An oocyte in vitro maturation culture medium additive and application thereof

Abstract

The application belongs to the technical field of cell biology, and provides an oocyte in vitro maturation culture medium additive and application thereof. The experimental results of the application show that coumarin 106 can improve the meiosis process blockage of oocytes, reduce the morphological proportion of abnormal spindle and the proportion of misaligned chromosomes, restore the level of acetylated tubulin, improve microtubule stability, improve the expression level of actin in the spindle of oocytes, improve the misdistribution and expression decrease of mitochondria in oocytes, improve the level of mitochondrial membrane potential, improve the fluorescence signal of cortical granules and specific cortical granule proteases in oocytes, improve the fertilization rate, reduce the level of reactive oxygen species and DNA damage in oocytes, and reduce the occurrence of early apoptosis in oocytes. The culture medium prepared by using coumarin 106 can be used for promoting oocyte in vitro maturation.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to an additive for oocyte in vitro maturation culture medium and its application. Background Technology

[0002] In vitro maturation (IVM) is a technique that induces the maturation of the cumulus-oocyte complex to the MII stage in vitro. To improve the IVM rate of immature oocytes, IVM culture systems have been continuously improved, including modifications to culture protocols, culture media, and optimization of the culture environment. A dual-phase IVM culture protocol with a higher oocyte maturation rate than conventional IVM protocols has been developed, and the clinical pregnancy rate of IVM has also been effectively improved. Because IVM avoids ovarian hyperstimulation syndrome, it is now widely used in patients with polycystic ovary syndrome; it is also suitable for patients with ovarian resistance syndrome who have ovarian reserve but are unresponsive to exogenous gonadotropins; furthermore, IVM can be combined with fertility preservation techniques, making it suitable for cancer patients who require fertility preservation.

[0003] Nevertheless, the clinical application of IVM still suffers from problems such as lower oocyte maturation rate, fertilization rate, cleavage rate, and clinical pregnancy rate compared to conventional in vitro fertilization / intracytoplasmic sperm injection (IVF / ICSI), which has prevented its widespread use.

[0004] Because porcine oocytes are closer to human oocytes in terms of size, maturation time, and other physiological developmental indicators than mouse oocytes, this invention uses porcine oocytes as a research model. An in vitro oxidative stress model is established through exogenous hydrogen peroxide (H2O2) treatment to accurately simulate core pathological phenotypes in the physiological aging process—including abnormal accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, and apoptosis. Based on this model, the role of the natural bioactive molecule coumarin 106 in the maturation process of porcine oocytes is systematically investigated. The aim is to find a new component that can improve the in vitro maturation efficiency of oocytes, thereby improving the in vitro developmental potential of nuclear transfer embryos. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an additive for oocyte in vitro maturation culture medium and its application.

[0006] In a first aspect, the present invention provides the application of coumarin 106 in the preparation of culture medium or culture medium additive for in vitro maturation of oocytes.

[0007] Secondly, the present invention provides a culture medium additive for oocyte in vitro maturation, the additive comprising coumarin 106.

[0008] Secondly, a culture medium for in vitro maturation of oocytes, said culture medium containing the aforementioned additives.

[0009] Preferably, the culture medium further comprises IVM maturation culture medium, and the concentration of coumarin 106 is in the range of 10–200 μM.

[0010] Preferably, the IVM maturation culture medium is composed of functionalized TCM199 culture medium, equine chorionic gonadotropin solution and human chorionic gonadotropin solution in a volume ratio of 98:1:1. The functionalized TCM199 culture medium is composed of TCM199, kanamycin solution, porcine follicular fluid, sodium pyruvate solution, cysteine ​​solution, epidermal growth factor solution and insulin solution in a volume ratio of 4500:7.5:500:11:145:5:5.

[0011] Preferably, the concentration of the kanamycin solution is 0.75 mg / mL, the concentration of the sodium pyruvate solution is 20 mg / mL, the concentration of the cysteine ​​solution is 3.5 mg / mL, the concentration of the epidermal growth factor solution is 1 mg / mL, and the solvent is physiological saline; the concentration of the insulin solution is 5 mg / mL, and the solvent is 1% (v / v) glacial acetic acid solution; the concentration of the equine chorionic gonadotropin solution is 500 IU / mL, and the solvent is TCM199; the concentration of the human chorionic gonadotropin solution is 400 IU / mL, and the solvent is TCM199.

[0012] Fourthly, the present invention also provides the application of the above-mentioned oocyte in vitro maturation culture medium additive or the above-mentioned oocyte in vitro maturation culture medium in the preparation of a culture medium for promoting oocyte in vitro maturation.

[0013] Fifthly, the present invention also provides the use of the above-mentioned oocyte in vitro maturation culture medium additive or the above-mentioned oocyte in vitro maturation culture medium in the preparation of reagents for promoting oocyte in vitro maturation.

[0014] In a sixth aspect, the present invention also provides the use of an additive for an in vitro maturation culture medium for oocytes or the above-mentioned in vitro maturation culture medium for oocytes in the preparation of a drug for promoting in vitro maturation of oocytes.

[0015] In a seventh aspect, the present invention also provides a method for promoting in vitro maturation of oocytes, comprising first equilibrating the culture medium for in vitro maturation of oocytes, then placing the oocytes into the equilibrated culture medium, and then placing them in an incubator with a CO2 volume concentration of 4%-6% for maturation culture for 44-48 hours.

