A sperm damage repair model and a method for establishing the same

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a sperm damage repair model and a method for establishing the same. The method comprises the following steps: step one, establishing a sperm lead poisoning model; step two, establishing a lead poisoning sperm repair model. The step two in which a lead poisoning sperm repair model is established comprises the following steps: step (1) preparing a preparation that optimizes sperm function by an extracellular vesicle pathway; step (2) repairing damaged sperm by an extracellular vesicle pathway-optimized preparation; and step (3) detecting the activity of the repaired sperm. In the present invention, by establishing a sperm damage repair model and adopting extracellular vesicle modification technology, key functional proteins are introduced into extracellular vesicles, which is of great significance for auxiliary research on improving sperm damage and improving sperm motility in vitro in patients with asthenospermia.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a sperm damage repair model and an establishment method thereof. Background Art

[0002] Environmental, social, and psychological factors can all affect male reproductive function. With the increasing level of industrialization and the resulting increasing environmental pollution, a growing number of researchers are focusing on the impact of industrial pollution on male reproduction. Recent studies have found that sustained exposure to environmental chemicals can increase the risk of reproductive disorders. The impact of lead pollution on the male reproductive system is gaining increasing attention, as lead can cause damage to multiple systems, including the hematopoietic, nervous, skeletal, and reproductive systems. Lead affects sperm motility, sperm capacitation, and the acrosome reaction, among other fertilization processes, thereby impacting male reproductive function. Therefore, establishing a sperm damage repair model is of great significance for supporting research on improving sperm damage and developing drugs to enhance sperm motility in vitro in patients with asthenospermia. Summary of the Invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a sperm damage repair model and its establishment method, so as to realize auxiliary research on male infertility treatment, such as related drug development research.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] The present invention discloses a method for establishing a sperm damage repair model, comprising the following steps:

[0006] Step 1: Establish a sperm lead poisoning model;

[0007] Step 2: Establish a lead poisoning sperm repair model.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] In the present invention, by adopting the extracellular vesicle modification technology, the key functional protein SKAP2 was introduced into extracellular vesicles, and a sperm damage repair model was established. The improvement of the motility activity of lead-contaminated sperm and the improvement of the sperm motility of patients with asthenospermia in vitro were achieved, providing a theoretical basis for auxiliary research on male infertility treatment, such as related drug development research. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a graph showing the changes in rat body weight over time of lead exposure in Example 1; Figure 2 This is a graph showing the effect of lead poisoning on the sperm quality of rats in the epididymis in Example 1; Figure 3 This is a diagram showing the results of sperm morphology examination of lead-exposed rats in Example 1; Figure 4 This is a diagram of the testicular structure of lead-exposed rats in Example 1; Figure 5 This is a transmission electron microscopy identification result of extracellular vesicles extracted from the seminal plasma of healthy male rats in Example 2; Figure 6 This is a diagram showing the diameter and concentration distribution of extracellular vesicles extracted from healthy male rat seminal plasma in Example 2 obtained by nanoparticle tracking analysis; Figure 7 This is a diagram showing the protein marker identification results of extracellular vesicles extracted from healthy male rat seminal plasma by Western immunoblotting in Example 2; Figure 8 This is a graph showing the effect of extracellular vesicles of seminal plasma on rat sperm motility in Example 2; Figure 9 This is a graph showing the effect of extracellular vesicles of seminal plasma on human sperm motility in Example 2; Figure 10 This is a graph showing the effect of extracellular vesicles of seminal plasma on human sperm capacitation in Example 2; Figure 11 This is a graph showing the effect of extracellular vesicles of seminal plasma on the acrosome reaction of rat sperm in Example 2; Figure 12 This is a graph showing the effect of extracellular vesicles of seminal plasma on the acrosome reaction of human sperm in Example 2; Figure 13 This is a diagram showing the identification results of extracellular vesicle proteins in rat prostate fluid, seminal vesicle fluid, and epididymal fluid in Example 2; Figure 14 This is a graph showing the difference in proteins between the extracellular vesicles of rat epididymal fluid exposed to lead and the control group in Example 2; Figure 15 This is a graph showing the difference in proteins between the extracellular vesicles of rat seminal plasma in the lead-exposed group and the control group in Example 2; Figure 16 This is a cluster analysis diagram of 37 differentially expressed proteins in extracellular vesicles of rat epididymal fluid in Example 2; Figure 17 This is a cluster analysis diagram of 7 differentially expressed proteins in extracellular vesicles of rat seminal plasma in Example 2; Figure 18 This is a GO enrichment analysis diagram of differential proteins in extracellular vesicles of rat seminal vesicle fluid in Example 2; Figure 19 This is the GO enrichment analysis diagram of differential proteins in extracellular vesicles of rat prostatic fluid in Example 2; Figure 20 This is a GO enrichment analysis diagram of differential proteins in extracellular vesicles of rat epididymal fluid in Example 2; Figure 21 This is the GO enrichment analysis diagram of differential proteins in rat seminal plasma extracellular vesicles in Example 2; Figure 22 This is a KEGG enrichment analysis diagram of differential proteins in extracellular vesicles of rat seminal plasma in Example 2; Figure 23 This is a KEGG enrichment analysis diagram of differential proteins in extracellular vesicles of rat seminal vesicle fluid in Example 2; Figure 24 This is a KEGG enrichment analysis diagram of differential proteins in extracellular vesicles of rat prostatic fluid in Example 2; Figure 25 This is a KEGG enrichment analysis diagram of differential proteins in extracellular vesicles of rat epididymal fluid in Example 2; Figure 26 This is a diagram showing the results of mouse sperm damage repair in Example 4; Figure 27 This is a diagram showing the results of human sperm damage repair in Example 5; Figure 28 This is a diagram showing the results of human sperm damage repair in Example 6; Figure 29 This is a standard curve of SKAP2 protein concentration using the BCA method when measuring the concentration of SKAP2 supernatant in Example 6. DETAILED DESCRIPTION

[0011] Example 1

[0012] This example discloses a method for establishing a rat chronic lead acetate exposure model, comprising the following steps:

[0013] Step (1), preparation of reagents:

[0014] 1.1 Prepare lead acetate poisoning solution: Taking 0.2% lead acetate solution as an example, weigh 2.00g of anhydrous lead acetate, dissolve it in 100mL of distilled water, transfer it to a volumetric flask and dilute to 1L to obtain 0.2% lead acetate solution;

[0015] The preparation method of 0.04% lead acetate solution, 0.4% lead acetate solution and 0.8% lead acetate solution is the same as that of 0.2% lead acetate solution;

[0016] Label 0.04% lead acetate solution, 0.2% lead acetate solution, 0.4% lead acetate solution, and 0.8% lead acetate solution, mark the date, and wait for use;

[0017] 1.2 Prepare SDS-PAGE electrophoresis buffer: Place a 1L bottle of Biyuntian SDS-PAGE electrophoresis buffer powder in a clean beaker, add 900mL of double-distilled water, mix and dissolve with a magnetic stirrer, transfer to a volumetric flask and dilute to 1L to obtain SDS-PAGE electrophoresis buffer;

[0018] Label the SDS-PAGE electrophoresis buffer, mark the date, and store at 4°C until use;

[0019] 1.3 Prepare transfer solution: Take a 1L bottle of Biyuntian semi-dry transfer solution powder and place it in a clean beaker. Add 700mL of double-distilled water and mix well with a magnetic stirrer to dissolve. Add 200mL of anhydrous ethanol and mix well. Transfer to a volumetric flask and dilute to 1L to obtain transfer solution.

[0020] Label the transfer solution, mark the date, and store at 4°C until use.

[0021] 1.4 Prepare membrane washing solution (PBST washing solution): Take 18.7g of PBS powder from a bag and place it in a clean beaker. Add 1900mL of double-distilled water and mix well with a magnetic stirrer to dissolve. Add 2mL of Tween-20 and mix well. Transfer to a volumetric flask and dilute to 2L to obtain the membrane washing solution.

[0022] Label the membrane washing solution, mark the date, and store at 4°C until use;

[0023] 1.5 Prepare the standard curve solution:

[0024] 1.5.1 Dilute the internal standard: Use the gradient dilution method to dilute the internal standard stock solution with a concentration of 10 ppm to the internal standard application solution with a concentration of 100 ppb;

[0025] 1.5.2 Dilute the external standard: Use the gradient dilution method to dilute the 10 ppm external standard stock solution into the external standard application solutions with concentrations of 1 ppm, 100 ppb, 10 ppb, and 1 ppb respectively;

[0026] 1.5.3 The final concentration gradient of the external standard in the standard curve solution is: 50 ppb, 10 ppb, 2 ppb, 0.4 ppb, 0.2 ppb, the final concentration of the internal standard is 10 ppb, and the remaining mass percentage is 3% nitric acid aqueous solution. The total volume of the standard curve solution is 8 mL;

[0027] Step (2): Establishing a rat model of chronic lead acetate exposure

[0028] 2.1 Selection of experimental subjects

[0029] Healthy male Sprague-Dawley rats (3-4 weeks old) weighing 60-110 g SPF (Specific Pathogen Free) were purchased from the Experimental Animal Center of the Army Medical University and acclimatized for 1 week in a laboratory animal room with a temperature of 26 ± 1°C, a relative humidity of 45%-65% RH, and a 12-h light-dark cycle (lights on at 8:00 am and lights off at 8:00 pm).

[0030] Animal maintenance was carried out in strict accordance with the manual of the Laboratory Animal Experiment Committee of the Army Medical University;

[0031] 2.2 The rats selected in 2.1 were randomly divided into six groups, with 12 rats in each group. The rats were fed with the corresponding lead acetate exposure dose through drinking water to establish a chronic exposure model. The six groups of lead acetate exposure experiments were: negative control group (control group), sodium acetate control group (0.4% sodium acetate, NaAC), low lead exposure group (0.04% lead acetate, PbAC), medium lead exposure group (0.2% lead acetate), high lead exposure group (0.4% lead acetate), and ultra-high exposure group (0.8% lead acetate). Diet and growth and development were recorded, and body weight was measured every week. The exposure experiment lasted for 4 months. The changes in rat body weight with exposure time are shown in Tables 1 and Figure 1 As shown:

[0032] Table 1

[0033]

[0034] From Table 1 and Figure 1 The sodium acetate control group showed a significant increase in appetite after 1 week of exposure, while the 0.80% lead-exposed group showed a significant decrease in appetite after 3 weeks of exposure. The other lead-exposed groups showed no change in appetite, but some experienced symptoms such as diarrhea and mental stress. After 5 weeks of lead exposure, the rats showed darkening of body color, loosening of hair, chlorosis, and even hair loss. Compared with the control group, the 0.80% PbAC group showed a significant decrease in body weight. Although the weight of the rats in the other lead-exposed groups changed less than that of the control and NaAC groups over time, no statistically significant effect of lead exposure on the weights of the rats' brain, testicles, prostate, or seminal vesicles was observed compared with the control group. Changes in the rat visceral coefficient (visceral coefficient = tissue / organ weight / animal weight) were analyzed. Only the prostate coefficient of the 0.04% PbAC group was significantly higher than that of the control group. No statistically significant differences in the changes in the organ coefficients of the other lead-exposed groups were found. The rat tissue and organ weights are shown in Table 2.

