Epitesticular corpuscle-specific sorting markers and epitesticular corpuscle sorting kits and methods

Abstract

The present application relates to the technical field of extracellular vesicle sorting, and particularly relates to a epididymal body specific sorting marker, and an epididymal body sorting kit and method. The present application finds that CD52 can be used as a specific marker for sorting epididymal bodies. The present application is the first time to directly separate epididymal bodies from human seminal plasma, the sample is easy to obtain, and the operation process is non-invasive, safe, and the separation path is simple and easy to operate. In addition, it provides a method for the separation of other tissue-specific EV subpopulations. The present application can contribute to further explore the effect of epididymal bodies on sperm function and improve sperm function, and epididymal bodies as a marker have great potential for clinical diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle sorting technology, and in particular to an epididymal corpuscle-specific sorting marker, an epididymal corpuscle sorting kit, and a method. Background Technology

[0002] Epididymal corpora are extracellular vesicles (EVs) secreted by epididymal epithelial cells. Numerous studies have reported that epididymal corpora can bind to sperm and deliver proteins and other substances, playing a crucial role in sperm maturation and fertilization. These roles include promoting sperm motility, protecting sperm from damage, eliminating abnormal sperm, participating in the binding of sperm and egg membranes, and the zona pellucida reaction. Furthermore, the non-coding small RNAs they deliver to sperm may be involved in the father's influence on offspring embryonic development and health. Epididymal corpora are of great significance to male reproduction. However, current robust human studies on epididymal corpora are limited. Clinically, obtaining epididymal tissue or epididymal fluid without prostatic fluid and seminal vesicle fluid is difficult. Therefore, developing a method for directly isolating epididymal corpora from human seminal plasma is essential.

[0003] Flow cytometry is an emerging technology based on the immunoaffinity principle of antigen-fluorescent antibody binding and has been used for cell sorting. However, due to factors such as small size and complex composition, and especially the difficulty in determining the specific membrane markers of EV subpopulations, studies on the separation of tissue-specific EV subpopulations using flow cytometry have not yet been reported. Summary of the Invention

[0004] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to screen for specific membrane markers of epididymal corpuscles and to develop a method for separating epididymal corpuscles based on the specific markers.

[0005] To achieve the above objectives, the present invention provides the application of CD52 as a specific marker for sorting epididymal corpuscles.

[0006] In a preferred embodiment of the present invention, the present invention provides an application of a composition for preparing a reagent for sorting epididymal corpora, said composition comprising a substance that specifically binds to CD52.

[0007] Preferably, the composition further includes substances that specifically bind to CD63, CD9 and CD81.

[0008] Preferably, the substance that specifically binds to CD52, CD63, CD9 and CD81 is a monoclonal antibody or a polyclonal antibody.

[0009] Preferably, the epididymal corpuscles are derived from humans, cattle, sheep, monkeys, and mice.

[0010] In a preferred embodiment of the present invention, a method for separating epididymal corpora from seminal plasma is provided, comprising the following steps:

[0011] 1) Isolate total EVs from semen samples;

[0012] 2) Flow cytometry was used to separate exosomes from total EVs in seminal plasma;

[0013] 3) Epididymal corpuscles were sorted using flow cytometry with CD52 as a marker;

[0014] 4) The epididymal corpora obtained by ultrafiltration are concentrated.

[0015] Preferably, the specific markers used in the exosome flow cytometry sorting include CD63, CD9, and CD81.

[0016] Preferably, step 1) includes: centrifugation to remove sperm; centrifugation to remove cell debris and other impurities; further centrifugation to remove cell debris using centrifugation parameters that are faster and longer than the previous centrifugation; ultracentrifugation for 50-90 min, discarding the supernatant, and resuspending the precipitate to obtain EVs.

[0017] Preferably, the ultracentrifugation step is repeated 1-3 times.

[0018] Preferably, the ultracentrifugation speed is 100,000 g and the temperature is 4℃±1℃.

[0019] Preferably, the antibody used for exosome flow cytometry sorting is a CD63 antibody; more preferably, the CD63 antibody is Biolegend 353004.

[0020] Preferably, the concentration of the CD63 antibody is 0.0625-1 μg.

[0021] Preferably, the antibody used for flow cytometry sorting of epididymal corpuscles is a CD52 antibody; more preferably, the CD52 antibody is Biolegend 316004.

[0022] Preferably, the concentration of the CD63 antibody is 0.0625-0.5 μg.

