Application of meiosis factor in diagnosis and treatment of fertility defects

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 UBE2J2 of a tested individual. The invention also provides kits and instruments for diagnosing or predicting fertility defects, such as infertility disorders, in mammalian individuals by detecting UBE2J2. 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] In men with non-obstructive azoospermia, the seminiferous tubules are intact, but spermatogenesis is impaired, resulting in the absence of sperm in the ejaculated semen. Spermatogenesis involves a series of precisely regulated sequential processes, including spermatogonia self-renewal and differentiation, spermatocyte meiosis, and sperm maturation.

[0003] The incidence of azoospermia in male infertility is 10-15%, which can be divided into nonobstructive azoospermia (NOA) and obstructive azoospermia (OA). NOA accounts for about 60% of azoospermia and manifests as spermatogenesis disorder. During spermatogenesis, a large amount of protein synthesis and degradation, the degradation of proteins and organelles is closely related to the ubiquitin-proteasome pathway.

[0004] Meiosis is the cell division process that transforms diploid germ cells into haploid gametes. The DNA of spermatogonia is replicated once, and the cells divide twice continuously. Finally, one diploid spermatogonia forms four haploid sperm. Meiosis is a conservative and special form of cell division that separates chromosomes and produces gametes with genetic diversity. Homologous chromosome separation occurs in the late stage of meiosis I, and sister chromatid separation occurs in the late stage of meiosis II. In order to ensure the correct separation of homologous chromosomes with maternal and paternal origins in the early stage of meiosis I, cells undergo a series of orderly events, including the formation of DNA double strand breaks (DSBs), homologous chromosome pairing and synapsis, homologous recombination (HR) and crossovers (COs) formation. The correct and orderly occurrence of these events plays a decisive role in the normal sexual reproduction of organisms. In this study, ubiquitin-conjugating enzyme E2 J2 (UBE2J2) is involved in the degradation of endoplasmic reticulum-related proteins. Its structure contains a ubiquitin-binding domain and a transmembrane domain. The ubiquitin-binding domain is involved in the transport of ubiquitin molecules, and the transmembrane domain can position itself on the endoplasmic reticulum membrane.

[0005] Ubiquitin-conjugating enzyme E2 J2 (UBE2J2) is a highly conserved E2 protein among species, involved in the degradation of endoplasmic reticulum-associated proteins (ERAD). The structure of UBE2J2 contains a ubiquitin-binding domain and a transmembrane domain. The ubiquitin-binding domain is the core domain of all E2 enzymes, in which the ubiquitin-binding domain is involved in the transport of ubiquitin molecules, and the transmembrane domain can locate itself on the endoplasmic reticulum membrane. The degradation of endoplasmic reticulum-associated proteins is achieved by recognition, extraction, and ubiquitination, and the delivery of endoplasmic reticulum membrane proteins or secretory proteins to the proteasome. Another protein in the UBE2J family, UBE2J1 (Ubiquitin-conjugating enzyme E2 J1), has been constructed with knockout mice. The knockout mice have no obvious morphological or behavioral abnormalities, but male mice are infertile due to defects in sperm flagella and acrosome function, as well as defects in cytoplasm removal in elongated spermatids. The research on UBE2J2 mainly focuses on the following aspects, including the degradation of human leukocyte antigen (HLA) molecules by binding to E3 TMEM129 (Transmembrane protein 129) in the immune aspect; regulating the degradation of squalene monooxygenase (SQLE) by binding to E3 MARCH6 (Membrane-associated ring-CH-typefinger 6) in the metabolic aspect to maintain cholesterol metabolic homeostasis; regulating the polyubiquitination of Wnt cargo receptor protein Evi by binding to E3 CGRRF1 (Cell growth regulator with ring finger domain 1) to degrade it and control its expression; and promoting epithelial-mesenchymal transition and invasion of hepatocellular carcinoma in vitro. At present, there are no reports on the role of UBE2J2 in reproduction.

