CN111896740A - Method and kit for diagnosing or predicting fertility defects - Google Patents
Method and kit for diagnosing or predicting fertility defects Download PDFInfo
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- CN111896740A CN111896740A CN201910373598.1A CN201910373598A CN111896740A CN 111896740 A CN111896740 A CN 111896740A CN 201910373598 A CN201910373598 A CN 201910373598A CN 111896740 A CN111896740 A CN 111896740A
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- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
Abstract
The invention provides a method for diagnosing or predicting fertility defects such as infertility disorder of a mammalian individual by detecting ZCWPW1, wherein the risk of the disease is also judged according to the sex and age of the subject individual. The invention also provides kits and devices for diagnosing or predicting fertility defects such as infertility disorders in a mammalian subject by detecting ZCWPW 1.
Description
Technical Field
The present invention relates to the field of disease and diagnosis. In particular, the invention relates to methods and kits for diagnosing or predicting fertility defects in mammals, particularly humans.
Background
Spermatogenesis and oogenesis are composed of a series of events, including mitosis and meiosis, each step being critical for successful breeding. Reproductive senescence is a natural process of reduced fertility and gonadal decline in the production of various steroid hormones, which are more prominent in women than in men (Donnez & dolams, 2013). Among the many steps of spermatogenesis and oogenesis, meiosis is a special type of cell division that produces haploid germ cells from diploid progenitor cells and ensures their genetic diversity by homologous recombination (Handel and Schimenti, 2010). During meiosis, recombination is initiated by programmed DNA Double Strand Breaks (DSBs) produced by the evolutionarily conserved Spo11 protein at sites determined by higher chromosome structure and local chromatin changes (Keeney, Giroux, & Kleckner, 1997). Meiotic, programmed DNA double strand breaks recruit a series of recombinant proteins to form recombination sites, promoting homologous chromosomal associations at the zygotic stage (Baudat, Imai, & de Massy, 2013).
ZCWPW1(zinc finger CW-type and PWWP domain-containing 1) is one of the members of the protein family containing the CW domain. The zinc finger CW (zf-CW) domain is a zinc binding domain of about 60 amino acids with three conserved tryptophanes and four conserved cysteine residues (Perry and Zhao, 2003). The PWWP domain is involved in epigenetic regulation and is present in a variety of proteins involved in chromatin function, including histone modification enzymes, DNA modification 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 with a trimethylated K4(H3K4me3) histone H3 tail (He et al, 2010). In the structure of the complex of human zf-CW domain and H3K4me3 peptide, the H3K4me3 peptide forms a three-strand antiparallel β -sheet together with the two β -strands of the zf-CW domain (He et al, 2010). Chromatin drop analysis confirmed that the zf-CW domain recognized H3K4me3 in vitro (Hoppmann et al, 2011).
Although zf-CW domain-containing proteins have been found to mediate epigenetic control in various species, the function of ZCWPW1 in mammalian development and physiology, particularly in meiosis and reproduction, is unknown.
There is also a need in the art for greater understanding of the genetic regulation of meiosis in mammals, and thus for the detection and prognosis of fertility disorders such as infertility disorders.
Disclosure of Invention
The inventors have for the first time found that deletion of Zcwpw1 disrupts mammalian premeiotic I and results in male (male) sterility and female (female) premature ovarian failure (POI). The inventors have found that ZCWPW1 is essential for the meiotic union of male germ cells and is an important factor in maintaining normal meiotic progression in females, and thus ZCWPW1 is essential for male (male) and female (female) fertility. Thus, the inventors provide a method for diagnosing fertility defects, such as infertility disorders, in mammals, particularly humans.
In particular, the invention provides a method for diagnosing or predicting a fertility deficiency, such as infertility disorder, in a mammalian subject, comprising detecting ZCWPW1 in the subject.
In one of its aspects the invention wherein said method comprises the step of knowing and/or selecting the sex of said subject. The sex of the subject is known, for example, by interrogation or observation or detection (e.g., detection of a sex-specific gene in the subject). When the subject is female (female), the method further comprises the step of knowing and considering the age of the subject, for example by interrogating or observing or detecting (e.g., detecting an age-related gene in the subject) the age of the subject. The inventors found that ZCWPW1 is essential for the meiotic union of male germ cells, and that deletion of ZCWPW1 directly results in fertility defects in male individuals. Meanwhile, the inventor finds that ZCWPW1 is an important factor for maintaining normal meiotic progression of female germ cells, and the deletion of ZCWPW1 has higher probability of causing fertility defects in older female individuals. The methods of the invention thus include knowing or considering the sex and age factors of the subject.
In the present invention, infertility disorders include, without limitation, azoospermia, oligospermia, asthenospermia, premature ovarian failure, polycystic ovary syndrome, endometriosis, premature labor, intrauterine growth restriction, and recurrent pregnancy loss, among others.
In the present invention, the mammal may be any mammal, including and not limited to rodents, canines, felines, equines, ovines, bovines, porcines, and primates. The mammal typically includes a human.
ZCWPW1(zinc finger CW-type and PWWP domain-containing 1) is one of the members of the protein family containing the CW domain. The Zcwpw1 gene is well conserved in mammals. Wherein the human Zcwpw1 Gene Gene ID is 55063; the ZCwpw1 Gene Gene ID of the mouse was 381678.
In one aspect of the invention, the method for diagnosing birth defects provided by the invention comprises the step of detecting the Zcwpw1 gene.