[0016] Preferably, the equilibration specifically involves: sealing the culture medium with paraffin oil and placing it in an incubator with a CO2 concentration of 4% to 6% for equilibration and preheating for at least 4 hours, wherein the temperature of the incubator is 38 to 39°C.

[0017] Preferably, the promotion of oocyte in vitro maturation includes any one or more of the following: improving the arrest of oocyte meiosis; reducing the proportion of abnormal spindle morphology and misaligned chromosomes in oocytes; restoring the level of acetylated tubulin; improving microtubule stability; increasing the expression level of actin in oocyte spindles; improving the misdistribution and decreased expression of mitochondria in oocytes; increasing the mitochondrial membrane potential; increasing the fluorescence signal of oocyte cortical granules and specific cortical granule proteases; improving fertilization rate; and reducing the level of reactive oxygen species and DNA damage in oocytes, thereby reducing the occurrence of early apoptosis in oocytes.

[0018] Preferably, the promoting oocytes are derived from non-human mammals or humans, including but not limited to pigs, cattle, and sheep.

[0019] The beneficial effects of this invention are:

[0020] The coumarin 106-containing culture medium provided by this invention can improve meiotic arrest in oocytes, reduce the proportion of abnormal spindle morphology and misaligned chromosomes, restore acetylated tubulin levels, and improve microtubule stability; increase actin expression levels in oocyte spindles; improve misdistribution and decreased expression of mitochondria in oocytes; increase mitochondrial membrane potential; enhance fluorescence signals of oocyte cortical granules and specific cortical granule proteases; improve fertilization rate; reduce reactive oxygen species levels and DNA damage levels in oocytes; and reduce early apoptosis in oocytes. Furthermore, the coumarin 106-containing culture medium provided by this invention is simple to prepare, easy to operate, and has significant application prospects. Attached Figure Description

[0021] Figure 1 The effect of coumarin 106 on H2O2-induced meiotic arrest in oocytes (A shows the first polar body statistics after oocyte maturation, B shows the in vitro development of oocytes; control is the control group, H2O2 is the H2O2 experimental group, in A, Coumarin 106+H2O2 are the experimental groups with different molar concentrations of coumarin 106, in B, Coumarin 106+H2O2 is the experimental group with 50 μM coumarin 106; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001, ns indicates no significant difference).

[0022] Figure 2The effect of coumarin 106 on H2O2-induced abnormal spindle assembly in oocytes (A is the result of immunofluorescence staining, B is the statistics of abnormal spindle assembly, C is the statistics of chromosome mismatch ratio, and D is the fluorescence signal of acetylated tubulin).

[0023] Figure 3 This is the effect of coumarin 106 on the decrease in actin expression in oocyte spindles caused by H2O2 (A is the result of actin immunofluorescence staining, and B is the fluorescence signal of actin).

[0024] Figure 4 The effect of coumarin 106 on the misdistribution of mitochondria in oocytes caused by H2O2 (A is the result of mitochondrial fluorescence staining, B is the statistical proportion of abnormal mitochondrial localization, and C is the fluorescence signal of mitochondria).

[0025] Figure 5 This study investigates the effect of coumarin 106 on the decrease in mitochondrial membrane potential in oocytes induced by H2O2 (A shows the results of fluorescence staining for mitochondrial membrane potential; B shows the fluorescence signal of mitochondrial membrane potential).

[0026] Figure 6 The effect of coumarin 106 on the decrease in cortical granules and specific cortical granule protease levels in oocytes caused by H2O2 (A is the result of immunofluorescence staining of cortical granules, B is the fluorescence signal of cortical granules, C is the result of immunofluorescence staining of specific cortical granule protease Ovastacin, and D is the fluorescence signal of specific cortical granule protease Ovastacin).

[0027] Figure 7 The effect of coumarin 106 on the impaired ability of oocytes to combine with sperm caused by H2O2 (A is the result of oocyte-sperm combination (the first row is the sperm cell nuclear fluorescence image, the second row is the sperm cell nuclear fluorescence image and the bright scene combined), B is the number of sperm combined with each oocyte, C is the pronuclear staining, and D is the fertilization rate).

[0028] Figure 8 The effect of coumarin 106 on the increase of reactive oxygen species (ROS) levels in oocytes induced by H2O2 (A is the result of ROS fluorescence staining, B is the fluorescence signal of ROS, C is the result of H2A.X antibody fluorescence staining for DNA damage, D is the fluorescence signal of H2A.X antibody, E is the result of apoptosis detection, and F is the fluorescence signal of apoptosis). Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] The experimental reagents used in the following examples are as follows:

[0033] 1) Experimental reagents

[0034] Coumarin 106, TCM199 liquid culture medium, H2O2, lanolin, Beyotime cell culture water, physiological saline, Triton-X100 immunostaining permeabilization buffer, glucose, sucrose, calcium chloride dihydrate, calcium chloride (CaCl2), sodium lactate, sodium trinitrate, magnesium chloride, cysteine, penicillin, sodium pyruvate, human chorionic gonadotropin (hCG), equine chorionic gonadotropin (eCG), insulin (INS), phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane buffer (TRIS), paraffin oil, kanamycin, magnesium chloride hexahydrate, hydroxyethylpiperazine ethanesulfonic acid (HEPES), phenol red, fetal bovine serum albumin (BSA), NCSU-23 (NC-S), Hoechst 33342, 16% paraformaldehyde (PFA), streptomycin, epidermal growth factor (EGF), sodium bicarbonate, potassium chloride, NaH2PO4, NaCl, glacial acetic acid, porcine follicular fluid (PFF), glycerol, glycine, dimethyl sulfoxide (DMSO), taurine, tris(hydroxymethyl)aminomethane (TRIS), hyaluronidase (HAase), polyvinyl alcohol (PVA).

