Deubiquitination enzyme complexes and factors during spermatogenesis and uses thereof

Abstract

The invention provides a kit for diagnosing fertility defects such as infertility in mammals with different sexes. The kit comprises a reagent for detecting UAF1 or UAF1 / USP1 deubiquitination enzyme complex of a tested individual. The invention also provides kits and instruments for diagnosing or predicting fertility defects, such as infertility, in a mammalian subject by detecting the UAF1 or UAF1 / USP1 deubiquitinating enzyme complex. Applicants thus provide a method of diagnosing fertility defects such as infertility in mammals, including humans, further facilitating the development of new therapeutic methods for male infertility individuals affected by gene mutations.

Description

Technical Field

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

[0002] Spermatogenesis is a complex morphogenetic differentiation process by which diploid spermatogonial stem cells are transformed into haploid spermatozoa within the seminiferous tubules of the testis. This differentiation process includes the self-renewal of spermatogonial stem cells, differentiation of spermatogonia into meiotic spermatocytes, and the morphogenesis of haploid spermatozoa known as spermiogenesis. Spermatogenesis includes five major processes: chromatin remodeling, acrosome development, mitochondrial rearrangement, flagellar assembly, and cytoplasm removal. These differentiation processes are tightly regulated by transcriptional, translational, and post-translational mechanisms, and alterations in these processes can lead to male infertility and reduced fertility.

[0003] Spermatogenesis requires precise temporal coordination of multiple important proteins, including KDM3A, BRDT, MORC3, etc. For example, KDM3A plays an important role in regulating the expression of transition protein 1 (TNP1) and protamine 1 (PRM1) to control sperm chromatin packaging and condensation. BRDT binds to the hyperacetylated H4 tail and recognizes H4K5 / K8ac through the BD1 domain, leading to the removal of histones in round spermatids. MORC3 is a new epigenetic regulatory protein that includes MIWI2 as a partner and can play a role in piRNA-dependent transposon silencing in male germ cells.

[0004] The level of a protein in a cell depends on its translation and degradation rates. Ubiquitination - one of the main pathways by which cells regulate protein degradation - is mediated by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). In contrast, the removal of ubiquitin modifications from substrate proteins is mediated by deubiquitinating enzymes (DUBs). In cells, the balance between ubiquitination and deubiquitination activities regulates many biological processes.

[0005] The field also needs to have a better understanding of the male spermatogenesis process in mammals, so as to detect and predict reproductive disorders such as infertility. Summary of the invention

[0006] The inventors first discovered that conditional knockout of Uaf1 leads to male sperm formation defects and infertility, but does not affect female fertility, indicating that UAF1 has different functions in male and female fertility. The inventors further discovered and demonstrated that the UAF1 / USP1 deubiquitinase complex plays a role in regulating the levels of multiple proteins related to spermatogenesis, including that UAF1 is expressed in mouse testis and localized to the nucleus during spermatogenesis; UAF1 interacts and co-localizes with USP1 in the testis. Male mice with conditional knockout of UAF1 in germ cells are completely infertile, have a significantly reduced number of epididymal sperm, and show defects in spermatogenesis. The inventors also discovered and demonstrated that the functional loss of the UAF1 / USP1 deubiquitinase complex reduces the levels of multiple proteins known to be involved in spermatogenesis in the testis, and the UAF1 / USP1 deubiquitinase complex is essential for normal spermatogenesis. Thus, the inventors provide a method for diagnosing or treating fertility defects such as infertility in mammals, especially humans.

[0007] In one aspect of the present invention, a method for diagnosing fertility defects such as infertility in mammals of different sexes by detecting UAF1 or UAF1 / USP1 deubiquitinase complex in a test subject is provided. The method may further include the step of detecting the sex of the test subject.

[0008] In one aspect of the present invention, a kit for diagnosing fertility defects such as infertility in mammals of different sexes is provided, which comprises a reagent for detecting UAF1 or UAF1 / USP1 deubiquitinase complex in a test subject. In the present invention, the kit also comprises a reagent for detecting the sex of the test subject, in particular, whether the test subject is male.

[0009] The present invention thus provides the use of a reagent for detecting UAF1 or UAF1 / USP1 deubiquitinase complex in a test subject in the preparation of a kit for diagnosing fertility defects such as infertility in mammals of different sexes. In the present invention, the kit also includes a reagent for detecting the sex of the test subject.

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

[0011] 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.

[0012] The human genome encodes more than 100 deubiquitinases, which are divided into six families. Among them, ubiquitin-specific proteases (USPs) are the largest family, and its member ubiquitin-specific protease 1 (USP1) is able to deubiquitinate a wide range of substrates. USP1-associated factor 1 (UAF1, also known as WDR48 or p80) can bind to UAF1, stabilizing its expression and mediating its access to substrates.