[0035] Table 2

[0036]

[0037]

[0038] Furthermore, the rats were tested and analyzed after the poisoning experiment, including:

[0039] (1) Whole blood lead test

[0040] After the poisoning experiment, the rats were placed in a rat holder and their tails were exposed. They were immersed in warm water at about 50°C to dilate the blood vessels, and the tails were visibly red. The tails were repeatedly wiped with alcohol cotton balls for disinfection, and then wiped dry with dry cotton balls. About 0.5 cm of the tail tip was cut off, and blood was allowed to flow out from the tail tip. The blood was allowed to drip into an EDTA blood collection tube. The collected whole blood samples of the rats were frozen at -80°C for later use.

[0041] The blood lead concentration in frozen rat whole blood samples was determined using inductively coupled plasma mass spectrometry (ICP-MS). The operating steps are as follows:

[0042] Blood sample processing: Take out frozen rat whole blood samples, keep them in a constant temperature water bath at 37°C for 5 minutes, vortex and shake to mix thoroughly, take 40 μL of rat whole blood samples into a 2 mL polytetrafluoroethylene digestion tube, add 150 μL of internal standard working solution with a concentration of 100 ppb, add 3 wt% nitric acid aqueous solution to make the volume 1.5 mL, turn upside down to mix thoroughly, sonicate for 1 hour, let it stand for 24 hours, centrifuge at 10,000 rpm for 20 minutes, and extract the supernatant for testing;

[0043] The lead (Pb) content in the supernatant was determined by ICP-MS, and the settings of the inductively coupled plasma mass spectrometer were: wavelength 283.3 nm, lamp current 8 mA, slit 0.7 L / nm;

[0044] (2) Analyze semen quality

[0045] 2.1 Extraction of Semen: After the exposure experiment, the epididymal tails of the rats were cut open longitudinally and placed in a 6-well plate with 1 mL of HTF medium. The HTF medium was placed on a 37°C thermostat for 2 minutes. During this time, the epididymal tails were gently pipetted 5 times to fully release the semen.

[0046] 2.2 Pipette 20 μL of semen and add it to 1 mL of HTF culture medium at 37°C. Mix thoroughly by inverting the tube three times. Quickly add 40 μL of semen to a disposable sperm analysis slide. Perform routine semen analysis using a sperm automatic detection and analysis system (CASA). Count 500 sperm per rat.

[0047] The detection indicators mainly include semen routine parameters and sperm motility parameters;

[0048] Conventional parameters include: semen volume, sperm concentration, total sperm count, sperm motility (percentage of progressively motile sperm PR, percentage of non-progressively motile sperm NP, percentage of immotile sperm IM), etc.

[0049] The main sperm motility parameters include: VCL average curvilinear velocity (μm / s), VSL average linear velocity (μm / s), VAP average path velocity (μm / s), LIN linearity (%), STR forwardness (%), WOB oscillation (%), ALH average lateral oscillation amplitude (μm), BCF average sperm whipping frequency (Hz). The test results are shown in Table 3 and Figure 2 As shown:

[0050] Table 3

[0051]

[0052]

[0053] From Table 3 and Figure 2 It can be seen that compared with the control group, the percentage of non-progressive motile sperm (NP) and grade C sperm (P=0.044) in the 0.20% and 0.80% PbAC-treated groups were significantly reduced, while the percentage of immotile sperm (IM), i.e. grade D sperm (P=0.002), was significantly increased. The grade A sperm percentage in the 0.80% PbAC-treated group was significantly reduced (P<0.001). The average linear velocity (VSL), oscillatory motion WOB, linear motion LIN, and forward motion STR of the 0.04% PbAC-treated group were significantly increased. The oscillatory motion WOB of the 0.20% PbAC-treated group was also significantly increased. The average whipping frequency (BCF) of the 0.40% PbAC-treated group was significantly reduced.

[0054] (3) Sperm morphology examination

[0055] The 2% eosin staining method was used to examine the abnormal sperm of rats. The experimental steps are as follows:

[0056] 3.1 Preparation of semen smear:

[0057] 3.1.1 Wipe both sides of the slide with lint-free paper;

[0058] 3.1.2. Use an HB pencil to record the rat number on the white painted area of the slide. Add 10 μL of the semen extracted in 2.1 to the edge of the slide.

[0059] 3.1.3 Use another slide to press the semen against the surface of the first slide and push the slide forward;

[0060] 3.1.4 Fix the slides with anhydrous ethanol and air dry them at room temperature to obtain fixed sperm smears;

[0061] 3.22% Eosin Staining: Place the fixed sperm smear on a slide rack and place it in a 2% Eosin staining solution. Let it stand for 48 hours, rinse with water to remove excess dye, and observe under a microscope.

[0062] 200 sperms in different visual fields were examined, and the proportions of normal sperm, head deformity, neck and mid-section deformity, tail deformity, and simultaneous deformity were counted. The sperm deformity index TZI and sperm deformity index SDI were calculated. The results of rat sperm morphology examination are shown in Table 4 and Figure 3 As shown:

[0063] Table 4

[0064]

[0065] From Table 4 and Figure 3 Results showed that the normal sperm rate in the 0.20%, 0.40%, and 0.80% PbAC-treated groups was significantly decreased (P<0.001), and the sperm deformity index (SDI) was significantly increased (P<0.01). The head deformity rate (P<0.001) and the neck and mid-section deformity rate (P<0.001) in the 0.80% PbAC-treated group were significantly increased. The tail deformity rate in the 0.20% and 0.40% PbAC-treated groups was significantly increased (P<0.001, P=0.028). However, the teratozoospermia index (TZI) in the 0.04% PbAC-treated group was significantly decreased (103.18±2.78 vs. 101.34±1.39, P<0.001).

[0066] (4) Testicular paraffin sections and HE staining

[0067] After the poisoning experiment, the testicular tissue of the rats was fixed with testicular tissue fixative, and the paraffin sections were stained with hematoxylin-eosin (HEstaining) to observe the testicular structure of the rats in each group. The experimental steps are as follows:

[0068] 4.1 Sample collection: Remove the rat testicles from the fixative and trim the target area with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in the embedding frame.

[0069] 4.2 Dewaxing: Rat testicular tissue paraffin sections were dewaxed with xylene for 10 min, then dewaxed with fresh xylene for another 10 min, and then soaked in anhydrous ethanol, 90% ethanol-water solution, 80% ethanol-water solution, 70% ethanol-water solution, and distilled water in that order for 5 min.

[0070] 4.3 Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and place it into the embedding frame according to the embedding surface requirements and affix the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0071] 4.4 Sectioning: Place the trimmed wax block on a paraffin microtome to slice the tissue to a thickness of 4 μm. Float the tissue on 40°C warm water on a microtome to flatten the tissue. Pick up the tissue on a glass slide and bake it in a 60°C oven. After the moisture is dried and the wax is melted, remove the tissue and store it at room temperature for later use.

[0072] 4.5 Staining:

[0073] 4.5.1 After dewaxing, stain the rat testicular tissue sections with hematoxylin staining solution for 10 min.

[0074] 4.5.2 Rinse with tap water for 10 minutes and then wash once with distilled water to remove impurities;

[0075] 4.5.3 Soak in 1% hydrochloric acid-ethanol differentiation solution (prepared by 1 mL of 37 wt% concentrated hydrochloric acid and 99 mL of 75 wt% ethanol aqueous solution) for 30 seconds, then rinse with tap water for 10 minutes;

[0076] 4.5.4 After staining with eosin solution for 2 minutes, rinse with tap water for 10 minutes;

[0077] 4.6 Dehydration: Dehydrate the sections using 70% ethanol aqueous solution, 80% ethanol aqueous solution, 90% ethanol aqueous solution, and anhydrous ethanol, respectively. The dehydration times are: 70% ethanol aqueous solution for 10 seconds, 80% ethanol aqueous solution for 10 seconds, 90% ethanol aqueous solution for 10 seconds, and anhydrous ethanol for 10 seconds.

[0078] 4.7 Clearing: Place the dehydrated rat testicular tissue sections in xylene for clearing for 5 min, then replace with fresh xylene and clear for another 5 min;

[0079] 4.8 Sealing: Use a sealing medium to seal the transparent rat testicular tissue slices. After sealing, set aside for use;

[0080] The stained rat testicular tissue sections were observed under a common optical microscope to observe the structure of the testicular spermatogenic epithelium and diagnose tissue damage; the HE staining results of rat testis were as follows: Figure 4 As shown by Figure 4It can be seen that the testicular structure of rats in the lead acetate exposure group showed obvious pathological changes: in the control group, normal 5-7 layers of spermatogenic cells were visible in the seminiferous tubules of the testes of rats, and the cells were poleward and arranged neatly. A small number of short spindle-shaped supporting cells were visible between the spermatogenic cells, and sperm production was seen in the middle of the tubule lumen; in the 0.20% PbAC exposure group, the spermatogenic cells in the seminiferous tubules of rats were significantly reduced, with only 2-3 layers, and a small number of sperm were seen in the middle of the tubule lumen; while in the 0.40% group, the spermatogenic cells in the seminiferous tubules disappeared, leaving only short spindle-shaped or oval supporting cells, the tubule wall was slightly collapsed, and no sperm was produced in the tubule lumen.

[0081] Example 2

[0082] This embodiment discloses a method for establishing a rat sperm lead poisoning model, comprising the following steps:

[0083] Step (1), extracting EVs from seminal plasma of healthy male rats;

[0084] The experimental subjects were the same as those in step 2.1 of Example 1;

[0085] EVs are double-membrane vesicles that are shed from the cell membrane or secreted by cells. They are 50-1000 nm in diameter and include exosomes, large vesicles, and apoptotic bodies. Therefore, EVs were extracted using a 50kD ultrafiltration tube centrifugation method. The extraction steps are as follows:

[0086] 1.1 Collection of seminal vesicle and prostatic fluid: Healthy male rats were sacrificed by cervical dislocation. The seminal vesicle and prostate were placed in PBS (pH 7.4) and cut open to allow the contents to flow out. The tissues were repeatedly rinsed and discarded. The collected fluids were seminal vesicle and prostatic fluid, respectively.

[0087] 1.2 Collection of epididymal fluid: After collecting seminal vesicle fluid and prostatic fluid, the intact epididymis of the rat was cut open longitudinally and repeatedly rinsed with PBS solution. The collected semen was centrifuged and the upper layer was the epididymal fluid.

[0088] 1.3 Prepare rat seminal plasma by mixing 65% seminal vesicle fluid, 25% prostatic fluid and 10% epididymal fluid;

[0089] 1.4 Extraction of rat seminal plasma EVs from rat seminal plasma

[0090] 1.4.1 Transfer rat seminal plasma into a sterile EP tube and centrifuge at 13,000 g for 10 min at 4°C to remove cells and other substances.

[0091] 1.4.2 Take the supernatant from the centrifugation and add it to an ultrafiltration tube. Then add 10 mL of exosome diluent and centrifuge using a swing-out rotor centrifuge. Centrifuge at 22°C and 3000 g until the volume of the liquid in the ultrafiltration tube is 2 mL to remove small molecules, salts, and other substances.