[0006] 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

[0007] The inventors first discovered the role of UBE2J2 in spermatogenesis. The inventors constructed Ube2j 2- / -The role of UBE2J2 in spermatogenesis was studied in mice, and it was found that Ube2j2 gene knockout caused azoospermia in male mice, and spermatocytes were arrested in the pachytene stage of the first prophase of meiosis. In order to exclude the possibility that UBE2J2 affects the reproductive system by affecting other tissues and organs, the inventors of the present application constructed a conditional knockout of Ube2j2 in germ cells. 2fl / - ; Stra8-Cre mice, whose phenotypes are consistent with those of whole-body knockout mice, further prove that UBE2J2 plays an important role in the prophase of the first meiotic division of spermatocytes. This study proves that UBE2J2 plays an important role in the first meiotic division of spermatogenesis, providing theoretical support for elucidating the pathological mechanism of UBE2J2 in meiosis.

[0008] Thus, the inventors provide a method for diagnosing or treating fertility defects such as infertility disorders in mammals, particularly humans.

[0009] In one aspect of the present invention, a method for diagnosing fertility defects such as infertility in mammals of different sexes by detecting UBE2J2 in a test subject is provided. The method may further include the step of detecting the sex of the test subject.

[0010] 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 UBE2J2 in a test subject. In the present invention, the kit also comprises a reagent for detecting the sex of the test subject, in particular, detecting whether the test subject is male.

[0011] The present invention thus provides the use of a reagent for detecting UBE2J2 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.

[0012] In the present invention, infertility disorders include but are not limited to teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, asthenozoospermia and the like.

[0013] 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.

[0014] UBE2J2, or ubiquitin-conjugating enzyme E2 J2 (UBE2J2), is a highly conserved E2 protein among species, involved in the degradation of endoplasmic reticulum-related proteins. The structure of UBE2J2 contains a ubiquitin binding domain and a transmembrane domain. The ubiquitin binding domain is the core domain of all E2 enzymes, in which the ubiquitin binding domain is involved in the transport of ubiquitin molecules, and the transmembrane domain can position itself on the endoplasmic reticulum membrane.

[0015] The Ube2j2 gene encoding UBE2J2 is highly conserved in mammals, wherein the Gene ID of the human Ube2j2 gene is 118424 and the Gene ID of the mouse Ube2j2 gene is 140499.

[0016] 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 UBE2J2 of the test individual, whether the UBE2J2 of the test individual is abnormal is detected, which includes comparing the activity of the subject's UBE2J2 gene or its regulatory factor, or the expression of UBE2J2 protein, or comparing with the situation of the gene or protein in normal people in the general or specific group. If there is a significant change, it is judged that an abnormality exists.

[0017] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include steps and reagents for detecting the genes of each component of the aforementioned complex, such as detecting the Ube2j2 gene encoding UBE2J2.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include a step of detecting UBE2J2 protein, such as a step of detecting UBE2J2 protein, which 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.

[0022] 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.

[0023] In yet another aspect of the present invention, the method and kit include steps and reagents for determining the activity of UBE2J2 protein.

[0024] The present invention also provides an apparatus (including a gene chip) for diagnosing or predicting fertility defects such as infertility in mammalian individuals.

[0025] The method and kit provided by the present invention are based on the discovery that the effect of UBE2J2 on the fertility of mammals is sex-dependent, so the diagnosis and prediction made by the method or kit provided by the present invention are different for mammals of different sexes. Therefore, in the method provided by the present invention, it is also included to detect the sex of the subject. In the kit provided by the present invention, it is also included to detect the sex of the subject. Methods and reagents for detecting the sex of the subject are well known in the art, for example, including detecting the sex-specific protein or gene of the subject, such as the specific nucleic acid sequence on the sex chromosome, etc.

[0026] The present invention also provides a method for treating or avoiding fertility defects in a mammalian individual, comprising treating an individual having a male UBE2J2 defect, such as the presence of UBE2J2 protein or a gene thereof.

