Application of germ cell specific pathogenic mutation in diagnosis of male fertility defects

Abstract

The invention provides a kit for diagnosing human male fertility defects such as infertility. The kit comprises a reagent for detecting TEX14 of a tested individual. The invention also provides a kit and an instrument for diagnosing or predicting human male fertility defects such as infertility by detecting TEX14, especially mutation of the pathogenic gene of TEX14. The applicant thus provides a method for diagnosing human male fertility defects, such as infertility, and further promotes the development of a new treatment method for male infertility individuals affected by gene mutation.

Description

Technical Field

[0001] The present invention relates to the field of diseases and diagnosis. Specifically, the present invention relates to methods and kits for diagnosing or treating fertility defects in mammals, especially humans. Background Art

[0002] Infertility affects 15% of fertile couples worldwide, with male diseases accounting for half of these cases. Oligozoospermia (OA) and azoospermia are two common phenotypes of male infertility, characterized by insufficient sperm levels due to failed spermatogenesis. Both oligozoospermia and azoospermia have typical high genetic heterogeneity, including karyotype abnormalities, Y chromosome microdeletions, and single gene mutations. Understanding these potential genetic defects is essential for providing accurate and effective genetic counseling for infertile couples and determining appropriate treatment strategies. In recent years, whole exome sequencing (WES) has become a powerful tool for investigating male infertility, and significant progress has been made in identifying pathogenic genes, such as SIX6OS1, TEX11, PNLDC1, KCTD19, and M1AP associated with oligozoospermia. However, the cause of more than 70% of cases remains unknown.

[0003] During gametogenesis, precursor germ cells undergo several rounds of mitosis and meiosis to produce mature gametes. In many species, these divisions are characterized by incomplete cytokinesis and the formation of stable intercellular bridges for the exchange of cytoplasmic materials, including RNA, proteins, and organelles, which is essential for the synchronous development and differentiation of germ cells. In males and females, intercellular bridges allow for the uniform distribution of important cytoplasmic components and synchronized cellular activities. In female animals, intercellular bridges play a key role during oogenesis. They allow cells to transfer cytoplasmic contents, such as RNA and proteins, to the developing oocyte. This transfer is essential for the growth and development of the oocyte, providing it with the resources required for maturation and subsequent embryonic development.

[0004] The formation and maintenance of intercellular bridges are regulated by a complex network of proteins. Key among them is TEX14 (testis expressed 14), a protein that is essential for the stability of these bridges. TEX14 maintains intercellular connections by interacting with other midbrains to prevent the eventual shedding of cytoplasmic bridges. Studies in mice have shown that mutations or defects can disrupt intercellular bridge formation, leading to germ cell development and infertility. However, the role of TEX14 in human reproduction, in particular its possible contribution to azoospermia and / or oligospermia, remains unclear.

[0005] This field also needs to have a better understanding of the human male spermatogenesis process, thereby detecting and predicting male infertility. Summary of the invention

[0006] The inventors found that TEX14 mutations lead to male spermatogenesis failure and complete infertility, proving that TEX14 is a pathogenic gene for male infertility. In addition, the inventors discovered multiple TEX14 mutations in male infertility for the first time in humans, including frameshift mutations, missense mutations, and splicing mutations. These mutations, especially frameshift mutations, lead to truncation of TEX14 protein and failure of intercellular bridge formation, resulting in meiotic arrest of human male germ cells. Thus, the inventors provide methods for diagnosing or treating fertility defects such as infertility disorders in human males.

[0007] In one aspect of the present invention, a method for diagnosing human male fertility defects such as infertility by detecting TEX14 in a subject is provided.

[0008] In one aspect of the present invention, a kit for diagnosing human male fertility defects such as infertility is provided, which comprises a reagent for detecting TEX14 in a test subject. In the present invention, the kit also comprises a reagent for detecting the gender of the test subject, in particular, detecting whether the test subject is male.

[0009] The present invention thus provides the use of a reagent for detecting TEX14 in a test subject in the preparation of a kit for diagnosing human male fertility defects such as infertility. In the present invention, the kit also includes a reagent for detecting the gender of the test subject.

[0010] In the present invention, infertility disorders include but are not limited to non-obstructive azoospermia, teratozoospermia, azoospermia, oligospermia, asthenozoospermia, asthenozoospermia and the like.

[0011] TEX14, or testis expressed 14, is a cell-to-cell bridge-forming factor. It is also known as CT113, SPGF23, SgK307, etc. The Ensembl database (http: / / asia.ensembl.org / index.html) shows that TEX14 is evolutionarily conserved in mammals such as humans. The Gene ID of the human Tex14 gene encoding TEX14 is 56155.

