Application of indole derivative in promoting in-vitro maturation of oocyte and embryonic development

Abstract

The invention discloses application of a compound shown in a formula I in promoting in-vitro cultured oocyte development or in-vitro embryo development, and in the formula I, R1 is selected from halogenated alkyl; r2 is selected from H, halogen or C1-6 alkyl; n is selected from 0, 1, 2 or 3. According to the application, the expansion area of cumulus cells in the cumulus-oocyte complex can be increased; the oxidative damage of the cumulus-oocyte complex is relieved; the first polar body discharge rate of the oocytes is improved; the abnormal distribution rate of mitochondria in oocytes is reduced; the membrane potential of mitochondria in the oocyte is improved; and the cleavage rate and blastocyst formation rate of fertilized eggs can be improved, and the apoptosis rate of oocytes can be reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of indole derivatives in promoting in vitro maturation of oocytes and embryonic development. Background Technology

[0002] In vitro maturation (IVM) refers to the technique of inducing immature oocytes (usually in the germinal follicle phase, GV phase) collected from the ovary to complete meiosis I under in vitro culture conditions, expel the first polar body, and develop to metaphase II (MII phase), thereby obtaining mature oocytes capable of fertilization. IVM technology not only avoids the hormonal stimulation required for superovulation in vivo, reducing costs and animal stress, but it is also a key step in assisted reproduction and livestock embryo engineering. Its goal is to simulate the in vivo environment in vitro, enabling immature oocytes to complete meiosis and acquire the ability to fertilize and subsequently develop. Although IVM technology has been developed for decades, its efficiency and stability are still far lower than in vivo maturation.

[0003] The main reasons are as follows: First, asynchronous nuclear and cytoplasmic maturation. Although most IVM oocytes can complete nuclear maturation (extrusion of the first polar body), cytoplasmic maturation is severely delayed. Cytoplasmic maturation involves complex processes such as cytoskeleton reorganization, mitochondrial redistribution and functional activation, migration of cortical granules to the cortex, and mRNA storage and translation regulation. These are crucial for subsequent sperm implantation, pronuclear formation, and early embryonic genome activation. Second, oxidative stress damage. The in vitro culture environment lacks the natural antioxidant system found in in vivo follicular fluid, leading to the accumulation of reactive oxygen species (ROS), causing DNA damage, decreased mitochondrial membrane potential, and increased apoptosis. Third, energy metabolism disorders. Oocytes are highly dependent on mitochondria for energy, but during IVM, abnormal mitochondrial distribution (aggregation in the cytoplasm rather than the cortex) and insufficient ATP synthesis often occur, affecting spindle assembly and chromosome segregation. Fourth, epigenetic reprogramming defects. The in vitro environment may interfere with epigenetic events such as histone modification and DNA methylation, thereby affecting embryonic gene expression programs and developmental fate.

[0004] Given the significant bottlenecks in current in vitro maturation technology, there is an urgent need to develop a novel additive that is structurally stable, highly efficient, and low in toxicity to improve the quality of oocyte maturation in vitro and the developmental potential of embryos after fertilization. Summary of the Invention

[0005] In order to overcome the problems existing in the prior art, the present invention aims to provide an application of a compound in promoting in vitro maturation of oocytes and embryonic development, so as to improve the quality of in vitro maturation of oocytes, reduce oxidative stress levels, enhance mitochondrial function, and increase blastocyst formation rate.

[0006] In a first aspect, the present invention provides the use of a compound of formula I in promoting the development of oocytes in vitro or promoting the development of embryos in vitro. Formula I, R1 is selected from alkyl halogenates; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2 or 3.

[0007] In some implementations, R1 is selected from C 1-6 Haloalkyl, more preferably halomethyl or haloethyl, and even more preferably -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2 or -CH2-CF3, more preferably -CF3; and / or n is selected from 0 or 1; and / or R2 is selected from H, F, Cl, or CH3; and / or The compound of formula I is Compound 1.

[0008] In some embodiments, the oocyte is a mammalian oocyte; the mammal is a human, pig, sheep, dog, cat, or rabbit, preferably a pig or sheep; and / or The concentration of the compound of formula I is 0.1~5 μmol / L, preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L; and / or The application is to increase the expansion area of ​​cumulus cells in the cumulus-oocyte complex; and / or The expanded area of ​​the cumulus cells can be increased by 1.18 to 1.40 times, preferably by 1.2 to 1.35 times; and / or The application is to reduce the reactive oxygen species level in the cumulus-oocyte complex; and / or The reactive oxygen species (ROS) level of the oocytes is 1-20 RFU, preferably 2-15 RFU; more preferably 1.5, 2, 3, 5, 7, or 10 RFU.

[0009] In some embodiments, the application is to increase the expulsion rate of the first polar body of the oocyte; and / or The oocyte first polar body expulsion rate is increased to 1.1 to 1.30 times, preferably to 1.15 to 1.25 times, more preferably to 1.18 times, 1.20 times, 1.22 times, or 1.24 times; and / or The application is to reduce the abnormal distribution rate of mitochondria in oocytes; and / or The abnormal mitochondrial distribution rate is 14-19%, preferably 15-18%, more preferably 16% or 17%; and / or The application is to increase the membrane potential of mitochondria in oocytes; and / or The membrane potential of the mitochondria in the oocyte is 1.5 to 5, preferably 1.7 to 4.5.