[0023] This invention represents the first direct isolation of epididymal corpuscles from human seminal plasma. Samples are readily available, the procedure is non-invasive, highly safe, and the isolation pathway is simple and easy to perform. Furthermore, it provides a method for isolating other tissue-specific EV subsets. This invention can contribute to further research on the role of epididymal corpuscles in sperm function and the improvement of sperm function, and epididymal corpuscles have significant potential as biomarkers for clinical diagnosis and treatment.

[0024] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0025] Figure 1 A is a transmission electron microscope image of seminal plasma EVs from normal men (NSP) and men who have undergone vasectomy (VAZ). Figure 1 B represents the NTA particle size analysis results of seminal plasma EVs from normal men (NSP) and men who have undergone vasectomy (VAZ). Figure 1 C represents the Western blot results of seminal plasma EVs from normal men (NSP) and men who have undergone vasectomy (VAZ). Figure 1 D is a laser confocal fluorescence image of seminal plasma EVs and epididymal fluid from normal men (NSP) and patients with vasectomy (VAZ);

[0026] Figure 2 A is a flow cytometry scatter plot of CD63-positive EVs at different PE-CD63 antibody concentrations; Figure 2 B is a bar chart showing the percentage of CD63-positive EVs in the antibody concentration gradient titration screening test; Figure 2 C represents the flow cytometry scattering of CD52-positive EVs at different FITC-CD52 antibody concentrations. Figure 2 D is a bar chart showing the percentage of CD52-positive EVs in the antibody concentration gradient titration screening test; Figure 2 E represents the results of epididymal corpuscle flow cytometry sorting.

[0027] Figure 3 A shows the Western blot results of seminal plasma EVs and ADAM7 expression in CD52+ and CD52- groups from normal men (NSP) and patients with vasectomy (VAZ), and the laser confocal fluorescence map of ADAM7 expression in CD52+ and CD52- groups;

[0028] Figure 4 This is a bar chart showing the effects of epididymal corpuscles (NSP+) from normal men, other seminal plasma EVs (NSP-) without epididymal corpuscles, and epididymal corpuscles (SA+) from patients with asthenospermia on the forward motility of sperm in asthenospermia at 15 min, 1 h, and 4 h. Detailed Implementation

[0029] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0030] Example 1

[0031] Screening of epididymal corpuscle-specific markers

[0032] By reviewing the literature and comparing the proteomic differences in seminal plasma between normal men and patients with vasectomy, and combining this with the protein expression data in tissues from The Human Protein Atlas website, we screened for CD52, CD36, CRISP1, and ADAM7 proteins specifically expressed in the epididymis of the male reproductive system. However, no published studies have shown that CD36 is present in epididymal bodies, and in subsequent flow cytometry experiments, we found that CD36 antibodies hardly bind to epididymal bodies. We observed CRISP1 expression in the seminal plasma EVs of patients with vasectomy using Western blot experiments, and recent studies report its possible presence in seminal vesicle fluid. Although ADAM7 protein has been shown to be specifically expressed in epididymal bodies, there is no commercially available flow cytometry antibody, making it impossible to screen epididymal bodies using ADAM7 protein. Therefore, CD36, CRISP1, and ADAM7 cannot be used as specific sorting markers for epididymal bodies.

[0033] After screening, CD52 was found to be a specific sorting marker for epididymal corpuscles.

[0034] 1. Separation of total EVs from human seminal plasma

[0035] Experimental Materials: Human semen samples were collected from the Reproductive Center of Union Hospital, Huazhong University of Science and Technology. Volunteers were aged between 25 and 35 years. Sample collection was conducted with the volunteers' consent and was reviewed by the Ethics Committee of Union Hospital. Volunteers abstained from ejaculation for 3-5 days before semen samples were obtained using a specific method and collected in disposable sterile collection cups.

[0036] Semen EV isolation method: Immediately after collection, the semen sample was placed at room temperature until complete liquefaction, followed by seminal plasma analysis. The collected semen sample was transferred to a 1.5 ml EP tube and centrifuged at 25°C, 300 g for 10 min to remove sperm. The supernatant was transferred to a new centrifuge tube and centrifuged at 4°C, 2000 g for 10 min to remove cell debris and other impurities, such as large particles. The supernatant was then transferred to a new centrifuge tube and centrifuged at 4°C, 10000 g for 30 min to remove cell debris. The supernatant was then transferred to an ultracentrifuge tube and centrifuged at 4°C, 100000 g for 70 min. The supernatant was discarded, and the precipitate was resuspended in PBS and centrifuged at 4°C, 100000 g for 70 min. The supernatant was discarded, and the precipitate, which was the EV, was resuspended in 200 μl of PBS and stored at -80°C for later use. EVs from patients with vasectomy (VAZ) were prepared using the same method.