[0012] In the application (including methods and related kits) provided by the present invention for diagnosing fertility defects or infertility in different genders of humans by detecting TEX14 of the test individual, whether the TEX14 of the test individual is abnormal is detected, including comparing the activity of the subject's TEX14 gene or its regulatory factor, or the expression of TEX14 protein, or comparing with the situation of the gene or protein in normal people in the general or specific group. If there is a significant change, it is judged that an abnormality exists.

[0013] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include steps and reagents for detecting the Tex14 gene encoding TEX14.

[0014] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include steps and reagents for detecting mutations in the gene encoding Tex14. In one embodiment, the mutation is a frameshift mutation, such as c.2908dup (p.Arg970ProfsTer4), c.1881dup (p.Gly628ArgfsTer8) or c.1728_1729del (p.Leu577ArgfsTer58). In one embodiment, the mutation is a missense mutation, such as c.865G>A (p.Glu289Lys) or c.1121A>G (p.Tyr374Cys). In one embodiment, the mutation is a splicing mutation, such as c.417+2T→C.

[0015] The method for checking genes used in the present invention, including the method for detecting gene mutations, is a method commonly used in the art for detecting genes, and is not particularly limited. For example, mass spectrometry, microarray method, sequencing method, detection method using base sequence amplification methods such as PCR (polymerase chain reaction), etc. can be cited. For example, in the PCR method for detecting gene mutations, a polynucleotide that is completely complementary to only the wild-type allele is used as a wild-type primer, and a polynucleotide that is completely complementary to only the mutant allele is used as a mutant primer. A nucleic acid containing a target mutation is used as a template to perform PCR using each primer, and the genotype can be identified by whether a PCR product can be obtained. Similarly, a polynucleotide that is completely complementary to only the wild-type allele is used as a wild-type probe, and a polynucleotide that is completely complementary to only the mutant allele is used as a mutant probe. By using a microarray fixed with a nucleic acid containing a target mutation and using each probe to hybridize, the genotype can be identified.

[0016] In addition, the detection of PCR products obtained when PCR is performed using primers specific to each gene can be performed by any method commonly used to detect and quantify PCR products. For example, it can be detected by electrophoresis, by real-time PCR using fluorescent intercalators such as SYBR Green, or by single molecule fluorescence analysis.

[0017] The polynucleotide that can be used as the primer or probe for detecting gene is not particularly limited as long as it can be a polynucleotide that hybridizes with the partial region comprising the gene or its complementary strand. The design of polynucleotide can be carried out by any one of the well-known methods in the technical field. For example, it can be designed simply by utilizing known genome sequence data and universal primer design tool. As this primer design tool, for example, Primer3 that can be utilized on the network is arranged. In addition, known genome sequence data can be obtained in NCBI as an international base sequence database usually.

[0018] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include a step of detecting TEX14 protein, such as a step of detecting TEX14 protein. This includes detecting the expression of the protein (including whether it exists, and whether there is a phenomenon of reduced expression relative to normal or standard) or its activity.

[0019] In another aspect of the present invention, the method and kit include steps and reagents for determining the expression of TEX14 protein by immunoassay. For example, the expression of the target protein is detected by immunofluorescence or western blotting with an antibody that specifically recognizes the protein. For another example, the expression of the target protein is detected by detecting the presence or amount of mRNA of the TEX14 protein, such as by detecting the amount of mRNA encoding the target protein in the sample by RT-PCR.

[0020] In yet another aspect of the present invention, the method and kit include steps and reagents for determining the activity of TEX14 protein.

[0021] The present invention also provides an apparatus (including a gene chip) for diagnosing or predicting fertility defects such as infertility in mammalian individuals.

[0022] In this article, protein symbols are not italicized and are all capitalized; gene symbols are italicized. For example, TEX14 is a protein, and the gene encoding the protein is written as Tex14. Sometimes in this article, protein symbols are not all capitalized. For example, sometimes "Tex14" in this article means TEX14 protein. Sometimes in this article, gene symbols are not italicized. For example, sometimes "Tex14" or "TEX14 gene" in this article means gene Tex14 encoding TEX14 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 1 shows the identification of TEX14 variants in patients with nonobstructive azoospermia (NOA) and oligospermia (OA). (A) Pedigree of four unrelated Chinese infertile patients with TEX14 variants. Arrows indicate family members examined by whole-exome sequencing. Sanger sequencing profiles of TEX14 variants are shown below the pedigree; arrows indicate the position of the variants. Short double horizontal lines indicate the absence of offspring. Squares indicate males; circles indicate females; solid symbols indicate affected family members; open symbols indicate unaffected family members. (B) TEX14 protein domain diagram with variant positions marked. Sequence alignment shows that the mutated residues remain consistent across species.

