Composition, culture solution, product and method for relieving aging of mature ova

Abstract

The invention relates to the technical field of biology, and provides a composition, a culture solution, a product and a method for relieving aging of mature ova. The composition is prepared from SB265610, MTK458 and 8-Br-cGMP (8-Br-cGMP). The composition can reduce the level of inflammatory factors and reduce the toxic effect of inflammatory reaction on ova. Meanwhile, the quantity and activity of mitochondria can be improved, and sufficient energy guarantee is provided for the ovum. In addition, the composition can effectively reduce the active oxygen level and reduce the damage of oxidative stress to ova. Through a series of combination effects, the composition significantly reduces the incidence rate of ovum apoptosis and the rate of spindle abnormality. By adopting the composition disclosed by the invention, the aging speed of the mature ovum is obviously relieved, and the development rate is increased by nearly 20 times. The invention provides a new strategy for animal embryo engineering and human assisted reproduction technology, and has a wide application prospect.

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 proposes a culture solution. According to an embodiment of the present invention, the culture solution includes the composition described in the first aspect. As mentioned above, the composition of the present invention 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, the number and activity of mitochondria can be increased to provide sufficient energy guarantee for eggs. In addition, the level of reactive oxygen can be effectively reduced, reducing the damage to eggs caused by oxidative stress. As a result, the culture solution of the present invention can alleviate the aging of mature eggs. Experimental results show that the use of the culture solution of this combination to cultivate mature eggs can increase the egg development rate 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 proposes a product. According to an embodiment of the present invention, the product includes the composition described in the first aspect or the culture solution described in the second aspect. As mentioned above, the composition of the present invention can reduce the level of inflammatory factors and inflammatory response, and provide a good culture environment for mature eggs. At the same time, the number and activity of mitochondria can be increased to provide sufficient energy guarantee for eggs. It can also effectively reduce the level of reactive oxygen and reduce the damage of oxidative stress to eggs. Thus, the product can alleviate the aging of mature eggs and improve the efficiency of development after fertilization. In addition, SB265610, MTK458 and 8-Br-cGMP are all safe and reliable ingredients with clear targets, which ensures the safety of the product during use. It can not only be applied to the in vitro culture of mature eggs after ovulation of various animals, but also provide reference for human assisted reproductive technology, such as accelerating the expansion of excellent varieties in the in vitro embryo production of cattle, pigs, horses, sheep and other animals, or improving the success rate of test-tube babies in the field of human assisted reproduction.

[0008] In the fourth aspect of the present invention, the present invention proposes 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 aging of mature eggs in vitro; improving the development 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 and apoptosis in eggs cultured in vitro; reducing the abnormal spindle assembly of eggs cultured in vitro.

[0009] In the fifth aspect of the present invention, the present invention proposes a method for alleviating the aging of mature eggs or preserving mature eggs in vitro. According to an embodiment of the present invention, the method comprises: using the composition described in the first aspect, the culture medium described in the second aspect, and at least one of the products described in the third aspect to culture and treat mature eggs of mammals. As mentioned above, the composition of the present invention can reduce the level of inflammatory factors and reduce the toxic effects of inflammatory reactions on eggs. At the same time, the number and activity of mitochondria can be increased to 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 the combined effect, the composition significantly reduces the incidence of egg apoptosis and the rate of spindle abnormalities. Therefore, the method of the present invention can alleviate the aging of mature eggs, preserve mature eggs, and improve their development rate.

[0010] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through 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 easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 is the statistical result of the abnormal spindle morphology ratio of each group of mature eggs after 12 hours of culture according to the embodiment of the present invention, wherein; A is an immunofluorescence staining image of the egg spindle, showing normal (left) and abnormal (right) spindle morphology; B is a statistical graph of the abnormal spindle morphology ratio of eggs cultured in vitro for 12 hours; Figure 2 is the fluorescence statistical result of mitochondrial membrane potential after culturing mature eggs of each group for 12 hours according to the embodiment of the present invention, wherein; A is a representative image of JC-1 red and green fluorescence in mature oocytes of the control group, aging group and experimental group after 12 h of in vitro culture; B is the statistical diagram of the relative ratio of mitochondrial membrane potential of the control group, aging group and experimental group after 12 hours of in vitro culture; Figure 3 is the statistical result of MitoTracker staining of the number of mitochondria in each group of mature eggs after 12 hours of culture according to the embodiment of the present invention, wherein; A is a representative image of MitoTracker staining of the control group, aging group, and experimental group oocytes after 12 h of in vitro culture; B is the statistical graph of MitoTracker staining fluorescence intensity of the control group, aging group and experimental group after 12 hours of in vitro culture; Figure 4is the statistical result of reactive oxygen species (ROS) staining after 12 hours of culture of each group of mature eggs according to the embodiment of the present invention, wherein; A is a representative image of intracellular ROS staining in the control group, aging group, and experimental group; B is the statistical graph of intracellular ROS staining fluorescence intensity in the control group, aging group and experimental group; Figure 5 is the statistical result of apoptosis TUNEL staining after 12 hours of culture of mature eggs in each group according to the embodiment of the present invention, wherein; A is a representative picture of TUNEL staining of apoptosis in the control group, aging group and experimental group; B is the statistical graph of TUNEL staining of apoptosis in the control group, aging group and experimental group; Figure 6 are the results of detecting the gene expression levels of chemokines and pro-inflammatory factors in the cells of the control group, the aging group and the test group according to the embodiments of the present invention; Figure 7 It is an implementation technology roadmap according to an embodiment of the present invention. DETAILED DESCRIPTION

[0012] The following embodiments of the technical solution of the present invention are described in detail. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0013] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0014] "Scope" disclosed in the present invention is defined in the form of lower limit and upper limit, and 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 special range. The scope defined in this way can be including end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 60-120 and 80-110 is listed for a specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3,4 and 5 are listed, the following scope can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present invention, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers, and the scope defined in this way can be including end values ​​a and b. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0015] If not otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution.

