Method for detecting the in vitro fertilization capacity of sperm by flow cytometry

Abstract

This invention discloses a method for detecting sperm in vitro fertilization capacity using flow cytometry, relating to the field of sperm in vitro fertilization technology. This method, after induction with H-ZP3, significantly reduces CB1 expression in capacitated sperm, indicating that H-ZP3 can induce the acrosome reaction. However, after the acrosome reaction occurs, CD46 can only detect the acrosome reaction induced by A23187. In the incomplete acrosome reaction induced by H-ZP3 protein, CD46 cannot detect the incomplete acrosome reaction. However, PSA can be used to detect the incomplete acrosome reaction using flow cytometry. Therefore, by using flow cytometry, PSA can detect the incomplete acrosome reaction induced by H-ZP3, more accurately determining the sperm acrosome reaction rate, which can provide some assistance for clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of in vitro fertilization technology, specifically a method for detecting sperm in vitro fertilization capacity using flow cytometry. Background Technology

[0002] The acrosome reaction refers to a series of changes that occur in the acrosome after sperm capacitation and upon encountering the egg. The reaction is a lengthy process, including activation of acrosome receptors, fusion of the acrosome membrane with the spermatocyte membrane, release of hydrolytic enzymes from the acrosome, and hydrolysis of the zona pellucida (outer membrane of the egg cell), ultimately leading to the fusion of the spermatocyte and egg cell membranes. The acrosome contains the acrosomal enzyme system, a complex enzyme system including hyaluronidase, corona radiata dispersase, acrosin, and arylsulfatase. Hyaluronidase's main function is to dissolve the hyaluronic acid between cumulus cells, dispersing the cumulus cells and allowing sperm to pass through these intercellular spaces. Corona radiata dispersase loosens the cells of the corona radiata. Acrosin, also known as sperm headgranule protein, has the function of dissolving the zona pellucida of the egg. Acrosin exists in the acrosome in the form of a zymogen, called pro-acrostin. During the acrosome reaction, acrosinogen is activated into active acrosin, which, along with other substances, participates in the mechanism by which sperm penetrate the zona pellucida to achieve fertilization. Arylsulfatase plays a role in dissolving the yolk membrane. The acrosome reaction is a prerequisite for fertilization, involving the fusion of the outer acrosome membrane and the sperm plasma membrane at many sites, forming numerous small vesicle-like structures. Finally, the outer acrosome membrane ruptures, releasing various enzymes within the acrosome and exposing the inner acrosome membrane. Sperm ejaculated from the epididymis enter the female reproductive tract and undergo capacitation and the acrosome reaction before fertilization. It is generally believed that cumulus cells and the zona pellucida are the main factors inducing the acrosome reaction. Under in vitro culture conditions, Ca2+, K+, and high-protein culture media can induce and promote the acrosome reaction.

[0003] Sperm capacitation and acrosome reaction are prerequisites for sperm's fertilization capacity. The sperm acrosome reaction is divided into complete and incomplete acrosome reactions, each detected by different markers. CD46 can detect a complete acrosome reaction induced by A23187, but it cannot detect incomplete acrosome reactions induced by H-PZ3 and progesterone. Under physiological conditions, the acrosome reaction after sperm capacitation is mostly incomplete. During fertilization, the H-ZP3 protein in the zona pellucida of the oocyte can induce the sperm acrosome reaction, thus completing the fertilization process. The common method for detecting the sperm acrosome reaction is fluorescence staining followed by observation of sperm fluorescence changes under a fluorescence microscope to determine whether an acrosome reaction has occurred. Fluorescence staining is a relatively reliable method of assessment.

[0004] Therefore, we propose a method for detecting sperm in vitro fertilization capacity using flow cytometry to address the aforementioned issues. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting sperm in vitro fertilization capacity by flow cytometry, aiming to solve the problem that CD46 can detect complete acrosome reactions induced by A23187, but cannot detect incomplete acrosome reactions induced by H-PZ3 and progesterone.

[0006] The present invention is implemented as follows: a method for detecting the in vitro fertilization capacity of sperm by flow cytometry, comprising the following steps: S1, preparing the relevant reagents required for the process; S2, extracting ZP3 protein; S3, inducing the acrosome reaction of sperm, performing flow cytometry analysis, and recording the analysis results; S4, analyzing the recorded data results.

