Method for constructing azoospermia mouse model by knocking out germ cell EIF5A gene and application

Abstract

The invention discloses a method for constructing an azoospermia mouse model by knocking out a germ cell EIF5A gene and application of the azoospermia mouse model, the construction method is to knock out the EIF5A gene on a mouse germ cell genome, and the knockout is carried out by using a Cre-LoxP system. The azoospermia mouse model constructed by the invention can be used for research on non-obstructive azoospermia and corresponding drug research and development. The model mouse shows that the testis weight and volume are reduced, the spermatogenic epithelium is atrophic, spermatogonium cells cannot complete meiosis completely, germ cells are massively deleted, and the sperm quantity is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and particularly relates to a method and application for constructing an azoospermia mouse model by knocking out the EIF5A gene in germ cells. Background Art

[0002] Infertility is a common disease that seriously affects human health, and its incidence has been increasing year by year in recent years. Infertility refers to a situation where a couple has normal sexual life and has not been pregnant for one year or more without taking any contraceptive measures.

[0003] The causes of male infertility are extremely complex, involving the anatomical structure and function of the reproductive system, infection, endocrine, genetic factors, etc. Azoospermia is an important pathogenic factor for male infertility, and its patients account for 10%-15% of male infertility patients. Azoospermia is defined as that after semen is centrifuged and the sediment is observed under a microscope, sperm are not found in all three observations. Clinically, it is generally classified into two types: obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) according to whether there is obstruction in the vas deferens of the patient. Among them, non-obstructive azoospermia is mostly caused by testicular spermatogenic dysfunction, resulting in reduced or absent sperm production. In recent years, the development of some assisted reproductive technologies has enabled some OA patients to achieve fertility, while NOA patients are difficult to benefit due to the lack of sperm. Therefore, it is imperative to study the pathogenic factors of NOA. Further studying the molecular biological basis of spermogenesis disorders in NOA patients, revealing the regulatory network of spermogenesis, and clarifying the causes of idiopathic male infertility have great clinical application value.

[0004] The eukaryotic translation initiation factor 5A encoded by the EIF5A gene is activated through a unique hypusine modification. This modification is catalyzed step by step by deoxyhypusine synthase (DHPS) and hypusine synthase (HPS), and it is the only known protein that depends on this modification. Its core function is to regulate the elongation stage of mRNA translation, especially playing a key role in the synthesis of proteins containing polyproline sequences (such as extracellular matrix proteins and signal transduction molecules), and improving translation efficiency by alleviating the "stuttering" of ribosomes in complex structural regions. There are two main subtypes of this gene: EIF5A1 is widely expressed in various tissues, participating in cell proliferation, differentiation, autophagy, and oxidative stress response, and affecting the G1 / S phase transition of the cell cycle; while EIF5A2 is lowly expressed in normal tissues but is abnormally highly expressed in various malignant tumors such as liver cancer, colorectal cancer, and ovarian cancer. It drives tumor invasion and metastasis by promoting epithelial-mesenchymal transition (EMT) and angiogenesis, and is regarded as a potential marker for poor cancer prognosis. In addition, the dysfunction of EIF5A is related to neurodegenerative diseases such as Alzheimer's disease (such as excessive phosphorylation of tau protein) and Parkinson's disease, and is involved in regulating the secretion of inflammatory factors such as IL-6 and TNF-α, and may mediate chronic inflammation and disease progression. Currently, anti-cancer strategies targeting the hypusine modification pathway (such as the DHPS inhibitor GC7) have shown efficacy in preclinical studies, and the functional studies of this gene in model organisms (such as Drosophila and yeast) have provided important clues for revealing the mechanisms of aging, viral infection (such as HIV depends on EIF5A to synthesize viral proteins), and metabolic regulation. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing an azoospermia mouse model by knocking out the EIF5A gene in germ cells and its application. The present invention for the first time reveals the important role of the EIF5A gene in the spermatogenesis process of mice. Male mice with knockout of the EIF5A gene in germ cells show testicular dysplasia and azoospermia. The mechanism by which the EIF5A gene regulates testicular development is expected to provide new ideas for the study of the mechanism of male infertility.

[0006] To achieve the above purpose, the present application adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a method for constructing an azoospermia mouse model. The construction method is to knock out the EIF5A gene on the genome of mouse germ cells, and the knockout is carried out using the Cre-LoxP system.