[0024] Figure 2 Shown are histological results of testicular biopsies from men carrying TEX14 mutations. (A) Representative images of hematoxylin and eosin staining of testicular sections from patient V1 diagnosed with obstructive azoospermia and a non-NOA control group, in whom spermatogenesis was completely normal in the seminiferous tubules. The scale bar represents 50 μm. (B) Immunofluorescence staining of the sperm marker PNA (red) in testicular sections from patient V1 and controls. Nuclei (blue) were stained with Hoechst33342. (C) Immunofluorescence staining of SYCP3 (red) and γH2AX (green) in testicular sections from controls and patient V1. Nuclei (blue) were stained with Hoechst. The scale bars represent 50 μm and 10 μm, respectively. (D) Spermatocyte smears from patient V1 and controls were labeled with antibodies to SYCP3 (red) and γH2AX (green). Pachytene and diplotene spermatocytes were not found in patient V1. The scale bar represents 10 μm. (E) Percentage of spermatocytes observed at various stages of meiotic prophase I in controls and patient V1. n indicates the number of meiotic prophase cells examined.

[0025] Figure 3Figure 3. Tex14 mutant mice show the human NOA phenotype. (A) Testes of 3-month-old WT (left) and Tex14 mutant (right) mice. (B) Testis-to-body weight ratio of 2- to 3-month-old WT and Tex14 mutant mice (three mice per genotype). (C) Western blot detection of TEX14 in testicular lysates of 14-day-old WT and Tex14 mutant mice. β-Actin was used as a control. (D) Hematoxylin staining of testicular and epididymal sections of adult Tex14 mutant and control mice. Scale bar, 50 μm. (E) PNA (red) immunofluorescence staining of testicular sections of adult Tex14 mutant and WT mice. Nuclei were stained with Hoechst (blue). Scale bar, 50 μm.

[0026] Figure 4 Showing that mutations in the mouse Tex14 gene cause meiotic arrest. (A) Immunofluorescence staining of γH2AX (green) in testicular sections of adult Tex14 mutant and WT mice. Cell nuclei were stained with Hoechst (blue). Scale bars are 50 μm and 10 μm, respectively. (B) Immunofluorescence staining of γH2AX (red) and SYCP3 (green) in spermatocyte smears of WT or Tex14 mutant mice. Scale bar is 10 μm. (C) Percentage of spermatocytes at various stages of meiotic prophase I. (D) WT and Tex14 mu1 / mu1 Representative smears of lateral elements SYCP3 (green) and central elements SYCP1 (red) in mouse spermatocytes. Data are presented as mean ± SD. Student's t-test, NS, p>0.05; *p<0.05.

[0027] Figure 5 The expression of intercellular bridge components in control and V1 patient testes is shown. (A) Immunofluorescence staining of SYCP3 (red) and TEX14 (green) in sections of patient V1 and control testes. Cell nuclei were stained with Hoechst (blue). Scale bars are 50 μm or 10 μm. (B) Immunofluorescence staining of MgRacGAP1 (red) in sections of patient V1 and control testes. Cell nuclei were stained with Hoechst33342 (blue). Scale bars are 50 μm or 10 μm.

[0028] Figure 6Working model showing the pathogenic effect of TEX14 variants in humans. During spermatogenesis, germ cells undergo multiple rounds of mitotic differentiation and meiosis to ultimately produce sperm. Throughout development, spermatogenic cells undergo incomplete cell divisions that form intercellular bridges. These specialized ring-like channels connect cells and ensure the exchange of biomacromolecules, organelles, and cytoplasm between germ cells. This exchange is essential for synchronized development and successful sperm production. Mutations in the TEX14 gene disrupt the formation of these intercellular bridges, leading to meiotic failure and causing male infertility. DETAILED DESCRIPTION

[0029] The essential contents and beneficial effects of the present invention will be further described below in conjunction with embodiments, which are only used to illustrate the present invention rather than to limit the present invention.

[0030] Example 1 Experimental methods and reagents

[0031] Experimental Ethics

[0032] Human experimentation: Patients and their parents provided informed consent to participate in this study, which was approved by the Medical Research Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University and the Ethics Committee of Reproductive Hospital Affiliated to Shandong University.

[0033] Animal experimentation: All experimental protocols involving mice were approved by the Animal Ethics Committee of Shandong University School of Medicine.

[0034] Patient selection

[0035] A total of 816 infertile men with nonobstructive azoospermia (NOA) or oligozoospermia (OA) were recruited from 2013 to 2023. All individuals had a normal somatic karyotype. No microdeletions of azoospermia factors were detected on the Y chromosome. Patients with other potential causes of infertility, such as medication use or exposure to gonadal toxic factors, were also excluded.

