vitro cultured oocytes; reducing the inflammatory response of in vitro cultured oocytes; reducing the levels of reactive oxygen species and apoptosis of in vitro cultured oocytes; reducing the abnormal spindle assembly of in vitro cultured oocytes.

[0009] In the fifth aspect of the present invention, a method for alleviating the aging of mature eggs or preserving mature eggs in vitro is proposed. According to an embodiment of the present invention, the method includes: culturing mammalian mature eggs with at least one of the composition described in the first aspect, the culture medium described in the second aspect, and the product described in the third aspect. As described above, the composition of the present invention can reduce the level of inflammatory factors and the toxic effect of the inflammatory response on eggs. At the same time, it can increase the number and activity of mitochondria to provide sufficient energy for eggs. In addition, this composition can also effectively reduce the level of reactive oxygen species and the damage of oxidative stress to eggs. Through the combined effect, this composition significantly reduces the incidence of egg apoptosis and the rate of spindle abnormalities. Therefore, by using the method of the present invention, the aging of mature eggs can be alleviated, mature eggs can be preserved, and their development rate can be increased.

[0010] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0012] Figure 1 is the statistical result of the abnormal ratio of spindle morphology after culturing mature eggs of each group according to the embodiment of the present invention for 12 h, where;

[0013] A is the immunofluorescence staining map of the egg spindle, showing normal (left) and abnormal (right) spindles;

[0014] B is the statistical chart of the abnormal morphology ratio of the egg spindle after culturing in vitro for 12 h;

[0015] Figure 2 is the statistical result of the fluorescence of mitochondrial membrane potential after culturing mature eggs of each group according to the embodiment of the present invention for 12 h, where;

[0016] A is the representative diagram of JC-1 red and green fluorescence of mature eggs in the control group, aging group, and experimental group after culturing in vitro for 12 h;

[0017] B is the statistical chart of the relative ratio of mitochondrial membrane potential of eggs in the control group, aging group, and experimental group after culturing in vitro for 12 h;

[0018] Figure 3 is the statistical result of MitoTracker staining of the number of mitochondria in mature eggs of each group according to the embodiment of the present invention after culturing for 12 h, where;

[0019] A is the representative diagram of MitoTracker staining of eggs in the control group, aging group, and experimental group after culturing in vitro for 12 h;

[0020] B is a statistical chart of the fluorescence intensity of MitoTracker staining after 12 hours of in vitro culture of eggs in the control group, aging group, and experimental group;

[0021] Figure 4 It is the statistical result of the staining of reactive oxygen species (ROS) after culturing the mature eggs of each group according to the embodiments of the present invention for 12 hours, where;

[0022] A is a representative diagram of intracellular ROS staining in the control group, aging group, and experimental group;

[0023] B is a statistical chart of the fluorescence intensity of intracellular ROS staining in the control group, aging group, and experimental group;

[0024] Figure 5 It is the statistical result of the apoptotic TUNEL staining after culturing the mature eggs of each group according to the embodiments of the present invention for 12 hours, where;

[0025] A is a representative diagram of apoptotic TUNEL staining in the control group, aging group, and experimental group;

[0026] B is a statistical chart of apoptotic TUNEL staining in the control group, aging group, and experimental group;

[0027] Figure 6 It is the detection result of the gene expression levels of chemokines and pro-inflammatory factors in the cells of the control group, aging group, and experimental group according to the embodiments of the present invention;

[0028] Figure 7 It is the implementation technical roadmap according to the embodiments of the present invention. Detailed implementation manners

[0029] The embodiments of the technical solutions of the present invention will be described in detail below. The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, so they are only examples and cannot be used to limit the protection scope of the present invention.

[0030] Referring to "embodiments" herein means that the specific features, structures, or characteristics described in connection with the embodiments can be included in at least one embodiment of the present invention. The phrase appears in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0031] The "range" disclosed by the present invention is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a specific range. The range defined in this way can include the end values and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if the ranges of 60 - 120 and 80 - 110 are listed for a specific parameter, it is understood that the ranges of 60 - 110 and 80 - 120 are also contemplated. In addition, if the minimum range values of 1 and 2 are listed, and if the maximum range values of 3, 4, and 5 are listed, then the following ranges are all contemplated: 1 - 3, 1 - 4, 1 - 5, 2 - 3, 2 - 4, and 2 - 5. In the present invention, unless otherwise specified, the numerical range "a - b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers, and the range defined in this way can include the end values a and b. For example, the numerical range "0 - 5" means that all real numbers between "0 - 5" are fully listed herein, and "0 - 5" is only an abbreviated representation of these numerical combinations. Additionally, when stating that a certain parameter is an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, the integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0032] If there is no special instruction, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution.

[0033] If there is no special instruction, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0034] If there is no special instruction, all steps of the present invention can be carried out sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out sequentially, or can also include steps (b) and (a) carried out sequentially. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b), and (c), or can also include steps (a), (c), and (b), or can also include steps (c), (a), and (b), etc.

[0035] The state of cumulus cells after ovulation is crucial for oocyte quality. If not fertilized after ovulation, the viability of cumulus cells decreases, producing harmful substances, secreting chemokines and pro-inflammatory factors, activating the inflammatory response pathway, accelerating the aging of mature oocytes, and reducing the developmental ability of oocytes. At the same time, adverse environmental factors in vitro, such as high oxygen, temperature changes, pH changes, nutritional deficiencies, etc., are also likely to cause damage to cumulus cells, and then damage the quality of mature oocytes. The aging of mature oocytes after ovulation is mainly reflected in the increase in the level of reactive oxygen species, the decrease in the number and membrane potential of mitochondria and dysfunction, the increase in the abnormal rate of spindle structure, and the decrease in developmental ability. However, it is currently uncertain whether the aging of mature oocytes after ovulation can be alleviated by inhibiting the inflammatory response and enhancing mitophagy.

