Construction method and application of male germ cell specific Agk knockout mouse model

Abstract

The invention relates to the field of animal models, in particular to a construction method and application of a male germ cell specific Agk knockout mouse model, and an Agkflox / flox mouse and a Stra8-Cre mouse are mated to obtain the male germ cell specific Agk knockout mouse model (Agk-CKO). The invention constructs a male germ cell specific Agk conditional knockout mouse model, and finds that the specific knockout of the Agk gene in the male germ cell leads to sterility, testicular development and spermatogenesis abnormality of an Agk-CKO adult male mouse, and the Agk-CKO spermatoblasts show abnormal cell state and damaged subsequent differentiation ability from the fine line phase. The animal model disclosed by the invention can be used for researching the regulation and control mechanism of the development and differentiation state and meiosis of male germ cells, and is expected to provide a new thought and a treatment target for clinical diagnosis and treatment and drug research and development of male infertility.

Description

Technical Field

[0001] The present invention relates to the field of animal models, and specifically, to a method for constructing a male germ cell-specific Agk knockout mouse model and its application. Background Art

[0002] AGK, whose full name is acylglycerol kinase, is a membrane protein encoded by a nuclear gene and contains a single N-terminal transmembrane domain. It was initially identified as a mitochondrial lipid kinase and serves as a subunit of the mitochondrial multiprotein translocase TIM22 complex, contributing to the translocation of proteins across and into the mitochondrial membrane, thereby maintaining normal mitochondrial structure and function. However, more and more studies suggest that AGK may exhibit different functions in specific cells through different mechanisms of action. For example, in CD8+ T cells, plasma membrane AGK can interact with the phosphatase PTEN (ectopically expressed upon TCR- and CD28 stimulation) that has translocated to the plasma membrane, and maintain the metabolic and functional status of CD8+ T cells by inhibiting the phosphatase activity of PTEN; in megakaryocytes, cytoplasmic AGK significantly enhances the activation of JAK2 / Stat3 mediated by TPO / Mpl in megakaryocytes by binding to JAK2, thereby promoting platelet production; in prostate cancer cells, the increased expression of AGK leads to the sustained activation of ERK1 / 2, thereby enhancing cell proliferation and migration; in human airway epithelial cells, AGK regulates the secretion of the inflammatory factor IL-8 through p38 MAPK / JNK / NF-κB signaling and EGFR transactivation, thus playing an important role in the innate immune regulation of airway epithelium and airway remodeling.

[0003] Multiple locus mutations of the human AGK gene have been confirmed to have a causal relationship with Sengers syndrome. Sengers syndrome is an autosomal recessive genetic disease first reported by Sengers et al. in 1975, characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis, but with normal intellectual development. The severity of the disease varies from person to person, with some dying in infancy and some surviving into adulthood. The main cause of death is heart failure caused by hypertrophic cardiomyopathy. So far, few case reports show that patients with Sengers syndrome can survive into the second decade, so there is little data on the natural history of the disease and little description of the fertility of patients with the disease.

[0004] The human AGK gene is located at 7q34 on the long arm of chromosome 7, with a full length of 104.06 kb. The main transcript is 3,628 bp, containing 16 exons, and encodes a protein of 422 amino acids. The mouse Agk gene is located on chromosome 6 (6B1), with a full length of 71.28 kb. The main transcript is 2,225 bp, containing 16 exons, and encodes a protein of 421 amino acids. Homology analysis of the amino acid sequences shows that the homology of the AGK protein between humans and mice is as high as 90%, indicating that this protein is highly conserved between humans and mice. However, the role of AGK in testicular development and spermatogenesis is still unclear, and there are few relevant literature reports.

[0005] Chinese patent document CN113881708A discloses a method for electrotransfection gene editing of animal fertilized eggs and its application. Chinese patent document CN109306359A discloses the application of a gene knockout animal model in male reproductive research. Chinese patent document CN112646841A discloses a method for constructing an azoospermia mouse model and its application. However, there is currently no report on the method for constructing a male germ cell-specific Agk knockout mouse model and its application. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for constructing a male germ cell-specific Agk knockout mouse model and its application.