The method for examining a gene of the present invention is a method generally used in the art for detecting a gene, and is not particularly limited. Examples of the method include a mass spectrometry method, a microarray method, a sequencing method, and a detection method using a base sequence amplification method such as PCR (polymerase chain reaction).
In addition, detection of a PCR product obtained when PCR is performed using primers specific to each gene can be performed by any method commonly used for detection and quantification of PCR products. For example, the detection can be carried out by electrophoresis, by real-time PCR using a fluorescent intercalator such as SYBRGreen, or by single molecule fluorescence analysis (single molecule fluorescence analysis).
The polynucleotide that can be used as a primer or a probe for detecting a gene is not particularly limited as long as it is a polynucleotide that can hybridize to a partial region containing the gene or a complementary strand thereof. The design of the polynucleotide can be carried out by any of the methods well known in the art. For example, it can be designed easily using known genome sequence data and a general-purpose primer design tool. Examples of the Primer design tool include Primer3 available on a network. In addition, publicly known genome sequence data is generally available in NCBI, which is an international base sequence database.
In one aspect of the invention, the method for diagnosing the fertility defect comprises a step of detecting the ZCWPW1 protein, for example, detecting the expression condition (including whether the protein exists or not and whether the expression is reduced relative to normal or standard) or the activity of the protein.
In yet another aspect of the invention, the method comprises the step of determining the expression of ZCWPW1 protein by immunoassay. Expression of ZCWPW1 is detected, for example, by immunofluorescence or western blotting with an antibody specifically recognizing the protein. In turn, the expression of ZCWPW1 is detected, for example, by detecting the presence or amount of mRNA from the protein, e.g., by RT-PCR, to detect the amount of mRNA encoding ZCWPW1 in a sample.
In yet another aspect of the invention, the method comprises the step of determining the activity of the ZCWPW1 protein.
The present invention also provides a kit or apparatus (including a gene chip) for diagnosing or predicting a fertility defect, such as infertility disorder, in a mammalian subject, comprising reagents for detecting ZCWPW1 in the subject.
In yet another aspect of the invention, the kit or apparatus comprises means or reagents for knowing and/or selecting the sex of the subject individual, e.g. for asking questions or indicating the corresponding markers to be selected for different sexes, e.g. for detecting sex-specific genes. In one aspect of the invention, the apparatus includes a means for querying, entering and recording whether the subject is male or female. For another example, the kit or apparatus includes a reagent for detecting a sex chromosome.
In yet another aspect of the invention, when the subject is female (female), the kit further comprises means or reagents for knowing the age of the subject, such as questions or indications of corresponding markers selected according to different ages, or such as reagents for detecting age-related genes. For example, the apparatus of the present invention may include means for interrogating, inputting and recording the age of the subject.
In yet another aspect of the 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 reagents for an immunoassay, such as a reagent for detecting the expression of ZCWPW1 by ELISA or western blot using an antibody that specifically recognizes the protein. For another example, the kit or apparatus comprises a reagent for detecting the expression of the ZCWPW1 by detecting the presence or amount of mRNA of the protein, for example, a reagent for detecting the amount of mRNA encoding ZCWPW1 in a sample by RT-PCR.
In yet another aspect of the invention, the kit or apparatus comprises reagents for detecting the Zcwpw1 gene.
In one aspect of the present invention, in the method and kit or apparatus of the present invention described above, the reagent for detecting a genetic marker in a sample comprises a primer or probe for detecting Zcwpw1, or a reagent for examining Zcwpw1, such as a specific antibody or a reagent for detecting mRNA of the protein.
In the present invention, there is also provided the use of an agent for detecting ZCWPW1 in the manufacture of a kit or device as hereinbefore described in the present aspect for diagnosing or predicting fertility defects, such as infertility disorders, in a mammalian subject. The reagent for detecting the genetic marker comprises a primer or a 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.
In this context, the protein symbols are not italicized and are all capitalized; the gene symbols are in italics. For example, ZCWPW1 is a protein and the gene encoding the protein is written as ZCwpw 1. Sometimes, however, italics is not used in the present context for the gene symbols. For example, sometimes "ZCWPW 1" or "ZCWPW 1 gene" herein denotes a gene ZCWPW1 encoding ZCWPW1 protein.
Drawings
FIG. 1 shows ZCWPW1 split at a fractional divisionExpression and dynamic localization in germ cells. Western blotting immunoblots, showing that during pre-meiosis I, ZCWPW1 expression increased during the thin to even line phase, but disappeared in long spermatids. Beta-actin was used as a control. Western blotting, cytoplasmic and nuclear fractionation analysis, only ZCWPW1 was detected in the nuclear fraction of mouse testis extracts. LaminB1 was used as a marker protein for the cytoplasm. FIGS. 1C-K, from postnatal 35 days (PD35) ZCwpw1+/+And Zcwpw1-/-Chromosome smear of spermatocytes of testis, immunostaining ZCWPW1 (green) and SYCP3 (red). ZCWPW1 is the diffusion (arrow) from the fine line period to the even line period (C-F, arrow). In pachytene cells, ZCWPW1 is located in XY body (G-H, white dotted circle). (I-K) at ZCwpw1-/-In spermatocytes, no ZCWPW1 signal was detected.