[0007] Further, S1 includes the following steps: S11, preparing OocyteStorageBuffer; S12, preparing NeutralizationBuffer; S121, weighing 1 M Tris + HCl and adjusting its pH to pH 9.0; S13, preparing 5 ml NaH2PO4; S131, weighing 5 ml NaH2PO4 and adjusting its pH to pH 2.5; S14, preparing 1X EBSS; S141, weighing 50 ml of 10X Earle's concentrated stock solution, 1.1 g of NaHCO3, and 450 ml of ddH2O respectively, and mixing the above components to prepare the solution.

[0008] S15. Prepare EBSS equilibration buffer with 0.3% BSA; S151. Weigh 50 ml of 1X Earle's concentrated stock solution and 0.15 g of BSA, then mix them together; S16. Prepare EBSS equilibration buffer with 3% BSA; S161. Weigh 50 ml of 1X Earle's concentrated stock solution and 1.5 g of BSA, then mix them together; S17. Prepare FACS Buffer; S171. Weigh 2 ml of EDTA, 1 g of BSA, 5 ml of NaN3, and 500 ml of PBS, then mix them together.

[0009] Further, S11 includes the following steps: S111, weigh 1 pack of PBS, 304.95 g of MgCl2·6H2O, 9.532 g of HEPES, 1.1629 g of PVP360 and 1 L of ddH2O respectively; S112, mix the components weighed in step S111, adjust the pH value to pH 7.2 after mixing, dissolve at 37°C, and filter out the undissolved particles, thus completing the preparation.

[0010] Further, S2 includes the following steps: S21, weigh 200 μl of NaH2PO4 and add 100 discarded oocytes to it, dissolve for 5 minutes; S22, centrifuge at 2000 rpm, after which take the supernatant and discard the precipitate; S23, add 1 / 10 volume of Neutralization Buffer for neutralization; S24, store the neutralized liquid at -20℃ for later use.

[0011] Further, S3 includes the following steps: S31, sperm upstream, fresh sperm upstream, to obtain sperm with good motility for subsequent operations; S32, inducing sperm capacitation and acrosome reaction, by inducing sperm capacitation and acrosome reaction with 3% BSA and H-ZP3 protein; S33, staining sperm with antibodies and detecting sperm acrosome reaction by flow cytometry.

[0012] Further, S31 includes the following steps: S311, after taking fresh semen and mixing it by blowing and blowing, divide one 0.5ml tube into multiple tubes; S312, carefully add 1ml of 0.3% BSA in EBSS equilibration solution, and incubate at 37°C with 5% CO2 for 1 hour; S313, circulate and aspirate about 500μl of the supernatant from the upper layer, mix the supernatant of the same sample, and count the samples.

[0013] Further, S32 includes the following steps: S321, after diluting the sperm concentration to 10^6 / mL with EBSS equilibration buffer, divide it into 4 portions of 200 μl each and transfer them to a 96-well plate; S322, centrifuge at 500×g for 5 minutes and discard the supernatant; S323, resuspend the control tube in 100 μl of EBSS equilibration buffer containing 0.3% BSA, and resuspend the other three groups in 100 μl of EBSS equilibration buffer containing 3% BSA at 37°C and 5% CO2 for 3 hours; S324, add H-ZP3 protein to the acrosome reaction induction tube to induce the acrosome reaction for 30 minutes, with a final concentration of 1 ng / μl.

[0014] Further, S33 includes the following steps: S331, after the acrosome reaction, add 1 mL of FACS Buffer, preheat at 37°C, centrifuge at 500×g for 5 minutes, and discard the supernatant; S332, resuspend and transfer to a 96-well plate, add 200 μl of FACS Buffer, and centrifuge at 500×g for 5 minutes; S333, prepare ZA antibody; S334, prepare PSA; S335, add 100 μl of Fixation / Permeabilization working solution to each well for resuspending, and incubate at 4°C in the dark for 60 minutes; S336, after incubation, add 150 μl of Permeabilization washing buffer, centrifuge at 500×g for 5 minutes at room temperature, and discard the supernatant. Supernatant; S337, Prepare CB1 antibody with 1×Permeabilization Buffer, 1:200, 100 μl per well, incubate at 4℃ in the dark for 60 minutes; S338, After incubation, add 2000 μl of FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat washing three times; S339, Resuspend in 200 μl of FACS Buffer, centrifuge at 500×g for 5 minutes at room temperature, then filter through a 70 μm filter and transfer to a flow cytometry tube, add 100 μl of FACS Buffer and mix well before flow cytometry analysis, and record the results.

