Molecular marker for later period of spermatogenesis and application of molecular marker

Abstract

The present invention provides methods for diagnosing or predicting fertility defects, such as infertility, in a mammalian male subject by detecting CCDC175. The invention also provides kits and instruments for diagnosing or predicting fertility defects, such as infertility disorders, in mammalian male individuals by detecting CCDC175. The invention finds and proves for the first time that deletion of CCDC175 destroys acrosome biogenesis of mammal sperms, resulting in serious spermatogenesis arrest and enhanced spermatogenic cell apoptosis, thereby providing a method for diagnosing infertility of mammals, including humans. And a new thought and a new method are provided for treatment of male sterility individuals influenced by gene mutation.

Description

Technical Field

[0001] The present invention relates to the fields of diseases and diagnostics. Specifically, the present invention relates to a molecular marker in the late stage of spermatogenesis and methods and kits for diagnosing or treating male fertility defects in mammals, particularly humans, using the molecular marker. Background Art

[0002] Spermatogenesis is the process by which male gametes are produced and continuously undergo cell renewal and differentiation, including spermatogonial mitosis, spermatocyte meiosis, and spermiogenesis. Spermiation refers to the process by which mature spermatids are released from Sertoli cells into the lumen of the seminiferous tubules. This process is a key determinant of the number of sperm entering the epididymis and the sperm content during ejaculation. Spermiation is completed at the end of stage VIII, at which time spermatids are released into the lumen and the residual cytoplasm of the spermatids, called residual bodies, are phagocytosed by Sertoli cells. Although the main goal of spermiation is to release spermatids from Sertoli cells, this process also promotes extensive reorganization and remodeling of spermatids to produce streamlined sperm. Spermiation involves multiple steps and changes in spermatids and Sertoli cells, and at the end of spermatogenesis, prepares for the final release of elongated sperm from Sertoli cells.

[0003] During these precisely timed and highly organized events, germ cells traverse the seminiferous epithelium from the basal to the apical side by adhering to somatic Sertoli cells. Sertoli cells play a key role in controlling spermatogenesis. Their functions include providing structural support and nutrients for germ cell development, mediating the self-renewal and differentiation of spermatogonial stem cells (SSCs), phagocytosing degenerating germ cells, protecting germ cells from autoreactive immune responses, and releasing spermatids during spermiogenesis and hormonal regulation. Throughout all stages of spermatogenesis, Sertoli cells and germ cells have a process of intercellular communication, which involves the attachment, displacement, denaturation, and cell-cell transfer of signaling molecules and cellular components. This Sertoli-germ cell communication is essential for the success of spermatogenesis. Morphologically, this process is characterized by the removal of an atypical adhesion junction called the ectoplasmic specialization (ES) and the formation of a transient endocytic apparatus called the tubulobulbar complex (TBCs), which requires cytoskeletal remodeling and recruitment of proteins required for endocytosis.

[0004] One of the key events in spermatogenesis is the remodeling of the sperm nucleus and cytoplasm to produce streamlined sperm, the removal of the Sertoli cell "ectoplasmic specialization" (ES) junctions and the contraction of the Sertoli cell cytoplasm, and the elongation of spermatids into the lumen. Spermiation ends with the detachment of spermatids into the lumen and the phagocytosis of the remainder of the residual body by the Sertoli cell. Spermiation begins at stage VII of the seminiferous tubules in rats and mice, when most late spermatids are aligned along the luminal edge. ES is crucial for Sertoli-germ cell communication to support all stages of germ cell development and maturation. Its formation and remodeling require rapid reorganization of the cytoskeleton. However, the molecular mechanisms regulating ES assembly remain largely unknown.

[0005] The tubulobulbar complex (TBC) is an interesting structure that is most prominent between late spermatids and Sertoli cells during spermiogenesis, but also appears in a stage-specific manner between Sertoli cells of the blood-testis barrier. Each TBC consists of an elongate plasma membrane projection of a Sertoli cell or a spermatid that extends into a corresponding tubule invagination of an adjacent Sertoli cell. A "bristle-coated" invagination appears at the apex of the structure, and the distal portion of the complex bulges to form a "globular" region. This swollen globular region lacks an associated network of actin filaments and is closely associated with smooth endoplasmic reticulum. The formation of TBCs begins when a small portion of spermatid cytoplasm starts to protrude into a Sertoli cell at a bristle-coated invagination on the Sertoli cell membrane, an area that lacks Sertoli cell ES but is usually located on either side of Sertoli cell ES.

[0006] Current studies have shown that the "bristle-coated" invagination contains clathrin, a protein involved in endocytosis. Clathrin may be involved in the initiation of TBC formation and the recruitment of the actin cytoskeleton during formation. A narrow protrusion of spermatid cytoplasm forms from the perinuclear region, invaginating the Sertoli cell cytoplasm to form a long, narrow double-membrane tubular structure that terminates in a bristle-coated invagination and is "coated" with clathrin.