[0016] Unless otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0017] If not otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0018] The state of cumulus cells after ovulation is crucial to the quality of eggs. If fertilization is not achieved after ovulation, the vitality of cumulus cells decreases, harmful substances are produced, chemokines and pro-inflammatory factors are secreted, the inflammatory response pathway is activated, the aging of mature eggs is accelerated, and the ability to develop eggs is reduced. At the same time, adverse environmental factors in vitro, such as high oxygen, temperature changes, pH changes, and malnutrition, can also easily cause cumulus cell damage, thereby damaging the quality of mature eggs. The aging of mature eggs after ovulation is mainly reflected in the increase in reactive oxygen levels, the decrease and dysfunction of the number and membrane potential of mitochondria, the increase in the rate of spindle structural abnormalities, and the decrease in developmental ability. However, it is not yet certain whether the aging of mature eggs after ovulation can be alleviated by inhibiting inflammatory responses and enhancing mitochondrial autophagy.

[0019] SB265610 is a chemokine receptor (CXCR2) blocker with a structure shown in Formula 1, which can reduce inflammatory damage to cells caused by chemokines in an in vitro environment; 8-Br-cGMP is a derivative of cGMP, which is synthesized by introducing a bromine atom (Br) on the 8-carbon atom of cGMP. It plays a role in regulating cell signal transduction and cell function in vivo; MTK458 is an orally active and brain-permeable PINK1 activator with a structure shown in Formula 2, which can activate mitochondrial autophagy, clear damaged mitochondria during in vitro culture, and maintain the stability of the intracellular environment.

[0020] Formula 1, Formula 2.

[0021] The inventors found in the experiment that the combined use of SB265610, MTK458 and 8-Br-cGMP has a great alleviating effect on the aging of mature eggs after ovulation. This combination not only significantly reduces the level of inflammatory factors, but also reduces the toxic effects of inflammatory reactions on eggs. At the same time, it can increase the number and activity of mitochondria, providing sufficient energy guarantee for eggs. In addition, the composition can also effectively reduce the level of reactive oxygen and reduce the damage of oxidative stress to eggs. Through a series of combined effects, the incidence of egg apoptosis and the rate of spindle abnormalities are significantly reduced, the aging rate of mature eggs in vitro is significantly alleviated, and the development rate is increased by nearly 20 times. Therefore, the combination alleviates the aging rate of mature eggs and significantly improves the development rate. This strategy is important for exploring the aging mechanism of mature eggs after ovulation in the future and further developing mature egg in vitro preservation technology. The present invention provides a composition, a culture medium, a product and its use, and a method for alleviating the aging of mature eggs or preserving mature eggs in vitro, which will be described in detail below.

[0022] Composition, culture medium, product 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 SB265610, MTK458 and 8-Br-cGMP composition of the present invention 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.

[0023] 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 numerical values. Thus, by making the molar ratio of SB265610, MTK458 and 8-Br-cGMP within the above range, it is possible to effectively reduce the toxic effects of inflammatory reactions on eggs, increase the number and activity of mitochondria, and provide sufficient energy guarantee for eggs. It effectively reduces the level of reactive oxygen and reduces the damage to eggs caused by oxidative stress.

[0024] In the second aspect of the present invention, the present invention proposes a culture solution. According to an embodiment of the present invention, the culture solution includes the composition described in the first aspect. As mentioned above, the composition of the present invention 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, the number and activity of mitochondria can be increased to provide sufficient energy guarantee for eggs. In addition, the level of reactive oxygen can be effectively reduced, reducing the damage to eggs caused by oxidative stress. As a result, the culture solution of the present invention can alleviate the aging of mature eggs. Experimental results show that the use of the culture solution of this combination to cultivate mature eggs can increase the egg development rate by nearly 20 times, which is much higher than the effect of the prior art.

[0025] In some embodiments of the present invention, the culture solution further includes a basal culture solution, thereby providing sufficient nutrition and physiological conditions for the mature eggs to maintain the normal survival of the eggs.

[0026] In some embodiments of the present invention, the basal culture medium is a commonly used basal culture medium for cell or egg culture, which can be purchased. Exemplarily, it includes but is not limited to one or a mixture of two or more commercial basal culture 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.

[0027] In some embodiments of the present invention, the final concentration of SB265610 in the culture medium is 0.1µmol / L-30µmol / L. For example, it can be 0.1µmol / L, 0.3µmol / L, 0.5µmol / L, 1µmol / L, 3µmol / L, 5µmol / L, 10µmol / L, 15µmol / L, 20µmol / L, 25µmol / L, 30µmol / L, etc., or it can be a range composed of any of the above values. Thus, by making the final concentration of SB265610 in the above range, the culture medium can effectively reduce the level of chemokines and inflammatory response when culturing mature eggs in vitro, 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 0.3µmol / L-3µmol / L.