[0015] Further, S333 includes the following steps: S3331, 1:500, 100 μl system per well, incubate at 4°C in the dark for 15 minutes; S3332, after incubation, add 2000 μl FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the washing three times.

[0016] Further, S334 includes the following steps: S3341, 1: 1:200, 100 μl system per well, incubate at 37°C in the dark for 10 minutes; S3342, after incubation, add 2000 μl FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the washing three times.

[0017] Compared with existing technologies, the method for detecting sperm in vitro fertilization capacity by flow cytometry provided by the present invention has the following beneficial effects:

[0018] After incubation in 3% BSA-EBSS at 37℃ for 3 hours, sperm capacitation occurred. The addition of H-ZP3 induced an acrosome reaction. CB1, a protein on the sperm membrane surface, is lost and its expression decreases during the acrosome reaction. Experimental results showed that H-ZP3 significantly reduced CB1 expression in capaciform sperm, indicating that H-ZP3 can induce an acrosome reaction. Furthermore, after the acrosome reaction, CD46 expression was only detected in A23187-induced acrosome reactions, while no significant change was observed in H-ZP3-induced acrosome reactions. (See attached image). The results show that CD46 cannot detect incomplete acrosome reactions. PSA, as another method for detecting sperm acrosome reactions, was found to be significantly more expressed in the H-ZP3-induced acrosome reaction than in the uninduced group. This indicates that PSA can be used to detect incomplete sperm acrosome reactions using flow cytometry. Therefore, this patent utilizes flow cytometry and PSA to detect H-ZP3-induced incomplete sperm acrosome reactions, providing a more accurate assessment of sperm acrosome reaction rates and offering some assistance for clinical diagnosis. Attached Figure Description

[0019] Figure 1 This is a flowchart of the method for detecting sperm in vitro fertilization capacity using flow cytometry according to the present invention.

[0020] Figure 2 This is a flowchart of the reagent preparation method related to the present invention.

[0021] Figure 3 This is a flowchart of the OocyteStorageBuffer preparation method of the present invention.

[0022] Figure 4 This is a flowchart of the ZP3 protein extraction method of the present invention.

[0023] Figure 5 This is a flowchart of the sperm induction and on-machine detection method of the present invention.

[0024] Figure 6 This is a flowchart of the sperm upstream method of the present invention.

[0025] Figure 7 This is a flowchart of the method for inducing sperm capacitation and acrosome reaction according to the present invention.

[0026] Figure 8 This is a flowchart of the sperm antibody staining and sperm acrosome reaction detection method of the present invention.

[0027] Figure 9 This is a flowchart of the ZA antibody preparation method of the present invention.

[0028] Figure 10 This is a flowchart of the PSA preparation method of the present invention.

[0029] Figure 11 This is a scatter plot of the sperm acrosome reaction detected by flow cytometry according to the present invention.

[0030] Figure 12 The image shows the results of CD46 detection of ZP3-induced incomplete acrosome reaction in sperm under 0.3% BSA and 3% BSA conditions according to this invention.

[0031] Figure 13 The image shows the results of CD46 detection of ZP3-induced incomplete acrosome reaction in sperm under 3% BSA+A23187 and 3% BSA+H-ZP3 conditions according to the present invention.

[0032] Figure 14 This is a graph showing the results of PSA detection of ZP3-induced incomplete acrosome reaction in sperm under 0.3% BSA according to the present invention.

[0033] Figure 15 This is a graph showing the results of PSA detection of ZP3-induced incomplete acrosome reaction in sperm under 3% BSA according to the present invention.

[0034] Figure 16 This is a graph showing the results of PSA detection of ZP3-induced incomplete acrosome reaction in sperm under 3% BSA+ZP3 conditions according to the present invention.

