Preparation method of spermatogenic dysfunction model of rat

Abstract

A preparation method of a rat spermatogenic dysfunction model belongs to the technical field of experimental animal production.The preparation method comprises the steps of Wistar rat selection and grouping, corresponding operation of rats in each group, observation of sperm related parameters and observation of tissue sections.The total sperm number and sperm motility of rats can be remarkably reduced, the sperm malformation rate of the rats can be remarkably increased, and the spermatogenic dysfunction rate of the rats can be remarkably increased. The spermatogenic capacity of the male Wistar rats is reduced, HE dyeing shows that spermatogenic tubules atrophy, the thickness of spermatogenic epithelium is reduced and spermatogenic cells are obviously reduced, lead acetate is low in cost, short in molding time and remarkable in effect, the spermatogenic capacity of the rats can be effectively reduced, and the method has the application values of low cost, short time consumption, simplicity in operation and the like in the using process. The method can provide reference values for establishment of other model animals, treatment of male infertility diseases and auxiliary development of new drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of experimental animal production and relates to a method for preparing a rat spermatogenesis disorder model, and specifically relates to a method for modeling Wistar rats with spermatogenesis disorder caused by using lead acetate. Background Art

[0002] Wistar rats are white closed colony rats that were bred by the Wistar Institute in the United States in 1907 and are now used in laboratories around the world. Their main characteristics are: (1) a wide head, long ears, and a tail that is shorter than the body; (2) a stable sexual cycle, strong reproductive capacity, large litter size, an average of about 10 pups per litter, and fast growth and development; (3) strong resistance to infectious diseases; (4) a low incidence of spontaneous tumors; and (5) high humidity requirements. They are prone to ringtail disease when the relative humidity of the air is below 40%.

[0003] Wistar rats are the most commonly used animal laboratory rats and have the longest history of use in biomedical research. Because this population of animals is sensitive to various nutrients and drugs, has a high absorption rate, sensitive physiological reactions, obvious behavioral changes, and a fast stress response, they are often used in various physiological endocrinology, pharmacology, nutrition, and behavioral experiments.

[0004] Male infertility has become a global problem, increasingly threatening the normal and healthy life of human beings. The World Health Organization has found through statistics that about 15% of couples of childbearing age have infertility problems. Studies have shown that testicular spermatogenesis dysfunction caused by various reasons is the main manifestation of male infertility. Spermatogenesis dysfunction can affect sperm production, and clinical manifestations include oligospermia, asthenospermia or azoospermia.

[0005] Lead, as a heavy metal environmental toxin, is one of the main causes of male infertility in recent years. Lead is widely distributed in the ecosystem and is one of the three most toxic heavy metals to humans announced by the Food and Agriculture Organization of the United Nations. The human body is contaminated through lead-contaminated food, drinking water, dust, etc. Long-term low-dose exposure to lead can affect the reproductive function of animals and humans. Lead has a significant effect on male reproductive function, especially on the growth and development, gonadal development and spermatogenesis of male animals. It is an important part of modern reproductive toxicology and reproductive epidemiology research. Lead can significantly reduce the relevant serum hormones, sperm density, sperm motility and sperm deformity rate of male animals, and cause obvious pathological changes in testicular tissue. As a model animal, Wistar rats can be used to make spermatogenic dysfunction models, so as to study the therapeutic effects of drugs on spermatogenic dysfunction models. It has a wide range of effective uses in the study of male infertility diseases, so the method of preparing rat spermatogenic dysfunction models is particularly important. Summary of the invention

[0006] The purpose of the present invention is to propose a method for preparing a rat spermatogenesis dysfunction model based on the physiological characteristics of Wistar rats and their relatively sensitive characteristics to drugs, and to use the preparation of the model for medical research and new drug development.

[0007] The method for preparing the rat spermatogenesis dysfunction model provided in this application adopts the following technical scheme: The method for preparing a rat spermatogenesis dysfunction model is characterized by comprising the following steps: S1. Select Wistar rats and group them Wistar rats were selected and divided into a model group and a normal group, with ten rats in each group; S2. Corresponding operations of rats in each group Each rat in the model group was continuously injected with lead acetate intraperitoneally, and each rat in the normal group was continuously injected with an equal amount of normal saline intraperitoneally; S3. Observation of sperm-related parameters After anesthetizing the rats, the epididymis was separated and cut into pieces in physiological saline. The sperm was incubated and diluted. The total sperm count, sperm motility and sperm deformity rate were detected using a hemocytometer. S4. Tissue section observation The rat testicles were fixed with paraformaldehyde and then dehydrated with sucrose gradient. The sections were sliced ​​with a freezing microtome and stained with HE.