Figure 2 shows the effect of Zcwpw1 on animal fertility. Figure 2A is a schematic of the construction of Zcwpw1 knockout mice using CRISPR/Cas9 genome editing technology. Grnas (arrows), corresponding coding exons (black and red bold lines) and non-coding exons (grey bold lines) are shown. The thick red line (coding exon) shows the deletion fragment (. about.3kb) from the wild-type allele. ATG is the initiation codon. Western blot analysis confirmed that compared to wild type, at ZCwpw1-/-The absence of ZCWPW1 protein in the testes of mice indicates the success of ZCwpw1 knockout mice. Beta-actin was used as a control. Co-immunoprecipitation analysis of ZCWPW1 and H3K4me3 from PD18 testis protein extract in fig. 2C and 2d. ZCWPW1 was immunoprecipitated with H3K4me 3. FIG. 2E from PD35ZCwpw1-/-And Zcwpw1-/-Testis from male mice. ZCwpw1-/-Male mice have a reduced testicle size at PD35 (n-3, Welch's t-test analysis: p<0.0001). FIG. 2F adult ZCwpw1+/+Hematoxylin and Eosin (HE) staining of testis. Figure 2G adult ZCwpw1-/-HE staining of testicular sections showed complete cessation of spermatogenesis. Arrow, apoptotic spermatocytes; arrow, empty seminal tubules; asterisk, seminiferous tubules lack meiotic spermatocytes. Fig. 2H the female mice accumulated pup numbers over the specified time period. Each genotype n ═3 mice. FIGS. 2I-2N from ZCwpw1+/+And Zcwpw1-/-Histological analysis of ovaries of females. FIGS. 2I-2J morphological Studies of ovaries show that at 3 months of age, ZCwpw1-/-Ovary (J) appeared to be similar to ZCwpw1+/+Ovary (I) similar morphology. FIGS. 2K-2L ZCwpw1 at 6 months of age-/-Ovary (L) contains fewer follicles and Corpus Luteum (CL). FIGS. 2M-2N premature ovarian (N) lack oocytes or follicles at about 8 months of age.
FIG. 3 shows ZCwpw1-/-Chromosome associations in spermatocytes are disrupted. FIGS. 3A-F from PD35 wild type (A-C) and ZCwpw1-/-(D-F) immunostaining of spermatocytes of male testis: lines SYCP1 (green) and SYCP3 (red). Spermatocytes are divided into the following stages: the thin line stage, the even line stage, the pachytene stage and the pachytene-like stage (defined as 5 or more synaptosomal pairs). The arrows indicate the chromosomes in a union, and the arrows indicate the individual chromosomes. FIG. 3G wild type spermatocyte and ZCwpw1-/-Number of synaptic chromosome pairs in spermatocytes. At ZCwpw1-/-In spermatocytes, the average number of chromosome pairs is 8. FIG. 3H ZCwpw1+/+And Zcwpw1-/-Frequency of meiotic stages in spermatocytes. At ZCwpw1-/-In mice 47.2% and 34.3% of spermatocytes arrested in the even line phase or pachytene-like phase, respectively, while 61.5% of ZCwpw1+/+Spermatocytes are in the pachytene stage. The numbers marked in the bar graph represent the percentage of cells at the indicated meiotic stage. Three mice were analyzed for each genotype. P values were calculated by Student's t-test.
FIG. 4 shows ZCwpw1-/-Spermatocytes exhibit defects in meiotic recombination. FIGS. 4A-F from ZCwpw1+/+(A-C) and ZCwpw1-/-(D-F) immunostaining of chromosome spreads of spermatocytes of males: SYCP3 (red) and γ H2AX (green). Representative images of oocytes at the thin line, even line and pachytene stages are shown. FIGS. 4G-HZcwwp 1+/+And Zcwpw1-/-Cells immunostained for SYCP3 (red) and MLH1 (green). ZCwpw1-/-Spermatocytes lack the MLH1 signal (H).
FIG. 5 shows the SC structure at Zcwpw1-/-Normal expression in spermatocytes. FIG. 5(A-D) SIM (ultra high resolution microscopy) images of sperm cell chromosomes of PD25 testis: immunostaining for SYCP3 (red) and C-SYCP1 (green). The arrow indicates the union region. FIG. 5A '-D' enlarged view of the union region, showing C-SYCP1 (arrow, green signal) co-located with SYCP3 (arrow, red signal). Figure 5 SIM image of sperm cell chromosome of E-H PD25 testis: immunostaining lines SYCP3 (red) and N-SYCP1 (green). The arrows indicate the union region and the arrows indicate the individual chromosomes. FIG. 5 is an enlarged view of the E '-H' union region showing N-SYCP1 positioned in a continuous pattern (arrows) in the center region of the SC. Fig. 5I-L SIM images of sperm cell chromosomes from PD25 testis: immunostaining SYCP3 (red) and TEX12 (green). The arrows indicate the union region and the arrows indicate the individual chromosomes. FIG. 5 is an enlarged view of the I '-L' union region showing TEX12 located in the middle region between LEs (arrows).