[0027] In this article, protein symbols are not italicized and are all capitalized; gene symbols are italicized. For example, UBE2J2 is a protein, and the gene encoding the protein is written as Ube2j2. Sometimes in this article, protein symbols are not all capitalized. For example, sometimes "Ube2j2" in this article means UBE2J2 protein. Sometimes in this article, gene symbols are not italicized. For example, sometimes "Ube2j2" or "UBE2J2 gene" in this article means the gene Ube2j2 encoding UBE2J2 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The expression profile of UBE2J2 in various tissues and testes of different ages is shown. (A) The protein expression of UBE2J2 in various tissues of mice was detected by Western Blot. The arrow indicates the target band of UBE2J2. (B) The expression of UBE2J2 in the testes of mice of different ages was detected by Western Blot. PD, days after birth.

[0029] Figure 2 The knockout scheme and knockout efficiency verification of Ube2j2 gene knockout mice are shown. (A) Schematic diagram of the knockout scheme of Ube2j2 gene knockout mice, which deleted 4977 bases including 4 exons. (B) Western blot was used to detect the knockout efficiency of UBE2J2 in mouse testis.

[0030] Figure 3 Shows the pathological results of the testis and epididymis of Ube2j2 knockout mice. (A) Comparison of the testis morphology and size of wild-type and Ube2j2 knockout mice at 60 days after birth. Below the testis is a ruler, and a small grid represents 1 mm. (B) Pathological sections of the testis and epididymis tail of wild-type and Ube2j2 knockout mice at 60 days after birth, and hematoxylin staining after fixation in Bouin's solution. Arrows indicate seminiferous tubules without spermatocytes, and triangular arrows indicate apoptotic pachytene spermatocytes. St, seminiferous tubules; Ep, epididymis.

[0031] Figure 4 Ube2j2 knockout spermatocytes are arrested at the mid-pachytene stage. (A) Adult control mice and Ube2j2 - / - The results of SYCP1 and SYCP3 immunofluorescence staining of mouse spermatocyte chromosome spreads, showing spermatocytes in the leptotene, zygotene, early pachytene, mid-pachytene, and late pachytene stages. (B) Adult control mice and Ube2j2 - / -Results of H1t and SYCP3 immunofluorescence staining of chromosome spreads of mouse spermatocytes.

[0032] Figure 5 The homologous recombination complexes in spermatocytes from Ube2j2 knockout mice are unstable, as shown in the figure for adult control mice and Ube2j2 - / - Mouse spermatocyte chromosome spreads for MSH4 and SYCP3 immunofluorescence staining. The image shows spermatocytes at the leptotene stage and early, mid, and late pachytene stages.

[0033] Figure 6 The results show that the homologous recombination complexes in spermatocytes from Ube2j2 knockout mice are abnormally processed and matured. (A) - / - Mouse spermatocyte chromosome spreads RNF212 and SYCP3 immunofluorescence staining results. The figure shows spermatocytes in the early and mid-pachytene stages. (B) Adult control mice and Ube2j2 - / - Mouse spermatocyte chromosome spreads HEI10 and SYCP3 immunofluorescence staining results. The figure shows spermatocytes in the early and mid-pachytene stages.

[0034] Figure 7 The results show that Ube2j2 knockout leads to reduced SUMOylation in spermatocytes. (A) Adult control mice and Ube2j2 - / - SUMO2 / 3 and SYCP3 immunofluorescence staining results of mouse spermatocyte chromosome spread. (B) Adult control mice and Ube2j2 - / - Mouse spermatocyte chromosome spreads for SUMO1 and SYCP3 immunofluorescence staining. The image shows spermatocytes at the leptotene stage and early, mid, and late pachytene stages.

