Construction method of dyszoospermia mouse model

Abstract

The invention discloses a method for constructing a dyszoospermia mouse model, which comprises the following steps: enabling a male mouse to drink 2-3wt% of dextran sodium sulfate solution for 6-8 days to obtain the dyszoospermia mouse model, the survival rate is smaller than or equal to 40%, the malformation rate is larger than or equal to 60%, and the pathological characteristics of the model are highly similar to those of human inflammation-related sterility. Compared with a traditional method, the method saves more than 80% of time and cost, and is especially suitable for anti-inflammatory drug screening and oxidative stress mechanism research.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and particularly to a method for constructing a spermatogenesis disorder mouse model. Background Art

[0002] Low sperm quality mainly includes teratospermia and testicular dysfunction, which are the key factors leading to male infertility. Most cases of male infertility are caused by abnormalities in factors such as sperm quantity, motility, morphology, and motility ability. Teratospermia refers to a normal sperm quantity, but the morphology, structure, or chromosomes of sperm are abnormal and cannot participate in the reproductive process normally, which is a manifestation of male infertility, and its incidence is about 20% - 30%. Testicular dysfunction refers to a disease state in which the testis cannot normally produce a sufficient quantity or quality of sperm and testosterone hormone. It can cause damage to male reproductive function, thereby affecting aspects such as sexual desire, erectile function, and fertility. The incidence of testicular dysfunction is relatively low globally, about 1% - 5%. Due to the complex etiology of low sperm quality, there are various modeling methods, generally including drug modeling (adenine, ornidazole, tripterygium glycosides, etc.), high-fat diet modeling, physical factor modeling, gene knockout modeling, etc. However, these methods are relatively complex to use, and the modeling time is long and the cost is high.

[0003] Therefore, in order to solve the disadvantages of long modeling time, low model stability, and high cost in the prior art, it is necessary to develop a method for constructing a spermatogenesis disorder mouse model. Summary of the Invention

[0004] The object of the present invention is to provide a method for constructing a spermatogenesis disorder mouse model, which uses dextran sulfate sodium solution (DSS) for modeling, has the advantages of simple operation, short modeling time, high model stability, and low cost. In addition, this model can also be used to explore the occurrence mechanism of low sperm quality and explore drug prevention and treatment.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, a method for constructing a spermatogenesis disorder mouse model is provided, and the method includes: Let the mice drink a 2wt% - 3wt% dextran sulfate sodium solution for 6 - 8 days to obtain a spermatogenesis disorder mouse model.

[0006] In the above technical solution, a control group can be set up, and the experimental animals are divided into a control group and a spermatogenesis disorder model group. The control group drinks water, and the model group replaces the drinking water with a 2wt% - 3wt% dextran sulfate sodium solution and models for 6 - 8 days to obtain a spermatogenesis disorder mouse model.

[0007] Furthermore, the mice include one of male C57BL / 6 and male BALB / c mice.

[0008] Further, the concentration of the sodium dextran sulfate solution is 2.5 wt%. The drinking amount of the sodium dextran sulfate solution is 1 ml / g body weight / day.

[0009] Further, the time for model establishment is 7 days.

[0010] Further, the method further includes: Collecting testicular and epididymal samples of the control group and the spermatogenesis disorder model group respectively, and evaluating the model through testicular weight measurement, HE staining, Tunel staining, PCNA staining and eosin Y staining.

[0011] Further, the HE staining includes: Putting testicular tissue blocks into a pre-prepared fixing solution to denature and coagulate the proteins of tissues and cells. After successful fixation, put them into embedding cassettes and rinse with running water; placing the tissue blocks in alcohol with gradually increasing concentrations for dehydration to gradually remove the water in the tissue blocks; Placing the dehydrated tissue blocks in xylene as a clearing agent to replace the alcohol in the tissue blocks with xylene; placing the cleared tissue blocks in melted paraffin to obtain wax-impregnated tissue blocks; Putting the wax-impregnated tissue blocks into an embedding frame, pouring melted paraffin, and forming wax blocks after the paraffin solidifies; after the embedded wax blocks harden, cutting them into thin slices on a microtome, flattening and attaching them to glass slides, drying and staining.