[0035] 2) Reagent preparation

[0036] Preparation of streptomycin solution: Dilute streptomycin with physiological saline to a concentration of 100 U / mL.

[0037] Preparation of penicillin solution: Dilute penicillin with physiological saline to a concentration of 100 U / mL.

[0038] Preparation of IVM operating solution: Take 300 mL of Beyotime cell culture water into a sterilized beaker, weigh the reagents according to Table 1, and finally add 0.5551 g of sodium chloride to maintain the osmotic pressure of the liquid at 300 mOsm. Stir magnetically until the reagents are completely dissolved, and bring the volume to 500 mL with Beyotime cell culture water. Filter the liquid through a 0.22 μm filter.

[0039] Table 1. Composition and proportions of IVM operating fluid

[0040]

[0041] Preparation of IVM maturation culture medium: Take 4.5 mL of TCM199 and add 7.5 μL of kanamycin solution, 500 μL of filtered porcine follicular fluid, 11 μL of sodium pyruvate solution, and 145 μL of cysteine ​​solution sequentially. Filter into a new 15 mL centrifuge tube, add 5 μL of EGF solution and 5 μL of insulin solution. Mix well, then add more fluid according to the ratio of 980 μL liquid + 20 μL eCG / hCG solution. Mix well to obtain the IVM maturation culture medium.

[0042] Preparation of sperm diluent (fertilization solution): Take 50 mL of Beyotime cell culture water into a sterilized beaker, add 0.0275 g sodium pyruvate, 0.0112 g potassium chloride, 0.3306 g sodium chloride, 0.0991 g glucose, 0.0551 g calcium chloride dihydrate and 0.1212 g TRIS, and let it stand at 4°C overnight to allow it to dissolve completely.

[0043] EGF solution: Weigh 0.1000g of EGF and dissolve it in 100mL of physiological saline.

[0044] Kanamycin solution: Weigh 0.0375g and dissolve in 50mL H2O.

[0045] eCG solution: Dissolve 2000 IU of eCG in 4 mL of TCM199.

[0046] hCG solution: Dissolve 2000 IU hCG in 5 mL TCM199.

[0047] eCG / hCG solution: Take 1 mL of eCG solution and 1 mL of HCG solution and mix thoroughly.

[0048] Sodium pyruvate solution: Weigh 0.1001g of sodium pyruvate and dissolve it in 5mL of H2O.

[0049] Cysteine ​​solution: Weigh 0.0173g of cysteine ​​and dissolve it in 5mL of H2O.

[0050] Insulin concentration: Weigh 5 mg of insulin and dissolve it in 1 mL of 1% glacial acetic acid solution.

[0051] Preparation of culture medium containing 50 μM H2O2: Measure 1 μL of 1 M H2O2 solution and dissolve it in 20 mL of IVM mature culture medium, then mix well.

[0052] Preparation of coumarin 106 culture medium: Dissolve coumarin 106 in Beyotime cell culture water to prepare a 1M solution. Dilute this solution with IVM maturation medium as needed to achieve the desired final concentrations (10, 20, 50, 100, and 200 μM).

[0053] Hyaluronidase solution: Add 0.05g of HAase to 1mL of TCM199 and mix well.

[0054] Immunofluorescence permeabilization solution: Weigh 0.0022g NaN3, 0.0476g HEPES, 1.026g sucrose, 0.02925g NaCl and 0.00285g MgCl2 sequentially and place them in 10mL PBS. Let stand at 4℃ until completely dissolved.

[0055] Immunofluorescence wash: Measure 1 μL of Triton X-100 and place it in 10 mL of PBS. Let it stand at 4°C until completely dissolved.

[0056] Immunofluorescence blocking solution: Take 0.3g BSA and place it in 10mL PBS, and let it stand at 4℃ until completely dissolved.

[0057] 3) Experimental antibodies

[0058] Mouse monoclonal anti-α-tubulin-FITC antibody, rabbit monoclonal acetyl-α-tubulin (Lys40) antibody, anti-phalloidin-TRITC antibody, and lentil lectin (LCA)-FITC were obtained from Sigma-Aldrich; Alexa 488 goat anti-rabbit IgG (H+L) is derived from Thermo Fisher Scientific; Alexa 594 goat anti-rabbit IgG (H+L) was obtained from Zhongshan Jinqiao Company; rabbit anti-human ovastacin polyclonal antibody was obtained from Dr. Jurrien Dean; Mito-Tracker probe was obtained from Invitrogen Company; rabbit monoclonal anti-γH2AX antibody was obtained from Cell Signaling Technology Company; and reactive oxygen species detection kit and Annexin V-FITC detection kit for early apoptosis were obtained from Beyotime Company.

