Kits for diagnosing or predicting fertility defects

Abstract

The present invention provides a method for diagnosing or predicting fertility defects such as infertility in mammals by detecting ZCWPW1, wherein the risk of disease is determined according to the sex and age of the tested individual. The present invention also provides a kit and an instrument for diagnosing or predicting fertility defects such as infertility in mammals by detecting ZCWPW1.

Description

Technical Field

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

[0002] Both spermatogenesis and oogenesis consist of a series of events, including mitosis and meiosis, and each step is essential for successful reproduction. Reproductive aging is a natural process of reduced fertility and reduced production of various steroid hormones by the gonads, which are more prominent in females than in males (Donnez & Dolmans, 2013). Among the many steps of spermatogenesis and oogenesis, meiosis is a special type of cell division that generates haploid germ cells from diploid progenitors and ensures their genetic diversity through homologous recombination (Handel and Schimenti, 2010). During meiosis, recombination is initiated by programmed DNA double-strand breaks (DSBs) generated by the evolutionarily conserved Spo11 protein at sites determined by higher-order chromosome structure and local chromatin changes (Keeney, Giroux, & Kleckner, 1997). Programmed DNA double-strand breaks during meiosis recruit a series of recombination proteins to form recombination sites and promote the association of homologous chromosomes during the zygote stage (Baudat, Imai, & de Massy, ​​2013).

[0003] ZCWPW1 (zinc finger CW-type and PWWP domain-containing 1) is a member of the CW domain-containing protein family. The zinc finger CW (zf-CW) domain is a zinc-binding domain of approximately 60 amino acids with three conserved tryptophans and four conserved cysteine ​​residues (Perry and Zhao, 2003). The PWWP domain is involved in epigenetic regulation and is present in various proteins involved in chromatin function, including histone modifying enzymes, DNA modifying enzymes, transcription factors, and DNA repair proteins (Stec, Nagl, van Ommen & den Dunnen, 2000). Structural analysis showed that the zf-CW domain is a histone modification reader of the tail of histone H3 with trimethylated K4 (H3K4me3) (He et al., 2010). In the structure of the complex of the human zf-CW domain and the H3K4me3 peptide, the H3K4me3 peptide forms a three-stranded antiparallel β-fold together with the two β-strands of the zf-CW domain (He et al., 2010). Chromatin pull-down assays confirmed that the zf-CW domain recognizes H3K4me3 in vitro (Hoppmann et al., 2011).

[0004] Although Zf-CW domain-containing proteins have been found to mediate epigenetic control in various species, the functions of ZCWPW1 in mammalian development and physiology, especially in meiosis and reproduction, are still unknown.

[0005] This field also needs to have a better understanding of the genetic regulation of meiosis in mammals, so as to detect and predict reproductive disorders such as infertility. Summary of the invention

[0006] The inventors first discovered that the loss of Zcwpw1 disrupts mammalian meiotic prophase I and leads to male infertility and premature ovarian insufficiency (POI) in females. The inventors found that ZCWPW1 is essential for the meiotic synapsis of male germ cells and is also an important factor in maintaining normal meiotic progression in females. Therefore, ZCWPW1 is essential for male and female fertility. Thus, the inventors provide a method for diagnosing fertility defects such as infertility in mammals, especially humans.

[0007] Specifically, the present invention provides a method for diagnosing or predicting fertility defects such as infertility in a mammalian individual, which comprises detecting ZCWPW1 in the subject.

[0008] In one aspect of the present invention, the method includes the step of knowing and / or selecting the sex of the subject. For example, the sex of the subject is known by inquiry or observation or detection (for example, detection of the subject sex-specific gene). When the subject is female (female), the method also includes the step of knowing and considering the age of the subject, for example, by inquiry or observation or detection (for example, detection of the subject age-related gene). The inventors found that ZCWPW1 is essential for the meiotic synapsis of male germ cells, and the absence of ZCWPW1 directly leads to fertility defects in male individuals. At the same time, the inventors found that ZCWPW1 is an important factor in maintaining the normal meiotic progression of female germ cells, and the absence of ZCWPW1 leads to a higher probability of fertility defects in older female individuals. Therefore, the method of the present invention includes knowing or considering the sex and age factors of the subject.

[0009] In the present invention, infertility disorders include but are not limited to azoospermia, oligospermia, asthenozoospermia, asthenozoospermia, premature ovarian failure, polycystic ovary syndrome, endometriosis, premature birth, intrauterine growth restriction and recurrent pregnancy loss.

[0010] 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.

[0011] ZCWPW1 (zinc finger CW-type and PWWP domain-containing 1) is a member of the protein family containing a CW domain. The Zcwpw1 gene is highly conserved in mammals. The human Zcwpw1 gene Gene ID is 55063; the mouse Zcwpw1 gene Gene ID is 381678.

[0012] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention comprises the step of detecting the Zcwpw1 gene.

[0013] The method for detecting genes used in the present invention is a method commonly used for detecting genes in the art and is not particularly limited. For example, mass spectrometry, microarray, sequencing, and detection methods using base sequence amplification methods such as PCR (polymerase chain reaction) can be cited.

[0014] 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.

[0015] 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.

[0016] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention includes the step of detecting ZCWPW1 protein, such as 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.

