Composition, culture medium, product and method for promoting in vitro maturation of immature oocytes

Abstract

The present invention relates to the field of biotechnology and provides a composition, culture medium, product, and method for promoting in vitro maturation of immature oocytes. The composition comprises KT5823 and gastrodin. The composition can reduce the levels of pro-inflammatory factors in oocytes, alleviating the toxic effects of inflammation and autophagy on oocytes. Furthermore, it can effectively reduce reactive oxygen species (ROS) levels and increase the antioxidant glutathione content, thereby alleviating oxidative stress damage to oocytes. Through these synergistic effects, the composition significantly reduces oocyte apoptosis, significantly improves the quality of in vitro oocyte maturation, and significantly increases the development rate after in vitro fertilization.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition, culture medium, product and method for promoting in vitro maturation of immature oocytes. Background Art

[0002] In vitro production (IVP) is an embryo engineering technology of great industrial value, enabling rapid propagation of superior varieties and accelerating the development of new varieties. Currently, renowned multinational breeding companies such as ABS, SEMEX, and Alta have established independent subsidiaries or departments dedicated to the research and development of IVP technology. IVP involves in vitro oocyte maturation, in vitro fertilization, and in vitro embryo culture. In vitro oocyte maturation is the most significant step affecting IVP efficiency. Poor quality of in vitro oocyte maturation is a significant challenge facing IVP, resulting in 60% to 90% of oocytes being unusable. Furthermore, in vitro oocyte maturation is a key method in human assisted reproduction, offering significant advantages in reducing hormone use, lowering treatment costs, streamlining treatment procedures, and improving safety and pregnancy rates. However, the development rate of human oocytes after in vitro maturation is only 10-30%, with no significant improvement over the past few decades.

[0003] Therefore, how to improve the efficiency of IVP technology by promoting the quality of in vitro maturation of oocytes is a major and prominent issue that technicians in this field urgently need to solve. Summary of the Invention

[0004] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. Therefore, through extensive screening experiments, the inventors discovered that the combined use of KT5823 and gastrodin can improve the quality of oocyte maturation in vitro, reduce intracellular reactive oxygen species (ROS) levels and inflammatory responses, and effectively increase oocyte development rate.

[0005] Therefore, in its first aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises KT5823 and gastrodin. After extensive experiments, the inventors discovered that the KT5823 and gastrodin combination can improve the quality of oocyte maturation in vitro. This composition can reduce the levels of pro-inflammatory factors in oocytes and alleviate the toxic effects of inflammatory and autophagic reactions on oocytes. Furthermore, it can effectively reduce the level of reactive oxygen species, increase the content of the antioxidant glutathione, and alleviate the damage caused by oxidative stress to oocytes. Through these synergistic effects, the composition significantly reduces the apoptosis rate of oocytes, significantly improves the quality of oocyte maturation in vitro, and enhances the development rate of oocytes after in vitro fertilization.

[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 pro-inflammatory factors in oocytes and reduce the toxic effects of inflammatory reactions and autophagic reactions on oocytes. At the same time, it can effectively reduce the level of reactive oxygen species, reduce the damage of oxidative stress to oocytes, and significantly reduce the apoptosis rate of oocytes. Thus, the culture solution of the present invention can promote the quality of in vitro maturation of oocytes and improve the development rate of mature oocytes after fertilization. Experimental results show that when the culture solution of this combination is used for in vitro maturation of oocytes, the oocyte development rate is greatly improved by more than 30%, which is much higher than the effect of the prior art.

[0007] In its third aspect, 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 previously mentioned, the composition of the present invention can reduce the levels of pro-inflammatory factors in oocytes, reducing the toxic effects of inflammatory and autophagic reactions on oocytes. It can also effectively reduce the level of reactive oxygen species, minimizing oxidative stress damage to oocytes. Consequently, the product can promote in vitro maturation of oocytes and improve the development rate of mature oocytes after fertilization. Furthermore, KT5823 and gastrodin are both safe and reliable ingredients, ensuring the safety of the product during use. This product is not only suitable for in vitro maturation of oocytes in a variety of animals, but can also provide a reference for human assisted reproductive technology, such as accelerating the propagation of superior breeds in in vitro embryo production in animals such as cattle, pigs, horses, and sheep, or improving the success rate of in vitro fertilization in 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: improving the in vitro maturation rate of oocytes; improving the development rate of oocytes after in vitro fertilization; reducing the level of reactive oxygen species (ROS) in oocytes; increasing the content of glutathione (GSH) in oocytes; reducing the level of inflammatory factors in oocytes; and reducing the level of apoptosis in oocytes.

