Application of CDKN3 protein in preparation of kit for diagnosing non-obstructive azoospermia

Abstract

The invention discloses an application of a CDKN3 protein in preparation of a kit for diagnosing non-obstructive azoospermia, and belongs to the field of reproductive medicine, and the kit takes the CDNK3 protein with low expression in tissues as a marker for diagnosing the non-obstructive azoospermia. The kit is used for diagnosing the non-obstructive azoospermia by detecting the expression level of 54-67 amino acids (sequence: KDVRRNVQKDTEEL) at the N end of the CDKN3 in a sample tissue of a subject. Compared with the expression level of the CDKN3 protein in a normal control, the expression level of the CDKN3 protein in a sample tissue of a patient with non-obstructive azoospermia is remarkably reduced. Whether a subject suffers from non-obstructive azoospermia or not can be effectively judged by detecting the expression level of the CDKN3 protein in the sample tissue of the subject. The method has important research significance and clinical application value for non-obstructive azoospermia.

Description

Technical Field

[0001] The invention belongs to the field of reproductive medicine, and in particular, relates to the application of CDKN3 protein in preparing a kit for diagnosing non-obstructive azoospermia. Background Art

[0002] Male infertility is a common disease that seriously affects male reproductive health. It is mainly manifested as oligospermia, asthenozoospermia, teratozoospermia and azoospermia. Azoospermia is the most serious type clinically, accounting for about 5%-20% of male infertility. According to whether the vas deferens is obstructed, it can be further divided into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). NOA refers to the patient's sperm production being damaged due to testicular spermatogenesis dysfunction. It is the main type of azoospermia, accounting for about 60%. The absolute number of male infertility patients caused by NOA is huge, and it has become a group that cannot be ignored. However, the pathogenic mechanism of NOA is complex, and there is a lack of effective treatment methods. The main way to give birth to offspring is through sperm donation. Single gene mutation is one of the important causes of NOA. According to statistics, more than 20 genes have been identified as missing, which can cause NOA and severe oligospermia. In Genecards, nearly 1,000 genes that may be related to NOA are listed, which has promoted the research and development of male infertility diagnosis and treatment. However, the cause of 60% of NOA patients is still unknown.

[0003] Spermatogenesis refers to a continuous and complex biological process from spermatogonia to mature sperm, including the self-renewal and differentiation of spermatogonial stem cells, meiosis of spermatocytes, and deformation and release of sperm. Any abnormality in any of these links will lead to spermatogenesis disorders. The characteristic of mammalian meiosis is the ability to produce haploid gametes. Before the separation of homologous chromosomes, chromosomes will spontaneously form double-strand breaks (DSBs) and undergo homologous recombination repair to ensure the stability of the genetic material of the offspring; at the same time, cross-links are formed between homologous chromosomes, which promotes the exchange of part of the genetic information between homologous chromosomes, ensures the genetic diversity of species, and plays an important role in species evolution. Errors in the meiotic process can cause abnormal gametes and lead to male infertility. Spermatocyte development block caused by meiotic abnormalities is the most common pathological type in testicular biopsies of NOA patients. Therefore, the study of the meiotic mechanism of male germ cells can provide new ideas for the study of the mechanism of spermatogenesis and the diagnosis and treatment of male infertility.

[0004] The spontaneous formation of DSBs is a unique molecular biological event during meiosis. After the start of meiosis, HORMAD1 protein localizes to chromosomes and recruits REC114 and MEI4 proteins at the site where DSBs are about to be formed to form a pre-DSB complex. Then, under the catalysis of pre-DSB, the multi-species conserved nuclease SPO11 protein is recruited to the site to cut DNA and form DSBs. After DSBs are formed, a 1kb long single-stranded nucleotide (ssDNA) is removed at the break site by 5-' exonuclease, leaving a complementary ssDNA overhang. This ssDNA overhang is bound by the single-stranded binding protein RPA, and under the joint action of RAD51 and DMC1, it invades the double strands of homologous chromosomes, thereby initiating recombination.