[0012] Further, the method for eosin Y staining includes: Adding 1 drop of fresh semen and 1 drop of eosin Y staining solution on a glass slide and gently mixing them with a pipette; Covering the mixed sample with a coverslip and standing for 10 - 40 seconds; Using an optical microscope to observe sperm survival rate and malformation rate under high magnification.

[0013] Further, the method for testicular Tunel staining includes: Putting testicular tissue into 4% paraformaldehyde for fixation at room temperature for 2 hours or overnight at 4°C, dehydrating with gradient ethanol (70%, 80%, 90%, 95%, 100% ethanol for 1 hour each), clearing with xylene, embedding in paraffin, and sectioning; After dewaxing and hydrating the sections, performing antigen repair with proteinase K or sodium citrate buffer; then, adding a TUNEL reaction mixture (containing TdT enzyme and labeled dUTP) to the tissue and incubating at 37°C in the dark for 60 minutes; After washing with PBS, counterstaining with DAPI and mounting the slides to observe apoptotic cells.

[0014] Furthermore, the method for testicular PCNA staining includes: Fix fresh testicular tissue with 4% paraformaldehyde and then prepare paraffin sections or frozen sections. After dewaxing and hydration, perform antigen retrieval (it is recommended to use EDTA or sodium citrate buffer for heat-induced antigen retrieval); Block with 5% BSA or goat serum for 30 minutes, add PCNA primary antibody (such as mouse anti-PCNA monoclonal antibody, diluted 1:100 - 1:200), and incubate overnight at 4°C; after washing with PBS, add fluorescently labeled secondary antibody (such as FITC- or Cy3-labeled anti-mouse IgG, diluted 1:500), and incubate for 1 hour at room temperature in the dark; counterstain cell nuclei with DAPI for 5 minutes, and mount with an anti-fluorescence quenching mounting medium; Observe under a fluorescence microscope. PCNA-positive cell nuclei show red fluorescence.

[0015] Furthermore, the testicular weight of the model group decreased by ≥20% compared with the control group.

[0016] Furthermore, eosin Y staining showed that the sperm survival rate in the model group was ≤40%.

[0017] Furthermore, Tunel staining showed that the testicular apoptosis level in the model group was >30%.

[0018] Furthermore, PCNA staining showed that the testicular proliferation level in the model group was <20%.

[0019] The evaluation in the method also includes pathological features, including increased seminiferous tubule spacing, epithelial vacuolization, and interstitial fibrosis; simultaneously detect oxidative stress indices (MDA content ≥12 μmol / g, SOD activity ≤40 U / g).

[0020] In the second aspect of the present invention, there is provided the use of dextran sulfate sodium in the preparation of experimental reagents or tool drugs for inducing a spermatogenesis disorder mouse model.

[0021] Furthermore, the dextran sulfate sodium induces testicular inflammatory responses through drinking, and the specific manifestations are as follows: (1) The testicular weight decreased by ≥20% 7 days after model establishment; (2) The sperm survival rate was ≤40%, and the malformation rate was ≥60%; (3) Pathological features included increased seminiferous tubule spacing, epithelial vacuolization, and interstitial fibrosis; (4) There was an increase in the content of oxidative stress marker MDA by ≥12 μmol / g and a decrease in the activity of antioxidant enzyme SOD by ≥40%.

[0022] In the second aspect of the present invention, there is provided a method for screening a drug for treating spermatogenesis disorders, and the method includes the following steps: performing a drug efficacy test using the spermatogenesis disorder animal model constructed by the present invention.

[0023] Specifically, a drug screening group and a model control group are set up. By comparing the improvement degree of testicular histopathology and the recovery of sperm kinetics parameters of the experimental subjects in the two groups, the therapeutic effect of the candidate drug is evaluated.

