A method for establishing oligoasthenospermia model by using trimethylamine oxide

Abstract

This invention discloses a method for establishing a model of oligoasthenospermia using trimethylamine N-oxide. The method involves injecting trimethylamine N-oxide into the peritoneal cavity of an animal to obtain the oligoasthenospermia model. This invention successfully established a mouse model of oligoasthenospermia by intraperitoneal injection of trimethylamine N-oxide (TMAO) into male mice. This provides new ideas and clues for in-depth research on the pathogenesis of oligoasthenospermia and for researchers studying male reproductive health. It also has significant scientific and medical value for understanding the pathogenesis of male infertility, developing new therapies, evaluating treatment effects, and promoting related clinical research.

Description

Technical Field

[0001] This invention belongs to the fields of reproductive biology and clinical medicine, specifically relating to a method for establishing an oligoasthenospermia model using trimethylamine oxide. Background Technology

[0002] Oligospermia and asthenospermia, a collective term for oligospermia and asthenospermia, are common causes of male infertility. According to the World Health Organization (WHO) standards, oligospermia is defined as a sperm density in semen of less than 15 × 10⁻⁶. ^6 sperm count / mL or total sperm count less than 39 × 10⁹ / mL ^6 Asthenospermia refers to a condition where the percentage of actively motile sperm in semen is less than 32%, while oligospermia refers to a condition where the percentage of actively motile sperm in the semen is less than 32%. These two conditions often occur simultaneously and are collectively known as asthenospermia. With the fast pace of modern life and increasing pressure on men, male infertility has become a global health problem, affecting approximately 200 million people, and the number of patients is on the rise. Among infertile couples, about half are due to male semen abnormalities, and this problem is increasingly affecting younger men.

[0003] Male infertility is a multifactorial and complex disease with an incompletely understood mechanism. Current treatments are largely based on experience, making in-depth research into the pathogenesis of oligoasthenospermia crucial for developing effective treatments. While clinical treatments for male infertility have achieved some success in recent years, establishing suitable animal models remains essential for further research into the pathogenesis of oligoasthenospermia and the development of therapeutic drugs. Mouse models of oligoasthenospermia can mimic the pathological characteristics of human oligoasthenospermia, providing an experimental basis for further research into the pathogenesis and enabling researchers to gain a deeper understanding of the physiological and molecular mechanisms of spermatogenesis disorders.

[0004] The etiology of oligoasthenospermia is complex, thus various animal models are used, including those based on conventional drugs, chemotherapy drugs, high-fat diets, physical factors, and gene knockout. TMAO is a metabolite produced by gut microbiota and synthesized in the host's liver. Studies have shown a correlation between TMAO and reproductive health. For example, TMAO has been reported in the follicular fluid of normally fertilized and developing human embryos, therefore, TMAO can be used to predict embryo transfer outcomes. 3,3-Dimethyl-1-butanol (DMB) is an organic compound that, as a choline analog, effectively inhibits the conversion of choline to trimethylamine (TMA) in the intestine, thereby reducing plasma TMAO levels, making it an effective TMAO inhibitor. Summary of the Invention:

[0005] The purpose of this invention is to provide a method for establishing an oligoasthenospermia model using trimethylamine oxide.

[0006] The present invention provides a method for establishing an oligoasthenospermia model using trimethylamine oxide, which involves injecting trimethylamine oxide into the peritoneal cavity of an animal to obtain the oligoasthenospermia model.

[0007] Preferably, the animal is a mouse.

[0008] Preferably, the dosage is 40 mg per kilogram of body weight, administered daily.

[0009] Further optimization involves 5 weeks of injections.

[0010] This invention also provides the application of trimethylamine oxide in establishing a model of oligoasthenospermia.

[0011] Preferably, the animal is a mouse.

[0012] This invention also provides the application of trimethylamine oxide in the preparation of drugs for establishing oligoasthenospermia models.

[0013] In this invention, intraperitoneal injection of trimethylamine N-oxide (TMAO) into mice significantly reduced sperm motility and semen density, thus 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 studying male reproductive health. It also has significant scientific and medical value for understanding the pathogenesis of male infertility, developing new therapies, evaluating treatment effects, and promoting related clinical research. Attached image description:

[0014] Figure 1 This is a conceptual diagram based on the research conducted for this invention.

[0015] Figure 2 The results show the sperm motility test results of three groups of mouse samples.

[0016] Figure 3 The results show the statistical results of semen density in three groups of mouse samples.

[0017] Figure 4 The results of WB detection of germ cell marker proteins in testicular samples from three groups of mice are shown. Detailed implementation method:

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

[0019] Example 1

[0020] 1. Laboratory animal handling and reagent preparation

[0021] This invention used 3-week-old male ICR mice as experimental subjects. The mice were housed separately in a clean-grade animal facility under strict standard mouse rearing conditions. They were subjected to a 12-hour light-dark cycle, with a room temperature of 23±1℃ and an ambient humidity of 50%. They had free access to water and food. All procedures complied with animal ethics standards and experimental protocols. A normal control group (Con), a trimethylamine oxide treatment group (TMAO), and a 3,3-dimethyl-1-butanol (DMB) inhibitor rescue group (TMAO+DMB) were established. Specific treatment methods for mice: Three-week-old male ICR mice were used in this experiment. The Con group received a single intraperitoneal injection of physiological saline at a dose of 4 μL per gram of body weight every morning for five weeks. The TMAO group received a single intraperitoneal injection of 10 mg / mL TMAO at a dose of 40 mg per kilogram of body weight every morning for five weeks. The TMAO+DMB group was fed DMB in drinking water and received a single intraperitoneal injection of 10 mg / mL TMAO at a dose of 40 mg per kilogram of body weight every morning for five weeks. DMB reagent was added to the drinking water of this group at a mass fraction of 1%. The other two groups had normal drinking water.

