阅读说明：本技术 一种***顶体反应检测方法 (Sperm acrosome reaction detection method ) 是由 吕旭楠 李翼飞 仪菲菲 焦安军 梁栋 于 2020-06-02 设计创作，主要内容包括：本发明公开了一种精子顶体反应检测试剂,其特征在于：包括精子获能处理液,顶体反应诱导液以及染色液；其中,所述精子获能处理液含有干酪乳杆菌LC-N235发酵上清液。本发明还提供了用所述精子顶体反应试剂做成的试剂盒。本发明还提供了用所述精子顶体反应试剂做成的试剂盒进行顶体检测的方法。本发明的优点：(1)突破性缩短了精子获能所需要的时间,提高了效率。(2)保护精子,提高精子的活力从而使得样本保存不当引起的偏差减少。(The invention discloses a sperm acrosome reaction detection reagent, which is characterized in that: comprises sperm capacitation treatment liquid, acrosome reaction inducing liquid and staining liquid; wherein the sperm capacitation treatment liquid contains a lactobacillus casei LC-N235 fermentation supernatant. The invention also provides a kit prepared from the sperm acrosome reaction reagent. The invention also provides a method for detecting acrosome by using the kit made of the sperm acrosome reaction reagent. The invention has the advantages that: (1) the time required by sperm capacitation is shortened in a breakthrough manner, and the efficiency is improved. (2) Protecting sperm, improving sperm motility and reducing bias caused by sample improper storage.)

1. A sperm acrosome reaction detection reagent is characterized in that: comprises sperm capacitation treatment liquid, acrosome reaction inducing liquid and staining liquid; wherein the sperm capacitation treatment liquid contains a lactobacillus casei LC-N235 fermentation supernatant.

2. The detection reagent of claim 1, wherein the lactobacillus casei LC-N235 fermentation supernatant is prepared by:

(1) inoculating lactobacillus casei LC-N235 into an MRS solid culture medium, streaking, culturing at 37 ℃ for 24h, selecting a single colony with better growth in a flat plate, inoculating into an MRS liquid culture medium, and activating under the culture conditions: culturing at 32 deg.C and 220r/min for 24 hr to obtain 1.0 x 10 seed solution of Lactobacillus casei LC-N235⁷(CFU/ml)；

(2) Inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 3%, fermenting at the temperature of 30 ℃, performing fermentation culture for 25h, centrifuging at 1000rpm for 20min, taking the supernatant, filtering again with 200 meshes, and performing ultraviolet disinfection to obtain the seed solution;

the fermentation medium comprises the following components in percentage by weight: 10% of glucose, 2% of trehalose, 0.1% of tryptone, 2% of yeast extract, 0.05% of magnesium sulfate heptahydrate, 8% of calcium carbonate and the balance of distilled water, wherein the pH value is 6.5.

3. The detection reagent of claim 2, wherein the sperm capacitation treatment fluid is specifically formulated as: 10g/L of fermentation supernatant of Lactobacillus casei LC-N235, 0.15mol/L of sodium chloride, 1.2mmol/L of monopotassium phosphate, 5g/L of glucose, 0.04mol/L of N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid disodium salt, 3.2mmol/L of sodium bicarbonate, 0.3mmol/L of sodium pyruvate and 5g/L of bovine serum albumin, and the pH is adjusted to 7.0.

4. The detection reagent according to claim 2 or 3, wherein the staining solution is a FITC-PSA fluorescent staining solution and an propidium iodide staining solution, and the acrosome reaction inducing solution is Ca 5mg²⁺Carrier A23187 was dissolved in 9.56mL of DMSO.

5. A sperm acrosome reaction kit comprising the detection reagent of claims 1-4.

6. A method for detection of acrosomes, characterized in that the detection is carried out using the detection reagent according to claims 1 to 4 or the kit according to claim 5.