[0013] The Usp1 and Uaf1 genes encoding USP1 and UAF1 are highly conserved in mammals. The Gene ID of the human Usp1 gene is 7398; the Gene ID of the mouse Usp1 gene is 230484; the Gene ID of the human Uaf1 gene is 57599; and the Gene ID of the mouse Uaf1 gene is 67561.

[0014] In the application (including methods and related kits) provided by the present invention for diagnosing fertility defects or infertility in mammals of different sexes by detecting UAF1 or UAF1 / USP1 deubiquitinase complex of the test individual, whether the UAF1 or UAF1 / USP1 deubiquitinase complex of the test individual is abnormal is detected, which includes comparing the activity of the UAF1 or UAF1 / USP1 gene or its regulatory factor, or the expression of UAF1 or USP1 protein of the subject, or comparing with the situation of the gene or protein in normal people in the general or specific group, and if there is a significant change, it is judged that there is an abnormality.

[0015] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include steps and reagents for detecting genes of each component of the aforementioned complex, such as steps and reagents for detecting Uaf1 and / or Usp1 genes encoding UAF1 and / or USP1. In one embodiment of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include steps and reagents for detecting genes encoding Uaf1. In one embodiment of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include steps and reagents for detecting genes encoding Uaf1 and Usp1.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include the step of detecting each component protein in UAF1 or UAF1 / USP1 deubiquitinase complex, such as detecting UAF1, or detecting UAF1 and USP1 proteins. This includes detecting the expression of the protein (including whether it exists, and whether the expression is reduced relative to the normal or standard) or its activity.

[0020] In another aspect of the present invention, the method and kit include steps and reagents for immunoassaying the expression of each component protein in the aforementioned complex. For example, the expression of the target protein is detected by immunofluorescence or Western blotting with an antibody that specifically recognizes the protein. For another example, the expression of the target protein is detected by detecting the presence or amount of the mRNA of the protein, such as by RT-PCR to detect the amount of mRNA encoding the target protein in the sample. In another aspect of the present invention, the method includes a method for determining whether the protein forms a complex, such as an immunoprecipitation method.

[0021] In yet another aspect of the present invention, the method and kit include steps and reagents for determining the activity of UAF1, or UAF1 and USP1 proteins.

[0022] The present invention also provides an apparatus (including a gene chip) for diagnosing or predicting fertility defects such as infertility in mammalian individuals.

[0023] The method and kit provided by the present invention are based on the discovery that the effect of UAF1 or UAF1 / USP1 deubiquitinase complex on the fertility of mammals is gender-dependent, so the diagnosis and prediction made by the method or kit provided by the present invention are different for mammals of different genders. Therefore, in the method provided by the present invention, it is also included to detect the gender of the subject. In the kit provided by the present invention, it is also included to detect the gender of the subject. Methods and reagents for detecting the gender of the subject are well known in the art, for example, including detecting the gender-specific proteins or genes of the subject, such as specific nucleic acid sequences on sex chromosomes, etc.

[0024] The present invention also provides a method for treating or avoiding fertility defects in mammalian individuals, which comprises treating individuals having male UAF1 or UAF1 / USP1 deubiquitinase complex defects, such as defects in the individual components of the UAF1 / USP1 complex or their genes, and the rRNA bound thereto.

[0025] In this article, protein symbols are not italicized and are all capitalized; gene symbols are italicized. For example, UAF1 is a protein, and the gene encoding the protein is written as Uaf1. Sometimes in this article, protein symbols are not all capitalized. For example, sometimes "Uaf1" in this article refers to UAF1 protein. Sometimes in this article, gene symbols are not italicized. For example, sometimes "Uaf1" or "UAF1 gene" in this article refers to the gene Uaf1 encoding UAF1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 showed that UAF1 interacts with USP1 in the testis.

[0027] A. Molecular weight (MW) of each indicated protein colocalized with UAF1 in WT mouse testes and the number of peptides detected by mass spectrometry.

[0028] B. Molecular weight of each indicated protein and number of peptides detected by mass spectrometry that colocalize with USP1 in WT mouse testes.

[0029] CD. Co-immunoprecipitation (Co-IP) analysis of UAF1 and USP1 in adult testis protein extracts.

[0030] E. Co-IP analysis of UAF1 and USP1 in UAF1-C-HA testicular protein extracts.

[0031] Figure 2 Showing the expression and localization of UAF1 in testis.

[0032] A. Western blots for UAF1 and USP1 in the cytoplasmic and nuclear fractions of adult WT testes, showing that UAF1 and USP1 are present in the cytoplasm and nucleus. Lamin B1 was used as a marker for the nuclear fraction. β-Actin was used as a marker for the cytoplasmic fraction.

[0033] B. UAF1 expression begins in testis at PD 8. β-actin was used as a loading control.

[0034] C. Double immunostaining of WT germ cells for UAF1 and PNA in testis sections at the indicated stages shows the dynamic expression of UAF1 during adult spermatogenesis. DNA was counterstained with DAPI. Scale bar, 5 μm.