[0059] Example 1: Effect of coumarin 106 on H2O2-induced meiotic arrest in oocytes

[0060] During in vitro oocyte culture, H2O2 treatment simulated the oxidative stress phenotype during aging. H2O2 treatment significantly inhibited the developmental capacity of cumulus cells, with some cumulus cells not spreading or only spreading to a low degree, remaining close to the oocyte. Normal oocyte maturation is a prerequisite for maintaining oocyte quality; therefore, this example statistically analyzes the first polar body PB1 extruded by the oocyte during meiosis I.

[0061] I. Experimental Methods

[0062] (1) Collection of pig eggs

[0063] 1) Add the preheated IVM maturation culture medium to the cell culture wells (100 μL / well), cover with a layer of paraffin oil (50 μL), and place in a constant temperature incubator set at 38.5℃ and containing 5% CO2 for at least 4 hours to obtain well-balanced enzyme-labeled wells (culture medium).

[0064] 2) Gently wash fresh pig ovaries collected from local slaughterhouses multiple times with penicillin and streptomycin solutions until no blood flows out of the ovary nest. After washing two to three more times with preheated saline, place the beaker containing the fresh pig ovaries in a 38°C water bath for later use.

[0065] 3) Using a 10mL syringe with a 0.7mm needle, extract the follicular fluid from the pig ovary and slowly inject it into a 50mL centrifuge tube. After collecting the follicular fluid, let it stand for 20 minutes. After observing that there is a clear precipitate in the centrifuge tube, use a pipette or syringe to aspirate the supernatant to obtain the follicular fluid precipitate.

[0066] 4) Add IVM manipulator solution at a ratio of 1 mL of follicular fluid precipitate to 7 mL of IVM manipulator solution. Gently and thoroughly mix the solution, then observe and collect the oocytes under a microscope. Use a pipette to transfer the oocyte complexes (COCs) with uniform cytoplasm and well-encapsulated by multiple layers of granulosa cells into a small dish to complete the collection of oocytes (COCs).

[0067] (2) In vitro culture

[0068] Control group: COCs containing granulosa cells were washed once with IVM processing solution and three times with IVM maturation culture solution before being transferred into pre-balanced enzyme-labeled wells, 50 COCs per well. The wells were then placed in a constant temperature incubator. After 44-48 hours, the oocytes matured and entered the MII arrest phase.

[0069] Experimental Groups: Oocytes containing granulosa cells were washed with IVM maturation medium and then transferred to a culture medium containing 50 μM H2O2. They were incubated in a CO2 incubator for 30 min. Afterward, they were washed again with IVM maturation medium and divided into six equal groups. One group was placed in IVM maturation medium (designated the H2O2 experimental group), and the remaining groups were divided into groups containing 10, 20, 50, 100, and 200 μM coumarin 106, respectively (designated the coumarin 106+H2O2 experimental groups). All groups were then returned to an incubator at 5% CO2, 38.5℃, and saturated humidity for further culture. Oocytes matured after 44-48 h.

[0070] The diffusion of cumulus ova and the expulsion of polar bodies in the control and experimental groups were observed under a microscope, and representative images of in vitro development were taken. The maturation data of each group were statistically analyzed.

[0071] II. Experimental Results

[0072] Analysis of oocytes compared with the control group showed that H2O2 treatment significantly reduced the maturation rate, while the addition of 50 μM coumarin 106 improved the maturation rate and restored the in vitro maturation process of oocytes (see...). Figure 1 Therefore, coumarin 106 can improve the arrest of oocyte meiosis caused by H2O2.

[0073] Subsequent experiments were conducted using oocytes (MII arrest phase) cultured for 48 h in the control group (Control), H2O2 experimental group (H2O2), and the coumarin 106+H2O2 experimental group with 50 μM coumarin 106 added.

[0074] Example 2: Effect of coumarin 106 on H2O2-induced abnormal oocyte spindle assembly

[0075] In vitro maturation arrest of oocytes is usually highly correlated with abnormal spindle / chromosomal structure. The assembly and stability of the spindle mainly depend on the microtubule organizing center (MTOC) and related microtubule proteins dispersed in the cell for joint regulation. Since the acetylation level of α-tubulin is an important indicator of microtubule stability, this example uses immunofluorescence staining to observe this.

[0076] I. Experimental Methods

[0077] (1) Immunofluorescence staining

[0078] 1) Digestion: Add 100 μL of IVM processing solution to 25 μL of hyaluronidase solution and mix thoroughly by pipetting. Then add the three groups of COCs cultured for 48 h in Example 1, shake for 5 min to digest the granulosa cells surrounding the oocytes, and collect naked oocytes (DOs).

[0079] 2) Fixation: Wash the naked eggs collected in step 1) once with IVM handling solution, transfer them to 50 μL of 4% PFA (pH 7.4) droplet, and let stand at room temperature for 1 h.

[0080] 3) Permeabilization: Transfer the fixed oocytes from step 2) into 50 μL of immunofluorescence permeabilization solution and incubate at room temperature in a humidified chamber for at least 8 hours or overnight at 4°C.

[0081] 4) Blocking: After permeabilization, oocytes were transferred to 50 μL of immunofluorescence blocking solution and blocked in a humidified chamber at room temperature for 1 hour.

