Protein for regulating pig sperm capacitation and application thereof

Abstract

The invention belongs to the field of biology, and discloses a protein for regulating pig sperm capacitation, the protein is Rac1 protein, and the amino acid sequence is shown as SEQ ID NO: 1; in addition, the invention further discloses application of the protein to adjustment of pig sperm capacitation, the proportion of pig capacitated sperms can be increased by adding the protein into capacitation liquid, and the concentration of the protein in the capacitation liquid is 0.2-0.4 microgram / mL.

Description

Technical Field

[0001] The present application relates to the biological field, and in particular to a protein for regulating pig sperm capacitation and its application. Background Art

[0002] Pork is an indispensable source of meat in the dietary structure of Chinese residents, and it has always been at the top of the meat consumption list. Ensuring the supply of pork is not only a need for national strategic security, but also a need to meet the basic needs of people's lives. The continuous updating of modern breeding technologies such as artificial insemination, sex sorting, and embryo transplantation will help to obtain high-quality offspring in a continuous and stable manner, achieve stable production and supply of animal products, and have great application potential in animal production and breeding. Semen quality is the foundation and basis of the above-mentioned breeding technologies. How to improve sperm motility and quality is a difficult problem that my country's pig breeding industry needs to explore and solve in the long term.

[0003] Sperm capacitation is a functional modification of sperm to acquire fertilization ability. It is essential for increasing sperm motility so that it can successfully pass through the viscous fallopian tube environment, penetrate the cumulus cells and zona pellucida, and finally fuse with the oocyte membrane. The invention of in vitro sperm capacitation technology in pigs has led to the rapid development of in vitro fertilization and embryo transfer technology. At present, research on the regulatory mechanism of sperm capacitation is still relatively scarce. Actin cytoskeleton remodeling is a key link in the process of sperm capacitation. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) is involved in the polymerization of actin. Rac1 is also expressed in mammalian sperm, but its role in mammalian sperm is still unclear.

[0004] Chinese patent application 202110543785.7 discloses an application of NSC668394 in the preparation of a reagent for improving the fertilization ability of pig sperm, wherein the reagent for improving the fertilization ability of pig sperm is a mixture of a pig sperm capacitation solution containing BSA and sodium benzoate caffeine and NSC668394; the pig sperm capacitation solution containing BSA and sodium benzoate caffeine comprises the following components: KCl 0.224g / L, CaCl2·2H2O 1.102g / L, Na Cl 6.611g / L, Tr is 2.324g / L, D-glucose 1.982g / L, sodium pyruvate 0.550g / L, BSA 2g / L, and sodium benzoate caffeine 0.68g / L.

[0005] The method treats pig sperm by adding different concentrations of the small molecule compound NSC668394, detects the fertilization ability of the pig sperm, and finally obtains a concentration of the small molecule compound NSC668394 of 10 to 30 μM.

[0006] Chinese patent application 200710046321.5 discloses a protein for regulating sperm capacitation and its use. The protein is epididymis-specific expression protein HongrES1 protein. By making the protein into an agonist or antagonist, it can effectively regulate the sperm capacitation of mammals, thereby affecting the fertility of mammals.

[0007] The method can effectively inhibit or promote sperm capacitation by selecting appropriate administration time and administration site, and combining an agonist or antagonist containing 0.00001 to 0.01 g / 60 kg body weight / day of HongrES1 protein.

[0008] The problem that this program needs to solve: How to increase the proportion of capacitated sperm in pigs through Rac1 protein. Summary of the invention

[0009] The purpose of the present application is to provide a protein for regulating porcine sperm capacitation and its application, which can induce porcine sperm capacitation in vitro, optimize the in vitro capacitation effect, increase the proportion of porcine sperm capacitation in vitro, and can supplement and expand the existing capacitation solution.

[0010] To achieve the above-mentioned purpose, the present application discloses a protein for regulating pig sperm capacitation, wherein the protein is Rac1 protein.

[0011] Preferably, the amino acid sequence of the Rac1 protein is as shown in SEQ ID NO:1.

[0012] Preferably, the porcine sperm is derived from Duroc pigs.

[0013] In addition, the invention also discloses the use of the above protein in regulating the capacitation of pig sperm. The protein can increase the proportion of capacitation sperm in pigs by adding it into the capacitation liquid.