[0010] In some embodiments, the application is to reduce DNA damage in oocytes; and / or The DNA damage in the oocyte is reduced to 0.2-0.75 times, preferably 0.3-0.65 times; more preferably 0.35 times, 0.4 times, 0.45 times, 0.5 times or 0.6 times; and / or The application is to reduce the incidence of abnormal spindle structures in oocytes; and / or The incidence of abnormal spindle structures in the oocytes is 10-15%, preferably 11-14%, and more preferably 12-13%.

[0011] In some embodiments, the application is to improve the cleavage rate of fertilized eggs; and / or The cleavage rate of the fertilized egg is increased to 1.1 to 1.5 times, preferably 1.15 to 1.45 times, more preferably 1.2 times, 1.25 times, 1.3 times, 1.35 times or 1.4 times; and / or The application is to improve the blastocyst formation rate of fertilized eggs; and / or The blastocyst formation rate of the fertilized egg is increased to 1.15 to 2.5 times, preferably 1.2 to 2.3 times, and more preferably 1.3 times, 1.5 times, 1.7 times, 1.9 times or 2.1 times.

[0012] Secondly, the present invention provides the use of a compound of formula I in the preparation of a medicament for promoting the development of oocytes in vitro or promoting the development of embryos in vitro. Formula I, R1 is selected from alkyl halogenates; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2 or 3.

[0013] Thirdly, the present invention provides an in vitro culture method for oocytes, wherein immature oocytes are cultured in an oocyte culture medium containing a compound of formula I; Formula I, R1 is selected from alkyl halogenates; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2, or 3; and / or The in vitro culture temperature is 36.5~39℃ and the CO2 concentration is 4~6%; preferably, the temperature is 37℃, 37.5℃, or 38.5℃; even more preferably, the CO2 concentration is 5%; and / or The concentration of the compound of formula I is 0.1~5 μmol / L, preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L; and / or The immature oocytes are either in the form of a cumulus-oocyte complex or in the form of a naked oocyte without a cumulus.

[0014] Fourthly, the present invention provides an in vitro culture medium for oocytes, wherein the culture medium is an oocyte culture medium containing a compound of formula I. Formula I, R1 is selected from alkyl halogenates; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2, or 3; Preferably, the concentration of the compound of formula I is 0.1~5 μmol / L, more preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L.

[0015] The compound of Formula I of this invention can promote the development of oocytes in vitro, such as increasing the expansion area of ​​cumulus cells in the cumulus-oocyte complex; reducing oxidative damage to the cumulus-oocyte complex; increasing the extrusion rate of the first polar body in oocytes; reducing the rate of abnormal mitochondrial distribution in oocytes; increasing the membrane potential of mitochondria in oocytes; reducing DNA damage in oocytes; and reducing the incidence of abnormal spindle structures in oocytes. The compound of Formula I of this invention can also promote in vitro embryonic development, such as increasing the cleavage rate and blastocyst formation rate of fertilized eggs, and reducing the oocyte apoptosis rate. Attached Figure Description

[0016] Figure 1 The effects of compound 1 on cumulus cell expansion in the porcine cumulus-oocyte complex; its effects on the first polar body expulsion rate and cleavage rate of porcine oocytes; and its effects on the blastocyst formation rate of porcine fertilized eggs.

[0017] Figure 2 Effects of compound 1 on reactive oxygen species levels in the porcine cumulus-oocyte complex; effects on mitochondrial distribution and membrane potential in oocytes.

[0018] Figure 3 Effects of compound 1 on the incidence of abnormal spindle structures and DNA damage in porcine oocytes.

[0019] Figure 4Effect of compound 1 on apoptosis levels in porcine oocytes.

[0020] Figure 5 Effect of compound 1 on cumulus cell expansion in sheep cumulus-oocyte complex.

[0021] Figure 6 Effects of compound 1 on the cleavage rate of sheep oocytes and on the blastocyst formation rate of sheep fertilized eggs.

[0022] Note: Compound 1 is represented as CF3-MT in the figure. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0024] <Application> This application provides the use of a compound of formula I in promoting the development of oocytes or embryos in vitro. Formula I, R1 is selected from alkyl halogenates; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2 or 3.

[0025] In some implementations, R1 is selected from C 1-6 The alkyl group is more preferably a methyl halo, ethyl halo, and even more preferably -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2 or -CH2-CF3, more preferably -CF3. In some embodiments, n is selected from 0 or 1. In some embodiments, R2 is selected from H, F, Cl or CH3. The present invention unexpectedly discovered that a trifluoromethyl group as R1 can enhance the antioxidant activity of the compound, reduce oxidative damage, thereby maintaining the stability of the cumulus-oocyte microenvironment; it can also regulate mitochondrial homeostasis, improve the spatial distribution of mitochondria in oocytes and increase mitochondrial membrane potential, which helps maintain mitochondrial function and improve energy metabolism efficiency.

[0026] In some implementations, Equation I is Compound 1.

[0027] In some embodiments, the oocyte is a mammalian oocyte; the mammal is a human, pig, sheep, dog, cat, or rabbit, preferably a pig or sheep.

[0028] In some embodiments, the pigs include Duroc pigs, Landrace pigs, Yorkshire pigs, or local breeds.

[0029] In some embodiments, the sheep includes sheep, goats, Hu sheep, or local breeds.

[0030] In some embodiments, the cattle include dairy cattle, beef cattle, Holstein cattle, Simmental cattle, or local breeds.

[0031] In some embodiments, the concentration of the compound of formula I is 0.1~5 μmol / L, preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L; in some embodiments, the concentration of the compound of formula I can be 0.5 μmol / L, 0.9 μmol / L, 1.1 μmol / L, 1.3 μmol / L, 1.5 μmol / L or 2 μmol / L.