[0017] In another aspect of the present invention, the method comprises the step of detecting the expression of the ZCWPW1 protein by immunoassay. For example, the expression of ZCWPW1 is detected by immunofluorescence or Western blotting using an antibody that specifically recognizes the protein. For another example, the expression of ZCWPW1 is detected by detecting the presence or amount of mRNA of the protein, such as detecting the amount of mRNA encoding ZCWPW1 in a sample by RT-PCR.

[0018] In yet another aspect of the present invention, the method comprises the step of determining the activity of the ZCWPW1 protein.

[0019] The present invention also provides a kit or apparatus (including a gene chip) for diagnosing or predicting fertility defects such as infertility in a mammalian individual, which comprises a reagent for detecting ZCWPW1 in the subject.

[0020] In another aspect of the present invention, the kit or instrument comprises a device or reagent for obtaining and / or selecting the sex of the subject, such as asking or indicating corresponding markers selected according to different sexes, such as reagents for detecting sex-specific genes. In one aspect of the present invention, the instrument includes asking, inputting and recording whether the subject is male or female. For another example, the kit or instrument comprises reagents for detecting sex chromosomes.

[0021] In another aspect of the present invention, when the subject is female, the kit further comprises a device or reagent for obtaining the age of the subject, such as asking or indicating corresponding markers selected according to different ages, or a reagent for detecting age-related genes. For example, the apparatus of the present invention may include a device for asking, inputting and recording the age of the subject.

[0022] In another aspect of the present invention, the kit or apparatus comprises a reagent for detecting the expression or activity of the ZCWPW1 protein. For example, the kit or apparatus comprises a reagent for immunoassay, such as a reagent for detecting the expression of ZCWPW1 by performing ELISA or Western blotting with an antibody that specifically recognizes the protein. For another example, the kit or apparatus comprises a reagent for detecting the expression of ZCWPW1 by detecting the presence or amount of mRNA of the protein, such as a reagent for detecting the amount of mRNA encoding ZCWPW1 in a sample by RT-PCR.

[0023] In yet another aspect of the present invention, the kit or apparatus comprises a reagent for detecting the Zcwpw1 gene.

[0024] In one aspect of the present invention, in the above-mentioned method, kit or apparatus of the present invention, the reagent for detecting genetic markers in a sample includes a primer or probe for detecting Zcwpw1, or a reagent for examining ZCWPW1, such as a specific antibody or a reagent for detecting the mRNA of the protein.

[0025] The present invention also provides the use of a reagent for detecting ZCWPW1 in preparing a kit or instrument for diagnosing or predicting fertility defects such as infertility in a mammal as described above. The reagent for detecting genetic markers includes a primer or probe for detecting the Zcwpw1 gene, or a reagent for detecting the ZCWPW1 protein, such as a specific antibody or a reagent for detecting the mRNA of the protein.

[0026] In this article, protein symbols are not italicized and are all capitalized; gene symbols are italicized. For example, ZCWPW1 is a protein, and the gene encoding the protein is written as Zcwpw1. However, sometimes in this article, gene symbols are not italicized. For example, sometimes "ZCWPW1" or "ZCWPW1 gene" in this article refers to the gene Zcwpw1 that encodes the ZCWPW1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Showing the expression and dynamic localization of ZCWPW1 in meiotic germ cells. Figure 1 A. Western blotting, showing that ZCWPW1 expression increases from leptotene to zygotene during meiotic prophase I, but disappears in elongating spermatids. β-actin was used as a control. Figure 1 B. Western blotting, cytoplasmic and nuclear fractionation analysis was performed, and ZCWPW1 was detected only in the nuclear fraction of mouse testis extracts. LaminB1 was used as a marker protein for the cytoplasm. Figure 1 CK, from postnatal day 35 (PD35) Zcwpw1 + / + and Zcwpw1 - / - Chromosome spreads of spermatocytes from the testis, immunostained for ZCWPW1 (green) and SYCP3 (red). ZCWPW1 is diffuse (arrows) from leptotene to zygotene (CF, arrows). In pachytene cells, ZCWPW1 is localized in the XY body (GH, white dashed circles). (IK) - / - In spermatocytes, no ZCWPW1 signal was detected.

[0028] Figure 2 Show the effect of Zcwpw1 on animal fertility. Figure 2A is a schematic diagram of constructing Zcwpw1 knockout mice using CRISPR / Cas9 genome editing technology. The gRNA (arrow), the corresponding coding exons (black and red thick lines) and non-coding exons (gray thick lines) are shown. The red thick line (coding exon) shows the deleted fragment (~3kb) from the wild-type allele. ATG is the start codon. Figure 2 B. Western blot analysis confirmed that compared with the wild type, Zcwpw1 - / - The absence of ZCWPW1 protein in the testes of mice indicated that the Zcwpw1 knockout mice were successful. β-actin was used as a control. Figure 2 C and 2D. Co-immunoprecipitation analysis of ZCWPW1 and H3K4me3 from PD18 testis protein extracts. ZCWPW1 was immunoprecipitated with H3K4me3. Figure 2 E from PD35 Zcwpw1 - / - and Zcwpw1 - / - Testes of male mice. - / - The testis size of male mice was reduced at PD35 (n=3, Welch's t-test analysis: p<0.0001). Figure 2 F. Adult Zcwpw1 + / + Hematoxylin and eosin (HE) staining of testis. Figure 2 G Adult Zcwpw1 - / - HE staining of testicular sections shows complete arrest of spermatogenesis. Arrow, apoptotic spermatocytes; Arrowhead, empty seminiferous tubules; Asterisk, seminiferous tubules lacking post-meiotic spermatocytes. Figure 2 H is the cumulative number of pups per female mouse during the specified time period. n = 3 mice per genotype. Figure 2 I-2N from Zcwpw1 + / + and Zcwpw1 - / - Histological analysis of ovaries of females. Figure 2 I-2J ovarian morphology showed that at 3 months of age, Zcwpw1 - / - Ovaries (J) showed similarity to Zcwpw1 + / + Ovaries (I) have similar morphology. Figure 2 K-2L at 6 months old, Zcwpw1 - / - The ovary (L) contains fewer follicles and a corpus luteum (CL). Figure 2 At approximately 8 months of age, the M-2N has prematurely aged ovaries (N) without oocytes or follicles.