[0092] 1.4.3 Add another 15 mL of exosome dilution solution to the ultrafiltration tube and repeat the above steps until the volume of the liquid in the ultrafiltration tube is approximately 250 μL. Pipet the ultrafiltration tube to wash out the liquid to obtain healthy male rat seminal plasma EVs.

[0093] Healthy male rat seminal plasma EVs were frozen at -80°C;

[0094] Step (2): Identification of EVs in seminal plasma of healthy male rats

[0095] 2.1 Determination of the morphology of EVs in seminal plasma of healthy male rats

[0096] Transmission electron microscopy (TEM) was used to observe the double-layered membrane ultrastructure of EVs in seminal plasma of healthy male rats.

[0097] Use tweezers to clamp the edge of a 300-mesh copper mesh, place the copper mesh on an inclined silica gel plate and fix it. Resuspend the healthy male rat seminal plasma EVs extracted by ultrafiltration in 300 μL PBS aqueous solution (PH = 7.4), take 100 μL of the resuspension, and drop it on the front of a carbon-loaded copper mesh with a diameter of 2 mm. After drying in a fume hood, add 3 wt% sodium phosphotungstate aqueous solution (pH = 6.8), negatively stain at room temperature for 5 minutes, gently wash once with ultrapure water droplets, dry at room temperature, dry under an incandescent lamp for 2 minutes, observe with a microscope, and observe the morphology of male rat seminal plasma EVs at 80 FkV and take pictures. Figure 5 It can be seen that the extracted vesicles are spherical and have a double-layer plasma membrane structure, which is consistent with the structural characteristics of extracellular vesicles;

[0098] 2.2 Nanoparticle Tracking Analysis (NTA)

[0099] The Brownian motion of each particle was tracked and analyzed, and the size distribution and concentration of EVs in male rat seminal plasma were calculated using the Stockes-Einstein equation:

[0100] 2 μL of rat seminal plasma EVs was pipetted into a 5 mL EP tube. 2 mL of ultrapure water was added to dilute the sample 1000-fold. The diluted sample was mixed and passed through a 0.22 μm filter membrane using a 1 mL syringe. The filtrate was centrifuged at 5000 × g for 1 min to remove small bubbles in the sample. The centrifuged sample was injected into an NS300 nanoparticle tracking analyzer (NS300, Malvern). After the particle count page stabilized, five different fields of view were selected for measurement, and the data were analyzed using NTA3.2 software.

[0101] The setting parameters of the NS300 nanoparticle tracking analyzer are as follows: CameraLevel is set to 13, TimeofVideoRecords is set to 60s, DetectThreshold is set to 6, and other parameters use the system default parameters; Figure 6 It can be seen that the diameter of the vesicles extracted from each group is in the range of 50-150 nm, the particle size peak is narrow and the size is uniform, which is consistent with the size characteristics of extracellular vesicles. The concentration distribution is 5.82×10 8 -2.57×10 9 ;

[0102] 2.3 Protein marker identification

[0103] Western blotting was used to identify the surface proteins CD9, CD63, and CD81 of EVs in male rat seminal plasma.

[0104] 2.3.1 Protein sample preparation

[0105] Dissolve RIPA lysis buffer (strong), add PMSF (phenylmethylsulfonyl fluoride) at a volume ratio of RIPA:PMSF = 100:1 to a final concentration of 1 mM PMSF, and mix thoroughly to obtain cell lysate;

[0106] Take 50 μL of rat seminal plasma EVs, add 150 μL of prepared cell lysis buffer, mix well, lyse on ice for 30 min, centrifuge at 13,000 × g for 15 min at 4°C, aspirate the supernatant and place it in a new 1.5 mL EP tube to obtain the protein stock solution;

[0107] During the aspiration process, be careful not to aspirate the precipitate, and all operations must be performed on ice;

[0108] 2.3.2 Protein concentration determination

[0109] The protein concentration of the protein stock solution was detected using the BCA protein concentration assay (enhanced, Beyotime). The operation steps are as follows:

[0110] 2.3.2.1 Place the protein stock solution in a 1.5 mL EP tube and dilute it 2-fold with PBS (pH 7.4) to obtain the sample to be tested.

[0111] The samples to be tested were placed on ice for later use;

[0112] 2.3.2.2 Prepare 0.5 mg / mL BSA protein standard solution and set aside;

[0113] 2.3.2.3 Prepare BCA working solution by mixing BCA Solution A and BCA Solution B in a 50:1 ratio based on the required sample volume (200 μL per sample). Mix thoroughly and set aside.

[0114] 2.3.2.4 Add 0.5 mg / mL BSA protein standard solution to a 96-well plate at the following times: 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL. Then, add the corresponding PBS aqueous solution (pH = 7.4) to adjust the standard concentrations to: 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL. Add 2 μL of sample to each well. Make up the volume to 20 μL in all wells with PBS aqueous solution (pH = 7.4).

[0115] 2.3.2.5 Add 200 μL of the prepared BCA working solution to each well, gently tap to mix, and incubate in a constant temperature oven at 37°C for 30 min. Stop incubation when the standard develops a clear blue color.

[0116] 2.3.2.6 Place the 96-well plate in a multifunctional microplate reader and measure the absorbance of each well at a wavelength of 562 nm;

[0117] 2.3.2.7 Use ELISAcalc software to create a standard curve equation. Substitute the absorbance value of the test sample into the equation to calculate the protein concentration in the test sample. The actual protein concentration is the calculated protein concentration × 10;

[0118] 2.3.3 Leveling and denaturation

[0119] The protein concentration of male rat seminal plasma EVs determined by the BCA protein concentration assay in 2.3.2 was uniformly mixed with 5× protein loading buffer at a ratio of 4:1, and the concentration was added at 100°C. The protein concentration of the seminal plasma EVs sample was adjusted with PBS aqueous solution (pH = 7.4) to obtain a leveled protein dilution. The protein concentration of the leveled protein dilution was 4 mg / mL. The leveled protein dilution was heated for 15 minutes to denature the protein, cooled to room temperature, and stored in a -20°C refrigerator for use.

[0120] 2.3.4SDS-PAGE electrophoresis

[0121] 2.3.4.1 Gel Preparation: Prepare 12 wt% separating gel and 5 wt% stacking gel according to the instructions of the SDS-PAGE gel preparation kit (Biyuntian);

[0122] 2.3.4.2 Glue Filling: Clean the glass plate for gel preparation and dry it in a constant temperature oven. Align the bottoms of the long and short plates and place them in the gel rack. Clamp the device. Use a 1 mL pipette to add the prepared 12% separating gel to the glass plate along the edge (add to 2 / 3 of the short plate). Then add a small amount of anhydrous ethanol to seal the edge to prevent the gel from contacting with air. Let it stand at room temperature for 30 minutes. After the separating gel solidifies, pour out the alcohol and absorb it with filter paper. Use the same method to fill the glass plate with the prepared 5% concentrated gel. Insert the comb at an angle to avoid bubbles. Let it solidify at room temperature for 30 minutes.

[0123] 2.3.4.3 SDS-PAGE Electrophoresis: Remove the comb and fill it with the prepared 1× electrophoresis buffer. Add 5 μL of the colored pre-stained protein marker (170 kD) to the leftmost lane and 40 μg of the leveled protein diluent sample to the sample well. Install the electrophoresis tank and fill it with running buffer. Connect the power supply and run the electrophoresis at 80 V for 30 min. Once the sample has entered the separating gel and formed a straight line, run the electrophoresis at 120 V for 80 min.

[0124] 2.3.5 Transfer (semi-dry method) and blocking

[0125] 2.3.5.1 Cut polyvinylidene fluoride (PVDF) membrane to a size of 5 cm × 8 cm;

[0126] 2.3.5.2 Activate the PVDF membrane by immersing it in anhydrous ethanol for 2 minutes.

[0127] 2.3.5.3 Transfer the activated PVDF membrane, filter paper, and gel to the transfer buffer prepared in step 1.4 of Example 1 and soak at room temperature for 30 seconds to fully wet them. Turn on the semi-dry transfer apparatus, cut the gel at the position of the protein marker, and place it in the order of filter paper (negative) - PVDF membrane - gel - filter paper (positive). Use a roller to compact the layers to remove air bubbles.

[0128] 2.3.5.4 Cover the transfer apparatus with the cover and transfer at a constant voltage of 20V for 20 minutes;

[0129] 2.3.5.5 Use rapid blocking solution (PBSTw, Biyuntian) as the blocking solution. After transfer, wash the membrane with PBST washing solution on a decolorizing shaker for 2 minutes and then block in blocking solution at room temperature for 1 hour.

[0130] 2.3.6 Incubation with primary and secondary antibodies

[0131] 2.3.6.1 Prepare primary antibodies for CD9, CD63, and CD81 at a volume ratio of primary antibody diluent to antibody of 1000:1. Blot the PVDF membrane dry with filter paper, place it in the primary antibody box, and incubate overnight in a shaker in a cold storage (4.3°C).

[0132] 2.3.6.2 After the primary antibody incubation is complete, wash the membrane three times with PBST on a shaker, each wash lasting 15 minutes.

[0133] 2.3.6.3 Prepare the corresponding secondary antibodies (HRP-labeled) at a volume ratio of secondary antibody diluent:antibody = 2000:1. Blot the membrane dry with filter paper, place in the secondary antibody box, and incubate at room temperature for 1 hour.

[0134] 2.3.6.4 After secondary antibody incubation, wash the membrane three times with PBST on a shaker, each wash lasting 15 minutes.

[0135] 2.3.7 Gel imaging system analysis

[0136] ECL supersensitive luminescent solution A solution: B solution = 1:1 equal volume mixed thoroughly, the washed PVDF membrane was placed in a dish containing luminescent solution, placed in a gel imaging system (Fusion FX) for exposure and photography, and the bands were analyzed using ImageJ software; Figure 7 It can be seen that the three protein indicators CD9, CD63 and CD81 were all expressed in the extracted samples, which is consistent with the characteristics of extracellular vesicle proteins;

[0137] Step (3) determining the effects of seminal plasma EVs of rats exposed to different lead concentrations on normal rat sperm:

[0138] EVs from the seminal plasma of rats exposed to different lead concentrations were extracted using the same extraction method as in step (1) and co-cultured with normal sperm of male rats;

[0139] 3.1 Determination of the effects of seminal plasma EVs on sperm motility in normal rats exposed to different lead concentrations:

[0140] Sperm from the cauda epididymis of healthy rats was extracted using the same extraction method as in 2.1 of Example 1. The cells were liquefied in a 37°C water bath for 20 min. 3 mL of the prepared BWW medium was placed at the bottom of a 15 mL sterile centrifuge tube. 2 mL of liquefied semen was slowly added along the tube wall. The cells were centrifuged at 900 × g for 5 min to separate the seminal plasma from the sperm. The supernatant liquid was discarded, and the sperm pellet was resuspended in 2 mL of BWW medium to prepare a sperm suspension. 5 μL of the suspension was dripped onto a disposable standard slide for sperm analysis (Warehouse 4, Spain). The sperm suspension concentration was assessed using the SSA-II automatic sperm detection and analysis system. The sperm suspension was adjusted with BWW medium to a sperm cell concentration close to 1 × 10 7 / mL, 1mL sperm suspension was taken twice each time in a 24-well plate, and rat seminal plasma EVs exposed to different lead concentrations and healthy male rat seminal plasma EVs were added to make the content of rat seminal plasma EVs in 1mL sperm suspension 10μg / μL, and placed in an incubator at 37°C, 5% CO2, and 95% humidity for incubation. Sperm motility was detected after 1h and 4h, respectively. The detection index was the same as 2.2 in the analysis of semen quality in Example 1; the test results of the effect of healthy male rat seminal plasma EVs on normal rat sperm motility are shown in Table 5:

