Application of RBBP7 gene / protein as a drug target in the preparation of products for diagnosis and treatment of male infertility

Abstract

The present invention provides the use of the RBBP7 gene / protein as a drug target in the preparation of products for the diagnosis and treatment of male infertility. Experiments have shown that mutant RBBP7 genes / proteins lead to male infertility, spermatogenesis disorders, spermatogenesis arrest, and azoospermia. The mutant RBBP7 gene / protein of the present invention and one of its pathogenic mutation sites can be used as target genes for the diagnosis of male infertility. Furthermore, the expression level of RBBP7 in the RBBP7 gene / protein is significantly reduced in patients with non-obstructive azoospermia. By increasing the activity and / or expression of the RBBP7 protein, male infertility can be prevented and / or treated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of the RBBP7 gene and / or protein as a molecular marker in the diagnosis of male infertility, and to a method for detecting pathogenic mutation sites in human male infertility genes. Background Art

[0002] In recent decades, male reproductive capacity has steadily declined worldwide, with both sperm count and quality declining. The prevalence of infertility among couples of childbearing age is as high as 15%, with male factors accounting for approximately 50%. Male infertility places a heavy burden on individuals, families, and society. Clinically, male infertility primarily manifests as oligoasthenozoospermia or azoospermia. Non-obstructive azoospermia (NOA), primarily located in the testicles, results in an absence of detectable sperm in semen and is one of the most severe forms of male infertility. The causes of male infertility are complex, including factors related to the anatomy and function of the reproductive system, infections, endocrine and immunological factors, genetic factors, and psychological factors. Genetic defects leading to testicular spermatogenesis dysfunction account for approximately 10%-15% of male infertility. Some patients with oligoasthenozoospermia and azoospermia are able to conceive through assisted reproductive technology (ART). However, for male infertility or sperm abnormalities caused by genetic defects, ART can cause vertical transmission, potentially impacting the reproductive health of offspring.

[0003] The RBBP7 gene, located on the X chromosome, belongs to the highly conserved WD-repeat protein subfamily. It is a key component of numerous histone modification and chromatin remodeling complexes, participating in intracellular processes such as histone acetylation, deacetylation, and DNA methylation, regulating numerous physiological and pathological processes. Clinically, abnormal RBBP7 expression is known to be associated with some cancers, such as retinoblastoma and Wilms' tumor 1 (Wilms 1), but no familial or other direct evidence has been found linking mutations in this gene to the disease. RBBP7 has diverse functions. In Wilms' tumor, RBBP7 is a target gene of the tumor suppressor WT1, inhibiting cell growth. In non-small cell lung cancer cells, knockdown of RBBP7 inhibits cell migration. Knockdown of RBBP7 expression in mouse oocytes impairs meiotic progression and chromosome segregation. Because RBBP7 interacts with VCP, which regulates spindle disassembly, knockdown disrupts spindle disassembly during the first meiotic division, affecting the precise segregation of homologous chromosomes and leading to meiotic arrest. Furthermore, knockdown of RBBP7 disrupts histone acetylation, affecting the proper positioning of the meiotic chromosome passenger complex (CPC), also leading to meiotic arrest. Expression profiling studies of meiosis and spermatogenesis in male rats have identified RBBP7 as a candidate gene regulating meiosis. Among the genes that interact with RBBP7, its upstream gene, WT1, regulates cell polarity in Sertoli cells in the testis. Deletion of WT1 in mice results in germ cell death, with only Sertoli cells remaining in the vas deferens. Despite significant research on RBBP7, clinical cases of male infertility caused by RBBP7 mutations resulting in spermatogenesis impairment have not been reported.

[0004] RBBP7 protein is conserved across multiple species and highly expressed in mammalian testis. However, the biological function and related molecular mechanisms of RBBP7 in spermatogenesis remain unclear. Summary of the Invention

[0005] The present invention aims to provide the use of RBBP7 as a molecular marker for the diagnosis of male infertility. The present invention discovers that the occurrence of idiopathic non-obstructive azoospermia (NOA) is associated with abnormalities in the RBBP7 gene or abnormal expression levels of the protein encoded by the RBBP7 gene. By detecting the RBBP7 gene sequence or RBBP7 protein expression levels, it can be clearly determined that idiopathic NOA is associated with the RBBP7 gene. Furthermore, a mutation in the last exon of the RBBP7 gene is provided, wherein the nucleotide sequence is a T insertion at position 16,863,960 of the RBBP7 gene sequence; or the amino acid sequence is a mutation of amino acid 401 of the RBBP7 polypeptide from W to M, and amino acids 402-425 are truncated to AGE. The RBBP7 gene and its one pathogenic mutation site provide a new biomarker and a new target for the treatment of male infertility in existing diagnostic kits.