[0036] Whole Exome Sequencing (WES)

[0037] Genomic DNA samples were extracted from the peripheral blood of 673 patients with non-obstructive azoospermia from the Reproductive Hospital of Shandong University and Renji Hospital of Shanghai Jiao Tong University using the TIANamp Blood DNA Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., China) for whole exome sequencing. Genomic DNA was fragmented using Covaris E220; whole genome libraries were constructed by end repair, 3' end poly A addition, and PCR amplification. Target gene fragments were captured and enriched from the whole genome library using target gene-specific probe hybridization, streptavidin magnetic bead capture, and PCR amplification to construct target gene libraries, which were sequenced using the Illumina HiSeq 2000 platform.

[0038] Variant analysis

[0039] After WES analysis of the entire study group, individuals with TEX14 variants were extracted for analysis. The variant filtering process of WES data was as follows: (1) variants with allele frequency (MAF) > 0.01 in 1000 Genomes, ESP6500, ExAC, and gnomAD; (2) variants with recessive inheritance and DNM were retained; (3) variants that may affect protein sequence (nonsense, missense, splice site variants, and coding); (4) 14 software programs (Sorting Intolerant From Tolerant, PolyPhen-2, LRT, MutationTaster, MutationAssessor, FATHMM, PROVEAN, MetaSVM, MetaLR, M-CAP, fathmm-MKL, CADD, GERP++, and SiPhy) were used to predict the function of deletion variants or potentially harmful missense variants, and variants predicted as harmless in more than half of the software were excluded; (5) variants within genes expressed in the testis. After filtering, candidate variants identified as potentially causing spermatogenesis arrest were subsequently validated by Sanger sequencing.

[0040] Construction of mice carrying the Tex14 c.2908dupC mutation

[0041] Tex14(c.2908dupC) mice were generated by Cyagen Biosciences' CRISPR / Cas9 genome editing technology. Single guide RNA (sgRNA) targeting the mouse Tex14 gene, donor oligonucleotides containing c.2908dupC and Cas9 mRNA were co-injected into mouse fertilized eggs to generate targeted knock-in offspring, which were then sequenced. F0 mice were bred to wild-type mice to assess germline transmission and F1 animal generation.

[0042] Cell culture and transfection

[0043] HEK293T cells were cultured in DMEM supplemented with 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin in a 5% carbon dioxide incubator at 37° C. Cells were transfected with the expression vector using Lipofectamine 3000 Transfection Reagent (L3000015).

[0044] Western blotting

[0045] Proteins were denatured and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted onto 0.2 μm polyvinylidene difluoride membranes (PVDF) and incubated with anti-tex14 antibodies (1:3000 dilution; protein), MgcRacGAP (1:3000; protein), and β-Actin antibodies (1:3000; protein) at 4°C overnight. The membranes were then incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies for 1.5 h at room temperature and visualized using enhanced chemiluminescence (Pierce). Immunoreactive bands were detected and analyzed using the BIO-RAD ChemiDoc MP imaging system and Image Lab software (BIO-RAD, USA).

[0046] Quantitative PCR

[0047] Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Japan), and cDNA was synthesized using PrimeScript RT reagent kit (TaKaRa, Japan) according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed using FastStart Universal SYBR Green Master (Rox) reagent (Roche, Cat#04913914001) in a LightCycler 96 Real-Time PCR System (Roche).

[0048] Histological analysis

[0049] Testicular tissue was fixed in Bouin solution overnight, then embedded in paraffin and sliced ​​at a thickness of 5 μm. Tissue sections were deaffinity with xylene, rehydrated with graded ethanol, and then stained with hematoxylin and / or eosin (H&E) in sequence. Tissue sections were sealed with neutral resin after dehydration and transparency. Images were captured using a fluorescence microscope (BX52, Olympus) and processed using Photoshop (Adobe) software package.

[0050] Immunofluorescence analysis of chromosome spreads

[0051] Preparation for spermatocyte spread and immunofluorescence staining. To spread spermatocytes, seminiferous tubules were placed in hypotonic extraction buffer [30 mM Tris (pH 8.2), 50 mM sucrose, 17 mM trisodium citrate, 5 mM EDTA, 2.5 mM dithiothreitol (DTT) and 1 mM PMSF] and incubated at room temperature for 20-25 min. Subsequently, cell suspensions were made in 100 mM sucrose solution and spread on slides in 1% PFA (pH 9.2) containing 0.1% Triton X-100. The slides were then placed in a humidified chamber for at least 3 h. They were then rinsed once with 0.4% Photoflo (Kodak). The sections were blocked with 1× PBST containing 3% skim milk for 30 min. The slides were then incubated with primary antibodies in a humidified chamber at 37°C overnight. After washing three times with 1× PBST, secondary antibodies were applied for 1 h in a humidified 37°C chamber. Both primary and secondary antibodies were diluted in 1× PBS containing 3% skim milk. After secondary antibody incubation, slides were washed 3-5 times in 1× PBST. Images were captured using a confocal microscope. Projection images were then prepared using Bitplane Imaris (version 8.1) software.