[0028] In some embodiments of the present invention, the final concentration of 8-Br-cGMP in the culture medium is 0.1mmol / L-100mmol / L. For example, it can be 0.1mmol / L, 0.3mmol / L, 1mmol / L, 3mmol / L, 5mmol / L, 10mmol / L, 30mmol / L, 50mmol / L, 80mmol / L, 100mmol / L, etc., or it can be a range composed of any of the above numerical values. Thus, by making the final concentration of 8-Br-cGMP in the above range, the culture medium can effectively reduce the level of pro-inflammatory factors when culturing mature eggs in vitro, and reduce the damage caused by inflammatory factors and reactive oxygen to eggs. In some embodiments of the present invention, the final concentration of 8-Br-cGMP in the culture medium is 1mmol / L-10mmol / L.

[0029] In some embodiments of the present invention, the final concentration of MTK458 in the culture medium is 0.1µmol / L-100µmol / L. For example, it can be 0.1µmol / L, 0.3µmol / L, 0.5µmol / L, 1µmol / L, 3µmol / L, 5µmol / L, 10µmol / L, 15µmol / L, 30µmol / L, 50µmol / L, 100µmol / L, etc., or it can be a range composed of any of the above numerical values. Thus, by making the final concentration of MTK458 in the above range, mitochondrial autophagy can be effectively activated to maintain the homeostasis of the internal environment of the egg. In some embodiments of the present invention, the final concentration of MTK458 in the culture medium is 3-30µmol / L.

[0030] In the third aspect of the present invention, the present invention proposes a product. According to an embodiment of the present invention, the product includes the composition described in the first aspect or the culture solution described in the second aspect. As mentioned above, the composition of the present invention can reduce the level of inflammatory factors and inflammatory response, and provide a good culture environment for mature eggs. At the same time, the number and activity of mitochondria can be increased to provide sufficient energy guarantee for eggs. It can also effectively reduce the level of reactive oxygen and reduce the damage of oxidative stress to eggs. Thus, the product can alleviate the aging of mature eggs and improve the efficiency of development after fertilization. In addition, SB265610, MTK458 and 8-Br-cGMP are all safe and reliable ingredients, which ensure the safety of the product during use, and can not only be applied to the in vitro culture of mature eggs after ovulation of various animals, but also provide reference for human assisted reproductive technology, such as accelerating the expansion of excellent varieties in the in vitro embryo production of animals such as cattle, pigs, horses, sheep, etc., or improving the success rate of test-tube babies in the field of human assisted reproduction.

[0031] In some embodiments of the present invention, the product comprises a kit or a medicament.

[0032] In some embodiments of the present invention, the product is a drug, and the product may further include a pharmaceutically acceptable carrier or excipient.

[0033] In the present invention, "pharmaceutically acceptable carrier" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by relevant government regulatory authorities as acceptable for human or livestock use.

[0034] In the present invention, "excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. In addition to any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention. Examples of the types of the term "excipient" include, but are not limited to, adhesives, disintegrants, lubricants, glidants, stabilizers, fillers and diluents, etc. Excipients can enhance the handling characteristics of pharmaceutical preparations, i.e., make the preparations more suitable for direct compression by increasing fluidity and / or adhesion.

[0035] use In the fourth aspect of the present invention, the present invention proposes 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 aging of mature eggs in vitro; improving the development 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 and apoptosis in eggs cultured in vitro; reducing the abnormal spindle assembly of eggs cultured in vitro.

[0036] In some embodiments of the present invention, the mature ovum is derived from a mammal.

[0037] In some embodiments of the present invention, the mammal includes but is not limited to at least one of mice, pigs, cows, horses, sheep, humans, and other non-human primates.

[0038] method In the fifth aspect of the present invention, the present invention proposes a method for alleviating the aging of mature eggs or preserving mature eggs in vitro. According to an embodiment of the present invention, the method comprises: using the composition described in the first aspect, the culture medium described in the second aspect, and at least one of the products described in the third aspect to culture and treat mature eggs of mammals. As mentioned above, the composition of the present invention can reduce the level of inflammatory factors and reduce the toxic effects of inflammatory reactions on eggs. At the same time, the number and activity of mitochondria can be increased to 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 the combined effect, the composition significantly reduces the incidence of egg apoptosis and the rate of spindle abnormalities. Therefore, the method of the present invention can alleviate the aging of mature eggs, preserve mature eggs, and improve their development rate.

[0039] In some embodiments of the present invention, the present invention does not specifically limit the source of the mature eggs, which may be eggs matured in vivo or eggs matured in vitro.

[0040] In some embodiments of the present invention, the mammal includes but is not limited to at least one of mice, pigs, cows, horses, sheep, humans, and other non-human primates.

[0041] In some embodiments of the present invention, the culture treatment conditions are: CO2 concentration of 2-5%, humidity of 70-100%, and temperature of 30-39°C.

[0042] 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 it according to specific needs. For example, the time of the mature egg culture treatment of the present invention can be 1-12 hours.