[0036] SB265610 is a chemokine receptor (CXCR2) blocker with the structure shown in Formula 1, which can reduce the inflammatory damage caused by chemokines in the in vitro environment; 8-Br-cGMP is a derivative of cGMP, synthesized by introducing a bromine atom (Br) at the 8th carbon atom of cGMP, and it plays a role in regulating cell signal transduction and cell function in vivo; MTK458 is an orally active and brain-penetrant PINK1 activator with the structure shown in Formula 2, which can activate mitophagy, remove damaged mitochondria during in vitro cell culture, and maintain the stability of the intracellular environment.

[0037] Formula 1, Formula 2.

[0038] The inventors found in experiments that the combined use of SB265610, MTK458, and 8-Br-cGMP had a great alleviating effect on the aging of mature oocytes after ovulation. This combination not only significantly reduced the level of inflammatory factors and the toxic effect of the inflammatory response on oocytes. At the same time, it can increase the number and activity of mitochondria, providing sufficient energy guarantee for oocytes. In addition, this composition can also effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to oocytes. Through a series of combined effects, the incidence of oocyte apoptosis and the abnormal rate of spindles were significantly reduced, and the in vitro aging rate of mature oocytes was significantly alleviated, and the developmental rate was increased by nearly 20 times. Therefore, this combination alleviated the aging rate of mature oocytes and significantly increased the developmental rate. This strategy plays an important role in exploring the aging mechanism of mature oocytes after ovulation and further developing in vitro preservation techniques for mature oocytes in the future.

[0039] The present invention provides a composition, a culture medium, a product, their uses, and a method for alleviating the aging of mature oocytes or preserving mature oocytes in vitro, which will be described in detail below.

[0040] Composition, culture medium, product

[0041] In the first aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises: SB265610, MTK458, and 8-Br-cGMP. Through a large number of experiments, the inventors found that the use of the composition of SB265610, MTK458, and 8-Br-cGMP of the present invention can alleviate the aging of mature eggs. This composition can reduce the level of inflammatory factors, reduce the toxic effect of the inflammatory response on eggs, and provide a good culture environment for mature eggs. At the same time, it can increase the number and activity of mitochondria, providing sufficient energy guarantee for eggs. In addition, this composition can also effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to eggs. Through these synergistic effects, this composition significantly reduces the incidence of egg apoptosis and the rate of spindle abnormalities.

[0042] In some embodiments of the present invention, the molar ratio or molar concentration ratio of SB265610, MTK458, and 8-Br-cGMP is 1:10:(1000 - 3000). For example, it can be 1:10:1000, 1:10:1200, 1:10:1500, 1:10:1700, 1:10:2000, 1:10:2200, 1:10:2500, 1:10:2800, 1:10:3000, etc., or it can be a range composed of any of the above values. Thus, by making the molar ratio of SB265610, MTK458, and 8-Br-cGMP within the above range, it can effectively reduce the toxic effect of the inflammatory response on eggs, increase the number and activity of mitochondria, and provide sufficient energy guarantee for eggs. It can effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to eggs.

[0043] In the second aspect of the present invention, the present invention provides a culture medium. According to an embodiment of the present invention, the culture medium comprises the composition described in the first aspect. As described above, the composition of the present invention can reduce the level of inflammatory factors, reduce the toxic effect of the inflammatory response on eggs, and provide a good culture environment for mature eggs. At the same time, it can increase the number and activity of mitochondria, providing sufficient energy guarantee for eggs. In addition, it can also effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to eggs. Thus, the culture medium of the present invention can alleviate the aging of mature eggs. Experimental results show that using the culture medium of this combination to culture mature eggs can increase the egg development rate by nearly 20 times, which is much higher than the effect of the prior art.

[0044] In some embodiments of the present invention, the culture medium further comprises a basal culture medium. Thus, it can provide sufficient nutrients and physiological conditions for mature eggs to maintain the normal survival of eggs.

[0045] In some embodiments of the present invention, the basal culture medium is a commonly used basal medium for cell or egg culture and can be obtained by purchase. Exemplarily, it includes, but is not limited to, one or a mixture of two or more of commercially available basal media such as KSOM culture medium, MEMα culture medium, DMEM / F12 culture medium, M2 culture medium, M16 culture medium, CZB culture medium, HTF culture medium, TYH culture medium, TCM199 culture medium, etc.

[0046] In some embodiments of the present invention, the final concentration of SB265610 in the culture medium is 0.1 μmol / L - 30 μmol / L. For example, it can be 0.1 μmol / L, 0.3 μmol / L, 0.5 μmol / L, 1 μmol / L, 3 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 25 μmol / L, 30 μmol / L, etc., or can be a range composed of any of the above values. Thus, by making the final concentration of SB265610 within the above range, when the culture medium is used to culture mature eggs in vitro, the chemokine level and inflammatory response can be effectively reduced, providing a good culture environment for the eggs. In some embodiments of the present invention, the final concentration of SB265610 in the culture medium is 0.3 μmol / L - 3 μmol / L.

[0047] In some embodiments of the present invention, the final concentration of 8-Br-cGMP in the culture medium is 0.1 mmol / L - 100 mmol / L. For example, it can be 0.1 mmol / L, 0.3 mmol / L, 1 mmol / L, 3 mmol / L, 5 mmol / L, 10 mmol / L, 30 mmol / L, 50 mmol / L, 80 mmol / L, 100 mmol / L, etc., or can be a range composed of any of the above values. Thus, by making the final concentration of 8-Br-cGMP within the above range, when the culture medium is used to culture mature eggs in vitro, the pro-inflammatory factor level can be effectively reduced, and the damage caused by inflammatory factors and reactive oxygen species to the eggs can be reduced. In some embodiments of the present invention, the final concentration of 8-Br-cGMP in the culture medium is 1 mmol / L - 10 mmol / L.

