CN108815152B - Construction method of azoospermia mouse model - Google Patents

Abstract

The invention discloses a method for constructing an azoospermia mouse model, which comprises the following steps: (1) providing a sexually mature male mouse with the weight of 25-35 g; (2) dissolving Busulfan in DMSO, and diluting with normal saline to obtain diluent; (3) disinfecting the lower abdomen of the mouse with alcohol, airing, sucking the diluent obtained in the step (2), inserting a needle, and finishing the first intraperitoneal injection after no error is confirmed by back pumping; and then injecting for 3 times at intervals in the same day, and obtaining the azoospermia mouse model after 35-37 days. The method has high molding rate and good stability; according to the invention, 84 accumulated C57BL/6 mice are modeled for azoospermia, the total number of the models is 84/84 and the molding rate is 100% through testis HE staining identification; by adopting the method for constructing the model, the death rate of the mouse is 0 percent; the total number of deaths of 84C 57BL/6 mice injected cumulatively according to the method of the present invention was 0, and the mortality was 0%.

Description

Construction method of azoospermia mouse model

Technical Field

The invention relates to the technical field of animal model construction, in particular to a construction method of an azoospermia mouse model.

Background

Azoospermia can be classified into obstructive azoospermia and non-obstructive azoospermia according to whether the vas deferens is unobstructed or not. The non-obstructive azoospermia is mainly caused by testicular spermatogenic dysfunction, the clinical treatment is relatively troublesome, no effective drug treatment exists for the part of patients at present, and semen collection is mainly carried out by means of operations, such as testicular puncture biopsy, open biopsy or microscopic semen collection operation. In recent years, there have been more and more reports of methods for treating non-obstructive azoospermia, such as spermatogonial stem cell transplantation. With the development of cell replacement therapy studies for non-obstructive azoospermia, there is a need to obtain stable animal models of azoospermia for experimentation.

Currently, the non-obstructive azoospermia model of mice based on Busulfan is more commonly used. Busulfan, also known as malan (myleran), is a chemical name of 1, 4-butanediol disulfonate, a sulfonate that undergoes alkylation upon dissociation in vivo. The medicine starts to be used for treating the leukemia in the 50 s, can obviously inhibit the generation of the granulocytes with small dose, and has obvious curative effect on the chronic granulocytic leukemia (the remission rate is 80-90%). The whole blood picture can be inhibited by increasing dosage. Has no effect on chronic granulocytic leukemia acute lesion and acute leukemia. Has bone marrow inhibiting effect. Prolonged use can cause amenorrhea or testicular atrophy. According to the reproductive toxicity effect of busulfan, researchers establish an azoospermia mouse model based on busulfan, and then gradually apply the model to the scientific research of non-obstructive azoospermia.

At present, methods for establishing an azoospermia mouse model based on busulfan are different, the dosage is different from 10mg to 50mg per kilogram of body weight, and the modeling period is different for 28 days, 40 days, 60 days and the like. Although the existing methods are different, the method has the characteristics of low molding rate, poor stability, high death rate and the like. Lack of a construction scheme with high molding rate and good stability seriously restricts the research progress of the non-obstructive azoospermia.

Disclosure of Invention

Based on the problems, the invention aims to overcome the defects of the prior art and provide the method for constructing the azoospermia mouse model, and the method has high molding rate and good stability.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for constructing an azoospermia mouse model comprises the following steps:

(1) providing a sexually mature male mouse with the weight of 25-35 g;

(2) dissolving Busulfan in DMSO, and diluting with normal saline to obtain diluent;

(3) disinfecting the lower abdomen of the mouse in the step (1) with alcohol, airing, sucking the diluent obtained in the step (2), inserting a needle, and finishing the first intraperitoneal injection after no error is confirmed by back pumping; and then injecting for 3 times at intervals in the same day, and obtaining the azoospermia mouse model after 35-37 days.

Preferably, the dose of busulfan is: busulfan (40 mg) was injected into 1kg of mice.

Preferably, in the step (3), the intraperitoneal injection is carried out in 4 times of the same day, the liquid volume of each injection is 1/4 which is the same and is the volume of the diluent, and the interval between the two injections is 3 hours. Wherein the volume of the liquid volume injected in each time is not more than 0.7 ml.

Preferably, in the step (3), the mouse model of azoospermia is obtained after 36 days.

Preferably, the male mouse is a C57BL/6 male mouse.

Preferably, in the step (2), the volume ratio of DMSO in the diluent is 2.5%.

Preferably, in the step (1), the weight of the mouse is 25-30 g.

Preferably, the week age of the mouse is 8-12.

Preferably, the step (2) adopts an insulin needle to suck the diluent obtained in the step (2).

As another aspect of the present invention, the present invention provides a mouse model of azoospermia constructed by the above method.