[0009] In the fifth aspect of the present invention, the present invention proposes a method for promoting in vitro maturation of immature oocytes or improving the quality of in vitro maturation of oocytes. 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 perform in vitro culture treatment on immature oocytes of mammals. As mentioned above, the composition of the present invention can significantly reduce the inflammatory response, autophagy response, oxidative stress and apoptosis rate of oocytes through a synergistic effect, significantly improve the quality of in vitro maturation of oocytes, and effectively improve the development rate after fertilization.

[0010] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by 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 readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0012] Figure 1 The present invention relates to the effects of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation medium on the level of reactive oxygen species (ROS) in mature oocytes, wherein:

[0013] A is the reactive oxygen species (ROS) staining image of mature oocytes in the control group and the treatment group;

[0014] B is the statistical results of reactive oxygen species (ROS) levels in mature oocytes of the control group and the treatment group.

[0015] Figure 2 The present invention relates to the effect of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation medium on apoptosis of mature oocytes, wherein:

[0016] A is the TUNEL staining of mature oocyte apoptosis in the control group and the treatment group;

[0017] B is the statistics of apoptosis rate of mature oocytes in the control group and the treatment group.

[0018] Figure 3 The present invention relates to the effects of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation medium on the expression levels of apoptosis genes and autophagy genes in mature oocytes, wherein:

[0019] A is the fluorescence quantitative PCR results of apoptosis genes in mature oocytes of the control group and the treatment group;

[0020] B is the results of fluorescence quantitative PCR of autophagy genes in mature oocytes of the control group and the treatment group.

[0021] Figure 4 The present invention relates to the effects of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation fluid on in vitro fertilized embryonic cells, wherein:

[0022] A is the immunofluorescence staining of SOX2 and CDX2 in mature oocytes and in vitro fertilized embryos of the control group and the treatment group;

[0023] B is the statistical proportion of trophoblast cells (CDX2 positive) and inner cell mass cells (SOX2 positive) in in vitro fertilized embryos of mature oocytes in the control group and the treatment group.

[0024] Figure 5 The present invention relates to the effect of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation medium on glutathione (GSH) levels in mature oocytes, wherein:

[0025] A is the glutathione staining image of mature oocytes in the control group and the treatment group;

[0026] B is the statistical results of glutathione levels in mature oocytes of the control group and the treatment group. DETAILED DESCRIPTION

[0027] 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 are not intended to limit the scope of protection of the present invention.

[0028] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute a separate or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0029] " scope " disclosed in the present invention is limited in the form of lower limit and upper limit, and given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of special range. The scope that this mode limits can be to include end value, and can be combined arbitrarily, and promptly any lower limit can form a scope with any upper limit combination. For example, if the scope of 60-120 and 80-110 is listed for specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range value 1 and 2 are listed, and if the maximum range value 3,4 and 5 are listed, then 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, 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 that this mode limits can be to include end value a and b. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this 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.

[0030] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0031] 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.

[0032] Unless 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.

[0033] Protein kinase G (PKG) is a serine / threonine protein kinase expressed in most somatic tissues. PKG is involved in the regulation of numerous physiological and pathological processes, including bone remodeling, cardiac fibrosis, memory formation, neural repair, and tumorigenesis. The PKG inhibitor KT5823 has been shown to alleviate dopamine neuron degeneration and death, reduce lipid oxidation in adipocytes, inhibit cancer cell metastasis, and alleviate inflammatory responses.

[0034] Gastrodin is a naturally active compound extracted from the dried root of Gastrodia elata, a plant of the orchid family. It has significant neuroprotective effects, maintaining homeostasis; sedative, hypnotic, and anxiolytic properties; improving hemodynamics and cardiovascular protection; regulating bone metabolism; and inhibiting inflammatory signals and scavenging free radicals, protecting cells from oxidative stress.

[0035] In their experiments, the inventors discovered that the combined use of KT5823 and gastrodin significantly promoted the in vitro maturation of oocytes. This combination not only significantly reduced the levels of pro-inflammatory factors in oocytes and alleviated the toxic effects of inflammatory and autophagic reactions on oocytes, but also effectively reduced the levels of reactive oxygen species and increased the content of the antioxidant glutathione, thereby reducing the damage to oocytes caused by oxidative stress. Through a synergistic effect, KT5823 and gastrodin significantly reduced the apoptosis rate of oocytes, thereby improving the quality of oocyte maturation in vitro. In in vitro fertilization experiments, the development rate of oocytes was also significantly improved. This strategy will play an important role in the future exploration of the mechanism of oocyte maturation in vitro and the further development of technologies to promote the quality of oocyte maturation in vitro.

[0036] The present invention provides a composition, a culture medium, a product and its use, and a method for promoting the in vitro maturation quality and development rate of oocytes, which will be described in detail below.