[0141] Table 5

[0142]

[0143] Note: * indicates P < 0.05, ** indicates P < 0.01;

[0144] The test results of the effects of EVs from seminal plasma of rats exposed to different lead concentrations on sperm motility of normal rats (incubated for 15 minutes) are shown in Table 6:

[0145] Table 6

[0146]

[0147] The test results of the effects of EVs from seminal plasma of rats exposed to different lead concentrations on sperm motility of normal rats (incubated for 1 h) are shown in Table 7:

[0148] Table 7

[0149]

[0150] The test results of the effects of EVs from seminal plasma of rats exposed to different lead concentrations on sperm motility of normal rats (incubated for 2 hours) are shown in Table 8:

[0151] Table 8

[0152]

[0153] The test results of the effects of EVs from seminal plasma of rats exposed to different lead concentrations on sperm motility of normal rats (incubated for 4 hours) are shown in Table 9:

[0154] Table 9

[0155]

[0156]

[0157] From Table 5-9 and Figure 8It can be seen that at 15 minutes of incubation, no significant differences were found in the motility parameters of sperm co-cultured with EVs from rat seminal plasma in the control group and the 0.20% PbAC-exposed group. However, after 1 hour of incubation, the percentage of grade A sperm in the 0.20% PbAC-exposed group co-cultured with EVs was significantly reduced compared with the control group (P=0.047). After 2 hours of incubation, the percentage of grade A sperm in the 0.20% PbAC-exposed group co-cultured with EVs was significantly reduced (P=0.015), and the percentage of immotile sperm (IM), i.e., the percentage of grade D sperm, and the mean curvilinear velocity (VCL) were significantly increased (P=0.039). After 4 hours of incubation, the percentage of progressively motile sperm (PR) (P=0.022), non-progressively motile sperm (PRNP) (P=0.044), grade B sperm (P=0.020), and grade C sperm (P=0.044) in the 0.20% PbAC-exposed group EVs-co-cultured sperm were significantly reduced; the percentage of immotile sperm (IM), i.e., grade D sperm (P=0.044), was significantly increased. At the above four time points, no statistical differences were found in the motility indicators between the 0.04% PbAC-exposed group and the control group when the sperm were co-cultured with seminal plasma EVs. Figure 8 Middle: A: Effect of extracellular vesicles from the seminal plasma of healthy rats on sperm motility; B: Effect of extracellular vesicles from the seminal plasma of lead-exposed rats on sperm motility after 1 hour of incubation; C: Effect of extracellular vesicles from the seminal plasma of lead-exposed rats on sperm motility after 2 hours of incubation; D: Effect of extracellular vesicles from the seminal plasma of lead-exposed rats on sperm motility after 4 hours of incubation;

[0158] 3.2 Determine the effect of seminal plasma EVs from rats exposed to different lead concentrations on sperm capacitation in normal rats;

[0159] Sperm capacitation can be assessed by measuring sperm tyrosine phosphorylation (P-Tvr) levels using Western blotting with rat anti-phosphotyrosine antibody clone 4G10 (Millipore). The procedure is as follows:

[0160] 3.2.1 Sperm Capacitation: Prepare a sperm suspension according to the method in step 3.1. When culturing rat sperm, add 3 mg / mL bovine serum albumin (BSA) to the BWW medium to facilitate sperm capacitation. Incubate in a 37°C, 5% CO2 incubator for 4 h to obtain capacitated sperm.

[0161] 3.2.2 Sperm Protein Lysis: Collect capacitated sperm and centrifuge at 12,000 x g for 5 min at 4°C. A small amount of sperm sediment will be visible at the bottom of the EP tube. Carefully remove the supernatant and lyse the sperm proteins as in step 2.3.2. Add 50 μL of phosphatase inhibitor to the lysis buffer. Keep the entire process on ice.

[0162] 3.2.3 Western blotting: The remaining steps were the same as step 2.3, except that the PBS / PBST solution was replaced with TBS / TBST solution. The cells were blocked with 5% BSA for 1 h at room temperature. The primary antibody was rat anti-phosphotyrosine 4G10 (1:1000 dilution; Merck, Germany). Western blot intensity was analyzed using ImageJ software.

[0163] Depend on Figure 10 It can be seen that compared with the blank group, the sperm protein tyrosine phosphorylation level of the healthy male rat seminal plasma EVs supplemented was significantly increased at around 70KD (P = 0.028), while it was significantly reduced at 40-55KD and 35-40KD (P = 0.009, P = 0.009); there was no significant difference in the sperm protein tyrosine phosphorylation levels of the rats supplemented with the control group EVs and the 0.20% group EVs at 70KD, 40-55KD and 35-40KD, as shown in Figure 2. Figure 10 (C, D); Detection of Ca in sperm cells 2+ The calcium ion concentration in sperm of the EVs-supplemented group was significantly higher than that of the blank group (P=0.001); the calcium ion concentration in sperm of the control group supplemented with EVs was significantly higher than that of sperm of the 0.20% EVs-supplemented group (P<0.001); Figure 10 Middle: A: Western blotting to detect tyrosine phosphorylation of sperm proteins in the blank group and the EVs-supplemented group; B: Difference in tyrosine phosphorylation levels of sperm proteins in the blank group and the EVs-supplemented group; C: Western blotting to detect tyrosine phosphorylation of sperm proteins in the rats supplemented with EVs in the control group and the 0.20% group; D: Difference in tyrosine phosphorylation levels of sperm proteins in the rats supplemented with EVs in the control group and the 0.20% group; E: Left: Ca2+ in sperm of rats in the blank group and the EVs-supplemented group 2+ Level difference, right: rat sperm Ca supplemented with control group EVs and 0.20% group EVs 2+ Level differences;

[0164] 3.3 Determine the effect of seminal plasma EVs from rats exposed to different lead concentrations on the acrosome reaction of normal rat sperm;

[0165] The acrosome reaction test is a stable parameter of sperm function, which is mainly evaluated by the structural integrity of the acrosome. The acrosome reaction is induced and evaluated. The operation steps are as follows:

[0166] 3.3.1 Sperm Capacitation: Prepare a sperm suspension according to the method in step 3.1 and incubate it in a 37°C, 5% CO2 incubator for 3 h to obtain capacitated sperm;

[0167] 3.3.2 Acrosome reaction: After capacitation, the sperm suspension was added with Ca2+ dissolved in DMSO. 2+ Carrier A23187, Ca2+ The final concentration of vector A23187 was 10 μmol / L and the cells were incubated in a 37°C, 5% CO incubator for 1 h to induce acrosome reaction in sperm;

[0168] 3.3.3 Centrifugation and washing: The semen suspension after acrosome reaction was centrifuged at 600×g for 5 min. The supernatant was removed and 1 mL of GENMED cleaning solution (Reagent A) was added to mix the pellet. The sperm cells were counted on a disposable sperm analysis standard slide and adjusted to 2×10 7 / mL;

[0169] 3.3.4 Staining and Fixation: Pipette 10 μL of sperm cells into a new 1.5 mL EP tube, add 10 μL of GENMED staining solution (Reagent B), mix well, incubate at room temperature in the dark for 30 min, then add 1 μL of GENMED fixation solution (Reagent C) and mix well.

[0170] 3.3.5 Pushing and sealing: Immediately transfer 10 μL of the sample to one end of a clean adhesive slide. Use another slide tilted 45° to push the slide. Add 20 μL of anti-fluorescence quenching sealing solution and cover with a coverslip.

[0171] 3.3.6 Observation and counting: Immediately observe and count under a (confocal) fluorescence microscope (400X), observing green fluorescence - filter excitation wavelength 395nm, emission wavelength 510nm;

[0172] Under high-power microscope, the entire acrosome of sperm that has not undergone acrosome reaction will show uniform bright green fluorescence, and only the equatorial plane of the acrosome will show green fluorescence. Sperm heads with no or low fluorescence are reactive acrosomes. At least 200 sperm should be randomly counted to calculate the rate of sperm undergoing acrosome reaction.

[0173] The results of the determination of the effect of healthy male rat seminal plasma EVs on the acrosome reaction of normal rat sperm are shown in Table 10:

[0174] Table 10

[0175]

[0176] The results of the determination of the effects of seminal plasma EVs of rats exposed to different lead concentrations on the acrosome reaction of normal rat sperm are shown in Table 11:

[0177] Table 11

[0178]

[0179] From Table 10-11 and Figure 11It can be seen that compared with the blank group, the sperm reactive acrosome (P=0.026) and acrosome reaction rate (12.65±2.62VS16.89±2.82, P=0.036) of the sperm supplemented with EVs from the seminal plasma of healthy rats were significantly increased, indicating that extracellular vesicles may induce the premature acrosome reaction of sperm. The analysis of the effect of extracellular vesicles from the seminal plasma of lead-exposed rats on the acrosome reaction of sperm showed that compared with the control group, extracellular vesicles from the seminal plasma of rats in the 0.20% PbAC-exposed group significantly increased the number of uncapacitated acrosomes of sperm (P=0.002), and significantly decreased the number of reactive acrosomes (P=0.001) and acrosome reaction rate (11.88±1.98VS 6.34±0.96, P<0.001), indicating that extracellular vesicles in this group may have an inhibitory effect on the acrosome reaction of sperm. Figure 11 Middle: A: Acrosome reaction of rat sperm: CTC fluorescence staining. The uniform green fluorescence of the head in the left image indicates an uncapacitated acrosome, and the no fluorescence of the head in the right image indicates a reactive acrosome; B: The effect of extracellular vesicles in the seminal plasma of healthy rats on the acrosome reaction of rat sperm; C: The effect of extracellular vesicles in the seminal plasma of lead-exposed rats on the acrosome reaction of rat sperm;

[0180] 3.4 Determination of protein profiles of seminal plasma EVs in rats exposed to different lead concentrations

[0181] 3.4.1 Protein extraction and quantification: Prepare protein samples and determine protein concentration using the same method as in step 2.3 Protein marker identification in Example 2;

[0182] 3.4.2 Protein reductive alkylation and enzymatic hydrolysis:

[0183] 3.4.2.1 Take 30 μg of protein solution from each sample and dilute to 100 μL with 25 mM ammonium bicarbonate aqueous solution to obtain a protein solution;

[0184] 3.4.2.2 Reductive Alkylation: First, add dithiothreitol (DTT) (Sigma) to the protein solution and mix thoroughly to a final concentration of 10 mM. Reduce the solution at 95°C for 5 min. Then, add iodoacetamide (IAA) (Sigma) at a volume ratio of DDT:IAA = 1:5, mix thoroughly, and incubate in the dark at room temperature for 30 min for alkylation. After alkylation, centrifuge at 20,000 x g for 10 min to obtain the reduced alkylated protein solution.