[0035] Figure 8 The results showed that spermatocytes from Ube2j2 knockout mice could not form chiasmata, which was consistent with the results of adult control mice and Ube2j2 - / - Mouse spermatocyte chromosome spreads MLH1 and SYCP3 immunofluorescence staining results. The picture shows spermatocytes in the early and mid-pachytene stages.

[0036] Fig. 9 The results show that there is no change in the ubiquitination level in Ube2j2 knockout mice. (A) Adult control mice and Ube2j2 - / - Results of immunofluorescence staining of FK2 and SYCP3 in chromosome spreads of mouse spermatocytes. FK2 can detect ubiquitinated proteins and ubiquitin chains. (B) Western Blot detection of control mice and Ube2j2 at 18 days after birth - / - Ubiquitin content in mouse testis.

[0037] Fig.10The Ube2j2 knockout mice showed significant phenotype differences at PD14. (A) Control mice and Ube2j2 knockout mice aged from PD12 to PD14 - / - Body weight of mice. (B) Control mice and Ube2j2 mice aged PD12 to PD14 - / - Testis weight of mice. (C) Control mice and Ube2j2 mice aged PD12 to PD14. - / - Body-to-testis ratio of mice. Statistical method is T test, Error Bar represents standard error, ** represents P < 0.01. (D) PD13 and PD14 age control mice and Ube2j2 - / - Mouse testis pathological section, hematoxylin staining after Bouin's solution fixation. Arrows indicate empty seminiferous tubules without spermatocytes.

[0038] Fig.11 The results of GO enrichment analysis and GSEA analysis of differentially expressed proteins in Ube2j2 knockout mice are shown. (A) Ube2j2 - / - GO enrichment analysis of differentially expressed down-regulated proteins in mouse testis, with less than 1 / 1.5 fold as the threshold for significant down-regulation. BP, biological process; CC, cellular component; MF, molecular function. (B, C) Ube2j2 - / - GSEA analysis of all protein expressions in mouse testis (P<0.01, FDR<0.1).

[0039] Fig.12 The results show that Ube2j2 knockout leads to abnormal recruitment of CDK1 and PLK1 to chromosome axes in spermatocytes. (A) Adult control mice and Ube2j2 - / - PLK1 and SYCP3 immunofluorescence staining results of spermatocyte chromosome spreads of mice. (B) Adult control mice and Ube2j2 - / - Mouse spermatocyte chromosome spread CDK1 and SYCP3 immunofluorescence staining results. The picture shows spermatocytes in the early and mid-pachytene stages.

[0040] Fig.13 We show that Ube2j2 knockout results in a failure of HSPA2 recruitment to chromosome periphery in spermatocytes. - / - Mouse spermatocyte chromosome spreads for HSPA2 and SYCP3 immunofluorescence staining. The figure shows spermatocytes in the early, mid, and late pachytene stages.

[0041] Fig.14UBE2J2 is localized in the nucleus. (A) Western Blot was used to detect the protein expression of HSPA2 in the testis of PD14 mice. (B) Western Blot was used to detect the expression of UBE2J2 in the cytoplasm and nucleus of the testis of mice. β-actin indicates the cytoplasm, and Lamin B1 indicates the nucleus. DETAILED DESCRIPTION

[0042] 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.