[0007] As the continuous formation of TBCs progresses, an expanded region appears at the end of the tubular structure. The ARP3 complex is a seven-subunit protein that regulates the actin cytoskeleton by serving as the nucleation core for actin branching from the mother filament. Proteins associated with the Arp3 complex, along with N-WASP, which localizes to TBCs and is involved in the activation of ARAP2 / 3, and the dendritic actin assembly mediated by the ARAP2 / 3 complex, may play a major role in TBC extension. Interestingly, during the formation of TBCs, N-WASP plays a role in the activation of the ARAP2 / 3 complex in actin dynamics. The role of actin filaments in TBCs is not fully understood, although it may play a role in the formation and maintenance of the tubular region.

[0008] There is still a need in the art for a greater understanding of the regulation of spermatogenesis in mammals, including humans, for the detection and prediction of male fertility disorders. Summary of the Invention

[0009] The inventors have for the first time discovered and demonstrated that coiled-coil domain containing 175 (CCDC175) is expressed only in the testes of adult mice and is essential for the biogenesis of the sperm tubule-vacuole complex during spermatogenesis. Deletion of CCDC175 disrupts the acrosome of mammalian sperm, impairs spermatogenesis, and results in the absence of sperm in the epididymis, leading to severe spermatogenic arrest and germ cell apoptosis. Therefore, abnormal expression of CCDC175 leads to abnormal spermatogenesis and even male infertility. At the same time, the inventors have found that CCDC175 is essential for male fertility but has no effect on female fertility. Thus, the inventors provide methods for diagnosing or treating male fertility defects in mammals, especially humans.

[0010] Specifically, the present invention provides a method for diagnosing or predicting fertility defects, such as infertility disorders, in male individuals of mammals, which includes detecting the gene or protein of coiled-coil domain containing 175 (CCDC175) in a test male individual.

[0011] In the present invention, the infertility disorders of the male individuals include, but are not limited to, teratospermia, azoospermia, oligospermia, asthenospermia, asthenozoospermia, etc.

[0012] In the present invention, the mammal can be any mammal, including and not limited to rodents, canines, felines, equines, ovines, bovines, porcines, and primates. The mammal typically includes humans.

[0013] Coiled-coil domain containing 175 (CCDC175), also known as Spiral Coiling Domain 175. The Ccdc175 gene encoding CCDC175 has a conserved coding sequence in various mammals. The GeneID of the mouse Ccdc175 gene is 73936. The Gene ID of human Ccdc175 is 729665.

[0014] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention includes the step of detecting the Ccdc175 gene.

[0015] The method for examining genes used in the present invention is a method commonly used in the art for gene detection and is not particularly limited. For example, mass spectrometry, microarray, sequencing, detection methods using base sequence amplification methods such as PCR (polymerase chain reaction), etc. can be cited.

[0016] In addition, the detection of the PCR product obtained when performing PCR using primers specific to each gene can be carried out by any method commonly used for detecting and quantifying PCR products. For example, it can be detected by electrophoresis, detected by real-time PCR using a fluorescence intercalator such as SYBR Green, or detected by single molecule fluorescence analysis.

[0017] The polynucleotide that can be used as a primer or probe for gene detection is not particularly limited as long as it can hybridize with a partial region of the gene or its complementary strand. The design of the polynucleotide can be carried out using any of the well-known methods in this technical field. For example, using publicly known genomic sequence data and general primer design tools, it can be easily designed. As such a primer design tool, for example, there is Primer3 that can be used on the Internet. In addition, publicly known genomic sequence data can usually be obtained from NCBI, an international base sequence database.

[0018] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention includes the step of detecting the CCDC175 protein, such as detecting the expression of the protein (including its presence or absence, and whether there is a decrease in expression relative to normal or standard) or its activity.

[0019] In yet another aspect of the present invention, the method includes a step of detecting the expression of CCDC175 protein by immunoassay. For example, the expression of CCDC175 is detected by immunofluorescence or Western blot using an antibody that specifically recognizes the protein. Also for example, the expression of CCDC175 is detected by detecting the presence or amount of the mRNA of the protein, such as by detecting the amount of mRNA encoding CCDC175 in a sample by RT-PCR.

[0020] In yet another aspect of the present invention, the method includes a step of determining the activity of CCDC175 protein.

[0021] This aspect also provides a kit or instrument (including gene chip) for diagnosing or predicting fertility defects such as infertility disorders in a mammalian individual, which includes a reagent for detecting the Ccdc175 gene of the subject individual.

[0022] In yet another aspect of the present invention, the kit or instrument includes a reagent for detecting the expression or activity of CCDC175 protein. For example, the kit or instrument includes reagents for immunoassay, such as reagents for detecting the expression of CCDC175 by ELISA or Western blot using an antibody that specifically recognizes the protein. Also for example, the kit or instrument includes a reagent for detecting the expression of CCDC175 by detecting the presence or amount of the mRNA of the protein, such as a reagent for detecting the amount of mRNA encoding CCDC175 in a sample by RT-PCR.

