Method for establishing oligoasthenospermia model by using alpha-ketoglutaric acid

Abstract

The invention discloses a method for establishing an oligoasthenospermia model by using alpha-ketoglutaric acid. Alpha-ketoglutaric acid is added into drinking water of animals to obtain the oligoasthenospermia model. According to the invention, alpha-ketoglutarate (alpha-KG) is added into drinking water of 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 application belongs to the field of reproductive biology technology and clinical medicine, and particularly relates to a method for establishing oligoasthenospermia model by using alpha-ketoglutarate. BACKGROUND

[0002] Oligoasthenospermia, as a common type of male infertility, includes oligospermia and asthenospermia, and is an important factor leading to male infertility. According to the definition of the World Health Organization (WHO), oligospermia refers to the sperm density in semen being less than 1500 million / mL or the total number of sperm being less than 39 million / time, and asthenospermia refers to the proportion of sperm with active forward motility in semen being less than 32%. These two diseases often coexist and are collectively referred to as oligoasthenospermia. Nowadays, with the acceleration of the pace of modern society and the increasing pressure faced by men, male infertility has become a global health challenge, affecting about 200 million people, and the number of patients is increasing. According to reports, among infertile couples, about half of the problems are caused by abnormal semen in men, and this problem is also showing a trend of youth.

[0003] Male infertility is a disease involving many factors and very complex etiology. So far, the detailed pathogenesis has not been completely revealed, and most of the current treatment methods are based on experience. Therefore, in-depth exploration of the pathogenesis of oligoasthenospermia is crucial for developing more effective treatment methods. In recent years, although certain progress has been made in the treatment of male infertility, in order to more deeply study the pathogenesis of oligoasthenospermia and develop new treatment methods, it is particularly important to construct a suitable animal model. The oligoasthenospermia mouse model can reflect the pathological characteristics of human oligoasthenospermia, provide an experimental basis for studying the pathogenesis, and enable researchers to more deeply explore the molecular mechanism of spermatogenic disorders.

[0004] Due to the diverse causes of oligoasthenospermia, the methods of constructing animal models for this disease are also diverse, including ordinary drug-induced models, chemotherapy drug-induced models, high-fat diet-induced models, physical factor-induced models, and gene knockout models. The establishment of these models provides an important tool and platform for studying the causes, pathology, and treatment of oligoasthenospermia. Through these models, researchers can simulate different pathogenic conditions and explore how various factors affect sperm production and function, which is of great significance for revealing the pathogenesis of oligoasthenospermia and finding possible prevention and treatment targets. At the same time, these models also provide an experimental platform for screening and efficacy evaluation of new drugs, which helps to accelerate the development process of new drugs and provides scientific basis for clinical treatment. As a metabolic product in the tricarboxylic acid cycle, α-KG can participate in multiple cellular metabolic pathways and can act as an energy donor, amino acid precursor, and epigenetic regulator. Succinic acid (Succ) is also an important metabolic product in the tricarboxylic acid cycle, and α-ketoglutarate dehydrogenase complex (OGDH) is a rate-limiting enzyme that can convert α-ketoglutarate to succinate. The accumulation of succinate inhibits the activity of α-ketoglutarate dehydrogenase, thereby affecting the metabolism of α-ketoglutarate. SUMMARY

[0005] The purpose of the present application is to provide a method for establishing an oligoasthenospermia model using α-ketoglutarate.

[0006] The method for establishing an oligoasthenospermia model using α-ketoglutarate of the present application involves adding α-ketoglutarate to the drinking water of animals to obtain an oligoasthenospermia model.

[0007] Preferably, the animals are mice.

[0008] Preferably, the α-ketoglutarate is added to the drinking water in an amount of 5% by mass fraction.

[0009] Further preferably, the drinking lasts for 5 weeks.

[0010] The present application also provides the use of α-ketoglutarate in establishing an oligoasthenospermia model.

[0011] Preferably, the animals are mice.

[0012] The present application also provides the use of α-ketoglutarate in the preparation of a drug for establishing an oligoasthenospermia model.

[0013] In the present application, the mice are given alpha-ketoglutarate to drink, which can significantly reduce the sperm motility and semen density of male mice, thereby successfully establishing an oligoasthenozoospermia mouse model. This provides new ideas and clues for researchers in the study of the pathogenesis of oligoasthenozoospermia and male reproductive health, and has important scientific and medical value for understanding the mechanism of male infertility, developing new therapies, evaluating treatment effectiveness, and promoting related clinical research. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The research of the present application is schematically shown in the figure.

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

[0016] Figure 3 The results of semen density statistics for the three groups of mouse samples. DETAILED DESCRIPTION

[0017] The method of the present application will be further described below in conjunction with the drawings and through specific examples.