Technical Field

The invention relates to the field of sperm detection, in particular to a sperm acrosome reaction detection method suitable for high-efficiency detection of a flow cytometer.

Background

Acrosome is the specific structure of sperm, which can undergo Acrosome Reaction (AR) only if the sperm has a complete acrosome, thereby passing through the zona pellucida. Research shows that the acrosome reaction percentage of live sperms is in strong positive correlation with the transparence zone penetration rate of sperms, and the acrosome reaction rate of sperms induced by the transparence zone becomes a reliable index for evaluating the functions of the sperms. The normal sperm acrosome reaction with fertilization capability is bound to undergo fusion and rupture of a plasma membrane and an acrosome outer membrane of a sperm head to release contents, and the sign of the completion of the acrosome reaction is that the complete fusion of the acrosome outer membrane is based on the characteristic, so that the condition of the acrosome reaction of the sperm can be judged by some means, and then the fertilization capability of the sperm is evaluated.

Relatively mature acrosome detection methods have been established, wherein fluorescent staining, such as the aureomycin (CTC) staining, is highly fluorescent and easily distinguishable; the Coomassie Brilliant Blue (CBB) dyeing method is simple, convenient and economic and can be popularized in general laboratories; the acid phosphatase (ACP) detection method is objective and sensitive, and can detect multiple samples simultaneously.

Chinese patent also discloses a sperm acrosome reaction detection reagent based on flow cytometry, which comprises acrosome reaction inducing liquid and staining liquid and is characterized by also comprising sperm capacitation liquid. The detection method comprises the following steps: and standing the just taken semen for 30-60 minutes to completely liquefy the semen. Diluting semen with sperm capacitation solution to obtain about 1mL sperm suspension in the detection tube, and placing the sperm suspension at 37 deg.C and CO₂Incubate for 3 hours in the incubator to induce capacitation. 10. mu.L of the acrosome reaction-inducing solution at a concentration of 1mmol/L was added to the test tube to give a final concentration of 10. mu. mol/L, 10. mu.L of DMSO was added to the control tube, and all the tubes were incubated at 37 ℃ for 15 minutes. In the detection, 10. mu.L of PSA-FITC staining solution is added into the tube, and the tube is stained at 37 ℃ for more than 1 hour (or overnight at 4 ℃). And adding 10 mu L of PI staining solution into the detection tube, and staining for 10-15 min. And (4) detecting by using a flow cytometer, and detecting a green light signal and a red light signal by using 488nm exciting light. Sperm motility and acrosome reactivity are enhanced by the action of the sperm capacitation fluid. The disadvantage of this invention is that the energy-obtaining solution needs to be incubated for 3 hours, which has a large limitation for rapid detection.

The invention aims to provide a novel sperm acrosome reaction detection method, which shortens the time required by sperm capacitation in a breakthrough manner and improves the efficiency. In addition, sperm are protected and the motility of the sperm is improved, so that the deviation caused by improper sample preservation is reduced.

Disclosure of Invention

The invention aims to provide a novel sperm acrosome reaction detection method, which shortens the time required by sperm capacitation in a breakthrough manner and improves the efficiency. In addition, sperm are protected and the motility of the sperm is improved, so that the deviation caused by improper sample preservation is reduced.

The south-Jiang university of the Chinese patent applicant has disclosed a composition for improving the sperm motility of an isolated animal, wherein the composition comprises Enterococcus Faecium (Enterococcus faecalis). The method utilizes microorganism for the first time to improve sperm motility. However, the method needs the use of a live bacteria preparation, is difficult to store, has great use limitation, and does not find any influence on sperm capacitation.

However, the research and development team of the company is inspired by the research and development team to screen some probiotics, and the inventor finds that the fermentation supernatant of some bacteria also has the effect of improving the sperm motility, but most of the improvement effects are not obvious, and the inventor screens a strain of bacteria, and the fermentation supernatant of the strain has the very obvious effect of improving the sperm motility. Further, we have found that it can greatly reduce the time required for sperm capacitation.