[0052] Immunofluorescence staining of testis sections

[0053] Testicular tissue was fixed with 4% paraformaldehyde and then embedded in paraffin and sectioned (5 μm thickness). For immunofluorescence detection, slides were blocked with 3% skim milk in PBS for 30 min and then incubated with primary antibodies in a humidified chamber at 37°C overnight. After washing three times in PBS containing 0.05% Triton X100 (PBST), secondary antibodies were applied for 1 hour at 37°C. Subsequently, slides were washed three times in PBST. Immunostained slides were imaged using confocal microscopy. Projected images were then prepared using Photoshop (Adobe) software package or Bitplane Imaris (version 8.1) software.

[0054] Statistical analysis

[0055] Data were evaluated using GraphPad Prism 8. Data are presented as mean ± SEM, and differences were considered statistically significant when *p < 0.05.

[0056] Example 2 Identification of TEX14 Pathogenic Variants

[0057] Exome sequencing was performed on genomic DNA from a study group that included 673 men with non-obstructive azoospermia (NOA), 143 men with oligozoospermia, and 100 fertile men as controls. Screening identified four men from two different centers who carried potential pathogenic variants in TEX14 ( Figure 1 A, Table 1).

[0058] Table 1 TEX14 variants identified by WES and bioinformatics prediction.

[0059]

[0060] Patient V1 carried a homozygous duplication: (Chr 17[GRCh37]:56663324dup; NM_198393.4[TEX14]:c.2908dup)(Mu1), which resulted in premature translation termination and truncation of the TEX14 protein ( Figure 1 Patient V2 carried two compound heterozygous mutations, including a likely pathogenic insertion that could result in a frameshifted allele: p.Gly628ArgfsTer8 (chr17[GRCh37]:g.56676825dup; NM_198393.4[TEX14]:c.1881dup) (Mu2), which ultimately truncates the protein product, and another allele with a pathogenic deletion: p.Leu577ArgfsTer58 (chr17[GRCh37]:g.56676979-56676980del; NM_198393.4[TEX14]:c.1728_1729del) (Mu3), which could also result in a truncation of the protein product ( Figure 1 B). Patient V3 also carries two compound heterozygous variants, one of which is in the kinase domain at a highly conserved site in mammals ( Figure 1 B) pathogenic missense mutation: p.Tyr374Cys (chr6[GRCh37]:g.56688585T→C; NM_198393.4[TEX14]:c.1121A>G; CADD score, 26.5)(Mu4). On the other allele, the patient had a new base substitution at the canonical splice donor site between exon 4 and introns 4-5: (chr17[GRCh37]:g.56700206A→G; NM_198393.4[TEX14]:c.417+2T→C)(Mu5)( Figure 1B). This variant disrupts the splicing sequence recognized by the splicing machinery. Patient V4 carries two compound heterozygous variants, including a pathogenic insertion causing a frameshift: p.Gly628ArgTer8 (chr17[GRCh37]:g.56676825dup(Mu2); NM_198393.4[TEX14]:c.1881dup), which can cause protein truncation on one allele, and a pathogenic missense variant: p.Glu289Lys (chr17[GRCh37]:56692609C→T; NM_198393.4[TEX14]:c.865G>A; CADD score, 32)(Mu6), which disrupts a conserved site in the kinase domain of the other allele of TEX14 ( Figure 1 B).

[0061] None of the six TEX14 variants were detected in the control group. The presence of each variant was confirmed by Sanger sequencing using gDNA templates from patients ( Figure 1 A).

[0062] The results suggest that biallelic variants in TEX14 are pathogenic for male infertility in these individuals.

[0063] Example 3 Clinical manifestations of males carrying TEX14 gene mutations

[0064] Two patients (one from family 1 and one from family 2) were diagnosed with NOA by physical examination or histological examination according to the AUA-ASRM criteria and the diagnostic criteria in the EAU guidelines. Neither of the two men had risk factors for testicular obstruction, and both of them presented a normal male phenotype (i.e., no gynecological enlargement, normal adult pubic hair distribution, no hypospadias, and testicles located low in the normal scrotum). Their testicles were slightly reduced in size, and the epididymis in each patient was not atrophic, and the vas deferens were palpable on both sides, which is consistent with NOA cases. Patients V3 and V4 were diagnosed with oligospermia based on physical examination. All four men had a normal karyotype of 46 chromosomes XY, no Y chromosome microdeletions, azoospermia, and no sperm were detected in the ejaculate after high-speed centrifugation. Fertility treatment was unsuccessful in all patients, and morphologically abnormal sperm were observed only in the testicular sperm extracted from patient V3.