[0043] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0044] Example 1: Collection and culture of mature eggs after mouse ovulation, in vitro fertilization and embryo culture 1. Collection of mature eggs after mouse ovulation The mice used in the present invention are 8-week-old ICR strains, all purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Each mouse is intraperitoneally injected with 5 IU pregnant mare serum gonadotropin (PMSG), and 48 hours later, each mouse is intraperitoneally injected with 5 IU human chorionic gonadotropin (hCG). 14-16 hours after the injection of hCG, the mice are killed by cervical dislocation, the abdomen is cut open to remove the fallopian tube, and the fallopian tube is placed in a mouse egg in vitro operation fluid M2 preheated at 37°C. Under a stereo microscope, the fallopian tube dilatation is scratched with a 1 mL syringe needle to release mature cumulus oocyte complexes (COCs) for standby use. The formula of the mouse egg in vitro operation fluid M2 is shown in Table 1.

[0045] Table 1 Formula of mouse oocyte in vitro manipulation fluid M2

[0046] 2. Cultivation of mature eggs after ovulation in mice The mouse mature cumulus oocyte complexes (COCs) were washed twice with the egg basal culture medium KSOM (MR-106, Sigma), and then transferred to different culture media pre-equilibrated at 37°C for 2 hours. 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 cultured in an incubator at 37.0°C, 5% CO2, and 100% humidity for 12 hours.

[0047] 3. In vitro fertilization of eggs Prepare 100 μL mouse sperm capacitation solution TYH droplets, cover the droplets with mineral oil, and place them in a 37 ℃ incubator for equilibration for 1 hour. Sterilize the instruments used in the experiment in advance. 10-12 week old adult healthy male mice were killed by cervical dislocation, and the abdominal cavity was carefully cut open with sterilized scissors. The epididymis was gently removed with forceps, and the blood vessels and adipose tissue were cleaned and placed on sterile filter paper. Use forceps to gently squeeze the sperm in the epididymis to one side of the epididymis, keep the forceps clamped and not move, and use scissors to gently cut the epididymis on the side with sperm in the mineral oil of the capacitation solution. After squeezing out the sperm, put it into the capacitation solution TYH. Place it in a 37 ℃ carbon dioxide incubator for 1 hour. The formula of mouse sperm capacitation solution TYH is shown in Table 2.

[0048] Table 2 Mouse sperm capacitation solution TYH formula

[0049] The mature cumulus oocyte complexes (COCs) obtained in step 1 or the COCs cultured for 12 hours in step 2 were washed twice with the egg basal culture medium KSOM, and then transferred to the egg fertilization medium mHTF droplets pre-equilibrated at 37°C for 1 hour.

[0050] After sperm capacitation for 1 hour, observe sperm motility, select sperm with stronger motility, use a pipette to draw 5 µL of sperm from the edge of the TYH capacitation drop, and then add it to the mHTF drop of egg fertilization solution containing COCs from the edge of the fertilization drop. Place in a 37 ℃, 5% CO2, saturated humidity incubator to incubate sperm and eggs for 3-4 hours. The formula of mouse egg fertilization solution mHTF is shown in Table 3.

[0051] Table 3 Formula of mouse egg fertilization fluid mHTF

[0052] 4. Mouse embryo culture in vitro After 3-4 hours of co-incubation, the fertilized eggs were aspirated and gently blown to remove the residual cumulus cells and sperm on the surface of the fertilized eggs. The fertilized eggs were first washed 3 times with egg fertilization solution mHTF, and then washed 3 times with KSOM culture medium (MR-106, Sigma), transferred to KSOM basal culture medium that had been pre-equilibrated in the incubator for more than 2 hours, and placed in a 37.0℃, 5% CO2, saturated humidity incubator for culture. 30 fertilized eggs were placed in 100 µL KSOM culture medium, and the blastocyst rate was counted after 96 hours of culture.

[0053] The mature mouse cumulus oocyte complexes (COCs) taken out in step 1 were divided into three groups, namely, a fresh group (control group), an aged group, and an experimental group (treatment group). The three groups of COCs were treated differently, such as Figure 7 As shown, COCs in the fresh group were directly subjected to in vitro fertilization and embryo culture (steps 3 and 4), COCs in the aged group were placed in KSOM basal culture medium for in vitro culture for 12 h (step 2) and then subjected to in vitro fertilization and embryo culture (steps 3 and 4), and COCs in the experimental group were placed in KSOM culture medium containing different concentrations of SB265610, MTK458 or 8-Br-cGMP or combined additives for in vitro culture for 12 h (step 2) and then subjected to in vitro fertilization and embryo culture (steps 3 and 4).

[0054] Example 2: Study on the effect of SB265610 on alleviating the aging of mature mouse oocytes in vitro This example shows the application of adding different concentrations of SB265610 to the in vitro basal culture medium KSOM for eggs in alleviating the aging of mature eggs.

[0055] 1. Experimental design and experimental methods (1) Experimental design Fresh group: non-aged fresh mature eggs; Aging group: fresh mature oocytes were aged in vitro in KSOM basal culture medium for 12 hours; Treatment groups 1-6: Fresh mature oocytes were aged in vitro for 12 h in KSOM basal culture medium containing 0.1µmol / L (treatment group 1), 0.3µmol / L (treatment group 2), 1µmol / L (treatment group 3), 3µmol / L (treatment group 4), 10µmol / L (treatment group 5), and 30µmol / L (treatment group 6) SB265610.