[0023] In yet another aspect of the present invention, the kit or instrument includes a reagent for detecting the Ccdc175 gene.

[0024] In one aspect of the present invention, in the above-mentioned method, kit or instrument of the present invention, the reagent for detecting a genetic marker in a sample includes a primer or probe for detecting Ccdc175, or a reagent for examining CCDC175, such as a specific antibody or a reagent for detecting the mRNA of the protein, etc.

[0025] In the present invention, there is also provided the use of a reagent for detecting CCDC175 in the preparation of a kit or instrument for diagnosing or predicting fertility defects such as infertility disorders in a mammalian individual as described above. The reagent for detecting a genetic marker includes a primer or probe for detecting the Ccdc175 gene, or a reagent for examining the CCDC175 protein, such as a specific antibody or a reagent for detecting the mRNA of the protein, etc.

[0026] This aspect also provides a method for treating or avoiding fertility defects in a mammalian individual, which includes treating an individual with a Ccdc175 defect such as a Ccdc175 mutation.

[0027] In this article, protein symbols are not italicized and are all in uppercase; gene symbols are italicized. For example, CCDC175 is a protein, and the gene encoding this protein is written as Ccdc175. Sometimes in this article, protein symbols are also not italicized. For example, sometimes in this article, "Ccdc175" represents the CCDC175 protein. Sometimes in this article, gene symbols are also not italicized. For example, sometimes in this article, "CCDC175" or "CCDC175 gene" represents the gene Ccdc175 encoding the CCDC175 protein. Brief Description of the Drawings

[0028] Figure 1 It shows that CCDC175 is expressed in the testicular tubular body complexes (TBCs).

[0029] A. CCDC175 is specifically expressed in the testis and is verified using the Mouse Cell Atlas (MCA) (https: / / bis.zju.edu.cn / MCA / search3.html).

[0030] B. The expression of CCDC175 is significantly increased in the testis at PD21 days and remains stable in the adult testis, verified using the Mouse Cell Atlas (MCA).

[0031] C and D. Uniform manifold approximation and projection (UMAP) plots from the Male Health Atlas (MHA) (http: / / malehealthatlas.cn / ) show the expression of CCDC175 in different cell types. SPG: spermatogonia; SPC: spermatocytes; SPT: spermatids / spermatozoa; SC: Sertoli cells; LC: Leydig cells; PTM: peritubular myoid cells; EC: endothelial cells; SMC: vascular smooth muscle cells.

[0032] E - G. UMAP - plots and violin plots show the expression of CCDC175 in different mouse germ cells, using the Male Health Atlas (MHA). SSC1 - 3: spermatogonial stem cells I - III; Progenitor: spermatogonial progenitor cells; A1 - 4: A1 - 4 spermatogonia; In: intermediate spermatogonia; B: type B spermatogonia; PI: pre - leptotene; L: leptotene; Z: zygotene; P: pachytene; D: diplotene; M: metaphase; RS: round spermatids. E: elongated spermatids.

[0033] The H-J.UMAP plot shows the expression of CCDC175 in different mouse cells using the Mouse Cell Atlas (MCA).

[0034] K. CCDC175 accumulates in the tubular body complex. Immunofluorescence analysis of Ccdc175+ / + and Ccdc175- / - testes was performed using F-actin (red, labeled by phalloidin), CCDC175 (green), and ARAP3 (gray).

[0035] L. Compared with germ cells, CCDC175 accumulates more significantly in somatic cells of the testis. Immunoblot analysis of CCDC175 in FSPCs and FSCs was performed in testes at PD18. Tubulin was used as an internal control.

[0036] Figure 2 Showing Ccdc175 - / - Absence of sperm in the epididymis of male mice

[0037] A. Schematic of the knockout strategy for constructing Ccdc175 - / - mice. gRNA1 and gRNA2 were designed to delete exon3 of the Ccdc175 locus.

[0038] B. Immunoblot of Ccdc175 - / - mice testis showed that the CCDC175 protein was not detected; while it was present in the testes of Ccdc175 + / + control mice. Tubulin was used as an internal control.

[0039] C. Ccdc175 - / - Female mice showed normal fertility. The Y-axis represents the average number of offspring per litter during a 3-month observation window (n = 7). Data are presented as mean ± SEM. Two-tailed Student's t-test; ns indicates no significance.

[0040] D. Ccdc175 - / - Male mice were infertile. When mating with Ccdc175 + / + male mice, all 3 female mice became pregnant, while when mating with Ccdc175 - / - male mice, no female mice became pregnant. The Y-axis represents the average number of offspring per litter over 3 months (n = 7). Data are presented as mean ± SEM. Two-sided Student's t-test; P < 0.0001.

[0041] E. Quantification of Ccdc175 + / + and Ccdc175 - / -Body weight of male mice. Data are presented as mean ± SEM (n = 6). Two-tailed Student's t-test; ns indicates not significant.