[0032] In some implementations, the Formula I compound is provided in the form of a kit, culture medium, or drug.

[0033] The applications of the compound of Formula I are described in the following sections: [Application of Formula I in promoting the maturation of oocytes in vitro] and [Application of Formula I in promoting in vitro embryonic development]. The details are as follows.

[0034] [Application of Formula I in promoting the maturation of oocytes in vitro] Application of a compound of formula I in promoting the maturation of oocytes in vitro.

[0035] In some embodiments, the application is an application to promote in vitro maturation of oocytes. In some embodiments, the application is an application to promote cytoplasmic maturation of oocytes.

[0036] Compound of Formula I can promote the expansion of cumulus cells in the cumulus-oocyte complex. In some embodiments, the application is to increase the expansion area of ​​cumulus cells in the cumulus-oocyte complex.

[0037] In some embodiments, the expanded area of ​​the cumulus cells can be 46,000 to 54,000 μm. 2 Preferably 48000~52000μm 2 More preferably, it is 49000~51000μm 2 In some embodiments, the expanded area of ​​the cumulus cells is greater than 50,000 μm. 2 ; In some embodiments, the expanded area of ​​the cumulus cells can be increased to 1.18 to 1.40 times, preferably 1.2 to 1.35 times, and more preferably 1.23 times, 1.25 times, 1.26 times, 1.28 times or 1.30 times.

[0038] Compound of Formula I can promote meiosis I in oocytes and the expulsion of the first polar body, thereby promoting nuclear maturation of the oocyte. In some embodiments, the application is to increase the expulsion rate of the first polar body in oocytes. In some embodiments, the expulsion rate of the first polar body in oocytes is 75-95%, preferably 77-92%, more preferably 78-90%, and even more preferably 79-88%. In some embodiments, the expulsion rate of the first polar body in oocytes is increased by 1.1-1.30 times, preferably by 1.15-1.25 times, and more preferably by 1.18 times, 1.20 times, 1.22 times, or 1.24 times.

[0039] Compound of Formula I can alleviate oxidative damage to the cumulus-oocyte complex. In some embodiments, the application is to reduce the reactive oxygen species (ROS) level in the cumulus-oocyte complex. The ROS level results are expressed as relative fluorescence intensity (RFU). In some embodiments, the ROS level of the cumulus-oocyte complex is 1 to 20 RFU, preferably 2 to 15 RFU; more preferably 1.5, 2, 3, 5, 7, or 10 RFU.

[0040] Compound I can improve the spatial distribution of mitochondria in oocytes, reduce abnormal mitochondrial aggregation, and significantly increase mitochondrial membrane potential, thereby maintaining mitochondrial function in oocytes and improving energy metabolism efficiency. In some embodiments, the application is to reduce the rate of abnormal mitochondrial distribution in oocytes. In some embodiments, the abnormal mitochondrial distribution rate is 14-19%, preferably 15-18%, more preferably 16% or 17%. In some embodiments, the application is to increase the membrane potential of mitochondria in oocytes. In some embodiments, the mitochondrial membrane potential is determined by measuring the red / green fluorescence ratio using the JC-1 staining method. In some embodiments, the mitochondrial membrane potential in oocytes is 1.5-5, preferably 1.7-4.5. In some embodiments, the mitochondrial membrane potential in oocytes is 2, 2.2, 2.5, 2.8, 3, 3.3, 3.5, 3.7, or 4.0.

[0041] The compound of Formula I has a protective effect of reducing DNA damage and maintaining genome stability. In some embodiments, the application is to reduce DNA damage in oocytes. In some embodiments, the degree of reduction in DNA damage in oocytes is determined by detecting fluorescence intensity using γH2AX immunofluorescence. In some embodiments, the fluorescence intensity is 4-15, preferably 6-13; more preferably 7, 8, 9, 10, 11 or 12. In some embodiments, the DNA damage in oocytes is reduced to 0.2-0.75 times, preferably 0.3-0.65 times; more preferably 0.35 times, 0.4 times, 0.45 times, 0.5 times or 0.6 times.

[0042] Compound of Formula I can improve the maturation quality of oocytes. In some embodiments, the application is to reduce the incidence of abnormal spindle structures in oocytes. In some embodiments, the incidence of abnormal spindle structures is detected by immunofluorescence staining. The detection targets are the spindle morphology and chromosome arrangement of oocytes in metaphase II (MII) of meiosis. In some embodiments, the incidence of abnormal spindle structures in oocytes is 10-15%, preferably 11-14%, and more preferably 12-13%.

[0043] In some embodiments, the application is to reduce the oocyte apoptosis rate. In some embodiments, the effect of reducing oocyte apoptosis is confirmed by Annexin V-FITC staining and / or TUNEL assay. In some embodiments, compared with the untreated group, the oocyte apoptosis rate is reduced to 0.2-5%, preferably 1-4%; in some embodiments, the oocyte apoptosis rate is reduced to 0.5%, 1.1%, 1.3%, 1.5%, 1.8%, and 2.1%.

[0044] [Application of Formula I in Promoting In Vitro Embryo Development] Application of a compound of formula I in promoting in vitro embryonic development.

[0045] Compound of Formula I promotes early embryonic development. In some embodiments, the application is to increase the cleavage rate of fertilized eggs. In some embodiments, the cleavage rate of the fertilized eggs is 65-83%, preferably 67-81%, more preferably 69-79%, and even more preferably 71%, 73%, 75%, or 77%. In some embodiments, the cleavage rate of the fertilized eggs is increased by 1.1-1.5 times, preferably 1.15-1.45 times, more preferably 1.2 times, 1.25 times, 1.3 times, 1.35 times, or 1.4 times.