[0006] The present invention also provides a kit for diagnosing male infertility, which comprises a reagent for detecting the nucleotide sequence shown in SEQ ID NO.1 or the amino acid sequence shown in SEQ ID NO.2.

[0007] As a preferred embodiment, the reagent includes a pair of primers, and the nucleotide sequences of the primers are shown as SEQ ID NO.3 to SEQ ID NO.4.

[0008] As a preferred embodiment, the method further comprises a carrier recording a judgment criterion; the judgment criterion may be: if the mutated RBBP7 gene / protein exists in the sperm of the subject being tested, the subject being tested is or is suspected to be a male infertility patient.

[0009] The present invention also provides the use of a detection reagent for detecting RBBP7 gene / protein abnormality in preparing a kit for diagnosing male infertility.

[0010] The RBBP7 gene abnormality includes at least one of the following abnormalities: mutation of the gene promoter or enhancer sequence, single or multiple base sequence deletion, insertion or substitution in the gene coding sequence;

[0011] The abnormal expression level of RBBP7 protein includes at least one of the following abnormalities: decreased expression of RBBP7 protein, premature termination or loss of expression of RBBP7 protein, and deletion, insertion or substitution of amino acids in important functional domains of RBBP7 protein.

[0012] Furthermore, a hemizygous mutation occurs in the last exon of the RBBP7 gene, in which the nucleotide sequence is an insertion of a T base at position 16863960 of the RBBP7 gene sequence, resulting in premature termination of translation; or its amino acid sequence is a mutation of the 401st amino acid of the RBBP7 polypeptide from W to M, and the amino acids 402-425 are truncated to AGE, resulting in premature termination.

[0013] Furthermore, the detection reagent includes a reagent for detecting RBBP7 protein or RBBP7 DNA, mRNA, RNA sequence or miRNA targeting RBBP7.

[0014] The present invention also provides the use of the RBBP7 gene / protein as a drug target in the preparation of a product, wherein the product may have at least one of the following functions A1) to A9):

[0015] A1) Prevent male infertility; A2) Treat male infertility; A3) Improve male fertility; A4) Promote sperm maturation; A5) Promote spermatogenic cell mitosis; A6) Promote spermatogenic cell meiosis; A7) Reduce spermatogenic cell apoptosis; A8) Promote spermatogenic cell differentiation; A9) Repair spermatogenic cell cell cycle.

[0016] Experiments have shown that mutated RBBP7 genes / proteins can lead to spermatogenesis failure, abnormal spermatogenic cell proliferation, differentiation, and apoptosis, and cell cycle arrest. By detecting RBBP7 gene and / or RBBP7 protein levels, and whether mutated RBBP7 genes / proteins are present, the genetic causes of male infertility can be diagnosed, thus demonstrating significant application value. Furthermore, the expression level of RBBP7, a member of the RBBP7 gene / protein, is significantly reduced in NOA patients. By increasing RBBP7 protein activity and / or expression, male infertility can be prevented and / or treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below with reference to the accompanying drawings and examples.

[0018] Figure 1 Screening and identification of the causative gene in a family with non-obstructive azoospermia. (A) A pedigree with non-obstructive azoospermia. Arrows indicate the proband, squares indicate males, circles indicate females, and black symbols indicate affected individuals. (B) Flowchart for whole-exome sequencing of the two NOA brothers and their parents in this family to identify the causative gene. (C) Sanger sequencing confirmed the RBBP7 (NM_002893.4, C.1201ins1) variant in this family. The mother is heterozygous for RBBP7, the father is wild-type, and the two brothers are hemizygous for the RBBP7 (NM_002893.4, C.1201ins1) variant.