[0056] (2) Test methods Mouse mature eggs were collected according to the method described in Example 1, and the mature eggs were cultured in vitro using the culture medium of the above groupings. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the blastocyst rate was counted.

[0057] 2. Test results 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 aged group and different treatment groups were significantly reduced (p<0.05). The blastocyst rate of treatment group 2, i.e., group 0.3µmol / L SB265610 addition (7.5%±0.5%), group 3, i.e., group 1µmol / L SB265610 addition (8.1%±1.1%), and group 4, i.e., group 3µmol / L SB265610 addition (6.3%±0.9%) were significantly higher than the blastocyst rate of the aged group (1.8%±0.3%) (p<0.05). The blastocyst rate of treatment group 1 (adding 0.1µmol / L SB265610) (2.6%±0.7%), treatment group 5 (adding 10µmol / L SB265610) (1.7%±0.4%), and treatment group 6 (adding 30µmol / L SB265610) (1.1%±0.2%) was not different from that of the aging group (1.8%±0.3%) (p>0.05). Among them, the blastocyst rate of treatment group 3 (adding 1µmol / L SB265610) was the highest.

[0058] Table 4 Effect of SB265610 on the development rate of mouse mature oocytes after alleviating in vitro aging

[0059] Different superscript letters (a, b, c) in the same column in the table indicate significant differences among the groups, p < 0.05.

[0060] Example 3: Study on the effect of MTK458 on alleviating the aging of mature mouse oocytes in vitro This example shows the application of adding different concentrations of MTK458 to the oocyte in vitro basal culture medium KSOM in alleviating the aging of mature oocytes.

[0061] 1. Experimental design and experimental methods (1) Experimental design Fresh group: non-aged fresh mature eggs; Aging group: fresh mature oocytes were aged in vitro in KSOM basal culture medium for 12 hours; Treatment groups 1-7: Fresh mature oocytes were aged in vitro for 12 h in KSOM basal culture medium containing 0.1µmol / L (treatment group 1), 0.3µmol / L (treatment group 2), 1µmol / L (treatment group 3), 3µmol / L (treatment group 4), 10µmol / L (treatment group 5), 30µmol / L (treatment group 6), and 100µmol / L (treatment group 7) MTK458.

[0062] (2) Test methods Mouse mature eggs were collected according to the method described in Example 1, and the mature eggs were cultured in vitro using the culture medium of the above groupings. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the blastocyst rate was counted.

[0063] 2. Test results The statistical results of in vitro embryo development rate are shown in Table 5. The data in Table 5 show that compared with the cleavage rate (81.3%±5.3%) and blastocyst rate (30.3%±3.9%) of the fresh group, the cleavage rate and blastocyst rate of the aged group and different treatment groups were significantly reduced (p<0.05). The blastocyst rate of treatment group 4, i.e., the group with 3µmol / L MTK458 added (6.3%±0.3%), the blastocyst rate of treatment group 5, i.e., the group with 10µmol / L MTK458 added (9.5%±0.7%), and the blastocyst rate of treatment group 6, i.e., the group with 30µmol / L MTK458 added (5.1%±0.3%) were significantly higher than the blastocyst rate of the aged group (1.9%±0.2%) (p<0.05). The blastocyst rate of treatment group 1 (adding 0.1µmol / L MTK458) (1.9%±0.4%), treatment group 2 (adding 0.3µmol / L MTK458) (2.6%±0.4%), treatment group 3 (adding 1µmol / L MTK458) (2.7%±0.2%), and treatment group 7 (adding 100µmol / L MTK458) (1.8%±0.1%) were not different from the blastocyst rate of the aging group (1.9%±0.2%) (p>0.05). Among them, the blastocyst rate of treatment group 5 (adding 10µmol / L MTK458) was the highest.

[0064] Table 5 Effect of MTK458 on the development rate of mature mouse oocytes after aging in vitro

[0065] Different superscript letters (a, b, c) in the same column in the table indicate significant differences among the groups, p < 0.05.

[0066] Example 4: Study on the effect of 8-Br-cGMP on alleviating the aging of mature mouse oocytes in vitro This example shows the application of adding different concentrations of 8-Br-cGMP to the in vitro basal culture medium KSOM for alleviating the aging of mature eggs.

[0067] 1. Experimental design and experimental methods (1) Experimental design Fresh group: non-aged fresh mature eggs; Aging group: fresh mature oocytes were aged in vitro in KSOM basal culture medium for 12 hours; Treatment groups 1-7: Fresh mature oocytes were aged in vitro for 12 hours in KSOM basal culture medium containing 0.1mmol / L (treatment group 1), 0.3mmol / L (treatment group 2), 1mmol / L (treatment group 3), 3mmol / L (treatment group 4), 10mmol / L (treatment group 5), 30mmol / L (treatment group 6), and 100mmol / L (treatment group 7) 8-Br-cGMP.

[0068] (2) Test methods Mouse mature eggs were collected according to the method described in Example 1, and the mature eggs were cultured in vitro using the culture medium of the above groupings. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the blastocyst rate was counted.