[0018] Example 1

[0019] 1. Experimental animal handling and reagent preparation

[0020] The present application uses 3-week-old ICR male mice as experimental objects, and the mice are caged and fed in a clean level animal room, and are fed in strict accordance with standard mouse feeding conditions. The mice are placed in a 12-hour light and 12-hour dark cycle, the room temperature is 23±1℃, the environmental humidity is 50%, and the mice can drink water and eat freely. All procedures comply with animal ethics standards and experimental specifications. A normal control group (Con), an alpha-ketoglutarate (α-KG) treatment group (α-KG), and a succinic acid (Succ) inhibitor rescue group (α-KG+Succ) are set up. The specific treatment method for the mice: In this experiment, 3-week-old ICR male mice are used. The Con group is given a single intraperitoneal injection of normal saline at a dose of 1 μL per gram of body weight every morning, and the injection is continued for five weeks. The group drinks normally; the α-KG group is fed with drinking water. The α-KG reagent is added to the drinking water at a mass fraction of 5%. The α-KG+Succ group drinks water with the addition of the α-KG reagent at a mass fraction of 5%, and at the same time, a single intraperitoneal injection of 2 mg / mL Succ is performed every morning at a dose of 20 mg per kilogram of body weight, and the injection is continued for five weeks.

[0021] Succ solution preparation: 50 mg metabolites (A1453553, Ambeed) were transferred to a 50 mL centrifuge tube, 25 mL of sterile saline was added to completely dissolve, the concentration was 2 mg / mL. According to the dose of 20 mg per kilogram of body weight, 1 ml syringe was used for intraperitoneal injection, and 1 g of mouse body weight was ensured to correspond to 10 μL of inhibitor working solution.

[0022] α-ketoglutarate (α-KG) solution configuration: α-KG reagent (75890, Sigma) was added to the drinking water of the α-KG group and the α-KG+Succ group at a ratio of 5%. That is, 12.5 g of α-KG was added to 250 mL of drinking water.

[0023] 2. Sample collection

[0024] After 5 weeks of continuous intraperitoneal injection, semen samples of different treatment groups were collected for comparative detection experiment. After the mice were decapitated, the epididymis of the mice was picked up, the sperm was released for in vitro capacitation, and the sperm motility data was measured.

[0025] 3. Sperm motility and semen density detection

[0026] The sperm capacitation solution was prepared in advance, α-MEM / F12 and FBS were mixed according to the volume ratio of 9:1, and preheated on a 37°C hot plate. The mice were decapitated, the epididymal tail tissue was carefully removed and placed in a 3 cm dish containing 200 μL of sperm capacitation solution, the epididymal tail was cut open with a blade to release fresh sperm and timed for 5 minutes, then 20 μL of turbid liquid was diluted in a new 200 μL of sperm capacitation solution, mixed evenly, and the sperm liquid was obtained. Finally, 4 μL of sperm liquid was dropped on a sperm slide to make the sperm quickly spread in the slide, and the SCA sperm quality analyzer was used for photography for sperm motility detection and semen density analysis.

[0027] 5. Result explanation

[0028] Figure 1 The research of the present application is schematically shown. The experiment is divided into three groups: normal control group (Con), α-ketoglutarate group (α-KG), and succinic acid (Succ) inhibitor rescue group (α-KG+Succ). The control group (Con) was injected with physiological saline intraperitoneally at a dose of 1 μL per gram of body weight once a day in the morning, and the injection was continued for five weeks. The drinking water of this group was normal; the α-KG group was fed with drinking water, and α-KG reagent was added to the drinking water at a ratio of 5%. The α-KG+Succ group was injected with 2 mg / mL Succ intraperitoneally at a dose of 20 mg per kilogram of body weight once a day in the morning, and the injection was continued for five weeks. The drinking water of this group was also added with α-KG reagent at a ratio of 5%. Figure 2The results of sperm motility detection of three groups of mice are shown in the table. The results show that compared with the control group (Con), adding α-KG in drinking water can significantly reduce sperm motility (P<0.05), but after intraperitoneal injection of Succ for 5 weeks, the sperm motility of mice is significantly improved compared with the α-KG group (P<0.05). Figure 3 The results of sperm density statistics of three groups of mice are shown in the table. The results show that compared with the control group (Con), adding α-KG in drinking water can significantly reduce sperm density (P<0.05), but after intraperitoneal injection of Succ for 5 weeks, the sperm density of mice is significantly improved compared with the α-KG group (P<0.05).

[0029] The present application finds that α-ketoglutaric acid can be used to construct oligoasthenospermia model mice. This provides new ideas and clues for future in-depth study of the pathogenesis of oligoasthenospermia and researchers of male reproductive health.

Claims

1. A method for establishing an oligoasthenospermia model by using α-ketoglutaric acid, characterized in that, The animal is added with α-ketoglutaric acid in drinking water to obtain the oligoasthenospermia model.

2. The method of claim 1, wherein, The animal is a mouse.

3. The method of claim 2, wherein, The α-ketoglutaric acid is added into the drinking water in a mass fraction of 5% for drinking.

4. The method of claim 3, wherein, The drinking lasts for 5 weeks.

5. Application of α-ketoglutaric acid in establishing an oligoasthenospermia model.

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

7. Application of α-ketoglutaric acid in preparing a drug for establishing an oligoasthenospermia model.

Images