[0046] Compound of Formula I can increase the proportion of fertilized eggs that develop to the blastocyst stage and increase the total number of cells in the blastocyst. In some embodiments, the application is to increase the blastocyst formation rate of fertilized eggs. In some embodiments, the blastocyst formation rate of the fertilized eggs is 26-40%, preferably 28-38%, more preferably 30%, 32%, 34%, or 36%. In some embodiments, the blastocyst formation rate of the fertilized eggs is increased by 1.15-2.5 times, preferably 1.2-2.3 times, more preferably 1.3 times, 1.5 times, 1.7 times, 1.9 times, or 2.1 times.

[0047] <Applications in Drug Preparation> This application provides the use of the compound of formula I in the preparation of a medicament for promoting the maturation of cultured oocytes or promoting the development of embryos in vitro.

[0048] The applications defined above will not be repeated here.

[0049] <In vitro culture methods for oocytes> This application provides a method for in vitro culture of oocytes, in which immature oocytes are cultured in an oocyte culture medium containing a compound of formula I.

[0050] In some embodiments, the in vitro culture temperature is 36.5~39°C and the CO2 concentration is 4~6%; preferably, the temperature is 37°C, 37.5°C, or 38.5°C; and even more preferably, the CO2 concentration is 5%.

[0051] In some embodiments, the concentration of the compound of formula I is 0.1~5 μmol / L, preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L.

[0052] In some embodiments, the immature oocyte is in the form of a cumulus-oocyte complex or a naked oocyte without a cumulus.

[0053] In some embodiments, the oocyte culture medium contains a basal culture medium, and preferably, also contains biological supplements, hormones, growth factors, and antibiotics.

[0054] The basal culture medium is M199, TCM-199, DMEM, DMEM / F12, α-MEM, Ham's F12, or RPMI-1640, with M199 being preferred.

[0055] The biological supplement is fetal bovine serum, newborn calf serum, porcine follicular fluid, or ovine follicular fluid, preferably porcine follicular fluid or fetal bovine serum. The concentration of the biological supplement is 8-12%, preferably 10%.

[0056] The hormone is one or more of serum gonadotropins, human chorionic gonadotropins, follicle-stimulating hormone, luteinizing hormone, or estradiol.

[0057] The growth factor is selected from one or more of epidermal growth factor (EGF), leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), and insulin-like growth factor (IGF).

[0058] The antibiotic is selected from one or more of β-lactam antibiotics and aminoglycoside antibiotics, preferably including but not limited to one or more of penicillin, streptomycin, gentamicin, and kanamycin; more preferably, it contains at least one or two of penicillin and streptomycin.

[0059] In some embodiments, the oocyte culture medium further comprises basal culture medium, cysteine, sodium pyruvate, growth factors, follicular fluid, insulin, serum gonadotropins, human chorionic gonadotropin (hCG), and antibiotics. The follicular fluid is derived from mammals, preferably humans, pigs, sheep, dogs, cats, and rabbits. In some embodiments, the oocyte culture medium further comprises basal culture medium, 1 mg / mL cysteine, 0.44 mg / mL sodium pyruvate, 10 ng / mL epidermal growth factor, 40 ng / mL leukemia inhibitory factor, 20 ng / mL fibroblast growth factor, 20 ng / mL insulin-like growth factor, 10% porcine follicular fluid (v / v), 50 ng / mL insulin, 10 IU / mL serum gonadotropins, 10 IU / mL hCG, 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0060] In some embodiments, the oocyte culture medium is a porcine oocyte culture medium.

[0061] In some embodiments, the oocyte culture medium further comprises basal culture medium, fetal bovine serum, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, sodium pyruvate, growth factors, and antibiotics. In some embodiments, the oocyte culture medium further comprises basal culture medium, 10% fetal bovine serum, 10 μg / mL FSH, 10 μg / mL LH, 1 μg / mL estradiol, 2 mM sodium pyruvate, 100 ng / mL epidermal growth factor, 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0062] In some embodiments, the oocyte culture medium is a sheep oocyte culture medium.

[0063] The definition of the compound of formula I is as described above.

[0064] In some embodiments, the in vitro culture method for oocytes can promote the development of in vitro cultured oocytes. The definition of promoting the development of in vitro cultured oocytes is as described above.

[0065] <In vitro culture medium for oocytes> This application provides an in vitro culture medium for oocytes, wherein the culture medium is an oocyte culture medium containing a compound of formula I.

[0066] In some embodiments, the oocyte culture medium can promote the maturation of oocytes cultured in vitro.

[0067] In some embodiments, the oocyte culture medium can promote in vitro embryonic development.

[0068] The definitions of the compound of Formula I, the oocyte culture medium, and the definition of promoting the development of oocytes in vitro are as described above.

[0069] In some embodiments, the oocyte culture medium can promote in vitro maturation of oocytes. In some embodiments, the oocyte culture medium can increase the expansion area of ​​cumulus cells in the cumulus-oocyte complex. In some embodiments, the oocyte culture medium can reduce oxidative damage to the cumulus-oocyte complex. In some embodiments, the oocyte culture medium can increase the oocyte first polar body extrusion rate. In some embodiments, the oocyte culture medium can reduce the rate of abnormal mitochondrial distribution in oocytes. In some embodiments, the oocyte culture medium can increase the membrane potential of mitochondria in oocytes. In some embodiments, the oocyte culture medium can reduce DNA damage in oocytes. In some embodiments, the oocyte culture medium can reduce the incidence of abnormal spindle structures in oocytes.