[0042] F. Ccdc175 - / - The testes of male mice are smaller than those of Ccdc175 + / + male mice. Ccdc175 + / + and Ccdc175 - / - Quantitative analysis of testis weight in male mice (n = 6). Data are presented as mean ± SEM. Two-tailed Student's t-test; ns indicates P < 0.001.

[0043] G. Ccdc175 + / + and Ccdc175 - / - Testis weight / body weight ratio of mice. Data are presented as mean ± SEM (n = 6). Two-tailed Student's t-test; P < 0.01.

[0044] H. Ccdc175 + / + and Ccdc175 - / - The testes of mice are similar in size.

[0045] I. Ccdc175 + / + and Ccdc175 - / - HE staining of testis sections from mice. * indicates large vesicles in the seminiferous tubules of Ccdc175 - / - mice. P (red): pachytene spermatocytes, rSt (blue): round spermatids, spz (green): spermatozoa.

[0046] J. Ccdc175 + / + and Ccdc175 - / - HE staining of the cauda epididymis from mice. Red arrowheads indicate apoptotic bodies in the cauda epididymis of Ccdc175 - / - mice.

[0047] K. Ccdc175 - / - The sperm count in the epididymis of male mice is significantly reduced (n = 5). Data are presented as mean ± SEM. Two-tailed Student's t-test; P < 0.01.

[0048] Figure 3 Shown in Ccdc175 - / - testes, sperm are not released

[0049] A. For Ccdc175 + / + and Ccdc175 - / -The cross-sections of mouse seminiferous tubules were analyzed by PAS and hematoxylin staining. Arrows indicate germ cells at different stages of spermatogenesis. In Ccdc175 - / - In stage VIII seminiferous tubules (red circle) of Ccdc175

[0050] B. In Ccdc175 - / - In stage VIII (red circle) of seminiferous tubules of male mice, the ejaculation process of mature sperm was blocked. IF staining was performed on testes of Ccdc175 + / + and Ccdc175 - / - mice using F-actin (red), PNA (green), and DAPI (blue). P: pachytene spermatocytes, D: diplotene spermatocytes, rST: round spermatids, eST: elongated spermatids, spz: mature sperm. Scale bar, 10 μm.

[0051] C. In Ccdc175 + / + and Ccdc175 - / - testes, the structure of F-actin was disordered and severely aggregated into punctate patterns. IF analysis was performed on testes of Ccdc175 + / + (upper) and Ccdc175 - / - mice (lower) using phalloidin (green). Nuclei were stained with DAPI (blue).

[0052] D. Compared with Ccdc175 + / + mice, the number of unreleased sperm increased in Ccdc175 - / - male mice. In Ccdc175 - / - male mice, sperm stagnated at stage IX (n = 3). Data are presented as mean ± SEM. Two-tailed Student's t-test; ns indicates P < 0.01.

[0053] E. In Ccdc175 - / - male mice, the proportion of stage IX was significantly higher than that in Ccdc175 + / + . While the proportion of stages XIII - XVI was significantly lower in Ccdc175 - / - male mice than in Ccdc175 + / + . Data are presented as mean ± SEM. Two-tailed Student's t-test; ns indicates P < 0.01.

[0054] Figure 4Show the localization of CCDC175 in TBCs. CCDC175 accumulates in the tubular body complex (TBC) and plays a key role in the formation and assembly of TBC. For Ccdc175 + / + and Ccdc175 - / - mouse testes were subjected to IF analysis of F-actin (red), CCDC175 (green), and ARAP3 (gray).

[0055] Figure 5 Show that in Ccdc175 - / - mouse testes, the TBC structure is affected

[0056] (A) The distribution of TBC is disrupted in Ccdc175 - / - mice. ARAP3 localizes to TBC in Ccdc175 + / + mice. For Ccdc175 + / + and Ccdc175 - / - mouse testes were subjected to IF analysis of F-actin (red), ARAP3 (green).

[0057] (B) ARAP3 is highly expressed in stage VIII of Ccdc175 + / + mouse testes. In Ccdc175 - / - mouse testes, the distribution of TBC is disrupted. For Ccdc175 + / + and Ccdc175 - / - mouse testes were subjected to IF analysis of F-actin (red), ARAP3 (green).

[0058] (C) DYNAMIN2 localizes to TBC in Ccdc175 + / + mice. For Ccdc175 + / + and Ccdc175 - / - mouse testes were subjected to IF analysis of F-actin (red), DYNAMIN2 (green).

[0059] (D) In Ccdc175 - / - mouse testes, the formation of TBC is disrupted. In Ccdc175 - / - sperm of stage 15-16 in male mice, the TBC structure disappears (n = 9). Data are presented as mean ± SEM. Two-tailed Student's t-test; ns indicates P < 0.0001. DETAILED DESCRIPTION

[0060] The following will further illustrate the essential content and beneficial effects of the present invention in combination with embodiments. These embodiments are only used to illustrate the present invention and not to limit the present invention.