[0185] 3.4.2.3 Add the reductively alkylated protein solution to a 10 kD ultrafiltration tube. Wash three times with 200 μL of 8 M urea (centrifugation at 13,500 x g), then wash twice with 200 μL of 50 mM ammonium bicarbonate (centrifugation at 13,500 x g). Discard the solution at the bottom of the ultrafiltration tube.

[0186] 3.4.2.4 Add the protein solution in the ultrafiltration tube to 150 μL of 50 mM ammonium bicarbonate solution, add trypsin (Promega) and Lys-C (intracellular protease) at a ratio of 1:50, and digest at 37°C for 16 h.

[0187] 3.4.2.5 After digestion is complete, collect the enzymatically digested peptide fragments by centrifugation: Recover the peptide fragments by centrifugation at 13500 x g into a new collection tube. To improve the recovery rate of the peptide fragments, wash the filter membrane twice with 200 μL of 50 mM ammonium bicarbonate aqueous solution, concentrate and dry by centrifugation to obtain the peptide fragments.

[0188] 3.4.3 Desalting with Monospin Desalting Column: Desalt the peptide fragments through Monospin desalting column, dry them and prepare them for mass spectrometry analysis. The desalting method is as follows:

[0189] 3.4.3.1 Dissolve the dried peptide fragments in 0.1% trifluoroacetic acid (TFA) solution to obtain a reconstituted sample;

[0190] 3.4.3.2 Activate the desalting column using 100% acetonitrile;

[0191] 3.4.3.3 Equilibrate the desalting column with 0.1% TFA solution;

[0192] 3.4.3.4 Add the reconstituted sample to the desalting column and centrifuge;

[0193] 3.4.3.5 Add 0.1% TFA solution to clean the desalting column;

[0194] 3.4.3.6 Add 50% acetonitrile solution, centrifuge to elute the peptide fragments, and collect the eluted solution in a new EP tube;

[0195] 3.4.3.7 Centrifuge and concentrate the eluted solution to remove acetonitrile;

[0196] The dried peptide fragments were dissolved in 0.1% trifluoroacetic acid (TFA), desalted using a C-18 column, and concentrated by vacuum centrifugation.

[0197] 3.4.4 Liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection:

[0198] 3.4.4.1 The peptide fragments after vacuum desalting and concentration were reconstituted with mass spectrometry solution A and centrifuged at 13,000 rpm for 5 min. The supernatant was aspirated and slowly added to the sample loading bottle. Chromatographic separation was performed using a nano-flow HPLC system EASY-nLC1200 (Thermo Scientific, USA);

[0199] 3.4.4.2 Analytical column (C-18, 1.9 μm, 75 μm × 20 cm) was used for gradient elution using 0.1% formic acid in acetonitrile in water (B) (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL / min.

[0200] 3.4.4.3 An Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific, USA) was used with positive ion detection. The primary mass spectrometer resolution was 70,000, the primary mass spectrometer scan range was 300–1800 m / z, the automatic gain control (AGC) was set to 1e6, the maximum ion injection time was 50 ms, the dynamic exclusion (DE) time was 60 s, the secondary mass spectrometer resolution was 17,500, the isolation window was 2.0 m / z, the higher energy collision dissociation (HCD) mode was used, and the normalized collision energy (NCE) was set to 30 eV.

[0201] 3.4.5 Protein Identification and Quantification Analysis: The data were searched using Thermo Fisher Scientific's Proteome Discoverer 2.5 to obtain protein ID sequences. Quality control was performed based on enzymatic digestion efficiency. Protein and peptide characteristics were analyzed using: a. protein relative molecular mass distribution; b. peptide sequence length distribution; c. distribution of the number of identified peptides; and d. protein coverage distribution.

[0202] For relative quantification of polypeptide peptides, the more accurate primary mass spectrometry-related peptide peak intensity (PrecursorIntensity) is used as the label-free quantitative parameter. When multiple peptides are detected for a protein in the search library, the peak intensities of all peptides detected for the protein are weighted to obtain the peak intensity (Abundance) of the protein expression. Figure 13 It can be seen that the relative molecular weight of proteins is mainly distributed in the range of 20-120KDa ( Figure 13 A); the number of proteins containing ≥2 unique peptides was 2954, accounting for 73.45% of the total number of proteins ( Figure 13 B); the maximum peptide length is 9, and the average length is 12.63, which is in line with the reasonable range of peptide length ( Figure 13 C); the percentage of proteins with identification coverage in the range of [0,10%] was 38.59%, the percentage of proteins with coverage ≥20% accounted for 41.42% of the total proteins, and the average protein identification coverage was 20.14% ( Figure 13 D); Figure 13Middle: A: Protein relative molecular weight distribution; B: Unique peptide number distribution; C: Peptide sequence length distribution; D: Protein coverage distribution

[0203] Furthermore, the peak intensities of each protein in each peptide sample were normalized to obtain Abundance (Normalized), and the proteins in all samples were quantitatively analyzed by Abundance.

[0204] When comparing two groups, the normalized signal mean of all samples in each group was calculated to calculate the intergroup ratio Foldchange, and the Student t-test was used to calculate the p-value of the two groups;

[0205] 3.4.5.1 For intra-group biological replicates that do not meet the statistical difference of ≥3, proteins that meet the following two conditions are selected as differentially expressed proteins between groups: a. Fold change ratio between groups ≥4 or ≤0.25 (i.e., 1 / 4); b. #Unique Peptides ≥2;

[0206] 3.4.5.2 Statistics of differences in biological replicates within the group (≥3) were used to screen proteins that met the following two conditions as differentially expressed proteins between groups: a. Fold change between groups ≥1.2 or ≤0.8333 (i.e., 1 / 1.2); bp-value <0.05. The enzyme digestion efficiency of each sample was statistically analyzed using Proteome Discoverer 2.4 database search results. The statistical results are shown in Table 12:

[0207] Table 12

[0208]

[0209] The statistical results of the total mass spectrometry database search are shown in Table 13:

[0210] Table 13

[0211]

[0212] The number of protein types in each sample obtained by searching the database is shown in Table 14:

[0213] Table 14

[0214]

[0215]

[0216] 3.4.6 Bioinformatics analysis:

[0217] 3.4.6.1 Significant difference analysis: The data of peptide samples detected by mass spectrometry are statistically calculated by T test / U test between groups, and two parameter values, p-value and Foldchange, are obtained. These two parameters are used to draw a volcano plot. The up-regulated differential proteins that meet certain Foldchange and p-value are represented by red dots, the down-regulated differential proteins are represented by green dots, and the other proteins are represented by gray dots, such as Figure 14 and Figure 15 As shown;

[0218] 3.4.6.2 Cluster analysis: For differentially regulated proteins between groups, unsupervised hierarchical cluster analysis (Hierarchical cluster) was performed. The distances between multiple samples were calculated based on protein expression data to form a distance matrix. The two closest classes were merged into a new class. The distances between the new class and the current classes were calculated. The new class was merged and calculated again until only one class remained. The expression of the selected differentially regulated proteins was used to calculate the direct correlation between the samples. In the cluster diagram, the horizontal axis represents the sample names between the groups, and the vertical axis represents the differentially regulated genes. Red represents differentially regulated genes with high expression values in the grouped samples, and blue represents differentially regulated proteins with low expression values in the grouped samples. Figure 16 and Figure 17 As shown;

[0219] 3.4.6.3 GO enrichment analysis: GO has three ontologies, which respectively describe the molecular function, cellular location, and biological process of genes. GO analysis was performed on the differentially expressed proteins between groups. The 10 entries with the smallest p-values were selected to draw a barplot (all entries were displayed if there were less than 10 entries). In the barplot, the vertical axis is the number of counts (the number of genes matching the entry), and the horizontal axis is the entry. Figure 18-21 As shown;

[0220] 3.4.6.4 KEGG Pathway Enrichment Analysis: When performing pathway enrichment analysis, differentially expressed proteins are first mapped to pathways included in the KEGG database. Further, the probability of differentially expressed proteins being mapped to different pathways is calculated using algorithms such as Fisher. This is pathway enrichment analysis. A P-value < 0.05 indicates that the pathway is significantly enriched and is not a low-probability event, but a pathway affected by the experimental treatment. Pathway analysis is performed on differentially expressed proteins between groups, and the 15 entries with the smallest p-value are selected to draw a dotplot (all entries are displayed if there are less than 15 entries). In the dotplot, the horizontal axis is GeneRatio (the number of genes (count) matching the entry in the input list / the total number of genes identified in the corresponding category in the input list), and the vertical axis is the entry. The color of the dot indicates the p-value (the statistical significance level of the enrichment analysis), and the size of the dot indicates the count (the number of genes matching the entry). Figure 22-25 As shown;

[0221] The differential proteins in the extracellular vesicles of the epididymis of lead-exposed rats and the control group are shown in Table 15:

[0222] Table 15

[0223]

[0224]

[0225] The differential proteins in the extracellular vesicles of the seminal plasma of lead-exposed rats and the control group are shown in Table 16:

[0226] Table 16

[0227]

[0228] As shown in Tables 15 and 16, compared with the control group, there were 397 differential proteins in the extracellular vesicles of the prostate fluid of the rats in the lead exposure group, of which 186 were upregulated and 211 were downregulated; there were 841 differential proteins in the extracellular vesicles of the seminal vesicle fluid, of which 341 were upregulated and 500 were downregulated; there were 37 differential proteins in the extracellular vesicles of the epididymal fluid, of which 14 were upregulated and 23 were downregulated; there were 7 differential proteins in the extracellular vesicles of the seminal plasma, of which 2 were upregulated proteins, namely very long chain acyl-CoA dehydrogenase (ACADVL) and cilium formation-related protein (TTC17), and 5 were downregulated proteins, namely nonspecific serine / threonine protein kinase, choline transporter-like protein (CTL), Src kinase-associated phosphoprotein 2 (SKAP2), Ras-associated (RalGDS / AF-6) and pleckstrin homology domain 1 (RAPH1), and heme binding protein 2 (HEBP2).

[0229] Example 3

[0230] This embodiment discloses a method for establishing a human sperm lead poisoning model, comprising the following steps:

[0231] Step (1), extraction of human seminal plasma EVs

[0232] 1.1 Selection of experimental subjects (sperm donation volunteers)

[0233] Selection criteria: ① Age 18 years or older; ② No urogenital inflammation such as orchitis or epididymitis; ③ No reproductive system trauma; ④ No obvious abnormalities of the testicles, epididymis, and accessory glands (including cryptorchidism, spermatic cord torsion, epididymal nodules, varicocele, etc.); ⑤ No fertility-related genetic diseases;

[0234] Exclusion criteria: ① Abuse of tobacco and alcohol; ② Excessive and frequent staying up late; ③ Long-term high mental stress; ④ Insufficient sex hormone secretion; ⑤ Recent use of drugs that damage the reproductive system;

[0235] The research in this invention was approved by the Ethics Committee of Xinxiang Medical College (Ethics Review Approval No.: XYLL-20170311);

[0236] Human semen donated by volunteers was frozen at -80°C;

[0237] 1.2 Extraction of human seminal plasma EVs

[0238] 1.2.1 Thaw human semen stored at -80°C naturally, transfer to a sterile EP tube, and centrifuge at 13,000 g for 10 min at 4°C to remove sperm cells and other substances.