[0069] 2. Test results The statistical results of in vitro embryo development rate are shown in Table 6. The data in Table 6 show 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 aged group and different treatment groups were significantly reduced (p<0.05). The blastocyst rate of treatment group 3, i.e., the group with 1mmol / L 8-Br-cGMP added (11.5%±1.8%), the blastocyst rate of treatment group 4, i.e., the group with 3mmol / L 8-Br-cGMP added (12.3%±2.1%), and the blastocyst rate of treatment group 5, i.e., the group with 10mmol / L 8-Br-cGMP added (8.7%±1.1%) were significantly higher than the blastocyst rate of the aged group (3.4%±0.5%) (p<0.05). The blastocyst rates of treatment group 1 (adding 0.1mmol / L 8-Br-cGMP) (1.9%±0.3%), treatment group 2 (adding 0.3mmol / L 8-Br-cGMP) (5.2%±0.6%), treatment group 6 (adding 30mmol / L 8-Br-cGMP) (2.1%±0.9%), and treatment group 7 (adding 100mmol / L 8-Br-cGMP) (1.2%±0.4%) were not different from those of the aging group (3.4%±0.5%) (p>0.05). Among them, the blastocyst rates of treatment group 3 (adding 1mmol / L 8-Br-cGMP) and treatment group 4 (adding 3mmol / L 8-Br-cGMP) were similar and the highest.

[0070] Table 6 Effect of 8-Br-cGMP on the development rate of mouse mature oocytes after aging in vitro

[0071] Different superscript letters (a, b, c) in the same column in the table indicate significant differences among the groups, p < 0.05.

[0072] Example 5: Study on the effect of SB265610, MTK458 and 8-Br-cGMP combination in alleviating the aging of mature mouse oocytes in vitro This example shows the application of adding SB265610, MTK458 and 8-Br-cGMP in combination to the oocyte in vitro basal culture medium KSOM in alleviating the aging of mature oocytes.

[0073] 1. Experimental design and experimental methods (1) Experimental design Fresh group: non-aged fresh mature eggs; Aging group: fresh mature oocytes were aged in vitro in KSOM basal culture medium for 12 hours; Treatment group 1: fresh mature oocytes were aged in vitro in KSOM basal medium containing 1µmol / L SB265610 and 10µmol / L MTK458 for 12 h; Treatment group 2: fresh mature oocytes were aged in vitro for 12 h in KSOM basal medium containing 1 µmol / L SB265610 and 1 mmol / L 8-Br-cGMP; Treatment group 3: fresh mature oocytes were aged in vitro for 12 h in KSOM basal medium containing 10 µmol / L MTK458 and 1 mmol / L 8-Br-cGMP; Treatment group 4: Fresh mature oocytes were aged in vitro for 12 hours in KSOM basal medium containing 1µmol / L SB265610, 10µmol / L MTK458 and 1mmol / L 8-Br-cGMP.

[0074] (2) Test methods Mouse mature eggs were collected according to the method described in Example 1, and the mature eggs were cultured in vitro using the culture medium of the above groupings. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the blastocyst rate was counted.

[0075] 2. Test results The statistical results of in vitro embryo development rate are shown in Table 7. The data in Table 7 show that compared with the fresh group cleavage rate (80.4% ± 7.4%) In comparison, 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 significantly decreased (p<0.05), while the cleavage rate of treatment group 4 (SB265610, MTK458 and 8-Br-cGMP added simultaneously) (77.3%±4.6%) was not different from that of the fresh group (p>0.05). In addition, the blastocyst rate 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 aged control group (2.3%±0.3%), and lower than that of the fresh group (42.7%±5.2%), but the blastocyst rate of treatment group 4 (SB265610, MTK458, and 8-Br-cGMP co-added group) (37.3%±4.2%) was not different from that of the fresh group (p>0.05). Therefore, the effect of the co-added group of SB265610, MTK458, and 8-Br-cGMP was better than the combined effect of any two.

[0076] Table 7 Effect of SB265610, MTK458 and 8-Br-cGMP combination on the development rate of mouse mature oocytes in vitro aging

[0077] Different superscript letters (a, b, c) in the same column in the table indicate significant differences among the groups, p < 0.05.

[0078] Example 6: Study on the effect of SB265610, MTK458 and 8-Br-cGMP combination on the quality of mature oocyte aging (1) Experimental design Fresh group (control group): non-aged fresh mature eggs; Aging group: fresh mature oocytes were aged in vitro in KSOM basal culture medium for 12 hours; Treatment group (experimental group): Fresh mature oocytes were aged in vitro for 12 hours in KSOM basal culture medium containing 1µmol / L SB265610, 10µmol / L MTK458 and 1mmol / L 8-Br-cGMP.

[0079] (2) Mature egg collection and culture, in vitro fertilization and embryo culture Mouse mature eggs were collected according to the method described in Example 1, and the mature eggs were cultured in vitro using the culture medium of the above groupings. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1.