[0070] In some embodiments, the oocyte culture medium can increase the cleavage rate or blastocyst formation rate of fertilized eggs. In some embodiments, the oocyte culture medium can reduce the oocyte apoptosis rate.

[0071] Preparation Example 1: Preparation of Compound 1 N-(2-(5-methoxy-2-(trifluoromethyl)-1H-indol-3-yl)ethyl)acetamide Compound 1 Melatonin (464.6 mg, 2 mmol), sodium trifluoromethyl sulfinate (632.3 mg, 4 mmol), ferric chloride (32.4 mg, 0.2 mmol), and potassium fluoride (58.1 mg, 1 mmol) were added sequentially to a Schlenk tube. N,N-dimethylacetamide (10.0 mL) was injected under an argon atmosphere, and the mixture was stirred at room temperature while tert-butyl hydroperoxide (70% aqueous solution, 10 mmol) was slowly added dropwise. The reaction mixture was then heated to 85 °C and stirred continuously for 2 hours. After the reaction was complete, the solvent was removed by vacuum distillation. The crude product was purified by rapid column chromatography (ethyl acetate / petroleum ether) to give compound 1, with a yield of 312.3 mg, or 52%.

[0072] Example 1: Acquisition and sorting of cumulus-oocyte complexes (COCs) In this embodiment, the ovaries of healthy sows of reproductive age were selected as the source of oocytes. The ovaries were harvested immediately after slaughter and placed in physiological saline containing antibiotics at 37°C, then transported to the laboratory within 1 hour. Follicular fluid of 3-6 mm was aspirated from the ovaries using an 18G needle. Under a stereomicroscope, cumulus-oocyte complexes with dense, intact, and homogeneous follicular cells were selected from the follicular fluid for subsequent in vitro maturation (IVM) experiments.

[0073] Example 2: Experiment on the effect of compound 1 on cumulus cell expansion during in vitro oocyte maturation The cumulus-oocyte complexes from Example 1 were divided into four groups: ① Control group (0 μmol / L compound 1) ② Group 1 of 0.1 μmol / L compounds ③ Group 1 of 1 μmol / L compounds ④ Group 1 of 10 μmol / L compounds Each group of cumulus-oocyte complexes was placed in a porcine in vitro maturation culture system, with the corresponding concentration of Compound 1 added, and cultured at 38.5℃ and 5% CO2 for 44 h, maintaining the corresponding concentration of Compound 1 throughout the culture. After maturation culture, each group of cumulus-oocyte complexes was imaged using a fluorescence inverted microscope, and the cumulus cell expansion area was measured using ImageJ image analysis software.

[0074] The porcine in vitro maturation culture system consisted of M199 culture medium (GIBCO, C11150500BT) supplemented with 1 mg / mL cysteine, 0.44 mg / mL sodium pyruvate, 10 ng / mL epidermal growth factor, 40 ng / mL leukemia inhibitory factor, 20 ng / mL fibroblast growth factor, 20 ng / mL insulin-like growth factor, 10% porcine follicular fluid, 10 IU / mL serum gonadotropin, 10 IU / mL human chorionic gonadotropin, 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0075] Experimental results are as follows Figure 1 As shown. The area of ​​cumulus cell expansion in the control group was 40899 ± 1268 μm. 2 The area of ​​cumulus cells in group 1 with 0.1 μmol / L compound was 45978 ± 1342 μm. 2 The concentration was slightly higher than that in the control group; the area of ​​cumulus cell expansion in group 1 (1 μmol / L compound) was 50307 ± 1268 μm. 2 Significantly higher than the control group ( P <0.05), and the cumulus-oocyte complex showed a more obvious radial expansion morphology; the area of ​​cumulus cells in group 1 with 10 μmol / L compound was 40466 ±1470 μm. 2 There was no significant difference compared to the control group.

[0076] The results indicate that compound 1 at a concentration of 1 μmol / L can significantly promote the expansion of cumulus cells, compound 1 at a concentration of 0.1 μmol / L can promote the expansion of cumulus cells to a certain extent, while excessively high concentrations (10 μmol / L) do not show any promoting effect.

[0077] Example 3: Experiment on the effect of compound 1 on the expulsion rate of the first polar body of oocytes Four groups of mature cumulus-oocyte complexes were prepared using the same method as in Example 2.

[0078] Cumulus cells were removed from the cumulus-oocyte complex in each group to obtain oocytes. The first polar body extrusion rate (PBE) of the oocytes was then observed and calculated under an inverted microscope.

[0079] Experimental results are as follows Figure 1As shown. The first polarity expulsion rate of the control group was 71.60 ± 0.51%. The first polarity expulsion rate of the 0.1 μmol / L compound 1 group was 72.00 ± 0.45%, which was not significantly different from the control group. The first polarity expulsion rate of the 1 μmol / L compound 1 group was 83.14 ± 0.40%, which was significantly higher than that of the control group. The first polarity expulsion rate of the 10 μmol / L compound 1 group was 72.10 ± 0.11%, which was not significantly different from that of the control group.

[0080] The above results indicate that a concentration of 1 μmol / L of compound 1 significantly promotes the completion of meiosis I and the expulsion of the first polar body from oocytes, while concentrations of 0.1 μmol / L and 10 μmol / L did not produce a significant promoting effect. A concentration of 1 μmol / L of compound 1 helps improve the efficiency of oocyte nuclear maturation.