FIG. 6 shows ZCwpw1-/-Meiotic prophase I of the oocyte occurs successfully but appears delayed. FIG. 6A embryonic stage 17.5 day (E17.5) ZCwpw1+/+And Zcwpw1-/-Frequency of chromosome meiotic stages in oocytes. At ZCwpw1-/-Of the oocytes in female mice, 75% and 20% were in the even and pachytene phase, respectively, while 70% of the oocytes were in the Zcwpw1 phase+/+The oocyte is in pachytene. The numbers marked by the bar graph represent the percentage of cells at the indicated meiotic stage. Three mice were analyzed for each genotype. P values were calculated by Student's t-test. Zcwwp 1 of PD1 in FIG. 6B+/+And Zcwpw1-/-Frequency of meiotic stages in oocytes. The numbers marked by the bar graph represent the percentage of cells at the indicated meiotic stage. Three mice were analyzed for each genotype. P values were calculated by Student's t-test. FIGS. 6C-H from wild-type (C-E) and ZCwpw1-/-(F-H) chromosome smear of E17.5 ovary of female oocyte: SYCP3 (red) and SYCP1 (green), showing representative images of oocytes at the thin line, even line and pachytene stages. The arrow indicates the synaptogue chromosome. FIG. 6I-L E17.5.5 chromosome spreads of ovaries, observed at the indicated stage using SIM: SYCP3(Red) and N-SYCP1 (green). The arrows indicate the area of the union. FIG. 6 is an enlarged view of the I '-L' union region showing N-SYCP1 positioned in a continuous pattern (arrows) in the center region of the SC. FIG. 6M-P E17.5.5 chromosome smear of ovary, observed at the indicated stage using SIM: SYCP3 (red) and TEX12 (green). The arrows indicate the area of the union. Fig. 6 is an enlarged view of the M '-P' union region showing that TEX12 is positioned in a continuous pattern (arrows).
FIG. 7 shows ZCwpw1-/-The pool of female follicles decreases and leads to premature ovarian failure (POI). FIGS. 7A and B representative ZCwpw1 from E13.5 female mice+/+(A) And Zcwpw1-/-(B) Ovarian sections were dissected and MVH immunostained with hematoxylin counterstain. FIG. 7C oocyte count, shown at ZCwpw1 at E13.5+/+And Zcwpw1-/-Similar numbers of oocytes are present in females. MVH positive cells were counted. FIGS. 7D and E representative ZCwpw1 from PD1 female mice+/+(D) And Zcwpw1-/-(E) Ovarian sections were dissected and MVH immunostained with hematoxylin counterstain. FIG. 7F oocyte count, showing ZCwpw1-/-The ovary contains less oocytes than ZCwpw1+/+And (4) ovaries. MVH positive cells were counted. FIGS. 7G and H representative ZCwpw1 from PD8 female mice+/+(G) And Zcwpw1-/-(H) Ovary sections were counterstained with hematoxylin. MVH positive cells were counted. FIG. 7I oocyte count show ZCwpw1-/-The ovary contains fewer follicles than Zcwpw1+/+And (4) ovaries. MVH positive oocyte nuclei with characteristics of the surrounding granular cell layer were counted. In all cases, each section (8 μm per section) was counted and the total number of oocytes per ovary was calculated. Six ovaries from three mice were analyzed for each genotype. P values were calculated by Student's t-test.
Detailed Description
The spirit and advantages of the present invention will be further illustrated by the following examples, which are provided by way of illustration and are not intended to be limiting.
Example 1 Experimental methods and reagents
Preparation of mouse ZCWPW1 antibody
Antibodies to mouse ZCWPW1 were produced in house in the laboratory. Briefly, the cDNA fragment encoding amino acid 488-622 of mouse ZCwpw1 was inserted into the p-ET-32a + vector (EMD Millipore) and transfected into BL21-CodonPlus (DE3) E.coli cells. Cells were cultured overnight at 37 ℃ and induced by the addition of 0.2mM IPTG (Sigma-Aldrich) at 28 ℃ for 4 hours. Cells were harvested by centrifugation and disrupted by sonication and purified by Ni-NTA agarose (QIAGEN) according to the manufacturer's instructions. The proteins were dialyzed in PBS and used to immunize rabbits, and the antisera were affinity purified on antigen-conjugated CNBr-activated agarose (GE Healthcare).
Purification of male germ cells
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 was assessed under light microscopy based on their diameter and morphological characteristics.
Tissue Collection and histological analysis
Testes from at least three mice of each genotype were dissected immediately after euthanasia of the mice, fixed in 4% paraformaldehyde (Solarbio, beijing, P1110) for 24 hours, stored in 70% ethanol, and embedded in paraffin. 5 μm sections were prepared and mounted on glass slides. After deparaffinization, slides were stained with hematoxylin and eosin for histological analysis.
Immunocytology and antibodies
Sperm cell smears were prepared as described in Peters, Plug, van Vugt, & de Boer, 1997. Oocytes from embryonic ovary (E17.5 embryo) and PD1 ovary were digested with collagenase, incubated in hypotonic buffer, fixed in paraformaldehyde, and incubated with the indicated antibodies for immunofluorescence. Primary antibodies for immunofluorescence were as follows: rabbit anti-SYCP 3 (1: 500 dilution; Abcam # ab15093), mouse anti-SYCP 3 (1: 500 dilution; Abcam # ab97672), rabbit anti-SYCP 1 (C-terminal) (1: 2,000 dilution; Abcam # ab15090), rabbit anti-SYCP 1 (N-terminal) (1: 2,000 dilution; Abclonal # A12139), rabbit anti-RPA 2 (1: 200 dilution; Abcam # ab76420), rabbit anti-RAD 51 (1: 200 dilution; Thermo Fisher scientific # PA5-27195), rabbit anti-DMC 1(1: 100 dilution; Santa Cruz Biotechnology # sc-22768), mouse anti-gamma H2AX (1: 300 dilution; Millipore #05-636), and mouse anti-MLH 1(1:50 dilution; BDsciences # 550838). Primary antibodies were detected with Alexa Fluor 488 or 594-conjugated secondary antibodies (1: 500 dilution; Abcam # ab150084, # ab150077, # ab150113, # ab150120) incubated for 1 hour at room temperature. Slides were washed several times with PBS and fixed using VECTASHIELD medium with DAPI (Vector Laboratories, # H-1200).
