A sperm protein marker (LYPD4) closely related to human sperm hyperactivation movement and application thereof

Abstract

The application discloses a sperm protein marker LYPD4 closely related to human sperm hyperactivation movement and an application thereof, and belongs to the technical field of biology.The application first proves that the LYPD4 protein is closely related to human sperm hyperactivation movement, and the relative content of the LYPD4 protein is significantly positively correlated with the human sperm hyperactivation rate; the LYPD4 antibody can significantly inhibit the total activity, forward movement, hyperactivation movement, penetration mucus capacity and sperm capacitation level of human sperm.The application provides a new application of the LYPD4 as a sperm function evaluation biomarker, and can be used for preparing a human sperm hyperactivation capacity detection kit, screening high-quality sperm for assisted reproduction, male infertility diagnosis and therapeutic drugs.The application fills a research blank of specific molecular markers of sperm hyperactivation movement, provides a new target and a new method for male reproductive health diagnosis and treatment and assisted reproduction technology, and has important scientific research value and clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to LYPD4, a sperm protein marker closely related to the hyperactivation of human sperm motility, and its application. Background Technology

[0002] In mammalian fertilization, sperm must undergo key steps such as capacitation, hyperactivation, and acrosome reaction to penetrate the fallopian tube mucus and zona pellucida of the egg to complete fertilization. Hyperactivated motility, characterized by high-amplitude, asymmetric flagellar beating patterns, is essential for penetrating the highly viscous fallopian tube mucus and achieving fertilization. Clinically, abnormal sperm hyperactivation is a significant factor in unexplained infertility and assisted reproductive technology failure, but the specific molecular markers and regulatory mechanisms remain unclear.

[0003] LYPD4 (LY6 / PLAUR Domain Containing 4) protein is the protein product encoded by the LYPD4 gene, located on human chromosome 19q13.2, belonging to the LY6 / PLAUR Domain Containing family. LYPD4 is an acrosomal localized protein, specifically expressed in human and mouse testes and sperm. Previous studies have shown that LYPD4 gene knockout male mice are infertile, with sperm unable to pass through the uterine-fallopian tube junction (UTJ), but the molecular mechanism remains unclear, and its regulatory role in human sperm function, its relationship with hyperactivated motility, and its clinical application value are all unknown. Summary of the Invention

[0004] Technical problem to be solved: In view of the above-mentioned technical problems, the present invention provides a sperm protein marker LYPD4 that is closely related to the hyperactivation and motility of human sperm and its application. For the first time, the regulatory role of LYPD4 in the hyperactivation, motility, capacitation and mucus penetration of human sperm is clearly defined, and a sperm function evaluation method based on LYPD4 is established.

[0005] Technical solution: In a first aspect, the present invention provides an application of the sperm protein marker LYPD4 in the preparation of human sperm function assessment products, wherein the LYPD4 protein is closely related to the hyperactivated motility of human sperm, and the application includes determining the hyperactivated motility of sperm by detecting the relative content of LYPD4 protein in human sperm.

[0006] Preferably, the human sperm function assessment product is used to assess the fertilization capacity of human sperm or to screen for high-quality sperm in assisted reproduction.

[0007] Preferably, the relative content of the LYPD4 protein is positively correlated with the hyperactivation rate of human sperm.

[0008] Secondly, the present invention provides an application of the sperm protein marker LYPD4 in regulating human sperm function, wherein LYPD4 is used to positively regulate at least one of the following sperm functions: (1) Sperm motility and forward movement ability; (2) Sperm hyperactivation and motility; (3) Sperm's ability to penetrate mucus; (4) Sperm capacitation.

[0009] Preferably, the application is to promote sperm hyperactivation, improve sperm penetration ability through mucus, or enhance sperm capacitation level.

[0010] Thirdly, the present invention provides the application of a sperm function modulator targeting LYPD4 in the preparation of a drug, the drug being used to improve sperm function or increase the success rate of assisted reproduction.