[0035] Figure 17 This is a formulation diagram of the FACSBuffer of the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0038] In the accompanying drawings of this embodiment, the same or similar reference numerals correspond to the same or similar components. In the description of this invention, it should be understood that if terms such as "upper," "lower," "left," and "right" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, they are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the accompanying drawings are only for illustrative purposes and should not be construed as limiting this patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0039] Reference Figure 1-17 The image shown is a preferred embodiment of the present invention.

[0040] The method for detecting sperm in vitro fertilization capacity by flow cytometry includes the following steps: S1, preparing the relevant reagents required for the process; S2, extracting ZP3 protein; S3, inducing sperm acrosome reaction, performing flow cytometry analysis, and recording the analysis results; S4, analyzing the recorded data results.

[0041] Specifically, S1 includes the following steps: S11, preparing OocyteStorageBuffer; S11 includes the following steps: S111, weighing 1 pack of PBS, 304.95g of MgCl2·6H2O, 9.532g of HEPES, 1.1629g of PVP360, and 1L of ddH2O respectively; S112, mixing the components weighed in step S111, adjusting the pH value to pH 7.2 after mixing, dissolving at 37°C, and filtering out undissolved particles, thus completing the preparation; S12, preparing NeutralizationBuffer; S121, weighing... S13. Prepare 5 ml of NaH2PO4 by adding 1 M Tris and HCl to adjust the pH to 9.0; S131. Weigh 5 ml of NaH2PO4 and adjust the pH to 2.5; S14. Prepare 1 X EBSS; S141. Weigh 50 ml of 10 X Earle's concentrated stock solution, 1.1 g of NaHCO3, and 450 ml of ddH2O, and mix the above components; S15. Prepare 0.3% BSA EBSS equilibration solution; S151. Weigh 50 ml of 1 X Earle's concentrated stock solution and 0.15 g of BSA, and then mix them.

[0042] S16. Prepare EBSS equilibration buffer with 3% BSA; S161. Weigh 50 ml of 1X Earle's concentrated stock solution and 1.5 g of BSA, and then mix them; S17. Prepare FACS Buffer; S171. Weigh 2 ml of EDTA, 1 g of BSA, 5 ml of NaN3 and 500 ml of PBS, and then mix them.

[0043] In this implementation scheme, OocyteStorageBuffer is an oocyte storage buffer, PBS is a buffer solution whose main components are Na2HPO4, KH2PO4, NaCl and KCl, and it is generally used as a solvent to dissolve and protect the reagents. HEPES is a nonionic amphoteric buffer, PVP360 is polyvinylpyrrolidone, also known as PNVP, which is a water-soluble polymer. ddH2O is double-distilled water. Neutralization Buffer is a neutralization buffer. Tris+HCl is a buffer solution (pH 7.5), which is a premixed, sterile filtered solution with pH adjusted. EBSS is a salt solution associated with a weaker phosphate buffer. BSA is bovine serum albumin. Steps S15 and S16 are performed in a clean bench to prevent contamination. 1X Earle's solution can be prepared first, and then BSA can be added for dissolution. FACS buffer is a buffer salt solution that can be used in immunofluorescence staining protocols, antibody and cell dilution steps, surface staining and washing steps.

[0044] Specifically, S2 includes the following steps: S21, weigh 200 μl of NaH2PO4 and add 100 discarded oocytes to it, dissolve for 5 minutes; S22, centrifuge at 2000 rpm, after which take the supernatant and discard the precipitate; S23, add 1 / 10 volume of Neutralization Buffer for neutralization; S24, store the neutralized liquid at -20℃ for later use.

[0045] In this implementation scheme, NaH2PO4 is sodium dihydrogen phosphate, an acidic salt of phosphoric acid. It is a raw material for manufacturing sodium hexametaphosphate and sodium pyrophosphate, mainly used in leather making, boiler water treatment, as a quality improver and baking powder, and as a buffer and baking powder raw material in the food and fermentation industries. It is also used as a feed additive, detergent, and dyeing auxiliary agent. 2000 rpm is the speed of the centrifuge, which is 2000 revolutions per minute. This step is used to extract ZP3 protein.