In conclusion, the beneficial effects of the invention are as follows:

the method has high molding rate and good stability; according to the invention, 84 accumulated C57BL/6 mice are modeled for azoospermia, the total number of the models is 84/84 and the molding rate is 100% through testis HE staining identification; the experiment is completed in 3 batches, so that the stability is very good;

by adopting the method for constructing the model, the death rate of the mouse is 0 percent; according to the method of the present invention, 84C 57BL/6 mice that had been injected cumulatively had a total mortality of 0 and a mortality of 0%; the weight changes of 84 mice are +/-1.5 g within 72 hours, and the weight changes of 82 mice are +/-1.0 g, which indicates that the invention is safe and feasible in the acute stage.

Drawings

FIG. 1 is a graph showing the staining of mouse testis HE at different dosages and the results of the change of testis, epididymal head and body weight coefficient;

FIG. 2 is a graph showing the staining pattern of testis HE and the result of testis/body weight coefficient change at different stages under Busulfan 40mg/kg treatment;

FIG. 3 is a graph showing the HE staining pattern of epididymal heads and the result of the change of epididymal heads/body weight coefficients at different stages under Busulfan 40mg/kg treatment;

FIG. 4 is a diagram showing the long-term observation and variation results of Busulfan 40mg/kg processed testis, epididymis head and seminal vesicle;

wherein BW represents body weight, TW represents testis, EW represents epididymal head, and SW represents seminal vesicle.

Detailed Description

In order to determine the appropriate molding dose and time, the inventors considered the following 3 factors: 1) the damage to the mouse spermatogenic function cannot be too large or too small, otherwise the effectiveness of the drug or the cells is easily interfered; 2) after the administration is stopped, the model rat can recover self under the conventional feeding state, and the influence of self recovery on the model is reduced to the maximum extent; 3) the most efficient combination of molding dose and time is selected.

The spermatogenic cycle of the mice was 34.4 days, consisting of 4 equally divided phases (8.6 days/phase). Based on the mouse's spermatogenic cycle, in some embodiments, 36 days are used as modeling cycles (spanning 1 spermatogenic cycle), and the modeling effects of three different doses of 20mg/kg, 30mg/kg, and 40mg/kg are compared. Furthermore, model observation was carried out for the 40mg/kg dose group (0 days, 9 days, 18 days, 27 days, 36 days) with a long-term effect for 60 days in a 9-day period.

Busulfan is first dissolved in DMSO, diluted with physiological saline and administered by intraperitoneal injection. Theoretically, the solubility of busulfan in DMSO is 49 mg/ml; in some embodiments, busulfan has been found experimentally to have a maximum solubility in DMSO of only 20mg/ml at both room temperature and 37 ℃. DMSO is widely used as a solvent, although the DMSO belongs to low toxic substances, when the dosage is too large, the possibility of acute toxicity, cytotoxicity, genetic toxicity, reproductive development toxicity and the like still exists in the aspect of animal toxicity. According to the literature, the half Lethal Dose (Lethal Dose 50%, LD50) of DMSO injected intravenously in mice is 3.94mg/kg, the intraperitoneal injection concentration of the mice can be calculated according to the administration volume of 0.1mL/10g by referring to intravenous injection, the maximum intraperitoneal injection concentration of the DMSO injected in mice should not exceed 1/2 of LD50, namely 0.19mg/mL, and the concentration of the DMSO calculated according to the DMSO density of 1.100g/mL, namely the ratio of the DMSO to the total liquid volume after dilution should not exceed 0.17mL/1mL in principle. The body weight of male mice with sexual maturity of C57BL/6 at 8-12 weeks is about 25-30g, generally not more than 35 g. The liquid amount injected into the abdominal cavity of the mouse is preferably 20ml/kg, and preferably not more than 80 ml/kg. In the case of 25g mice, a single injection dose of 0.5ml is preferred.

Based on the findings, the invention provides a construction method of a mouse model for azoospermia based on busulfan by combining multiple factors such as a mouse spermatogenic cycle, a common dosage and actual solubility of busulfan, low toxicity of DMSO (dimethyl sulfoxide), and an amount of liquid injected into an abdominal cavity of a mouse, so that the molding rate and stability are effectively improved, and the death rate is reduced.

The invention aims to construct an azoospermia mouse model with high modeling rate, good stability and low mortality by utilizing busulfan, and forms a set of technical scheme which is reasonable and can be popularized and applied in the research of non-obstructive azoospermia. In some embodiments, the invention establishes a mouse model of azoospermia by using an insulin needle to inject 4 times (3 hours apart) of busulfan (40mg/kg, dissolved in DMSO and having a final concentration of 2.5%) intraperitoneally every day (the maximum liquid amount does not exceed 0.7 ml/time); wherein the modeling dose of the busulfan C57BL/6 mouse azoospermia model is 40mg/kg, and the busulfan is dissolved in DMSO at 20 mg/ml; insulin injections were given 4 times a day at time points of 8:30, 11:30, 14:30, 17: 30.

to better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise stated, the concentrations of the reagents in the present invention are mass concentrations, and the experimental methods in the present invention are all the methods commonly used in the art.