[0005] Inappropriate DSBs formation can cause fatal damage to spermatocytes: 1. Failure to repair in time leads to cell apoptosis; 2. Incorrect repair methods, such as non-homologous end joining or non-homologous gene recombination repair, lead to genetic information errors and loss. Therefore, the DSBs process of meiosis is subject to strict molecular regulation, including the site specificity and number of DSBs. The formation of a DSBs site will inhibit the formation of other DSBs at nearby sites. The DNA damage sensor ATM\ATR kinase is currently known as the DSBs controller, and its function is conserved in species such as mammals, fruit flies and yeast. When a break occurs at a certain site on the chromosome, ATM\ATR promotes phosphorylation reactions at the damage site to inhibit the further formation of DSBs. Therefore, ATM\ATR is a DSBs damage inhibitor conserved in multiple species. In mammals, it is not clear whether such a phospho-inhibitory enzyme exists.

[0006] CDKN3 is a testis-dominantly expressed gene in humans and mice, with a homology of 87.94%. Human CDKN3 contains 212 amino acids, of which the 33rd to 201st amino acids are conserved protein phosphatase domains, suggesting that it may be a dephosphorylation regulatory enzyme.

[0007] Studies have shown that CDKN3 can bind to CDK1 / 2 kinases in tumors and inhibit the activity of CDK1 / 2 in promoting phosphorylation of substrate proteins. As a potential dephosphorylation regulatory enzyme, CDKN3 protein may maintain phosphorylation homeostasis and DSBs formation during meiosis by binding to and inhibiting protein phosphorylation during the formation of DSBs in spermatocytes. Summary of the invention

[0008] The purpose of the present invention is to provide a use of CDKN3 protein in the preparation of a kit for the diagnosis of non-obstructive azoospermia. CDKN3 protein is one of the differentially expressed proteins identified by proteomics technology in the early stage. CDKN3 is significantly downregulated in the testicular tissue of patients with non-obstructive azoospermia, and is very likely to become a clinical target for non-obstructive azoospermia.

[0009] The present invention adopts the following technical solution to solve the above technical problems: application of CDKN3 protein in the preparation of a kit for diagnosing non-obstructive azoospermia, wherein the kit uses CDKN3 protein lowly expressed in tissues as a marker for diagnosing non-obstructive azoospermia.

[0010] Furthermore, the kit uses the prepared CDKN3-specific antibody to detect the expression level of the N-terminal 54-67 amino acids of CDKN3 (sequence: KDVRRNVQKDTEEL) in the sample tissue of the subject.

[0011] Furthermore, the expression level of the N-terminal 54-67 amino acids of CDKN3 (sequence: KDVRRNVQKDTEEL) in the sample tissues of patients with non-obstructive azoospermia was significantly downregulated.

[0012] Furthermore, the sample tissue is testicular puncture tissue.

[0013] Furthermore, the kit can use immunoblotting, immunofluorescence, and immunohistochemistry to analyze and detect the expression level of CDKN3 protein in sample tissues.

[0014] So far, there is no report on the involvement of CDKN3 in the occurrence of NOA, nor on the role and mechanism of CDKN3 in the formation of DSBs in spermatocytes. Therefore, the present invention is original and innovative.

[0015] Beneficial effects of the present invention: The present invention experimentally found that the expression level of CDKN3 protein is significantly downregulated in the testicular tissue of patients with non-obstructive azoospermia, and is very likely to become a clinical target for non-obstructive azoospermia; by detecting the expression level of CDKN3 protein in the sample tissue of the subject, it can be effectively determined whether the subject suffers from non-obstructive azoospermia; it has important research significance and clinical application value for non-obstructive azoospermia. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the expression level of CDKN3 protein in the testes of normal men and patients with non-obstructive azoospermia; Figure 1 The results of immunoblot analysis of CDKN3 protein in human testis: expression is lost in testicular tissue of patients with non-obstructive azoospermia.

[0017] Figure 2 The expression and localization of CDKN3 in mouse testis tissue; because CDKN3 is a testis dominantly expressed gene homologous to humans and mice, with a homology of 87.94%, the specific antibody of CDKN3 protein can also detect the expression level of CDKN3 protein in mouse testis tissue; Figure 2 A in the middle shows the results of CDKN3 immunohistochemical analysis of testicular tissues of mice in the control group (left) and experimental group (right): CDKN3 expression is absent in cdkn3 knockout mice; Figure 2 B shows the immunohistochemistry results of CDKN3 in the testicular tissue of mice in the control group (left) and experimental group (right). CDKN3 protein is highly expressed in the nuclei of spermatocytes in the preleptotene, leptotene, and zygotene stages of mouse testis.