[0035] D. Schematic diagram of the localization of UAF1 during spermatogenesis in adult testis. Spg, spermatogonia; Pl, preleptotene; L, leptotene; Z, zygotene; EP, early pachytene; P, pachytene; D, diplotene; Rs, round spermatid; Es, elongated spermatid; S, sperm.

[0036] Figure 3 showed that conditional knockout of Uaf1 in germ cells resulted in reduced testis size and male infertility.

[0037] A. Methods used to generate Uaf1 sKO mice, in which Uaf1 is specifically knocked out in germ cells.

[0038] B. Western blot analysis for UAF1 in 8-week-old WT and Uaf1 sKO testes (n=3). β-Actin served as a loading control.

[0039] C. UAF1 immunofluorescence in WT and Uaf1 sKO adult testes. Scale bar, 100 μm.

[0040] D. Number of pups per litter 6 months after natural cross between male mice (>8 weeks old) and WT female mice. Uaf1 sKO male mice showed complete infertility. Data are presented as mean ± SEM, n = 4. ***P < 0.001 by Student's t-test.

[0041] E. Gross morphology of representative testes of WT and Uaf1 sKO mice. Scale bar, 1 mm.

[0042] F. Testis weights of Uaf1 sKO and age-matched control mice. Data are shown as mean ± SEM, n = 3. **P < 0.01 by Student's t-test.

[0043] Figure 4showed that Uaf1 sKO mice have defects in sperm development.

[0044] A. H staining of testis histological sections from 12-week-old WT and Uaf1 sKO mice. Scale bar, 50 μm.

[0045] B. Immunofluorescence staining against mouse VASA homolog (MVH) in testes of 12-week-old WT and Uaf1 sKO mice. Scale bar: 50 μm.

[0046] C. PAS-hematoxylin staining of testicular sections from 12-week-old WT and Uaf1 sKO mice. The cell cycle phase of the seminal epithelium was determined by the morphology of spermatocytes and round spermatids. Scale bar, 20 μm.

[0047] D. The ratio of sperm and Sertoli cells in the tubule cross section at specific stages of the seminal epithelial cycle and the corresponding steps of sperm development are shown. The number of late spermatozoa is reduced in Uaf1 sKO testes.

[0048] E. Left: TUNEL assay in testis sections from 12-week-old WT and Uaf1 sKO mice. Scale bar, 50 μm. Right: Quantification of the number of TUNEL-positive cells per tubule. Data are shown as mean ± SEM, n = 3. ***P < 0.001 by Student's t-test.

[0049] Figure 5 showed that late steps of sperm development were disrupted in Uaf1 sKO mice.

[0050] A. H staining of histological sections of the caudal epididymis of 12-week-old WT and Uaf1 sKO mice. Scale bar, 20 μm.

[0051] B. Sperm counts in the caudal epididymis of WT and Uaf1 sKO mice at 12 weeks of age. Data are shown as mean ± SEM, n = 3. **P < 0.01 by Student's t test.

[0052] C. HE staining of spermatozoa from 12-week-old WT and Uaf1 sKO mice. Scale bar, 10 μm. Note that most spermatozoa exhibited abnormal head morphology in the caudal epididymis of Uaf1 sKO.

[0053] D. Spermatids The figure shows the number of abnormal sperms in WT and Uaf1 sKO mice. Data are presented as mean ± SEM, n = 3. ***P < 0.001 by Student's t-test.

[0054] E. Representative transmission electron micrographs of sperm heads from 12-week-old WT and Uaf1 sKO mice. Scale bar, 1 μm.

[0055] F. Representative transmission electron micrographs of the ultrastructure of spermatozoa in cross sections from 12-week-old WT and Uaf1 sKO mice. Scale bar, 200 μm.

[0056] Figure 6 .Proteomic analysis of round spermatozoa from WT and Uaf1 sKO mice.

[0057] A. Volcano plot of protein levels in wild-type and Uaf1 sKO round spermatozoa. The average protein expression ratios of 3 replicates between 8-week-old WT and Uaf1 sKO mouse round spermatozoa (log2 transformed) are plotted against the t-test p value (-log10 transformed). The cutoff values ​​for expression were at least p=0.05 and 1.3-fold change in WT or Uaf1 sKO round spermatozoa. The table shows the total number of proteins identified, as well as the number of up- and down-regulated proteins.

[0058] B. Heat map of 6312 proteins differentially expressed between wild-type and Uaf1 sKO round spermatids. These differentially expressed proteins were selected based on the following criteria: fold change ≥ 1.3 and q value < 0.05.

[0059] C. Venn diagram showing the overlap between differentially expressed proteins identified and quantified in WT and Uaf1 sKO round spermatids.

[0060] D. GO analysis of significantly changed proteins found in biological processes, molecular functions, and KEGG pathways.