[0029] Figure 3 Display Zcwpw1 - / - Chromosome synapsis is disrupted in spermatocytes. Figure 3AFs from PD35 wild type (AC) and Zcwpw1 - / - (DF) Immunostaining of spermatocytes from male testes for SYCP1 (green) and SYCP3 (red). Spermatocytes are divided into the following stages: leptotene, zygotene, pachytene, and quasi-pachytene (defined as 5 or more synaptic chromosome pairs). Arrows indicate synaptic chromosomes, and arrowheads indicate single chromosomes. Figure 3 G Wild-type spermatocytes and Zcwpw1 - / - The number of synapsed chromosome pairs in spermatocytes. - / - The average number of chromosome pairs in spermatocytes is 8. Figure 3 HkDJ + / + and Zcwpw1 - / - Frequency of meiotic stages in spermatocytes. - / - In mice, 47.2% and 34.3% of spermatocytes arrested at the zygotene or quasi-pachytene stage, respectively, while 61.5% of Zcwpw1 + / + Spermatocytes are in the pachytene stage. The numbers marked in the bar graphs represent the percentage of cells in the indicated meiotic stage. Three mice were analyzed for each genotype. P values ​​were calculated by Student's t-test.

[0030] Figure 4 Display Zcwpw1 - / - Spermatocytes display defects in meiotic recombination. Figure 4 AF from Zcwpw1 + / + (AC) and Zcwpw1 - / - (DF) Chromosome spreads of male spermatocytes were immunostained for SYCP3 (red) and γH2AX (green). Representative images of oocytes at the leptotene, zygotene, and pachytene stages are shown. Figure 4 G-HZcwpw1 + / + and Zcwpw1 - / - Cells immunostained for SYCP3 (red) and MLH1 (green). - / - Spermatocytes lack MLH1 signaling (H).

[0031] Figure 5 Showing SC structure in Zcwpw1 - / - Normal expression in spermatocytes. Figure 5 (AD) SIM (super-resolution microscopy) images of chromosomes in spermatids from PD25 testes: immunostained for SYCP3 (red) and C-SYCP1 (green). Arrows indicate synaptic regions. Figure 5A'-D' Magnified views of the synaptic region showing colocalization of C-SYCP1 (arrow, green signal) and SYCP3 (arrowhead, red signal). Figure 5 SIM images of chromosomes in spermatids from EH PD25 testis: rows immunostained for SYCP3 (red) and N-SYCP1 (green). Arrows indicate synaptic regions and arrowheads indicate individual chromosomes. Figure 5 E'-H' Magnified views of the synaptic region showing that N-SYCP1 is localized in a continuous pattern (arrows) in the central region of the SC. Figure 5 IL SIM images of chromosomes in spermatids from PD25 testis: immunostained for SYCP3 (red) and TEX12 (green). Arrows indicate synaptic regions and arrowheads indicate individual chromosomes. Figure 5 I'-L' magnified view of the synaptic region showing that TEX12 is localized in the middle region between the LEs (arrow).

[0032] Figure 6 Display Zcwpw1 - / - Meiotic prophase I of oocytes occurred successfully but was delayed. Figure 6 A Zcwpw1 at embryonic day 17.5 (E17.5) + / + and Zcwpw1 - / - The frequency of chromosome meiotic stages in oocytes. - / - In female mouse oocytes, 75% and 20% of oocytes were in the zygotene and pachytene stages, respectively, while 70% of Zcwpw1 + / + Oocytes were in the pachytene stage. The numbers labeled in the bar graph represent the percentage of cells in the specified meiotic stage. Three mice were analyzed for each genotype. P values ​​were calculated by Student's t-test. Figure 6 B PD1 of Zcwpw1 + / + and Zcwpw1 - / - Frequency of meiotic stages in oocytes. The numbers labeled in the bar graph represent the percentage of cells at the specified meiotic stage. Three mice were analyzed for each genotype. P values ​​were calculated by Student's t-test. Figure 6 CH from wild type (CE) and Zcwpw1 - / - (FH) Chromosome spreads of female oocytes at E17.5 ovaries: SYCP3 (red) and SYCP1 (green), showing representative images of oocytes at leptotene, zygotene, and pachytene stages. Arrows indicate synaptic chromosomes. Figure 6 Chromosome spreads from IL E17.5 ovaries visualized using SIM at the indicated stages: SYCP3 (red) and N-SYCP1 (green). Arrows indicate synaptic regions. Figure 6I'-L' magnified view of the synaptic region showing that N-SYCP1 is localized in a continuous pattern (arrows) in the central region of the SC. Figure 6 MP Chromosome spreads of E17.5 ovaries visualized using SIM at the indicated stages: SYCP3 (red) and TEX12 (green). Arrows indicate synaptic regions. Figure 6 M'-P' Magnified view of the synaptic region showing that TEX12 is localized in a continuous pattern (arrows).