[0048] In some embodiments of the present invention, the final concentration of MTK458 in the culture medium is 0.1 μmol / L - 100 μmol / L. For example, it can be 0.1 μmol / L, 0.3 μmol / L, 0.5 μmol / L, 1 μmol / L, 3 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 30 μmol / L, 50 μmol / L, 100 μmol / L, etc., or can be a range composed of any of the above values. Thus, by making the final concentration of MTK458 within the above range, mitochondrial autophagy can be effectively activated and the internal environment homeostasis of the oocytes can be maintained. In some embodiments of the present invention, the final concentration of MTK458 in the culture medium is 3 - 30 μmol / L.

[0049] In the third aspect of the present invention, the present invention provides a product. According to the embodiments of the present invention, the product includes the composition described in the first aspect or the culture medium described in the second aspect. As described above, the composition of the present invention can reduce the levels of inflammatory factors and inflammatory responses, providing a good culture environment for mature oocytes. At the same time, it can increase the number and activity of mitochondria, providing sufficient energy guarantee for the oocytes. It can also effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to the oocytes. Thus, this product can alleviate the aging of mature oocytes and improve the development efficiency after fertilization. In addition, SB265610, MTK458, and 8-Br-cGMP are all safe and reliable components, ensuring the safety of this product during use. It can not only be applicable to the in vitro culture of mature oocytes after ovulation in various animals, but also provide a reference for human assisted reproductive technology. For example, it can accelerate the propagation of excellent varieties in the in vitro embryo production of animals such as cattle, pigs, horses, and sheep, or improve the success rate of in vitro fertilization in the field of human assisted reproduction.

[0050] In some embodiments of the present invention, the product includes a kit or a drug.

[0051] In some embodiments of the present invention, the product is a drug, and the product may further include a pharmaceutically acceptable carrier or excipient.

[0052] In the present invention, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that is permitted by the relevant government regulatory authorities for use in humans or livestock.

[0053] In the present invention, "excipients" can include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for the range where any conventional excipient is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of a pharmaceutically acceptable composition in a harmful manner, their uses are also within the scope contemplated by the present invention. Non-limiting examples of the types of "excipients" include binders, disintegrants, lubricants, glidants, stabilizers, fillers and diluents, etc. Excipients can enhance the handling characteristics of pharmaceutical preparations, that is, make the preparations more suitable for direct compression by increasing fluidity and / or adhesiveness.

[0054] Use

[0055] In the fourth aspect of the present invention, the present invention provides that the composition described in the first aspect, the culture medium described in the second aspect or the product described in the third aspect has at least one of the following uses: preserving mature eggs in vitro; delaying the in vitro aging of mature eggs; increasing the developmental rate of eggs cultured in vitro; increasing the number or activity of mitochondria in eggs cultured in vitro; reducing the inflammatory response of eggs cultured in vitro; reducing the levels of reactive oxygen species and apoptosis in eggs cultured in vitro; reducing the abnormal spindle assembly of eggs cultured in vitro.

[0056] In some embodiments of the present invention, the mature eggs are derived from mammals.

[0057] In some embodiments of the present invention, the mammals include at least one of, but are not limited to, mice, pigs, cows, horses, sheep, humans, and other non-human primates.

[0058] Method

[0059] In the fifth aspect of the present invention, the present invention provides a method for alleviating the aging of mature eggs or preserving mature eggs in vitro. According to the embodiments of the present invention, the method includes: culturing and treating mature eggs of mammals with at least one of the composition described in the first aspect, the culture medium described in the second aspect, and the product described in the third aspect. As described above, the composition of the present invention can reduce the level of inflammatory factors and reduce the toxic effect of the inflammatory response on eggs. At the same time, it can increase the number and activity of mitochondria and provide sufficient energy guarantee for eggs. In addition, this composition can also effectively reduce the level of reactive oxygen species and reduce the damage of oxidative stress to eggs. Through the combined effect, this composition significantly reduces the incidence of egg apoptosis and the abnormal rate of spindles. Thus, by using the method of the present invention, the aging of mature eggs can be alleviated, mature eggs can be preserved, and their developmental rate can be increased.

[0060] In some embodiments of the present invention, the source of mature eggs is not specifically limited, and they can be eggs matured in vivo or eggs matured in vitro.

[0061] In some embodiments of the present invention, the mammals include, but are not limited to, at least one of mouse, pig, cow, horse, sheep, human, and other non-human primates.

[0062] In some embodiments of the present invention, the conditions for the culture treatment are: the CO2 concentration is 2 - 5%, the humidity is 70 - 100%, and the temperature is 30 - 39 °C.

[0063] It should be noted that the present invention does not specifically limit the time of the culture treatment, and those skilled in the art can select according to specific needs. Exemplarily, the time for culturing mature eggs in the present invention can be 1 - 12 hours.

[0064] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0065] Example 1: Collection, culture, in vitro fertilization and embryo culture of mature eggs after ovulation in mice

[0066] 1. Collection of mature eggs after ovulation in mice

[0067] The mice used in the present invention are 8-week-old ICR strains, all purchased from SPF (Beijing) Biotechnology Co., Ltd. Each mouse was intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG). 48 hours later, each mouse was intraperitoneally injected with 5 IU of human chorionic gonadotropin (hCG). 14 - 16 hours after the injection of hCG, the mice were sacrificed by cervical dislocation. The abdominal cavity was opened to take out the oviducts, which were placed in pre-warmed mouse oocyte manipulation medium M2 at 37 °C. Under a stereomicroscope, the ampulla of the oviduct was punctured with a 1 mL syringe needle to release the mature cumulus oocyte complexes (COCs) for use. The formula of mouse oocyte manipulation medium M2 is shown in Table 1.

[0068] Table 1 Formula of mouse oocyte manipulation medium M2

[0069]

[0070] 2. Culture of mature eggs after ovulation in mice

[0071] The retrieved mouse mature cumulus oocyte complexes (COCs) were washed twice with the basic oocyte culture medium KSOM (MR-106, Sigma), and then transferred to different culture media pre-equilibrated at 37 °C for 2 hours. Among them, 500 μL of culture medium was placed in each well of a 4-well plate, and 10 mouse COCs were placed at the same time, and then cultured in an incubator at 37.0 °C, 5% CO2, and 100% humidity for 12 hours.

