Method for establishing oligoasthenospermia model by using trimethylamine oxide

Abstract

The invention discloses a method for establishing an oligoasthenospermia model by using trimethylamine oxide. According to the method, trimethylamine oxide is injected into the abdominal cavity of an animal to obtain the oligoasthenospermia model. According to the invention, trimethylamine oxide (TMAO) intraperitoneal injection is carried out on a male mouse, so that the oligoasthenospermia mouse model is successfully established. The invention provides a new thought and clue for deeply researching the pathogenesis of oligoasthenospermia and researchers of male reproductive health, and has important scientific and medical values for understanding the mechanism of male infertility, developing a new therapy, evaluating the treatment effect and promoting related clinical research.

Description

Technical Field

[0001] The present invention belongs to the fields of reproductive biology technology and clinical medicine, and particularly relates to a method for establishing a model of oligoasthenospermia by using trimethylamine N-oxide. Background Art

[0002] Oligoasthenospermia, as a general term for oligospermia and asthenospermia, is a common cause of male infertility. According to the standards of the World Health Organization (WHO), oligospermia is defined as a sperm density in semen lower than 15×10 ^6 per mL or a total sperm count lower than 39×10 ^6 per ejaculation, while asthenospermia refers to a proportion of actively forward-moving sperm in semen lower than 32%. These two conditions often occur simultaneously and are collectively called oligoasthenospermia. With the acceleration of the modern social rhythm and the increase of male stress, male infertility has become a global health problem, affecting the lives of about 200 million people, and the number of patients is on the rise. Among infertile couples, about half are caused by abnormal semen in men, and this problem is gradually getting younger.

[0003] Male infertility is a multi-factor and complex disease, and its specific mechanism has not been fully understood. At present, most treatments are based on experience. Therefore, in-depth research on the pathogenesis of oligoasthenospermia is crucial for developing effective treatment methods. In recent years, certain achievements have been made in the clinical treatment of male infertility, but constructing a reasonable animal model is still very crucial for further studying the pathogenesis of oligoasthenospermia and developing therapeutic drugs. The oligoasthenospermia mouse model can simulate the pathological characteristics of human oligoasthenospermia and provide an experimental basis for further studying the pathogenesis, so that researchers can more deeply understand the physiological and molecular mechanisms of spermatogenesis disorders.

[0004] The etiology of oligoasthenospermia is complex, so there are various modeling methods for its animal model, including general drug modeling, chemotherapy drug modeling, high-fat diet modeling, physical factor modeling, gene knockout modeling, etc. TMAO is a metabolite produced by intestinal flora and can be synthesized in the host's liver. Research shows that TMAO is related to reproductive health. For example, it has been reported that the content of TMAO in the follicular fluid of normally fertilized and developed human embryos, so TMAO can be used to predict the outcome of embryo transfer. 3,3-Dimethyl-1-butanol (DMB) is an organic compound. As a choline analogue, it can effectively inhibit the conversion of choline in the intestine into trimethylamine (TMA), thereby reducing the level of TMAO in plasma and is an effective TMAO inhibitor. Summary of the Invention

[0005] The object of the present invention is to provide a method for establishing a model of oligoasthenospermia by using trimethylamine N-oxide.

[0006] The method for establishing a model of oligoasthenospermia by using trimethylamine N-oxide in the present invention is to inject trimethylamine N-oxide into the abdominal cavity of an animal to obtain a model of oligoasthenospermia.

[0007] Preferably, the animal is a mouse.

[0008] Preferably, it is injected daily at a dose of 40 mg per kilogram of body weight.

[0009] More preferably, it is injected for 5 weeks.

[0010] The present invention also provides the application of trimethylamine N-oxide in establishing a model of oligoasthenospermia.

[0011] Preferably, the animal is a mouse.

[0012] The present invention also provides the application of trimethylamine N-oxide in the preparation of a drug for establishing a model of oligoasthenospermia.