[0037] Composition, culture medium, product

[0038] In its first aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises KT5823 and gastrodin. After extensive experiments, the inventors discovered that the KT5823 and gastrodin composition of the present invention can promote the quality of oocyte maturation in vitro and increase the development rate of mature oocytes after fertilization. The composition can reduce the levels of pro-inflammatory factors in oocytes and alleviate the toxic effects of inflammatory and autophagic reactions on oocytes. Simultaneously, it can effectively reduce the level of reactive oxygen species and alleviate the damage to oocytes caused by oxidative stress. Through these synergistic effects, the composition significantly reduces the apoptosis rate of oocytes, significantly improves the quality of oocyte maturation in vitro, and increases the development rate of oocytes after in vitro fertilization.

[0039] In some embodiments of the present invention, the molar ratio or molar concentration ratio of KT5823 to gastrodin is (0.03-0.1):1. For example, it can be 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, etc., or it can be any range consisting of the above values. Thus, by adjusting the molar ratio of KT5823 to gastrodin within the above range, it is possible to effectively improve the quality of in vitro maturation of oocytes and the development rate of mature oocytes after fertilization, reduce the level of pro-inflammatory factors in oocytes, and alleviate the toxic effects of inflammatory and autophagic reactions on oocytes. At the same time, it can effectively reduce the level of reactive oxygen species, increase glutathione content, alleviate the damage of oxidative stress to oocytes, and significantly reduce the rate of oocyte apoptosis.

[0040] 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 pro-inflammatory factors in oocytes and reduce the toxic effects of inflammatory reactions and autophagic reactions on oocytes. At the same time, it can effectively reduce the level of reactive oxygen species, increase the content of glutathione, reduce the damage of oxidative stress to oocytes, and significantly reduce the apoptosis rate of oocytes. Thus, the culture solution of the present invention can promote the quality of in vitro maturation of oocytes and improve the development rate of mature oocytes after fertilization. Experimental results show that when the culture solution of this combination is used for in vitro maturation of oocytes, the oocyte development rate is greatly improved by more than 30%, which is much higher than the effect of the prior art.

[0041] In some embodiments of the present invention, the culture fluid further includes a basal culture fluid. Thus, a nutritional and physiological condition simulating an in vivo environment can be provided for the oocyte to support the normal maturation of the oocyte. According to an embodiment of the present invention, the basal culture fluid includes: 80-95% TCM199 culture fluid, 8-12 IU / mL pregnant mare serum gonadotropin, 8-12 IU / mL luteinizing hormone, 1-20 ng / mL estrogen, 95-105 IU / mL penicillin, 95-105 μg / mL streptomycin, 5-20% follicular fluid, 0.4-0.6 mg / mL glucose, 1.8-2.3 mg / mL sodium bicarbonate, 0.04-0.08 mg / mL sodium pyruvate, and 0.4-0.7 mg / mL cysteine.

[0042] According to an embodiment of the present invention, the basal culture medium includes: 90% TCM199 culture medium, 10 IU / mL pregnant mare serum gonadotropin, 10 IU / mL luteinizing hormone, 10 ng / mL estrogen, 100 IU / mL penicillin, 100 μg / mL streptomycin, 10% follicular fluid, 0.50 mg / mL glucose, 2.10 mg / mL sodium bicarbonate, 0.06 mg / mL sodium pyruvate, and 0.57 mg / mL cysteine.

[0043] In some embodiments of the present invention, the final concentration of KT5823 in the culture medium is 0.1 µmol / L-10 µmol / L. For example, it can be 0.1 µmol / L, 0.3 µmol / L, 1 µmol / L, 3 µmol / L, 5 µmol / L, 10 µmol / L, etc., or can be a range consisting of any of the above values. Thus, by ensuring that the final concentration of KT5823 is within the above range, the culture medium can effectively reduce the level of inflammatory factors in oocytes during in vitro culturing of oocytes, thereby preventing damage to the oocytes caused by inflammatory reactions.

[0044] In some embodiments of the present invention, the final concentration of gastrodin 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, 70 µmol / L, 100 µmol / L, or any range thereof. Thus, by ensuring that the final concentration of gastrodin is within the above range, the level of reactive oxygen species in the oocyte can be effectively reduced, the content of the antioxidant glutathione can be increased, and the occurrence of oxidative stress damage in the oocyte can be alleviated.