Real-time RT-PCR
Total RNA was isolated from various tissues of wild-type or knockout adult mice. To analyze the expression levels of Zcwpw1mRNA in various tissues, equivalent 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 F5'-GGG GAC ATC TAA GCT AGG CC-3' and R5' -CTG CAC TCA CGG CCA TCTT-3 ". Primers β -actin F5'-CAT CCG TAA AGA CCT CTA TGC CAA C-3' and β -actin R5'-ATG GAG CCA CCG ATC CAC A-3' were used to amplify β -actin as a housekeeping gene. All RT-PCR reactions were initially denatured at 95 ℃ for 10 min, then subjected to 25 cycles, denatured at 95 ℃ for 30 sec, annealed at 60 ℃ for 30 sec, extended at 72 ℃ for 30 sec, and finally extended at 72 ℃ for 5 min using a T100 thermocycler (Bio-Rad).
Western Blot
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 extract was centrifuged at 20,000 Xg for 20 minutes at 4 ℃. 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). Relative protein levels in each sample were normalized to β -actin to normalize loading changes. Primary antibodies used for immunoblotting included anti-beta-actin (1: 10,000 dilution; Cell Signaling Technology, #4970) and anti-ZCWPW 1(1: 1,000 dilution; as described above in "preparation of mouse ZCWPW1 antibody").
Confocal microscope
The immunolabeled chromosomes were imaged by confocal microscopy using a Leica TCS SP5 resonance scanning confocal microscope driven by Leica Application Suite software or an Andor Dragonfly spinning disk confocal microscope driven by Fusion software. Projection images were then prepared using ImageJ software (NIH, version 1.6.0-65) or biplane imaging (version 8.1) software. Histological samples were analyzed with epifluorescence microscopy (BX52, Olympus) and processed using photoshop (adobe). SIM analysis was performed using the 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 the ultra-high resolution optical series part.
Statistical analysis
All data are expressed as mean ± SEM. Statistical significance of differences between the mean values of different genotypes was measured by the paired two-tailed distribution Welch's t-test. Data were considered significant when p values were less than 0.05 (. sup.), or 0.01 (. sup.).
Example 2 expression and dynamic localization of ZCWPW1 in meiotic germ cells
ZCWPW1 was found to be expressed in various organs of mice by RT-PCR and Western blotting detection, and was highly expressed in adult testis and embryonic ovary in pre-meiosis I. To further study the expression pattern of ZCWPW1 in germ cells, different types of male germ cells were isolated by BSA gradient sedimentation (Gan et al, 2013). By Western blotting, it was found that during pre-meiotic stage I, expression of ZCWPW1 increased from the fine line phase to the even line phase, but became barely detectable in elongated spermatids (fig. 1A). To determine the subcellular localization of ZCWPW1 in germ cells, a cytoplasmic and nuclear fractionation analysis was performed and it was found that ZCWPW1 was detected only in the nuclear fraction of mouse testis extracts, indicating that ZCWPW1 plays a role in the nucleus (fig. 1B).
By immunostaining of the expanded spermatocyte nuclei, scattered ZCWPW1 immunofluorescence signals were found from the thin line phase (FIGS. 1, C and D, green signal, arrow) to the even line phase (FIGS. 1, E and F, green signal, arrow). The signal disappeared from the autosome (FIG. 1H, red signal) and appeared only in the pachytene of the XY body (FIGS. 1, G and H, green signal, white circle). These phenomena suggest that ZCPWW 1 may only function in the fine line phase to the even line phase on autosomes.
Example 3 preparation of a Zcwpw1 deficient mouse model
To study the in vivo function of ZCWPW1, a ZCWPW1 deficient mouse model lacking exon 3 through exon 7 was generated using the CRISPR/Cas9 system (Cyagen Biosciences). Fig. 2A is a schematic of the CRISPR/Cas9 genome editing system at the Zcwpw1 locus, showing the grnas (arrows), the corresponding coding exons (black and red bold lines) and non-coding exons (grey bold lines). The thick red line (coding exon) shows the deletion fragment (. about.3kb) from the wild-type allele.
The PCR primers for the mutant allele of Zcwpw1 were: positive 5'-AGC TGC TGG GAT TAAATG TCT GTT CC-3' and negative 5'-CTA CAC TGT GCC TTC TAC CTT CTT TGA GA-3' primers were used to generate a 690bp fragment using genomic DNA from a mouse tail sample as a template. The PCR primers for the wild type allele of Zcwpw1 were positive 5'-CAA GAT GGA GGA GAT ATG CAG TAC ATG-3' and negative 5'-CTA CAC TGT GCC TTC TAC CTT CTT TGA GA-3', resulting in a 617bp fragment.
Western blot analysis confirmed that compared to wild type, the mutant is at ZCwpw1-/-The ZCWPW1 protein was not present in the testis of mice (fig. 2B). Beta-actin was used as loading control. From heterozygote (ZCwpw 1)+/-) Progeny from male and female mouse crosses exhibit a normal Mendelian genotype distribution, ZCwpw1-/-Mice were able to survive and appeared to develop normally.