[0061] E. Protein levels of KDM3A, BRDT, and MORC in testes of WT and Uaf1 sKO mice (n=3). β-Actin was used as a loading control. DETAILED DESCRIPTION

[0062] 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.

[0063] Example 1 Experimental methods and reagents

[0064] Mouse

[0065] Uaf1 in C57BL / 6J background flox / flox Mice were produced by Saiye (Guangzhou) Biotechnology Co., Ltd. using CRISPR-Pro technology. Stra8-GFPCre strain mice were produced by Shanghai Model Organisms Technology Co., Ltd. All of the above mice were raised on the C57BL / 6J (B6) background. Uaf1 flox / floxMice were crossed with Stra8-GFPCre knockout mice to obtain Uaf1 fl / -; Stra8-Cre mice, which lack Uaf1 in their germ cells, thus obtaining Uaf1-deficient mice (Uaf1 sKO mice), lacking 984 base pairs (6350-7334, including the third exon of the Uaf1 gene NM_026236.3). For animal experiments with Uaf1sKO mice, a sibling control group with normal Uaf1 expression (Uaf1 fl / fl ). All animal experiments were performed in accordance with the Health Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Science and Technology Committee of Shandong University School of Medicine. UAF1-C-HA mice were provided by the Center for Genome Tagging Project (GTP) of the Genetic Resources Center of the Chinese Academy of Sciences, which was funded by the Shanghai Science and Technology Commission (19411951800). The HA tag protein gene sequence was inserted into the C-terminus of the UAF1 protein by CRISPR / Cas9 technology, and the HA protein was co-expressed with the UAF1 protein without affecting the expression of the UAF1 protein. All animals were produced under specific pathogen-free (SPF) levels, with a humidity of 40–70%, and a daily cycle of 12 h of light, 23 °C, and 12 h of darkness, 21 °C.

[0066] Histology, immunostaining, electron microscopy, TUNEL assay

[0067] For histology, 5 μm paraffin-embedded sections of testis and epididymis were prepared and stained with periodic acid-paraffin (PAS) and / or hematoxylin.

[0068] The testicular samples were observed and photographed using a transmission electron microscope (TEM) (TECNAI-10, Philips) at an accelerating voltage of 80 kV.

[0069] For immunostaining, testicular sections were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.2% Triton X-100, and incubated with primary antibodies. The primary antibodies used for immunofluorescence were as follows: rabbit anti-UAF1 (1:200 dilution; Millipore #HPA058015), rabbit anti-UAF1 (1:200 dilution; PTG #16503-1-AP), rabbit anti-USP1 (1:200 dilution; PTG #14346-1-AP). Primary antibodies were detected with Alexa Fluor 488 or 594-conjugated secondary antibodies (1:500 dilution; Abcam #ab150084, #ab150077, #ab150113, and #ab150120) at room temperature for 1 hour. IF images were immediately captured using an LSM 780 / 710 microscope (Zeiss) or an SP8 microscope (Leica).

[0070] For TUNEL staining, the manufacturer's instructions (keyGEN BioTECH, #KGA7072) were followed.

[0071] CASA

[0072] Sperm motility was assessed using an Olympus B×51 microscope (Olympus, Tokyo, Japan) with a 20× phase objective on sperm samples from WT and Uaf1 sKO mice, with at least three mice per genotype. Ten μL of sperm samples were placed in an 80-mm-deep glass cell chamber (Hamilton Thorne, 80 mm 2X-CEl) and photographed by a CCD camera. More than 200 spermatozoa were analyzed by computer-dependent semen analysis using the Animal Motility system (Hamilton Thorne, Beverly, MA, USA). Semen analysis was repeated at least five times.

[0073] Western blotting

[0074] The immunoreactivity was detected and analyzed using the Bio-Rad ChemiDoc MP imaging system and Image Lab software (Bio-Rad, USA). The relative protein level in each sample was normalized to β-actin to standardize for loading variations. Primary antibodies for immunoblotting included anti-β-actin (1:10,000 dilution; PTG, #66009-Ig), anti-Lamin-B1 (1:3,000 dilution; PTG#12987-1-AP), anti-UAF1 (1:1,000 dilution; PTG#16503-1-AP), anti-USP1 (1:1,000 dilution; PTG#14346-1-AP), anti-KDM3A (1:1,000, PTG#12835-1-AP), anti-BRDT (1:1,000, GeneTex#GTX100201), anti-MORC3 (1:1,000 dilution; PTG#24994-1-AP), anti-FANCD2 (1:1,000 dilution; abcam#ab108928), and anti-PCNA (1:1,000 dilution; Santa Cruz Biotechnology Co., Ltd.). cruz#SC56).