[0065] Example 4 Effect of mutation on TEX14 expression

[0066] The relative expression of TEX14 in the patient's testis was analyzed by qPCR to detect mRNA degradation or truncated protein caused by the homozygous TEX14 variant (c.2908dupC).

[0067] In patient V1, who carried the homozygous variant, the level of TEX14 mRNA in the testis was 11.63% of that in the control group (P = 0.003). To determine whether these variants resulted in truncated protein, Western blot was used to assess TEX14 protein levels in the testis of patient V1 and the control group. This analysis showed no protein band of the expected size, indicating that the variant (c.2908dupC) did result in the loss of TEX14 protein.

[0068] To evaluate the effects of other variants on protein expression, wild-type (WT) TEX14 and three mutant TEX14s (c.2908dup[p.Arg970ProfsTer4], c.1881dup[p.Gly628ArgfsTer8], and c.1121A>G[p.Tyr374Cys]) with N-terminal fusion of EGFP or two mutant TEX14s (c.865G>A[p.Glu289Lys and c.1728_1729del[p.Leu577ArgfsTer58]) with N-terminal fusion of mCherry were expressed in 293T cells. Western blot analysis showed that frameshift mutations (p.Arg970ProfsTer4, p.Gly628ArgfsTer8 and p.Leu577ArgfsTer58) resulted in the expression of truncated TEX14 protein in the cell lines.

[0069] Example 5 TEX14 deletion leads to spermatocyte meiosis arrest in patients with homozygous variant p.Arg970Profs*4

[0070] To characterize the meiotic defects associated with homozygous variants in TEX14, hematoxylin and eosin (H&E) stained testicular sections from patient V1 and samples from another male diagnosed with obstructive azoospermia (OA) were used as controls ( Figure 2 A). Spermatogenic cells at various stages of development were observed in the seminiferous tubules of the control patients. However, only spermatogonia and spermatocytes were observed in the seminiferous tubules of patient V1. It is noteworthy that the most advanced spermatogenic cells that can be found in the patient are spermatocytes, and no late meiotic cells can be detected. To confirm this observation, PNA immunofluorescence staining was performed on testicular sections of patient V1 and the OA control group ( Figure 2 B), PNA is a marker for sperm and sperm acrosome. The results showed that there was no PNA signal in the testicular samples of V1 patients, while a large number of PNA-positive sperm could be observed in the testicles of the control group ( Figure 2B) These results indicate that spermatogenesis in this NOA patient is arrested at the spermatocyte stage. These results strongly suggest that NOA is caused by the failure of spermatocytes in patients with homozygous TEX14 variants to complete meiosis.

[0071] Next, testicular sections from patient V1 and a man diagnosed with obstructive azoospermia were immunofluorescently stained with antibodies against γH2AX (DSB marker) and SYCP3 (meiotic chromosome axis) to detect the progression of meiotic prophase I ( Figure 2 C). In the seminiferous tubules of the control group, γH2AX was localized in the spermatocyte nuclei, with diffuse signals in leptotene and zygotene spermatids, and could be observed in the typical sex bodies of pachytene spermatocytes. In addition, spermatocytes with typical sex bodies were easily found in the OA control samples, but such cells were not found in any of the seminiferous tubule samples of patient V1 with a homozygous TEX14 frameshift mutation (c.2908dupC).

[0072] To further examine how TEX14 variants affect meiotic processes, testicular biopsy samples were obtained from patient V1, and spermatocyte smears were prepared and subsequently immunofluorescently stained for SYCP3 and γH2AX ( Figure 2 D). The progress of spermatocyte meiotic prophase I was detected by immunofluorescence staining of SYCP3 and γH2AX. The results showed that 11.48% and 25.68% of spermatocytes were in the leptotene and zygotene stages, with small spots and bright γH2AX signal foci in the nucleus, respectively. In addition, 63.93% of pachytene spermatocytes showed typical sex bodies, with strong γH2AX signals around the X and Y chromosomes ( Figure 2 E). In contrast, in patient V1, analysis of 108 spermatocytes revealed that leptotene and zygotene nuclei were similar to control samples; however, the most advanced spermatocytes showed persistent γH2AX signals on autosomes and failed to form sex bodies ( Figure 2 D).