[0061] Example 1 Experimental Methods and Reagents

[0062] Animals

[0063] The 3rd exon was selected as the target site, and a Ccdc175 knockout mouse was created using the CRISPR / cas9-mediated genome editing system from Cyagen Biosciences. Founder animals and offspring animals were identified by DNA genomic sequence analysis. All mouse and animal care protocols were approved by the Regional Ethics Committee of the School of Medicine, Shandong University.

[0064] Tissue Collection and Histological Analysis

[0065] Ccdc175 + / + and Ccdc175 - / - Male mice of each genotype (n = 5) were used in this experiment. After euthanasia, all samples were fixed overnight in 4% PFA or Bouin's. All samples were dehydrated in 75% ethanol, paraffin-embedded, sectioned at 5 μm, mounted on slides for HE, PAS, and IF staining.

[0066] Fixed Frozen Sections

[0067] Pfa-fixed testes were placed in a pool of OCT compound (abs9756, Absin) and frozen at the optimal cutting temperature. Using a cryostat, the frozen tissue was sectioned into 5 mM slices and then collected on polylysine-coated slides. The sections were immersed in cold acetone for 1 min and then air-dried for IF.

[0068] Fix epithelial debris material.

[0069] PFA-fixed and shelled in PBS, the testes were minced into small pieces with a scalpel. The tubule segments were aspirated through 18-gauge and 21-gauge syringe needles. After sedimentation for 10 min, the upper supernatant was transferred to a new 15-ml centrifuge tube and then concentrated by low-speed centrifugation. The upper solution was gently removed, and the lower pellet was mixed with 50 - 100 μl PBS and then placed on a poly-l-lysine-coated slide. The slide was placed in cold acetone for 1 min and then air-dried for IF.

[0070] Antibodies

[0071] Primary antibodies were from the following companies and used at the specified working concentrations for IF and immunoblotting: CCDC175 (1:200 dilution, γH2AX, 1:200 dilution, 1,200 dilution), γH2AX (1:500 dilution, 05,63), Sox9 (1:200 dilution, 18200 dilution, VINCUL, 1:200), Conjugated Lectin PNA (1:500, L21409), VINCUL (1:500, R415)

[0072] Immunofluorescence

[0073] After dropping to room temperature, the glass slides were blocked with 5% BSA or 5% bovine serum albumin for 1 h. They were permeabilized with 0.5% (v / v) Triton-X 100, and these sections were incubated with the primary antibody at 4 °C for 14 - 16 h. After washing three times with PBST, these sections were incubated with the secondary antibody at 37 °C for 1 h and with DAPI for 10 min. These glass slides were covered with coverslips and then imaged with a confocal microscope.

[0074] Western blotting

[0075] Total testicular and isolated spermatogenic epithelial lysates were used to extract proteins with lysis buffer, followed by grinding and incubation on ice for 25 min. After centrifugation at 12,000 rpm for 25 min at 4 °C, the supernatant of the extract was used for Western blotting. Separated by SDS-PAGE, these proteins were electrotransferred onto nitrocellulose membranes. After soaking with 5% non-fat milk for 1 h, the membranes were incubated with the corresponding primary and secondary antibodies for 1 h.

[0076] Statistical analysis

[0077] All experiments were repeated at least three times. Using GraphPad Prism, all statistical analyses were performed using Student's t-test, with a paired two-tailed distribution, and p-values were < 0.05 (*), 0.01 (**), or 0.001 (***).

[0078] Example 2 CCDC175 is expressed in TBCs of the testis

[0079] Single-cell RNA sequencing data analysis of the Mouse Cell Atlas (MCA) project (https: / / bis.zju.edu.cn / MCA / search3.html) showed that Ccdc175 RNA was specifically expressed in the testis ( Figure 1 A), with PD1 as the baseline level, Ccdc175 RNA was highly expressed in the testes of PD21-day-old and adult mice ( Figure 1 B), indicating that Ccdc175 may play a role during spermatogenesis. Using the Male Health Atlas (MHA) to plot the Uniform Manifold Approximation and Projection (UMAP) map (http: / / malehealthatlas.cn / ), the plot showed that CCDC175 was highly expressed in spermatocytes, spermatids, and sperm clusters ( Figure 1 C - 1D). UMAP and violin plots showed that CCDC175 was initially detected in pachytene, continued to increase during diplotene, and reached the highest level in elongated spermatids ( Figure 1E-1G). The UMAP-plot shows that CCDC175 is mainly expressed in the testis ( Figure 1 H-1J).