[0075] Immunoprecipitation

[0076] 8-week-old testicular protein extracts were immunoprecipitated using anti-UAF1 (1:100 dilution), anti-USP1 (1:100 dilution), and anti-HA (1:100 dilution). Antibodies were incubated with WT and Uaf1 sKO testicular extracts overnight at 4°C. Separated proteins were detected by Western blotting with the indicated antibodies. Immunoprecipitation was performed using rabbit anti-HA antibody (Cell Signaling Technology #3724) and rabbit anti-IgG antibody (Abmart #B30011S).

[0077] Fluorescence activated cell sorting (FACS) of round spermatids

[0078] Round spermatozoa were collected from 8-week-old WT and Uaf1 sKO mice for purification by fluorescence-activated cell sorting for proteomics studies as described by Bastos et al., 2005; Gaysinskaya et al., 2014; Lima et al., 2017. After removal of the tunica albuginea, the testes were placed in 5 ml of PBS containing type I collagenase (120 U / ml) and mixed at 35°C for 10 min. The testes were placed in 5 ml of 0.25% trypsin plus 0.1 ml of deoxyribonuclease I (5 mg / ml) and digested at 35°C for 8 min, then stopped by adding 0.5 ml of fetal bovine serum (FBS). The suspension was passed through a 70 μm filter. The cell suspension was centrifuged at 500 g for 5 min at 4°C and the supernatant was removed. The cell pellet was then resuspended in 1 ml of DMEM and 40 μg of Hoechst33342, 2 μl of Zombie NIR TM Dye and 5 μl DNase I. The cell suspension was stirred at 34°C for 20 minutes, then centrifuged at 500 g for 5 minutes at 4°C, resuspended in PBS at a concentration of 105 ml for sorting. Using FACS, the fluorescently labeled harvested cell population was collected into a 1.5 ml microcentrifuge tube (Eppendorf, 022431021) containing 0.5 ml PBS according to the sorting channel. The cell suspension was centrifuged and part of the supernatant was removed, leaving about 10 μl PBS.

[0079] Proteomic analysis

[0080] Round spermatozoa for proteomic analysis were obtained from 8-week-old C57BL / 6 male mice and subjected to LC-MS / MS analysis according to standard protocols. In brief: (1) Round spermatozoa from 8-week-old WT and Uaf1 sKO mice were collected by FACS purification. After homogenization, cell extracts were centrifuged at 13,000 g for 20 min at 4°C. The supernatant was collected after centrifugation and the protein concentration was determined. After trypsin digestion and purification, the peptides were reconstituted in 0.5 M TEAB and processed using the TMT kit according to the manufacturer's instructions. The released peptides were identified and quantified by LCMS / MS on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (ThermoFisher Scientific). The raw data were processed using Domain Annotation (InterProScan), GO Annotation (http: / / www.ebi.ac.uk / GOA / ), and KEGG Pathway Annotation (KEGG online service tool KAAS mapper).

[0081] Bioinformatics analysis

[0082] Differentially expressed proteins were determined using a 1.3-fold change and a threshold of P < 0.05 in the Wilcoxon test. Subsequently, these proteins were analyzed using a variety of bioinformatics tools. The KEGG database was used to analyze the enriched pathways. Rpackage clusterProfiler and Metascape (http: / / metascape.org) were used to perform GO and KEGG analysis. For GO and KEGG enrichment analysis, two-tailed Fisher's exact test was applied to test the differentially expressed proteins against all identified proteins. Results with a corrected P < 0.05 were considered significant.

[0083] Statistical analysis

[0084] Statistical data are presented as mean ± standard error. Most experiments included at least three independent samples and were repeated at least three times. Two-tailed unpaired Student's t-test was used to compare the results of two experimental groups. Two-tailed Student's t-test, p < 0.05, p < 0.01, p < 0.001 were considered statistically significant and are indicated by asterisks (*), (**), and (***), respectively. "ns" indicates not significant.

[0085] Example 2 UAF1 interacts with USP1 in mouse testis

[0086] UAF1 binds to and activates a complex of three deubiquitinases, including USP1, USP12, and USP46. To determine which deubiquitinase binds to UAF1 in the testis, testicular protein extracts were immunoprecipitated and analyzed by mass spectrometry using a UAF1 antibody. Mass spectrometry detected the presence of USP1 and USP12 in the UAF1 complex purified from mouse testis ( Figure 1 A). Next, testicular protein extracts were immunoprecipitated and analyzed by mass spectrometry using USP1 antibodies. USP1 was identified as a UAF1 interacting partner in mouse testis ( Figure 1 B). The interaction between UAF1 and USP1 in testis was verified by co-immunoprecipitation and immunoblotting, and it was confirmed that i) UAF1 immunoprecipitation was pulled to USP1 in WT mouse testis, and ii) USP1 immunoprecipitation was pulled to UAF1 in WT mouse testis ( Figure 1 CD).

[0087] In addition, Uaf1-HA tag knock-in mice were constructed and co-immunoprecipitation was performed in UAF1-HA mouse testis, again indicating an interaction between UAF1 and USP1 ( Figure 1 E).