[0037] Experiment: Transmission electron microscopy was used to photograph the seminal plasma EVs of normal males (NSP) and patients with vasectomy (VAZ) to observe their morphology; NTA particle size analysis was performed on the EVs; Western blot was used to identify and analyze the proteins in the EVs; CD52 and PKH26 were stained and fluorescence images were taken using laser confocal microscopy.

[0038] Results: Transmission electron microscopy results are as follows Figure 1 As shown in Figure A, seminal plasma EVs from normal males (NSP) and patients with vasectomy (VAZ) both exhibit a saucer-like, round or oval shape, with clearly visible membrane structures; NTA, as Figure 1 As shown in Figure B, the results indicate that the average size of seminal plasma EVs in normal men is 147.2 nm, while the average size of seminal plasma EVs in patients with vasectomy is 154.7 nm; Western blot analysis shows... Figure 1 As shown in Figure C, EVs expressed common biomarkers: CD9, CD63, and Alix, but did not express the endoplasmic reticulum biomarker Calnexin (sperm was used as a positive control). These results indicate that total EVs in seminal plasma were separated. Normal male seminal plasma EVs expressed CD52, while seminal plasma EVs from patients with vasectomy (excluding epididymal corpuscles) did not express CD52. Laser confocal microscopy results are shown below. Figure 1 As shown in Figure D, CD52 (green fluorescence) and PKH26 (red fluorescence, EVs-specific dye) are extensively colocalized in epididymal fluid; CD52 is positive in normal male seminal plasma exosomes (NSP-EVs) and colocalizes with PKH26; almost no CD52 is expressed in seminal plasma exosomes (OA-EVs, without epididymal corpora) of patients with obstructive azoospermia; the above results indicate that CD52 is specifically expressed in epididymal corpora and can be used as a sorting marker for epididymal corpora.

[0039] Example 2

[0040] 2. Separation of epididymal corpuscles from seminal plasma EVs

[0041] 1) Antibody concentration gradient titration assay: Take a flow cytometry tube, add 10 μl of total EVs from seminal plasma, and label with different concentration gradients of PE-CD63 (Biolegend, 353004) and FITC-CD52 (Biolegend, 316004) antibodies. Vortex mix in the flow cytometry tube, add 290 μl of PBS, and incubate on ice in the dark for 30 min. Use a flow cytometer (CytoFLEX, Beckman) to detect the percentage of positive rate of EVs under different concentration gradients of CD63. Take the antibody concentration with the highest percentage as the antibody concentration used. After labeling EVs with this antibody concentration, label EVs with different concentrations of CD52 antibody to determine the CD52 antibody concentration.

[0042] 2) Co-incubation of seminal plasma exosomes with flow cytometry antibodies: Take flow cytometry tubes and label them as the isotype control group and the CD63+CD52+EVs group, respectively. For the isotype control group: take 300 μl of EVs and add PE isotype control antibody and FITC isotype control antibody. For the CD63+CD52+EVs group: take 300 μl of EVs and add PE-CD63 antibody and FITC-CD52 antibody. Vortex mix in the flow cytometry tube, add 300 μl of PBS, and incubate on ice in the dark for 30 min.

[0043] 3) Flow cytometry sorting of epididymal corpora: Replace the 405 / 450nm filter and use the PB450 channel. Select small particles in the diameter range of 0-1000nm, designated as gate P1; based on the isotype control, circle the PE-CD63 positive group in gate P1, designated as gate P2; based on the isotype control, circle the FITC-CD52 negative (gate P3) and positive (gate P4) groups in gate P2, and collect the CD52-negative and CD52-positive EV subgroups. The CD63 / CD52 double-positive EV group (gate P4) is the epididymal corpora.

[0044] 4) Concentration of epididymal corpuscles: Add the sorted liquid to ultrafiltration concentration tubes, with the volume added to each tube not exceeding the top mark; pre-cool the centrifuge before centrifugation, adjust the acceleration to the lowest setting, centrifuge at 4°C, 3000g for 15 minutes; collect the retained concentrate.

[0045] result: Figure 2 A shows a flow scatter plot of CD63-positive EVs at different PE-CD63 antibody concentrations, using the isotype control antibody as a standard. Statistical analysis shows that... Figure 2 As shown in B, the highest proportion of CD63-positive EVs was found in the antibody concentration gradient titration screening test. The concentration of PE-CD63 antibody used was 0.25 μg, at which the proportion of CD63-positive EVs was approximately 15%.