[0007] To achieve the above purpose, the present invention adopts the following technical means:

[0008] In the early stage of the present invention, when constructing a disease animal model of human Sengers syndrome by knocking out the Agk gene in mice, it was found that Agk - / - homozygous mice died in large numbers within a few days after birth (usually 24 - 48 h after birth). At birth, the genotype ratio of the mice conforms to Mendelian genetic laws, but due to the large number of deaths of homozygous mice, the proportion of Agk - / - genotypes in adult mice decreased significantly. These mice not only showed typical clinical manifestations similar to Sengers syndrome, such as hypertrophic cardiomyopathy and skeletal myopathy, but also showed abnormal phenotypes of testicular dysplasia and male infertility. However, the role of AGK in testicular development and spermatogenesis is still unclear, and there are few relevant literature reports.

[0009] Transcriptome data indicate that the tissue expression profiles of the AGK gene in humans and mice are very similar. By analyzing the RNA-seq expression profile data of different types of mouse testicular spermatogenic cells (spermatogonia, spermatocytes, and round spermatids), the results suggest that the expression level of the Agk gene is relatively high in testicular spermatocytes. To further investigate the biological functions of AGK in testicular tissue development and spermatogenesis, the present invention crossed Agk flox / flox mice with Stra8-Cre mice to obtain male germ cell-specific Agk conditional knockout mice (Agk-CKO). Compared with control mice, adult male Agk-CKO mice are infertile, with abnormal testicular development and spermatogenesis, specifically manifested as testicular atrophy, a significant decrease in the number of sperm in the cauda epididymis, and almost no intact acrosome structure in these sperm. Further analysis shows that testicular development stagnates in Agk-CKO male mice after 20 days post-partum (20-dpp), the testis-to-body weight ratio decreases significantly, the number of round spermatids in the seminiferous tubules of the testis decreases significantly, and the blank area in the lumen increases significantly. By detecting the expression levels of marker molecules of different types of cells in the testicular tissue samples of the Ctrl and Agk-CKO groups, the results show that there is no significant difference in the expression of the spermatogonia marker Plzf between the two groups, while the expression levels of the spermatocyte marker molecules Spo11, Ovol2, Nme8, and Ccna1 decrease significantly in the testicular tissue of Agk-CKO after 20-dpp. Among them, Spo11 is a marker for leptotene / zygotene spermatocytes, and its expression level decreases by about 21% at 20-dpp and by about 35% after 26-dpp, while Ovol2, Nme8, and Ccna1 are markers for pachytene / diplotene spermatocytes, and their expression levels decrease by about 60% at 20-dpp, by more than 82% at 26-dpp, and by more than 96% at 4 months of age. There is no significant difference in the expression level of the marker molecule Stra8, which represents the entry of spermatogonia into the meiotic state, between the two groups. In summary, the specific knockout of the Agk gene in male germ cells leads to abnormal cell state and impaired subsequent differentiation ability of spermatocytes starting from the leptotene stage. The above research shows that AGK plays an important role in regulating the development, differentiation, and meiosis of spermatocytes, and is expected to provide new ideas and therapeutic targets for the clinical diagnosis and treatment of male infertility and drug development.

[0010] Based on the above technical solutions, in the first aspect of the present invention, there is provided a method for constructing a male germ cell-specific Agk knockout mouse model, which is to cross Agk flox / flox mice with Stra8-Cre mice to obtain a male germ cell-specific Agk knockout mouse model (Agk-CKO).

[0011] Furthermore, the construction method includes the following steps:

[0012] First, mate Agk flox / flox mice with Stra8-Cre mice to obtain Agk flox / + ;Stra8-Cre + mice. Then, mate Agk flox / + ;Stra8-Cre + male mice with Agk flox / flox female mice to obtain Agk flox / - ;Stra8-Cre + 、Agk flox / - ;Stra8-Cre - 、Agk flox / + ;Stra8-Cre + or Agk flox / + ;Stra8-Cre - male offspring mice, where Agk flox / - ;Stra8-Cre + male mice are the male germ cell-specific Agk knockout mouse model.

[0013] Furthermore, the Agk flox / flox mice are constructed by inserting a loxP site on both sides of exon 3 of the Agk gene based on the ET cloning and ES cell targeting methods.

[0014] Furthermore, the Stra8-Cre mice are from the Jackson Laboratory. For the specific preparation method, refer to the literature: Genesis. 2008 Dec;46(12):738-42. doi: 10.1002 / dvg.20437.