[0014] The energy-acquisition solution comprises: 3.0 mM KCl, 113.1 mM NaCl, 20 mM Trizma base, 11.0 mM D-glucose, 5.0 mM Sodium pyrurate, 7.5 mM CaCl2·2H2O concentrate, and 100 ug / mL heparin sodium.

[0015] Preferably, the concentration of the protein in the energy-harvesting solution is 0.2-0.4 ug / mL;

[0016] Further preferably, the concentration of the protein in the energy-harvesting solution is 0.31 ug / mL.

[0017] The beneficial effects of this application are:

[0018] The present application provides Western Blotting and CTC staining detection methods, which found that Rac1 inhibitor treatment can significantly reduce the level of protein tyrosine phosphorylation during porcine sperm capacitation, and lead to a significant decrease in the sperm capacitation ratio. In vitro application of recombinant human Rac1 protein to treat porcine sperm has an enhancing effect on porcine sperm capacitation, proving that Rac1 is a potential porcine sperm capacitation protein, which will lay a foundation for improving the efficiency and vitality of sperm preservation in vitro and accelerating the research progress of the mechanism of porcine sperm-egg binding, and provide new ideas and strategies for the diagnosis and treatment of abnormal sperm vitality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the phosphorylation pattern diagram of sperm capacitation treated with NSC23766. NC represents before capacitation, C-0.5h and C-1h represent 0.5h and 1h after capacitation, respectively; 0, 50, and 100 represent the treatment concentrations of NSC23766 of 0μM, 50μM, and 100μM, respectively;

[0020] Figure 2 The CTC staining results of sperm treated with NSC23766 0.5h after capacitation, A is the CTC fluorescence staining results of sperm after different treatments, A1 represents the non-capacitation treatment group, A2, A3, A4 represent NSC23766 treatment concentrations of 0μM, 50μM, 100μM; B is the fluorescence staining pattern of different sperm, B1, B2, B3 represent non-capacitation sperm, capacitation sperm, sperm with acrosome reaction, respectively; C is the quantitative results of sperm of various patterns after CTC staining;

[0021] Figure 3 The tyrosine phosphorylation results of sperm treated with human recombinant Rac1 protein for 1 hour after capacitation. NC represents no capacitation for 1 hour, C-1h represents capacitation for 1 hour, and rhRac1 represents the addition of human recombinant Rac1 protein during capacitation. DETAILED DESCRIPTION

[0022] In the description of this application, it should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0023] Example 1 Semen collection and sperm motility analysis

[0024] Prepare BTS (Beltsville Thawing Solution) pig semen diluent, in which the final concentrations of BTS components are: glucose 37.15g / L, sodium citrate 6.00g / L, sodium bicarbonate 1.25g / L, potassium chloride 0.75g / L, EDTA 1.25g / L. Dissolve in ultrapure water and adjust the pH to 7.2, add 1% double antibody, filter with 0.22uM filter membrane and store at 4°C. Collect Duroc pig semen by hand-holding method. The semen collection volume is greater than 100mL, the sperm density is greater than 200 million / mL, the sperm motility is greater than 65%, and the morphology is normal greater than 80%. The collected semen is transferred to the laboratory in a thermos cup. Centrifuge the semen at 800g for 5min at room temperature, wash and resuspend the precipitate with BTS, and adjust the sperm density to 200 million / mL.

[0025] Sperm motility was measured using the Pig Fairy sperm analyzer software. 6 μl of sample was added to a 6-chamber slide and preheated at 37°C for 5 min. Sperm motility was immediately analyzed under the SCA system, and more than 200 sperm were counted for each measurement.

[0026] Example 2 Pig sperm capacitation and inhibitor treatment

[0027] The reagents and formulas used are as follows:

[0028] Sperm washing: 0.1% BSA (m / v, sigma) PBS solution, after preparation, filter it through 0.22um filter membrane between cells, prepare it before use, and wrap it with sealing film and tin foil.

[0029] Fertilization fluid (mTBM): KCl 3.0mM, NaCl 113.1mM, Trizma.base 20mM, D-glucose 11.0mM, Sodium pyrurate 5.0mM, CaCl2·2H2O concentrate 7.5mM, filtered through a 0.22um filter membrane. Wrap with sealing film and tin foil, store at 4℃, and use within one week.