[0046] For CD63-positive EVs obtained by screening with 0.25 μg PE-CD63 antibody, a gradient titration screening experiment was conducted using different concentrations of FITC-CD52 antibody. The flow cytometry scatter plots of CD52-positive EVs at different FITC-CD52 antibody concentrations are shown below. Figure 2 As shown in Figure C, the bar chart of the percentage of CD52-positive EVs is as follows: Figure 2 As shown in D, the concentration of FITC-CD52 antibody used to achieve the highest proportion of CD52-positive EVs was 0.125 μg, at which concentration CD63-positive EVs accounted for approximately 30%.

[0047] Antibody concentrations determined by antibody titration assays were used for flow cytometry sorting of epididymal corpora, such as... Figure 2As shown in E, P4 gate represents CD63 / CD52 double-positive seminal plasma EVs, i.e., epididymal corpuscles; P3 gate represents CD63 positive / CD52 negative seminal plasma EVs, i.e., seminal plasma EVs without epididymal corpuscles.

[0048] Example 3

[0049] 3. Verification of epididymal corpuscles

[0050] Experiment: ADAM7 has been shown to be specifically expressed in the epididymis of the male reproductive system and can be transferred to sperm via epididymal corpora. Seminal plasma EVs from normal men and patients with vasectomy were used to verify the specific expression of ADAM7 in epididymal corpora. ADAM7 was selected as a specific marker to verify the expression of ADAM7 in the epididymal corpora obtained in Example 2. Western blot and laser confocal microscopy experiments were used to observe the expression of ADAM7 in the sorted CD63 / CD52 double-positive EVs (CD52+) and CD63 positive / CD52 negative EVs (CD52-) obtained in Example 2.

[0051] Results: Western blot results Figure 3 A indicates that ADAM7 is present in the seminal plasma EVs of normal men but not in the seminal plasma EVs of patients with vasectomy, indicating that ADAM7 is specifically present in the epididymal corpuscles; Figure 3 A also indicates that ADAM7 is expressed in the CD52+ group but not in the CD52- group, suggesting that the EVs in the CD52+ group obtained by flow cytometry sorting are epididymal corpora.

[0052] The results of the laser confocal experiment are as follows Figure 3 As shown in Figure B, the CD52+ group contained a large amount of ADAM7 (red fluorescence), which co-localized with CD63 (green fluorescence), while the CD52- group had almost no ADAM7 fluorescence, consistent with the results of Western blot. The results of laser confocal microscopy also proved that the CD52+ group contained epididymal corpuscles, while the CD52- group contained seminal plasma exosomes without epididymal corpuscles, indicating that the epididymal corpuscles were successfully sorted.

[0053] Example 4

[0054] 4. The effect of epididymal corpuscles on sperm forward motility in patients with severe asthenospermia

[0055] Experimental Materials: Human semen samples were collected from the Reproductive Center of Union Hospital, Huazhong University of Science and Technology. Volunteers were aged 25-35 years. Sample collection was conducted with the volunteers' consent and was reviewed by the Ethics Committee of Union Hospital. Volunteers abstained from ejaculation for 3-5 days before semen samples were obtained using a specific method and collected in disposable sterile collection cups. Immediately after collection, the semen samples were placed at room temperature until complete liquefaction, followed by seminal plasma analysis. Normal males: Semen parameters were within the normal range. Patients with asthenospermia: The percentage of progressively motile sperm in the semen was less than 32%, while other semen parameters were within the normal range. Two or more semen analyses were recommended. Individuals with varicocele, acute inflammation of the urogenital system, and smokers were excluded.

[0056] Experiment: 1) Sperm separation and counting: Preheat sperm gradient centrifugation buffer and gamete buffer in a 37°C water bath; add 1 ml of 80% gradient centrifugation buffer (lower layer) to a centrifuge tube, then add 1 ml of 40% gradient centrifugation buffer (upper layer), and then add 1 ml of semen sample; centrifuge at 510g for 15 min, and remove the supernatant; add 1 ml of gamete buffer to the pellet, centrifuge at 310g for 10 min, and remove the supernatant; add an appropriate amount of sperm cell BWW culture medium to resuspend the pellet; gently pipette and mix the sperm suspension, then drop 10 μL of the sperm suspension onto a glass slide, gently cover with a coverslip, and let stand for 1 min to allow the liquid to stop drifting; observe under an optical microscope at 400x magnification, and count the number of sperm in each field of view using a hemocytometer. Observe at least 5 fields of view for each sample, and count 200 or more sperm. Calculate the average sperm count in each field of view, and then use the formula: Sperm Concentration = Average * 1 / 4 * 10 6 / ml, calculate sperm concentration. When sperm concentration is too high, dilute by the following ratios before counting: >101 sperm per field of view under 400x magnification, dilute 1:20; 16-100 sperm, dilute 1:5; 2-15 sperm, dilute 1:2.