[0073] These results suggest that meiosis of V1 in NOA patients is arrested at the zygotene stage.

[0074] Example 6 Tex14 mutant mice recapitulate the NOA phenotype of human patients with homozygous Tex14 mutations

[0075] CRISPR / Cas9 gene editing was used to generate TEX14 knock-in (Tex14 mu1 / mu1) mice carrying a homozygous mutation equivalent to p.Arg970Profs*4. The Tex14 mutation in the mutant mouse model was confirmed by Sanger sequencing. To explore whether the Tex14 mutant caused mRNA degradation or protein truncation, RT-qPCR analysis of Tex14 mRNA in the mutant mice was first performed. This analysis showed that Tex14 mu1 / mu1 Transcription in the testes of mice was reduced to approximately 10.52% of that in wild-type mice. Subsequent WB analysis using anti-TEX14 antibody showed that Tex14 was expressed at postnatal day 14. mu1 / mu1 TEX14 protein was not detected in the testicular lysates of mice, whereas the full-length protein was present in samples from control mice ( Figure 3 A). Tex14 mu1 / mu1 and Tex14 + / mu1 Immunofluorescence staining of mouse testis sections further revealed that there were no TEX14-positive cells in the seminiferous tubules of mutant mice, while abundant TEX14 signals could be observed in the seminiferous tubules of control mice. mu1 / mu1 Compared with wild-type littermates, male mice were viable and showed no obvious developmental abnormalities, but had smaller testes and a significantly reduced testis-to-body weight ratio (p < 0.0001; Figure 3 B and C). Histological analysis showed that Tex14 mu1 / mu1 The seminiferous tubules of mice contain spermatogonia and primary spermatocytes, but lack post-meiotic germ cells. Similarly, no sperm was observed in the cauda epididymis of the mutant mouse model ( Figure 3 D) Consistent with these results, mu1 / mu1 Immunofluorescence staining of PNA in testicular sections of Tex14 mutant mice and wild-type mice showed that no sperm was found in the testes of Tex14 mutant mice ( Figure 3 E), indicating a complete arrest of meiosis. Together, these results suggest that the c.2908dupC mutation in Tex14 mice results in its loss at both the mRNA and protein levels, and that this loss-of-function effect results in the loss of Tex14 mu1 / mu1 NOA phenotype in mice.

[0076] Example 7 TEX14 deficiency causes spermatocytes of mice carrying homozygous p.Arg970Profs*4 to arrest at the zygotene stage

[0077] To determine the Tex14 mu1 / mu1 The exact subphase of meiotic arrest in male mice is critical for Tex14 mu1 / mu1 γH2AX immunofluorescence staining was performed on the testes of Tex14 and wild-type mice. mu1 / mu1In the testicular sections of mice, almost no spermatocytes with developed sex bodies were found, while in the WT littermate control mice, a large number of spermatocytes in the leptotene and zygotene stages containing sex bodies were found, and the nuclei of these spermatocytes were localized with γH2AX signals ( Figure 4 A). These results indicate that TEX14 mutation causes meiotic arrest at prophase I in mice. To further confirm the progression of prophase I, we performed a double-blind analysis of control and Tex14 mice. mu1 / mu1 Co-staining of SYCP3 and γ-H2AX in mouse spermatocyte smears Figure 4 B). In the testes of control mice, spermatocytes were observed in all substages of meiotic prophase I, including leptotene, pachytene, zygotene, and diplotene, with percentages of (6.86±0.88%), (9.23±0.96%), (60.62±1.52%), (21.84±0.10%), and (1.46±0.20%), respectively. In contrast, in Tex14 mu1 / mu1 In mice, spermatogonia in the leptotene, pachytene, zygotene, and diplotene stages were observed, but significantly higher proportions of spermatocytes in the leptotene (27.36±3.24%) and pachytene (63.72±2.55%) stages were found, while the proportions of spermatocytes in the zygotene (7.63±2.72%) and diplotene (1.16±0.26%) stages were significantly lower than those in the control group ( Figure 4 C) These results indicate that meiosis is arrested at the diplotene stage. mu1 / mu1 In mouse spermatocyte smears, SYCP3 and SYCP1, the core elements of the synaptic complex, were co-stained. mu1 / mu1 There were no differences in synapses between the control mice ( Figure 4 D). Taken together, these results indicate that the Tex14c.2908dupC mutation causes meiotic diplotene arrest in the mouse model.