[0024] Among them, the model control group refers to a spermatogenesis disorder animal model constructed by the method of the present invention; the drug screening group is, on the basis of the model control group, intervened by administering a candidate drug through oral administration, injection and other drug administration routes. During the experiment, through technical means such as tissue section analysis and computer-aided semen analysis, the changes in core indexes such as the structural integrity of seminiferous tubules, the number of spermatogenic cell layers, sperm concentration and motility between the two groups are quantitatively compared.

[0025] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: A method for constructing a spermatogenesis disorder mouse model provided by the present invention uses dextran sulfate sodium (DSS) to establish a model. Compared with previous construction methods (such as chemical induction, physical injury, gene editing, etc.), this method is simple to operate. DSS is administered orally and does not require complex surgeries or equipment. In addition, this method has high modeling stability. Since the dose and cycle of DSS can be precisely controlled, a stable and reproducible spermatogenesis disorder model can be established, while traditional modeling methods (such as heat stress, chemical induction) may lead to inconsistent models due to operation differences. At the same time, the inflammatory response induced by DSS is closer to some pathological states (such as autoimmune orchitis) and is suitable for studying inflammation-related spermatogenesis disorders. Traditional methods (such as hormone intervention) cannot fully simulate inflammation or oxidative stress-related spermatogenic dysfunction. In summary, this method has the advantages of strong specificity, simple operation, high model stability, etc., and provides a new method and idea for inducing spermatogenesis disorders in animals. Description of the Drawings

[0026] Figure 1 It is a flowchart of the method for constructing a spermatogenesis disorder mouse model provided by the embodiment of the present invention.

[0027] Figure 2 They are relevant evaluation indexes for inducing low sperm quality in 8-week-old male mice by exposing to 2.5 wt% dextran sulfate sodium for one week. Among them, A is the comparison of testicular weights of two groups of mice, B is the comparison of sperm survival rates of two groups of mice, C is the comparison of sperm malformation rates of two groups of mice, D is the HE staining of testes of two groups of mice, and E is the eosin Y water staining of sperm of two groups of mice.

[0028] Figure 3Apoptosis and proliferation changes in the testes of mice after 1-week exposure to 2.5 wt% dextran sulfate sodium. Among them, A shows the Tunel staining results of the testes of mice in the spermatogenic disorder group and the control group, and B shows the PCNA staining results of the testes of mice in the spermatogenic disorder group and the control group. Detailed implementation manners

[0029] The following will combine the detailed implementation manners and examples to specifically elaborate on the embodiments of the present invention, and the advantages and various effects of the embodiments of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the embodiments of the present invention, rather than limiting the embodiments of the present invention.

[0030] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the embodiments of the present invention belong. In case of conflict, this specification shall prevail.

[0031] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the embodiments of the present invention can be obtained through market purchase or by existing methods.

[0032] DSS, full name dextran sulfate sodium, and the CAS number of DSS is 68528-80-3. The DSS solution in the embodiments of the present invention was purchased from Nanjing Experimental Valley Information Technology Development Co., Ltd., and the product number is SYG2024-333430-2.

[0033] The following will combine examples and experimental data to elaborate in detail on a spermatogenic disorder mouse model and its construction method of the present application.

[0034] Example 1: Construction method of spermatogenic disorder mouse model 1. Divide the experimental animals into a control group and a model group, perform spermatogenic disorder modeling, and collect testis and epididymis samples. The specific operations are as follows: (1) Adaptively raise 30 8-10-week-old C57BL / 6 male mice in an SPF-level environment for one week, and randomly divide them into 2 groups, including a control group and a spermatogenic disorder model group, with 15 mice in each group.

[0035] In the model group, each mouse drinks 30 ml of 2.5 wt% DSS solution per day for 7 consecutive days; the drinking volume is related to the body weight of the mouse, specifically 1 ml / g. Based on the average body weight of the mouse being 30 g, the daily drinking volume is calculated to be 30 ml.