[0008] The Cre-loxP system is a gene editing tool based on bacteriophage P1, consisting of the Cre recombinase and loxP sites: The Cre enzyme can specifically recognize and cleave the 34-bp loxP site, and through the recombination mechanism, achieve the deletion, inversion, or translocation of DNA fragments. Among them, STRA8-Cre is a tool designed based on the characteristics of the STRA8 gene, a key regulator of germ cell meiosis: The Cre recombinase gene is placed under the control of the STRA8 promoter, enabling it to be specifically expressed only before germ cells enter meiosis.

[0009] In the above technical solution, the GeneID of the EIF5A gene is: 276770.

[0010] In the above technical solution, the specific method of knockout is as follows: In mouse embryonic stem cells, a floxed EIF5A allele is generated through homologous recombination to obtain flox / + heterozygous mice. After the flox / + heterozygous mice are mated with each other to obtain flox / flox homozygous mice, they are then mated with STRA8-Cre tool mice. The Cre recombinase is used to target and excise the exon between the two loxP sites, resulting in the loss of function of the EIF5A gene in mouse germ cells, thereby achieving conditional knockout of EIF5A in germ cells.

[0011] In the second aspect, the present invention provides the application of the non-obstructive azoospermia mouse model constructed by the above construction method in the study of non-obstructive azoospermia.

[0012] The beneficial effects of the present invention are as follows: The present invention uses the Cre-LoxP system to knockout the EIF5A gene on the genome of germ cells of C57BL / 6J mice to construct a non-obstructive azoospermia mouse model, which can be used for the research of non-obstructive azoospermia and the development of corresponding drugs. The model mice show reduced testicular weight and volume, atrophy of the seminiferous epithelium, incomplete meiosis of spermatogonia, a large number of missing germ cells, and a decrease in sperm count. Description of the Drawings

[0013] Figure 1 : Expression of EIF5A in various tissues and its expression pattern in testicular tissue; among them, A: Expression level of the EIF5A gene in various organs of 56-day-old mice; B: Expression level of EIF5A in the testes of wild-type mice aged 0-8 weeks, with higher EIF5A expression levels at P14 and P21; C: Detection of the expression level of EIF5A in testicular sections of 56-day-old wild male mice by immunofluorescence method, and EIF5A is expressed in both germ cells and Sertoli cells. Figure 2 : Schematic diagram of the targeting strategy for generating a floxed EIF5A allele through homologous recombination in mouse embryonic stem cells.

[0014] Figure 3: Mouse genotype identification result diagram.

[0015] Figure 4 : Appearance photo of P56 mice.

[0016] Figure 5 : Conditional knockout mice of EIF5A were successfully constructed; among them, A, the appearance of testes of adult wild-type and conditional knockout mice, the testes of conditional knockout mice were significantly smaller; B, detecting the expression level of EIF5A in the testes of wild-type and conditional knockout mice, the expression level of EIF5A in conditional knockout mice was low; C, fertility test of wild-type and conditional knockout mice; D, testis-body ratio (mg / g) of wild-type and conditional knockout mice.

[0017] Figure 6 : A, HE sections of testes of adult wild-type and conditional knockout mice, no complete spermatogenesis process was seen in the testes of conditional knockout mice, and sperm were lacking; B, HE sections of epididymides of adult wild-type and conditional knockout mice, there were no sperm in the epididymides of conditional knockout mice while the epididymides of wild-type mice were filled with sperm.

[0018] Figure 7 : A, detecting apoptotic cells in the testes of adult wild-type and conditional knockout mice by tunnel staining, the apoptotic signal in the testes of conditional knockout mice was significantly increased; B, detecting the proliferation signal PCNA in the testis sections of adult wild-type and conditional knockout mice, the PCNA signal in conditional knockout mice was significantly decreased; C, detecting the germ cell signal MVH in the testis sections of adult wild-type and conditional knockout mice, obvious germ cell deficiency was seen in the testis sections of conditional knockout mice.

[0019] Figure 8 : Statistical chart of detection of marker molecules during testicular meiosis.

[0020] Figure 9 : Detection result diagram of synaptonemal complex formation in the prophase of the first meiosis.

[0021] Figure 10 : Detection result diagram of γ-H2AX signal in the prophase of the first meiosis.

[0022] Figure 11 : PCR program. Detailed implementation manners

[0023] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will convey the concept of the present invention fully to those skilled in the art. The present invention will be defined only by the claims.