[0239] 1.2.2 Take the centrifuged supernatant and add it to an ultrafiltration tube. Then add 10 mL of exosome diluent and centrifuge using a swing-out rotor centrifuge. Centrifuge at 22°C and 3000 g until the volume of the liquid in the ultrafiltration tube is 2 mL to remove small molecules, salts, and other substances.

[0240] 1.2.3 Add another 15 mL of exosome dilution solution to the ultrafiltration tube and repeat the above steps until the volume of the liquid in the ultrafiltration tube is approximately 250 μL. Pipet the ultrafiltration tube to wash out the liquid to obtain human seminal plasma EVs.

[0241] Human seminal plasma EVs were frozen at −80°C;

[0242] Step (2), identifying human seminal plasma EVs, using the same identification method as the identification method for healthy male rat seminal plasma EVs in step (2) of implementation 2; Step (3), determining the effects of rat seminal plasma EVs exposed to different lead concentrations on human sperm;

[0243] 3.1 Determination of the effect of rat seminal plasma EVs exposed to different lead concentrations on human sperm motility: The determination method is the same as step 3.1 of implementation 2;

[0244] The test results of the effects of human seminal plasma EVs on human sperm motility are shown in Table 17:

[0245] Table 17

[0246]

[0247] The test results of the effects of rat seminal plasma EVs exposed to different lead concentrations on human sperm motility (incubation for 1 h) are shown in Table 18:

[0248] Table 18

[0249]

[0250]

[0251] The test results of the effects of rat seminal plasma EVs exposed to different lead concentrations on human sperm motility (incubated for 4 hours) are shown in Table 19:

[0252] Table 19

[0253]

[0254] From Tables 17-19 and Figure 9 It can be seen that compared with the control group, after incubation for 1 hour, the forward motility sperm PR (P=0.035), total motility percentage PR+NP (P=0.037), grade B sperm percentage (P=0.013) and average lateral swing amplitude ALH (P=0.022) of human sperm in the 0.20% extracellular vesicle supplemented group were significantly reduced, and the immotile sperm percentage IM (P=0.034) or grade D sperm percentage (P=0.041) was significantly increased, and the differences were statistically significant. After incubation for 4 hours, the human sperm in the 0.20% group were significantly increased. The PR of sperm with forward motility (P=0.005), the percentage of total motility (PR+NP) (P=0.025), the percentage of superactivated sperm (P=0.012), the percentage of grade B sperm (P=0.022), the mean linear velocity (VSL) (P=0.032), the forward motion (STR) (P=0.026), and the mean whipping frequency (BCF) (P=0.028) of the sperm were significantly decreased, while the percentage of immotile sperm (IM) (P=0.026), i.e. the percentage of grade D sperm (P=0.027), was significantly increased. Figure 9 Middle: A: Effects of extracellular vesicles from seminal plasma of healthy men on sperm motility (incubation for 4 hours); B: Effects of extracellular vesicles from seminal plasma of lead-exposed men on human sperm motility (incubation for 1 hour); C: Effects of extracellular vesicles from seminal plasma of lead-exposed men on human sperm motility (incubation for 4 hours);

[0255] 3.2 The effects of EVs from seminal plasma of rats exposed to different lead concentrations on human sperm capacitation were determined using the following steps:

[0256] 3.2.1 Sperm capacitation: A sperm suspension was prepared according to the method in step (3) of Example 2. When culturing human sperm, 3 mg / mL human serum albumin (HSA) was added to the BWW medium to facilitate sperm capacitation. The suspension was incubated in an incubator at 37°C and 5% CO2 for 4 h to obtain capacitated sperm.

[0257] 3.2.2 Sperm protein lysis: the method is the same as step 3.2.2 of Example 2;

[0258] 3.2.3 Western blotting: The method is the same as step 3.2.3 of Example 2; Figure 10 It can be seen that the detection of Ca in sperm cells 2+ The calcium ion concentration in sperm of the EVs-supplemented group was significantly higher than that of the blank group; the calcium ion concentration in sperm of the control group supplemented with EVs was significantly higher than that of sperm of the 0.20% EVs-supplemented group. Figure 10 Middle, F: Left: Ca in human sperm cells of blank group and group supplemented with EVs from healthy men 2+ Level difference, right panel: Human sperm Ca2+ supplemented with control EVs and 0.20% EVs 2+ Level differences;

[0259] 3.3 Determine the effect of seminal plasma EVs from rats exposed to different lead concentrations on the acrosome reaction of normal rat sperm;

[0260] The determination method is the same as step 3.3 of Example 2;

[0261] The results of the determination of the effect of human seminal plasma EVs on the acrosome reaction of human sperm are shown in Table 20:

[0262] Table 20

[0263]

[0264] The results of the determination of the effects of rat seminal plasma EVs exposed to different lead concentrations on the acrosome reaction of human sperm are shown in Table 21

[0265] Table 21

[0266]

[0267] From Table 20-21 and Figure 12 It can be seen that compared with the blank group, the number of uncapacitated acrosomes in sperm supplemented with EVs from healthy male seminal plasma was significantly reduced (P = 0.027), and the number of reactive acrosomes (P < 0.001) and acrosome reaction rate (20.98 ± 1.08 VS 31.90 ± 4.34, P = 0.004) were significantly increased, indicating that extracellular vesicles may induce premature acrosome reaction in human sperm, as shown in Table 20. Figure 12 (B); if Figure 12 (C) Compared with the control group, the number of uncapacitated acrosomes in human sperm of rat seminal plasma EVs supplemented with 0.20% PbAC was significantly reduced (P=0.003), and the number of reactive acrosomes (P=0.001) and acrosome reaction rate (29.88±2.88 vs 20.32±2.54, P=0.001) were significantly increased, indicating that lead-poisoned extracellular vesicles may pass through, leading to a decrease in the acrosome reaction rate of human sperm by some mechanism. Figure 12 Middle: A: Acrosome reaction of human sperm; B: Effect of extracellular vesicles from healthy male seminal plasma on the acrosome reaction of human sperm; C: Effect of extracellular vesicles from lead-exposed seminal plasma on the acrosome reaction of human sperm.

[0268] Example 4

[0269] This embodiment discloses a method for establishing a sperm damage repair model, comprising the following steps:

[0270] Step (1) Preparation of a preparation for optimizing sperm function via the extracellular vesicle pathway

[0271] 1.1 Construction of plasmids;

[0272] The mouse SKAP2-CDS sequence was amplified by PCR from mouse cDNA;

[0273] The components of the PCR reaction system are: 2× PhantaMax Master Mix 25 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, mouse cDNA template 10 ng, DNA polymerase 1 μL, and the balance is deionized water, added to 50 μL;

[0274] The PCR reaction procedure is as follows: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 1 min; (3) annealing at 55°C for 2 min; (4) extension at 72°C for 90 s per kb (DNA kilobase pair) DNA fragment; (5) cycling (2)-(4) for 35 cycles; (6) full extension at 72°C for 5 min to ensure that all DNA segments are fully extended.

[0275] The mouse SKAP2 PCR reaction product (mouse SKAP2-CDS sequence) was stored at 10°C;

[0276] The sequence of the mouse SKAP2-CDS forward primer is shown in SEQ ID NO. 1:

[0277] 5'-TGATAAGGCCATTGCCGTGGATCCAATGCCCAACCCCAGCTGTACCTCTT-3';

[0278] The sequence of the mouse SKAP2-CDS reverse primer is shown in SEQ ID NO. 2:

[0279] 5'-CGCCGCTGCCGCCACCGCCGAATTCAATATCATACATCTCCATTAGGTAG-3';

[0280] The mouse SKAP2-CDS sequence was inserted into the PHY-GFP plasmid between the BamHI and EcoRI sites to obtain a recombinant plasmid;

[0281] The protein sequence of mouse SKAP2 is shown in SEQ ID NO.3:

[0282]

[0283] The process of inserting the mouse SKAP2-CDS sequence into the PHY-GFP plasmid between the BamHI and EcoRI sites includes: using BamHI and EcoRI endonucleases to perform double enzyme digestion on the mouse SKAP2 PCR reaction product and the PHY-GFP plasmid, respectively, the double enzyme digestion reaction is reacted in a 37°C water bath for 2 hours, and the target gene fragments are respectively recovered by 1% agarose gel electrophoresis, and the target gene fragments are reacted with the plasmid fragment using DNA ligase to connect the target gene fragments with the plasmid fragments, and the reaction conditions are 16°C for 12 hours to obtain a recombinant plasmid;

[0284] Furthermore, the successful construction of the recombinant plasmid and the presence of the target gene can be confirmed by culture, screening, and identification steps, including the following steps: taking 10 μL of the recombinant plasmid and mixing it with 100 μL of DH5a competent bacteria, ice bathing for 30 minutes, heat shocking at 42°C for 90 seconds, and immediately placing it on ice for 5 minutes, adding 700 μL of LB medium preheated to room temperature, and incubating it at 37°C in a constant temperature shaker for 50 minutes, taking 200 μL of the bacterial solution, mixing it with a pipette, and evenly spreading it on an LB plate containing 100 μg / mL Ampicillin resistance, incubating it inverted at 37°C in a constant temperature incubator overnight, picking 5 single colonies and inoculating them in 5 mL of LB culture medium containing 100 μg / mL Ampicillin resistance, incubating it at 300 rpm and 37°C in a constant temperature shaker overnight, amplifying the overnight bacterial solution, selecting the positive bacterial solution, extracting the plasmid using a plasmid mini-preparation kit, and then sequencing and verifying it;

[0285] 1.2 Cell transfection and cell line construction;

[0286] (1) Using EZ Transfection Reagent for recombinant plasmid transfection: dilute EZ Trans transfection reagent into serum-free high-glucose DMEM medium, mix well, and obtain diluted transfection reagent; dilute recombinant plasmid DNA into serum-free high-glucose DMEM medium, mix well, add diluted transfection reagent, mix well, and let stand at room temperature for 15 minutes to complete recombinant plasmid transfection and form EZ Trans-DNA complex, i.e., recombinant plasmid product;

[0287] The EZ Trans-DNA complex was evenly added to the culture dish containing HEK293T cells and gently shaken to evenly disperse the EZ Trans-DNA complex. The cells were co-incubated in a 37°C, 5% CO2 incubator for 18 hours. Fresh high-glucose DMEM medium containing serum was replaced and the cells were co-incubated for 24 hours to analyze the expression of the transferred gene fragments. After transfection, the cells were centrifuged at 4000 rpm for 5 minutes at 4°C and the supernatant was collected.