[0013] In the present invention, intraperitoneal injection of trimethylamine N-oxide (TMAO) into mice can significantly reduce the sperm motility and semen density of male mice, thereby successfully establishing a mouse model of oligoasthenospermia. This provides new ideas and clues for in-depth research on the pathogenesis of oligoasthenospermia and for researchers in male reproductive health. At the same time, it has important scientific and medical value for understanding the mechanism of male infertility, developing new therapies, evaluating treatment effects, and promoting related clinical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a conceptual diagram of the research conducted for the present invention.

[0015] Figure 2 It is the detection result of sperm motility of three groups of mouse samples.

[0016] Figure 3 It is the statistical result of semen density of three groups of mouse samples.

[0017] Figure 4 It is the WB detection result of germ cell marker proteins in testicular samples of three groups of mice. DETAILED DESCRIPTION OF THE INVENTION

[0018] The method of the present invention will be further described below with reference to the drawings and through specific examples.

[0019] Example 1

[0020] 1. Experimental animal treatment and reagent preparation

[0021] In this invention, 3-week-old male ICR mice were used as experimental subjects. The mice were housed separately in a clean-grade animal room and strictly raised according to the standard mouse breeding conditions. The mice were placed in a 12-hour light and 12-hour dark cycle, with a room temperature of 23 ± 1°C and an environmental humidity of 50%. They had free access to water and food. All procedures complied with animal ethics standards and experimental norms. A normal control group (Con), a trimethylamine N-oxide treatment group (TMAO), and a 3,3-dimethyl-1-butanol (DMB) inhibitor rescue group (TMAO + DMB) were set up. Specific treatment methods for the mice: In this experiment, 3-week-old male ICR mice were used. In the Con group, a single intraperitoneal injection of normal saline was performed every morning at a dose of 4 μL per gram of body weight for five consecutive weeks; in the TMAO group, a single intraperitoneal injection of 10 mg / mL TMAO was performed every morning at a dose of 40 mg per kilogram of body weight for five consecutive weeks; in the TMAO + DMB group, DMB was fed by drinking water. A single intraperitoneal injection of 10 mg / mL TMAO was performed every morning at a dose of 40 mg per kilogram of body weight for five consecutive weeks. The drinking water in this group was added with DMB reagent at a mass fraction of 1%, and the other two groups drank normal water.

[0022] Preparation of TMAO solution: Transfer all 500 mg of the metabolite TMAO (A292360, Ambeed, China) to a 50 mL centrifuge tube, add 50 mL of sterilized normal saline to completely dissolve it, and the concentration is 10 mg / mL. Perform intraperitoneal injection with a 1 mL syringe at a dose of 40 mg per kilogram of body weight to ensure that 40 μL of the metabolite working solution corresponds to 1 g of mouse body weight.

[0023] Preparation of DMB solution: Add DMB reagent (A280873, Ambeed, China) to the drinking water in the TAMO + DMB group at a ratio of 1%. That is, take 2.5 g of DMB and add it to 250 mL of drinking water.

[0024] 2. Sample collection

[0025] After continuous intraperitoneal injection for 5 weeks, samples such as testes and semen of different treatment groups were collected for comparative detection experiments. After decapitating and sacrificing the mice, spray alcohol on the abdominal skin of the mice and expose the position of their testes with sterilized surgical scissors. Place the testes in a centrifuge tube and store them at -80°C for subsequent Western Blot experiments; pick up the epididymis of the mice, release the sperm for in vitro capacitation and measure the sperm motility data.

[0026] 3. Sperm motility and semen density detection

[0027] Prepare the sperm capacitation medium in advance. Mix α-MEM / F12 and FBS evenly at a volume ratio of 9:1 and preheat it on a 37°C heating table. Sacrifice the mice by cervical dislocation, carefully remove the cauda epididymis tissue and place it in a 3 cm dish containing 200 μL of sperm capacitation medium. Use a blade to cut open the cauda epididymis to release fresh sperm and time for 5 minutes. Then, aspirate 20 μL of the turbid liquid and dilute it in a new 200 μL of sperm capacitation medium and mix evenly to obtain sperm liquid. Finally, aspirate 4 μL of sperm liquid and drop it on a sperm slide to allow the sperm to quickly spread into the slide. Use an SCA sperm quality analyzer to take pictures for sperm motility detection and semen density analysis.