[0046] Specifically, S3 includes the following steps: S31, sperm upstream, fresh sperm upstream, to obtain sperm with good motility for subsequent operations; S32, inducing sperm capacitation and acrosome reaction, using 3% BSA and H-ZP3 protein to induce sperm capacitation and acrosome reaction; S33, antibody staining of sperm, and flow cytometry detection of sperm acrosome reaction.

[0047] In this implementation scheme, the acrosome reaction refers to the process by which sperm release acrosomal enzymes after capacitation, dissolving the corona radiata and zona pellucida. The acrosome is a sac-like organelle located in front of the sperm head cell nucleus and between the nucleus and plasma membrane. It is essentially a specialized lysosome derived from the Golgi apparatus, enclosed by a single membrane, and is flat and sac-like. It contains glycoproteins and various hydrolytic enzymes and serves as a storage site for enzymes related to the acrosome reaction. Under in vitro conditions, serum albumin (BSA), high-density lipoprotein, glycosaminoglycans, progesterone, calcium ion carriers, sperm-egg-binding protein (ZP), CO2, etc., can all promote sperm capacitation.

[0048] Specifically, S31 includes the following steps: S311, take fresh semen, mix it by pipetting, and divide 0.5ml tubes into multiple tubes; S312, carefully add 1ml of 0.3% BSA in EBSS equilibration solution, and incubate at 37°C with 5% CO2 for 1 hour; S313, circulate and aspirate about 500μl of the supernatant from the upper layer, mix the supernatant of the same sample, and count the samples.

[0049] In this embodiment, in step S312, the EBSS is carefully added along the tube wall to separate the EBSS and semen into two layers to avoid mixing. In the EBSS equilibration solution with 0.3% BSA and under a 5% CO2 environment, the sperm with good motility are allowed to swim to the upper culture medium by the upstream method. This method can remove seminal plasma and substances that inhibit fertilization in seminal plasma, achieving in vitro capacitation. It is simple to operate, low in cost, has few adverse reactions, and can obtain sperm with larger particle size and lower DNA fragmentation rate. The upper clear liquid is aspirated to obtain sperm with good motility. After aspiration and mixing, the sperm count is performed.

[0050] Specifically, S32 includes the following steps: S321, after diluting the sperm concentration to 10^6 / mL with EBSS equilibration buffer, divide it into 4 portions of 200 μl each and transfer them to a 96-well plate; S322, centrifuge at 500×g for 5 minutes and discard the supernatant; S323, resuspend the control tube in 100 μl of EBSS equilibration buffer containing 0.3% BSA, and resuspend the other three groups in 100 μl of EBSS equilibration buffer containing 3% BSA, and incubate at 37°C with 5% CO2 for 3 hours; S324, add H-ZP3 protein to the acrosome reaction induction tube to induce the acrosome reaction for 30 minutes, with a final concentration of 1 ng / μl.

[0051] In this implementation scheme, multiple groups of sperm were capacitation was performed in EBSS equilibrium solution with different concentrations of BSA. The results of multiple reactions were recorded and compared to conclude that CD46 could not detect ZP3-induced incomplete acrosome reaction in sperm.

[0052] Specifically, S33 includes the following steps: S331, after the acrosome reaction, add 1 mL of FACSBuffer, preheat at 37°C, centrifuge at 500×g for 5 minutes, and discard the supernatant; S332, resuspend and transfer to a 96-well plate, add 200 μl of FACSBuffer, and centrifuge at 500×g for 5 minutes; S333, prepare ZA antibody; S333 includes the following steps: S3331, 1:500, 100 μl system per well, incubate at 4°C in the dark for 1 minute. 5 minutes; S3332. After incubation, add 2000 μl of FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the washing three times; S334. Prepare PSA; S334 includes the following steps: S3341. 1:200, 100 μl system per well, incubate at 37℃ in the dark for 10 minutes; S3342. After incubation, add 2000 μl of FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the washing three times. S335. Resuspend 100 μl of Fixation / Permeabilization working solution in each well and incubate at 4°C in the dark for 60 minutes. S336. After incubation, add 150 μl of Permeabilization wash buffer, centrifuge at 500×g for 5 minutes at room temperature, and discard the supernatant. S337. Prepare CB1 antibody with 1×Permeabilization Buffer at a 1:200 ratio, add 100 μl to each well, and incubate at 4°C in the dark for 60 minutes. S338. After incubation, add 2000 μl of FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the wash three times. S339. Resuspend 200 μl of FACS Buffer, centrifuge at 500×g for 5 minutes at room temperature, then filter through a 70 μm filter and transfer to a flow cytometry tube. Add 100 μl of FACS Buffer, mix well, and wait for flow cytometry analysis. Record the results.