[0072] 3. In vitro fertilization of oocytes

[0073] Prepare 100 μL of mouse sperm capacitation solution TYH droplets, cover the droplets with mineral oil, and equilibrate them in an incubator at 37 °C for 1 hour. Sterilize the instruments used in the experiment in advance. Decapitate and sacrifice adult healthy male mice aged 10-12 weeks, carefully cut open the abdominal cavity with sterilized scissors, gently remove the epididymis with forceps, clean the blood vessels and adipose tissue, and place it on a sterile filter paper. Gently squeeze the sperm in the epididymis to one side of the epididymis with forceps, keep the forceps clamped, and gently cut open the side of the epididymis with sperm in the mineral oil of the capacitation solution with scissors. After squeezing out the sperm, put it into the capacitation solution TYH. Place it in a carbon dioxide incubator at 37 °C, and the sperm capacitation time is 1 hour. The formula of mouse sperm capacitation solution TYH is shown in Table 2.

[0074] Table 2 Formula of mouse sperm capacitation solution TYH

[0075]

[0076] Wash the mature cumulus oocyte complexes (COCs) obtained in step 1 or the COCs cultured for 12 hours in step 2 twice with the basic oocyte culture medium KSOM, and then transfer them to the oocyte fertilization solution mHTF droplets pre-equilibrated at 37 °C for 1 hour.

[0077] After 1 hour of sperm capacitation, observe the sperm motility, select sperm with stronger motility, use a pipette to aspirate 5 μL of sperm from the edge of the TYH capacitation droplet, and then add it to the oocyte fertilization solution mHTF droplet containing COCs from the edge of the fertilization droplet. Place it in an incubator at 37 °C, 5% CO2, and saturated humidity, and incubate the sperm and oocytes for 3-4 hours. The formula of mouse oocyte fertilization solution mHTF is shown in Table 3.

[0078] Table 3 Formula of mouse oocyte fertilization solution mHTF

[0079]

[0080] 4. In vitro culture of mouse embryos

[0081] After co-incubating sperm and eggs for 3-4 hours, the fertilized eggs were aspirated, and the residual cumulus cells and sperm on the surface of the fertilized eggs were gently pipetted off. The fertilized eggs were first washed 3 times with oocyte fertilization medium mHTF, then washed 3 times with KSOM culture medium (MR-106, Sigma), transferred into KSOM basal culture medium pre-equilibrated in the incubator for more than 2 hours, and cultured in an incubator at 37.0°C, 5% CO2, and saturated humidity. 30 fertilized eggs were placed in 100 µL KSOM culture medium, and the blastocyst rate was counted after 96 hours of culture.

[0082] Among them, the mouse mature cumulus oocyte complexes (COCs) taken out in step 1 were divided into three groups, namely the fresh group (control group), the aging group, and the experimental group (treatment group). The three groups of COCs were treated differently, such as Figure 7 shown. The COCs in the fresh group were directly subjected to in vitro fertilization and embryo culture (steps 3 and 4). The COCs in the aging group were cultured in vitro in KSOM basal culture medium for 12 h (step 2) and then subjected to in vitro fertilization and embryo culture (steps 3 and 4). The COCs in the experimental group were cultured in vitro in KSOM culture medium containing different concentrations of SB265610, MTK458, or 8-Br-cGMP or combined additives for 12 h (step 2) and then subjected to in vitro fertilization and embryo culture (steps 3 and 4).

[0083] Example 2: Study on the effect of SB265610 in alleviating in vitro aging of mouse mature oocytes

[0084] This example shows the application of adding different concentrations of SB265610 in the oocyte in vitro basal culture medium KSOM in alleviating the aging of mature oocytes.

[0085] 1. Experimental design and experimental method

[0086] (1) Experimental design

[0087] Fresh group: non-aged fresh mature oocytes;

[0088] Aging group: fresh mature oocytes were cultured in vitro in KSOM basal culture medium for 12 hours;

[0089] Treatment groups 1-6: fresh mature oocytes were cultured in vitro in KSOM basal culture medium containing 0.1 µmol / L (treatment group 1), 0.3 µmol / L (treatment group 2), 1 µmol / L (treatment group 3), 3 µmol / L (treatment group 4), 10 µmol / L (treatment group 5), 30 µmol / L (treatment group 6) SB265610 for 12 hours.

[0090] (2) Experimental method

[0091] Collect mature mouse oocytes according to the method described in Example 1, culture the mature oocytes in vitro using the culture media of the above-mentioned groups, perform in vitro fertilization and embryo culture according to the method described in Example 1 at the same time, and count the blastocyst rate.

[0092] 2. Test results

[0093] The statistical results of in vitro embryo development rate are shown in Table 4. The data in Table 4 show that compared with the cleavage rate (83.3% ± 2.3%) and blastocyst rate (33.2% ± 5.3%) of the fresh group, the cleavage rate and blastocyst rate of the aging group and different treatment groups are significantly reduced (p < 0.05). The blastocyst rate of treatment group 2 (i.e., the group added with 0.3 μmol / L SB265610) (7.5% ± 0.5%), the blastocyst rate of treatment group 3 (i.e., the group added with 1 μmol / L SB265610) (8.1% ± 1.1%) and the blastocyst rate of treatment group 4 (i.e., the group added with 3 μmol / L SB265610) (6.3% ± 0.9%) are significantly higher than the blastocyst rate of the aging group (1.8% ± 0.3%) (p < 0.05). The blastocyst rate of treatment group 1 (i.e., the group added with 0.1 μmol / L SB265610) (2.6% ± 0.7%), the blastocyst rate of treatment group 5 (i.e., the group added with 10 μmol / L SB265610) (1.7% ± 0.4%) and the blastocyst rate of treatment group 6 (i.e., the group added with 30 μmol / L SB265610) (1.1% ± 0.2%) have no difference with the blastocyst rate of the aging group (1.8% ± 0.3%) (p > 0.05). Among them, the blastocyst rate of treatment group 3 (i.e., the group added with 1 μmol / L SB265610) is the highest.