[0043] Example 1 Experimental methods and reagents

[0044] Experimental animals

[0045] The mouse Ube2j2 gene (NCBI sequence number: NM_021402, Ensembl: ENSMUSG00000023286) is located on mouse chromosome 4 and consists of 8 exons, with the ATG start codon located in exon 2 and the TGA stop codon located in exon 8 (transcript number: ENSMUST00000024056). The Ube2j2 gene knockout mouse was constructed by Saiye (Suzhou) Biotechnology Co., Ltd., using CRISPR-cas9 technology to knock out a 4977bp base fragment in exons 4 to 7 of the Ube2j2 gene in C57BL / 6 background mice, and the gene knockout positive mice were constructed by extracting mouse paw or mouse tail genomic DNA for PCR and sequencing. The PCR identification of Ube2j2 knockout mice shared an upstream primer 5'-GTTTATGCACCTGTCTCCTCCAG-3', and the upstream primer and downstream primer 5'-CCATTTGGGAGCAGAACTCAAG-3' produced a 556bp fragment for identifying gene knockout (Knockout, KO), and the downstream primer 5'-TCTAGAACTCCAGATACTGACCC-3' produced a 699bp fragment for identifying wild type (Wildtype, WT). By hybridizing with mice with ICR background, the offspring of mice with C57BL / 6 and ICR mixed background were obtained. Wild-type C57BL / 6 and ICR mice used for expansion were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0046] Ube2j2 fl / + The mice were constructed by Saiye (Suzhou) Biotechnology Co., Ltd., and the 2402bp base fragment of exons 5 to 8 was selected as the conditional gene knockout (cKO) region. Stra8-Cre transgenic mice were from Tong Minghan's laboratory at the Chinese Academy of Sciences. fl / +Mice were crossed with Stra8-Cre transgenic mice to obtain Ube2j2 fl / - Stra8-Cre mice, i.e., Ube2j2 cKO mice with specific knockout of Ube2j2 gene in germ cells. The constructed starting mice were identified by PCR and sequenced for sequence analysis. The PCR identification of Ube2j2 cKO mice shared an upstream primer 5'-TACATAGAGATTCTCCACACACGG-3'. The upstream primer and the downstream primer 5'-AGTCGAGTAACGAGCTACCAAAAG-3' produced a 380 bp fragment for identifying cKO, and the downstream primer 5'-ACAAAAGGTCTGGCACTACAGTTT-3' produced a 169 bp fragment for identifying WT and a 229 bp fragment for identifying flox successfully inserted. The PCR identification of Stra8-Cre transgenic mice shared a common upstream primer 5'-ACTCCAAGCACTGGGCAGAA-3', and the upstream primer was combined with the downstream primer 5'-GCCACCATAGCAGCATCAAA-3' and the downstream primer 5'-CGTTTACGTCGCCGTCCAG-3' to generate a 400 bp fragment for identifying the successful knock-in of Stra8 and a 702 bp fragment for identifying the WT.

[0047] All experimental mice were housed in an SPF-level animal room with a 12-h light (6:00-18:00) and 12-h dark cycle per day, with sufficient sterile food and double-distilled water for drinking, and the temperature was maintained at 22-24°C.

[0048] All animal experiments were approved by the Animal Ethics Committee of Shandong University and conducted in strict accordance with the relevant regulations and operating procedures established by the Animal Protection Association of Shandong University.

[0049] Antibodies used

[0050] Rabbit anti-UBE2J2 polyclonal antibody was prepared by Dion Biotech (Wuhan, China) and used at a dilution of 1:1,000.

[0051] Antibodies used for Western blotting also include Beta Actin (Proteintech, 66009-1-Ig), Lamin B1 (Proteintech, 12987-1-AP), and Ubiquitin P4D1 (Cell Signaling Technology, 3936S).

[0052] Antibodies used for immunofluorescence staining included SYCP3 (Abcam, ab15093, ab97672), SYCP1 (Abcam, ab15090), H1t (Abcam, ab81498), γH2AX (Millipore, 05-636), ATR (Santa Cruz, sc-515173), pATM (Sigma-Aldrich, 05-740), RPA2 (Abcam, ab76420), RAD51 (Thermo Fisher Scientific, PA5-27195), MSH4 (Abcam, ab58666), RNF212 (Wuhan University, gifted from Dr. Mengcheng Luo), HEI10 (Abcam, ab71977), MLH1 (BD Biosciences, Inc., Boston College of Science). Biosciences, 550838), FK2 (Ubiquigent, 68-0121-500), HSPA2 (Proteintech, 12797-1-AP), CDK1 (Abcam, ab18), PLK1 (Abcam, ab17056). Secondary antibodies were Alexa Fluor 488- or 594-conjugated secondary antibodies (Abcam, ab150077, ab150120, ab150080, ab150117).