Example 4 ZCwpw1 is essential for maintaining male fertility
Using ZCWPW1 antibody immunoprecipitated testis protein extract, H3K4me3 was found to be present in immunoprecipitated proteins from wild-type testis, but ZCwpw1-/-Absent from testis (fig. 2C). Similarly, immunoprecipitation using the H3K4me3 antibody pulled down ZCWPW1 protein from wild-type testis protein extract, but not ZCWPW1-/-ZCWPW1 protein was pulled down in testis (fig. 2D). It was demonstrated that deletion of Zcwpw1 disrupted the binding of Zcwpw1 to H3K4me 3.
Although ZCwpw1-/-Mice develop and look normal, but ZCwpw1-/-Male mice were found to be completely sterile. As shown, ZCwpw1 at 35 days after birth (PD)-/-Male mouse testis is much smaller than control males (n-3, Welch's t-test analysis: p<0.0001) (fig. 2E). Histological analysis showed Zcwpw1 compared to wild-type males (FIG. 2F, hematoxylin and eosin staining)-/-Spermatogenesis in male mice is disturbed: ZCwpw1-/-In male mice, the seminiferous tubules lacked meiotic spermatocytes (fig. 2G, asterisks) and contained apoptotic cells (fig. 2G, arrows) or were almost empty (fig. 2G, arrows). ZCwpw1-/-Male mice show pre-mature death of germ cells, demonstrating that Zcwpw1 is essential for maintaining male germ cell development and male fertility in mice.
Example 5 Effect of ZCwpw1 on female fertility is age-related
Zcwpw1 was found to be comparable to males-/-Fertility of female mice up to mid-adult (5-6 months of age) vs. ZCwpw1+/+Female mice were normal compared with comparable numbers of pups (n-3 for each genotype) (fig. 2H). Morphological analysis of the ovaries revealed that at 3 months, ZCwpw1-/-Female mouse ovary (FIG. 2, J) appeared similar to ZCwpw1+/+Female mouse ovaries similar healthy ovarian morphology (fig. 2, I). However, at 6 months of age, at ZCwpw1-/-Very few follicles and corpus luteum were seen in female mouse ovaries (fig. 2L), indicating no normal ovarian function, whereas Zcwpw1+/+The female mouse ovary contained healthy follicles (fig. 2K, arrow) and the Corpus Luteum (CL) (fig. 2K). At 6 months ZCwpw1-/-Of female miceOvarian morphology confirmed fertility observations. As expected, ZCwpw1-/-The female mouse ovary did not have any follicles in the later adult stage of approximately 8 months of age (fig. 2N), and thus became sterile.
Example 6 ZCwpw1-/-Chromosome association in spermatocytes is disrupted
To determine ZCwpw1-/-The cause of disruption of meiosis in male mouse spermatocytes was first visualized for chromosomes in the pre-meiotic stage by immunostaining for Synaptonemal Complex (SC) protein. SC consists of two transverse elements (LE) and one Central Element (CE) (Fraune, Schramm, Alsheimer,&Benavente，2012；Schucker，Holm，Franke，Sauer，&Benavente，2015)。
at ZCwpw1+/+In male mouse spermatocytes, LEs marked by SYCP3 begin to form at the fine line phase (fig. 3A, red signal), but lack of SYCP1 signaling (green signal) indicates CE is not yet present (fig. 3A). In the idol phase, the LE was completely formed and the chromosome gradually mutated as shown by the appearance of the SYCP1 signal (fig. 3B, arrows). As shown by the continuous SYCP1 signal, wild type spermatocytes reached complete autosomal synaptation at the pachytene stage (fig. 3C, arrows).
At ZCwpw1-/-In male mouse spermatocytes, LEs were formed at the fine line phase (fig. 3D, red signal) and SYCP1 began to appear on paired chromosomes at the adventitial phase, indicating that synaptogenesis was initiated to some extent (fig. 3E, arrows). However, at ZCwpw1-/-In spermatocytes, there are always a large number of non-synaptogenic autosomes (FIG. 3F, arrow, red signal) compared to synaptogenic chromosomes (FIG. 3F, arrow), and these spermatocytes remain in a state that states they claim to be "pachytene-like" (5 or more synaptogenic chromosome pairs are present). For 35 days of Postnatal (PD) ZCwpw1+/+And Zcwpw1-/-Quantitative observations were made of synaptosomal pairs in each nucleus in the male mouse testis (fig. 3G): at ZCwpw1+/+In testis, complete association of all chromosome pairs of 153 cells was observed, with only 16 cells having 4-18 association chromosomes; however, at ZCwpw1-/-In the testis, every cellOn average, only 8 synaptosomal pairs, and is shown at ZCwpw1-/-None of the 164 spermatocytes observed in the testis had a complete association.
The percentage of sperm cells at different stages of meiosis was further analyzed using Postnatal (PD)28 day mouse testes and found to be ZCwpw1-/-None of the spermatocyte development was able to reach pachytene (FIG. 3H), 34.3% of which ceased at pachytene-like stage (FIG. 3H, pachytene-like). This is in contrast to ZCwpw1 which has reached 61.5% of the pachytene stage+/+Spermatocytes contrast sharply (FIG. 3H, bold line).