[0015] In the second aspect of the present invention, a male germ cell-specific Agk knockout mouse model is provided, which is constructed by the construction method described above.

[0016] In the third aspect of the present invention, an application of a male germ cell-specific Agk knockout mouse model constructed by the construction method described above in the clinical diagnosis and treatment of male infertility and drug research and development is provided.

[0017] In the fourth aspect of the present invention, an application of a male germ cell-specific Agk knockout mouse model constructed by the construction method described above in the study of the regulation mechanism of male germ cell development, differentiation status and meiosis is provided.

[0018] In the fifth aspect of the present invention, there is provided an application of a reagent for knocking out the Agk gene in the preparation of an animal model with testicular development arrest, oligospermia and male infertility.

[0019] The advantages of the present invention are as follows:

[0020] The present invention constructs a male germ cell-specific Agk knockout mouse model (Agk-CKO). Through technical means such as fertility assessment, histopathological analysis of testes and sperm, and differential gene expression detection, it is found that the specific knockout of the Agk gene in male germ cells leads to infertility in adult male Agk-CKO mice, abnormal testicular development and spermatogenesis, and abnormal cell state and impaired subsequent differentiation ability of Agk-CKO spermatocytes starting from the leptotene stage. This model can be used for the study of the regulatory mechanism of spermatocyte development and differentiation status and meiosis, and is expected to provide new ideas and therapeutic targets for the clinical diagnosis and treatment of male infertility and drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 . Specific knockout of the Agk gene in male germ cells leads to testicular development arrest, oligospermia and male infertility. (A, B) The AGK gene has similar tissue expression characteristics between humans and mice and is widely expressed in multiple tissues including testicular tissue; (A) Tissue expression profile of the human AGK gene shown by HPA RNA-seq data; (B) Tissue expression profile of the mouse Agk gene shown by mouse ENCODE transcriptome data; (C) The GSE100964 dataset shows that the expression level of the mouse Agk gene in testicular spermatocytes is relatively high; (D) Immunohistochemical analysis of mouse testicular tissue sections with anti-AGK antibody indicates strong AGK immunohistochemical staining signals in spermatocytes; (E, F) Mating Agk flox / flox mice with Stra8-Cre mice to obtain male germ cell-specific Agk conditional knockout mice (Agk-CKO); (E) qRT-PCR results confirm that the Agk mRNA level in Agk-CKO testicular tissue is significantly reduced; (F) WB results confirm that the AGK protein level in Agk-CKO testicular tissue is significantly reduced; (G) Comparison of the morphological sizes of the testes and epididymides of Ctrl and Agk-CKO adult male mice shows that the Agk-CKO testes are significantly smaller and the epididymides are not full; (H) The testis-to-body weight ratio of Agk-CKO male mice is significantly reduced; (I) The number of sperm in the cauda epididymidis of Agk-CKO male mice is significantly reduced; (J) Agk-CKO adult male mice are infertile.

[0022] Figure 2. Spermatogenesis in Agk-CKO male mice is abnormal. (A) H&E staining results of testicular and cauda epididymidis tissue sections showed that spermatogenic cells in Agk-CKO testicular tissue were disorderly arranged, with a large number of vacuoles, few round spermatids, and very few sperm in the cauda epididymidis; (B) PNA staining analysis of testicular tissue sections of Ctrl and Agk-CKO male mice. PNA is an acrosome marker that can indicate the gradually formed acrosome structure in round spermatids; (C) qRT-PCR analysis of genes Acrv1, Prss37, and Prss54 specifically expressed in round spermatids, suggesting that almost no round spermatids could be detected in Agk-CKO testicular tissue; (D) PNA staining of sperm smears from the cauda epididymidis, suggesting that sperm in the cauda epididymidis of Agk-CKO had almost no intact acrosome structure.

