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

Abstract

The invention relates to the technical field of biology, and provides a composition, a culture solution, a product and a method for promoting in-vitro maturation of immature oocytes. The composition is prepared from KT5823 (KT5823) and Gastrodin (Gastrodin). The composition can reduce the proinflammatory factor level of oocytes and relieve the toxic effect of inflammatory response and autophagy response on the oocytes. Meanwhile, the active oxygen level can be effectively reduced, the content of glutathione serving as an antioxidant substance is increased, and damage of oxidative stress to oocytes is relieved. Through the synergistic effects, the composition obviously reduces the apoptosis rate of oocytes, obviously improves the in-vitro maturation quality of the oocytes, and greatly improves 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, a culture medium, a product and a method for promoting in vitro maturation of immature oocytes. Background Art

[0002] In vitro embryo production (IVP) technology can quickly achieve the expansion of excellent varieties and accelerate the breeding process of new varieties. It is an embryo engineering technology with great industrial value. At present, the world's well-known multinational breeding companies such as ABS, SEMEX, and Alta have established independent branches or departments specializing in the research and development of IVP technology. IVP technology includes steps such as in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos. In vitro maturation of oocytes is the most important link affecting the efficiency of IVP technology. The poor quality of in vitro maturation of oocytes is a huge problem faced by IVP technology, which results in 60% to 90% of oocytes not being effectively utilized. In addition, in vitro maturation of oocytes is also an important method for human assisted reproduction, which has significant advantages in reducing the use of hormones, reducing treatment costs, simplifying treatment processes, and improving safety and chances of pregnancy. However, the development rate of human oocytes after in vitro maturation is only 10-30%, and there has been no significant improvement in 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 technical personnel in this field urgently 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 KT5823 and Gastrodin can improve the quality of oocyte maturation in vitro, reduce the level of reactive oxygen and inflammatory response in oocytes, and effectively improve the oocyte 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: KT5823 and gastrodin. After a large number of experiments, the inventors found that the use of KT5823 and gastrodin composition can improve the quality of oocyte maturation in vitro. The composition 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, increase the content of antioxidant glutathione, and reduce the damage of oxidative stress to oocytes. Through these synergistic effects, the composition significantly reduces the apoptosis rate of oocytes, significantly improves the quality of in vitro maturation of oocytes, and improves 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, 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 the oocyte development rate is greatly improved by more than 30% when the culture solution of this combination is used for in vitro maturation of oocytes, 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 medium described in the second 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 and reduce the damage of oxidative stress to oocytes. Thus, the product can promote the in vitro maturation of oocytes and improve the development rate of mature oocytes after fertilization. In addition, KT5823 and Gastrodin are both safe and reliable ingredients, which ensure the safety of the product during use. It can not only be applied to the in vitro maturation of oocytes 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: 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 with immature oocytes of mammals. As mentioned above, the composition of the present invention can significantly reduce the inflammatory response, autophagic 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 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 The present invention relates to the effect of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation fluid on the level of reactive oxygen species (ROS) in mature oocytes, wherein: A is the reactive oxygen species (ROS) staining of mature oocytes in the control group and the treatment group; B is the statistical results of reactive oxygen species (ROS) levels in mature oocytes of the control group and the treatment group.

[0012] Figure 2 The invention relates to the effect of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation solution on apoptosis of mature oocytes according to an embodiment of the invention, wherein: A is the TUNEL staining of mature oocyte apoptosis in the control group and treatment group; B is the statistics of apoptosis rate of mature oocytes in the control group and the treatment group.

[0013] Figure 3 The present invention relates to the effect of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation fluid on the expression levels of apoptosis genes and autophagy genes in mature oocytes, wherein: A is the fluorescence quantitative PCR results of apoptosis genes in mature oocytes of the control group and the treatment group; B is the results of fluorescence quantitative PCR of autophagy genes in mature oocytes of the control group and the treatment group.

[0014] 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: A is the immunofluorescence staining of SOX2 and CDX2 in mature oocytes and in vitro fertilized embryos in the control group and the treatment group; B is the statistical proportion of trophoblast cells (CDX2 positive) and inner cell mass cells (SOX2 positive) in mature oocytes in vitro fertilized embryos in the control group and the treatment group.

[0015] Figure 5The present invention relates to the effect of adding KT5823 and Gastrodin in combination to oocyte in vitro maturation fluid on the level of glutathione (GSH) in mature oocytes, wherein: A is the glutathione staining image of mature oocytes in the control group and the treatment group; B is the statistical results of glutathione levels in mature oocytes of the control group and the treatment group. DETAILED DESCRIPTION

[0016] 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.