[0080] IF detection of anti-CCDC175 (green) antibody, F-actin (red), Arap3 (gray), and DAPI (blue) was performed on sperm of wild-type mice. When the apical processes of Sertoli cells containing mature sperm were mechanically disrupted from the seminiferous epithelium, the localization of CCDC175 in the area containing clusters of tubulobulbar complexes (TBCs) was very obvious ( Figure 1 K). Germ cells and somatic cells were purified from P18 testes and immunoblotted. CCDC175 was expressed in both somatic cells and spermatogenic cells (FSPCs) ( Figure 1 J).

[0081] Example 3 Construction of Ccdc175 KO mice

[0082] The testis-specific Ccdc175 gene is located on chromosome 12. The ATG start codon is in exon 1, and the TGA stop codon is in exon 121. To characterize the potential function of Ccdc175 during spermatogenesis, Ccdc175 - / - mice were created using the CRISPR / Cas9-mediated genome editing system from Cyagen Biosciences. Hybridization of heterozygous genome-edited mice produced healthy offspring at the expected Mendelian ratio. Exon 3 was selected as the target site, and 112 bp was deleted ( Figure 2 A). As expected, no CCDC175 protein was detected by immunoblotting of protein extracts from the testes of Ccdc175 - / - mice ( Figure 2 B).

[0083] Example 4 Ccdc175 KO does not affect testis size but causes male infertility

[0084] The fertility of male and female Ccdc175 knockout mice was examined. Female Ccdc175 knockout mice produced offspring after mating with age-matched wild-type males ( Figure 2 C). In contrast, male Ccdc175 knockout mice formed mating plugs after mating with wild-type adult female mice but did not produce offspring ( Figure 2 D). At the gross and histological levels, phenotypic changes in Ccdc175 knockout male mice were identified by examining the testicular structure of adult male mice. There were no significant differences in testis size, testis weight, body weight, or the ratio of testis weight to body weight between Ccdc175 knockout mice and wild-type (WT) mice ( Figure 2 E–2H).

[0085] Histological examination of testicular sections by hematoxylin-eosin staining showed that Ccdc175 - / - mice had significant differences in sperm bound to the testis in the seminiferous tubules. In the testes of Ccdc175-deficient mice, two types of seminiferous tubules were detected: one with the expected tubule structure, while the second was disordered with large vacuoles ( Figure 2 I, asterisk). Failure of normal sperm cytoplasmic removal may lead to the absence or reduction of the number of residual bodies in stage VIII. Different from the normal cytoplasmic removal of wild-type (WT) sperm, sperm cells with abnormal cytoplasmic retention were present in Ccdc175 - / - .

[0086] Ccdc175 - / - mice had a large number of cell deaths, apoptosis, and residual bodies in the epididymis, but no sperm ( Figure 2 j, arrow). Sperm released from the caudal epididymis were examined and found that the number of sperm in Ccdc175 - / - was significantly reduced compared to WT mice. Next, the number of sperm in the caudal epididymis was quantified and found that Ccdc175 - / - mice did not produce sperm ( Figure 2 K).

[0087] These results indicate that Ccdc175 is essential for male fertility but has no effect on female fertility.

[0088] Example 5 Knocking out Ccdc175 affects the formation of normal acrosomes.

[0089] Spermatogenesis is a process in which haploid round spermatids complete a series of special events to become motile streamlined sperm. Different stages of spermatogenesis are distinguished by changes in the morphological appearance of the developing acrosome and the shape of the nucleus. To characterize the potential function of Ccdc175 during spermatogenesis, PAS and HE staining experiments were further performed.

[0090] Ccdc175 + / + and Ccdc175 - / - PAS analysis of spermatids at different stages in male mice. In spermatids from step 1 to step 10, the acrosome morphology of sperm in Ccdc175 - / - male mice was generally normal. During the sperm head formation stage from step 11 to step 18, sperm in Ccdc175 + / + and Ccdc175 - / - male mice had abnormal rod-shaped sperm head morphology.

[0091] In summary, knocking out Ccdc175 does not affect normal spermatogenesis, but rather affects normal acrosome formation.

[0092] Example 6 Knocking out Ccdc175 affects normal fertilization.

[0093] PAS analysis showed that Ccdc175 + / + and Ccdc175 - / - male mice could detect all spermatid epithelial components and stages ( Figure 3 A). Ccdc175 + / + testes had normal spermatid formation in stages VII and VIII, and the cross-section of the tubules would have spermatid heads arranged along the lumen edge, ready for spermatid formation. However, in Ccdc175 - / - mouse testes, the failure of spermatidization led to many (if not all) elongated spermatids (step 16) being unable to align along the lumen edge at the beginning of stages VIII to XI.

[0094] In the ninth stage of Ccdc175 - / - mouse testes, the average number of retained elongated spermatids (step 16) was significantly higher than that of wild-type ( Figure 3 D). Ccdc175 - / - mice compared with wild-type mice showed a significant increase in the proportion at stage IX after spermatid release ( Figure 3 E). Compared with the control group, the number of elongated spermatids in steps 13 to 16 was significantly reduced in testes lacking Ccdc175 ( Figure 3 E).