[0045] In its third aspect, 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 previously mentioned, the composition of the present invention can reduce the levels of pro-inflammatory factors in oocytes and mitigate the toxic effects of inflammatory and autophagic reactions on oocytes. It can also effectively reduce the level of reactive oxygen species, mitigate oxidative stress damage to oocytes, and significantly reduce the rate of oocyte apoptosis. Thus, the product can promote the quality of in vitro oocyte maturation and improve the development rate of mature oocytes after fertilization. Furthermore, KT5823 and gastrodin are both safe and reliable ingredients, ensuring the safety of the product during use. This product is not only suitable for in vitro maturation of oocytes in a variety of animals, but also provides a reference for human assisted reproductive technology. For example, it can accelerate the propagation of superior breeds in in vitro embryo production in animals such as cattle, pigs, horses, and sheep, and improve the success rate of in vitro fertilization in human assisted reproduction.

[0046] In some embodiments of the present invention, the product comprises a kit or a medicament.

[0047] In some embodiments of the present invention, the product is a drug, and the product may further include a pharmaceutically acceptable carrier or excipient.

[0048] In the present invention, "pharmaceutically acceptable carrier" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant government regulatory authorities as acceptable for use by humans or livestock.

[0049] In the present invention, "excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for the scope of any conventional excipient incompatibility with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component 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. Excipients can enhance the handling characteristics of pharmaceutical preparations, i.e., make the preparation more suitable for direct compression by increasing fluidity and / or adhesion.

[0050] use

[0051] 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: improving the in vitro maturation rate of oocytes; improving the oocyte development rate; reducing the level of reactive oxygen species in oocytes; increasing the glutathione content in oocytes; reducing the level of inflammatory factors in oocytes; and reducing the level of oocyte apoptosis.

[0052] In some embodiments of the present invention, the oocyte is derived from a mammal.

[0053] 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.

[0054] method

[0055] In the fifth aspect of the present invention, the present invention proposes a method for promoting the in vitro maturation of immature oocytes. 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 mammalian oocytes. As mentioned above, the composition of the present invention can reduce the level of pro-inflammatory factors in oocytes, reduce the toxic effects of inflammatory reactions and autophagic reactions on oocytes, and can also effectively reduce the level of reactive oxygen species and reduce the damage to oocytes caused by oxidative stress. By applying the method of the present invention, the apoptosis rate of oocytes is significantly reduced, the quality of in vitro maturation is improved, and the development rate after fertilization is significantly improved. The method is simple to operate, safe and non-toxic, and can effectively promote the in vitro maturation of immature oocytes and enhance their developmental potential.

[0056] In the sixth aspect of the present invention, the present invention proposes a method for improving the in vitro development rate or in vitro maturation quality of oocytes. 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 mammalian oocytes. As mentioned above, the composition of the present invention can reduce the level of pro-inflammatory factors in oocytes, reduce the toxic effects of inflammatory reactions and autophagic reactions on oocytes, and can also effectively reduce the level of reactive oxygen species and reduce the damage to oocytes caused by oxidative stress. By applying the method of the present invention, the apoptosis rate of oocytes is significantly reduced, the quality of in vitro maturation is improved, and the development rate after fertilization is significantly improved. The method is simple to operate, safe and non-toxic, and can effectively promote the in vitro maturation of immature oocytes and enhance their developmental potential.

[0057] In some embodiments of the present invention, the oocyte is derived from a mammal.

[0058] 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.

[0059] 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.

[0060] It should be noted that the present invention does not impose any specific limitation on the duration of the culture treatment, and those skilled in the art may select a duration according to specific needs. For example, the oocyte culture treatment time of the present invention may be 12-48 hours.

[0061] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0062] Example 1: Oocyte in vitro maturation, in vitro fertilization and embryo culture

[0063] 1. Oocyte maturation in vitro

[0064] Ovary ovaries were collected from the slaughterhouse, placed in saline at 30–35°C, and transported to the laboratory within 2 hours. After three washes with saline, the follicles were punctured with a 20G disposable syringe containing 5 mL of oocyte aspiration solution to release the cumulus-oocyte complexes. Ovary complexes with uniform cytoplasm and at least three layers of cumulus cells were collected under a stereomicroscope. Selected oocyte complexes were washed three times with oocyte aspiration solution and then three times with in vitro maturation basal medium that had been equilibrated in an incubator for 3 hours. The complexes were then transferred to four-well plates containing 600 μL of the different in vitro maturation mediums and 300 μL of mineral oil per well for culture. The plates were then incubated in a humidified incubator at 38.5°C with 5% CO₂ for 22–24 hours.

[0065] Egg aspiration fluid composition: 49 mL TCM199 culture medium + 1 mL FBS (fetal bovine serum).

[0066] The composition of the basal culture medium for in vitro maturation of oocytes is as follows: 90% TCM199 culture medium, 10 IU / mL pregnant mare serum gonadotropin, 10 IU / mL luteinizing hormone, 10 ng / mL estrogen, 100 IU / mL penicillin, 100 μg / mL streptomycin, 10% follicular fluid, 0.50 mg / mL glucose, 2.10 mg / mL sodium bicarbonate, 0.06 mg / mL sodium pyruvate, and 0.57 mg / mL cysteine.