[0019] Figure 2 .Situation of two NOA patients in this pedigree with RBBP7 mutation; (A) H&E staining of cross-sections of seminiferous tubules from testicular biopsy samples of a control and patient (II:1). (B) Immunohistochemical staining analysis of the supporting cell marker WT1 in cross-sections of seminiferous tubules from testicular biopsy samples of a control and patient (II:1); white arrows indicate supporting cells, and black arrows indicate spermatogonia. (C) The upper figure shows the genomic structure of RBBP7, in which the 1bp insertion mutation is located in isoform 1 (NM_002893.4, c.1201ins1). The lower figure shows the genomic structure of RBBP7 wild type (425aa) and RBBP7 mutant (RBBP7 △ ) protein structure. The missense mutation is located in the final WD40 domain of RBBP7. (D) RFLP analysis of affected siblings II:1 and II:2 and their parents. (E) RBBP7 immunohistochemical analysis of cross-sections of seminiferous tubules from testicular biopsies of controls and the patient (II:1). (F) Figure 2 In E, image J was used to quantitatively analyze the expression level of RBBP7.

[0020] Figure 3 Figure 1. Effects of knockdown of RBBP7 expression in spermatogenic cells on their biological behavior. (A) After knockdown of RBBP7 expression in GC-1 cells, RBBP7 mRNA expression was decreased. (B) After knockdown of RBBP7 expression in GC-2 cells, RBBP7 mRNA expression was decreased. (C) After knockdown of RBBP7 expression, RBBP7 protein expression was decreased in both GC-1 and GC-2 cells. (D) Image J quantitative analysis of RBBP7 expression in Figure C. (E, F) CCK-8 assay for proliferation of GC-1 (E) and GC-2 (F) cells after knockdown of RBBP7 expression. (G) Flow cytometric analysis of apoptosis in GC-1 and GC-2 cells after knockdown of RBBP7 expression. (H) Percentage of apoptotic cells in control and si-RBBP7-treated GC-1 and GC-2 cells. (I) Flow cytometric analysis of cell cycle distribution in control and si-RBBP7-treated GC-1 and GC-2 cells. (J) Percentage of cells in G1, S, and G2 / M phases.

[0021] Figure 4Knockdown of the Drosophila ortholog of RBBP7, Caf1-55, results in male sterility. (A) Western blot analysis of Drosophila w1118, da-Gal4 > Caf1-55-IR1, and da-Gal4 > Caf1-55-IR2 genotypes, with GAPDH as the internal control protein. (B) Image J quantitative analysis of Caf-55 expression levels in Figure A. (C) Statistical analysis of fertility in Drosophila of various genotypes. (DH) White light images of the testes of late third instar larvae of various genotypes. The translucent oval circled in the center represents the testis, and the surrounding white area represents adipocytes. (D) W1118, (E) Nos-Gal4 > Caf1-55-IR1, (F) Tj-Gal4 > Caf1-55-IR1, (G) Nos-Gal4 > Caf1-55-IR2, (H) Tj-Gal4 > Caf1-55-IR2. The testes of EH larvae are significantly smaller. Scale bar: 80 μm. (D'-H') White light images of the testes of 2-day-old adults of each genotype; TS represents testis, SV represents spermatocyst, AG represents epididymis, and ED represents vas deferens. Scale bar: 200 μm. (D"-H") White light images of the spermatocyst of 2-day-old adults of each genotype. Scale bar: 50 μm. (I) Statistical analysis of testis area in third-instar larvae. ** indicates P < 0.01, *** indicates P < 0.001. (J) Statistical analysis of testis length in adults. *** indicates P < 0.001.