[0053] In this implementation scheme, ZA antibody can be used for fluorescence experiments. PSA is prostate-specific antigen, which is a tryptophan protease produced by the epithelium of the prostate duct. PSA is usually secreted into prostatic fluid and semen in an active free form. PSA is organ-specific and is synthesized only by prostate epithelial cells. Fixation / Permeabilization working solution is a fixation / permeabilization working solution. Cells usually need to be fixed and permeabilized before flow cytometry. CB1 antibody is cannabinoid receptor 1 antibody. After mixing the prepared liquid, flow cytometry analysis can be performed to obtain the result that PSA can detect ZP3-induced incomplete acrosome reaction in sperm.

[0054] The technical solutions described in the embodiments of this application above have at least the following technical effects or advantages:

[0055] After incubation in 3% BSA-EBSS at 37℃ for 3 hours, sperm capacitation occurred. The addition of H-ZP3 induced an acrosome reaction. CB1, a protein on the sperm membrane surface, is lost and its expression decreases during the acrosome reaction. Experimental results showed that H-ZP3 significantly reduced CB1 expression in capaciform sperm, indicating that H-ZP3 can induce an acrosome reaction. Furthermore, after the acrosome reaction, CD46 expression was only detected in A23187-induced acrosome reactions, while no significant change was observed in H-ZP3-induced acrosome reactions. (See attached image). The results show that CD46 cannot detect incomplete acrosome reactions. PSA, as another method for detecting sperm acrosome reactions, was found to be significantly more expressed in the H-ZP3-induced acrosome reaction than in the uninduced group. This indicates that PSA can be used to detect incomplete sperm acrosome reactions using flow cytometry. Therefore, this patent utilizes flow cytometry and PSA to detect H-ZP3-induced incomplete sperm acrosome reactions, providing a more accurate assessment of sperm acrosome reaction rates and offering some assistance for clinical diagnosis.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting sperm in vitro fertilization capacity using flow cytometry, comprising the following steps: S1. Prepare the necessary reagents for the process; S2. Extraction of ZP3 protein; S3. Induce the sperm acrosome reaction, perform instrumental analysis, and record the results. S4. Analyze the recorded data results; S2 includes the following steps: S21. Weigh 200µl of NaH2PO4 and add 100 discarded oocytes to it, then dissolve for 5 minutes. Centrifuge at 2000 rpm for 22 seconds. After the operation, take the supernatant and discard the precipitate. S23. Add 1 / 10 volume of Neutralization Buffer for neutralization; S24. Store the neutralized liquid at -20°C for later use; S3 includes the following steps: S31. Sperm upstream: Fresh sperm are upstreamed to obtain sperm with good motility for subsequent operations; S32, Inducing sperm capacitation and acrosome reaction, using 3% BSA and H-ZP3 protein to induce sperm capacitation and acrosome reaction; S33. Sperm were stained with antibodies and the acrosome reaction of sperm was detected by flow cytometry; S33 includes the following steps: S331. After the acrosome reaction is complete, add 1 mL of FACS Buffer, preheat at 37°C, centrifuge at 500×g for 5 minutes, and discard the supernatant. S332, resuspend and transfer to a 96-well plate, add 200µl FACS Buffer, and centrifuge at 500×g for 5 minutes; S333, Prepare ZA antibody; S334, Prepare PSA; S335, add 100µl of Fixation / Permeabilization working solution to each well and resuspend, then incubate at 4°C in the dark for 60 minutes; S336. After incubation, add 150µl of Permeabilization washing buffer, centrifuge at 500×g for 5 minutes at room temperature, and discard the supernatant. Prepare CB1 antibody with S337 and 1×Permeabilization Buffer, 1:200, 100µl per well, and incubate at 4°C in the dark for 60 minutes; S338. After incubation, add 2000µl of FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the washing three times. S339. Add 200µl of FACS Buffer to resuspend, centrifuge at 500×g for 5 minutes at room temperature, then filter through a 70μm filter and transfer to a flow cytometer. Add 100µl of FACS Buffer and mix well before running the flow cytometer and recording the results.