[0008] By adopting the above technical scheme, since the testicles and epididymis are important spermatogenic organs of rats, lead will cause various oxidative damages to the spermatogenic organs and produce various organ physiological lesions after entering the body. Therefore, a model group and a normal group were used for comparison. The model group was intraperitoneally injected with lead acetate to observe the spermatogenic dysfunction of rats, and the normal group was used as a control group to observe the lesions of the spermatogenic organs of rats.

[0009] The Wistar male rats described in step S1 are eight-week-old male rats, weighing between 200-240 g.

[0010] By adopting the above technical solution, Since Wistar rats become adults at eight weeks of age and their sexual organs mature, eight-week-old rats were used and their body weight was maintained between 200-240 g to prevent excessive individual differences from affecting the experimental results.

[0011] The dosage of lead acetate in step S2 is 20 mg / kg per day, and the duration is not less than 10 days.

[0012] By adopting the above technical scheme, different doses and different administration methods of lead acetate can lead to reduced reproductive capacity in male rats, but the most significant effect is caused by intraperitoneal injection of lead exposure. Intraperitoneal injection of 25 mg / kg lead acetate for seven days can cause spermatogenesis dysfunction in rats. In order to stabilize the model, the dosage is reduced and the time is extended, so 20 mg / kg is used for intraperitoneal injection for no less than ten days.

[0013] The specific method of isolating the epididymis in step S3 is: chopping the sperm in 2.5 ml of 0.9% saline, incubating the sperm in a 37° water bath for one minute, and diluting ten times.

[0014] By adopting the above technical solution, 0.9% physiological saline can maintain the osmotic pressure balance of sperm cells and prevent sperm death; the 37° water bath simulates the in vivo environment to prevent the sperm from reducing its vitality due to the decrease in temperature after leaving the body, and even causing sperm death.

[0015] The fixation in step S4 is 4% paraformaldehyde fixation for 48 hours, and the gradient dehydration is carried out by sinking in 25% sucrose and then sinking again in 30% sucrose. The sections are sliced ​​with a freezing microtome and HE staining is performed.

[0016] By adopting the above technical solution, paraformaldehyde is used to form a stable three-dimensional structure by cross-linking with proteins in cells or tissues, thereby fixing the morphology and structure of the sample, which helps to maintain the original state of cells or tissues, making subsequent observation and analysis more accurate; gradient dehydration is to prevent the water in the tissue from forming ice crystals, which will break the cells, causing the cells to distort and deform, affecting the subsequent observation and analysis of the tissue. In the process of frozen sectioning, gradient dehydration can make the tissue better combined with the embedding agent, ensuring the smooth progress of the sectioning process.

[0017] In summary, the present invention includes the following beneficial technical effects: 1. The present invention can significantly reduce the total sperm count and sperm motility, improve the sperm deformity rate, and reduce the spermatogenic ability of male Wistar rats. HE staining shows that the seminiferous tubules are atrophied, the thickness of the seminiferous epithelium is reduced, and the number of spermatogenic cells is significantly reduced. The present invention is a reliable and rapid modeling method.

[0018] 2. The lead acetate used in the present invention has low cost, short modeling time and significant effect, can effectively reduce the spermatogenesis ability of rats, and has application value such as low cost, short time consumption and simple operation during use.

[0019] 3. The model preparation method of the present invention can provide reference value for the establishment of other model animals, the treatment of male infertility diseases and the auxiliary development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1It is a graph of the total sperm count in the present invention.

[0021] Figure 2 It is a sperm motility diagram of the present invention.

[0022] Figure 3 It is a sperm deformity rate diagram of the present invention.

[0023] Figure 4 This is the HE staining picture of the testis section of the common group in the present invention.

[0024] Figure 5 This is a HE staining picture of the testis section of the model group in the present invention. DETAILED DESCRIPTION

[0025] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art fall within the scope defined by the claims attached to this application. Example

[0026] A method for modeling spermatogenesis dysfunction in male Wistar rats caused by lead acetate, wherein 20 mg / kg lead acetate is injected intraperitoneally for ten days to make a model, and after ten days, the rats are anesthetized to separate the epididymis, and the sperm is cut into pieces in 2.5 ml of saline and incubated at 37° for one minute, and diluted ten times to detect the total sperm count, sperm motility and sperm deformity rate. A rat lateral testicle is fixed with 4% paraformaldehyde for 48 hours, then dehydrated with 25% and 30% sucrose gradient, and sliced ​​with a cryostat and HE stained.