[0022] Figure 5 Knockdown of the Drosophila homolog of RBBP7, Caf1-55, results in abnormal germ cell proliferation, differentiation, and apoptosis. (AC) Immunofluorescence staining of eya in the testes of adult male Drosophila flies of various genotypes. (A'-C') Immunofluorescence staining of vasa in the testes of adult male Drosophila flies of various genotypes. (A"-C") Immunofluorescence staining of 1B1 in the testes of adult male Drosophila flies of various genotypes. (AA") 1118 ; (BB") Nos-Gal4>Caf1-55-IR1, (CC") Tj-Gal4>Caf1-55-IR1. (D-D') Statistical analysis of the number of punctate fusion bodies in the testes of Drosophila of each genotype (** indicates P<0.01, *** indicates P<0.001). (EE") Capase 3 immunofluorescence staining of the testes of Drosophila of each genotype. (FH) PH3 immunofluorescence staining of the testes of adult male Drosophila of each genotype. (F'-H') PH3 fluorescence staining of the apex of the testes of adult male Drosophila of each genotype. (F"-H") TUNEL fluorescence staining of the apex of the testes of adult male Drosophila of each genotype. (FF") W 1118; (GG”) Nos-Gal4 > Caf1-55-IR1; (H-H”) Tj-Gal4 > Caf1-55-IR1. (I) Statistical analysis of PH3-positive cells in the testis head of each genotype. ** indicates P < 0.01, *** indicates P < 0.001. (J) Statistical analysis of TUNEL-positive cells in the testis head of each genotype. *** indicates P < 0.001. (KK”) Immunofluorescence staining of γH2Av in the testes of adult male Drosophila melanogaster of each genotype.

[0023] Figure 6 Statistics of male fruit fly fertility of each genotype; (A) The top two rows show the RBBP7 wild type (425aa) and RBBP7 mutant (RBBP7 Δ ) protein structure - the missense mutation is located at amino acid 401 of the last WD40 domain of RBBP7, and then a stop codon appears after the production of 4 new amino acids, resulting in protein truncation; the bottom two rows show the Caf1-55 wild type (430aa) and Caf1-55 mutant (Caf1-55 Δ ) Protein structure—The introduced mutation creates a stop codon after amino acid position 405, resulting in a truncated protein. (B) Statistical graph of fertility rates for each Drosophila genotype (25°C). DETAILED DESCRIPTION

[0024] The present invention provides the use of the RBBP7 gene / protein. The RBBP7 gene / protein can be used as a molecular marker to prepare a kit for diagnosing male infertility, and can also be used as a drug target to prepare a product that can prevent male infertility; treat male infertility; promote sperm maturation; improve male fertility; promote spermatogenic cell mitosis; promote spermatogenic cell meiosis; reduce spermatogenic cell apoptosis; promote spermatogenic cell differentiation; and repair one or more functions in the cell cycle of spermatogenic cells.

[0025] The present invention also provides the use of a pathogenic mutation site (NM_002893.4, C.1201ins1) of RBBP7 as a molecular marker for diagnosing male infertility. This mutation occurs in the nucleotide sequence shown in SEQ ID NO. 1 or in the amino acid sequence of the polypeptide shown in SEQ ID NO. 2. The present invention discloses the use of this RBBP7 mutation site as a molecular marker in the preparation of a kit for diagnosing male infertility, providing a new molecular marker and therapeutic target for existing kits for diagnosing male infertility.

[0026] The present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings. The embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] The experimental methods in the following examples, unless otherwise specified, are all conventional methods; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0028] Example 1 Screening and identification of pathogenic genes in a family with non-obstructive azoospermia

[0029] This study was approved by the Ethics Committee of the Affiliated Hospital of Obstetrics and Gynecology, Zhejiang University School of Medicine, and the patients gave their informed consent. Figure 1 A) Two brothers in this family have NOA. Causes of the disease have been ruled out as organic lesions of the reproductive system, chromosomal abnormalities, AZF abnormalities, cryptorchidism, or mumps. Therefore, 0.2 ml of blood was collected from the patient with idiopathic NOA, and DNA was extracted using the TIANamp Genomic DNA Kit (according to the manufacturer's instructions - Genomic DNA Extraction from Whole Blood).

[0030] Whole-exome sequencing was performed on DNA samples from the family members using the Illumina platform PE150 mode. After sequencing, adapters and low-quality sequences were removed from the raw data to obtain clean data. Sequence alignment of the clean data columns with G37(h19) was performed using BWA software. Variant sites were detected using GATK software, and ANNOVAR software was used to annotate and predict the deleteriousness of the detected genetic variants. The inheritance pattern was based on the published inheritance pattern of the patient's family. To screen for mutations most relevant to the family's NOA, candidate genes must meet the following criteria: ① the mutation region is located in the exon region; ② non-synonymous mutations; ③ the mutation frequency is less than 5%; ④ at least two of them are harmful in the SIFT, Polyphen2_HDIV, Polyphen2_HVAR, LRT, Mutation_Taster, PROVEIN, CRAVAT, and SIFT_indels mutation prediction databases; ④ genes that have been found to be associated with known diseases and phenotypes in the Online Mendelian Inheritance in Man (OMIM, https: / / www.omim.org / ) and the Mouse Phenotype Database (IMPC, https: / / www.mousephenotype.org / ) must be excluded; ⑤ they must be specifically expressed or highly expressed in the testis in the gene expression database. Finally, a new rare frameshift mutation in RBBP7 (frameshift insertion: chrX:16863960-T) was screened out. Figure 1 B).