[0036] The control group drinks an equal amount of pure water, and the feed for each group is normal.

[0037] (2) Then anesthetize and sacrifice the mice, and take out the testes and epididymides.

[0038] 2. Preparation of testicular HE-stained sections The specific method is as follows: Take a testicular tissue block from the animal body, generally with a thickness not exceeding 0.5 cm. Immerse the tissue block in a pre-prepared fixing solution (such as 10% formalin) to denature and coagulate the proteins of the tissue and cells. After successful fixation, place it in an embedding cassette and rinse with running water for 30 minutes to remove the fixing solution in the tissue. Dehydrate the tissue block in alcohol with increasing concentrations, gradually removing the water in the tissue block. Generally, soak it in 70%, 80%, 90%, 95% and 100% alcohol for a certain period of time (such as 1 hour or adjusted according to the specific situation). Place the dehydrated tissue block in xylene, a clearing agent, to replace the alcohol in the tissue block with xylene. The clearing time depends on the size of the tissue and the penetration ability of xylene, generally requiring soaking for several minutes until the tissue block is completely clear. Place the cleared tissue block in melted paraffin and keep it warm in a paraffin melting box. After the paraffin has completely penetrated the tissue block, proceed to the next step. Place the paraffin-impregnated tissue block in an embedding frame and pour in melted paraffin. After the paraffin solidifies, a wax block is formed. After the embedded wax block hardens, it can be cut into thin slices on a microtome. Fix the embedded wax block on the microtome and cut it into thin slices, generally 5 - 8 microns thick. The cut thin slices often wrinkle and need to be flattened in heated water. Affix the flattened thin slices to a glass slide and dry them in an incubator at 45°C. Before staining, the paraffin in the sections must be removed with xylene. Generally, soak in xylene I and xylene II for 10 minutes each (or adjust the time according to the specific situation). Pass the sections through alcohol with decreasing concentrations and finally into distilled water. Generally, soak in 100%, 95%, 90%, 80%, 70% alcohol for a certain period of time (such as 5 minutes) each, and then into distilled water for a moment. Hematoxylin staining: Immerse the sections in a hematoxylin aqueous solution for staining for several minutes (such as 3 - 8 minutes) to stain the chromatin in the nucleus and the nucleic acid in the cytoplasm purple-blue. The staining time can be adjusted according to the tissue type and staining effect. After staining, treat the sections with a differentiating solution for several seconds or minutes (depending on the differentiating solution) to remove the excess hematoxylin dye. Then rinse the sections with tap water and treat them with a blueing solution for several seconds or minutes (depending on the blueing solution) to restore the blue color of the sections. Immerse the sections in eosin staining solution for staining for several minutes (such as 1 - 3 minutes) to stain the components in the cytoplasm and extracellular matrix red. The staining time can also be adjusted according to the tissue type and staining effect. Immerse the stained sections in 95% alcohol I, 95% alcohol II, absolute ethanol I, absolute ethanol II for a certain period of time (such as 5 minutes) each to remove the water in the sections. Immerse the dehydrated sections in xylene I and xylene II for a certain period of time (such as 5 minutes) each to make the sections transparent. Drop neutral balsam on the transparent sections and cover them with a coverslip for sealing. After the balsam is slightly dry, attach a label, and the section specimens can be used. 3. Observation of sperm survival rate and malformation rate by eosin Y water staining of sperm The specific method of eosin Y water staining of sperm is as follows: Add 1 drop of fresh semen and 1 drop of eosin Y staining solution on a glass slide, and gently mix them with a pipette. Cover the mixed sample with a cover slip and let it stand for about 30 seconds. Observe the sperm survival rate and malformation rate under a high-power optical microscope. The heads of live sperm are white or light pink and do not stain, while the heads of dead sperm are red or dark pink. Count at least 100 sperm and calculate the percentage of non-stained (live sperm) and observe whether there are any abnormalities in the sperm morphology.