[0288] When the HEK-293T cells grew to 80%, the collected supernatant and an equal volume of fresh MEM medium (containing 10% exosome-free fetal bovine serum) were used to incubate the HEK293T cells in a 37°C, 5% CO2 incubator. After incubation for 48 hours, the HEK293T cells were transferred to MEM medium containing 1 μg / mL puromycin for further culture. After further culture for 48 hours, the HEK293T cells were transferred to MEM medium containing 0.5 μg / mL puromycin for subsequent culture to obtain a cell line;

[0289] 1.3 Isolation of exosomes;

[0290] The cell line was expanded and cultured in 10 culture dishes with a diameter of 15 cm. After the cell concentration reached 70%, the cells were rinsed three times with 1x PBS and then continued to be cultured in serum-free MEM medium. After culturing for 48 hours, the supernatant was collected; the supernatant was centrifuged at 2000g for 30 minutes at 4°C and then at 12000g for 30 minutes at 4°C. The supernatant was filtered through a 0.22μm sterilizing filter, and the supernatant was concentrated to 500μL using a 100kD ultrafiltration tube at 4000g at 4°C. 10mL of PBS was added to the concentrate, and the concentrate was concentrated again to 500μL to obtain a mouse SKAP2-HEK2293T exosome suspension, i.e., a preparation for optimizing sperm function through the extracellular vesicle pathway;

[0291] Step (2) Repairing damaged mouse sperm

[0292] 2.1 Isolation of damaged mouse sperm:

[0293] The semen of lead-exposed mice was centrifuged to separate seminal plasma and sperm. Spermatid BWW culture medium was added to the semen and centrifuged at 900 rpm for 5 minutes to obtain damaged mouse sperm.

[0294] If the seminal plasma and sperm are not completely separated, remove the supernatant and add sperm cell BWW medium again, and centrifuge at 900 rpm for 5 min until the seminal plasma and sperm are separated;

[0295] The lead poisoning process of mice was as follows: male mice were exposed to 0.1 wt% lead acetate aqueous solution through drinking water for 3 months;

[0296] 2.2 Repair incubation:

[0297] Use 24-well plates and add 2.5 × 10 samples per well. 6Place damaged mouse sperm in 1 mL of BWW medium, add 1.72 μL of the extracellular vesicle pathway-optimized sperm function preparation to each well, and incubate in a 37°C, 5% CO2, 95% humidity incubator for 1 h.

[0298] Furthermore, after the incubation was completed, the motility of the sperm was tested, and the test results are shown in Table 22:

[0299] Table 22 Improvement of mouse sperm motility level stimulated in vitro

[0300]

[0301]

[0302] a-The results were presented as the mean±SD; b-Compared with theControl,P<0.05; c-Compared with the EVs-Empty,P<0.05; d-Compared with theControl,P<0.01; e-Compared with the EVs-Empty,P<0.01;

[0303] In the table, sperm motility-related indicators include: PR (Progressive Motility, percentage of progressively motile sperm): blank control group: 14.94±2.97%, negative control group: 15.14±1.97%; treatment group: 25.92±4.92% (compared with the blank and negative control groups, P<0.001); Analysis: The percentage of progressively motile sperm in the treatment group was significantly higher than that in the blank and negative control groups, indicating that the treatment may have enhanced sperm progressive motility; PRNP (Progressive and Non-Progressive Motility, percentage of progressively and non-progressive motility): blank control group: 22.57±4.55%; negative control group: 22.96±3.13%; treatment group: 33.91±6.76% (compared with the blank and negative control groups, P<0.001); Analysis: The percentage of progressively and non-progressive motility sperm in the treatment group was also significantly higher than that in the blank and negative control groups, indicating that the treatment had a positive effect on overall sperm motility.

[0304] Sperm motility index: Hyperactivation (hyperactivation percentage): blank control group: 2.79±1.04%; negative control group: 1.68±0.73%; treatment group: 4.08±1.18% (compared with blank and negative control groups, P<0.01). Analysis: The hyperactivation percentage increased in the treatment group, indicating that treatment may promote sperm hyperactivation, a key factor in successful sperm fertilization.

[0305] Sperm motility related indicators: VCL (Curvilinear Velocity): blank control group: 21.98±3.18μm / s; negative control group: 23.50±3.91μm / s; treatment group: 41.24±8.21μm / s (compared with blank and negative control groups, P<0.001); Analysis: The curvilinear velocity of the treatment group increased significantly, indicating that the treatment improved the overall motility of sperm; VSL (Straight Line Velocity): blank control group: 4.64±1.45μm / s; negative control group: 4.54±1.06μm / s; treatment group: 8.68±2.60μm / s (compared with blank and negative control groups, P<0.01); Analysis: The straight line velocity of the treatment group also increased significantly, indicating that the treatment made the sperm move faster in the straight direction; VAP (Average Path Velocity (average path speed): blank control group: 9.80±1.73μm / s; negative control group: 10.42±1.84μm / s; treatment group: 18.22±3.93μm / s (compared with blank and negative control groups, P<0.001); Analysis: The average path speed of the treatment group increased significantly, indicating that the treatment improved the stability and efficiency of sperm motility;

[0306] Sperm morphology and dynamics indicators: STR (Straightness): blank control group: 0.10±0.03; negative control group: 0.09±0.02; treatment group: 0.15±0.03 (compared with the blank control group, P<0.05); Analysis: The linearity of the treatment group improved, but only with a significant difference compared with the blank control group, indicating that treatment may make sperm more linear during movement; BCF (Beat Cross ALH (Alpha-Hypothetical Homologous Hypothesis) was measured in the blank control group (2.22±0.53 Hz) and the negative control group (2.14±0.38 Hz). The ALH value was defined as the maximum lateral oscillation amplitude of the sperm head along its trajectory and is an important indicator of sperm dynamics and exploratory ability. The values were 2.22±0.28 μm in the blank control group (2.44±0.43 μm in the negative control group) and 4.08±0.73 μm in the treatment group (P<0.001 compared with the blank and negative control groups). The results indicate that the ALH value in the treatment group was significantly higher than that in the blank and negative control groups, suggesting that treatment may enhance lateral oscillation of the sperm head, thereby improving sperm dynamics and exploratory ability in complex environments. The clinical significance of ALH is that increased ALH may help improve sperm fertilization ability, as greater lateral head displacement may make sperm more flexible as they traverse the female reproductive tract, making it easier for them to find the egg and complete fertilization. Furthermore, ALH is an important parameter for assessing sperm quality and male fertility.

[0307] Depend on Figure 26 It can be seen that mouse SKAP2-HEK2293T exosomes can improve mouse sperm motility indicators; after mouse SKAP2-HEK2293T exosomes stimulated and damaged mouse sperm in vitro, compared with the blank control (Blank Control) and empty HEK293T exosomes (EVs-Empty), sperm anterior motility and sperm vitality were significantly increased, and each case was higher than the original control. In addition, its motility indicators ALH, BCF, STR, VAP, VCL, VSL and sperm Hyperactivation (superactivation percentage) were significantly improved compared with the two control groups.

[0308] In the figure, PR stands for preterm motility rate, which is the sum of grade A and grade B sperm; PRNP is sperm motility, which represents the sum of grade A, grade B and grade C sperm.

[0309] Example 5

[0310] This embodiment discloses a method for establishing a sperm damage repair model, comprising the following steps:

[0311] Step (1) Preparation of a preparation for optimizing sperm function via the extracellular vesicle pathway

[0312] 1.1 Construction of plasmids;

[0313] The human SKAP2-CDS sequence was amplified from human cDNA by polymerase chain reaction;

[0314] The components and reaction procedures of the PCR reaction system were the same as those in Example 4;

[0315] The sequence of the forward primer of human SKAP2-CDS is shown in SEQ ID NO.4:

[0316] 5'-TGATAAGGCCATTGCCGTGGATCCAATGCCCAACCCCAGCAGCACCTCCT-3';

[0317] The sequence of the reverse primer of human SKAP2-CDS is shown in SEQ ID NO.5:

[0318] 5'-CGCCGCTGCCGCCACCGCCGAATTCAATATCATACATCTCCATTATGTAG-3';

[0319] The SKAP2-CDS sequence was inserted into the PHY-GFP plasmid between the BamHI and EcoRI sites to obtain a recombinant plasmid;

[0320] The protein sequence of human SKAP2 is shown in SEQ ID NO.6:

[0321]

[0322] The process of inserting the human SKAP2-CDS sequence into the PHY-GFP plasmid between the BamHI and EcoRI sites was the same as the process of inserting the mouse SKAP2-CDS sequence into the PHY-GFP plasmid between the BamHI and EcoRI sites in Example 4;

[0323] 1.2 Cell transfection and cell line construction;

[0324] The procedures for cell transfection and cell line construction were the same as those in Example 4;

[0325] 1.3 Isolate exosomes to obtain human SKAP2-HEK2293T exosome suspension, i.e., a preparation for optimizing sperm function through the extracellular vesicle pathway;

[0326] The operation process of isolating exosomes is the same as that of Example 4;

[0327] Step (2) Repairing damaged human sperm

[0328] 2.1 Isolation of damaged human sperm;

[0329] Semen from humans with asthenozoospermia was centrifuged to separate seminal plasma and spermatozoa, and spermatid BWW culture medium was added to the semen, and the mixture was centrifuged at 900 rpm for 5 minutes to obtain damaged human sperm.

[0330] If the seminal plasma and sperm are not completely separated, remove the supernatant and add sperm cell BWW medium again, and centrifuge at 900 rpm for 5 min until the seminal plasma and sperm are separated;

[0331] 2.2 Repair incubation;

[0332] Use 24-well plates and add 2.5 × 10 samples per well. 6 Place damaged human sperm in 1 mL of BWW medium, add 1.72 μL of the extracellular vesicle pathway-optimized sperm function preparation to each well, and incubate in a 37°C, 5% CO2, 95% humidity incubator for 1 h.

[0333] Furthermore, after the incubation was completed, the motility of the sperm was tested, and the test results are shown in Table 23:

[0334] Table 23 Improvement of human sperm motility level stimulated in vitro

[0335]

[0336] a-The results were presented as the mean±SD; d-Comparedwith the blankcontrol,P<0.05; e-Compared with the negative control,P<0.05;

[0337] Depend on Figure 27 It can be seen that human SKAP2-HEK2293T exosomes can improve sperm motility indicators in asthenozoospermia; after human SKAP2-HEK2293T exosomes stimulated and damaged human sperm in vitro, compared with blank controls and empty HEK2293T exosomes, sperm motility and sperm vitality were significantly increased, and each case was higher than the original control, while the rate of grade D dead sperm was significantly reduced;

[0338] In the figure, A+B represents the preceding motility rate, which is the sum of the sperm rates of grade A and grade B; A+B+C is the sperm motility.

[0339] Example 6

[0340] This embodiment discloses a method for establishing a sperm damage repair model, comprising the following steps:

[0341] Step (1) Preparation of a preparation for optimizing sperm function via the extracellular vesicle pathway

[0342] 1.1 Extraction of milk-derived exosomes:

[0343] The milk was centrifuged for the first time to remove fat globules, cells and cell debris; the first centrifugation was performed at 4°C and 13,000 x g for 30 min, the supernatant of the first centrifugation was collected, the supernatant of the first centrifugation was centrifuged for the second time to remove large particles and microvesicles, the second centrifugation was performed at 4°C and 100,000 x g for 60 min, the supernatant of the second centrifugation was collected, the supernatant of the second centrifugation was centrifuged for the third time, the third centrifugation was performed at 4°C and 145,000 x g for 90 min, the centrifugal precipitate was collected, washed three times with PBS, and filtered through a 0.22 μm filter membrane to obtain an exosome suspension, i.e., milk-derived exosomes (mEXOs);

[0344] 1.2 Exosome protein coating;

[0345] Milk-derived exosomes were loaded onto SKAP2 protein using an ultrasonic system: milk-derived exosomes and SKAP2 protein were mixed in PBS at a mass ratio of 1:1. The final concentration of milk-derived exosomes in the mixture was 4 μg / mL. After ultrasonication at an amplitude of 20% for 30 seconds, the mixture was cooled for 2 minutes. The ultrasonication-cooling process was repeated 6 times. After the ultrasonication was completed, the mixture was incubated at 37°C for 60 minutes to obtain SKAP2 protein-coated milk-derived exosomes.