[0082] 5) Antibody incubation:

[0083] Primary antibody incubation: Oocytes were transferred into 50 μL of diluted primary antibody solution and incubated overnight at 4°C. They were then washed three times with immunofluorescence washing buffer, 5 min each time.

[0084] Secondary antibody incubation: Oocytes incubated with primary antibody were transferred into secondary antibody dilution solution, stained in a humidified chamber, and incubated at room temperature for 1 hour. They were then washed three times with immunofluorescence washing solution for 5 minutes each time.

[0085] 6) Nucleus staining: Transfer the cleaned oocytes into 50 μL of Hoechst 33342 nucleus staining solution (1:10000 dilution) and incubate at room temperature in a humidified chamber for 10 min.

[0086] 7) Mounting: Apply a small amount of glycerin evenly to the center of the slide, transfer the oocytes into the glycerin, apply lanolin to the four corners of the coverslip, then invert the coverslip and slowly lower it to cover the glycerin area containing the cells. Store the prepared slide at -20℃.

[0087] In this example, when the primary antibody dilution is mouse monoclonal anti-α-tubulin-FITC dilution (1:200), secondary antibody incubation is not required; when the primary antibody dilution is rabbit monoclonal acetyl-α-tubulin (Lys-40) dilution (1:100), the secondary antibody dilution is Alexa. 594 Goat anti-rabbit IgG dilution (1:200 dilution).

[0088] (2) Measurement

[0089] The slides were placed under a laser confocal microscope, and the prepared oocytes were imaged and observed using confocal imaging (LSM 900META, Zeiss). The images were then labeled and saved on the computer for later analysis and processing.

[0090] During the observation and recording process using laser confocal microscopy, appropriate values ​​were set for different parameters in the ZEN software according to the strength of the positive signal. To ensure the comparability of the results between the control and experimental groups, the same parameters were maintained when observing and recording different groups of samples. The obtained images were analyzed for fluorescence using ImageJ software. When measuring the fluorescence intensity in the cortical area, the Threshold color function module was used for analysis.

[0091] II. Experimental Results

[0092] H2O2 treatment increased the proportion of abnormal spindle assembly, the proportion of chromosomes with discrete arrangement, and the level of acetylated tubulin, i.e., hyperacetylation. Adding coumarin 106 reduced the morphology of abnormal spindles, significantly decreased the proportion of misaligned chromosomes, restored the level of acetylated tubulin, and improved microtubule stability (see...). Figure 2 ).

[0093] Example 3: Effect of coumarin 106 on H2O2-induced decrease in actin expression in oocyte spindles

[0094] Oocyte maturation consists of nuclear maturation and cytoplasmic maturation. The experimental results of Examples 1-2 have shown that coumarin 106 can improve nuclear maturation, while high-quality oocyte development is inseparable from complete cytoplasmic maturation. This example further explores the effect of coumarin 106 on oocyte cytoplasmic development.

[0095] Except for the primary antibody dilution solution being the anti-phalloidin-TRITC antibody dilution solution (1:200) and the fact that secondary antibody incubation is not required, the other steps, parameters, and measurement methods of immunofluorescence staining are the same as in Example 2.

[0096] During in vitro oocyte culture, H2O2 treatment significantly weakened actin signaling, which recovered somewhat after the addition of coumarin 106 (see...). Figure 3 In other words, coumarin 106 improved the decrease in actin expression in the oocyte spindle caused by H2O2.

[0097] Example 4: Effect of coumarin 106 on H2O2-induced misdistribution of mitochondria in oocytes

[0098] A key phenotype of oxidative stress during aging is mitochondrial damage, which disrupts energy metabolism. Therefore, this embodiment evaluates the distribution and function of mitochondria.

[0099] I. Experimental Methods

[0100] Mitochondrial fluorescent staining:

[0101] 1) Digestion: Consistent with the "digestion" step in the immunofluorescence staining in Example 2;

[0102] 2) Staining: Place in a drop of 500 nM mitochondrial working solution prepared with IVM operating solution and incubate at 37°C in the dark for 30 min.

[0103] 3) Washing: After staining, wash 3 times (5 min / time) with preheated IVM operating solution.

[0104] 4) Imaging: Transfer to 10 μL of IVM operating droplet in a staining dish and image using a Zeiss LSM 900 confocal microscope with unified parameters within 1 hour. Acquire images using ZEN software. Finally, analyze the fluorescence intensity of the cortical area using ImageJ (Thresholdcolor module) and perform statistical analysis using GraphPad Prism 9.

[0105] II. Experimental Results

[0106] During in vitro oocyte culture, H2O2 treatment increased the proportion of abnormal mitochondrial localization and significantly decreased fluorescence signal (see...). Figure 4 Coumarin 106 improved the misdistribution and decreased expression of mitochondria in oocytes caused by H2O2.

[0107] Example 5: Effect of coumarin 106 on H2O2-induced decrease in mitochondrial membrane potential during oocyte spinning.

[0108] Observations of mitochondrial membrane potential (JC-1) revealed a significant decrease in membrane potential levels.

[0109] In this embodiment, except that "placed in a drop of 500 nM mitochondrial working solution prepared with IVM operating solution" in Example 4 is replaced with "placed in Mito-Tracker probe dilution solution diluted 1:100 with IVM operating solution", all other steps and parameters are the same as those in Example 5 for mitochondrial fluorescence staining.