[0031] Sanger sequencing was used to verify the mutation; a pair of primers were designed for the RBBP7 gene locus, and the RBBP7 gene fragment was amplified by PCR using the patient's genomic DNA as a template. After sequencing, sequence alignment was performed to confirm that the NOA patient had a T base insertion at position 16863960 of the RBBP7 gene sequence, which resulted in premature translation termination; or the amino acid sequence was that the 401st amino acid of the RBBP7 polypeptide was mutated from W to M, and the amino acids at positions 402-425 were truncated to AGE, resulting in premature translation termination ( Figure 1 C).

[0032] The primers used are as follows:

[0033] h RBBP7-F-1: 5'-AAACAGTGGACGAAGCACCA-3' (SEQ ID NO.3)

[0034] h RBBP7-R-1: 5'-CACCCACCCCCAGTTGAAAT-3' (SEQ ID NO.4)

[0035] Example 2: Two NOA patients in the RBBP7 mutation family

[0036] H&E staining: After dewaxing and hydration, the paraffin sections of testicular puncture tissue were stained with hematoxylin for 5 minutes, and the excess stain on the slides was washed with water. Then, 0.5-1wt% hydrochloric acid alcohol (70vol% alcohol) was used for color separation for 10 seconds, and then rinsed with running water for 15-30 minutes. After a short period of alkalization in a saturated lithium carbonate solution, the cell nuclei turned blue, and the sections were briefly washed with distilled water; stained with 0.1-0.5wt% eosin stain for 1-5 minutes, and then dehydrated with gradient ethanol (70vol% ethanol 2s → 80vol% ethanol 2s → 90vol% ethanol 2s → 95vol% ethanol 5min×2 times → anhydrous ethanol 5min×2 times); transparentized with xylene, and finally sealed with neutral gum for observation and photography under an optical microscope ( Figure 2 A) The results showed that testicular spermatogenesis was blocked and only spermatogonia and Sertoli cells remained in the seminiferous tubules.

[0037] Immunohistochemical staining: After dewaxing and hydration, paraffin sections of testicular puncture tissue were subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at high pressure for 15 minutes, cooled to room temperature, washed three times with PBS, incubated in 3 vol% H2O2 in the dark for 10 minutes at room temperature, washed three times with PBS to remove endogenous catalase, and then blocked with 0.5% BSA; primary antibodies (WT1 antibody, Elabscience, catalog number #PA6533, dilution 1:200; RBBP7 antibody, Abbexa, catalog number #141930, dilution 1:200) were added, incubated in a humidified box at 4°C overnight, washed three times with PBS, and corresponding peroxidase-labeled secondary antibodies were added. The sections were incubated in a humidified box at room temperature for 2 hours, washed three times with PBS, and then developed with DAB; after hematoxylin counterstaining and color separation, the sections were dehydrated with graded alcohols, transparentized with xylene, mounted with resin, and observed and photographed under a microscope ( Figure 2 B, E), and the expression level of RBBP7 was quantitatively analyzed using Image J ( Figure 2 F). Figure 2 Figure B shows that only spermatogonia and Sertoli cells remain in the seminiferous tubules. Figures 2E and 2F show that RBBP7 expression is significantly decreased in the proband (II:1).

[0038] Figure 2 C RBBP7 reference sequence, this gene mutation is the insertion of 1 base in the last WD40 domain of RBBP7, resulting in the termination of protein translation extraction (NM_002893.4, c.1201ins1, pW401M fs*4).

[0039] Restriction fragment length polymorphism (RFLP) technology was used to identify RBBP7 gene mutations ( Figure 2D) The results showed that the mutant RBBP7 amplification product could not be digested by Bgl I enzyme, while the wild-type RBBP7 amplification product could be digested by Bgl I enzyme into fragments of 19 bp and 207 in size.