[0088] Together, these results suggest that UAF1 and USP1 form a deubiquitinase complex in the testis and are involved in male spermatogenesis.

[0089] To explore the potential function of UAF1 in mammalian male spermatogenesis, we first examined the expression pattern of UAF1 in mouse testis. Immunoblotting results showed that UAF1 was expressed in different mouse organs, including ovaries and testes. Cytoplasmic-nuclear separation of testicular proteins was performed, and it was found that UAF1 and USP1 were expressed in both the cytoplasm and the nucleus ( Figure 2 A). Germ cell development is strictly defined during mouse embryonic and postnatal development. UAF1 was detected in the testes of postnatal day 8 mice, and its accumulation level was observed to increase continuously during postnatal development until adulthood ( Figure 2 B) These results suggest that UAF1 may play a role in male spermatogenesis.

[0090] Subsequently, immunofluorescence staining was performed on paraffin sections of wild-type (WT) adult mouse testes to assess the expression and subcellular localization of UAF1 protein during male spermatogenesis. Within the spermatic tubule epithelium, spermatogenesis is described as a sequence of 12 spatially synchronized stages (I-XII) along the seminiferous tubules, with individual stages represented in a cross-section of one seminiferous tubule. Spermatogenesis is further divided into 16 steps (Sp.1-16), defined by the post-meiotic morphology of the sperm acrosome and nucleus. Combined staining with UAF1 antibody and acrosomal antigen PNA showed that UAF1 is present in most germ cell development stages, including mitotic spermatogonia, meiotic spermatocytes, and early round spermatids ( Figure 2 C). UAF1 is abundant in the cytoplasm of early spermatocytes and subsequently translocates to the nuclei of late spermatocytes and round spermatids, with the highest levels of UAF1 detected in the nuclei of 1-2 spermatids ( Figure 2 C). In addition, it was found that UAF1 and USP1 showed similar dynamic translocation expression patterns in male germ cells of different subtypes.

[0091] Immunofluorescence analysis of Uaf1-HA-tagged knock-in mouse testes showed that the same UAF1 accumulation pattern as in WT animals was observed using anti-UAF1 antibodies. In addition, the nuclear localization of UAF1 and USP1 during meiotic prophase was confirmed by immunostaining of chromosome spreads. Together, these results reveal that UAF1 and USP1 exhibit dynamic nuclear and cytoplasmic expression patterns in postnatal male germ cells and may be involved in the formation of male sperm.

[0092] Example 3 UAF1 is essential for male fertility

[0093] In order to clarify the physiological function of UAF1 in male spermatogenesis, a conditional knockout method was used to knock out Uaf1 fl / fl The mutant strain (in which exons 2 and 4 of the Uaf1 gene are framed by loxP sites) was mated with Stra8-Cre knock-in mice, which express CRE recombinase in male germ cells at PD3 (i.e., before they initiate meiosis). Figure 3 A). Immunoblotting results showed that the UAF1 protein level in the testes of Uaf1 sKO male mice was significantly decreased compared with WT ( Figure 3 B). In addition, immunofluorescence staining results showed that UAF1 was absent in the germ cells of Uaf1 sKO males ( Figure 3 C) These results indicate that the UAF1 gene was successfully knocked out in male germ cells.

[0094] Although Uaf1 sKO male mice were viable and appeared normal, fertility tests after 5 months showed that they were completely infertile ( Figure 3 D). Consistent with this infertility phenotype, the testis size of Uaf1 sKO mice was significantly smaller than that of WT, approximately 50% of that of WT ( Figure 3 E), and the testis / body weight ratio was significantly reduced ( Figure 3 F). Unlike males, Uaf1 sKO female mice have normal fertility. Ovarian morphological analysis showed that Uaf1 sKO ovaries exhibited normal ovarian morphology at 3 and 6 months of age.

[0095] Thus, conditional knockout of Uaf1 resulted in spermatogenesis defects and infertility in males but did not affect female fertility, suggesting that UAF1 has distinct functions in male and female fertility.

[0096] Example 4 Haploid germ cell development defects in Uaf1 sKO mice

[0097] To further explore the function of UAF1 in male spermatogenesis, hematoxylin staining was performed on the testicular tissues of 12-week-old WT and Uaf1 sKO mice. The testicular sperm of WT mice developed normally, with elongated sperm in almost all tubules, while the testes of adult Uaf1 sKO mice showed a decrease in the diameter of the seminiferous tubules and a decrease in the number of germ cells ( Figure 4 A). Immunofluorescence staining of testicular tissue showed that staining for the germ cell-specific marker MVH indicated that Uaf1sKO mice had a large loss of germ cells in the testes ( Figure 4 B).