[0023] Figure 3 . Meiotic arrest of spermatocytes in Agk-CKO testes, and a significant decrease in pachytene spermatocytes. (A) qRT-PCR was used to detect the expression changes of the Agk gene in testicular tissues of mice at different postnatal ages. The results showed that the expression level of Agk in the testicular group was relatively stable, but there was an obvious upregulation around 20-dpp; (B) Analysis of the testis-to-body weight ratio of Ctrl and Agk-CKO male mice at different postnatal ages. The results showed that there was no significant difference in the testis-to-body weight ratio between Ctrl and Agk-CKO male mice at 20-dpp, but there were significant differences at 26-dpp, 32-dpp, and in adult mice; (C) H&E staining analysis of testicular tissue sections of Ctrl and Agk-CKO male mice at different postnatal ages. The results showed that the number of round spermatids in the seminiferous tubules of Agk-CKO testes began to decrease from 20-dpp, and the blank area in the middle of the lumen increased significantly with the increase of mouse age; (D-I) qRT-PCR analysis of the expression levels of marker molecules of different types of cells in Ctrl and Agk-CKO testicular tissues; (D) There was no significant difference in the spermatogonial marker Plzf between the two groups; (E) There was no significant difference in the expression level of the marker molecule Stra8 representing the entry of spermatogonia into the meiotic state between the two groups; (F) The expression level of the leptotene / zygotene spermatocyte marker molecule Spo11 decreased by about 21% at 20-dpp and by about 35% after 26-dpp; (G-I) The expression levels of the pachytene / diplotene spermatocyte marker molecules Ovol2, Nme8, and Ccna1 decreased by about 60% at 20-dpp, by more than 82% at 26-dpp, and by more than 96% at 4 months of age. Detailed implementation manners

[0024] The following provides a detailed description of the specific implementation manners of the present invention in combination with embodiments.

[0025] Example:

[0026] Experimental materials and methods:

[0027] (1) Construction of the Agk-CKO mouse model

[0028] Agk flox / flox mice were constructed based on the ET cloning and ES cell targeting methods, with a loxP site inserted on both sides of exon 3 of the Agk gene (for the specific construction method, refer to the literature: Chen Yan. Using gene knockout mouse models to study the biological functions of MrgF and Agk [D]. Shanghai: Shanghai Jiao Tong University, 2014.).

[0029] Moreover, Agk flox / flox mice have been backcrossed with C57BL / 6J mice for more than 10 generations, and their genetic background is C57BL / 6J. Stra8-Cre mice are from Jackson Laboratory. For the specific preparation method, refer to the literature: Genesis. 2008 Dec;46(12):738-42. doi: 10.1002 / dvg.20437.

[0030] Stra8 is a molecular marker representing the entry of spermatogonia into the meiotic state, mainly expressed during the transition from spermatogonia to spermatocytes, and can drive the expression of Cre recombinase in testicular tissues after 3 days postpartum (3-dpp). First, Agk flox / flox mice were mated with Stra8-Cre mice to obtain Agk flox / + ;Stra8-Cre + mice. Then, Agk flox / + ;Stra8-Cre + male mice were mated with Agk flox / flox female mice to obtain Agk flox / - ;Stra8-Cre + , Agk flox / - ;Stra8-Cre - , Agk flox / + ;Stra8-Cre + or Agk flox / + ;Stra8-Cre - male offspring. Among them, Agk flox / - ;Stra8-Cre + male mice are the Agk-CKO male mice in the present invention, and Agk flox / + ;Stra8-Cre -Male mice were used as controls (Ctrl). The genotypes of each mouse were identified by PCR, and primers P1 and P2 were used to identify Agk flox / flox For mice, the flox band is 558 bp and the wt band is 471 bp; primers P3 and P4 are used to identify Stra-Cre mice, and the Cre-positive band is 155 bp; primers P5 and P6 are used to identify Agk gene knockout, and the ko band is 403 bp and the wt band is 685 bp. The primer sequences are as follows:

[0031] P1, 5'-tgggctcatcgttccttctgtct-3' (SEQ ID No. 1);

[0032] P2, 5′-ttcttgacaggctgctcttcttag-3′ (SEQ ID No. 2);

[0033] P3, 5'-GTGCAAGCTGAACAACAGGA-3' (SEQ ID No. 3);

[0034] P4, 5'-AGGGACACAGCATTGGAGTC-3' (SEQ ID No. 4);

[0035] P5, 5'-tgggctcatcgttccttctgtct-3' (SEQ ID No. 5);

[0036] P6, 5'-gtgggcgtcagcactgttcttc-3' (SEQ ID No. 6).