[0053] Histology, immunostaining, electron microscopy

[0054] For histology, 5 μm paraffin-embedded sections of testis and epididymis were prepared and stained with periodic acid-paraffin (PAS) and / or hematoxylin.

[0055] The testicular samples were observed and photographed using a transmission electron microscope (TEM) (TECNAI-10, Philips) at an accelerating voltage of 80 kV.

[0056] For immunostaining, testicular sections were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.2% Triton X-100, and incubated with primary antibodies. Primary antibodies were detected with secondary antibodies (1:500 dilution) for 1 h at room temperature. IF images were immediately captured using an LSM 780 / 710 microscope (Zeiss) or an SP8 microscope (Leica).

[0057] Western blotting

[0058] 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.

[0059] Proteomic analysis

[0060] The samples were sent to Hangzhou Jingjie Biotechnology Co., Ltd. on dry ice for whole-protein ubiquitination modification and whole-protein 4D Label-free quantitative proteomics determination of mouse testis samples.

[0061] Data Analysis:

[0062] ① Analysis of protein differential expression and ubiquitination modification site differences: First, select the samples to be compared, calculate the average quantitative value of modification sites / proteins in repeated samples, and finally calculate the fold change (FC) of the comparison group. In order to determine the significance of the difference, the relative quantitative value of each modification site / protein in the two comparison pairs of samples was subjected to T-test, and P≤0.05 was used as the significance index by default. In order to make the test data conform to the normal distribution required by the T test. Before the test, the relative quantitative value of the modification site / protein needs to be transformed by Log2 logarithm. Through the above difference analysis, when P≤0.05, the difference expression change of more than 1.5 is used as the threshold of significant upregulation, and less than 1 / 1.5 is used as the threshold of significant downregulation. In order to obtain proteins / ubiquitination modifications specifically expressed in WT or KO, all WT group and all KO group proteins are summed, and proteins specifically expressed in WT / KO group are obtained and Venn diagrams are drawn.

[0063] ②Gene Ontology (GO) and pathway enrichment (Kyoto Encyclopedia of Genes and Genomes, KEGG) analysis of differentially expressed proteins was completed by clusterProfiler (4.0.5) and Metascape (https: / / metascape.org). Fisher test was performed and significant enriched GO entries with P < 0.01 and Min Overlap = 3 were retained. KEGG (https: / / www.kegg.jp / ) is a database that integrates molecular features, especially data generated by genome sequencing, to elucidate these functions and interactions. Metascape was used to analyze the enrichment of differentially expressed genes in KEGG pathways. The enrichment analysis results were plotted using R packages clusterProfiler and ggplot2 (3.3.6).

[0064] ③Gene set enrichment analysis (GSEA) was performed using the GSEA (version 4.2.3) method with all protein expression data and phenotypic classification as materials to find out whether the overall expression of the pathway is inhibited or activated. In the results, gene sets with P < 0.01 and FDR < 0.1 were considered to be significantly enriched in a certain pathway.

[0065] Immunoprecipitation-Mass Spectrometry (IP-MS)

[0066] ①IP, add UBE2J2 antibody, and the operation steps are the same as described in the previous IP part.

[0067] ② When performing Western Blot electrophoresis, stop electrophoresis when the colored loading buffer runs into the separation gel for about 1.5 cm.

[0068] ③After Coomassie Brilliant Blue staining and decolorization, cut off the protein-containing gel strip, add some ddH2O to keep it moist, and place it in an EP tube.

[0069] ④ Send samples. Subsequent sample processing and data analysis by IP-MS will be performed by Jingjie Biotechnology Co., Ltd., which will provide information on the proteins contained in the strips, that is, proteins that may interact with UBE2J2.

[0070] Statistical analysis

[0071] Statistical data are presented as mean ± standard error. 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.