[0027] The specific steps are as follows: Twenty eight-week-old Wistar male rats weighing between 200-240g were selected, and ten were randomly selected as the model group, and the rest were selected as the normal group. Each rat in the model group was intraperitoneally injected with 20mg / kg lead acetate for ten days, and each rat in the normal group was injected with an equal amount of normal saline.

[0028] Ten days later, the rats were anesthetized and the epididymis was isolated. The sperm was cut into pieces in 2.5 ml of saline and incubated in a water bath at 37° for one minute. The sperm was then diluted tenfold and the total sperm count, sperm motility and sperm deformity rate were detected under an optical microscope using a blood cell counting plate. One side of the testis was fixed with paraformaldehyde for 48 hours. For sucrose gradient dehydration, the testis was first precipitated in 25% sucrose and then placed in 30% sucrose and precipitated again before sectioning and HE staining.

[0029] Total sperm count Figure 1 As shown in Figure 2, the total sperm count in the model group was significantly lower than that in the normal group (p < 0.01); the sperm motility was Figure 2As shown in Figure 2, the sperm motility in the model group was significantly lower than that in the model group (p < 0.01); the sperm deformity rate was Figure 3 The normal group was significantly lower than the model group (p<0.01); HE staining Figure 4 , Figure 5 As shown in the figure, the seminiferous tubules in the normal group were arranged closely, the various types of spermatogenic cells in the tubules were arranged closely, the seminiferous epithelium was thick, and the spermatogenic cells were dense; while the model group had obvious lesions, and the main changes in the cases were: the seminiferous tubules were atrophied and sparsely arranged, the thickness of the seminiferous epithelium was significantly reduced, and the spermatogenic cells were significantly reduced and unevenly distributed.

[0030] In summary, the lead acetate-induced spermatogenic dysfunction modeling method provided in this embodiment can significantly reduce the total sperm count and sperm motility of Wistar rats, improve the sperm deformity rate, and reduce the spermatogenic ability of male Wistar rats by intraperitoneal injection of 20 mg / kg lead acetate for ten days. HE staining shows that the seminiferous tubules are atrophied, the thickness of the seminiferous epithelium is reduced, and the number of spermatogenic cells is significantly reduced. It is a reliable and rapid modeling method; the lead acetate used has a low cost, a short modeling time and a significant effect, and can effectively reduce the spermatogenic ability of rats. It has the application value of low cost, short time consumption, and simple operation during use, and has a practical reference basis and broad application prospects in the treatment of spermatogenic dysfunction in male animals and the development of new male infertility drugs.

Claims

1. A method for preparing a rat spermatogenesis dysfunction model, characterized in that: The model preparation method comprises the following steps: S1. Selection of Wistar rats and grouping Wistar rats were selected and divided into a model group and a normal group, with ten rats in each group; S2. Corresponding operations of rats in each group Each rat in the model group was continuously injected with lead acetate intraperitoneally, and each rat in the normal group was continuously injected with an equal amount of normal saline intraperitoneally; S3. Observation of sperm-related parameters After anesthetizing the rats, the epididymis was separated and cut into pieces in physiological saline. The sperm was incubated and diluted. The total sperm count, sperm motility and sperm deformity rate were detected using a hemocytometer. S4. Tissue section observation The rat testicles were fixed with paraformaldehyde and then dehydrated with sucrose gradient. The sections were sliced ​​with a freezing microtome and stained with HE.

2. The method for preparing a rat spermatogenesis dysfunction model according to claim 1, characterized in that: The Wistar male rats described in step S1 are eight-week-old male rats, weighing between 200-240 g.

3. The method for preparing a rat spermatogenesis dysfunction model according to claim 1, characterized in that: The dosage of lead acetate in step S2 is 20 mg / kg per day, and the duration is not less than 10 days.

4. The method for preparing a rat spermatogenesis dysfunction model according to claim 1, characterized in that: The specific method of isolating the epididymis in step S3 is: chopping the sperm in 2.5 ml of 0.9% saline, incubating the sperm in a 37° water bath for one minute, and diluting ten times.

5. The method for preparing a rat spermatogenesis dysfunction model according to claim 1, characterized in that: The specific method of tissue section observation in step S4 is: fix with 4% paraformaldehyde for 48 hours, sink to the bottom in 25% sucrose and then sink to the bottom again in 30% sucrose for gradient dehydration, and slice with a freezing microtome and perform HE staining.

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