[0022] TMAO solution preparation: Transfer 500 mg of metabolite TMAO (A292360, Ambeed, China) to a 50 mL centrifuge tube, add 50 mL of sterile physiological saline to completely dissolve it, achieving a concentration of 10 mg / mL. Administer intraperitoneally at a dose of 40 mg per kilogram of body weight using a 1 mL syringe, ensuring that 1 g of mouse body weight corresponds to 40 μL of working solution of the metabolite.

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

[0024] 2. Sample collection

[0025] After 5 weeks of continuous intraperitoneal injection, testicular and semen samples were collected from different treatment groups for comparative testing. Mice were euthanized by cervical dislocation, and alcohol was sprayed onto the abdominal skin. The testes were then exposed using sterile surgical scissors and placed in centrifuge tubes for cryopreservation at -80°C for subsequent Western blotting experiments. The epididymis was collected, sperm were released, and in vitro capacitation and sperm motility data were measured.

[0026] 3. Sperm motility and semen density testing

[0027] Prepare sperm capacitation solution in advance by mixing α-MEM / F12 and FBS at a volume ratio of 9:1 and preheating on a 37°C hot plate. Euthanize mice by cervical dislocation, carefully remove epididymal tail tissue and place it in a 3cm dish containing 200μL of sperm capacitation solution. Use a blade to incise the epididymal tail to release fresh sperm and time the process for 5 minutes. Then, aspirate 20μL of the turbid solution and dilute it in a fresh 200μL of sperm capacitation solution, mixing thoroughly to obtain sperm fluid. Finally, aspirate 4μL of the sperm fluid and drop it onto a sperm slide to allow the sperm to quickly diffuse into the slide. Image the sample using an SCA sperm quality analyzer for sperm motility and semen density analysis.

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

[0029] Collected testicular samples were added to an appropriate amount of RIPA tissue lysis buffer and lysed using a tissue homogenizer. The mixture was vortexed every 5 minutes and centrifuged at 12000 rpm for 1 minute, repeating this step 3 times. The supernatant was transferred to a 1.5 mL centrifuge tube, and 1 / 4 volume of 5×SDS was added. After vortexing and mixing, the sample was boiled in water for 5 minutes to obtain protein samples suitable for subsequent electrophoresis. A 10% separating gel and a 4% stacking gel were prepared for SDS-polyacrylamide gel electrophoresis. During electrophoresis, the pre-stained protein markers were first run at a constant voltage of 80V, then the voltage was increased to 120V until the end. Wet transfer was then performed, followed by blocking in TBST blocking buffer containing 5% BSA and antibody incubation. After incubation, the bands were chemiluminescently analyzed using a chemiluminescent solution, and the protein expression level was analyzed based on the band gray values.

[0030] 5. Results Explanation

[0031] Figure 1 This is a conceptual diagram of the research conducted in this invention. The experiment consisted of three groups: a normal control group (Con), a trimethylamine oxide treatment group (TMAO), and a 3,3-dimethyl-1-butanol (DMB) inhibitor rescue group (TMAO+DMB). The control group (Con) received a single intraperitoneal injection of physiological saline at a dose of 4 μL / g body weight every morning for five weeks. The TMAO group received a single intraperitoneal injection of 10 mg / mL TMAO at a dose of 40 mg / kg body weight every morning for five weeks. DMB was administered via drinking water. The TMAO+DMB group received a single intraperitoneal injection of 10 mg / mL TMAO at a dose of 40 mg / kg body weight every morning for five weeks. DMB reagent was added to the drinking water of this group at a ratio of 1%, while the other two groups received normal drinking water. Figure 2The results show the sperm motility of three groups of mice. The results showed that, compared with the control group (Con), injection of TMAO significantly reduced sperm motility (P<0.05), but after adding DMB to the drinking water for 5 weeks, the sperm motility of the mice was significantly higher than that of the TMAO group (P<0.05). Figure 3 The results show 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), but after adding DMB to the drinking water for 5 weeks, the semen density of mice was significantly higher than that of the TMAO group (P<0.05). Figure 4 The results of Western blot analysis of germ cell marker proteins in testicular samples from three groups of mice are shown. The results showed that, compared with the control group (Con), the protein levels of VASA and STRA8 in the testicular tissue of the TMAO-injected group were significantly lower (P<0.05). However, after adding DMB to the drinking water for 5 weeks, the protein levels of VASA and STRA8 in the testicular tissue were significantly higher than those in the TMAO group (P<0.05).

[0032] This invention discovers that TMAO can be used to construct mouse models of oligoasthenospermia, providing new ideas and clues for future in-depth research on the pathogenesis of oligoasthenospermia and for researchers studying male reproductive health.

Claims

1. A method for establishing an oligoasthenospermia model using trimethylamine oxide, characterized in that, The oligoasthenospermia model was obtained by injecting trimethylamine oxide into the peritoneal cavity of animals. The injection of trimethylamine oxide into the peritoneal cavity of the animals was carried out at a dose of 40 mg trimethylamine oxide per kilogram of body weight per day for 5 weeks. The animals were 3-week-old male ICR mice.

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