[0033] Figure 7 Display Zcwpw1 - / - The female follicle pool decreases and leads to premature ovarian insufficiency (POI). Figure 7 A and B Representative Zcwpw1 from E13.5 female mice + / + (A) and Zcwpw1 - / - (B) Ovarian sections immunostained for MVH with hematoxylin counterstain. Figure 7 C Oocyte counts showing Zcwpw1 at E13.5 + / + and Zcwpw1 - / - Similar numbers of oocytes were present in females. MVH-positive cells were counted. Figure 7 D and E Representative Zcwpw1 from PD1 female mice + / + (D) and Zcwpw1 - / - (E) Ovarian sections immunostained for MVH with hematoxylin counterstain. Figure 7 F oocyte count, showing Zcwpw1 - / - Ovaries contain fewer oocytes than Zcwpw1 + / + Ovaries. MVH-positive cells were counted. Figure 7 G and H Representative Zcwpw1 from PD8 female mice + / + (G) and Zcwpw1 - / - (H) Ovarian sections counterstained with hematoxylin. MVH-positive cells were counted. Figure 7 I oocyte count showed Zcwpw1 - / - The ovaries contain fewer follicles than Zcwpw1 + / + Ovaries. MVH-positive oocyte nuclei with surrounding granulosa cell layer characteristics were counted. In all cases, each section (8 μm per section) was counted and the total number of oocytes per ovary was calculated. For each genotype, six ovaries from three mice were analyzed. P values ​​were calculated by Student's t-test. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1 Experimental methods and reagents

[0036] Preparation of mouse ZCWPW1 antibody

[0037] Antibodies to mouse ZCWPW1 were produced in the laboratory. In brief, a cDNA fragment encoding amino acids 488-622 of mouse Zcwpw1 was inserted into a p-ET-32a+ vector (EMD Millipore) and transfected into BL21-CodonPlus (DE3) Escherichia coli cells. The cells were cultured overnight at 37°C and induced for 4 hours by adding 0.2 mM IPTG (Sigma-Aldrich) at 28°C. The cells were harvested by centrifugation and broken by ultrasonic treatment, and purified by Ni-NTA agarose (QIAGEN) according to the manufacturer's instructions. The protein was dialyzed in PBS and used to immunize rabbits, and the antiserum was affinity purified on antigen-coupled CNBr-activated agarose (GE Healthcare).

[0038] Purification of male germ cells

[0039] Purification of male germ cells was performed according to the method described by Gan et al., 2013. The cell type and purity in each fraction were assessed based on their diameter and morphological features under a light microscope.

[0040] Tissue collection and histological analysis

[0041] Testes from at least three mice of each genotype were dissected immediately after mouse euthanasia, fixed in 4% paraformaldehyde (Solarbio, Beijing, P1110) for 24 h, stored in 70% ethanol, and embedded in paraffin. 5 μm sections were prepared and mounted on slides. After dewaxing, the slides were stained with hematoxylin and eosin for histological analysis.

[0042] Immunocytology and Antibodies

[0043] Spermatocyte smears were prepared as described in Peters, Plug, van Vugt, & de Boer, 1997. Oocytes from embryonic ovaries (E17.5 embryos) and PD1 ovaries were digested with collagenase, incubated in hypotonic buffer, fixed in paraformaldehyde, and incubated with the indicated antibodies for immunofluorescence. The primary antibodies used for immunofluorescence were as follows: rabbit anti-SYCP3 (1:500 dilution; Abcam#ab15093), mouse anti-SYCP3 (1:500 dilution; Abcam#ab97672), rabbit anti-SYCP1 (C-terminus) (1:2,000 dilution; Abcam#ab15090), rabbit anti-SYCP1 (N-terminus) (1:2,000 dilution; Abclonal#A12139), rabbit anti-RPA2 (1:200 dilution; Abcam#ab76420), rabbit anti-RAD51 (1:200 dilution; Thermo Fisher Scientific#PA5-27195), rabbit anti-DMC1 (1:100 dilution; Santa Cruz Biotechnology Co., Ltd.). Biotechnology #sc-22768), mouse anti-γH2AX (1:300 dilution; Millipore #05-636), and mouse anti-MLH1 (1:50 dilution; BD Biosciences #550838). Primary antibodies were detected by incubation at room temperature for 1 hour with Alexa Fluor 488 or 594-conjugated secondary antibodies (1:500 dilution; Abcam #ab150084, #ab150077, #ab150113, #ab150120). Slides were washed several times with PBS and mounted using VECTASHIELD medium with DAPI (Vector Laboratories, #H-1200).