[0094] Table 4 Effects of SB265610 on alleviating the in vitro aging of mature mouse oocytes on the development rate

[0095]

[0096] Among them, different letter superscripts (a, b, c) in the same column of the table indicate significant differences among groups (p < 0.05).

[0097] Example 3: Study on the effect of MTK458 on alleviating the in vitro aging of mature mouse oocytes

[0098] This example shows the application of adding different concentrations of MTK458 in the basic in vitro culture medium KSOM of oocytes in alleviating the aging of mature oocytes.

[0099] 1. Experimental design and methods

[0100] (1) Experimental design

[0101] Fresh group: Unaged fresh mature oocytes;

[0102] Aging group: Fresh mature oocytes were cultured in vitro for 12 hours in KSOM basal culture medium.

[0103] Treatment groups 1 - 7: Fresh mature oocytes were cultured in vitro for 12 hours in KSOM basal culture medium containing 0.1 µmol / L (treatment group 1), 0.3 µmol / L (treatment group 2), 1 µmol / L (treatment group 3), 3 µmol / L (treatment group 4), 10 µmol / L (treatment group 5), 30 µmol / L (treatment group 6), and 100 µmol / L (treatment group 7) of MTK458 respectively.

[0104] (2) Test method

[0105] Mouse mature oocytes were collected according to the method described in Example 1, and the mature oocytes were cultured in vitro using the culture media of the above - mentioned groups. At the same time, in vitro fertilization and embryo culture were carried out according to the method described in Example 1, and the blastocyst rate was counted.

[0106] 2. Test results

[0107] The statistical results of in vitro embryo development rate are shown in Table 5. The data in Table 5 indicate that compared with the cleavage rate (81.3% ± 5.3%) and blastocyst rate (30.3% ± 3.9%) of the fresh group, the cleavage rates and blastocyst rates of the aging group and different treatment groups were significantly reduced (p < 0.05). The blastocyst rate of treatment group 4 (i.e., the group added with 3 µmol / L MTK458) (6.3% ± 0.3%), the blastocyst rate of treatment group 5 (i.e., the group added with 10 µmol / L MTK458) (9.5% ± 0.7%), and the blastocyst rate of treatment group 6 (i.e., the group added with 30 µmol / L MTK458) (5.1% ± 0.3%) were significantly higher than the blastocyst rate of the aging group (1.9% ± 0.2%) (p < 0.05). The blastocyst rate of treatment group 1 (i.e., the group added with 0.1 µmol / L MTK458) (1.9% ± 0.4%), the blastocyst rate of treatment group 2 (i.e., the group added with 0.3 µmol / L MTK458) (2.6% ± 0.4%), the blastocyst rate of treatment group 3 (i.e., the group added with 1 µmol / L MTK458) (2.7% ± 0.2%), and the blastocyst rate of treatment group 7 (i.e., the group added with 100 µmol / L MTK458) (1.8% ± 0.1%) had no difference with the blastocyst rate of the aging group (1.9% ± 0.2%) (p > 0.05). Among them, the blastocyst rate of treatment group 5 (i.e., the group added with 10 µmol / L MTK458) was the highest.

[0108] Table 5 Effects of MTK458 on alleviating in vitro aging of mouse mature oocytes on development rate

[0109]

[0110] Among them, different letter superscripts (a, b, c) in the same column of the table indicate significant differences between groups (p < 0.05).

[0111] Example 4: Study on the effect of 8-Br-cGMP in alleviating in vitro aging of mouse mature oocytes

[0112] This example shows the application of adding different concentrations of 8-Br-cGMP to the basic in vitro culture medium KSOM for oocytes in alleviating the aging of mature oocytes.

[0113] 1. Experimental design and methods

[0114] (1) Experimental design

[0115] Fresh group: Fresh, non-aged mature oocytes;

[0116] Aging group: Fresh mature oocytes were cultured in vitro in the basic KSOM culture medium for 12 hours for aging.

[0117] Treatment groups 1 - 7: Fresh mature oocytes were respectively cultured in vitro in the basic KSOM culture medium containing 0.1 mmol / L (treatment group 1), 0.3 mmol / L (treatment group 2), 1 mmol / L (treatment group 3), 3 mmol / L (treatment group 4), 10 mmol / L (treatment group 5), 30 mmol / L (treatment group 6), 100 mmol / L (treatment group 7) of 8-Br-cGMP for 12 hours for aging.

[0118] (2) Experimental methods

[0119] Mouse mature oocytes were collected according to the method described in Example 1, and the mature oocytes were cultured in vitro using the culture media of the above groups. At the same time, in vitro fertilization and embryo culture were carried out according to the method described in Example 1, and the blastocyst rate was counted.

[0120] 2. Experimental results

[0121] The statistical results of in vitro embryo development rate are shown in Table 6. The data in Table 6 indicate that compared with the cleavage rate (85.8%±6.1%) and blastocyst rate (38.5%±4.2%) of the fresh group, the cleavage rate and blastocyst rate of the aging group and different treatment groups are significantly decreased (p<0.05). The blastocyst rate of treatment group 3 (adding 1 mmol / L 8-Br-cGMP) (11.5%±1.8%), the blastocyst rate of treatment group 4 (adding 3 mmol / L 8-Br-cGMP) (12.3%±2.1%), and the blastocyst rate of treatment group 5 (adding 10 mmol / L 8-Br-cGMP) (8.7%±1.1%) are significantly higher than the blastocyst rate of the aging group (3.4%±0.5%) (p<0.05). The blastocyst rate of treatment group 1 (adding 0.1 mmol / L 8-Br-cGMP) (1.9%±0.3%), the blastocyst rate of treatment group 2 (adding 0.3 mmol / L 8-Br-cGMP) (5.2%±0.6%), the blastocyst rate of treatment group 6 (adding 30 mmol / L 8-Br-cGMP) (2.1%±0.9%), and the blastocyst rate of treatment group 7 (adding 100 mmol / L 8-Br-cGMP) (1.2%±0.4%) have no difference with the blastocyst rate of the aging group (3.4%±0.5%) (p>0.05). Among them, the blastocyst rate of treatment group 3 (adding 1 mmol / L 8-Br-cGMP) and the blastocyst rate of treatment group 4 (adding 3 mmol / L 8-Br-cGMP) are similar and the highest.