[0110] The results showed that the addition of coumarin 106 improved the abnormal distribution pattern of mitochondria, enhanced fluorescence signal, and significantly increased mitochondrial membrane potential. Coumarin 106 can improve the decrease in mitochondrial membrane potential in oocytes induced by H2O2.

[0111] Example 6: Effect of coumarin 106 on the decrease in cortical granules and specific cortical granule protease levels in oocytes caused by H2O2.

[0112] Cortical granules (CGs) are vesicle-like structures unique to the cortical region of oocyte cytoplasm and are one of the standards for measuring oocyte cytoplasmic maturation. Ovastacin, the first component identified in mammalian cortical granules, plays a role in cleaving the sperm binding site in the zona pellucida after fertilization and is an important component in cortical granules for preventing polyspermia. Therefore, this example investigated the effects of coumarin 106 on the distribution of CGs and ovastacin in oocytes.

[0113] I. Experimental Methods

[0114] (1) Immunofluorescence staining of CGs

[0115] 1) Digestion: Add 100 μL of IVM operating solution to 25 μL of hyaluronidase working solution and mix thoroughly by pipetting. Then add the three groups of COCs cultured for 48 h in Example 1 and shake for 5 min.

[0116] 2) Remove the transparent zone

[0117] Transfer the digested granulosa cells into a T-shaped acid droplet and repeatedly pipette until the zona pellucida disappears completely. Then quickly transfer the oocytes into an IVM operating droplet and wash 2-3 times.

[0118] 3) Fixation: Add 4% PFA to the glass reaction plate, 100 μL per well, transfer naked oocytes (DOs) without granulosa cells into the fixation solution, and fix in a humidified chamber at room temperature for 30 min;

[0119] 4) Washing: Wash 3 times with 0.3% BSA (100mM glycine), 5 min each time; wash 5 min with 0.1% Triton-100; wash twice with PBS, 5 min each time.

[0120] 5) Primary antibody incubation: Dilute the lentil lectin (LCA)-FITC antibody with blocking buffer according to the ratio recommended in the instructions. Use a pipette to add 50 μL of the diluted antibody to each glass reaction plate. Transfer the blocked oocytes into the plate and incubate at room temperature for 1 h. Wash with 0.3% BSA for 5 min and 0.1% Triton-100 for 5 min.

[0121] 6) Secondary antibody incubation: Incubate Alexa with blocking solution 488 goat anti-rabbit IgG (H+L) antibody was diluted according to the appropriate ratio, and the washed oocytes were transferred into the solution and incubated in a humidified box at room temperature for 1 hour; washed with 0.3% BSA for 5 minutes; washed with 0.1% Triton-100 for 5 minutes (with thorough stirring using a glass needle);

[0122] 7) Nuclear staining: Transfer the eggs into the nuclear dye Hoechst 33342 and incubate at room temperature for 10 min;

[0123] 8) Mounting: Apply lanolin to the four corners of the coverslip. Draw a circle of appropriate size in the center of the slide using an immunohistochemistry pen, and apply a small amount of glycerin to the center. Transfer the eggs into the glycerin using a pipette, and then mount the coverslip. Record the relevant information on the ground side of the slide and store it properly at -20℃ for later use.

[0124] (2) Ovastacin immunofluorescence staining

[0125] 1) Digestion: Consistent with the "digestion" step in the immunofluorescence staining in Example 2;

[0126] 2) Fixation: Transfer the naked oocytes from step 1) to 4% PFA and fix them in a humidified chamber at room temperature for 1 hour;

[0127] 3) Washing: Wash 3 times with washing solution prepared with 100μL 0.3% BSA + 100mM glycine PBS, 5min / time; treat with 100μL 0.1% Triton X-100PBS for 5min; then wash twice with 100μL PBS, 5min / time.

[0128] 4) Primary antibody incubation: Dilute rabbit anti-human Ovastacin polyclonal antibody with PBS according to the instructions, add it to oocytes, and fix in a humidified chamber at room temperature for 1 hour; wash 3 times with 100 μL of washing buffer prepared with 0.3% BSA + 0.01% Triton X-100, 5 min each time;

[0129] 5) Secondary antibody incubation: Add Alexa to PBS according to the instructions. Dilute 594 goat anti-rabbit IgG secondary antibody and fix in a humidified chamber at room temperature for 1 hour; wash 3 times with 100 μL of 0.3% BSA + 0.01% Triton X-100 washing buffer, 5 min each time;

[0130] 6) Mounting: Same as the "Mounting" content in CGs immunofluorescence staining.

[0131] II. Experimental Results

[0132] During in vitro culture of oocytes, H2O2 treatment significantly reduced the fluorescence signals of cortical granules and ovastacin, while the addition of coumarin 106 improved the fluorescence signals of cortical granules and ovastacin.

[0133] Example 7: Effect of coumarin 106 on H2O2-induced impairment of oocyte-sperm binding ability

[0134] The correct identification and binding of the egg and sperm is a crucial prerequisite for fertilization. The results of Example 7 indicate that the addition of coumarin 106 can improve the abnormal state of CGs and ovastacin in oocytes caused by H2O2 treatment. Ovastacin is mainly responsible for cutting the sperm binding site after fertilization to prevent polyspermia. The damage to CGs caused by H2O2 treatment, leading to premature release of ovastacin, may affect sperm-oocyte binding. To further investigate whether coumarin 106 has a certain improving effect on this process, this example uses a sperm-oocyte binding experiment for evaluation.