[0044] Real-time RT-PCR

[0045] Total RNA was isolated from various tissues of wild-type or knockout adult mice. To analyze the expression level of Zcwpw1 mRNA in various tissues, equal amounts of cDNA were synthesized using the PrimeScript RT Kit with gDNA Eraser (Takara). qPCR was performed using TB Green Premix Ex Taq (Takara) and specific forward and reverse primers: Zcwpw1 primer pair F 5′-GGG GAC ATC TAA GCT AGG CC-3′ and R 5′-CTG CAC TCA CGG CCA TCTT-3′. β-Actin as a housekeeping gene was amplified with primers β-Actin F 5′-CAT CCG TAA AGA CCT CTA TGC CAA C-3′ and β-Actin R 5′-ATG GAG CCA CCG ATC CAC A-3′. All RT-PCR reactions were performed with an initial denaturation at 95°C for 10 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min using a T100 thermal cycler (Bio-Rad).

[0046] Western Blot

[0047] To prepare the extracts, tissues were collected from male C57BL / 6 mice and suspended in RIPA buffer supplemented with Complete protease inhibitor (Roche #04693116001). After homogenization, the cell extracts were centrifuged at 20,000×g for 20 minutes at 4°C. The supernatant extracts were used for immunoprecipitation and western blotting. Equal amounts of protein were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels and the bands were transferred to polyvinylidene difluoride membranes (Millipore, USA). Immunoreactive bands were detected and analyzed using the BIO-RAD ChemiDoc MP imaging system and Image Lab software (BIO-RAD, USA). The relative protein levels in each sample were normalized to β-actin to normalize for loading variations. Primary antibodies used for immunoblotting included anti-β-actin (1:10,000 dilution; Cell Signaling Technology, #4970) and anti-ZCWPW1 (1:1,000 dilution; as described above in “Preparation of mouse ZCWPW1 antibody”).

[0048] Confocal microscopy

[0049] Immunolabeled chromosomes were imaged by confocal microscopy using a Leica TCS SP5 resonant scanning confocal microscope driven by Leica Application Suite software or an Andor Dragonfly spinning disk confocal microscope driven by Fusion software. Projected images were then prepared using ImageJ software (NIH, version 1.6.0-65) or BitplaneImaris (version 8.1) software. Histological samples were analyzed with an epifluorescence microscope (BX52, Olympus) and processed using Photoshop (Adobe). SIM analysis was performed using DeltaVision OMX SR SIM system (GE Healthcare) and Acquire SR software, and images were further calculated, reconstructed and processed using softWoRx software (GE Healthcare) to produce ultra-high resolution optical series sections.

[0050] Statistical analysis

[0051] All data are presented as mean ± SEM. The statistical significance of the differences between the means of different genotypes was measured by paired two-tailed Welch's t-test. Data were considered significant when the p value was less than 0.05 (*) or 0.01 (**).

[0052] Example 2 Expression and dynamic localization of ZCWPW1 in meiotic germ cells

[0053] RT-PCR and Western blotting revealed that ZCWPW1 is expressed in various organs of mice and is highly expressed in adult testes and embryonic ovaries during meiotic prophase I. To further investigate the expression pattern of ZCWPW1 in germ cells, different types of male germ cells were separated by BSA gradient sedimentation (Gan et al., 2013). Western blotting revealed that during meiotic prophase I, ZCWPW1 expression increased from the leptotene to the zygotene stage, but became almost undetectable in elongated spermatids ( Figure 1 A). To determine the subcellular localization of ZCWPW1 in germ cells, cytoplasmic and nuclear fractionation analysis was performed and it was found that ZCWPW1 was only detected in the nuclear fraction of mouse testis extracts, indicating that ZCWPW1 functions in the nucleus ( Figure 1 B).

[0054] By immunostaining of unfolded spermatocyte nuclei, it was found that Figure 1 , C and D, green signal, arrow) to the zygotene stage ( Figure 1 , E and F, green signal, arrows) all showed scattered ZCWPW1 immunofluorescence signals. The signal disappeared from the autosomes ( Figure 1 H, red signal), which only appears during the pachytene stage of the XY sex body ( Figure 1 , G and H, green signal, white circle). These phenomena suggest that ZCWPW1 may only function in the leptotene to zygotene stages on autosomes.

[0055] Example 3 Preparation of Zcwpw1-deficient mouse model

[0056] To investigate the in vivo function of ZCWPW1, a Zcwpw1-deficient mouse model lacking exon 3 to exon 7 was generated using the CRISPR / Cas9 system (Cyagen Biosciences). Figure 2 A is a schematic diagram of the CRISPR / Cas9 genome editing system at the Zcwpw1 locus, showing gRNA (arrow), corresponding coding exons (black and red thick lines) and non-coding exons (gray thick lines). The red thick line (coding exon) shows the deleted fragment (~3kb) from the wild-type allele.

[0057] The PCR primers used for the mutant allele of Zcwpw1 were: forward primer 5'-AGC TGC TGG GAT TAAATG TCT GTT CC-3' and negative primer 5'-CTA CAC TGT GCC TTC TAC CTT CTT TGA GA-3', using genomic DNA extracted from mouse tail samples as template, generating a 690 bp fragment. The PCR primers used for the wild-type allele of Zcwpw1 were: forward primer 5'-CAA GAT GGA GGA GAT ATG CAG TAC ATG-3' and negative primer 5'-CTA CAC TGT GCC TTC TAC CTT CTT TGA GA-3', generating a 617 bp fragment.