[0039] The results are as Figure 2 shown: Figure 2 As shown in A, the testicular weight of the DSS group was significantly decreased compared with the control group (P<0.01). The testicular weight of the control group was 12.3 mg, and that of the DSS group was 10.1 mg, with a decrease of 18%, indicating DSS-induced testicular atrophy.

[0040] Figure 2 As shown in B and Figure 2 As shown in C: The sperm survival rate decreased from 85.2% in the control group to 38.7% (P<0.05), and the malformation rate increased from 4.1% to 62.4% (P<0.01).

[0041] Figure 2 As shown in D: In the histological results, HE staining showed that the seminiferous tubule spacing in the DSS group increased, the lumen collapsed, and epithelial vacuolization occurred, which was highly consistent with the typical pathological features of human inflammatory infertility (such as spermatogenic arrest and Sertoli cell vacuolization).

[0042] Figure 2 As shown in E: Eosin Y staining showed that the proportion of dead sperm in the DSS group increased significantly and the live sperm decreased, verifying the loss of sperm membrane integrity.

[0043] In summary, the mouse model of spermatogenic disorder was successfully constructed. HE staining showed damage to the structure of seminiferous tubules and epithelial vacuolization, indicating histological evidence supporting spermatogenic disorder. The low sperm survival rate and high malformation rate indicated functional evidence of impaired fertility.

[0044] 4. Preparation of testicular TUNEL staining sections, the specific method is as follows: First, fix fresh testicular tissue with 4% paraformaldehyde or 10% neutral formalin for 24 - 48 hours, then perform gradient ethanol dehydration, xylene clearing, and paraffin embedding, and prepare 4 - 5 μm thick sections. After dewaxing and hydrating the sections, perform antigen repair (treatment with proteinase K or sodium citrate buffer), and block endogenous peroxidase with 3% H2O2. Then, add TUNEL reaction mixture (containing TdT enzyme and fluorescein or HRP - labeled dUTP) to the tissue sections, incubate at 37°C in the dark for 1 hour, after washing with PBS, directly mount the slides for fluorescence microscopy (DAPI counterstaining) for observation, or for the HRP method, perform microscopy after DAB color development and hematoxylin counterstaining. Negative (without adding TdT enzyme) and positive (DNase I pretreatment) controls should be set for the experiment. Finally, observe apoptotic cells (green signal) under a microscope and calculate the positive rate.

[0045] 5. Preparation of testicular PCNA staining sections, the specific method is as follows: Take fresh testicular tissue, fix it with 4% paraformaldehyde for 24 hours, then perform paraffin embedding, prepare 4 - 5 μm sections, after dewaxing and hydrating, perform antigen repair with sodium citrate buffer (pH 6.0), wash with PBS, and then block with 5% BSA at room temperature for 30 minutes; dropwise add PCNA primary antibody (such as mouse anti - PCNA monoclonal antibody, diluted 1:200), incubate overnight at 4°C, after rinsing with PBS, add fluorescein - labeled secondary antibody (such as FITC - labeled goat anti - mouse IgG, diluted 1:500), incubate in the dark for 1 hour, counterstain the cell nuclei with DAPI for 5 minutes, and mount the slides with an anti - fluorescence quenching mounting medium; for the negative control, use PBS instead of the primary antibody. When observing under a fluorescence microscope, PCNA - positive cell nuclei show red fluorescence (FITC), and all cell nuclei show blue (DAPI). Evaluate the proliferative activity of the spermatogenic epithelium by calculating the proportion of positive cells. Note that the entire experiment should be carried out in the dark.

[0046] Figure 3 As shown in Figure A, in the results of TUNEL staining, the green fluorescence signal (apoptotic cells) in the DSS group increased significantly, indicating that the apoptosis level of testicular cells > 30%.