[0018] Figure 3 The phenotype of CDKN3 deficiency in non-obstructive azoospermia: male patients with CDKN3 protein deficiency have spermatogenesis arrested at the zygotene and zygotene stages; Figure 3 A in the middle is the result of HE staining analysis: the seminiferous tubule epithelium of male patients lacking CDKN3 protein has almost no sperm cells after meiosis. Figure 3 A1 control group (left) and experimental group (right), Figure 3 a2 in A is a partial enlarged view of a1, showing the control group (left) and the experimental group (right); Figure 3 The control group (left) and experimental group (right) in B are shown in Figure 1. b1 is the immunofluorescence staining results of γH2AX in paraffin sections of testicular tissue. Figure 3 Figure) and experimental group (right Figure 3 Figure): The spermatocytes of males lacking CDKN3 protein do not have cells that have completed DSB repair, and the spermatocytes are blocked in the thin and zygotene stages. b2 is a partial enlargement of the corresponding figure of b1, and b3 is a partial enlargement of the corresponding figure of b2. DETAILED DESCRIPTION

[0019] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be interpreted as limiting the present invention.

[0020] The present invention adopts the following technical solutions to solve the above technical problems: Example 1 Analysis of the expression pattern of CDKN3 in non-obstructive azoospermia Western blotting and immunohistochemical staining were used to observe the expression level and localization of CDKN3 in the testicular tissue of the control group and the experimental group.

[0021] (1) Specimen collection: Patients with non-obstructive azoospermia and CDKN3 knockout mice were selected as the experimental group, and men with normal fertility and mice with normal fertility were selected as the control group. The selected samples had no chromosomal abnormalities and no evidence of recent infection.

[0022] Obtaining male testicular tissue - testicular puncture: complete routine preoperative examinations, prepare the surgical area, disinfect the surgical area with iodine, perform lidocaine local anesthesia, insert the puncture needle into the testicle, extract the testicular seminiferous tubules through suction negative pressure, and apply pressure bandage to the testicle gauze. The blood was rinsed with saline in the two groups of testicular tissues. One tissue was stored in a -80℃ refrigerator for real-time fluorescence quantitative PCR, protein immunoblotting and other experiments; one tissue was embedded in paraffin and stored for immunofluorescence, immunohistochemistry and other experiments.

[0023] Obtaining mouse testicular tissue: Male mice were killed by cervical dislocation, and the skin and muscle layers were cut to expose the internal organs. At the lowest end of the abdomen, the fat layer was gently pulled to see the testicular tissue. One testicular tissue was removed from the capsule and stored in a -80℃ refrigerator for real-time fluorescence quantitative PCR, protein immunoblotting and other experiments; one testicular tissue was embedded in paraffin and stored for immunofluorescence, immunohistochemistry and other experiments.

[0024] (2) Preparation of CDKN3-specific antibodies: Based on the amino acid sequence encoded by the human CDKN3 gene, the signal peptide, hydrophilicity and antigenicity of the CDKN3 protein were analyzed to determine the amino acid sequence of the antigenic peptide: the amino acid sequence at positions 54-67 at the N-terminus (KDVRRNVQKDTEEL, a peptide synthesized by Shanghai Bioengineering and coupled to keyhole limpet hemocyanin (KLH)) was used to immunize healthy male New Zealand white rabbits. After multiple booster immunizations, polyclonal antibodies were prepared using the collected rabbit antisera and Western Blot analysis was performed to obtain CDKN3-specific antibodies.

[0025] (3) Western blotting The total protein of testicular tissue was extracted with RIPA lysis buffer, and the protein concentration was determined by BCA protein concentration determination kit. The loading buffer was added, heated at 95℃ for 10min, 20ul total protein was loaded, SDS-PAGE gel electrophoresis, PVDF membrane transfer, 5% skim milk powder was blocked for 1 hour, diluted CDKN3 specific antibody was used to shake at 4℃ overnight, TBST was washed for 5min*3 times, the secondary antibody was shaken at room temperature for 1 hour, TBST was washed for 5min*3 times, and ECL chemiluminescence was used for color development.