[0017] 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.

[0018] "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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] Protein kinase G (PKG) is a serine / threonine protein kinase that is expressed in most somatic tissues. PKG is involved in the regulation of a variety of physiological and pathological processes, such as bone remodeling, cardiac fibrosis, memory formation, nerve damage repair, tumorigenesis, etc. The PKG inhibitor KT5823 has the effects of alleviating dopamine neuron degeneration and death, reducing lipid oxidation levels in adipocytes, inhibiting cancer cell metastasis, and alleviating inflammatory responses.

[0023] Gastrodin is a natural active compound extracted from the dried root of Gastrodia elata, an orchid plant. Gastrodin has significant nervous system protection, maintaining the dynamic balance of the nervous system; sedative, hypnotic and anti-anxiety effects; improving hemodynamics, cardiovascular and cerebrovascular protection; regulating bone metabolism; inhibiting inflammatory signals and scavenging free radicals, protecting cells from oxidative stress damage.

[0024] The inventors found in the experiment that the combined use of KT5823 and gastrodin has a significant promoting effect on the in vitro maturation of oocytes. This combination not only significantly reduces the level of pro-inflammatory factors in oocytes, reduces the toxic effects of inflammatory and autophagic reactions on oocytes, but also effectively reduces the level of reactive oxygen species and increases the content of the antioxidant glutathione, thereby reducing the damage of oxidative stress to oocytes. Through the synergistic effect, KT5823 and gastrodin significantly reduced the apoptosis rate of oocytes, thereby improving the in vitro maturation quality of oocytes. In in vitro fertilization experiments, the development rate of oocytes was also significantly improved. This strategy is important for exploring the mechanism of in vitro maturation of oocytes in the future and further developing technologies to promote the quality of in vitro maturation of oocytes. 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.

[0025] 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: KT5823 and gastrodin. After a large number of experiments, the inventors found that the KT5823 and gastrodin composition of the present invention can promote the quality of in vitro maturation of oocytes and improve the development rate of mature oocytes after fertilization. The composition 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 and reduce the damage of oxidative stress to oocytes. Through these synergistic effects, the composition significantly reduces the apoptosis rate of oocytes, significantly improves the quality of in vitro maturation of oocytes, and improves the development rate of oocytes after in vitro fertilization.

[0026] In some embodiments of the present invention, the molar ratio or molar concentration ratio of KT5823 and 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 a range composed of any of the above numerical values. Thus, by making the molar ratio of KT5823 and gastrodin (Gastrodin) in 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 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, increase the content of glutathione, reduce the damage of oxidative stress to oocytes, and significantly reduce the apoptosis rate of oocytes.

[0027] 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, 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 the oocyte development rate is greatly improved by more than 30% when the culture solution of this combination is used for in vitro maturation of oocytes, which is much higher than the effect of the prior art.

[0028] 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, 0.4-0.7 mg / mL cysteine.

[0029] According to an embodiment of the present invention, the basic 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.

[0030] In some embodiments of the present invention, the final concentration of KT5823 in the culture medium is 0.1 µmol / L-10µmol / L. For example, it can be 0.1µmol / L, 0.3µmol / L, 1µmol / L, 3µmol / L, 5µmol / L, 10µmol / L, etc., or it can be a range composed of any of the above values. Thus, by making the final concentration of KT5823 within the above range, the culture medium can effectively reduce the level of inflammatory factors in oocytes when culturing oocytes in vitro, and prevent the damage of inflammatory response to oocytes.

[0031] In some embodiments of the present invention, the final concentration of the 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, etc., or it can be a range composed of any of the above values. Therefore, by making the final concentration of gastrodin in the above range, the level of active oxygen 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.

[0032] 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 medium described in the second 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, reduce the damage of oxidative stress to oocytes, and significantly reduce the apoptosis rate of oocytes. Thus, the product can promote the quality of in vitro maturation of oocytes and improve the development rate of mature oocytes after fertilization. In addition, KT5823 and Gastrodin are both safe and reliable ingredients, which ensure the safety of the product during use. It can not only be applied to the in vitro maturation of oocytes 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.

[0033] In some embodiments of the present invention, the product comprises a kit or a medicament.

[0034] In some embodiments of the present invention, the product is a drug, and the product may further include a pharmaceutically acceptable carrier or excipient.

[0035] 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.

[0036] 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.

[0037] 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: improving the in vitro maturation rate of oocytes; improving the oocyte development rate; reducing the level of active oxygen in oocytes; increasing the glutathione content in oocytes; reducing the level of inflammatory factors in oocytes; and reducing the level of oocyte apoptosis.