[0078] Example 8 Deletion of intercellular bridge protein in homologous p.Arg970Profs*4 germline cells

[0079] Next, we studied the effect of TEX14 mutation on intercellular bridge proteins in human germ cells. Immunofluorescence staining of intercellular bridge proteins TEX14 and SYCP3 in testicular sections of patient V1 and OA control group revealed that there was no TEX14 signal in the sample of patient V1, while there was a ring signal between spermatogonia in the sample of the control group ( Figure 5 A). Consistent with this result, immunofluorescence staining of another intercellular bridge protein, MgcRacGAP1, in testicular sections from patient V1 and OA controls revealed that the MgcRacGAP1 signal was attenuated in samples from patient V1 compared with controls ( Figure 5B) Further validation by WB analysis confirmed that MgcRacGAP1 was undetectable in patient V1, while a band of the expected size was observed in control samples, further demonstrating that the homologous p.Arg970Profs*4 variant resulted in the loss of the intercellular bridge.

[0080] These results suggest that TEX14 deficiency disrupts intercellular bridge formation in human male germ cells, leading to meiotic arrest at the diplotene stage and ultimately causing the NOA phenotype ( Figure 6 ).

[0081] The inventors of the present application have discovered and proved for the first time that TEX14 mutations lead to male spermatogenesis failure and complete infertility. In addition, the inventors have discovered multiple TEX14 mutations in male infertility for the first time in humans, including three frameshift mutations c.2908dup, p.Arg970ProfsTer4; c.1881dup, p.Gly628ArgfsTer8; c.1728_1729del, p.Leu577ArgfsTer58, two missense mutations c.865G>A, p.Glu289Lys; c.1121A>G, p.Tyr374Cys, and a splicing mutation c.417+2T→C. These mutations, especially frameshift mutations, lead to truncation of TEX14 protein and failure of intercellular bridge formation, ultimately leading to meiotic arrest of human male germ cells, thereby proving that TEX14 is a pathogenic gene for male infertility, especially NOA. Applicants have thus provided methods for diagnosing fertility defects such as infertility disorders in mammals, including humans, and further facilitated the development of new treatments for male infertile individuals affected by gene mutations.

[0082] The above is an explanation of the present invention and cannot be regarded as a limitation of the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that the present invention can also be realized in other ways except that it is particularly described in the above description and embodiments. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

Claims

1. A method for diagnosing male human male fertility defects such as infertility, comprising detecting TEX14 in a test subject, And, use of a reagent for detecting TEX14 in a test individual in the preparation of a kit for diagnosing human male fertility defects such as infertility.

2. The method according to claim 1, wherein the infertility disorder is selected from the group consisting of non-obstructive azoospermia, teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, and asthenozoospermia.

3. The use according to claim 1 or 2, wherein the detection reagent is a reagent for detecting the Tex14 gene, preferably a reagent for detecting the Tex14 gene mutation.

4. The use according to claim 3, wherein the mutation is selected from: (a) frameshift mutation, such as c.2908dup (p.Arg970ProfsTer4), c.1881dup (p.Gly628ArgfsTer8), or c.1728_1729del (p.Leu577ArgfsTer58); (b) missense variants, such as c.865G>A (p.Glu289Lys) or c.1121A>G (p.Tyr374Cys); or (c) Splicing mutation, for example, c.417+2T→C.

5. The use according to claim 1 or 2, comprising a reagent for detecting TEX14 protein, preferably, further comprising a reagent for determining the activity of TEX14 protein.

6. The use according to claim 5, comprising a reagent for detecting the expression of the TEX14 protein by immunoassay, such as a reagent for detecting the expression by ELISA or Western blotting using an antibody that specifically recognizes the protein; or These include reagents for detecting the presence or amount of mRNA of the TEX14 protein, such as reagents for detecting the amount of mRNA encoding the protein in a sample by RT-PCR.

7. A kit for diagnosing human male fertility defects such as infertility, comprising a reagent for detecting TEX14 in a subject and a reagent for detecting the sex of the subject.

8. The kit of claim 7, wherein the infertility disorder is selected from the group consisting of non-obstructive azoospermia, teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, and asthenozoospermia.

9. The kit according to claim 7, wherein the reagent for detecting TEX14 in the subject is a reagent for detecting the Tex14 gene, preferably a reagent for detecting a Tex14 gene mutation, More preferably, the mutation is selected from: (a) frameshift mutation, such as c.2908dup (p.Arg970ProfsTer4), c.1881dup (p.Gly628ArgfsTer8), or c.1728_1729del (p.Leu577ArgfsTer58); (b) missense variants, such as c.865G>A (p.Glu289Lys) or c.1121A>G (p.Tyr374Cys); or (c) Splicing mutation, for example, c.417+2T→C.

10. The kit according to claim 7, wherein the reagent for detecting TEX14 in the subject is a reagent for detecting TEX14 protein, and preferably, it also includes a reagent for measuring the activity of TEX14 protein.

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