[0030] Capacitation solution (prepared immediately and used within one week): Prepared on the basis of fertilization solution. Dissolve 100ug / mL of heparin sodium in fertilization solution (mTBM). Filter with 0.22um filter membrane and equilibrate in a 37℃ 5% CO2 incubator with half-open lid for more than 40 hours.

[0031] Sperm capacitation treatment (gentle movements, avoid light): 3 portions of pig semen from different ear numbers were transferred to a 50mL centrifuge tube. The sperm concentration was analyzed using a sperm motility analyzer. 50 million sperm were taken for each treatment, and the cells were washed by centrifugation at 800g for 3min, and the supernatant was removed. The cells were washed twice with sperm washing solution preheated at 37°C, and the supernatant was removed after centrifugation. According to the different treatments, fertilization solution (uncapacitation group, 1mL) and capacitation solution (capacitation drug treatment group, 1mL) were added respectively, and the tubes were gently blown and mixed 7 to 8 times. The lid was half-opened, tilted at a 45-degree angle, and cultured in a 37°C, 5% CO2, saturated humidity incubator for different periods of time, and the supernatant was removed after centrifugation. The cell pellets were collected and stored in a -80°C refrigerator until protein extraction.

[0032] Example 3 Detection of tyrosine phosphorylation level after capacitation:

[0033] Sperm protein extraction: After thawing the sperm with different treatments, 97uL of 1% SDS protein lysis solution (Beyot ime), 1u l of PMSF (Beyot ime, 100mM), and 2uL of phosphatase inhibitor (Beyot ime) were added. After oscillation and mixing, the sperm was lysed on ice for 15min. Ultrasonication was performed on ice at 100W for 4s with an interval of 30s. After repeating 5 times, the sperm was centrifuged at 12000rpm for 10min. The supernatant was collected and the protein concentration was determined using a BCA protein quantification kit (Beyot ime).

[0034] Western Blotting analysis: Use One-Step PAGE Gel Fast Preparation Kit (Vazyme, 12%) to prepare separation gel and concentration gel. Add protein to loading buffer and boil at 100℃ for 10min. Load 30ug of protein per well. Perform electrophoresis at 230V with prestained protein marker. After protein separation, transfer to membrane at 260mA for 60min. Block with 5% BSA blocking solution (prepared with 1×TBST) on a shaker at room temperature for 2h. Use antibody anti-Phospho-Tyrosine (CST, 1:1000, diluent is TBST containing 5% BSA) and internal reference antibody anti-βtubulin (servicebio, 1:1000, diluent is 5% BSA) and incubate overnight at 4℃. 10×TBST (NaCl 88g, Tris-HCl (1M pH=8.0) 200mL, Tween 20 5mL fixed to 1000mL) was diluted to 1×TBST, and the membrane was washed 3 times with 1×TBST buffer by shaking, each time for 10 minutes, and then the secondary antibody HRP-labeled goat anti-mouse (rabbit) IgG (H+L) (Beyotime, 1:2000, diluent containing 5% BSA) was used. The membrane was washed 3 times with 1×TBST buffer by shaking, and the ECL high-sensitive chemiluminescent reagent was used for color development and photography.

[0035] During the capacitation period, pig sperm was treated with Rac1 inhibitor (NSC23766), and the tyrosine phosphorylation level of pig sperm was detected by Western Blot after treatment with inhibitor at different concentrations (0, 50, 100 μM) and time (0.5 h, 1 h). Figure 1 The test results show that there is obvious tyrosine phosphorylation at 40kDa before and after sperm capacitation, but the tyrosine phosphorylation at 32kDa is significantly enhanced after capacitation. It can be seen that as the pig sperm capacitation process proceeds, the degree of tyrosine phosphorylation is also increasing.

[0036] Then, 50 μM and 100 μM Rac1 inhibitors were used to treat the capacitated pig sperm for 0.5 h and 1 h. Figure 1 It can be seen that the tyrosine phosphorylation level at 32kDa decreased significantly. Therefore, it can be seen that Rac1 inhibitor can effectively inhibit the capacitation of pig sperm, which can lead to a significant decrease in the tyrosine phosphorylation level at 32kDa.

[0037] Example 4 CTC staining analysis of sperm capacitation and acrosome reaction rate

[0038] The reagents and formulas used are as follows:

[0039] CTC buffer: Tris final concentration 20 mM, NaCl 130 mM, pH = 7.8.