[0038] In some embodiments of the present invention, the oocyte is derived from a mammal.

[0039] 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.

[0040] method In the fifth aspect of the present invention, the present invention proposes a method for promoting 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 and reduce the damage of oxidative stress to oocytes. 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.

[0041] 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 and treat 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 and reduce the damage of oxidative stress to oocytes. 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.

[0042] In some embodiments of the present invention, the oocyte is derived from a mammal.

[0043] 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.

[0044] 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.

[0045] 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. Exemplarily, the time of the oocyte culture treatment of the present invention can be 12-48 hours.

[0046] 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.

[0047] Example 1: Oocyte in vitro maturation, in vitro fertilization and embryo culture 1. Oocyte maturation in vitro Ovary was collected from slaughterhouse, placed in 30-35℃ saline, and transported back to the laboratory within 2 hours. After washing with saline three times, the follicle was punctured with a disposable syringe with a 20G needle containing 5mL of egg aspiration solution to release the cumulus oocyte complex. The oocyte cumulus oocyte complex with uniform oocyte cytoplasm and more than 3 layers of cumulus cells was collected under a stereoscope. The selected oocyte cumulus oocyte complex was washed three times with egg aspiration solution, and then washed three times with oocyte in vitro maturation basal culture medium pre-equilibrated in the incubator for 3 hours, and transferred to four-well plates containing 600 μL of different in vitro maturation culture medium and 300 μL of mineral oil for culture. Each well contained 30-35 oocyte cumulus oocyte complexes, and then the four-well plates were placed in a saturated humidity incubator with 5% CO2 and 38.5℃ for 22-24h.

[0048] Egg aspiration fluid composition: 49mL TCM199 culture medium + 1mL FBS (fetal bovine serum).

[0049] The components of the basic culture medium for in vitro maturation of oocytes are 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.

[0050] 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.

[0051] (2) In vitro fertilization The sheep cumulus oocyte complex matured in vitro for 22-24 hours was placed in 0.5% hyaluronidase, and most of the cumulus cells were removed by gentle and repeated blowing. The mature oocytes were washed three times with fertilization solution to obtain the mature oocytes, which were transferred into the fertilization solution that had been pre-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.

[0052] The frozen sperm was taken out of liquid nitrogen and thawed quickly in a 38°C water bath. The thawed sperm was then transferred to 600 μL of sperm upstream solution (purchased from IVF Bioscience) and incubated in an incubator for 30 minutes to allow the sperm to fully float. After that, 100 μL of supernatant was taken and added to a four-well plate containing mature oocytes, and the sperm and eggs were incubated for 19-21 hours. The fertilization conditions were saturated humidity, 38.5°C, 5% CO2, and 95% air.

[0053] The fertilization fluid was configured as follows: mSOFaa culture medium + 2% bovine serum by volume + 100 μg / mL streptomycin + 100 IU / mL penicillin. The mSOFaa culture medium was prepared as follows: 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% BME amino acid solution by volume, and 1% MEM non-essential amino acid solution by volume per 1 L. All reagents were purchased from Sigma-Aldrich.

[0054] (3) Embryo culture After 19-21 hours of co-incubation of sperm and eggs, aspirate the oocytes and gently blow to remove residual cumulus cells and sperm. Wash them three times with mSOFaa culture medium, transfer them to mSOFaa culture medium that has been equilibrated in the incubator for more than 2 hours, and place them in different wells of a four-well plate. Culture them in a saturated humidity incubator at 38.5°C, 5% O2, 5% CO2, and 90% N2. After 48 hours of culture, the cleavage rate was counted, and the culture was continued until the 7th day, and the blastocyst rate was counted.

[0055] Example 2: Effects of adding different concentrations of KT5823 to in vitro maturation fluid on embryonic development after fertilization 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.

[0056] 1. Experimental design and experimental methods (1) Experimental design Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h; 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.

[0057] (2) Test methods The sheep cumulus oocyte complex was collected according to the method described in Example 1, and the oocytes were cultured using the in vitro maturation culture medium of the above grouping. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were counted.

[0058] 2. Test results 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, i.e., the group supplemented with 1 µmol / L KT5823, 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 adding 1 µmol / L KT5823 to the basic culture medium for oocyte in vitro maturation can significantly improve the embryonic development rate after fertilization.

[0059] Table 1 Effects of adding KT5823 to in vitro maturation medium on embryonic development after fertilization

[0060] In the table, different superscript letters (a, b, c) in the same column indicate significant differences between the groups. p <0.05.