[0028] 4. Western Blot of germ cell marker proteins in testicular tissue samples

[0029] Add an appropriate amount of RIPA tissue lysate to the collected testicular samples and lyse them with a tissue grinder. Vortex every 5 minutes and centrifuge at 12,000 rpm for 1 minute, and repeat this step 3 times. Transfer the supernatant to a 1.5 mL centrifuge tube and add 1 / 4 volume of 5×SDS. After mixing by oscillation, boil in boiling water for 5 minutes to obtain protein samples that can be used for subsequent electrophoresis loading. Prepare 10% separating gel and 4% stacking gel for SDS polyacrylamide gel electrophoresis. During electrophoresis, first run the pre-stained protein Marker at a constant voltage of 80V and then switch to a constant voltage of 120V until the end; then transfer the membrane by wet transfer method, block it in a TBST blocking solution containing 5% BSA and then perform antibody incubation. After the incubation, use a chemiluminescent solution to perform chemiluminescence on the bands, and analyze the protein expression level according to the band gray value.

[0030] 5. Result description

[0031] Figure 1 This is the conceptual diagram of the research conducted for the present invention. This experiment was divided into three groups: a normal control group (Con), a trimethylamine N-oxide treatment group (TMAO), and a 3,3-dimethyl-1-butanol (DMB) inhibitor rescue group (TMAO + DMB). The control group (Con) was intraperitoneally injected with normal saline once a day in the morning at a dose of 4 μL per gram of body weight for five consecutive weeks; the TMAO group was intraperitoneally injected with 10 mg / mL TMAO once a day in the morning at a dose of 40 mg per kilogram of body weight for five consecutive weeks; DMB was fed in the form of drinking water. The TMAO + DMB group was intraperitoneally injected with 10 mg / mL TMAO once a day in the morning at a dose of 40 mg per kilogram of body weight for five consecutive weeks. The drinking water of this group was added with DMB reagent at a ratio of 1%, and the other two groups drank normal water. Figure 2These are the test results of sperm motility in three groups of mice. The results showed that compared with the control group (Con), injection of TMAO caused a significant decrease in sperm motility (P<0.05). However, after adding DMB to the drinking water for 5 weeks, the sperm motility of mice was significantly increased compared with the TMAO group (P<0.05). Figure 3 These are the statistical results of semen density in three groups of mice. The results showed that compared with the control group (Con), injection of TMAO caused a significant decrease in semen density (P<0.05). However, after adding DMB to the drinking water for 5 weeks, the semen density of mice was significantly increased compared with the TMAO group (P<0.05). Figure 4 These are the WB test results of germ cell marker proteins in testicular samples of three groups of mice. The results showed that compared with the control group (Con), the protein levels of VASA and STRA 8 in the testicular tissues of the TMAO injection group were significantly decreased (P<0.05). However, after adding DMB to the drinking water for 5 weeks, the protein levels of VASA and STRA8 in the testicular tissues were significantly increased compared with the TMAO group (P<0.05).

[0032] The present invention found that TMAO can be used to construct a mouse model of oligoasthenospermia, providing new ideas and clues for further research on the pathogenesis of oligoasthenospermia and for researchers in male reproductive health in the future.

Claims

1. A method for establishing an oligoasthenozoospermia model using trimethylamine oxide, characterized in that: The oligoasthenospermia model was obtained by injecting trimethylamine oxide into the abdominal cavity of animals.

2. The method according to claim 1, characterized in that: The animal is a mouse.

3. The method according to claim 2, characterized in that It is injected daily at a dose of 40 mg per kilogram of body weight.

4. The method according to claim 3, characterized in that The injections were given for 5 weeks.

5. Application of trimethylamine oxide in establishing oligoasthenozoospermia model.

6. The use according to claim 5, characterized in that: The animal is a mouse.

7. Application of trimethylamine oxide in the preparation of drugs for establishing oligoasthenozoospermia model.

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