[0037] (2) Bioinformatics analysis of Agk gene expression characteristics

[0038] The RNA-seq transcriptome data of normal human tissues and the mouse ENCODE transcriptome data were obtained from the NCBI database, such as Figure 1 As shown in A and 1B, the AGK gene has similar tissue expression characteristics between humans and mice, and is widely expressed in multiple tissues and organs including the testis. To further analyze the relative expression level of the Agk gene in different types of spermatogenic cells in testicular tissue, we analyzed the GEO dataset GSE100964, which used RNA-seq to detect RNA expression in isolated and purified spermatogonia, pachytene spermatocytes, and round spermatids. The results are shown in Figure 1 As shown in C, the expression level of Agk is relatively highest in pachytene spermatocytes.

[0039] (3) Immunohistochemical analysis

[0040] Paraffin sections of testicular tissues from adult wild-type male mice were dewaxed and antigen-retrieved, then treated with 3% H2O2-methanol solution for 5-7 min to eliminate the effect of endogenous catalase. Subsequently, the procedure was carried out according to the instructions of a commercial immunohistochemistry kit. The primary antibody was anti-AGK rabbit polyclonal antibody (Abcam, ab137616), and the control was without the primary antibody. DAB color development was performed under an optical microscope. After color development, the nuclei were stained with hematoxylin, differentiated with hydrochloric acid alcohol, and then dehydrated, cleared, mounted, and examined under the microscope. The results were as Figure 1 shown in D, and there were strong AGK immunohistochemical staining signals in spermatocytes.

[0041] (4)Real-time fluorescence quantitative qPCR to detect gene expression levels

[0042] Several age-matched Ctrl and Agk-CKO male mice were sacrificed, and the testicular tissues were dissected and separated. Total RNA was extracted from the tissues and reverse-transcribed into cDNA. Real-time fluorescence quantitative qPCR analysis was performed using gene-specific primers, with the Hprt gene as an internal reference for relative quantitative analysis. The sequences of the gene-specific primers are shown in Table 1.

[0043] Table 1. Primer sequences used for qPCR detection

[0044]

[0045] (5)Western blot to detect protein expression levels

[0046] Several age-matched Ctrl and Agk-CKO male mice were sacrificed, and the testicular tissues were dissected and separated. After removing the tunica albuginea, total tissue proteins were extracted with RIPA lysis buffer (strong) containing protease inhibitors, and the protein concentration was quantified by the BCA method. Equal amounts of protein (about 60 μg) were subjected to SDS-PAGE gel electrophoresis, and then the Western blot detection steps such as membrane transfer, primary antibody incubation, secondary antibody incubation, and ECL chemiluminescence detection were completed according to the conventional method.

[0047] (6)Observation of testicular and epididymal tissue morphology and analysis of testicular weight-to-body weight ratio

[0048] Five 2-month-old Ctrl and Agk-CKO male mice were weighed and then sacrificed. Bilateral testicular and epididymal tissues were collected, and the weights of the bilateral testes were measured and recorded. The morphological sizes of the testicular and epididymal tissues were recorded by taking pictures. The results were as Figure 1 shown in G, and the Agk-CKO testes were significantly smaller, and the epididymides were not full. The ratio between the testicular weight (mg) and the body weight (g) of the mice was statistically analyzed. The results were as Figure 1As shown in H, the testis-to-body weight ratio of Agk-CKO male mice was significantly decreased. The method for analyzing the testis-to-body weight ratio of mice at different postnatal ages was the same as above.

[0049] (7)Sperm count in the cauda epididymis

[0050] Three 2-month-old Ctrl and Agk-CKO male mice were sacrificed, and the bilateral cauda epididymides were dissected and placed in a 24-well plate containing 1 ml of TYH buffer. The cauda epididymis tissues were minced with scissors and incubated in an incubator at 37 °C for 30 min to release the sperm in the cauda epididymis. The sperm suspension was pipetted and mixed well, and after appropriate dilution, the sperm were counted under an optical microscope using a hemocytometer. The results are as Figure 1 shown in I. The number of sperm in the cauda epididymis of Agk-CKO male mice was significantly reduced.