[0135] I. Experimental Methods

[0136] 1. In vitro fertilization of oocytes

[0137] 1.1 Semen thawing

[0138] Quickly remove the frozen semen from the liquid nitrogen tank and place it in a 50°C water bath. After 16 seconds, add the frozen semen to a test tube containing 10 mL of sperm diluent through a top-bottom opening. Cap the tube and gently invert it three times to mix thoroughly. Centrifuge at 1900 RPM for 4 minutes, discard the supernatant, add 200 μL of sperm activation solution, and quickly tap the bottom of the test tube to mix. Open the cap and place the tube in a 38°C, 5% CO2 incubator until counting is complete.

[0139] 1.2 Sperm count and semen dilution

[0140] 1) Take 10 μL of washed sperm and add it to 990 μL of embryo culture water, then mix well by pipetting;

[0141] 2) Take 100 μL of the liquid from step 1) and add it to 900 μL of embryo culture water, then mix thoroughly by pipetting.

[0142] 3) Take 10 μL of the liquid from step 2) and spot it into a hemocytometer, then count the cells under a microscope to obtain N cells;

[0143] 4) Calculate the original sperm concentration, sperm count per millisperm = N × 10⁷;

[0144] 5) Dilute the semen to a final concentration of 1.0 × 10⁶, and use 50 μL of sperm per 30 eggs.

[0145] 1.3 In vitro fertilization

[0146] 1) Prepare culture drops of sperm capacitation solution and embryo culture medium 24 hours before fertilization and place them in an incubator for full equilibration;

[0147] 2) Transfer oocytes at the target stage into capacitation droplets, with a maximum of 30 oocytes placed in each capacitation droplet;

[0148] 3) Add the diluted semen to the capacitated drops using a pipette, 50 μL per drop (be careful to add it gently and lift the egg when adding);

[0149] 4) Place the culture droplets from step 3) into a 38℃, 5% CO2 incubator and incubate for 4–6 hours;

[0150] 5) Wash the fertilized eggs from step 4) 2-3 times with embryo culture medium on a heated platform and transfer them into a preheated embryo culture drop.

[0151] 2. Sperm binding count

[0152] After culturing oocytes and sperm in sperm capacitation drops for 1 hour, the oocytes were removed. The oocytes were picked out and placed in IVM manipulator drops, and gently pipetted under a microscope. Unbound sperm were removed and fixed. Fixation was performed with 4% paraformaldehyde at room temperature for 30 minutes; staining with Hoechst 33342 at room temperature for 10 minutes; and the slides were mounted and counted using laser confocal microscopy.

[0153] 3 Prokaryotic staining

[0154] After culturing fertilized eggs in embryo culture medium for 12 hours, they were removed and transferred into an IVM manipulator droplet using a pipette. Under a microscope, sperm adhering to the surface of the fertilized eggs were blown away with a pipette. The eggs were then fixed with 4% paraformaldehyde at room temperature for 30 minutes; Hoechst staining was performed at room temperature for 10 minutes; and the slides were mounted for imaging observation using laser confocal microscopy. For sperm binding and counting, ZEN used the Z Stack channel to scan individual samples, dividing each oocyte into 5-7 slides for counting and stacking.

[0155] II. Experimental Results

[0156] In the control group of unfertilized oocytes, a large number of sperm bound to the zona pellucida. However, the opposite was observed in the control group of two-cell embryos, where almost no sperm bound to the zona pellucida. This is because the sperm binding sites on the zona pellucida were cleaved after fertilization. After H2O2 treatment, the number of sperm binding to the zona pellucida in fertilized oocytes decreased significantly. The addition of coumarin 106 significantly increased the number of sperm binding to the zona pellucida, indicating an enhanced ability of oocytes to bind to sperm. Further analysis of fertilization rate showed that the pronuclear rate in oocytes significantly decreased after H2O2 treatment, but significantly increased after adding coumarin 106. This indicates that coumarin 106 improved the low fertilization rate caused by H2O2 treatment. Therefore, coumarin 106 can improve the impaired ability of oocytes to bind to sperm caused by H2O2, thereby increasing the fertilization rate.

[0157] Example 8: Effect of coumarin 106 on H2O2-induced increase in reactive oxygen species levels in oocytes

[0158] I. Experimental Methods

[0159] This embodiment included H2A.X fluorescent staining experiments, as well as ROS and Annexin-V fluorescent staining. The primary antibody dilution for H2A.X fluorescent staining was rabbit monoclonal anti-γH2AX dilution, and the secondary antibody dilution was Alexa. Except for the 594 goat anti-rabbit IgG (H+L) staining, the other steps were the same as the immunofluorescence staining parameters in Example 2. For ROS fluorescence staining, except that "placed in IVM operating solution to prepare 500nM mitochondrial working droplets" was replaced with "placed in IVM operating solution to prepare 10μM DCFH-DA fluorescent probe working droplets (from the reactive oxygen species detection kit)", the other steps were the same as the mitochondrial staining parameters in Example 4. For Annexin-V fluorescence staining, except that "placed in IVM operating solution to prepare 500nM mitochondrial working droplets" was replaced with "placed in IVM operating solution to prepare 10μM Annexin V-FITC working droplets (from the Annexin V-FITC early apoptosis detection kit)", the other steps were the same as the mitochondrial staining parameters in Example 4.