[0040] Example 3 RBBP7 gene knockdown leads to restricted spermatogonia and spermatocyte proliferation, cell cycle arrest, and increased apoptosis

[0041] The siRNA interference technique (siRNA sequence 5'-CCAUGAAGGAGAAGUGAAUTT-3' (SEQ ID NO. 5); control sequence: 5'-AUUCACUUCUCCUUCAUGGTT-3' (SEQ ID NO. 6)) was used to knock down the endogenous RBBP7 mRNA expression in GC-1 and GC-2 cells. The results showed that compared with the control group, both the RNA and protein levels of RBBP7 were significantly downregulated ( Figure 3 AD). CCK8 analysis of the growth curves of GC-1 and GC-2 cells with RBBP7 knockdown revealed that the proliferation capacity of GC-1 and GC-2 cells with knockdown was decreased compared with the control group ( Figure 3 E, F).

[0042] Flow cytometry was used to analyze the apoptosis of GC-1 and GC-2 cells with RBBP7 knockdown. The results showed that the apoptosis rate of GC-1 and GC-2 cells with RBBP7 knockdown was significantly higher than that of the control group ( Figure 3 G,H).

[0043] Flow cytometry was used to analyze the changes in the cell cycle of GC-1 and GC-2 cells with RBBP7 knockdown. The results showed that the proportion of cells in the S phase after RBBP7 knockdown (GC-1 cells 31.2%, GC-2 cells 28.4%) decreased compared with the control group (GC-1 cells 43.9%, GC-2 cells 46.3%). Figure 3 I, J), but the proportion of cells in the G2 / M phase increased significantly after RBBP7 knockdown, indicating that silencing RBBP7 led to cell cycle arrest in the G2 / M phase.

[0044] Example 4 Knockdown of the Drosophila homologous gene Caf1-55 of RBBP7 leads to male sterility

[0045] The Daughterless-Gal4 system was used to initiate Caf1-55 RNAi expression, protein was extracted, and immunoblotting analysis was performed. It was found that both RNAi Drosophila strains could effectively knock down Caf1-55 ( Figure 4 A, B).

[0046] Using a tissue-specific promoter, we knocked down the expression of Caf1-55 in the testes of Drosophila to analyze their male reproductive capacity. After knocking down Caf1-55 using Traffic Jam-Gal4 (abbreviated as tj-gal4, homologous to the cyst stem cells and cyst cell-specific expression of mammalian Sertoli cells), the fertility of male Drosophila was significantly reduced, but a small number of offspring could still be produced ( Figure 4 C); knocking down Caf1-55 with Nanos-Gal4 (hereinafter referred to as nos-gal4, mainly expressed in germline stem cells and spermatogonia) completely destroyed the fertility of male fruit flies ( Figure 4 C).

[0047] Observation of RBBP7 - / - The gonads of Drosophila; specifically, the testes of the late third instar larvae (LL3) stage of Caf1-55 knockdown showed no obvious morphological abnormalities compared with the control, but their size was significantly reduced ( Figure 4 DH, I). Testes of 2-day-old adult flies. It was found that the morphology of the testes of Drosophila with tj-Gal4 knockdown of Caf1-55 did not change significantly, but the testis length was shortened ( Figure 4 F', H', J); however, the testes of Drosophila in which Caf1-55 was knocked down using nos-Gal4 completely lost their coiled morphology, became short, and were even difficult to distinguish, showing structural collapse ( Figure 4 E', G', J). In addition, the epididymis of the fruit fly is swollen ( Figure 4 E'-G'). Magnifying the spermatophore of the fruit fly, we found that the spermatophore of the control fruit fly contained many sperm in an opaque brown state ( Figure 4 D”), while the spermatocysts after knocking down Caf1-55 by tj-Gal4 and nos-Gal4 were almost transparent ( Figure 4 E”-H”), indicating that there are no or very few mature sperm.

[0048] Example 5 Knockdown of the Drosophila homologous gene Caf1-55 of RBBP7 leads to abnormal proliferation, differentiation, and apoptosis of Drosophila germ cells

[0049] By immunofluorescence staining, after knocking down Caf1-55 by tj-Gal4 and nos-Gal4, compensatory increased cyst cells were observed in the testis compared with the control group ( Figure 5 AC).