The strain obtained by screening is lactobacillus casei (Lactobacillus casei) LC-N235 disclosed in Chinese patent 2012101725644 (failed), and is preserved in China Center for Type Culture Collection (CCTCC) in 2012 at 10/5 with the preservation number: CCTCC M2012157. Lactobacillus casei is a typical probiotic that is used without any biological risk. Moreover, research and development teams of the company find that lactobacillus casei of other strains has limited effect of improving sperm motility and shortening sperm capacitation time, and fermentation supernatant of the strain possibly contains specific metabolites and secondary metabolites. Since the known function of this strain is high production of L-lactic acid, we have speculated whether L-lactic acid has a positive effect on sperm capacitation, but later studies have found that L-lactic acid alone has no significant effect on sperm capacitation. Therefore, it is assumed that the main mechanism may be other metabolites, or the combined action of other metabolites and L-lactic acid.

The technical problem to be solved by the invention can be realized by the following technical scheme.

A sperm acrosome reaction detection reagent is characterized in that: comprises sperm capacitation treatment liquid, acrosome reaction inducing liquid and staining liquid; wherein the sperm capacitation treatment liquid contains a lactobacillus casei LC-N235 fermentation supernatant.

Preferably, the preparation method of the lactobacillus casei LC-N235 fermentation supernatant comprises the following steps:

(1) inoculating lactobacillus casei LC-N235 into an MRS solid culture medium, streaking, culturing at 37 ℃ for 24h, selecting a single colony with better growth in a flat plate, inoculating into an MRS liquid culture medium, and activating under the culture conditions: culturing at 32 deg.C and 220r/min for 24 hr to obtain 1.0 x 10 seed solution of Lactobacillus casei LC-N235⁷(CFU/ml)；

(2) Inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 3%, fermenting at the temperature of 30 ℃, performing fermentation culture for 25h, centrifuging at 1000rpm for 20min, taking the supernatant, filtering again with 200 meshes, and performing ultraviolet disinfection to obtain the seed solution; considering that excessive lactic acid can adversely affect sperm capacitation, we have used low temperature fermentation during fermentation, which can reduce the production of lactic acid and possibly increase the production of certain metabolites, thereby achieving the best sperm capacitation and energy enhancement effects.

The fermentation medium comprises the following components in percentage by weight: 10% of glucose, 2% of trehalose, 0.1% of tryptone, 2% of yeast extract, 0.05% of magnesium sulfate heptahydrate, 8% of calcium carbonate and the balance of distilled water, wherein the pH value is 6.5.

Preferably, the specific formula of the sperm capacitation treatment fluid is as follows: 10g/L of fermentation supernatant of Lactobacillus casei LC-N235, 0.15mol/L of sodium chloride, 1.2mmol/L of monopotassium phosphate, 5g/L of glucose, 0.04mol/L of N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid disodium salt, 3.2mmol/L of sodium bicarbonate, 0.3mmol/L of sodium pyruvate and 5g/L of bovine serum albumin, and the pH is adjusted to 7.0.

The staining solution is divided into FITC-PSA fluorescent staining solution and propidium iodide staining solution.

The acrosome reaction inducing liquid is 5mg Ca²⁺Carrier A23187 was dissolved in 9.56mL of DMSO.

The invention also provides a kit prepared from the sperm acrosome reaction reagent.

The invention also provides a method for detecting acrosome by using the kit made of the sperm acrosome reaction reagent.

The invention has the advantages that:

(1) the time required by sperm capacitation is shortened in a breakthrough manner, and the efficiency is improved.

(2) Protecting sperm, improving sperm motility and reducing bias caused by sample improper storage.

Drawings

FIG. 1 is a schematic representation of a lost cell image obtained by the sperm acrosome reaction detection method of the present invention.

Detailed Description

The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.

Specific examples of the present invention are described below.