[0024] Example 1 Expression of Eif5a in various tissues and its expression pattern in testes

[0025] The heart, brain, liver, spleen, lung, kidney, bladder, testis, and ovary of 56-day-old wild-type C57BL / 6J mice were obtained, and RNA was extracted, and the expression level of Eif5a gene was detected by qPCR. Figure 1 A. The results showed that Eif5a was highly expressed in spleen, lung, testis and ovary. Testicular tissues of wild-type mice were obtained at P0, P7, P14, P21, P28, P35, P42 and P56, RNA was extracted, and qPCR was performed to detect the expression level of Eif5a gene. Figure 1 B The results show that Eif5a is highly expressed in the testes of P14 and P21 mice. Immunofluorescence staining of Eif5a was performed using paraffin sections of the testes of 56-day-old wild-type mice. Figure 1 C The results showed that Eif5a was expressed in spermatogonia, pachytene spermatocytes, leptotene spermatocytes and supporting cells.

[0026] Example 2: Eif5a (flox / +) heterozygous mouse model (C57BL / 6) constructed.

[0027] In order to further clarify the role of Eif5a in testicular development, we commissioned Saiye (Suzhou) Biological Co., Ltd. to generate the floxed EIF5A allele in mouse embryonic stem cells by homologous recombination to obtain (flox / +) heterozygous mice. Figure 2 As shown, the same LoxP sites were inserted at both ends of the exon to obtain (flox / +) heterozygous mice. After mating with STRA8-Cre tool mice, the Cre recombinase was used to target and remove the exon between the two loxP sites, thereby conditionally knocking out the Eif5a gene in mouse germ cells.

[0028] Example 3 Mouse genotype identification

[0029] Primers were designed to identify the mouse genotype obtained in Example 2. The primer design sites were as follows: Figure 2 , the primer sequences are shown in Table 1. Genotyping of 10-day-old newborn mice: Obtain a small amount of mouse tail tissue, extract DNA using lysis solution (40mM NaOH+0.2mM EDTA), and stop lysis with an equal amount of neutralization solution (40mM Tris-Hcl). Perform PCR using the HotStart enzyme PCR reaction system, which is shown in Table 2. Prepare 2.5% agarose gel for DNA electrophoresis, Figure 3 This is the electrophoresis diagram for mouse genotype identification. Figure 3It can be seen that the wild-type mice have only the 301bp band, the homozygous mice have only the 364bp band, and the heterozygous mice have both the 364bp and 301bp bands. The 443bp band is for the STRA8-Cre transgenic mice, and the 263bp band is for the mice with the target gene knocked out.

[0030] Table 1

[0031]

[0032]

[0033] Table 2

[0034] Material Volume Premixed Taq Polymerase 10 μL Primer-F 1 μL Primer-R 1 μL DNA Template 2 μL ddH2O 6 μL

[0035] The PCR reaction procedure is as Figure 11 shown.

[0036] Example 4 Mouse Phenotype Identification

[0037] (1) There is no difference in the body weight of P56 mice (the day when the mouse is born is recorded as P0 day) ( Figure 4 ). There are significant differences in the size of the testes and epididymides between wild-type mice and knockout mice at P56 days ( Figure 5 A). RNA was extracted from the testes of 56-day-old wild-type mice and knockout mice, and qPCR was performed to detect the expression level of the Eif5a gene. The results showed that the expression level of the Eif5a gene in knockout mice was significantly decreased ( Figure 5 B). 56-day-old male wild-type mice and knockout mice were paired with fertile female mice respectively to observe fertility. After 6 months of fertility testing, the results showed that the knockout mice were completely infertile ( Figure 5 C). Male wild-type mice and knockout mice at P7, P14, P35, P56, and P120 were weighed and then sacrificed, and their testes were weighed to calculate the testis-body ratio (mg / g). The results showed that the testis-body ratio of knockout mice decreased compared with that of wild-type mice starting from P14 ( Figure 5 D). HE sections of the testes of 56-day-old wild-type mice showed that sperm appeared in the spermatogenic epithelium of the control mice, while sperm generation was not observed in the spermatogenic epithelium of knockout mice, and there were no secondary meiotic cells such as round spermatids, indicating that the testes of knockout mice failed to successfully enter the second meiotic division. Apoptotic cells could be observed in the testes of knockout mice ( Figure 6 A).

[0038] Further examination of the epididymis, the site of sperm maturation, found that the epididymis of wild-type mice was filled with sperm, while no sperm was observed in the epididymis of knockout mice, indicating that knockout mice are azoospermic mice ( Figure 6 B).