[0040] CTC staining solution: cysteine ​​5mM, CTC buffer, CTC 750uM, protect from light.

[0041] 12.5% ​​glutaraldehyde: dilute 50% glutaraldehyde with sterile water (or PBS) at a ratio of 1:3, protect from light, and pre-cool on ice before use.

[0042] CTC staining: After sperm was treated with different time and drug concentration, it was centrifuged at 2000g for 3min to remove the supernatant, 1mL PBS was added and mixed by blowing, 45uL semen was drawn and added to 45uL CTC dye solution (750uM ddH2O) to a 1.5mL centrifuge tube, mixed and wrapped with tin foil, and heated at 37℃ for 15min for staining. After staining, 15u l of the dye mixture was mixed with an equal volume of 12.5% ​​glutaraldehyde and dropped on a glass slide, anti-quenching sealing liquid was added to seal the slide, and photos were taken under a fluorescence microscope (Nikon TS100) to observe the sperm capacitation state. The experiment was repeated 3 times.

[0043] like Figure 2 The quantitative analysis results of Figure C in the figure show that when not capacitated, the proportions of capacitated sperm (A2) and non-capacitated sperm (A1) were 12.9% and 87.1%, respectively. 0.5h after capacitation, the proportion of capacitated sperm was significantly increased to 67.3%. After treatment with 50μM and 100μM Rac1 inhibitors for 0.5h, the proportion of capacitated sperm was significantly decreased to 51.4% and 48.8%. It can be seen that when the Rac1 inhibitor is added, the proportion of capacitated sperm can be significantly reduced, and the proportion of sperm undergoing acrosome reaction can also be reduced.

[0044] In summary, from Figure 1 , Figure 2 The results showed that Rac1 inhibitor could significantly reduce the tyrosine phosphorylation level during sperm capacitation and downregulate the proportion of capacitated sperm and sperm undergoing acrosome reaction. It can be seen that Rac1 protein can affect pig sperm capacitation.

[0045] Example 5 Effect of human recombinant Rac1 protein on porcine sperm capacitation

[0046] According to the above method, sperm were treated with non-capacitation, capacitation or capacitation + 0.31ug / mL human recombinant Rac1 protein. Protein was extracted from the treated sperm and the protein tyrosine phosphorylation level was detected by Western Blotting to analyze the effect of human recombinant Rac1 protein on porcine sperm capacitation. The experiment was repeated 3 times.

[0047] Among them, the amino acid sequence of Rac1 protein is:

[0048] MQAI KCVVVGDGAVGKTCLLISYTTNAFPGEEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYDRLRPLSYPQTVGETYGKDI TSRGKDKPIADVFLICFSLVSPASFENVRAKWYPEVRHHCPNTPI ILVGTKLDLRDDKDTI EKLKEKKLTPI TYPQGLAMAKEIGAVKYLECSALTQRGLKTVFDEAIRAVLCPPPVKKRKRKCLLL.

[0049] The results are as follows Figure 3 As shown, while it is known that Rac1 inhibitors can reduce the proportion of capacitated sperm, adding 0.31ug of human recombinant Rac1 protein can increase the tyrosine phosphorylation level, so Rac1 protein increases the capacitation ratio of sperm.

[0050] The above embodiments are preferred implementation modes of the present application, but the implementation modes of the present application are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be included in the protection scope of the present application.

Claims

1. A protein for regulating porcine sperm capacitation, characterized in that: The protein is Rac1 protein.

2. The protein according to claim 1, characterized in that The amino acid sequence of the Rac1 protein is shown in SEQ ID NO:

1.

3. The protein according to claim 1, characterized in that The pig sperm is derived from Duroc pigs.

4. Use of the protein according to claims 1-3 in regulating porcine sperm capacitation, characterized in that: The protein can increase the proportion of capacitated sperm in pigs by adding it to the capacitation solution; The energy-acquisition solution comprises: 3.0 mM KCl, 113.1 mM NaCl, 20 mM Trizma base, 11.0 mM D-glucose, 5.0 mM Sodium pyrurate, 7.5 mM CaCl2·2H2O concentrate, and 100 ug / mL heparin sodium.

5. The use according to claim 4, characterized in that The concentration of the protein in the energy-acquisition solution is 0.2-0.4 ug / mL.

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