For a better understanding of ZCwpw1-/-Association failure in spermatocytes, and ultra-high resolution structured fluorescence microscopy (SIM) was used to observe fine SC structures indistinguishable in confocal microscope images. Immunostaining was performed for SYCP3 and the CE-specific protein SYCP1 (both C-and N-terminal stained) and TEX 12. In SIM imaging, LE was shown as two independent strands by SYCP3 immunostaining (fig. 5A', arrows, red signal). SYCP1 formed a homodimer, with both C-termini of SCYP1 dimer localized in LE (Cahoon et al, 2017), which was observed in SIM imaging (fig. 5, a 'and C', green signals, arrows). The N-terminus of the SYCP1 dimer is located at CE (fig. 5, E 'and G', green signal, arrow) (Winkel, Alsheimer, Ollinger,&benavente, 2009), TEX12 is also located in the CE (fig. 5I 'and K', green signal, arrows). At ZCwpw1-/-In the episomal spermatocytes, association begins in some chromosomes. C-SYCP1 (FIG. 5B ', green signal, arrow) was found along the axis of SYCP3, and N-SYCP1 (FIG. 5F ', green signal, arrow) and TEX12 (FIG. 5J ', green signal, arrow) were found on the chromosomes in full synaptism.
However, surprisingly, there is an average of only 8 pairs of confluent chromosomes of pachypyle-like Zcwpw1-/-The cells of (2) did not differ significantly in the localization pattern of C-SYCP1 (FIG. 5D ', green signal, arrow), N-SYCP1 (FIG. 5H ', green signal, arrow) or TEX12 (FIG. 5L ', green signal, arrow). Meanwhile, ZCwpw1-/-The unionjugated autosomes in the cell are at the same timeAbsence of SYCP1 signal (fig. 5, D and H, arrow, red signal) and TEX12 signal (fig. 5L, arrow, red signal). Based on these results, the inventors concluded that Zcwpw1-/-Some autosomes in spermatocytes can initiate but not produce complete synaptogenesis.
Example 7 ZCwpw1-/-Shows a defect in meiotic recombination in spermatocytes
Meiotic programmed DNA Double Strand Break (DSBs) repair was assessed by analyzing the localization of phosphorylated γ -H2 AX. Similar to ZCwpw1+/+Signals of γ -H2AX in spermatocytes (FIGS. 4, A and B, arrows), at the fine line phase (FIG. 4D, arrow) and at the even line phase (FIG. 4E, arrow) ZCwpw1-/-gamma-H2 AX in spermatocytes marks the chromatin domain around the DSB site. In the thick line period, at ZCwpw1+/+In the cells, γ -H2AX signal was observed only in the non-confluent region of the sex chromosome (FIG. 4C, white dashed circle). However, at ZCwpw1-/-In spermatocytes, phosphorylated γ -H2AX signal persists in unassociated chromatin (fig. 4F, arrows), indicating failure of DSB repair.
In the late pachytene stage, the MLH1(MutL homolog 1) site is in ZCwpw1+/+Becomes evident in spermatocytes, indicating the presence of chromosomal exchange sites (FIG. 4G, arrows) (Marcon)&Moens, 2003). However, ZCwpw1-/-Spermatocytes failed to enter pachytene and no interchange occurred, as evidenced by deletion of MLH1 site (FIG. 4H).
Example 8ZCwpw1-/-Successful but delayed onset of meiotic prophase I in oocytes
And ZCwpw1-/-Zcwwp 1 at embryonic stage (E)17.5 days in comparison to spermatocytes-/-The oocytes can reach pachytene and develop into oocytes with normal fertility. However, they show a significant delay in meiotic progression. By calculating the percentage of oocytes at different meiotic stages, we found 70% Zcwpw1+/+The oocytes had formed SCs and reached pachytene (FIG. 6A), with only 20% of ZCwpw1-/-The oogonium cells have completed a union, approximately 75%ZCwpw1-/-The oocytes were still in the adventitious phase (FIG. 6A).
At 1 day Postnatal (PD), 45% of ZCwpw1+/+The blast cells had entered late doublet (FIG. 6B, late doublet), and about 15% of ZCwpw1+/+The oocyte is in the nuclear reticulum stage (FIG. 6B, nuclear reticulum stage). However, only 20% of ZCwpw1-/-The oocytes were in the late phase of the diplotene phase, with only 10% in the nuclear reticulum phase (FIG. 6B). From 38% of ZCwpw1-/-Oocytes in late pachytene and 32% of ZCwpw1-/-The oocytes are easily visible early in the diplotene phase, ZCwpw1-/-Meiosis was slower in oocytes (fig. 6B). Representative images of oocytes at the thin line, even line and pachytene stages are given (FIG. 6, C to H, arrows, synaptosomes).
To determine ZCwpw1 at embryonic stage (E) at 17.5 days-/-Whether the SC structures formed in the oogonium cells were intact and stable, the oogonium cell chromosomes were immunostained with antibodies against N-SYCP1 and TEX 12. It was found that ZCwpw1-/-N-SYCP1 and TEX12 were localized in CE in pairs of chromosomes at the amphibian and pachytene stages in the oocytes (N-SYCP1, FIGS. 6J, J 'and L, L', arrows; TEX12, FIGS. 6N, N 'and P, P', arrows). This is in contrast to ZCwpw1 at E17.5+/+Oocytes were very similar (N-SYCP1, FIGS. 6I, I 'and K, K', arrows; TEX12, FIGS. 6M, M 'and O, O', arrows). These results indicate that in female ZCwpw1-/-Normal association occurs in the oogonium.