2. The method for detecting the in vitro fertilizing ability of sperm by flow cytometry according to claim 1, characterized in that, S1 includes the following steps: S11. Prepare the Oocyte Storage Buffer; S12. Prepare the Neutralization Buffer; S121. Weigh 1 M Tris + HCl and adjust its pH value to 9.0; S13. Prepare 5ml of NaH2PO4; S131. Weigh 5ml of NaH2PO4 and adjust its pH value to 2.5; S14. Prepare 1X EBSS; S141. Weigh 50ml of 10X Earle's concentrated stock solution, 1.1g of NaHCO3 and 450ml of ddH2O respectively, and then mix them together. S15. Prepare an EBSS equilibration solution with 0.3% BSA. S151. Weigh 50ml of 1X Earle's concentrated stock solution and 0.15g of BSA, then mix them together. S16. Prepare an EBSS equilibration solution with 3% BSA. S161. Weigh 50ml of 1X Earle's concentrated stock solution and 1.5g of BSA respectively, and then mix them together. S17. Prepare the FACS Buffer; S171. Weigh 2 ml of EDTA, 1 g of BSA, 5 ml of NaN3, and 500 ml of PBS respectively, and then mix them together.

3. The method for detecting the in vitro fertilizing ability of sperm by flow cytometry according to claim 2, characterized in that, S11 includes the following steps: S111 Weigh 1 pack of PBS, 304.95g of MgCl2·6H2O, 9.532g of HEPES, 1.1629g of PVP360 and 1L of ddH2O respectively. S112. Mix the components weighed in step S111. After mixing, adjust the pH value to pH 7.2, dissolve at 37°C, and filter out any undissolved particles. The preparation is now complete.

4. The method for detecting the in vitro fertilizing ability of sperm by flow cytometry according to claim 1, characterized in that, S31 includes the following steps: S311. Take fresh semen, mix it thoroughly by blowing and pipetting, and divide one 0.5ml tube into multiple tubes. S312. Carefully add 1 ml of 0.3% BSA in EBSS equilibration solution, incubate at 37°C with 5% CO2 for 1 hour in the upper part of the middle ... S313. Draw approximately 500µl of supernatant from the upper layer by rotating the tube. Mix the supernatant from the same sample thoroughly and then count the samples.

5. The method for detecting the in vitro fertilizing ability of sperm by flow cytometry according to claim 1, characterized in that, S32 includes the following steps: S321. Dilute the sperm concentration to 10^6 / mL with EBSS equilibration buffer, divide into 4 portions of 200µl each, and transfer to 96-well plates. S322, 500×g, centrifuge for 5 minutes, discard the supernatant; S323, the control tube was resuspended in 100µl of EBSS equilibration solution containing 0.3% BSA, and the other three groups were resuspended in 100µl of EBSS equilibration solution containing 3% BSA, and subjected to 5% CO2 at 37℃ for 3h. S324. Add H-ZP3 protein to the acrosome reaction induction tube to induce the acrosome reaction for 30 minutes, with a final concentration of 1 ng / µl.

6. The method for detecting the in vitro fertilizing ability of sperm by flow cytometry according to claim 1, characterized in that, S333 includes the following steps: S3331, 1:500, 100µl system per well, incubated at 4°C in the dark for 15 minutes; S3332. After incubation, add 2000µl of FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the washing process three times.

7. The method for detecting the in vitro fertilizing ability of sperm by flow cytometry according to claim 1, characterized in that, S334 includes the following steps: S3341, 1:200, 100µl system per well, incubated at 37°C in the dark for 10 minutes; S3342. After incubation, add 2000µl of FACS buffer, centrifuge at 500×g for 5 minutes at room temperature, and repeat the washing three times.

Images