[0026] (4) Immunohistochemical staining The paraffin sections were placed in a 65°C oven for 1 hour, dewaxed and hydrated, 3% H2O2 was used to eliminate the activity of endogenous peroxidase, antigen repair, serum blocking, incubation and washing with diluted CDKN3-specific antibody, secondary antibody incubation and washing, horseradish enzyme labeling and washing, color development, hematoxylin counterstaining, sealing, and microscopic observation and photography.

[0027] Western blotting results showed that CDKN3 protein was completely absent in patients with non-obstructive azoospermia ( Figure 1 ), indicating that the expression level of CDKN3 protein was significantly downregulated in patients with non-obstructive azoospermia. Immunohistochemistry results showed that CDKN3 protein was highly expressed in the pre-leptotene, leptotene, and zygotene spermatocyte nuclei of mouse testis, while no expression was detected in the experimental group, indicating that CDKN3 protein was localized in the leptotene and zygotene cell nuclei of spermatocytes ( Figure 2 A-2B).

[0028] Example 2 Phenotypic analysis of CDKN3 deficiency in non-obstructive azoospermia (1) Eosin-hematoxylin staining The paraffin sections of testicular tissue were placed in a 65°C oven for 1 hour, dewaxed and hydrated, stained with hematoxylin, washed with water, washed with 1% hydrochloric acid alcohol, washed with water, stained with eosin, dehydrated, sealed, and observed and photographed under a microscope.

[0029] (2) Immunofluorescence staining The paraffin sections of testicular tissue were placed in an incubator at 60°C for 1 hour, washed with PBS, blocked with serum, incubated with primary antibody at 4°C overnight, washed with PBS, incubated with secondary antibody at room temperature for 1 hour, washed with PBS, nuclear stained, sealed, stored in a dark box, and photographed under a fluorescent staining microscope.

[0030] HE staining results of human testicular tissue sections showed that obvious round sperm and elongated sperm cells were visible in the seminiferous tubule lumen of the control group, but the seminiferous tubule epithelium of the testis of patients with non-obstructive azoospermia was almost completely devoid of meiotic sperm cells ( Figure 3 A in the middle), indicating that spermatogenesis is blocked at the spermatocyte stage after the lack of CDKN3. The results of immunofluorescence staining of human testicular tissue showed that the break signal protein γH2AX in spermatocytes of the control group was localized to sex chromosomes in spermatocytes at the pachytene stage, while the iconic pachytene spermatocyte localization was almost undetectable in the testes of patients with non-obstructive azoospermia. This suggests that after the loss of CDKN3, spermatocytes cannot complete double-strand break repair and are blocked at the thin and zygotene stages. These results indicate that CDKN3 protein plays an irreplaceable role in meiosis, and the loss of CDKN3 can lead to non-obstructive azoospermia.

[0031] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. The use of CDKN3 protein in the preparation of a kit for diagnosing non-obstructive azoospermia, characterized in that: The kit uses CDKN3 protein lowly expressed in tissue as a marker for diagnosing non-obstructive azoospermia.

2. Use of the CDKN3 protein according to claim 1 in preparing a kit for diagnosing non-obstructive azoospermia, characterized in that: The kit uses the prepared CDKN3 protein-specific antibody to detect the expression level of the N-terminal 54-67 amino acid sequence of CDKN3: KDVRRNVQKDTEEL in the sample tissue of the subject.

3. Use of the CDKN3 protein according to claim 2 in preparing a kit for diagnosing non-obstructive azoospermia, characterized in that: The sample tissue is testicular biopsy tissue from a patient with non-obstructive azoospermia.

4. Use of the CDKN3 protein according to claim 2 in preparing a kit for diagnosing non-obstructive azoospermia, characterized in that: The kit uses one or more of immunoblotting, immunofluorescence, and immunohistochemistry to analyze and detect the expression level of CDKN3 protein in sample tissues.

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