[0346] The SKAP2 protein-coated milk-derived exosomes (SKAP2-milk) are a preparation for optimizing sperm function through the extracellular vesicle pathway;

[0347] Furthermore, the loading rate of SKAP2 protein was determined using the following test method:

[0348] a) Milk-derived exosomes (exosome suspension) were labeled with a red fluorescent dye (Dil, Biotium, USA) as follows: the exosome suspension was mixed with Dil at a ratio of 1000 μg exosome suspension to 10 μL of Dil stock solution, and the mixture was slowly pipetted repeatedly to fully dissolve and avoid agglomeration; incubated in the dark at 37°C for 30 min, with slow repeated pipetting every 5 min (50 times), and then centrifuged at 140,000 g for 90 min. After centrifugation, the supernatant was removed, retaining the precipitate to remove the dye unbound to the exosomes. The precipitate was resuspended in sterile PBS and transferred to a light-proof EP tube to obtain Dil-mEXOs;

[0349] If the obtained Dil-mEXOs are to be used in the short term, they should be stored in a 4°C refrigerator. If they are to be stored for a long time, they should be kept in a -80°C refrigerator.

[0350] b) Dil-mEXOs and SKAP2 proteins were mixed in sterile PBS and repeatedly resuspended with a pipette to mix them evenly. The mixed system included 0.1 mL of sterile PBS, a mass ratio of Dil-mEXOs and SKAP2 proteins of 1:1, and a content of 0.4 μg / μL for each protein.

[0351] c) sonicating the mixture at 20% amplitude for 30 seconds, followed by cooling for 2 minutes, and then repeating the sonication-cooling process six times. After the sonication is complete, the mixture is incubated at 37° C. for 60 minutes. The sonication must be performed on ice.

[0352] d) Loading efficiency of SKAP2 protein: The concentration of unencapsulated SKAP2 protein in the supernatant was measured at 562 nm using a microplate reader. The SKAP2 protein loading efficiency was determined by taking the average value of multiple measurements. The determination was performed according to the following formula:

[0353] SKAP2 protein loading rate = (total SKAP2 protein concentration - SKAP2 supernatant concentration) / total SKAP2 protein concentration;

[0354] The loading rate of SKAP2 protein was measured to be 93.35%;

[0355] When determining the concentration of SKAP2 supernatant, the BCA protein concentration standard curve of SKAP2 is as follows: Figure 29 As shown;

[0356] Step (2) Repairing damaged human sperm

[0357] 2.1 Isolation of damaged human sperm;

[0358] Semen from humans with asthenozoospermia was centrifuged to separate seminal plasma and spermatozoa, and spermatid BWW culture medium was added to the semen, and the mixture was centrifuged at 900 rpm for 5 minutes to obtain damaged human sperm.

[0359] If the seminal plasma and sperm are not completely separated, remove the supernatant and add sperm cell BWW medium again, and centrifuge at 900 rpm for 5 min until the seminal plasma and sperm are separated;

[0360] 2.2 Repair incubation;

[0361] Use 24-well plates and add 2.5 × 10 samples per well. 6 Place damaged human sperm in 1 mL of BWW medium, add 1.72 μL of the extracellular vesicle pathway-optimized sperm function preparation to each well, and incubate in a 37°C, 5% CO2, 95% humidity incubator for 1 h.

[0362] Furthermore, after the incubation was completed, the motility of the sperm was tested, and the test results are shown in Table 25:

[0363] Table 24 Improvement of human sperm motility level stimulated in vitro

[0364]

[0365] a-The results were presented as the mean±SD; d-Comparedwith the blankcontrol,P<0.05; e-Compared with the negative control,P<0.05;

[0366] Depend on Figure 28It was found that SKAP2 protein-coated milk-derived exosomes can improve sperm motility in asthenozoospermia. After SKAP2 protein-coated milk-derived exosomes stimulated and damaged human sperm in vitro, sperm motility and sperm vitality were significantly increased compared to blank controls and empty milk exosomes (EVs-Empty), and in each case, the values were higher than those of the original controls.

[0367] In the figure, A+B represents the preceding motility rate, which is the sum of the sperm rates of grade A and grade B; A+B+C is the sperm motility.

[0368] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for establishing a sperm damage repair model, characterized in that: The following steps are involved: Step 1: Establish a sperm lead poisoning model; Step 2: Establish a lead poisoning sperm repair model; The method for establishing a lead poisoning sperm repair model comprises the following steps: Step (1) preparing a preparation for optimizing sperm function through the extracellular vesicle pathway; Step (2) the preparation for optimizing sperm function through the extracellular vesicle pathway repairs damaged sperm; Step (3) detecting the activity of the repaired sperm; The preparation for optimizing sperm function via the extracellular vesicle pathway is prepared by the following steps: Step (1) constructing a plasmid; The mouse SKAP2-CDS sequence was amplified by PCR from mouse cDNA; The mouse SKAP2-CDS sequence was inserted into the PHY-GFP plasmid between the BamHI and EcoRI sites to obtain a recombinant plasmid; Step (2) cell transfection and cell line construction; First, use EZ reverse transfection reagent to transfect the recombinant plasmid, and then use the recombinant plasmid product to transfect HEK293T cells. After transfection, centrifuge and collect the supernatant; HEK293T cells were cultured using the collected supernatant and an equal volume of fresh culture medium. After the culture was completed, the HEK293T cells were transferred to a culture medium containing 1 μg / mL puromycin for further culture. After further culture, the HEK293T cells were transferred to a culture medium containing 0.5 μg / mL puromycin for subsequent culture to obtain a cell line. Step (3) isolating exosomes; The cell line is expanded and cultured, washed, and then cultured in serum-free medium. After the culture is completed, the supernatant is collected; the supernatant is centrifuged, filtered, and concentrated, and PBS is added to the concentrate, which is concentrated again to obtain an exosome suspension; The exosome suspension is a preparation for optimizing sperm function through the extracellular vesicle pathway.

2. The method for establishing a sperm damage repair model according to claim 1, characterized in that: The step 1 of establishing a sperm lead poisoning model comprises the following steps: Step (1) conducting a chronic exposure experiment on rats by feeding them with lead acetate; Step (2) extracting extracellular vesicles from the seminal plasma of rats exposed to different lead concentrations and co-culturing them with normal sperm; Step (3) determining the effects of extracellular vesicles in seminal plasma of rats exposed to different lead concentrations on normal sperm; Wherein, the normal sperm includes normal rat sperm or normal human sperm.

3. The method for establishing a sperm damage repair model according to claim 1, characterized in that: The sequence of the mouse SKAP2-CDS forward primer is shown in SEQ ID NO.1; the sequence of the mouse SKAP2-CDS reverse primer is shown in SEQ ID NO.2; and the protein sequence of mouse SKAP2 is shown in SEQ ID NO.

3.

4. A method for establishing a sperm damage repair model, characterized in that: The following steps are involved: Step 1: Establish a sperm lead poisoning model; The method for establishing a sperm lead poisoning model comprises the following steps: Step (1) conducting a chronic exposure experiment on rats by feeding them with lead acetate; Step (2) extracting extracellular vesicles from the seminal plasma of rats exposed to different lead concentrations and co-culturing them with normal sperm; Step (3) determining the effects of extracellular vesicles in seminal plasma of rats exposed to different lead concentrations on normal sperm; Wherein, the normal sperm includes normal rat sperm or normal human sperm; Step 2: Establish a lead poisoning sperm repair model; The method for establishing a lead poisoning sperm repair model comprises the following steps: Step (1) preparing a preparation for optimizing sperm function through the extracellular vesicle pathway; Step (2) the preparation for optimizing sperm function through the extracellular vesicle pathway repairs damaged sperm; Step (3) detecting the activity of the repaired sperm; The preparation for optimizing sperm function via the extracellular vesicle pathway is prepared by the following steps: Step (1) constructing a plasmid; The human SKAP2-CDS sequence was amplified by PCR from human cDNA; The human SKAP2-CDS sequence was inserted into the PHY-GFP plasmid between the BamHI and EcoRI sites to obtain a recombinant plasmid; Step (2) cell transfection and cell line construction; First, use EZ reverse transfection reagent to transfect the recombinant plasmid, and then use the recombinant plasmid product to transfect HEK293T cells. After transfection, centrifuge and collect the supernatant; HEK293T cells were cultured using the collected supernatant and an equal volume of fresh culture medium. After the culture was completed, the HEK293T cells were transferred to a culture medium containing 1 μg / mL puromycin for further culture. After further culture, the HEK293T cells were transferred to a culture medium containing 0.5 μg / mL puromycin for subsequent culture to obtain a cell line. Step (3) isolating exosomes; The cell line is expanded and cultured, washed, and then cultured in serum-free medium. After the culture is completed, the supernatant is collected; the supernatant is centrifuged, filtered, and concentrated, and PBS is added to the concentrate, which is concentrated again to obtain an exosome suspension; The exosome suspension is a preparation for optimizing sperm function through the extracellular vesicle pathway.

5. The method for establishing a sperm damage repair model according to claim 4, characterized in that: The sequence of the forward primer of human SKAP2-CDS is shown in SEQ ID NO.4; the sequence of the reverse primer of human SKAP2-CDS is shown in SEQ ID NO.5; and the protein sequence of human SKAP2 is shown in SEQ ID NO.

6.

6. The method for establishing a sperm damage repair model according to claim 1 or 4, characterized in that: The preparation for optimizing sperm function through the extracellular vesicle pathway is prepared by the following steps: Step (1), extracting milk-derived exosomes; The milk is centrifuged for the first time, the supernatant of the first centrifugation is taken, the supernatant of the first centrifugation is centrifuged for the second time, the supernatant of the second centrifugation is taken, the supernatant of the second centrifugation is centrifuged for the third time, the centrifugal precipitate is collected, washed, and filtered to obtain an exosome suspension; The exosome suspension is milk-derived exosomes; Step (2), exosome protein coating; Milk-derived exosomes were loaded onto SKAP2 protein using an ultrasonic system: milk-derived exosomes and SKAP2 protein were mixed in PBS at a mass ratio of 1:

1. After ultrasonic treatment, the mixture was incubated to obtain SKAP2 protein-coated milk-derived exosomes. The SKAP2 protein-coated milk-derived exosomes are a preparation for optimizing sperm function through the extracellular vesicle pathway.

7. The method for establishing a sperm damage repair model according to claim 1 or 4, characterized in that: When establishing the lead poisoning sperm repair model, the preparation for optimizing sperm function through the extracellular vesicle pathway repairs the damaged sperm, comprising the following steps: The semen was centrifuged to separate the seminal plasma and sperm: sperm cell BWW culture medium was added to the semen and centrifuged to obtain damaged sperm; Repair incubation: Add sperm to BWW medium, add preparations for optimizing sperm function through the extracellular vesicle pathway, and incubate.

8. A sperm damage repair model obtained by using the method for establishing a sperm damage repair model according to any one of claims 1 to 7.

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