[0067] Among them, different in vitro maturation culture media included the basic culture medium for in vitro maturation of oocytes used in the control group, and the culture medium used in the experimental group was the basic culture medium for in vitro maturation of oocytes with different concentrations of KT5823 or Gastrodin or a combined additive group added to the basic culture medium for in vitro maturation of oocytes.

[0068] (2) In vitro fertilization

[0069] The sheep cumulus oocyte complex matured in vitro for 22-24 hours was placed in 0.5% hyaluronidase and gently and repeatedly blown to remove most of the cumulus cells. The oocytes were washed three times with fertilization solution to obtain mature oocytes. The oocytes were transferred into fertilization solution that had been equilibrated in the incubator for more than 2 hours and cultured in a four-well plate. Each well contained 400 μL of fertilization solution, covered with 300 μL of mineral oil, and 30 mature oocytes for in vitro fertilization.

[0070] Frozen sperm was removed from liquid nitrogen and rapidly thawed in a 38°C water bath. The thawed sperm was then transferred to 600 μL of sperm swim-up solution (purchased from IVF Bioscience) and incubated in an incubator for 30 minutes to allow the sperm to fully float. 100 μL of the supernatant was then added to a four-well plate containing mature oocytes and incubated for 19-21 hours. Fertilization conditions were saturated humidity, 38.5°C, 5% CO2, and 95% air.

[0071] The fertilization fluid was prepared as follows: mSOFaa culture medium + 2% bovine serum + 100 μg / mL streptomycin + 100 IU / mL penicillin.

[0072] The mSOFaa culture medium was prepared as follows per liter: 6.34 g NaCl, 0.54 g KCl, 0.23 g CaCl2·2H2O, 0.01 g MgSO4·7H2O, 0.17 g KH2PO4, 0.24 g NaHCO3, 0.30 g glucose, 0.04 g sodium pyruvate, 5.8 mL sodium lactate, 2% by volume BME amino acid solution, and 1% by volume MEM non-essential amino acid solution. All reagents were purchased from Sigma-Aldrich.

[0073] (3) Embryo culture

[0074] After 19-21 hours of co-incubation, the oocytes were aspirated and gently pipetted to remove any residual cumulus cells and sperm. The oocytes were washed three times with mSOFaa medium and then transferred to mSOFaa medium that had been equilibrated in the incubator for at least 2 hours. The oocytes were then placed in different wells of a four-well plate and cultured at 38.5°C in a saturated humidity incubator with 5% O₂, 5% CO₂, and 90% N₂. The cleavage rate was calculated after 48 hours of culture, and the blastocyst rate was calculated after culture was continued until day 7.

[0075] Example 2: Effects of adding different concentrations of KT5823 to in vitro maturation fluid on embryonic development after fertilization

[0076] This example shows the effect of adding different concentrations of KT5823 to the basal culture medium for in vitro maturation of oocytes on the development of embryos after fertilization.

[0077] 1. Experimental design and methods

[0078] (1) Experimental design

[0079] Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h;

[0080] Treatment groups 1-5: Immature oocytes were cultured in in vitro maturation basal medium containing KT5823 at concentrations of 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), and 10 µmol / L (treatment group 5) for 24 h.

[0081] (2) Test method

[0082] Sheep cumulus oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation culture medium of the above groupings. In vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were calculated.

[0083] 2. Test results

[0084] The statistical results of oocyte development rate are shown in Table 1. The data in Table 1 show that compared with the cleavage rate of the control group (72.3±5.7%), the cleavage rate of treatment group 3 (78.9±7.1%), i.e., the group supplemented with 1 µmol / L KT5823, and the cleavage rate of treatment group 4 (78.5±6.8%), i.e., the group supplemented with 3 µmol / L KT5823, were significantly increased ( p<0 .05), and the cleavage rates of the other treatment groups were similar to those of the control group. The blastocyst rate (28.1±3.5%) of treatment group 3 (1 µmol / L KT5823 supplemented) was significantly higher than that of the control group (20.8±2.3%) and the blastocyst rate of the other treatment groups ( p <0.05). The above results show that the addition of 1 µmol / L KT5823 to the basal culture medium for in vitro maturation of oocytes has the most significant effect on improving the embryonic development rate after fertilization.

[0085] Table 1 Effects of KT5823 added to in vitro maturation medium on embryonic development after fertilization

[0086]

[0087] In the table, different superscript letters (a, b, c) in the same column indicate significant differences between the groups. p <0.05.

[0088] Example 3: Effects of Gastrodin at Different Concentrations Added to In Vitro Maturation Fluid on Embryonic Development After Fertilization

[0089] This example shows the effect of adding different concentrations of gastrodin to the basal culture medium for in vitro maturation of oocytes on the development of embryos after fertilization.