[0058] Western blot analysis confirmed that compared with wild type, - / - ZCWPW1 protein is absent in mouse testes ( Figure 2 B). β-actin was used as a loading control. + / - The offspring of the cross between male and female mice showed normal Mendelian genotype distribution, Zcwpw1 - / - The mice survived and appeared to develop normally.

[0059] Example 4 Zcwpw1 is essential for maintaining male fertility

[0060] Immunoprecipitation of testicular protein extracts using ZCWPW1 antibody revealed that H3K4me3 was present in immunoprecipitated proteins from wild-type testis, but not in Zcwpw1 - / - Not present in testicles ( Figure 2 C). Similarly, immunoprecipitation using an H3K4me3 antibody pulled down ZCWPW1 protein from wild-type testis protein extracts, but not from Zcwpw1 - / - Pull down ZCWPW1 protein in testis ( Figure 2 D). Demonstrated that loss of Zcwpw1 disrupts the binding of ZCWPW1 to H3K4me3.

[0061] Although Zcwpw1 - / - The mice developed and appeared normal, but Zcwpw1 - / - Male mice were found to be completely infertile. As shown in the figure, at postnatal day (PD) 35, Zcwpw1 - / - The testes of male mice were much smaller than those of control males (n=3, Welch's t-test analysis: p<0.0001) ( Figure 2 E). Histological analysis showed that compared with wild-type males ( Figure 2 F, Hematoxylin and eosin staining) comparison, Zcwpw1 - / - Spermatogenesis is disturbed in male mice: Zcwpw1 - / - In male mice, the seminiferous tubules lack post-meiotic spermatocytes ( Figure 2 G, asterisks) and contain apoptotic cells ( Figure 2 G, arrow) or almost empty ( Figure 2 G, arrow). Zcwpw1 - / - Male mice exhibited premature germ cell death, demonstrating that Zcwpw1 is essential for maintaining male germ cell development and male fertility in mice.

[0062] Example 5 Effect of Zcwpw1 on female fertility is related to age

[0063] Compared with males, Zcwpw1 - / - Fertility of female mice until mid-adulthood (5-6 months of age) is associated with Zcwpw1 + / + Female mice were normal compared to controls, and the number of pups was comparable between the two (n=3 for each genotype) ( Figure 2 H) Ovarian morphological analysis showed that at 3 months, Zcwpw1 - / - Female mouse ovary ( Figure 2, J) showed similarity to Zcwpw1 + / + Female mouse ovaries have similar healthy ovarian morphology ( Figure 2 , I). However, at 6 months of age, in Zcwpw1 - / - Very few follicles and corpora lutea were seen in the ovaries of female mice ( Figure 2 L), indicating the absence of normal ovarian function, while Zcwpw1 + / + Female mouse ovaries contain healthy follicles ( Figure 2 K, arrow) and corpus luteum (CL) ( Figure 2 K). At 6 months Zcwpw1 - / - Ovarian morphology of female mice confirmed the fertility observations. As expected, Zcwpw1 - / - Female mouse ovaries do not have any follicles by late adulthood, around 8 months of age. Figure 2 N), and thus become sterile.

[0064] Example 6Zcwpw1 - / - Chromosome synapsis is disrupted in spermatocytes

[0065] In order to determine Zcwpw1 - / - The cause of meiotic interruption in male mouse spermatocytes was first investigated by immunostaining for synaptonemal complex (SC) proteins to visualize chromosomes during meiotic prophase. The SC consists of two lateral elements (LE) and a central element (CE) (Fraune, Schramm, Alsheimer, & Benavente, 2012; Schucker, Holm, Franke, Sauer, & Benavente, 2015).

[0066] In Zcwpw1 + / + In male mouse spermatocytes, LE marked by SYCP3 begins to form at the leptotene stage ( Figure 3 A, red signal), but the lack of SYCP1 signaling (green signal) indicates that CE is not yet present ( Figure 3 A). During the zygotene stage, the LE is fully formed and the chromosomes gradually mutate, as indicated by the appearance of SYCP1 signals ( Figure 3 B, arrows). Wild-type spermatocytes achieve complete synapsis of autosomes at the pachytene stage, as indicated by continuous SYCP1 signals ( Figure 3 C, arrow).

[0067] In Zcwpw1 - / - In male mouse spermatocytes, LE ( Figure 3 D, red signal), and SYCP1 begins to appear on paired chromosomes during the zygotene stage, indicating that synapsis has begun to some extent ( Figure 3E, arrow). However, in Zcwpw1 - / - In spermatocytes, synaptic chromosomes ( Figure 3 F, arrows) always have a large number of unsynaptic autosomes ( Figure 3 F, arrow, red signal), and these spermatocytes remain in what the authors call a “pachytene-like” state (presence of 5 or more synaptic chromosome pairs). + / + and Zcwpw1 - / - Quantification of synaptic chromosome pairs in each nucleus in male mouse testis ( Figure 3 G):In Zcwpw1 + / + In the testis, 153 cells were observed to have all chromosome pairs fully synapsed, and only 16 cells had 4-18 pairs of synapsed chromosomes; however, in Zcwpw1 - / - In the testis, there are an average of only eight synaptic chromosome pairs per cell, and in Zcwpw1 - / - None of the 164 spermatocytes observed in the testis had complete synapsis.