[0051] (8)Fertility evaluation of male mice

[0052] Four adult Ctrl and Agk-CKO male mice older than 2 months were caged with adult ICR female mice at a male-to-female sex ratio of 1:2. During the 3-month observation period, the number of litters born to the offspring of male mice in the two groups and the number of offspring per litter were counted. The results are as Figure 1 shown in J. The female mice mated with 4 Ctrl male mice gave birth to a total of 31 litters, with 8 - 16 offspring per litter, while the female mice mated with 4 Agk-CKO male mice did not give birth to any offspring, indicating that adult Agk-CKO male mice are infertile.

[0053] (9)H&E staining analysis of paraffin sections of testis and cauda epididymis

[0054] The testis and epididymis tissues of 2-month-old Ctrl and Agk-CKO male mice were dissected and fixed with picric acid fixative, then paraffin-embedded and sectioned. Subsequently, hematoxylin and eosin (H&E) staining was performed using conventional methods. The results are as Figure 2 shown in A. Compared with the testis and cauda epididymis tissues of Ctrl male mice, the spermatogenic cells in the testis tissue of Agk-CKO male mice were disorderly arranged, with a large number of vacuoles, and round spermatids were rarely seen. The sperm content in the cauda epididymis was very low. The method for H&E staining analysis of paraffin sections of testis from mice at different postnatal ages was the same as above.

[0055] (10)PNA fluorescence staining analysis of paraffin sections of testis and sperm smears

[0056] Paraffin sections of the testes of Ctrl and Agk-CKO adult male mice after dewaxing treatment, as well as sperm smears from the cauda epididymis, were washed three times with 1 x PBS, then Alexa Fluor 488-labeled PNA molecular probe was added dropwise. After incubation at room temperature for 1 h, they were washed three times with 1 x PBS, DAPI staining solution was added dropwise for nuclear staining, and after incubation at room temperature for 30 min, they were washed three times with 1 x PBS, and then mounted with an anti-fluorescence quenching DAKO mounting medium. Subsequently, they were observed and photographed under a fluorescence microscope. The results are as Figure 2 shown in Figures B and 2D. The acrosome structures gradually formed in round spermatids of Ctrl testicular tissue were strongly labeled by PNA fluorescence signals, while PNA fluorescence signals were sporadically distributed or even invisible in the seminiferous tubules of Agk-CKO testicular tissue. Similarly, in sperm from the cauda epididymis, the acrosome structures of Ctrl sperm were specifically labeled by PNA fluorescence signals, while almost no PNA fluorescence signals were detected in Agk-CKO sperm, indicating that they hardly had intact acrosome structures.

[0057] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for constructing a male germ cell-specific Agk knockout mouse model, characterized in that Agk flox / flox The mice were mated with Stra8-Cre mice to obtain male germ cell-specific Agk conditional knockout mice Agk-CKO, which is the male germ cell-specific Agk knockout mouse model.

2. The method for constructing a male germ cell-specific Agk knockout mouse model according to claim 1, characterized in that: The construction method comprises the following steps: firstly, flox / flox Mice were mated with Stra8-Cre mice to obtain Agk flox / + ;Stra8-Cre + mice, and then Agk flox / + ;Stra8-Cre + Agk flox / flox Female mice mate and obtain Agk flox / - ;Stra8-Cre + , Agk flox / - ;Stra8-Cre - , Agk flox / + ;Stra8-Cre + or Agk flox / + ;Stra8-Cre - In the male offspring, Agk flox / - ;Stra8-Cre + The male mouse is the male germ cell-specific Agk knockout mouse model.

3. The method for constructing a male germ cell-specific Agk knockout mouse model according to claim 1, characterized in that: The Agk flox / flox The mouse was constructed by inserting a loxP site on both sides of exon 3 of the Agk gene based on ET cloning and ES cell targeting methods.

4. An application of a male germ cell-specific Agk knockout mouse model constructed by the construction method as described in any one of claims 1 to 3 in the clinical diagnosis and treatment of male infertility and drug development.

5. An application of a male germ cell-specific Agk knockout mouse model constructed by the construction method as described in any one of claims 1 to 3 in the study of the developmental differentiation state of male germ cells and the regulatory mechanism of meiosis.

6. Application of reagents for knocking out the Agk gene in the preparation of animal models of testicular development arrest, sperm reduction and male infertility.

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