[0090] 1. Experimental design and methods

[0091] (1) Experimental design

[0092] Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h;

[0093] Treatment groups 1-7: Immature oocytes were cultured in in vitro maturation basal 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) gastrodin for 24 h.

[0094] (2) Test method

[0095] Sheep cumulus oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation culture medium of the above groupings. In vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were calculated.

[0096] 2. Test results

[0097] The statistical results of oocyte development rate are shown in Table 2. The data in Table 2 show that compared with the cleavage rate of the control group (71.3±5.3%), the cleavage rate of treatment group 5 (79.4±4.1%), i.e., the group supplemented with 10 µmol / L gastrodin, and the cleavage rate of treatment group 6 (79.2±4.5%), i.e., the group supplemented with 30 µmol / L gastrodin, were significantly increased ( p <0.05), and the cleavage rates of the other treatment groups were not significantly different from those of the control group. The blastocyst rate (29.5±4.7%) of treatment group 5 (10 µmol / L Gastrodin) was significantly higher than that of the control group (20.3±1.9%) and the blastocyst rate of the other treatment groups ( p <0.05, the blastocyst rate of treatment group 7 (11.8±1.1%) was lower than that of the control group and other treatment groups ( p <0.05). The above experimental results show that adding 10 µmol / L gastrodin to the basal culture medium for in vitro maturation of oocytes is most effective in improving embryonic development after fertilization.

[0098] Table 2 Effects of Gastrodin added to in vitro maturation fluid on embryonic development after fertilization

[0099]

[0100] In the table, different superscript letters (a, b, c) in the same column indicate significant differences between the groups. p <0.05.

[0101] Example 4: Effects of KT5823 and Gastrodin in vitro maturation fluid on embryonic development after fertilization

[0102] This example shows the effect of adding KT5823 and gastrodin in combination to the basal culture medium for in vitro maturation of oocytes on embryonic development after fertilization.

[0103] 1. Experimental design and methods

[0104] (1) Experimental design

[0105] Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h;

[0106] Treatment group 1: Immature oocytes were cultured in in vitro maturation basal medium containing only 1 µmol / L KT5823 for 24 h.

[0107] Treatment group 2: Immature oocytes were cultured in in vitro maturation basal medium containing only 10 µmol / L gastrodin for 24 h.

[0108] Treatment group 3: Immature oocytes were cultured in in vitro maturation basal medium supplemented with 1 µmol / L KT5823 and 10 µmol / L gastrodin for 24 h.

[0109] (2) Test method

[0110] Sheep cumulus oocyte complexes were collected according to the method described in Example 1, and oocytes were cultured using the in vitro maturation culture medium of the above groupings. In vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were calculated.

[0111] 2. Test results

[0112] The results of the experiment are shown in Table 3. The data in Table 3 show that the cleavage rate (79.1±4.2%) and blastocyst rate (26.9±1.8%) of treatment group 1, i.e., the group with only KT5823 added, the cleavage rate (78.8±3.3%) and blastocyst rate (28.2±1.5%) of treatment group 2, and the cleavage rate (92.9±3.1%) and blastocyst rate (52.5±3.1%) of treatment group 3, i.e., the group with combined addition of KT5823 and Gastrodin, were significantly higher than those of the control group (70.7±3.0%) and blastocyst rate (19.3±2.3%) ( p <0.05).

[0113] The cleavage rate and blastocyst rate of the group with only KT5823 increased by 8.4% and 7.6% compared with the control group.

[0114] The cleavage rate and blastocyst rate of the group supplemented with only Gastrodin increased by 8.1% and 8.9% compared with the control group.

[0115] The group with combined addition of KT5823 and Gastrodin had the highest cleavage rate and blastocyst rate, which were 22.2% and 33.2% higher than those in the control group.

[0116] The combined addition of KT5823 and gastrodin to the basic culture medium for in vitro maturation of oocytes is most effective in improving the efficiency of embryo development after fertilization.

[0117] Table 3 Effects of KT5823 and Gastrodin added to in vitro maturation fluid on embryonic development after fertilization

[0118]

[0119] In the table, different superscript letters (a, b, c) in the same column indicate significant differences between the groups. p <0.05.

[0120] Example 5: Effects of KT5823 and Gastrodin in combination with in vitro maturation fluid on oocyte maturation quality

[0121] 1. Experimental design and methods

[0122] (1) Experimental design

[0123] Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h;

[0124] Treatment group: Immature oocytes were cultured in in vitro maturation basal medium supplemented with 1 µmol / L KT5823 and 10 µmol / L gastrodin for 24 h.