[0011] Preferably, the sperm function regulator is a reagent that can upregulate LYPD4 expression or enhance LYPD4 function.

[0012] Fourthly, the present invention provides a human sperm superactivation ability detection kit, comprising an antibody that specifically recognizes LYPD4 protein, the kit being used to assess the level of sperm superactivation motility by detecting the relative content of LYPD4 protein in sperm.

[0013] Preferably, the kit is used for sperm screening or sperm quality evaluation in assisted reproduction.

[0014] Beneficial effects: 1) It clarifies the regulatory role of LYPD4 protein in human sperm hyperactivation motility, filling the research gap in protein markers related to this motility; its positive correlation with sperm hyperactivation motility provides a key target for elucidating the regulatory mechanism of sperm function, helps to deeply elucidate the molecular mechanism of fertilization, and provides new theoretical support and research directions for basic research in reproductive biology; 2) The LYPD4 protein is involved in the regulation of sperm motility, capacitation and acrosome response, and has the characteristic of multidimensional regulation of sperm fertilization ability. Targeted intervention methods can be developed based on this protein, providing new targets for improving sperm function and treating male infertility, breaking through the limitations of traditional treatment methods and improving the accuracy of male reproductive health diagnosis and treatment. 3) Based on the positive correlation between LYPD4 protein and sperm hyperactivation motility, it can be used as a specific biomarker for assessing sperm quality. By detecting the relative content of this protein, the ability of sperm hyperactivation motility can be quickly and accurately determined, providing a new indicator for clinical sperm function testing, optimizing the sperm screening process in assisted reproductive technology, and improving the success rate of conception. 4) It provides a new direction for the research and development of male reproductive health-related products. Based on the LYPD4 protein, products such as sperm function regulators and diagnostic kits can be designed. Its specific regulatory effect can ensure the targeting and effectiveness of the products and reduce interference with the normal reproductive system. It has broad application prospects and market value in the fields of assisted reproduction, male infertility diagnosis and treatment devices and drugs. Attached Figure Description

[0015] Figure 1 This is a graph showing the results of the inhibition of human sperm motility by the LYPD4 antibody in this application. A, F, and F represent the effects of the LYPD4 antibody on total sperm motility, progressive motility, hyperactivation rate (HA%), mean curve velocity (VCL), mean path velocity (VAP), and linear velocity (VSL), respectively. The HTF group served as a positive control, and the IgG group served as a control for the LYPD4 antibody group. *P<0.05, **P<0.01, one-way ANOVA was used. Figure 2 This is a graph showing the results of the LYPD4 antibody inhibiting the ability of human sperm to penetrate mucus. The graph shows the number of sperm at 2 cm from the capillary inlet, and the sperm count was standardized based on the control group. The IgG group served as the control for the LYPD4 antibody group. *P<0.05, one-way ANOVA was used. Figure 3 This is a graph showing the results of the inhibition of human sperm capacitation by the LYPD4 antibody in this application. The graph shows the statistical data of the percentage of sperm flagellar p-Tyr. The IgG group served as the control group for the LYPD4 antibody group. **P < 0.01, one-way ANOVA was used. Figure 4 Figure A shows the results of the positive correlation between the relative content of LYPD4 protein and the hyperactivated motility of human sperm, as presented in this application. Figure B shows the expression level of LYPD4 protein in human sperm samples with different HA percentages detected by Western blot. Figure B analyzes the correlation between LYPD4 protein levels and sperm HA percentage in 41 samples. The positive correlation expression is: Y = 13.07 * X + 23.07, and the correlation coefficient R is 1 / 2. 2 The value was 0.1951, P < 0.05. Detailed Implementation

[0016] The present invention will be described in detail below with reference to specific embodiments: Reagents and solutions: HS buffer: 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 20 mM HEPES, 5 mM sucrose, 10 mM lactate, 1 mM sodium pyruvate, pH adjusted to 7.4 with NaOH. HTF energy-generating solution: 93.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 21.4 mM lactic acid, 2.78 mM glucose, 21 mM HEPES, 4 mM NaHCO3, pH 7.4; add 330 mM sodium pyruvate (1000:1), HSA (25:1) and 21 mM NaHCO3 before use.