[0072] Example 2 UBE2J2 expression in testis increases with age

[0073] Analysis of the expression profile of UBE2J2 revealed that UBE2J2 protein is expressed in mouse liver, ovary, and testis. This suggests that in addition to its known role in hepatocellular carcinoma, UBE2J2 may also have important functions in the reproductive system (see Figure 1 A).

[0074] Western Blot analysis of the testes of mice of different ages revealed that the expression of UBE2J2 increased gradually after the initiation of meiosis on postnatal day 8 (PD8), suggesting that UBE2J2 may play a role in meiosis (see Figure 1 B).

[0075] Example 3 Ube2j2 gene knockout leads to azoospermia in male mice

[0076] To verify the hypothesis that UBE2J2 may play a role in meiosis, we generated Ube2j2 whole-body knockout mice by knocking out exons 4 to 7 of the Ube2j2 gene (see Figure 2 A). See the description in Example 1 for details. The knockout efficiency was good as verified by Western Blot (see Figure 2 B).

[0077] Compared with wild-type mice, Ube2j2 knockout mice have no obvious abnormalities in appearance, but their reproductive phenotype is drastic. Testicular samples were collected from two-month-old mice and it was found that the testicles of Ube2j2 knockout mice were significantly smaller (see Figure 3 A). There are no sperm in the epididymis of knockout mice, interstitial cells are present between the seminiferous tubules of the testis, supporting cells, spermatogonia, and early spermatocytes are present in the seminiferous tubules, but no spermatocytes in the late meiotic period are seen, spermatogenesis is blocked at the pachytene stage, followed by a large number of cell apoptosis, and some seminiferous tubules even have no spermatocytes (see Figure 3 B). This indicates that UBE2J2 plays an important role in spermatogenesis and is an indispensable protein for the normal progress of meiosis.

[0078] Then, the markers of each meiotic event were fluorescently stained by chromosome spreading. The pairing of chromosomes promotes the assembly of SC between homologous chromosomes. SC is a highly conserved protein structure among different species, which consists of three parts: axial elements (AEs), also known as lateral elements (LEs), transverse filaments (TFs) and central elements (CEs). AE mainly contains two component proteins, SYCP2 and SYCP3. SYCP2 and SYCP3 interact to form chromosome axes, provide a flexible scaffold for chromatin loop extrusion, and promote chromosome axis assembly. The main component of TF is SYCP1, and CE contains proteins such as SYCE1, SYCE2, SYCE3, SIX6OS1, TEX12, and SCRE. SYCP3 is the lateral axis component of SC, indicating the chromosome axis. The various periods of the first prophase of meiosis can be distinguished according to the morphology of SYCP3; SYCP1 is the TF element of SC, which is only located in the region where homologous chromosomes undergo synapsis.

[0079] About Ube2j 2- / -The synapsis of mice was investigated by co-staining SYCP3, a marker of chromosome axis, and SYCP1, which indicates synapsis, and finding that Ube2j 2- / - Unable to progress to the late pachytene stage, the stage that can be progressed to has no abnormal synapsis (see Figure 4 A). H1t protein is basically not expressed in the early pachytene stage, but begins to increase in the middle pachytene stage and is expressed in large quantities in the late pachytene stage, which can be used to distinguish the early, middle and late pachytene stages. 2- / - Mice were arrested at midpachytene stage (see Figure 4 B).

[0080] In summary, Ube2j2 gene knockout causes spermatocyte meiosis arrest at the mid-pachytene stage of the first prophase of meiotic division.

[0081] Example 4 Ube2j2 gene knockout leads to abnormal spermatocyte recombination

[0082] By staining the marker proteins of each event in the recombination process, the role of UBE2J2 in the meiotic recombination process was studied. 2- / - The recombination intermediates of mouse spermatocytes are unstable and cannot form crossovers (COs), indicating that UBE2J2 plays an important role in the meiotic recombination process.