[0047] Figure 3 As shown in Figure B, in the results of PCNA staining, the red fluorescence (proliferating cells) in the control group was densely distributed in the basal layer of the seminiferous tubules, while the signal in the DSS group weakened (proliferation level < 20%), indicating that the self - renewal ability of spermatogonial stem cells was impaired.

[0048] In summary, Figure 3 The results show that: in the spermatogenesis disorder group, the apoptosis level of the testis increased and the proliferation level decreased.

[0049] Comparative Example 1 In this Comparative Example 1, the concentration of the DSS solution was 1 wt%, and the remaining steps were the same as in Example 1.

[0050] Comparative Example 2 In this Comparative Example 2, the concentration of the DSS solution was 5 wt%, and the remaining steps were the same as those in Example 1.

[0051] Comparative Example 3 In this Comparative Example 3, the modeling time was 3 days, and the remaining steps were the same as those in Example 1.

[0052] Comparative Example 4 In this Comparative Example 4, the modeling time was 14 days, and the remaining steps were the same as those in Example 1.

[0053] Experimental Example 1. Statistical results of the modeling in each group The modeling results of Example 1 and Comparative Examples 1 - 2 are shown in Table 1; Table 1 - DSS concentration gradient experiment

[0054] As can be seen from Table 1: 2.5 wt% DSS was the optimal dose (the survival rate decreased by 52%, the malformation rate increased by 14 times, p < 0.001).

[0055] High concentration (5%) led to irreversible damage to the model and high mortality, not meeting the requirements of repeatability.

[0056] Experimental Example 2. Intervention time optimization experiment 1. The modeling results of Example 1 and Comparative Examples 3 - 4 are shown in Table 2: Table 2

[0057] As can be seen from Table 2: Intervention for 7 days resulted in irreversible damage (the best model stability). Prolonging the intervention to 14 days led to partial recovery and loss of research value.

[0058] 2. The method of the present invention is compared with the traditional method as shown in Table 3: Table 3

[0059] Compared with the previous modeling methods, inducing spermatogenic disorders in mice using DSS has the advantages of strong specificity, simple operation, high model stability, and low cost, and is particularly suitable for studying spermatogenic dysfunction related to inflammation and oxidative stress.

[0060] Finally, it should also be noted that the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0061] Although the preferred embodiments of the embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present invention.

[0062] Obviously, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if these modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalent technologies, the embodiments of the present invention are also intended to include these changes and modifications.

Claims

1. A method for constructing a spermatogenesis disorder mouse model, characterized in that, The method includes: Male mice are given a 2.5 wt% dextran sulfate sodium solution to drink for 7 days to obtain a spermatogenesis disorder mouse model. The method further includes: collecting testicular and epididymal samples of the mice, and evaluating the model by testicular weight measurement, HE staining, Tunel staining, PCNA staining and eosin Y staining. In the eosin Y staining, the sperm survival rate of the obtained spermatogenesis disorder mice is ≤ 40%. In the Tunel staining, the testicular apoptosis level is > 30%. In the PCNA staining, the testicular proliferation level is < 20%.

2. The construction method of a spermatogenesis disorder mouse model according to claim 1, characterized in that, The male mice are selected from one of male C57BL / 6 and male BALB / c mice.

3. Use of dextran sulfate sodium as an experimental reagent in the method for constructing a spermatogenesis disorder mouse model according to any one of claims 1-2.

4. The use according to claim 3, wherein, The concentration of the dextran sulfate sodium solution is 2 wt% - 3 wt%.

5. A screening method for drugs for treating spermatogenesis disorders, characterized in that, The method includes: performing a drug efficacy test based on the model obtained by constructing according to the method described in claims 1-2.

6. The screening method of a drug for treating spermatogenesis disorders according to claim 5, characterized in that, In the method, a drug screening group with a control and a model control group are set up, and the therapeutic effect of the candidate drug is evaluated by comparing the improvement degree of testicular histopathology and the recovery of sperm kinetic parameters between the two groups.

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