[0017] Example 1: LYPD4 regulates human sperm motility and hyperactivated movement 1) Collect semen samples from healthy volunteers and obtain a high-moisture sperm suspension using the upstream method; 2) Centrifuge at 1800 rpm for 6 min, discard the supernatant, resuspend in HTF capacitation solution to obtain sperm resuspension; 3) The sperm resuspension was divided into three groups: HTF control group, IgG control group, and LYPD4 antibody group. The sperm was cultured at 37℃ and 5% CO2 for 4 h to obtain sperm suspension. 4) The CASA system detects total vitality, forward motility, superactivation rate, VCL, VAP, and VSL.

[0018] The results are as follows Figure 1 As shown, the total sperm motility, forward motility, hyperactivation rate, and motility speed of the LYPD4 antibody group were significantly reduced, proving that LYPD4 positively regulates human sperm motility and hyperactivation.

[0019] Example 2: LYPD4 regulates the ability of human sperm to penetrate mucus 1) The sperm were treated using the same steps as in Example 1, and the sperm were cultured in three groups for 3 hours to obtain sperm suspensions; 2) Insert 1% methylcellulose capillaries into sperm suspensions of different groups and incubate at 37°C for 1 h; 3) Count the number of sperm at 2 cm in the capillary and standardize the count based on the number of sperm in the control group.

[0020] The results are as follows Figure 2 As shown, the number of sperm that penetrated the fallopian tube mucus was significantly reduced in the LYPD4 antibody group (P < 0.05), proving that LYPD4 is a key protein for sperm to penetrate the fallopian tube mucus.

[0021] Example 3: LYPD4 regulates human sperm capacitation 1) The sperm were treated using the same steps as in Example 1, and the sperm were cultured in three groups for 4 hours to obtain sperm suspensions; 2) Take 30 μL of sperm sample, spread it evenly on an adhesive glass slide, and let it air dry at room temperature; 3) Add 1 mL of 4% paraformaldehyde to each slide for 10 min to fix. After fixation, wash 3 times with HS buffer for 5 min each time. Add 1 mL of 0.5% Triton solution to permeate sperm for 15 min. After permeation, wash 3 times with HS buffer for 5 min each time. Add 100 μL of 10% goat serum to block for 1 h. Remove excess blocking solution, add 50 μL of primary antibody working solution (p-Tyr: 1:500), and place in a humidified chamber for overnight incubation at 4 ℃ in the dark. 4) The next morning, wash three times with HS buffer, 5 min each time. Add 100 μL of Alexa Fluor® 555 conjugated secondary antibody working solution and incubate at room temperature in the dark for 1 h; wash three times with HS buffer, 5 min each time; add 10 μL of fluorescence antiquencher containing DAPI, mount the slide, and use a fluorescence confocal microscope to photograph and count the samples.

[0022] The results are as follows Figure 3 As shown, the positive rate of p-Tyr in the LYPD4 antibody group was significantly reduced (P<0.01), proving that LYPD4 positively regulates sperm capacitation.