[0122] Table 6 Effects of 8-Br-cGMP on alleviating in vitro aging of mouse mature oocytes on development rate

[0123]

[0124] Among them, different superscripts (a, b, c) of the same column in the table indicate significant differences among groups (p<0.05).

[0125] Example 5: Study on the effect of the combination of SB265610, MTK458 and 8-Br-cGMP on alleviating in vitro aging of mouse mature oocytes

[0126] This example shows the application of combining SB265610, MTK458 and 8-Br-cGMP in the basic culture medium KSOM for oocytes in vitro in alleviating the aging of mature oocytes.

[0127] 1. Experimental design and experimental methods

[0128] (1) Experimental design

[0129] Fresh group: non-aged fresh mature oocytes;

[0130] Aging group: Fresh mature oocytes were cultured in vitro in KSOM basal medium for 12 hours of aging culture;

[0131] Treatment group 1: Fresh mature oocytes were cultured in vitro in KSOM basal medium containing 1 μmol / L SB265610 and 10 μmol / L MTK458 for 12 hours of aging culture;

[0132] Treatment group 2: Fresh mature oocytes were cultured in vitro in KSOM basal medium containing 1 μmol / L SB265610 and 1 mmol / L 8-Br-cGMP for 12 hours of aging culture;

[0133] Treatment group 3: Fresh mature oocytes were cultured in vitro in KSOM basal medium containing 10 μmol / L MTK458 and 1 mmol / L 8-Br-cGMP for 12 hours of aging culture;

[0134] Treatment group 4: Fresh mature oocytes were cultured in vitro in KSOM basal medium containing 1 μmol / L SB265610, 10 μmol / L MTK458 and 1 mmol / L 8-Br-cGMP simultaneously for 12 hours of aging culture.

[0135] (2) Test method

[0136] Mouse mature oocytes were collected according to the method described in Example 1, and the mature oocytes were cultured in vitro using the culture media of the above groups. At the same time, in vitro fertilization and embryo culture were carried out according to the method described in Example 1, and the blastocyst rate was counted.

[0137] 2. Test results

[0138] The statistical results of the in vitro embryo development rate are shown in Table 7. The data in Table 7 show that compared with the cleavage rate of the fresh group (80.4% ± 7.4%)

[0139] Compared with the fresh group, the cleavage rates of the aging group (25.7% ± 3.0%), treatment group 1 (36.1% ± 3.2%), treatment group 2 (23.8% ± 2.3%), and treatment group 3 (44.9% ± 3.1%) were all significantly decreased (p < 0.05). However, the cleavage rate of treatment group 4, that is, the group with the simultaneous addition of SB265610, MTK458, and 8-Br-cGMP (77.3% ± 4.6%) showed no difference from that of the fresh group (p > 0.05). In addition, the blastocyst rates of treatment group 1 (10.9% ± 0.8%), treatment group 2 (8.2% ± 0.5), and treatment group 3 (11.5% ± 1.1%) were all higher than that of the aging control group (2.3% ± 0.3%) and lower than that of the fresh group (42.7% ± 5.2%). However, the blastocyst rate of treatment group 4, that is, the group with the simultaneous addition of SB265610, MTK458, and 8-Br-cGMP (37.3% ± 4.2%) showed no difference from that of the fresh group (p > 0.05). Therefore, the effect of the group with the simultaneous addition of SB265610, MTK458, and 8-Br-cGMP is better than the combination effect of any two of them.

[0140] Table 7 Effects of the combination of SB265610, MTK458, and 8-Br-cGMP on alleviating the effect of in vitro aging of mouse mature oocytes on the development rate

[0141]

[0142] Among them, different superscripts (a, b, c) of the same column in the table indicate significant differences (p < 0.05) among groups.

[0143] Example 6: Study on the effect of the combination of SB265610, MTK458, and 8-Br-cGMP on the quality of aging mature oocytes

[0144] (1) Experimental design

[0145] Fresh group (control group): Unaged fresh mature oocytes;

[0146] Aging group: Fresh mature oocytes were cultured in vitro in KSOM basal medium for 12 hours;

[0147] Treatment group (experimental group): Fresh mature oocytes were cultured in vitro in KSOM basal medium containing 1 μmol / L SB265610, 10 μmol / L MTK458, and 1 mmol / L 8-Br-cGMP for 12 hours.

[0148] (2) Collection and culture of mature oocytes, in vitro fertilization, and embryo culture

[0149] Collect mature mouse oocytes according to the method described in Example 1, and culture the mature oocytes in vitro using the culture media of the above-mentioned groups. At the same time, perform in vitro fertilization and embryo culture according to the method described in Example 1.

[0150] (3)Staining of oocyte spindle structure

[0151] Place the mature oocytes that have been cultured in vitro for 12 hours of aging in 0.1% hyaluronidase for digestion for 2 minutes. Aspirate the oocytes with a mouth pipette and place them in KSOM. Subsequently, wash them twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). Then, fix the oocytes in 4% paraformaldehyde fixative at 4°C for 12 hours. Take the fixed oocytes, wash them 3 times with 0.1% PVA-PBS, and then place them in 0.1% PVA-PBS containing 0.1% TritonX 100 (0.1% TritonX 100-PVA-PBS) for permeabilization for 25 minutes. After the permeabilization is completed, place the oocytes in the immunofluorescence staining blocking solution and block them at 4°C for 12 hours. Incubate the blocked oocytes with Tubulin-FITC primary antibody at 4°C for 12 hours. After the incubation of the primary antibody, wash them 3 times with 0.1% PVA-PBS. Incubate the secondary antibody at room temperature for 1.5 hours, and then wash them 3 times with 0.1% PVA-PBS. Stain the cell nuclei with DAPI at room temperature for 5 minutes, and then wash them 3 times with 0.1% PVA-PBS. Subsequently, place the oocytes on a glass slide, add an anti-fluorescence quencher (DABCO) before pressing the coverslip, seal the coverslip with nail polish, and collect images through a fluorescence confocal microscope to count the abnormal rate of spindles.