[0098] Subsequent immunofluorescence staining of the nuclear chromosome synchronization marker SYCP3 was performed on the sections of seminiferous tubules of adult mice, and it was found that testicular spermatogenesis in Uaf1 sKO mice entered the meiotic stage. In addition, Uaf1 sKO spermatocytes were analyzed by chromosome unfolding stained with SYCP1, SYCP3, γH2AX, and MLH1. There were no significant differences between WT and Uaf1 sKO spermatocytes. The initiation of meiosis (marked by SYCP1), the repair of DNA double-strand breaks (marked by γH2AX), and recombination events (marked by MLH1) were not affected. These observations indicate that the sperm formation defect in Uaf1 sKO male mice occurs after meiosis.

[0099] Subsequently, postmeiotic development of the testes of Uaf1 sKO mice was assessed using PAS-hematoxylin staining ( Figure 4 C). The early elongated sperm state of the seminiferous tubules of Uaf1 sKO mice was not changed. However, late sperm development was affected in Uaf1 sKO mice: fewer sperm were identified in the condensed sperm (stage I, II-III), the number of mature sperm was sharply reduced (stage IV-VI, VII-VIII), and apoptosis signals were detected in late sperm ( Figure 4 D). By examining the histology of Uaf1 sKO and WT mice at different developmental time points (PD24, 28, 32, and 45), we found that Uaf1 sKO mice had abnormal sperm tubules and delayed development of round sperm starting from PD28. After testicular knockout of Uaf1, germ cells were gradually lost from PD28, and the onset of the first wave of germ cell development was significantly delayed in Uaf1 sKO mice.

[0100] These data indicate that haploid germ cell development is defective in Uaf1 sKO mice.

[0101] Example 5 Abnormal spermatogenesis in Uaf1 sKO mice

[0102] The epididymis was dissected and mature sperm was examined. It was found that there were very few sperm in the epididymal cavity of Uaf1 sKO mice. It was also observed that immature germ cells and abnormal sperm accumulated in the cauda epididymis of Uaf1 sKO mice ( Figure 5 A). Examination of sperm released from the cauda epididymis revealed a significant decrease in sperm counts in Uaf1 sKO mice compared with WT mice ( Figure 5 B) To determine sperm morphological characteristics, single sperm were analyzed by HE and found that Uaf1 sKO sperm exhibited many abnormalities, including abnormal head morphology, headless / headless sperm, and sperm with midpiece bending ( Figure 5C). Compared with WT, the ratio of sperm heads and flagella in Uaf1 sKO sperm was significantly increased ( Figure 5 D). In fact, transmission electron microscopy (TEM) examination of sperm further confirmed the abnormal morphology of the Uaf1 sKO sperm head ( Figure 5 E). Abnormal mitochondrial arrangement was also observed in Uaf1sKO spermatogonia ( Figure 5 F).

[0103] Since Uaf1 sKO mice were still able to produce some sperm, computationally assisted semen analysis (CASA) was used to characterize these sperm and revealed that the motility, or ability to move forward, of the sperm in Uaf1 sKO mice was poor; therefore, on top of the reduced sperm count, sperm function was also impaired in Uaf1 sKO mice. The irregularly shaped sperm heads, reduced sperm count, and decreased sperm motility collectively contributed to the infertility phenotype of Uaf1 sKO male mice.

[0104] Collectively, these observations suggest that the infertility of Uaf1 sKO male mice is due to defects in the post-meiotic period of spermatogenesis in males.

[0105] Example 6 UAF1 controls the stability and subcellular localization of USP1 in the testis

[0106] In this study, UAF1 and USP1 were found to form a deubiquitinase complex in the testis. During spermatogenesis, the expression of UAF1 showed a dynamic nuclear-cytoplasmic translocation expression pattern. Similarly, USP1 also had a similar dynamic expression pattern between the cytoplasm and nucleus of different types of male germ cells. Overall, UAF1 was found to interact and co-localize with USP1 in the testis.

[0107] To investigate whether UAF1 regulates spermatogenesis through USP1, immunofluorescence staining of USP1 was performed in the testes of Uaf1 sKO males. Compared with WT, it was found that the level of USP1 in the testes of Uaf1 sKO mice was significantly reduced, the level of intact USP1 was significantly reduced, and the N-terminal USP1 cleavage product was almost completely disappeared, indicating that UAF1 regulates the stability of USP1. To investigate the effect of UAF1 on the subcellular distribution of USP1 in the testis, the localization of USP1 in Uaf1 sKO and WT spermatids at different developmental stages was examined. It was observed that the abundance of USP1 was higher in the cytoplasm of Uaf1 sKO round spermatids.

[0108] These results reveal a requirement for UAF1 for the stabilization and subcellular localization of USP1 during spermatogenesis and that UAF1 regulates spermatogenesis through the deubiquitinase function of USP1.