[0080] (3) Oocyte spindle structure staining Mature oocytes that had been aged in vitro for 12 hours were digested in 0.1% hyaluronidase for 2 minutes, and then the oocytes were aspirated with a mouth pipette and placed in KSOM. They were then washed twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS), and then fixed in 4% paraformaldehyde fixative at 4°C for 12 hours. The fixed oocytes were washed three times with 0.1% PVA-PBS, and then permeabilized in 0.1% PVA-PBS containing 0.1% TritonX 100 (0.1%TritonX 100-PVA-PBS) for 25 minutes. The permeabilized oocytes were placed in immunofluorescence staining blocking solution at 4°C for 12 hours. The blocked oocytes were incubated with Tubulin-FITC primary antibody at 4°C for 12 hours. After the primary antibody incubation, they were washed three times with 0.1% PVA-PBS. The secondary antibody was incubated at room temperature for 1.5 hours, and then washed three times with 0.1% PVA-PBS. The nuclei were stained with DAPI at room temperature for 5 minutes, and then washed three times with 0.1% PVA-PBS. The eggs were then placed on a glass slide, and an anti-fluorescence quencher (DABCO) was added before pressing. The coverslip was sealed with nail polish, and images were collected using a fluorescence confocal microscope to calculate the spindle abnormality rate.

[0081] The mature eggs in the control group, aging group and experimental group were tested for spindle morphology abnormality according to the above method. The experimental results are as follows Figure 1 shown. Figure 1 The data showed that the spindle abnormality rate of the eggs in the aging group (72.10%±5.62%) was significantly higher than that in the control group (11.28%±2.94%) and the experimental group (18.63%±3.17%) (p<0.05). There was no significant difference between the experimental group and the control group (p>0.05), indicating that the method for alleviating the aging of eggs cultured in vitro provided by the present invention can well prevent the increase of the spindle abnormality rate of eggs.

[0082] (4) Oocyte mitochondrial activity detection Prepare JC-1 working solution: First, mix 800 μL of operating solution M2 and 200 μL of 5× JC-1 staining buffer vigorously, then add 5 μL of 200× JC-1 to the mixture. JC-1 working solution should be prepared and used immediately.

[0083] The mature oocytes that had been aged in vitro for 12 hours were placed in 0.1% hyaluronidase for digestion for 2 minutes. The oocytes were aspirated with a mouth pipette and placed in the operating solution M2, and then washed twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). The oocytes were then placed in the JC-1 working solution that had been pre-equilibrated at 37°C and incubated in a 37°C incubator for 15 minutes. The washing solution was prepared with 5×JC-1 staining buffer and operating solution M2 in a volume ratio of 1:4 and placed on ice for use. After the oocytes were incubated with the JC-1 working solution, they were washed three times with the washing solution. The oocytes were then pressed on a glass slide, photographed with a laser confocal microscope, and the mitochondrial membrane potential of the oocytes was analyzed using Image J software.

[0084] The mature eggs of the control group, aging group and experimental group were tested for mitochondrial activity according to the above method. The experimental results are as follows Figure 2 shown. Figure 2 The data showed that the mitochondrial membrane potential of the eggs in the aging group (0.41% ± 0.062%) was significantly lower than that of the control group (1.52% ± 0.21%) and 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 for alleviating the aging of eggs in vitro culture provided by the present invention can well prevent the decrease of the mitochondrial membrane potential of the eggs and protect the mitochondrial activity in the eggs.

[0085] (5) Oocyte mitochondrial staining Prepare Mito-Tracker Green working solution: Use DMSO to prepare Mito-Tracker Green concentrated stock solution with a concentration of 1 mM, and use egg in vitro operation solution M2 to prepare Mito-Tracker Green working solution at a volume ratio of 1:20,000 (Mito-Tracker Green concentrated stock solution: egg in vitro operation solution M2).

[0086] The mature oocytes that had been aged in vitro for 12 hours were placed in 0.1% hyaluronidase for digestion for 2 minutes. The oocytes were aspirated with a mouth pipette and placed in the operating solution M2, and then washed twice with PBS buffer containing 0.1% PVA (0.1% PVA-PBS). The oocytes were then placed in the Mito-Tracker Green working solution that had been pre-equilibrated at 37°C, and 30 oocytes were placed in 100μL Mito-Tracker Green working solution. The oocytes were placed in a 37°C carbon dioxide incubator for 30 minutes. After incubation, the oocytes were washed twice with the operating solution M2, and then the oocytes were pressed on a slide, photographed with a laser confocal microscope, and the fluorescence intensity of Mito-Tracker Green was analyzed using Image J software.

[0087] The mature oocytes of the control group, aging group and experimental group were stained with MitoTracker according to the above method to detect and evaluate the number of mitochondria. Figure 3 As shown, the data showed that the fluorescence intensity of mitochondrial MitoTracker staining of the eggs in the aging group (12.01% ± 2.32%) was significantly lower than that of the control group (36.33% ± 4.52%) and the experimental group (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 number of mitochondria in the aging group was significantly reduced, and the method for alleviating the aging of mature eggs provided by the present invention can significantly alleviate the reduction in the number of mitochondria.

[0088] (6) Cellular reactive oxygen species detection and cell apoptosis detection Specific process of cell reactive oxygen species (ROS) detection: Preparation of the working solution of the oxidation-sensitive fluorescent probe DCFH-DA: dilute DCFH-DA at a volume ratio of 1:1000 to a final concentration of 10 μM.