[0061] Example 3: Effects of adding different concentrations of Gastrodin to in vitro maturation fluid on embryonic development after fertilization This example shows the effect of adding different concentrations of gastrodin to the basic culture medium for in vitro maturation of oocytes on the development of embryos after fertilization.

[0062] 1. Experimental design and experimental methods (1) Experimental design Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h; 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) of gastrodin for 24 h.

[0063] (2) Test methods The sheep cumulus oocyte complex was collected according to the method described in Example 1, and the oocytes were cultured using the in vitro maturation culture medium of the above grouping. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were counted.

[0064] 2. Test results 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 with 10 µmol / L gastrodin, and treatment group 6 (79.2±4.5%), i.e., the group with 30 µmol / L gastrodin, were significantly increased ( p <0 .05, and there was no significant difference in cleavage rate between the other treatment groups and 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 other treatment groups ( p <0.05, the blastocyst rate of treatment group 7 (11.8±1.1%) with 100 µmol / L gastrodin 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 basic culture medium for oocyte in vitro maturation is most effective in improving embryonic development after fertilization.

[0065] Table 2 Effects of Gastrodin added to in vitro maturation fluid on embryonic development after fertilization

[0066] In the table, different superscript letters (a, b, c) in the same column indicate significant differences between the groups. p <0.05.

[0067] Example 4: Effects of adding KT5823 and Gastrodin to in vitro maturation fluid on embryonic development after fertilization This example shows the effect of adding KT5823 and gastrodin in combination to the basal culture medium for in vitro maturation of oocytes on the development of embryos after fertilization.

[0068] 1. Experimental design and experimental methods (1) Experimental design Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h; Treatment group 1: Immature oocytes were cultured in in vitro maturation basal medium containing only 1 µmol / L KT5823 for 24 h.

[0069] Treatment group 2: Immature oocytes were cultured in in vitro maturation basal medium containing only 10 µmol / L gastrodin for 24 h.

[0070] 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.

[0071] (2) Test methods The sheep cumulus oocyte complex was collected according to the method described in Example 1, and the oocytes were cultured using the in vitro maturation culture medium of the above grouping. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1, and the cleavage rate and blastocyst rate were counted.

[0072] 2. Test results 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., only adding KT5823, the cleavage rate (78.8±3.3%) and blastocyst rate (28.2±1.5%) of treatment group 2, i.e., only adding gastrodin, and the cleavage rate (92.9±3.1%) and blastocyst rate (52.5±3.1%) of treatment group 3, i.e., the 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).

[0073] The cleavage rate and blastocyst rate of the group supplemented with KT5823 alone were increased by 8.4% and 7.6% compared with the control group.

[0074] The cleavage rate and blastocyst rate of the group supplemented with gastrodin alone increased by 8.1% and 8.9% compared with the control group.

[0075] 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 of the control group.

[0076] 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 embryonic development after fertilization.

[0077] Table 3 Effects of KT5823 and Gastrodin added to in vitro maturation fluid on embryonic development after fertilization

[0078] In the table, different superscript letters (a, b, c) in the same column indicate significant differences between the groups. p <0.05.

[0079] Example 5: Effect of adding KT5823 and Gastrodin to in vitro maturation solution on the quality of oocyte maturation 1. Experimental design and experimental methods (1) Experimental design Control group: immature oocytes were cultured in vitro in the in vitro maturation basal medium for 24 h; 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.

[0080] (2) Oocyte in vitro maturation, in vitro fertilization and embryo culture The sheep cumulus oocyte complex was collected according to the method described in Example 1, and the oocyte was cultured using the in vitro maturation culture medium of the above grouping. At the same time, in vitro fertilization and embryo culture were performed according to the method described in Example 1.

[0081] (3) Detection of reactive oxygen species (ROS) in oocytes Preparation of the working solution of the oxidation-sensitive fluorescent probe DCFH-DA: DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM. The mature oocytes cultured in each group were placed in 0.1% hyaluronidase for digestion for 2 minutes, then washed twice with DPBS buffer, and the cells were counted using a counting plate. The cells were then added to 500 μL of 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 to fully contact. After incubation for 20 minutes, wash three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter. Laser confocal microscopy was then used to observe the cells, and the fluorescence intensity of reactive oxygen species (ROS) was analyzed using Image J software.

[0082] The content of reactive oxygen species (ROS) in mature oocytes of the control group and the 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 active oxygen in oocytes and avoid oxidative stress damage.