[0057] 2) Co-incubation of normal male epididymal corpora and sperm from patients with asthenospermia: Four groups were set up: Group 1: sperm from patients with asthenospermia; Group 2: sperm from patients with asthenospermia + normal male epididymal corpora; Group 3: sperm from patients with asthenospermia + epididymal corpora from patients with asthenospermia; Group 4: sperm from patients with asthenospermia + seminal plasma exosomes from normal men without epididymal corpora. 1 ml of sperm suspension was added to each of the four groups' EP tubes, at a ratio of 2.5 × 10⁻⁶. 6 Each sperm cell was supplemented with 50 μg of epididymal corpuscles or seminal plasma exosomes, and gently mixed by pipetting. All groups were incubated in BWW medium and placed in an incubator containing 5% CO2 at 37°C for 15 min, 1 h, and 4 h, with shaking every 30 min during the incubation period.

[0058] 3) Detection of forward sperm motility: Place a 10 μL sperm sample onto a glass slide, gently cover with a coverslip, and let stand at room temperature for 1 min. Observe under an optical microscope at 400x magnification. Quickly count the number of forward-moving, stationary, and immobile sperm in a specific order within each field of view. Observe at least 5 fields of view for each sample, counting 200 or more sperm. Estimate and record the percentage of sperm showing forward motility using the formula: Forward sperm motility rate = Number of forward-moving sperm / Total sperm count. Repeat the above operation, and record the average of three consecutive estimates as the final percentage of forward sperm motility (%).

[0059] result: Figure 4 The results showed that after 15 minutes of incubation, both epididymal corpora from normal men (NSP+) and other seminal plasma EVs without epididymal corpora (NSP-) improved sperm forward motility in patients with asthenospermia. After 1 hour of incubation, epididymal corpora from normal men (NSP+) significantly improved sperm forward motility in patients with asthenospermia. Other seminal plasma EVs without epididymal corpora from normal men (NSP-) improved sperm forward motility in patients with asthenospermia, but the effect was not as good as the NSP+ group. After 4 hours of incubation, epididymal corpora from normal men (NSP+) continued to improve sperm forward motility in patients with asthenospermia, while other seminal plasma EVs without epididymal corpora from normal men (NSP-) had no significant effect on sperm forward motility in patients with asthenospermia. Epididymal corpora from patients with asthenospermia (SA+) did not improve sperm forward motility in patients with asthenospermia, and even further impaired sperm forward motility after 1 hour and 4 hours of incubation.

[0060] The EVs subpopulation obtained by screening using CD52 as a specific marker in this application can significantly improve the forward motility of sperm in asthenospermia. This is consistent with current reports that epididymal corpuscles can improve the forward motility of sperm in asthenospermia, and can strongly demonstrate that CD52 is a specific marker of epididymal corpuscles.

[0061] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of CD52 as a specific marker for sorting epididymal corpuscles.

2. The use of a composition for preparing a reagent for sorting epididymal corpora, said composition comprising a substance that specifically binds to CD52.

3. The application according to claim 2, characterized in that, The composition also includes substances that specifically bind to CD63, CD9 and CD81.

4. The application according to claim 3, characterized in that, The substance that specifically binds to CD52, CD63, CD9, and CD81 is a monoclonal antibody or a polyclonal antibody.

5. The application according to any one of claims 2-4, characterized in that, The epididymal corpuscles are derived from humans, cattle, sheep, monkeys, and rats.

6. A method for separating epididymal corpora from seminal plasma, comprising the following steps: 1) Isolate total EVs from semen samples; 2) Flow cytometry was used to separate exosomes from total EVs in seminal plasma; 3) Epididymal corpuscles were sorted using flow cytometry with CD52 as a marker; 4) The epididymal corpora obtained by ultrafiltration are concentrated.

7. The method according to claim 6, characterized in that, The specific markers used in the flow cytometry sorting of exosomes include CD63, CD9, and CD81.

8. The method according to claim 7, characterized in that, The antibody used for the flow cytometry-based specific markers of exosomes is the CD63 antibody.

9. The method according to claim 8, characterized in that, The CD63 antibody used was Biolegend 353004.

10. The method according to claim 6, characterized in that, The antibody used for flow cytometry sorting of CD52 in the epididymal corpuscles is a CD52 antibody.

11. The method according to claim 10, characterized in that, The CD52 antibody used was Biolegend 316004.

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