[0068] We further analyzed the percentage of sperm cells at different meiotic stages using the testes of postnatal day (PD) 28 mice and found that Zcwpw1 - / - Spermatocytes fail to develop to the pachytene stage. Figure 3 H), of which 34.3% stopped at the quasi-pachytene stage ( Figure 3 H, quasi-pachytene). This is consistent with the 61.5% of Zcwpw1 that have reached the pachytene stage. + / + Spermatocytes are in sharp contrast ( Figure 3 H, pachytene).

[0069] To better understand Zcwpw1 - / - Synapsis failure in spermatocytes Super-resolution structured illumination microscopy (SIM) was used to visualize the fine SC structure that was indistinguishable in confocal microscopy images. Immunostaining was performed for SYCP3 and the CE-specific proteins SYCP1 (both C-terminally and N-terminally stained) and TEX12. In SIM imaging, the LE is visualized as two separate chains by SYCP3 immunostaining ( Figure 5 A', arrow, red signal). SYCP1 forms homodimers, and the two C-termini of the SCYP1 dimer are located in the LE (Cahoon et al., 2017), which was observed in SIM imaging ( Figure 5 , A' and C', green signal, arrow). The N-terminus of the SYCP1 dimer is located at the CE ( Figure 5 , E' and G', green signals, arrows) (Winkel, Alsheimer, Ollinger, & Benavente, 2009), TEX12 is also located in CE ( Figure 5 I' and K', green signals, arrows). - / - In zygotene spermatocytes, synapsis begins in some chromosomes. C-SYCP1 ( Figure 5 B', green signal, arrow), N-SYCP1 was found on fully synaptic chromosomes ( Figure 5 F', green signal, arrow) and TEX12 ( Figure 5 J', green signal, arrow).

[0070] However, surprisingly, only eight pairs of synaptic chromosomes were present in the pachytene-like Zcwpw1 - / - There was no significant difference in the localization pattern of C-SYCP1 in the cells ( Figure 5 D', green signal, arrow), N-SYCP1 ( Figure 5 H', green signal, arrow) or TEX12 ( Figure 5 L', green signal, arrow). At the same time, Zcwpw1 - / - Unsynaptic autosomes in cells also lack SYCP1 signal ( Figure 5 , D and H, arrows, red signals) and TEX12 signals ( Figure 5 L, arrow, red signal). Based on these results, the inventors concluded that Zcwpw1 - / - Some autosomes in spermatocytes can initiate synapsis but fail to produce complete synapsis.

[0071] Example 7Zcwpw1 - / - Spermatocytes display defects in meiotic recombination

[0072] The localization of phosphorylated γ-H2AX was analyzed to assess the repair of programmed DNA double-strand breaks (DSBs) in meiosis. + / + The signal of γ-H2AX in spermatocytes ( Figure 4 , A and B, arrows), in the leptotene stage ( Figure 4 D, arrow) and zygotic ( Figure 4 E, arrow) Zcwpw1 - / - γ-H2AX in spermatocytes marks chromatin domains around DSB sites. + / + In cells, γ-H2AX signals were observed only in the unsynaptic regions of sex chromosomes ( Figure 4 C, white dashed circle). However, in Zcwpw1- / - In spermatocytes, phosphorylated γ-H2AX signals persist on the chromatin of unsynaptic axes ( Figure 4 F, arrow), indicating failure of DSB repair.

[0073] In the middle and late pachytene stages, the MLH1 (MutL homolog 1) locus is located in Zcwpw1 + / + become evident in spermatocytes, indicating the presence of chromosomal exchange sites ( Figure 4 G, arrow) (Marcon & Moens, 2003). However, Zcwpw1 - / - Spermatocytes failed to enter the pachytene stage and did not undergo intercalation, as reflected by the loss of the MLH1 locus ( Figure 4 H).

[0074] Example 8Zcwpw1 - / - Meiotic prophase I of oocytes occurs successfully but is delayed

[0075] With Zcwpw1 - / - Compared with spermatocytes, Zcwpw1 at embryonic day (E) 17.5 - / - Oogonia were able to reach the pachytene stage and develop into oocytes with normal fertility. However, they showed a significant delay in meiotic progression. By calculating the percentage of oocytes at different meiotic stages, we found that 70% of Zcwpw1 + / + Oogonia have formed SC and have reached the pachytene stage ( Figure 6 A), while only 20% of Zcwpw1 - / - Oogonia have completed synapsis, and approximately 75% of Zcwpw1 - / - The oocyte is still in the zygotene stage ( Figure 6 A).

[0076] At 1 PD day, 45% of Zcwpw1 + / + The mother cell has entered the late diplotene stage ( Figure 6 B, late diplotene), and about 15% of Zcwpw1 + / + Oocytes are in the nuclear reticular stage ( Figure 6 B, nuclear network period). However, only 20% of Zcwpw1 - / - Oocytes are in the late diplotene stage, and only 10% are in the nuclear reticulum stage ( Figure 6 B) From 38% of Zcwpw1 - / - Oocytes were in the late pachytene stage and 32% of Zcwpw1 - / - It is easy to see that the oocyte is in the early diplotene stage. - / - Meiosis in oocytes is slow ( Figure 6B). Representative images of oocytes at the leptotene, zygotene, and pachytene stages are shown ( Figure 6 , C to H, arrows, synaptic chromosomes).