Example 1

One embodiment of the method for constructing the azoospermia mouse model comprises the following steps:

(1) taking a C57BL/6 male mouse which is sexually mature for 8-12 weeks as a model animal, wherein the weight of the male mouse is 25-30g and is not more than 35 g; the modeling dose (Busulfan) is 40mg/kg, and the period is 36 days;

(2) taking a mouse with the weight of 25g as an example, Busulfan 1mg is dissolved in 0.05ml of DMSO, and is diluted to 2ml by using physiological saline (the ratio of the DMSO to the total liquid is 2.5%) and transported to an animal center on ice for use;

(3) after a mouse enters an animal center through conventional disinfection, shaking up the diluent, sucking 0.5ml of diluent by using an insulin needle (specifically referring to table 1), disinfecting the lower abdomen by using alcohol, inserting the needle after the mouse is slightly dried, and finishing single intraperitoneal injection after the fact that no error exists in the drawing back confirmation;

(4) combining the work and rest time of an animal center, and simultaneously injecting the drugs into the animal center for 4 times in a single day in order to reduce the heart load of the mouse and facilitate the absorption and metabolism of the drugs of the mouse, wherein the injection is selected from the group consisting of 8:30, 11:30, 14:30 and 17: 30, the liquid volume for each injection is 0.5ml, and the interval is 3 hours;

(5) the mouse model was successfully constructed on day 0, day 36 of injection.

TABLE 1 conversion of mouse body weight to amount of drug solution

EXAMPLE 2 comparison of Molding Effect of Busulfan at different doses

Model mice were prepared using the modeling method of example 1, except that the dose of busulfan was compared to the modeling effect of three different doses, 20mg/kg, 30mg/kg, and 40mg/kg, respectively. As shown in FIG. 1, it is understood from FIG. 1 that spermatogenic cells were observed in the mouse testicular seminiferous tubules treated at 20mg/kg and 30mg/kg at day 36, while only supporting cells from the basal portion were observed in the mouse testicular seminiferous tubules treated at 40mg/kg, and no spermatogenic cells were observed, so that 40mg/kg of busulfan was suitable as the modeled dose.

Example 3 time effects on seminal tubules and testis in modeled mice

Model mice were prepared using the modeling method of example 1, except that the time after busulfan injection was varied, and the change in the mouse testis was compared after 0, 9, 18, 27, and 36 days. As shown in fig. 2, it can be seen from fig. 2 that the number of seminal tubules and testis/body weight changed dramatically from day 9 to day 18, suggesting that the model may be in a reversible phase before day 18 and gradually changed to an irreversible state after day 18.

Example 4 Effect of time on epididymal head of modeled mice

Model mice were prepared by the modeling method of example 1, except that the time after busulfan injection was varied, and the epididymal head changes were compared after 0, 9, 18, 27, and 36 days. As shown in fig. 3, it can be seen from fig. 3 that the epididymal head also shows changes such as the number of epididymal tubules decreasing with the extension of the modeling time, and the epididymal head/body mass coefficient tends to decrease continuously.

Example 5 Effect of time on aspects of the modeled mice

The model mouse was prepared by the modeling method of example 1, except that the time after busulfan injection was different, and the gross changes of testicular seminiferous tubules, epididymal heads and seminal vesicles of the mouse after the busulfan injection was performed for 0 days and 60 days, and the coefficient changes of each organ and the body weight were compared. As shown in fig. 4, it is clear from fig. 4 that the diameter of the seminiferous tubule body of testis became small and no spermatogenic cells were observed at day 60 of the modeling, indicating that the model did not self-recover in spermatogenic function. The ratio of the testis, epididymis head and weight coefficient is obviously reduced, and the ratio of the seminal vesicle and weight coefficient is obviously increased, which have statistical significance.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (6)

1. A method for constructing an azoospermia mouse model is characterized by comprising the following steps:

(1) providing a sexually mature male mouse with the weight of 25-35 g;

(2) dissolving busulfan in DMSO (dimethyl sulfoxide), and diluting with normal saline to obtain a diluent, wherein the volume ratio of the DMSO to the diluent is 2.5%, and the total volume of the diluent is not more than 2.8 mL;

(3) disinfecting the lower abdomen of the mouse in the step (1) with alcohol, airing, sucking the diluent obtained in the step (2), inserting a needle, and finishing the first intraperitoneal injection after no error is confirmed by back pumping; and then, injecting for 3 times at intervals in the same day, wherein the liquid amount injected for each time is 1/4 which is the same as the volume of the diluent, the interval between the two injections for the former time and the latter time is 3 hours, and obtaining the azoospermia mouse model after 35-37 days, wherein the dose of the busulfan is as follows: busulfan (40 mg) was injected into 1kg of mice.

2. The method according to claim 1, wherein the mouse model of azoospermia is obtained 36 days later in the step (3).

3. The method of constructing according to claim 1, wherein the male mouse is a C57BL/6 male mouse.

4. The method according to claim 1, wherein in the step (1), the weight of the mouse is 25-30 g.

5. The method according to claim 1, wherein the week-old of the mouse is 8 to 12.

6. The method for constructing a tissue culture medium according to claim 1, wherein the diluent obtained in the step (2) is sucked by an insulin needle in the step (2).

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