[0089] Mature oocytes aged in vitro for 12 hours were digested in 0.1% hyaluronidase for 2 minutes, and then washed twice with DPBS buffer. Then, the cells were added to 500 μL DCFH-DA working solution and incubated in a 37°C cell culture incubator for 20 minutes. Invert and mix every 3-5 minutes to allow the probe and cells to fully contact. After incubation for 20 minutes, the cells were washed 3 times to fully remove the DCFH-DA that did not enter the cells. Then, laser confocal microscopy was used to take pictures and observe, and ImageJ software was used to analyze the DCFH-DA fluorescence intensity.

[0090] Specific process of cell apoptosis detection: Mature oocytes that had been aged in vitro for 12 hours were collected and washed twice with 0.1% PVA in PBS buffer (0.1% PVA-PBS). After washing, the oocytes were placed in 4% PFA fixative and fixed in a 4 ℃ refrigerator for 2 hours, washed 3 times with 0.1% PVA-PBS, transferred to 0.5% TritonX100-0.1% PVA-PBS permeabilization solution for 1 hour, and then transferred to apoptosis TUNEL detection solution (C1086, Biyuntian) and incubated in a 37 ℃ incubator for 1 hour. After incubation, the oocytes were washed 3 times with 0.1% PBS-PVA, incubated in 20μL DAPI staining for 5 minutes, and photographed under a fluorescence microscope. The number of TUNEL-positive signal cells was taken as the number of apoptotic cells, and the number of DAPI-positive signal cells was taken as the total number of cells to calculate the cell apoptosis rate. The apoptosis rate was calculated as follows: apoptosis rate = (number of cells with TUNEL-positive signal / number of cells with DAPI-positive signal) × 100.

[0091] The mature oocytes of the control group, aging group and experimental group were tested for reactive oxygen species (ROS) content and cell apoptosis according to the above method. The results were as follows: Figure 4 and Figure 5 As shown. The data showed that the active oxygen intensity of the aging group was (13.18±0.95), and the apoptosis rate was (20.68%±1.78%), while the active oxygen intensity (3.96±0.29) and apoptosis rate (1.28%±0.09%) of the test group after 12 hours of in vitro culture using the culture solution provided by the present invention were significantly reduced, and there was a statistically significant difference (p<0.05). There was no significant difference between the test group and the control group (p﹥0.05). This shows that the method for alleviating the aging of mature eggs provided by the present invention can effectively reduce the intracellular active oxygen content, protect cells from oxidative stress damage, and prevent the occurrence of apoptosis.

[0092] (7) RNA extraction, reverse transcription and real-time fluorescence quantitative PCR The mature eggs of the control group, aging group and experimental group were collected by centrifugation and collected into 1.5 mL centrifuge tubes. The excess liquid was removed and stored in a -80°C refrigerator. Total RNA was extracted using Trizol. The reverse transcription system and procedure are as follows.

[0093] Configure the reverse transcription system:

[0094] Reverse transcription reaction procedure:

[0095] After reverse transcription, the cDNA samples were stored in a -80°C refrigerator for real-time fluorescence quantitative PCR.

[0096] Configure the real-time fluorescence quantitative PCR system:

[0097] Fluorescence quantitative PCR reaction procedure:

[0098] According to the real-time fluorescence quantitative PCR reaction system and procedure, the cDNA template was mixed with the kit premix and real-time fluorescence quantitative PCR was performed in a Bio-Rad CFX96 Touch Real-Time PCR instrument. GAPDH was used as the internal reference gene and 2 -△△Ct The relative expression levels of the target genes were calculated using the method.

[0099] The mature oocytes of the control group, aging group and experimental group were tested for the gene expression levels of chemokines and inflammatory factors according to the above method. The experimental results are as follows Figure 6 As shown. The data showed that in the test group cultured in vitro for 12 hours using the culture fluid 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 with the control group (p>0.05). This shows that the method for alleviating the aging of mature eggs provided by the present invention can well reduce the expression of chemokines and inflammatory factors, reduce inflammatory reactions, and protect cell activity.

[0100] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0101] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A composition, characterized in that include: SB265610, MTK458, and 8-Br-cGMP.

2. The composition according to claim 1, characterized in that The molar ratio of SB265610, MTK458 and 8-Br-cGMP is 1:10:(1000-3000).

3. A culture solution, characterized in that The invention comprises the composition according to any one of claims 1 to 2.

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

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

6. The culture solution according to claim 4, characterized in that The final concentration of MTK458 in the culture medium is 0.1µmol / L-100µmol / L.

7. The culture solution according to claim 4, characterized in that The final concentration of the 8-Br-cGMP in the culture medium is 0.1 mmol / L-100 mmol / L.

8. The culture solution according to claim 4, characterized in that The basic culture medium includes at least one of KSOM, MEMα, DMEM / F12, M2, M16, CZB, HTF, TYH, and TCM199.

9. A product, characterized in that The method comprises the composition according to any one of claims 1 to 2 or the culture solution according to any one of claims 3 to 8.

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

11. The composition of any one of claims 1 to 2, the culture solution of any one of claims 3 to 8, or the product of any one of claims 9 to 10 has at least one of the following uses: 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.

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

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

14. A method for alleviating the in vitro aging of mature eggs or preserving mature eggs in vitro, characterized in that: include: The mature ovum of a mammal is cultured with at least one of the composition according to any one of claims 1 to 2, the culture solution according to any one of claims 3 to 8, and the product according to any one of claims 9 to 10.

15. The method according to claim 14, 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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