[0109] Example 7: Changes in the levels of proteins related to spermatogenesis in the testis of Uaf1 sKO mice

[0110] Spermatogenesis is a complex developmental process that is regulated by gene transcription and translation as well as targeted protein degradation. Previous studies have shown that spermatogenesis depends on the balance between ubiquitination and deubiquitination. To understand the protein changes that occur during spermatogenesis in the testis of Uaf1 sKO mice, round spermatids were isolated from WT and Uaf1 sKO mouse testes using flow cytometry (FACS) sorting, followed by quantitative TMT-based proteomic analysis. A total of 684 differentially expressed proteins were identified (using a 1.3-fold change cutoff), of which 303 were downregulated and 381 were upregulated ( Figure 6 AC). Gene ontology analysis revealed that functionally enriched terms included "protein-DNA complex assembly", "spermatogenesis" and "histone modification" ( Figure 6 D).

[0111] Based on previously reported studies on knockout experiments affecting male fertility, the focus was on three proteins (KDM3A, BRDT, MORC3). Immunoblotting analysis showed that the levels of KDM3A, BRDT, and MORC3 were significantly reduced in round spermatogonia of Uaf1 sKO mice. In addition, the effects of UAF1 on the classic downstream molecules FANCD2 and PCNA were verified, and their expression was found to be reduced in Uaf1 sKO mice.

[0112] These results suggest that UAF1 is a regulator of normal reproductive function through its regulation of multiple proteins involved in spermatogenesis.

[0113] The inventors of this application first discovered that conditional knockout of Uaf1 leads to male spermatogenesis defects and infertility, but does not affect female fertility, suggesting that UAF1 has different functions in male and female fertility. In male mice, the deubiquitinase cofactor UAF1 is specifically involved in spermatogenesis. Uaf1 sKO male mice are completely infertile because the development of haploid germ cells is disrupted, accompanied by abnormal sperm morphology and function. The inventors' findings indicate that UAF1, as a scaffold protein with protease-specific peptidase activity, interacts with USP1 and participates in spermatogenesis. By performing proteomic studies on round spermatids, the inventors explored the function of UAF1 in spermatogenesis to elucidate the potential mechanism of UAF1 involvement in male infertility. The results showed that the levels of proteins that are essential for spermatogenesis and fertilization were significantly reduced in Uaf1 sKO mice. In addition, bioinformatics analysis and literature review also revealed that some important proteins that have recently been found to cause male infertility, such as KDM3A, BRDT, and MORC3, were significantly downregulated. Through the inventors' research, UAF1 was identified as a cofactor of a deubiquitinase, which is indispensable for guiding spermatogenesis in male mice. The formation of a complex between UAF1 and USP1 in the testis and the regulation of multiple proteins involved in spermatogenesis by UAF1 are of great significance for understanding the deubiquitinating regulatory mechanism of mammalian spermatogenesis.

[0114] Applicants have thus provided methods for diagnosing fertility defects such as infertility disorders in mammals, including humans, and further facilitated the development of new treatments for male infertile individuals affected by gene mutations.

[0115] The above is an explanation of the present invention and cannot be regarded as a limitation of the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that the present invention can also be realized in other ways except that it is particularly described in the above description and embodiments. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

Claims

1. Use of a reagent for detecting UAF1 or UAF1 / USP1 deubiquitinase complex in a subject in the preparation of a kit for diagnosing fertility defects such as infertility in mammals of different sexes.

2. The use according to claim 1, wherein the kit further comprises a reagent for detecting the gender of the subject.

3. The method according to claim 1, wherein the infertility disorder is selected from teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia and asthenozoospermia.

4. The use according to any one of claims 1 to 3, wherein the detection reagent is a reagent for detecting Uaf1, or Uaf1 and Usp1 genes.

5. The use according to any one of claims 1-3, comprising a reagent for detecting UAF1, or UAF1 and / or USP1 protein, preferably, further comprising a reagent for determining the activity of UAF1 and USP1 protein.

6. The use according to claim 5, comprising a reagent for detecting the expression of UAF1 and / or USP1 protein by immunoassay, such as a reagent for detecting the expression by ELISA or Western blotting using an antibody that specifically recognizes the protein; or These include reagents for detecting the presence or amount of mRNA of UAF1 and / or USP1 protein, such as reagents for detecting the amount of mRNA encoding the protein in a sample by RT-PCR.

7. A kit for diagnosing fertility defects such as infertility in mammals of different sexes, comprising a reagent for detecting UAF1 or UAF1 / USP1 deubiquitinase complex in a subject, and a reagent for detecting the sex of the subject.

8. The kit of claim 7, wherein the infertility disorder is selected from teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, and asthenozoospermia. 9 . The kit according to claim 7 , wherein the reagent for detecting UAF1 or UAF1 / USP1 deubiquitinase complex in the subject is a reagent for detecting Uaf1, or Uaf1 and Usp1 genes.

10. The kit according to claim 7, wherein the reagent for detecting UAF1 or UAF1 / USP1 deubiquitinase complex in the subject is a reagent for detecting UAF1, or UAF1 and / or USP1 protein, and preferably, it also includes a reagent for measuring the activity of UAF1 and USP1 protein.

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