[0081] Example 4: Effect of compound 1 on the cleavage rate of fertilized eggs Four groups of oocytes were prepared using the same method as in Example 3.

[0082] Oocytes from each group were transferred to a solution containing hyaluronidase. Residual granulosa cells were removed by pipetting. After washing three times, the washed oocytes were transferred to a fertilization tray and a suspension of floating sperm was added (10 μL of the original solution was diluted 5 times and 1 μL was added). After fertilization, the cells were cultured for 48 h and observed under a microscope to count the proportion of cleavage-stage embryos.

[0083] Experimental results are as follows Figure 1 As shown. The cleavage rate of the control group was 59.61 ± 1.92%. The cleavage rate of the 0.1 μmol / L compound 1 group was 61.66 ± 1.85%, showing a certain improvement, but the difference was not significant compared with the control group. The cleavage rate of the 1 μmol / L compound 1 group was 73.81 ± 2.38%, significantly higher than that of the control group (…). P <0.05). The cleavage rate of group 1 (10 μmol / L compound) was 59.92 ± 2.08%, which was not significantly different from the control group.

[0084] The results indicate that compound 1 at a concentration of 1 μmol / L significantly increased the cleavage rate of fertilized eggs, suggesting that it promotes early embryonic development; while concentrations of 0.1 μmol / L or 10 μmol / L did not show the same effect.

[0085] Example 5: Effect of compound 1 on the in vitro blastocyst formation rate of fertilized eggs Four groups of fertilized eggs were prepared using the same method as in Example 4, 48 hours after fertilization.

[0086] Each group of fertilized eggs was transferred to a culture dish containing TL solution, then pipetted and washed three times with PZM3 developmental solution, and then transferred to a developmental plate. The eggs were cultured in vitro until day 7 post-fertilization, and the blastocyst formation rate was observed and counted under a microscope.

[0087] Experimental results are as follows Figure 1 As shown. The blastocyst formation rate in the control group was 21.00 ± 0.58%. The blastocyst formation rate in the 0.1 μmol / L compound 1 group was 24.33 ± 0.33%, showing a slight increase. The blastocyst formation rate in the 1 μmol / L compound 1 group was 34.33 ± 0.33%, significantly higher than that in the control group (…). P <0.05). The blastocyst formation rate of group 1 with 10 μmol / L compound was 21.66 ± 0.55%, with no significant change.

[0088] The results indicate that a concentration of 1 μmol / L of compound 1 significantly increased the proportion of fertilized eggs that developed to the blastocyst stage, while concentrations of 0.1 μmol / L or 10 μmol / L of compound 1 did not produce the same promoting effect.

[0089] Example 6: Effect of compound 1 on oxidative stress levels in the cumulus-oocyte complex Mature cumulus-oocyte complexes were prepared using the same method as in Example 2 (2 groups: control group and 1 group with 1 μmol / L compound).

[0090] The levels of reactive oxygen species (ROS) in cumulus cells and oocytes in two groups of cumulus-oocyte complexes were detected using the DCF-DA probe.

[0091] Experimental results are as follows Figure 2 As shown. The reactive oxygen species (ROS) level in the control group oocytes was 66.64 ± 1.96 RFU. The ROS level in the cumulus-oocyte complex of group 1 (1 μmol / L compound 1) was significantly reduced to 3.482 ± 0.72 RFU. P <0.05).

[0092] The above results indicate that compound 1 maintains the stability of the cumulus-oocyte microenvironment by mitigating oxidative damage.

[0093] Example 7: Effects of Compound 1 on Mitochondrial Function in Oocytes Oocytes were prepared using the same method as in Example 3 (2 groups: control group and 1 group with 1 μmol / L compound).

[0094] MitoTracker was used to detect mitochondrial distribution in two groups of oocytes; and mitochondrial membrane potential was assessed by JC-1 staining. JC-1 forms red fluorescent J-aggregates at high membrane potentials and exists as green fluorescent monomers at low membrane potentials. The red / green fluorescence intensity ratio was used to quantitatively reflect the membrane potential level.

[0095] Experimental results are as follows Figure 2 As shown. (1) Mitochondrial distribution: The abnormal mitochondrial distribution rate of oocytes in the control group was 21.20 ± 0.58%; the abnormal mitochondrial distribution rate of oocytes in the 1 μmol / L compound group was 17.40 ± 0.51%, and the aggregation phenomenon was reduced; (2) Mitochondrial membrane potential: The red / green fluorescence ratio of the control group JC-1 was 0.92 ± 0.06, mainly showing monomeric green fluorescence, indicating a low membrane potential and weak function; the red / green fluorescence ratio of the 1 μmol / L compound group JC-1 was 3.16 ± 0.04, showing stronger red fluorescence, indicating a significantly increased mitochondrial membrane potential and stronger function.

[0096] The above results indicate that compound 1 at a concentration of 1 μmol / L can improve the spatial distribution of mitochondria in oocytes, reduce abnormal mitochondrial aggregation, and significantly increase mitochondrial membrane potential, suggesting that it helps maintain mitochondrial function and improve energy metabolism efficiency.

[0097] Example 8: Effect of compound 1 on DNA damage levels Oocytes were prepared using the same method as in Example 3 (2 groups: control group and 1 group with 1 μmol / L compound).

[0098] DNA damage in two groups of oocytes was detected using the γH2AX immunofluorescence method.