[0050] Using vasa (a marker specifically expressed by all germ cells) antibody immunofluorescence staining of Drosophila testes, we found that compared with the control group, after nos-Gal4 knockdown of Caf1-55, there were no vasa-positive cells at all ( Figure 5B'), showing complete disappearance of germline stem cells and germ cells;

[0051] After tj-Gal4 knockdown of Caf1-55, the distribution of germ cells became chaotic, with some abnormal germ cell clusters ( Figure 5 C').

[0052] Using 1B1 antibody to mark the germinal fusion bodies of adult worm testes, it was found that knocking down Caf1-55 in germ cells completely eliminated the fusion bodies ( Figure 5 B”); however, when Caf1-55 was knocked down in cyst cells, the number of punctate fusion bodies increased significantly, while the number of dendritic fusion bodies decreased significantly ( Figure 5 C”, D, D’), indicating that the abnormal germ cell clusters are composed of undifferentiated early germ cells.

[0053] Caspase 3 (which marks two structures in the testis during sperm differentiation: cystic bulges (cb) and waste bag (wb)) was used to analyze the process of sperm differentiation using immunofluorescence staining. It was found that after knocking down Caf1-55, the Caspase 3 signal at the top of the testis was not significantly different from that of the wild type ( Figure 5 EE”); however, no cystic protrusions and waste bag structures were found in the testes of Caf1-55 knockdown ( Figure 5 E', E'), the arrow in the wild type is clearly visible ( Figure 5 E, white arrowheads), indicating that spermatogenesis did not progress to the sperm differentiation stage after the loss of Caf1-55.

[0054] Phosphorylated histone H3 (PH3) was used to mark cells in mitosis or meiosis. Immunofluorescence staining results showed that in the control testis, a small number of mitotic cells were usually seen near the tip of the testis ( Figure 5 F-white arrow, F'), a meiotic cyst containing 16 PH3-positive spots can be observed at the posterior part of the tip ( Figure 5 F, thick white arrows), but no PH3-positive cells were seen at the end of the testis. After knocking down Caf1-55 with nos-Gal4 or tj-Gal4, the number of PH3-positive cells in the testis increased significantly and was distributed throughout the testis ( Figure 5G, H, thick white arrows, G', H'). As previously shown, knockdown of Caf1-55 by nos-Gal4 completely eliminates germ cells, and these PH3-positive cells are likely cyst cells. In the testes of tj-Gal4-knockdown testes, PH3-positive cells should include both germ cells and cyst cells. Statistical analysis of PH3-positive cells in the apical region of the Drosophila testes of various genotypes revealed a significantly higher number of PH3-positive cells in the Caf1-55-knockdown testes than in the control group ( Figure 5 I), suggesting that decreased Caf1-55 expression can cause abnormalities in the mitosis and meiosis of germ cells and Sertoli cells in the testis.

[0055] In the testis with knockdown of Caf1-55, Caspase 3 staining signal appeared very strong at the tail end of the testis ( Figure 5 E', E", white arrows). TUNEL (TdT-mediated dUTP Nick-End Labeling) staining was used to analyze cell apoptosis. It was found that when Caf1-55 was knocked down in cyst cells, the TUNEL signal in the testis head was significantly increased compared with the wild type ( Figure 5 H", J), indicating that cell apoptosis increased significantly, while the TUNEL signal was weak in the testis of nos-Gal4 knockdown of Caf1-55 ( Figure 5 G”, J).

[0056] γH2Av immunofluorescence staining was used to detect the effect of RBBP7's Drosophila homolog gene Caf1-55 on genome stability. The results showed that a very small number of γH2Av positive markers were occasionally seen in the head of the testis of the control group ( Figure 5 K), while after knocking down Caf1-55 in germ cells or cyst cells, the γH2Av-positive markers increased significantly and spread throughout the testis ( Figure 5 K, K”), indicating that knockdown of Caf1-55 produces DNA damage such as DNA double-strand breaks in testicular cells, indicating that Caf1-55 is involved in maintaining the stability of the Drosophila genome.