[0095] Example 7 Knocking out Ccdc175 impairs the cytoskeletal structure of f-actin during spermatid formation

[0096] During spermatogenesis, various cytoskeletal structures, including microtubules and microfilaments, are involved in the formation of the sperm head. Therefore, the cytoskeleton of Ccdc175 + / + and Ccdc175 - / - male mouse testes was analyzed by IF of PNA and phalloidin that labeled f-actin ( Figure 3 B). Similar to the PAS staining results, IF analysis showed that engulfed spermatid heads (steps 15 - step 16) could be seen at all stages, which may be due to the inability of sperm to leave the testes, such as abnormal efferent duct function or significantly reduced testicular testosterone levels ( Figure 3 B, circle).

[0097] F-actin (filamentous actin) is a polymer and the basic unit of microfilaments. It is an assembly of G-actin (globular actin) subunits and can cause rapid changes in cytoskeletal dynamics through polymerization, depolymerization, severing, capping, bundling, and branching with the help of actin-related proteins. During sperm head formation and spermiogenesis, a highly ordered stack of fluorinated rings surrounds the apical region of the elongated sperm nucleus. Compared with the control group, the depolymerization of the F-actin structure surrounding the elongated sperm nucleus in Ccdc175-deficient testes was disturbed and disordered ( Figure 3 C, circle). These results indicate that Ccdc175 affects the cytoskeletal structure during spermatogenesis.

[0098] Example 8 CCDC175 is located in the tubulo-vesicular complexes (TBCs) at the ventral bend of elongated spermatids

[0099] TBCs initiate at cell-cell junctions in the form of clathrin-coated pits; however, the pits do not break. Instead, the actin-related neck continues to elongate into the Sertoli cell. As each structure matures, a bulb starts to form near the end of the complex through local depletion of the actin network and the formation of extensive membrane contact sites with the endoplasmic reticulum cisternae. The bulb expands proximally and eventually breaks near the bottom of the structure.

[0100] Due to fertilization failure, mutations in CCDC175 in Sertoli cells mainly affect the formation and assembly of normal TBCs, but how the CCDC175 protein functions during fertilization and TBCs germination is unclear. Sperm attached to the Sertoli cell region were isolated from Ccdc175 + / + and Ccdc175 - / - mice, and then IF analysis of CCDC175, ARP3, F-actin, and DAPI was performed. DAPI staining indicated the position of the sperm nucleus, antibodies were used to detect the localization of CCDC175, and the exact position of TBCs was indicated by treating the segments with fluorescent phalloidin and ARP3. This was especially evident when the images of the CCDC175, ARP3, actin, and DAPI channels were merged together in the last row ( Figure 4 ). The localization of CCDC175 in the region containing clusters of TBCs was very obvious.

[0101] As each structure matures, globules start to form near the end of the complex through local depletion of the actin network and the formation of extensive membrane contact sites with the endoplasmic reticulum cisternae, while the fluorescence signal of CCD175 is diffusely distributed ( Figure 4 A). In the first and second rows, the fluorescence signal of CCDC175 is a small particle, similar to the pattern of clathrin-coated pitsFigure 4 lines 1-2 in ). In the third line, the CCD175 fluorescence signal is like a small cloud because the actin-related neck continues to elongate ( Figure 4 line 3 in ). As each structure matures, globules begin to form near the end of the complex through local deletion of the actin network and formation of extensive membrane contact sites with the endoplasmic reticulum cisternae, while the fluorescence signal of CCD175 is diffusely distributed ( Figure 4 lines 4-5 in ). The CCDC175 label is diffusely localized around the cluster and forms densely stained linear bundles between individual TBCs shown by actin staining ( Figure 4 line 6 in ). Similar CCDC175 staining was not detected in CCDC175-deficient mice ( Figure 4 lines 7-8 in ). In Ccdc175 - / - mice, the TBC structure is absent and the F-actin structure is disordered. These results indicate that CCDC175 promotes the formation and development of the tubules of TBCs. The CCDC175 protein network can provide a scaffold to support the growth and maintenance of the actin cuff around the membrane core of each complex and can connect adjacent complexes to each other.

[0102] Example 9 In Ccdc175-deficient Sertoli cells, the tubule-vesicle complex structure is disrupted

[0103] The tubule-vesicle complex (TBCs) is a cytoskeleton-related structure in Sertoli cells, consisting of filaments of the tubular part surrounded by actin and the endoplasmic reticulum of the double plasma membrane on both sides of the globular part of the double plasma membrane. TBCs are located at the Sertoli-Sertoli cell and Sertoli cell-spermatid interfaces and play important roles in ectoplasmic specialization reorganization, removal of excess sperm cytoplasm, sperm acrosome formation, and spermatogenesis. The formation of TBCs may involve regulators of clathrin-coated pit assembly, local regulation of actin polymerization, and drivers of dendritic actin assembly. Many proteins have been localized to specific sites within TBCs. TBCs are formed by actin-binding proteins (such as the ARAP2 / 3 complex, VINCULIN, EPS8, EPB41, and N-WASP), adhesion molecule proteins (such as NECTIN2 / 3), and endocytic proteins (such as DYNAMIN2 and APPLI1).