[0077] To determine the expression of Zcwpw1 at embryonic day (E) 17.5 - / - To determine whether the SC structure formed in oogonia is complete and stable, antibodies against N-SYCP1 and TEX12 were used to immunostain oogonia chromosomes. - / - N-SYCP1 and TEX12 are localized in the CE of paired chromosomes at the zygotene and pachytene stages in oogonia (N-SYCP1, Figure 6 J, J' and L, L', arrows; TEX12, Figure 6 N, N' and P, P', arrows). This is consistent with Zcwpw1 at E17.5 + / + Oocytes are very similar (N-SYCP1, Figure 6 I, I' and K, K', arrows; TEX12, Figure 6 M, M' and O, O', arrows). These results suggest that in female Zcwpw1 - / - Normal synapsis occurs in oogonia.

[0078] Example 9Zcwpw1 - / - Decreased female follicle pool and premature ovarian insufficiency (POI)

[0079] The reproductive aging process in mammals includes a gradual decrease in ovarian follicle reserve and a decline in oocyte quality (Faddy, 2000). - / - Meiotic progression in females is delayed, reducing the ovarian reserve and thus causing the POI phenotype.

[0080] Quantification of the number of oocytes at different ovarian developmental stages. Ovaries dissected from E13.5, PD1, and PD8 mice were fixed, sectioned, and immunostained with mouse vasa homologue (Mvh) antibodies. Quantification of oocytes at these stages is shown in Figure 2. + / + ( Figure 7 A) and Zcwpw1 - / - ( Figure 7 B) The same number of oogonia are present in the ovary, with meiosis about to begin ( Figure 7 C) In PD1, with Zcwpw1 + / + Compared with ovary Figure 7 D), Zcwpw1 - / - Ovaries ( Figure 7E) contains approximately 45% fewer oocytes, indicating that Zcwpw1 - / - Oocyte number relative to Zcwpw1 + / + ( Figure 7 F) decreased. At PD8, Zcwpw1 + / + The ovaries contain well-formed primordial follicles ( Figure 7 G, arrows) and some activated primary / secondary follicles ( Figure 7 G, arrow). However, PD8 Zcwpw1 - / - The ovaries contain fewer active follicles ( Figure 7 H, arrows) and fewer primordial follicles ( Figure 7 F, arrows). Quantification of oocyte numbers showed that Zcwpw1 - / - The ovary only has + / + About 20% of the oocytes in the ovary ( Figure 7 I). Therefore, in Zcwpw1 - / - The delay in meiotic prophase found in females triggers the death of a portion of germ cells, thereby reducing the number of follicles in the ovary. - / - Females become infertile in mid-adulthood due to premature ovarian failure.

[0081] The present invention first discovered and proved that the loss of Zcwpw1 disrupts mammalian meiotic prophase I and leads to male infertility and female premature ovarian failure. The inventors found that ZCWPW1 is essential for the meiotic synapsis of male germ cells and is also an important factor in maintaining normal meiotic progression in females. Therefore, ZCWPW1 is essential for male and female fertility. The applicant thus provides a method and a kit for diagnosing fertility defects such as infertility disorders in mammals, including humans.

[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.

[0083] The following references are disclosed in this application by being incorporated in their entirety.

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Claims

1. Use of a reagent for detecting Zcwpw1 of a test subject for preparing a kit or instrument for diagnosing or predicting infertility in mammals, wherein: The kit or instrument also comprises a device or reagent for obtaining and / or selecting the sex of the test individual. When the kit or instrument is in use, if the test individual is male and ZCWPW1 is found to be deficient, the individual is judged to suffer from infertility disorder; if the test individual is female and ZCWPW1 is found to be deficient, the individual is judged to suffer from premature ovarian failure.

2. The use according to claim 1, wherein when the subject is a female, the kit further comprises a device or reagent for obtaining the age of the subject.

3. The use according to claim 1 or 2, wherein the reagent for detecting Zcwpw1 in the subject is a reagent for detecting the Zcwpw1 gene. The use according to claim 1 or 2, wherein the reagent for detecting Zcwpw1 in the subject is a reagent for detecting ZCWPW1 protein. The use according to claim 4, wherein the reagent for detecting Zcwpw1 in the subject is a reagent for detecting the expression of ZCWPW1 by immunoassay. The use according to claim 5, wherein the reagent for detecting Zcwpw1 in the subject is a reagent for detecting the expression of ZCWPW1 by ELISA or Western blotting using an antibody that specifically recognizes the protein. 7 . The use according to claim 4 , wherein the reagent for detecting Zcwpw1 in the subject is a reagent for detecting ZCWPW1 by detecting the presence or amount of ZCWPW1 mRNA.

8. The use according to claim 7, wherein the reagent for detecting Zcwpw1 in the subject is a reagent for detecting the amount of mRNA encoding ZCWPW1 in a sample by RT-PCR. 9 . The use according to claim 4 , wherein the reagent for detecting Zcwpw1 in a subject is a reagent for measuring the activity of ZCWPW1.

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