[0039] (2) As a large number of cells were missing in the degenerated seminiferous tubules in the HE sections of the testes of the knockout mice, proliferation and apoptosis were further detected. Tunnel chromosome indicated a large number of apoptotic signals in the testis sections of the knockout mice ( Figure 7 A), and immunofluorescence detection of PCNA (proliferating cell nuclear antigen) indicated no significant difference in the proliferation signals in the testis sections of wild-type mice and knockout mice ( Figure 7 B); MVH is a marker molecule for germ cells, and its immunofluorescence detection indicated the absence of germ cells in the testes of the knockout mice ( Figure 7 C). Therefore, it is suggested that the apoptosis of germ cells in the testes of the knockout mice leads to the loss of germ cells, which also explains why the testis weight of the knockout mice is lower than that of wild-type mice.

[0040] (3) To clarify the cause of azoospermia in the knockout mice, marker molecules at various stages of meiosis were further detected by qPCR, such as Stra8 (differentiated spermatogonia), Sycp3 (spermatocytes in the first meiotic division), Dkkl1 (spermatocytes in the pachytene stage of the first meiotic division), and Tnp1 (spermatids). The results showed that the marker molecule Stra8 for differentiated spermatogonia and the marker molecule Sycp3 for the first meiotic division were upregulated in the knockout mice, the marker molecule Dkkl1 for spermatocytes in the pachytene stage of the first meiotic division showed no significant change, and the marker molecule Tnp1 for spermatids was not detected ( Figure 8 ). It is suggested that the germ cells of the knockout mice can enter the first meiotic division but cannot enter the second meiotic division, that is, the meiosis of germ cells in the testes of the knockout mice is blocked at the first meiotic division stage ( Figure 8 ).

[0041] (4) To further clarify the cause of meiotic failure in the knockout mice, we obtained the testes of 21-day-old mice and performed a chromosome spreading experiment to detect the formation of the synaptonemal complex (SC) in the prophase of the first meiotic division, where Sycp3 and Sycp1 were used as marker molecules for SC. Meiosis has a series of special and orderly chromosomal behaviors. According to the chronological order, the chromosomal behaviors in the first meiotic division include: the formation of the lateral axes of the synaptonemal complex and the cohesion between sister chromatids, the pairing, synapsis, recombination, and separation of homologous chromosomes, etc. According to the morphology of the synaptonemal complex, the prophase of the first meiotic division is divided into leptotene, zygotene, pachytene, diplotene, and diakinesis. The results showed that the sex chromosomes were separated in the knockout mice, indicating that the synapsis of spermatocytes in the first meiotic division of the knockout mice failed and the chromosomes could not be completely synapsed, thus failing to enter the pachytene stage ( Figure 9 ).

[0042] (5) To clarify the reason for the failure of strip knockout mice to enter the pachytene stage, we next detected the signal of γ-H2AX. During the first meiotic division, under the action of SPO11 molecules, DNA double-strand breaks (DSBs) occur on chromosomes. The formation of DSBs induces the formation of γ-H2AX signals. By the pachytene stage, the γ-H2AX signal is only present in the sex vesicle region, and the γ-H2AX signal on autosomes disappears as DSBs are repaired. Our results suggest that there is no significant difference in the γ-H2AX signal between control mice and strip knockout mice at the leptotene and zygotene stages. After entering the pachytene stage, the γ-H2AX signal in wild-type mice and strip knockout mice is only present in the sex vesicle region, indicating that the sex chromosomes cannot synapse completely.

[0043] Obviously, the above embodiments are only examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for constructing an azoospermia mouse model, characterized in that: The construction method is to knockout the EIF5A gene in the genome of mouse germ cells, and the knockout is carried out using the Cre-LoxP system.

2. The construction method according to claim 1, wherein: The GeneID of the EIF5A gene: 276770.

3. The construction method according to claim 1, characterized in that: The specific method of knockout is as follows: Generate floxed EIF5A alleles by homologous recombination in mouse embryonic stem cells to obtain flox / + heterozygous mice. After the flox / + heterozygous mice are mated with each other to obtain flox / flox homozygous mice, they are then mated with STRA8-Cre tool mice. The Cre recombinase is used to target and excise the exon between the two loxP sites, resulting in the loss of function of the EIF5A gene in mouse germ cells, thereby achieving conditional knockout of EIF5A in germ cells.

4. Application of the azoospermia mouse model constructed by the construction method described in any one of claims 1-3 in the study of non-obstructive azoospermia.

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