Example 9 ZCwpw1-/-Reduction of the pool of female follicles and resulting premature ovarian failure (POI)
The reproductive senescence process in mammals involves a gradual decrease in ovarian follicular reserve and a decrease in oocyte quality (Faddy, 2000). ZCwpw1-/-The female has delayed meiotic progression and reduced ovarian reserve, resulting in the POI phenotype.
The number of oocytes at different stages of ovarian development was quantified. Ovaries dissected from E13.5, PD1 and PD8 mice were fixed, sectioned, and immunostained with mouse vascular homolog (Mvh) antibody. The oocytes at these stages were processedQuantitative, shown in E13.5ZCwpw1+/+(FIG. 7A) and ZCwpw1-/-(FIG. 7B) the same number of oomycete cells were present in the ovary, wherein meiosis was about to begin (FIG. 7C). In PD1, with ZCwpw1+/+Ovarian phase comparison (FIG. 7D), ZCwpw1-/-The ovary (FIG. 7E) contained approximately 45% fewer oocytes, indicating that Zcwpw1 was about to complete when premeiotic I was about to complete-/-Oocyte number relative to ZCwpw1+/+(FIG. 7F) is decreased. In PD8, ZCwpw1+/+The ovary contains well-formed primordial follicles (fig. 7G, arrows) and some activated primary/secondary follicles (fig. 7G, arrows). However, PD8ZCwpw1-/-The ovary contained fewer activated follicles (fig. 7H, arrows) and fewer primordial follicles (fig. 7F, arrows). Quantification of oocyte number revealed ZCwpw1-/-Ovary has only ZCwpw1+/+About 20% of oocytes in the ovary (fig. 7I). Thus, at ZCwpw1-/-The delayed pre-meiosis phase found in females triggers the death of a proportion of the germ cells, thereby reducing the number of follicles in the ovaries. Due to insufficient ovarian reserve, ZCwpw1-/-Females are infertile in mid-adult life due to premature ovarian failure.
The present invention has for the first time discovered and demonstrated that deletion of Zcwpw1 disrupts mammalian premeiotic I and results in male (male) sterility and female (female) premature ovarian failure. The inventors have found that ZCWPW1 is essential for the meiotic union of male germ cells and is an important factor in maintaining normal meiotic progression in females, and thus ZCWPW1 is essential for male (male) and female (female) fertility. Applicants thus provide methods and kits for diagnosing fertility defects, such as infertility disorders, in mammals, including humans.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of organic chemistry, polymer chemistry, biotechnology and the like, and it will be apparent that the invention may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Many modifications and variations are possible in light of the above teaching and are therefore within the scope of the invention.
The following reference papers are disclosed in this application by way of full text.
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Claims (10)
1. Use of an agent for detecting Zcwpw1 in a subject for the manufacture of a kit or apparatus for diagnosing or predicting fertility defects, such as infertility disorders, in a mammal, such as a human.
2. The use of claim 1, wherein the infertility disorder is selected from azoospermia, oligospermia, asthenospermia, azoospermia, premature ovarian failure, polycystic ovary syndrome, endometriosis, preterm labor, intrauterine growth restriction, and recurrent pregnancy loss.
3. The use of claim 1, wherein said kit or apparatus further comprises a device or reagent for learning and/or selecting the sex of said subject individual; when the subject is female (female), the kit further comprises a device or agent for knowing the age of the subject.
4. The use of any one of claims 1-3, wherein the agent that detects ZCwpw1 in the subject is an agent that detects ZCwpw1 gene.
5. The use according to any one of claims 1 to 3, wherein the agent for detecting ZCwpw1 in a subject is an agent for detecting ZCWPW1 protein,
preferably, the kit comprises a reagent for immunoassay of expression of ZCWPW1, for example, a reagent for detection of expression of ZCWPW1 by ELISA or Western blotting using an antibody specifically recognizing the protein;
or
Including reagents for detecting ZCPWW 1 by detecting the presence or amount of mRNA of ZCPWW 1, e.g., reagents for detecting the amount of mRNA encoding ZCPWW 1 in a sample by RT-PCR,
more preferably, the kit further comprises an agent for determining the activity of ZCWPW 1.
6. A kit for diagnosing a fertility deficiency, such as infertility disorder, in a mammal, such as a human, comprising reagents for detecting ZCWPW1 in a subject.
7. The kit of claim 6, wherein the infertility disorder is selected from azoospermia, oligospermia, asthenospermia, azoospermia, premature ovarian failure, polycystic ovary syndrome, endometriosis, preterm labor, intrauterine growth restriction, and recurrent pregnancy loss.
8. The kit of claim 6, further comprising means or reagents for knowing and/or selecting the sex of the subject, such as for questioning or indicating the corresponding marker selected for the different sex, or such as for detecting sex-specific genes; when the subject is female (female), the kit further comprises means or reagents for knowing the age of the subject, such as questions or instructions for a corresponding marker selected for different ages, or such as reagents for detecting age-related genes.
9. The kit of claim 6, comprising reagents for detecting the Zcwpw1 gene in a subject.
10. The kit of claim 6, which comprises a reagent for detecting the ZCWPW1 protein of a subject,
preferably, reagents for immunoassay of ZCWPW1, such as antibodies specifically recognizing ZCWPW 1;
or
Wherein the reagent comprises a reagent for detecting the expression of the ZCPWW 1 by detecting the existence or the quantity of mRNA of the ZCPWW 1, for example, a reagent for detecting the mRNA coding for the ZCPWW 1 in a sample by RT-PCR,
more preferably, the kit further comprises an agent for determining the activity of ZCWPW 1.
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