[0152] Detect the abnormal rate of spindle morphology of the mature oocytes in the control group, aging group, and experimental group according to the above method. The experimental results are as Figure 1 shown. Figure 1 Data shows that the abnormal ratio of spindles in the aging group of oocytes (72.10% ± 5.62%) is significantly higher than that in the control group (11.28% ± 2.94%) and the experimental group (18.63% ± 3.17%) (p < 0.05). There is no significant difference between the experimental group and the control group (p > 0.05), indicating that the method provided by the present invention for alleviating the aging of oocytes in vitro culture can well prevent the increase in the abnormal ratio of oocyte spindles.

[0153] (4)Detection of oocyte mitochondrial activity

[0154] Prepare the JC-1 working solution: First, vigorously mix 800 μL of operating solution M2 and 200 μL of 5× JC-1 staining buffer, and then add 5 μL of 200× JC-1 to the mixture. The JC-1 working solution should be prepared and used immediately.

[0155] The mature oocytes aged in vitro for 12 hours were placed in 0.1% hyaluronidase for 2 minutes of digestion. The oocytes were aspirated with a mouth pipette and placed in operation fluid M2, and then washed twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). Subsequently, the oocytes were put into the JC-1 working fluid pre-equilibrated at 37°C and incubated in an incubator at 37°C for 15 minutes. The washing solution was prepared by mixing 5×JC-1 staining buffer and operation fluid M2 at a volume ratio of 1:4 and placed on ice for standby. After the incubation of the oocytes with the JC-1 working fluid, they were washed 3 times with the washing solution. Subsequently, the oocytes were pressed with a glass slide, photographed and observed with a laser confocal microscope, and the mitochondrial membrane potential of the oocytes was analyzed using Image J software.

[0156] The mitochondrial activities of the mature oocytes in the control group, aging group and experimental group were detected according to the above method, and the experimental results are as Figure 2 shown. Figure 2 Data showed that the mitochondrial membrane potential of the oocytes in the aging group (0.41% ± 0.062%) was significantly lower than that in the control group (1.52% ± 0.21%) and the mitochondrial membrane potential of the oocytes in the experimental group (1.33% ± 0.18%) (p < 0.05). There was no significant difference between the experimental group and the control group (p > 0.05), indicating that the method provided by the present invention for alleviating the aging of oocytes during in vitro culture can well prevent the decrease of the mitochondrial membrane potential of oocytes and protect the mitochondrial activity in oocytes.

[0157] (5) Mitochondrial staining of oocytes

[0158] Preparation of Mito-Tracker Green working fluid: The Mito-Tracker Green concentrated stock solution with a concentration of 1 mM was prepared using DMSO, and the Mito-Tracker Green working fluid was prepared by mixing the Mito-Tracker Green concentrated stock solution and operation fluid M2 for oocytes at a volume ratio of 1:20000 (Mito-Tracker Green concentrated stock solution: operation fluid M2 for oocytes).

[0159] The mature oocytes aged in vitro for 12 hours were placed in 0.1% hyaluronidase for 2 minutes of digestion, and the oocytes were aspirated with a mouth pipette and placed in the operating fluid M2. Subsequently, they were washed twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). Then the oocytes were put into the pre-equilibrated Mito-Tracker Green working fluid at 37°C. 100 μL of the Mito-Tracker Green working fluid was used for 30 oocytes. The oocytes were incubated in a 37°C carbon dioxide incubator for 30 minutes. After incubation, the oocytes were washed twice with the operating fluid M2. Subsequently, the oocytes were pressed on a glass slide, photographed and observed with a laser confocal microscope, and the fluorescence intensity of Mito-Tracker Green was analyzed using Image J software.

[0160] The mature oocytes in the control group, aging group and experimental group were subjected to mitochondrial MitoTracker staining according to the above method to detect and evaluate the mitochondrial quantity. The experimental results are as Figure 3 shown. The data show that the fluorescence intensity of mitochondrial MitoTracker staining in the aging group of oocytes (12.01% ± 2.32%) was significantly lower than that in the control group (36.33% ± 4.52%) and the fluorescence intensity of mitochondrial MitoTracker staining in the experimental group of oocytes (38.29% ± 5.18%) (p < 0.05). There was no significant difference between the experimental group and the control group (p > 0.05), indicating that the mitochondrial quantity in the aging group decreased significantly, and the method provided by the present invention for alleviating the aging of mature oocytes can significantly alleviate the reduction of mitochondrial quantity.

[0161] (6)Detection of cellular reactive oxygen species and detection of apoptosis

[0162] The specific process of detecting cellular reactive oxygen species (ROS):

[0163] Preparation of the oxidation-sensitive fluorescent probe DCFH-DA working fluid: DCFH-DA was diluted at a volume ratio of 1:1000 to make its final concentration 10 μM.

[0164] The mature oocytes aged in vitro for 12 hours were placed in 0.1% hyaluronidase for 2 minutes of digestion, and then washed twice with DPBS buffer. Subsequently, the cells were added to 500 μL of the DCFH-DA working fluid and incubated in a 37°C cell incubator for 20 minutes. They were inverted and mixed evenly every 3 - 5 minutes to ensure sufficient contact between the probe and the cells. After incubation for 20 minutes, the cells were washed 3 times to fully remove the DCFH-DA that did not enter the cells. Then they were photographed and observed with a laser confocal microscope, and the fluorescence intensity of DCFH-DA was analyzed using ImageJ software.