[0125] (2) Oocyte maturation in vitro, in vitro fertilization and embryo culture

[0126] The sheep cumulus oocyte complexes were collected according to the method described in Example 1, and oocyte culture was performed using the in vitro maturation 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.

[0127] (3) Detection of reactive oxygen species (ROS) in oocytes

[0128] To prepare the working solution of the oxidation-sensitive fluorescent probe DCFH-DA, dilute DCFH-DA 1:1000 in serum-free culture medium to a final concentration of 10 μM. Mature oocytes from each group were digested in 0.1% hyaluronidase for 2 minutes, washed twice with DPBS, and counted using a counting chamber. Cells were then added to 500 μL of the DCFH-DA working solution and incubated in a 37°C cell culture incubator for 20 minutes. Mix by inverting the cells every 3-5 minutes to ensure thorough contact with the probe. After a 20-minute incubation, cells were washed three times with serum-free culture medium to remove any remaining DCFH-DA. Cells were then photographed using a laser confocal microscope, and reactive oxygen species (ROS) fluorescence intensity was analyzed using Image J software.

[0129] The content of reactive oxygen species (ROS) in mature oocytes of the control group and treatment group was detected according to the above method. The results are as follows: Figure 1 shown. Figure 1 The data showed that the intensity of reactive oxygen species (ROS) in mature oocytes in the treatment group (0.52±0.03%) was significantly lower than that in the control group (0.65±0.05%) ( p<0.05). This indicates that the combination of KT5823 and Gastrodin provided by the present invention can significantly reduce the content of reactive oxygen species in oocytes and prevent oxidative stress damage.

[0130] (4) Cell apoptosis detection and RNA extraction, reverse transcription and real-time fluorescence quantitative PCR

[0131] The specific process of cell apoptosis detection is as follows:

[0132] Mature oocytes from each group were collected and washed twice with 0.1% PVA in PBS (0.1% PVA-PBS). After washing, oocytes were fixed in 4% PFA solution and placed in a 4°C refrigerator for 2 hours. They were then washed three times with 0.1% PVA-PBS and permeabilized in 0.5% TritonX100-0.1% PVA-PBS for 1 hour. They were then transferred to apoptosis assay solution (C1086, Bio-Tech) and incubated in a 37°C incubator for 1 hour. Following incubation, oocytes were washed three times with 0.1% PBS-PVA and incubated in 20 μL of DAPI staining solution for 5 minutes. The oocytes were then photographed under a fluorescence microscope. The number of cells with TUNEL-positive signals was used as the number of apoptotic cells, and the number of cells with DAPI-positive signals was used as the total cell number to calculate the apoptotic rate. The apoptosis rate was calculated as follows: apoptosis rate = (number of cells with TUNEL-positive signals / number of cells with DAPI-positive signals) × 100.

[0133] The specific process of RNA extraction, reverse transcription and real-time fluorescence quantitative PCR is as follows:

[0134] Mature oocytes from each group were collected by centrifugation and placed into 1.5 mL centrifuge tubes. Excess liquid was removed and the cells were stored at -80°C. Total RNA was extracted using Trizol. The reverse transcription system and procedure were as follows.

[0135] Configure the reverse transcription system:

[0136]

[0137] Reverse transcription reaction procedure:

[0138]

[0139] After reverse transcription, the cDNA samples were stored in a −80°C refrigerator and ready for real-time fluorescence quantitative PCR.

[0140] Configure the real-time fluorescence quantitative PCR system:

[0141]

[0142] Fluorescence quantitative PCR reaction procedure:

[0143]

[0144] 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 target genes were calculated using the method.

[0145] The mature oocytes of the control group and the treatment group were tested for apoptosis, apoptosis genes and autophagy genes according to the above method. The results are as follows: Figure 2 and Figure 3 shown. Figure 2 The data showed that the apoptosis rate of mature oocytes in the treatment group (1.81±0.62%) was significantly lower than that in the control group (6.57±0.51%) ( p <0.05). At the same time, Figure 3 The data showed that the expression levels of apoptosis genes and autophagy genes in mature oocytes of the treatment group were significantly lower than those in the control group ( p <0.05). This indicates that the combination of KT5823 and Gastrodin provided by the present invention can effectively prevent the damage of apoptosis and autophagy reactions to oocytes and protect the activity of oocytes.

[0146] (5) Immunofluorescence staining

[0147] Blastocysts were fixed with 4% paraformaldehyde for 1 hour, washed three times with 0.1% PVA-PBS, permeabilized in 0.5% Triton X100-PBS for 1 hour, and then washed again with 0.1% PVA-PBS. Embryos were incubated with primary antibodies against SOX2 and CDX2 overnight. Following the primary antibody incubation, the embryos were washed three times and incubated with secondary antibodies for 1 hour at room temperature in the dark. Embryos were then transferred to mounting medium containing 3 μL of DAPI dye and incubated at room temperature in the dark for 5 minutes. After mounting, the sections were observed using an inverted fluorescence microscope. The number of SOX2- and CDX2-positive cells was counted. SOX2-positive cells were identified as inner cell mass (ICM) cells, while CDX2-positive cells were identified as trophoblast (TE) cells. The ICM / TE ratio was calculated.