[0160] II. Experimental Results

[0161] H2O2 treatment impairs the proper positioning and function of mitochondria in oocytes, inducing high levels of reactive oxygen species (ROS) in the cytoplasm and exacerbating oxidative stress in oocytes. In the control group, the green fluorescence signal of ROS was weak; H2O2 treatment enhanced the fluorescence signal, resulting in a bright distribution in the cytoplasm; however, the addition of coumarin 106 reduced the enhanced fluorescence signal. Quantitative analysis results were consistent with fluorescence observations, showing that the addition of coumarin 106 reduced the H2O2-induced increase in ROS levels.

[0162] High levels of ROS inhibit the repair of DNA double-strand breaks, inducing oxidative DNA damage and leading to DNA damage accumulation. Further DNA damage detection was performed. After H2O2 treatment, the fluorescence signal of γ-H2A.X on chromosomes in oocytes was significantly enhanced; the addition of coumarin 106 reduced the level of green fluorescence signal.

[0163] Excessive ROS and high levels of DNA damage accumulation can induce early apoptosis in cells. Therefore, Annexin V-FITC labeling of oocytes was used to assess whether early apoptosis occurred. Fluorescence observation showed that the fluorescence signal on the plasma membrane in the control group was very weak and almost undetectable. In contrast, H2O2 treatment produced a clear and bright ring on the plasma membrane, and the addition of coumarin 106 effectively reduced the fluorescence signal on the plasma membrane. Subsequent measurement and statistical analysis of fluorescence expression levels showed that H2O2 treatment exacerbated early apoptosis in oocytes, while the addition of coumarin 106 effectively rescued this damage.

[0164] In summary, coumarin 106 reduced the increase in reactive oxygen species (ROS) and DNA damage levels in oocytes induced by H2O2, and further reduced the occurrence of early apoptosis in oocytes.

[0165] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The application of coumarin 106 in the preparation of culture medium or culture medium additives for in vitro maturation of oocytes, characterized in that, The concentration range of the coumarin 106 is 10~200 μM.

2. A culture medium for in vitro maturation of oocytes, characterized in that, The culture medium contains an additive containing coumarin 106 and an IVM maturation culture medium, wherein the concentration of coumarin 106 ranges from 10 to 200 μM.

3. The culture medium according to claim 2, characterized in that, The IVM maturation culture medium consists of functionalized TCM199 culture medium, equine chorionic gonadotropin solution, and human chorionic gonadotropin solution in a volume ratio of 98:1:

1. The functionalized TCM199 culture medium consists of TCM199, kanamycin solution, porcine follicular fluid, sodium pyruvate solution, cysteine ​​solution, epidermal growth factor solution, and insulin solution in a volume ratio of 4500:7.5:500:11:145:5:

5.

4. The culture medium according to claim 3, characterized in that, The concentration of the kanamycin solution is 0.75 mg / mL, the concentration of the sodium pyruvate solution is 20 mg / mL, the concentration of the cysteine ​​solution is 3.5 mg / mL, the concentration of the epidermal growth factor solution is 1 mg / mL, and the solvent is physiological saline; the concentration of the insulin solution is 5 mg / mL, and the solvent is 1% (v / v) glacial acetic acid solution; the concentration of the equine chorionic gonadotropin solution is 500 IU / mL, and the solvent is TCM199; the concentration of the human chorionic gonadotropin solution is 400 IU / mL, and the solvent is TCM199.

5. The use of the oocyte in vitro maturation culture medium according to any one of claims 2-4 in the preparation of reagents for improving the efficiency of oocyte in vitro maturation.

6. The use of the oocyte in vitro maturation culture medium according to any one of claims 2-4 in the preparation of a medicament for improving the efficiency of oocyte in vitro maturation.

7. A method for improving the efficiency of in vitro maturation of oocytes, characterized in that, The process involves first equilibrating the culture medium for in vitro maturation of oocytes as described in any one of claims 2-4, then placing the oocytes into the equilibrated culture medium, and finally placing them in an incubator with a CO2 volume concentration of 4%-6% for maturation culture for 44-48 hours.

8. The method according to claim 7, characterized in that, The equilibration process specifically involves sealing the culture medium with paraffin oil and placing it in an incubator with a CO2 concentration of 4% to 6% for at least 4 hours for equilibration and preheating. The temperature of the incubator is 38 to 39°C.

9. The method according to claim 7 or 8, characterized in that, The improvement of oocyte in vitro maturation efficiency includes improving the arrest of oocyte meiosis, reducing the proportion of abnormal spindle morphology and misaligned chromosomes, restoring the level of acetylated tubulin, improving microtubule stability, increasing the expression level of actin in oocyte spindle, improving the misdistribution and decreased expression of mitochondria in oocytes, increasing the mitochondrial membrane potential, increasing the fluorescence signal of oocyte cortical granules and specific cortical granule proteases, improving fertilization rate, and reducing the levels of reactive oxygen species and DNA damage in oocytes, and reducing the occurrence of early apoptosis in oocytes, among other things.

10. The method according to claim 9, characterized in that, The oocytes are derived from non-human mammals or humans, including pigs, cattle, or sheep.

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