[0099] Experimental results are as follows Figure 3 As shown. Oocytes in the control group exhibited strong and diffuse intranuclear fluorescence of γH2AX, with a fluorescence intensity of 21.94 ± 1.67. The γH2AX fluorescence signal in the 1 μmol / L compound 1 group was significantly weakened, with a fluorescence intensity of 8.92 ± 1.47, showing a significant difference compared to the control group. P <0.05).

[0100] The above results indicate that compound 1 at a concentration of 1 μmol / L has a protective effect in reducing DNA damage and maintaining genome stability.

[0101] Example 9: Effects of Compound 1 on Oocyte Spindle Assembly and Chromosome Alignment Oocytes were prepared using the same method as in Example 3 (2 groups: control group and 1 group with 1 μmol / L compound).

[0102] The morphology of the spindle apparatus in oocytes during metaphase II (MII) of meiosis was detected and analyzed using immunofluorescence staining.

[0103] According to the standard: A normal spindle exhibits a typical bipolar symmetrical structure, with chromosomes arranged closely and orderly on the equatorial plate; Abnormal spindle structures are characterized by broken spindle structures, multipolar formation, or diffuse structures, accompanied by chromosomes deviating from the equatorial plate or exhibiting a scattered distribution.

[0104] Experimental results are as follows Figure 3 As shown. In the control group, the incidence of abnormal spindles was 14.75 ± 0.37%; In group 1 of compound 1 (1 μmol / L), the incidence of abnormal spindles was 12.29 ± 0.29%, which was significantly lower than that in the control group; The above results indicate that a concentration of 1 μmol / L of compound 1 can reduce the incidence of abnormal spindle structures, suggesting that compound 1 helps to enhance the stability of the meiotic apparatus and improve the quality of oocyte maturation.

[0105] Example 10: Effect of compound 1 on apoptosis levels in in vitro mature oocytes Oocytes were prepared using the same method as in Example 3 (2 groups: control group and 1 group with 1 μmol / L compound).

[0106] Annexin V-FITC staining was used to assess the apoptosis level of oocytes in the two groups; TUNEL assay was used to assess the apoptosis level of oocytes in the two groups.

[0107] (1) Annexin V-FITC staining method: Oocytes were placed in Annexin V binding buffer, Annexin V-FITC working solution was added, and incubated in the dark for 15 min. Then, the cells were gently washed twice with binding buffer and transferred to a glass slide for microscopic imaging. The fluorescence intensity and the proportion of Annexin V positive signals of each oocyte were quantitatively analyzed using ImageJ software.

[0108] (2) TUNEL assay: Using a TUNEL assay kit, oocytes were fixed in 4% paraformaldehyde for 30 min, washed three times with PBS, and permeabilized. The TUNEL reaction mixture was then added and incubated at 37°C for 1 h. After the reaction, the cells were washed and the nuclei were counterstained with DAPI. FITC (TUNEL) channel images were acquired. ImageJ was used to calculate the TUNEL fluorescence value (i.e., the apoptosis index).

[0109] Experimental results are as follows Figure 4As shown. Annexin V-FITC staining method detection: The fluorescence intensity of oocytes in the control group was 14.40 ± 1.64 RFU, while the fluorescence intensity of group 1 (1 μmol / L compound) was 0.24 ± 0.14 RFU, significantly lower than that of the control group (…). P <0.05).

[0110] TUNEL assay: The apoptosis index of oocytes in the control group was 1.00 ± 0.001 RFU, while the apoptosis index of oocytes in group 1 (1 μmol / L compound) was 0.20 ± 0.002 RFU, significantly lower than that in the control group. P <0.05).

[0111] The above results indicate that the Annexin V assay was consistent with the TUNEL assay, both suggesting that a 1 μmol / L concentration of compound 1 could significantly reduce oocyte apoptosis levels.

[0112] Example 11: Effects of Compound 1 on in vitro maturation and embryonic development of sheep oocytes Ovaries of healthy female sheep were selected, and cumulus-oocyte complexes were collected from follicles with a diameter of 3-6 mm. After screening, sheep oocytes were obtained and then subjected to in vitro maturation experiments (IVM).

[0113] This experiment included a control group without compound 1, a group with 100 μmol / L compound 1, a group with 1 μmol / L compound 1, and a group with 0.00001 μmol / L compound 1.

[0114] The cumulus-oocyte complexes from each group were placed in an in vitro maturation culture system for sheep, and the corresponding concentration of compound 1 was added. The mixture was cultured at 38.5℃ and 5% CO2 for 24 h. The expansion status of the sheep cumulus cells was then observed to assess the quality of oocyte maturation in vitro. The mature oocytes were then used for subsequent in vitro fertilization (IVF).

[0115] The sheep in vitro maturation culture system consisted of M199 culture medium (GIBCO, C11150500BT) supplemented with 10% fetal bovine serum, 10 μg / mL follicle-stimulating hormone, 10 μg / mL luteinizing hormone, 1 μg / mL estradiol, 2 mM sodium pyruvate, 100 ng / mL epidermal growth factor, 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0116] Sheep sperm were treated with Percoll gradient centrifugation and adjusted to an appropriate concentration, and then co-incubated with mature sheep oocytes for 5 hours for complete fertilization. The fertilized sheep oocytes were transferred into an in vitro embryo culture system (prepared according to Tian X, et al. Int J Mol Sci. 2017.17;18(4):834) and cultured at 38.5℃ for 7 days. The cleavage rate (48 hours after fertilization) and blastocyst formation rate (day 7) were recorded.