[0148] The number of inner cell mass cells (ICM) and trophoblast cells (TE) in the embryos of the control and treatment groups after fertilization of mature oocytes was detected according to the above method. The results are as follows: Figure 4 The data showed that the ICM / TE ratio of embryos after fertilization of mature oocytes in the treatment group (57.32±4.08%) was significantly higher than that in the control group (32.40±2.57%) ( p<0.05). The ICM / TE ratio is one of the important criteria for evaluating embryo quality, indicating that the combination of KT5823 and Gastrodin provided by the present invention can effectively improve the embryo quality after oocyte fertilization.

[0149] (5) Glutathione (GSH) content detection

[0150] Mature oocytes were incubated with 5 µM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC, Invitrogen) for 30 minutes. Oocytes were then washed three times with 0.1% PVA-PBS, and the fluorescence intensity of each oocyte was analyzed. Images were taken using a laser confocal microscope, and glutathione (GSH) fluorescence intensity was analyzed using Image J software.

[0151] The glutathione (GSH) levels of mature oocytes in the control and treatment groups were detected according to the above method. The results are as follows: Figure 5 The data showed that the glutathione (GSH) content in mature oocytes in the treated group (67.45±5.04%) was significantly higher than that in the control group (51.26±3.59%) (p<0.05). Glutathione (GSH) is an antioxidant, indicating that the combination of KT5823 and gastrodin provided by the present invention has an ideal effect on enhancing the antioxidant capacity of oocytes.

[0152] In summary, by detecting a series of oocyte quality evaluation indicators, the composition, culture medium, product and method provided by the present invention for promoting the in vitro maturation of immature oocytes can significantly improve the quality of oocyte maturation and increase the development rate of mature oocytes after fertilization, which has practical value for optimizing the application of oocyte in vitro maturation technology in animal embryo production and human assisted reproduction.

[0153] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0154] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. The culture medium or a kit containing the culture medium is used for at least one of the following purposes, wherein the use is for non-diagnostic treatment purposes: Improve the maturation rate of oocytes in vitro; Improve the development rate of oocytes in vitro; Increased glutathione content in oocytes in vitro; Reduced reactive oxygen species levels in oocytes in vitro; Reduce the level of oocyte apoptosis in vitro; The culture medium consists of KT5823, gastrodin and basal culture medium; the final concentration of KT5823 in the culture medium is 1µmol / L; the final concentration of gastrodin in the culture medium is 10µmol / L.

2. The use according to claim 1, characterized in that The basic culture medium includes: 80-95% TCM199 culture medium, 8-12 IU / mL pregnant mare serum gonadotropin, 8-12 IU / mL luteinizing hormone, 1-20 ng / mL estrogen, 95-105 IU / mL penicillin, 95-105 μg / mL streptomycin, 5-20% follicular fluid, 0.4-0.6 mg / mL glucose, 1.8-2.3 mg / mL sodium bicarbonate, 0.04-0.08 mg / mL sodium pyruvate, and 0.4-0.7 mg / mL cysteine.

3. The use according to claim 1, characterized in that The oocyte is derived from a mammal.

4. The use according to claim 3, characterized in that The mammal includes at least one of mice, pigs, cows, horses, sheep, humans, and other non-human primates.

5. A method for promoting in vitro maturation of immature oocytes or improving the quality of in vitro maturation of oocytes, wherein the method is used for non-diagnostic treatment purposes, characterized in that: include: Performing in vitro culture treatment on mammalian immature oocytes using at least one of a culture medium or a kit containing the culture medium; The culture medium consists of KT5823, gastrodin and basal culture medium; the final concentration of KT5823 in the culture medium is 1µmol / L; the final concentration of gastrodin in the culture medium is 10µmol / L.

6. The method according to claim 5, wherein The basic culture medium includes: 80-95% TCM199 culture medium, 8-12 IU / mL pregnant mare serum gonadotropin, 8-12 IU / mL luteinizing hormone, 1-20 ng / mL estrogen, 95-105 IU / mL penicillin, 95-105 μg / mL streptomycin, 5-20% follicular fluid, 0.4-0.6 mg / mL glucose, 1.8-2.3 mg / mL sodium bicarbonate, 0.04-0.08 mg / mL sodium pyruvate, and 0.4-0.7 mg / mL cysteine.

7. The method according to claim 5, 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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