[0104] To test whether the disruption of the TBCs structure in Sertoli cells is affected, Ccdc175 was probed by IF analysis with linker molecules of TBCs components, including F-actin, ARAP3, and DYNAMIN2 + / + and Ccdc175 - / - TBCs in spermatids isolated from the testes of male mice and fixed epithelial fragments ( Figure 5)。In Ccdc175 + / + and Ccdc175 - / - ARP3 protein was detected in fixed epithelial fragments and fixed frozen sections of testes from Ccdc175 Figure 5 mice. The normal distribution of TBCs was found to be random, while F-actin was disordered (

[0105] To further confirm these results, spermatids attached to the Sertoli cell region were isolated from Ccdc175-deficient or control seminiferous tubules, and then IF analysis of ARP3 was performed. In Ccdc175 + / + mouse testes, ARP3 protein was detected at the ventral bend of elongating spermatids and co-localized with f-actin in the sperm head ( Figure 5 B). Compared with the well-organized TBCs in the control group, no ARP3 was detected in the testes of Ccdc175-deficient mice, the distribution of TBCs was absent, and F-actin was irregularly distributed in the sperm head ( Figure 5 C). During steps 15 to 16 in Ccdc175 + / + the proportion of normal TBCs disrupted was approximately 63.07%, while in the testes of Ccdc175-deficient mice, TBCs were absent ( Figure 5 D).

[0106] These results indicate that disruption of Ccdx175 leads to defects in the formation and assembly of normal TBCs. Failure of normal formation and assembly of TBCs results in failure of spermiogenesis.

[0107] The inventors of the present application, based on genetically engineered KO mice against Ccdx175, revealed the important role of coiled-coil domain containing 175 (CCDC175) in acrosome biogenesis and male fertility. The inventors' research results show that CCDC175 is only expressed in the testes of adult mice and demonstrate that CCDC175 is essential for the biogenesis of the spermatozoal-tubulobulbar complex. Male KO mice are completely infertile and there are no sperm in the epididymis. However, Ccdc175 deletion has no effect on female fertility.

[0108] The applicant thus provides a method for diagnosing male fertility defects in mammals including humans, and provides new ideas and methods for developing new treatments for male infertility individuals not affected by gene mutations.

[0109] The foregoing is a description of the present invention and should not be construed as a limitation thereon. Unless otherwise indicated, the practice of the present invention will employ conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc. Obviously, the present invention can be implemented in other ways than those specifically described in the foregoing description and examples. 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 pertains. Many changes and variations are possible in accordance with the teachings of the present invention and are therefore within the scope of the present invention.

Claims

1. Use of a reagent for detecting CCDC175 in a male subject to be tested for the preparation of a kit or instrument for diagnosing or predicting fertility defects such as infertility disorders in mammals such as humans.

2. The use according to claim 1, wherein the infertility disorder of the male subject is selected from teratospermia, azoospermia, oligospermia, asthenospermia, and asthenozoospermia.

3. The use according to claim 1 or 2, wherein the reagent for detecting CCDC175 in the male subject to be tested is a reagent for detecting the Ccdc175 gene.

4. The use according to claim 1 or 2, wherein the reagent for detecting CCDC175 in the male subject to be tested is a reagent for detecting the CCDC175 protein, and preferably, a reagent for measuring the activity of CCDC175 is also included.

5. The use according to claim 4, which includes a reagent for detecting the expression of CCDC175 by immunoassay, such as a reagent for detecting the expression of CCDC175 by ELISA or Western blot using an antibody that specifically recognizes the protein; or which includes a reagent for detecting the CCDC175 by detecting the presence or amount of its mRNA, such as a reagent for detecting the amount of mRNA encoding CCDC175 in a sample by RT-PCR.

6. A kit for diagnosing fertility defects such as infertility disorders in mammals such as humans, which includes a reagent for detecting CCDC175 in a male subject to be tested.

7. The kit according to claim 6, wherein the infertility disorder of the male subject is selected from teratospermia, azoospermia, oligospermia, asthenospermia, and asthenozoospermia.

8. The kit according to claim 6, which includes a reagent for detecting the Ccdc175 gene in the male subject to be tested.

9. The kit according to claim 6, which includes a reagent for detecting the CCDC175 protein in the male subject to be tested, and preferably, a reagent for measuring the activity of CCDC175 is also included.

10. The kit according to claim 9, which includes a reagent for detecting CCDC175 by immunoassay, such as an antibody that specifically recognizes CCDC175; or which includes a reagent for detecting the presence or amount of CCDC175 mRNA to detect the expression of CCDC175, such as a reagent for detecting mRNA encoding CCDC175 in a sample by RT-PCR.

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