[0165] The specific process of apoptosis detection:

[0166] Collect mature eggs that have been cultured in vitro for 12 hours of aging, and wash them twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). The washed eggs are placed in 4% PFA fixative and fixed in a refrigerator at 4 °C for 2 hours. Then wash them 3 times with 0.1% PVA-PBS, transfer them to a permeabilization solution of 0.5% TritonX100 - 0.1% PVA-PBS for 1 hour of permeabilization. After that, transfer them to a cell apoptosis TUNEL detection solution (C1086, Beyotime) and incubate them in an incubator at 37 °C for 1 hour. After the incubation is completed, wash the eggs 3 times with 0.1% PBS-PVA, place them in 20 μL of DAPI staining solution and incubate for 5 minutes, and take pictures of the eggs under a fluorescence microscope. Take the number of TUNEL-positive signal cells as the number of apoptotic cells and the number of DAPI-positive signal cells as the total number of cells, and calculate the cell apoptosis rate. The calculation method of the apoptosis rate is as follows: Apoptosis rate = (Number of TUNEL-positive signal cells / Number of DAPI-positive signal cells) × 100.

[0167] Detect the content of reactive oxygen species (ROS) and cell apoptosis of mature eggs in the control group, aging group and experimental group according to the above method, and the results are as Figure 4 and Figure 5 shown. The data shows that the ROS intensity of the aging group is (13.18 ± 0.95), and the apoptosis rate is (20.68% ± 1.78%). While the ROS intensity of the experimental group after culturing in vitro for 12 h with the culture liquid provided by the present invention is (3.96 ± 0.29), and the apoptosis rate is (1.28% ± 0.09%), which is significantly reduced, and there is a significant difference in statistical significance (p < 0.05). There is no significant difference between the experimental group and the control group (p > 0.05). It shows that the method provided by the present invention for alleviating the aging of mature eggs can well reduce the content of intracellular reactive oxygen species, protect cells from oxidative stress damage, and prevent apoptosis.

[0168] (7) RNA extraction, reverse transcription and real-time fluorescence quantitative PCR

[0169] Centrifuge to collect mature eggs in the control group, aging group and experimental group, collect them into 1.5 mL centrifuge tubes, suck out the excess liquid, store them in a refrigerator at -80 °C, and extract total RNA using Trizol. The reverse transcription system and procedures are as follows.

[0170] Configure the reverse transcription system:

[0171]

[0172] Reverse transcription reaction procedure:

[0173]

[0174] After reverse transcription was completed, the cDNA samples were stored in a -80 °C refrigerator for standby real-time fluorescence quantitative PCR.

[0175] Prepare the real-time fluorescence quantitative PCR system:

[0176]

[0177] Real-time fluorescence quantitative PCR reaction program:

[0178]

[0179] According to the real-time fluorescence quantitative PCR reaction system and program, mix the cDNA template with the kit premix, and perform real-time fluorescence quantitative PCR in a Bio-Rad CFX96 Touch Real-Time PCR instrument. Using GAPDH as the internal reference gene, calculate the relative expression content of the target gene by the method of 2 -△△Ct .

[0180] Detect the gene expression levels of chemokines and inflammatory factors in the mature oocytes of the control group, aging group and experimental group according to the above method, and the experimental results are as Figure 6 shown. The data show that in the experimental group cultured in vitro for 12 h using the culture medium provided by the present invention, the expression levels of chemokines Ccl5, Cxcl3, Ccl22, Cxcl1, Cxcl10, Ccr7 and inflammatory factors Il1β, Il8, IFN-γ and Tnfα were significantly lower than those in the aging group (p < 0.05), and there was no significant difference from the control group (p > 0.05). This indicates that the method provided by the present invention for alleviating the aging of mature oocytes can well reduce the expression of chemokines and inflammatory factors, reduce the inflammatory response, and protect cell activity.

[0181] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0182] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A composition, characterized in that include: SB265610, MTK458, and 8-Br-cGMP; The molar ratio of the SB265610, the MTK458 and the 8-Br-cGMP is 1:10:(1000-3000).

2. A culture solution, characterized in that The invention comprises the composition according to claim 1.

3. The culture solution according to claim 2, characterized in that Further comprising a basic culture medium.

4. The culture solution according to claim 3, characterized in that The final concentration of the SB265610 in the culture medium is 0.1µmol / L-30µmol / L.

5. The culture solution according to claim 3, characterized in that The final concentration of MTK458 in the culture medium is 0.1µmol / L-100µmol / L.

6. The culture solution according to claim 3, characterized in that The final concentration of the 8-Br-cGMP in the culture medium is 0.1 mmol / L-100 mmol / L.

7. The culture solution according to claim 3, characterized in that The basic culture medium includes at least one of KSOM, MEMα, DMEM / F12, M2, M16, CZB, HTF, TYH, and TCM199.

8. A product, characterized in that The method comprises the composition according to claim 1 or the culture solution according to any one of claims 2 to 7.

9. The product according to claim 8, characterized in that The products include: a kit or a medicine.

10. Use of the composition of claim 1, the culture solution of any one of claims 2 to 7, or the product of any one of claims 8 to 9, wherein the use is selected from at least one of the following: Preservation of mature eggs in vitro; Delaying the aging of mature eggs in vitro; Improve the development rate of in vitro cultured eggs; Increase the number or activity of mitochondria in oocytes cultured in vitro; Reduce the inflammatory response of in vitro cultured eggs; Reduce the level of reactive oxygen species and apoptosis in oocytes cultured in vitro; Reduce spindle assembly abnormalities in oocytes cultured in vitro.

11. The use according to claim 10, characterized in that The egg is derived from a mammal.

12. The use according to claim 11, characterized in that The mammal includes at least one of mice, pigs, cows, horses, sheep, humans, and other non-human primates.

13. A method for alleviating the in vitro aging of mature eggs or preserving mature eggs in vitro, characterized in that: include: The mature ova of a mammal are cultured with at least one of the composition of claim 1, the culture solution of any one of claims 2 to 7, and the product of any one of claims 8 to 9.

14. The method according to claim 13, characterized in that The mammal includes at least one of mice, pigs, cows, horses, sheep, humans, and other non-human primates.

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