[0023] Example 4: LYPD4 protein levels were significantly positively correlated with sperm hyperactivation rate. 1) Forty-one sperm samples with normal parameters were collected from the volunteer bank and divided into two parts. One part was washed twice and capacitated for 4 hours. The superactivation rate was detected by CASA. The other part was centrifuged at high speed to obtain sample precipitate. 2) Add an appropriate amount of strong RIPA lysis buffer containing protease inhibitor (PMSF) and phosphatase inhibitor (PIC) to the sample precipitate, sonicate for 10-15 s, and then place the sample on ice for lysis for 30 min. After lysis, centrifuge at 12000×g at 4℃ for 15-20 min, and collect the supernatant into a new EP tube. Take 20 μL of the supernatant and perform protein quantification according to the instructions of the BCA protein quantification kit. 3) After quantification, add an appropriate amount of loading buffer according to the sample loading amount of 40 μg for each sample, mix thoroughly by pipetting, denature on a metal plate at 100℃ for 5 min, and perform 12.5% ​​SDS-PAGE electrophoresis. 4) After PVDF transfer, block with 5% milk (5 g skim milk powder + 100 mL TBST) at room temperature for 1 h, and incubate with LYPD4 primary antibody at 4℃ overnight; recover the primary antibody the next day, wash the membrane with TBST buffer for 10 min, repeat 3 times; incubate with rabbit anti-secondary antibody working solution (1:10000) at room temperature in the dark for 1 h, discard the secondary antibody working solution, wash the membrane with TBST buffer for 10 min, repeat 3 times; 5) Scanning: The relative gray values ​​of the corresponding strips were measured and calculated using ImageJ software, and the correlation between the relative gray values ​​of LYPD4 and HA% was analyzed using Graphpadprism software.

[0024] The results are as follows Figure 4 As shown, the relative expression level of LYPD4 was significantly positively correlated with sperm hyperactivation rate (R²=0.1951, P<0.05), and it can be used as a specific marker for assessing sperm hyperactivation capacity.

[0025] This invention reveals for the first time that LYPD4 regulates the hyperactivated motility of human sperm, filling a gap in specific protein markers for sperm hyperactivation and providing a new target for research on the molecular mechanisms of fertilization. It also demonstrates multidimensional regulation of sperm function: simultaneously regulating motility, capacitation, hyperactivation, and mucus penetration, providing a novel therapeutic target for asthenospermia and unexplained infertility. Its clinical application value is clear: it can serve as a biomarker for precise sperm quality assessment, optimizing the sperm screening process in assisted reproductive technology and improving conception success rates. Furthermore, it has broad prospects for industrial transformation: it can be used to develop diagnostic kits, functional improvement agents, and targeted contraceptives, possessing significant medical and market value.

[0026] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a sperm protein marker LYPD4 in the preparation of human sperm function assessment products, characterized in that, The LYPD4 protein is closely related to the hyperactivated motility of human sperm, and the application includes determining the hyperactivated motility of sperm by detecting the relative content of LYPD4 protein in human sperm.

2. The application according to claim 1, characterized in that, The human sperm function assessment product is used to assess the fertilization capacity of human sperm or to screen for high-quality sperm in assisted reproduction.

3. The application according to claim 1, characterized in that, The relative content of the LYPD4 protein is positively correlated with the hyperactivation rate of human sperm.

4. The application of a sperm protein marker LYPD4 in regulating human sperm function, characterized in that, The LYPD4 is used to positively regulate at least one of the following sperm functions: (1) Sperm motility and forward movement ability; (2) Sperm hyperactivation; (3) Sperm's ability to penetrate mucus; (4) Sperm capacitation.

5. The application according to claim 4, characterized in that, The application is to promote sperm hyperactivation, improve sperm penetration ability through mucus, or enhance sperm capacitation level.

6. The application of a sperm function modulator targeting LYPD4 in drug preparation, characterized in that, The drug is used to improve sperm function or increase the success rate of assisted reproduction.

7. The application according to claim 6, characterized in that, The sperm function regulator is a reagent that can upregulate LYPD4 expression or enhance LYPD4 function.

8. A human sperm superactivation capacity detection kit, characterized in that, Containing an antibody that specifically recognizes the LYPD4 protein, the kit is used to assess the level of sperm hyperactivation by detecting the relative content of LYPD4 protein in sperm.

9. The reagent kit according to claim 8, characterized in that, The kit is used for sperm screening or sperm quality evaluation in assisted reproduction.

Images