[0057] Example 6 Verification of Functional Conservation of Human RBBP7 and Its Drosophila Homologous Gene Caf1-55

[0058] To construct transgenic fruit flies overexpressing RBBP7 mutations with the same site as the patient, transgenic fruit flies with normal RBBP7, transgenic fruit flies with normal Caf1-55, and transgenic fruit flies overexpressing Caf1-55 with premature termination of the patient homologous site, we amplified full-length RBBP7 cDNA, mutant RBBP7 cDNA, Caf1-55 cDNA, and mutant Caf1-55 cDNA and cloned them into the pUAST-attb vector. Then, a standard P element-mediated method was used to construct transgenic fruit fly models. The functional conservation of human RBBP7 and fruit fly Caf1-55 was further clarified. Figure 6 A). Overexpression of normal Caf1-55 or normal RBBP7 in a tj-Gal4-driven Caf1-55 knockdown background at 25°C or 29°C effectively rescued male reproductive capacity. However, overexpression of prematurely terminated Caf1-55 or mutant RBBP7 had no rescue effect, indicating that the functions of human RBBP7 and its Drosophila homolog Caf1-55 are conserved in Sertoli cells. Sequence Listing <110> Zhejiang University <120> Application of RBBP7 gene / protein as a drug target in the preparation of products for diagnosis and treatment of male infertility <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 406 <212> DNA <213> Homo sapiens <400> 1 cacccacccc cagttgaaat actgatagct ctaaaaactt agtaaaacct ttgttaggat 60 taacaggaga gtatatgaaa tcaaagcttt ttttgttctt gtaacagttt attcatggag 120 gacacactgc taagatttca gattttagct ggaaccccaa tgagccttgg gtcatttgct 180 cagtgtctga ggataacatc atgcagatat ggcaaatggt gagttaaaat gtttttcttg 240 aatgactgtc ttgtaaagga gagatttact acttacataa tgtaataaag ttatttcgaa 300 tatgtaggtg catattatgc caaacattta cagaaaccgt ttaaaagaac tttttccatt 360 tcaacaatac aatctttagt cccatatggt gcttcgtcca ctgttt 406 <210> 2 <211> 59 <212> PRT <213> Homo sapiens<l <400> 2 Phe Ile His Gly Gly His Thr Ala Lys Ile Ser Asp Phe Ser Trp Asn 1 5 10 15 Pro Asn Glu Pro Trp Val Ile Cys Ser Val Ser Glu Asp Asn Ile Met 20 25 30<0l Gln Ile Trp Gln Met Ala Glu Asn Ile Tyr Asn Asp Glu Glu Ser Asp 35 40 45 Val Thr Thr Ser Glu Leu Glu Gly Gln Gly Ser 50 55 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 aaacagtgga cgaagcacca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 cacccacccc cagttgaaat 20 <210> 5 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 5 ccaugaagga gaagugaaut t 21 <210> 6 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 6 auucacuucu ccuucauggt t 21

Claims

1. The use of a reagent for detecting abnormalities in the RBBP7 gene / protein in the preparation of a test kit for diagnosing male infertility, characterized in that: The RBBP7 gene uses the human genome GRCh37 / hg19 as a reference sequence. Its nucleotide sequence is a T base insertion at position 16863960 of the RBBP7 gene sequence, resulting in premature translation termination; or its amino acid sequence is a mutation of amino acid 401 of the RBBP7 polypeptide from W to M, and amino acids 402-425 are truncated to AGE, resulting in premature termination.

2. The use according to claim 1, characterized in that The male infertility detection kit includes reagents for detecting RBBP7 protein or RBBP7 DNA, mRNA, and RNA sequences.

3. The use according to claim 2, characterized in that Reagents for detecting RBBP7 DNA sequences include RBBP7 whole genome sequencing and RBBP7 whole exome sequencing detection reagents.

4. The use according to claim 2, characterized in that The kit includes reagents for detecting the nucleotide sequence shown in SEQ ID NO.1 or the amino acid sequence shown in SEQ ID NO.

2.

5. The use according to claim 4, characterized in that The reagents include a pair of primers, the nucleotide sequences of the primers are shown in SEQ ID NO.3 to SEQ ID NO.

4.

6. The use according to claim 2, characterized in that It also includes a carrier recording a judgment standard; the judgment standard is: if the RBBP7 mutant gene / protein exists in the sperm of the test subject, the test subject is suspected to be a male infertility patient.

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