[0117] Experimental results are as follows Figure 5 , Figure 6 As shown, the cleavage rate and blastocyst formation rate of the 1 μmol / L compound 1 group were significantly higher than those of the control group, the 100 μmol / L compound 1 group, and the 0.00001 μmol / L compound 1 group. P <0.05); the cleavage rate of the 100 μmol / L group was significantly lower than that of the control group and the 0.00001 μmol / L group ( P <0.05), suggesting that high concentrations of compound 1 may have an inhibitory effect on early embryonic development.

[0118] The results indicate that compound 1 has a significant concentration-dependent effect on in vitro maturation of sheep oocytes and subsequent embryonic development. Specifically, the 1 μmol / L compound 1 concentration effectively enhanced oocyte developmental potential and significantly improved the cleavage and blastocyst formation capabilities of post-fertilization embryos, while the 100 μmol / L or 0.00001 μmol / L compound 1 concentrations did not show the same promoting effect.

[0119] The technical solutions of this invention are not limited to the specific embodiments described above. It should be understood that any modifications and changes can be made without departing from the spirit of this invention. All technical variations made according to the technical solutions of this invention fall within the protection scope of this invention.

Claims

1. The application of a compound of formula I in promoting the development of oocytes or embryos in vitro. Equation I, in, R1 is selected from haloalkyl groups; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2 or 3.

2. The application according to claim 1, characterized in that, R1 is selected from C 1-6 Haloalkyl, more preferably halomethyl or haloethyl, and even more preferably -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2 or -CH2-CF3, more preferably -CF3; and / or n is selected from 0 or 1; and / or R2 is selected from H, F, Cl, or CH3; and / or The compound of formula I is Compound 1.

3. The application according to claim 1, characterized in that, The oocyte is a mammalian oocyte; the mammal is a human, pig, sheep, dog, cat, or rabbit, preferably a pig or sheep; and / or The concentration of the compound of formula I is 0.1~5 μmol / L, preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L; and / or The application is to increase the expansion area of ​​cumulus cells in the cumulus-oocyte complex; and / or The expanded area of ​​the cumulus cells can be increased by 1.18 to 1.40 times, preferably by 1.2 to 1.35 times; and / or The application is to reduce the reactive oxygen species level in the cumulus-oocyte complex; and / or The reactive oxygen species (ROS) level of the oocytes is 1-20 RFU, preferably 2-15 RFU; more preferably 1.5, 2, 3, 5, 7, or 10 RFU.

4. The application according to claim 1, characterized in that, The application is to increase the first polar body expulsion rate of oocytes; and / or The oocyte first polar body expulsion rate is increased to 1.1 to 1.30 times, preferably to 1.15 to 1.25 times, more preferably to 1.18 times, 1.20 times, 1.22 times, or 1.24 times; and / or The application is to reduce the abnormal distribution rate of mitochondria in oocytes; and / or The abnormal mitochondrial distribution rate is 14-19%, preferably 15-18%, more preferably 16% or 17%; and / or The application is to increase the membrane potential of mitochondria in oocytes; and / or The membrane potential of the mitochondria in the oocyte is 1.5 to 5, preferably 1.7 to 4.

5.

5. The application according to claim 1, characterized in that, The application is to reduce DNA damage in oocytes; and / or The DNA damage in the oocyte is reduced to 0.2-0.75 times, preferably 0.3-0.65 times; more preferably 0.35 times, 0.4 times, 0.45 times, 0.5 times or 0.6 times; and / or The application is to reduce the incidence of abnormal spindle structures in oocytes; and / or The incidence of abnormal spindle structures in the oocytes is 10-15%, preferably 11-14%, and more preferably 12-13%.

6. The application according to claim 1, characterized in that, The application is to improve the cleavage rate of fertilized eggs; and / or The cleavage rate of the fertilized egg is increased to 1.1 to 1.5 times, preferably 1.15 to 1.45 times, more preferably 1.2 times, 1.25 times, 1.3 times, 1.35 times or 1.4 times; and / or The application is to improve the blastocyst formation rate of fertilized eggs; and / or The blastocyst formation rate of the fertilized egg is increased to 1.15 to 2.5 times, preferably 1.2 to 2.3 times, and more preferably 1.3 times, 1.5 times, 1.7 times, 1.9 times or 2.1 times.

7. The use of a compound of formula I in the preparation of a drug that promotes the development of oocytes in vitro or promotes the development of embryos in vitro. Equation I, in, R1 is selected from haloalkyl groups; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2 or 3.

8. A method for in vitro culture of oocytes, characterized in that, Immature oocytes were cultured in an oocyte culture medium containing compound I. Equation I, R1 is selected from alkyl halogenates; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2, or 3; and / or The in vitro culture temperature is 36.5~39℃ and the CO2 concentration is 4~6%; preferably, the temperature is 37℃, 37.5℃, or 38.5℃; even more preferably, the CO2 concentration is 5%; and / or The concentration of the compound of formula I is 0.1~5 μmol / L, preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L; and / or The immature oocytes are either in the form of a cumulus-oocyte complex or in the form of a naked oocyte without a cumulus.

9. An in vitro culture medium for oocytes, characterized in that, The culture medium is an oocyte culture medium containing compound I. Equation I, R1 is selected from alkyl halogenates; R2 is selected from H, halogen, or C. 1-6 alkyl; n is selected from 0, 1, 2, or 3; Preferably, the concentration of the compound of formula I is 0.1~5 μmol / L, more preferably 0.3~5 μmol / L, more preferably 0.7~3 μmol / L, and even more preferably 1 μmol / L.

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