[0083] (4) Cell apoptosis detection and RNA extraction, reverse transcription and real-time fluorescence quantitative PCR The specific process of cell apoptosis detection is as follows: The mature oocytes cultured in each group were collected and washed twice with 0.1% PVA 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. They were washed three times with 0.1% PVA-PBS and transferred to 0.5% TritonX100-0.1% PVA-PBS permeabilization solution for 1 hour. They were transferred to apoptosis TUNEL detection solution (C1086, Biyuntian) and incubated in a 37 ℃ incubator for 1 hour. After incubation, the oocytes were washed three times with 0.1% PBS-PVA, incubated in 20μL DAPI staining solution for 5 minutes, and the oocytes were 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.

[0084] The specific process of RNA extraction, reverse transcription and real-time fluorescence quantitative PCR is as follows: The mature oocytes of each group were collected by centrifugation and placed in 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 were as follows.

[0085] Configure the reverse transcription system:

[0086] Reverse transcription reaction procedure:

[0087] After reverse transcription, the cDNA samples were stored in a -80°C refrigerator for real-time fluorescence quantitative PCR.

[0088] Configure the real-time fluorescence quantitative PCR system:

[0089] Fluorescence quantitative PCR reaction procedure:

[0090] 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.

[0091] The mature oocytes in 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 in 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 to oocytes and protect the activity of oocytes.

[0092] (5) Immunofluorescence staining The blastocysts were fixed with 4% paraformaldehyde for 1 h, then washed 3 times with 0.1% PVA-PBS, transferred to 0.5% Triton X100-PBS for permeabilization for 1 h, and then washed again with 0.1% PVA-PBS, incubated with SOX2 and CDX2 primary antibodies overnight, washed 3 times after the primary antibody incubation, and incubated with secondary antibodies at room temperature in the dark for 1 h. The embryos were then transferred into a mounting medium containing 3 μL DAPI dye, incubated at room temperature in the dark for 5 min, and observed under a fluorescent inverted microscope after mounting. The number of SOX2 and CDX2 positive cells was counted. SOX2 positive cells were inner cell mass cells (ICM), and CDX2 positive cells were trophoblast cells (TE), and the ICM / TE ratio was counted.

[0093] The number of inner cell mass cells (ICM) and trophoblast cells (TE) in the embryos of the mature oocytes in the control group and the treatment group was detected according to the above method. Figure 4The 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 well improve the embryo quality after oocyte fertilization.

[0094] (5) Glutathione (GSH) content detection Mature oocytes were incubated with 5µM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC, Invitrogen) for 30 minutes. Then, the oocytes were washed three times with 0.1% PVA-PBS, and the fluorescence intensity of each oocyte was examined and analyzed. Then, laser confocal microscopy was used to observe and the glutathione (GSH) fluorescence intensity was analyzed using Image J software.

[0095] The glutathione (GSH) levels of mature oocytes in the control group and treatment group were detected according to the above method. The results are as follows Figure 5 As shown. The data showed that the glutathione (GSH) content in the mature oocytes of the treatment group (67.45±5.04%) was significantly higher than that of the control group (51.26±3.59%) (p<0.05). Glutathione (GSH) is an antioxidant substance, indicating that the combination of KT5823 and gastrodin provided by the present invention has an ideal effect on enhancing the antioxidant capacity of oocytes.

[0096] In summary, by detecting a series of oocyte quality evaluation indicators, the composition, culture medium, product and method for promoting in vitro maturation of immature oocytes provided by the present invention 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 in vitro oocyte maturation technology in animal embryo production and human assisted reproduction.

[0097] 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.

[0098] 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: KT5823 and gastrodin.

2. The composition according to claim 1, characterized in that The molar ratio of the KT5823 to the gastrodin is (0.03-0.1):

1.

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 KT5823 in the culture medium is 0.1 µmol / L-10 µmol / L.

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

7. The culture solution according to claim 4, 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.

8. 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 7.

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

10. The composition of any one of claims 1 to 2, the culture solution of any one of claims 3 to 7 or the product of any one of claims 8 to 9 has at least one of the following uses: Improve the rate of oocyte maturation in vitro; Improve the oocyte development rate; Increase oocyte glutathione content; Reduce the level of reactive oxygen species in oocytes; Reduce the level of inflammatory factors in oocytes; Reduce the level of oocyte apoptosis.

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

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

13. A method for promoting in vitro maturation of immature oocytes or improving the quality of in vitro maturation of oocytes, characterized in that: include: At least one of the composition according to any one of claims 1 to 2, the culture medium according to any one of claims 3 to 7, and the product according to any one of claims 8 to 9 is used to perform in vitro culture treatment on immature oocytes of mammals.

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

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