[0083] Ube2j2 gene knockout leads to instability of homologous recombination intermediates in spermatocytes. After single-strand invasion forms homologous recombination intermediates, a series of recombination intermediate molecules are recruited to the homologous recombination repair site. In order to detect whether the homologous recombination intermediates are normal, we stained MSH4, RNF212, HEI10, and SUMO modification molecules SUMO1 and SUMO2 / 3. It was found that MSH4 is in Ube2j 2- / - The localization of the chromosomes in mice decreases from leptotene to pachytene (see Figure 5 ); HEI10 in Ube2j 2- / - There is almost no expression on the chromosomes of mice, while the expression of RNF212 increases and cannot be properly dissociated from the chromosomes at the mid-pachytene stage (see Figure 6 A and B); SUMO1, SUMO2 / 3 in Ube2j 2- / - The distribution of heterochromatin regions at centrioles in pachytene mouse spermatocytes is reduced (see Figure 7 A and B). This result shows that Ube2j 2- / - Homologous recombination intermediates in mice are unstable.

[0084] The next step of meiotic homologous recombination is to recruit MutLγ molecules to dissociate the D-loop and dHJs structures to produce COs and complete the meiotic homologous recombination process. MLH1 is a MutLγ molecule that marks the site where COs are produced on homologous chromosomes. Co-staining of MLH1 and SYCP3 revealed that MLH1 was localized in the SC of the mid-pachytene and late pachytene stages of control mice, while Ube2j 2- / - There is no MLH1 signal on the mouse chromosome, indicating that Ube2j 2- / - Mice are unable to form COs (see Figure 8 ).

[0085] In summary, Ube2j 2- / - Homologous recombination intermediates in mice are unstable and cannot form COs, resulting in abnormal recombination.

[0086] Example 5 UBE2J2 plays a role in meiotic prophase by affecting the expression of spermatogenesis-related proteins

[0087] About Ube2j 2- / - The ubiquitination level of mice was detected, and the ubiquitination proteomics analysis of mouse testes was performed. It was found that UBE2J2 affects biological processes such as chromosome dissociation, and it was verified that UBE2J2 interacts with HSPA2 (Heat shock-related 70kDa protein 2) to promote the disassembly of the synaptonemal complex, thereby promoting the continuation of meiosis.

[0088] The ubiquitination levels of spermatocytes at different stages of the first meiotic prophase were detected, and it was found that Ube2j 2- / - The ubiquitination level of spermatocytes in mice at different stages did not change significantly compared with that in control mice (see Fig. 9 A) Subsequently, Western Blot was performed to analyze the overall ubiquitination level in testis and found that Ube2j 2- / - The overall ubiquitination level in mouse testis was also unchanged (see Fig. 9 B).

[0089] In order to find the molecules and pathways that cause changes in specific protein expression and ubiquitination modification, we selected testes that were about to show phenotypes for ubiquitination proteomics analysis. It is generally believed that spermatocytes in the leptotene stage begin to appear at PD12, and spermatocytes in the pachytene stage begin to appear at PD14. 2- / - There was no significant difference in body weight between the mice and the control group (see Fig.10 A) Testis weight and body-to-testis ratio at PD14 2- / - The expression of Ube2j in PD12 and PD13 mice was significantly reduced compared with control mice. 2- / -There was no significant change in the mice compared with the control mice (see Fig.10 B and C).

[0090] Further observation of the histological phenotype by hematoxylin staining of paraffin sections of the testes of mice aged PD13 and PD14 revealed that Ube2j 2- / - The spermatogenesis in the seminiferous tubules of mice at PD13 was normal, with no significant difference compared with wild-type control mice; however, the Ube2j 2- / - Apoptotic cells began to appear in the testes of mice, and empty seminiferous tubules without spermatocytes were observed (see Fig.10 D) Based on the results of testicular ratio and testicular histology analysis, PD13 Ube2j without abnormal phenotype was selected 2- / - Ubiquitination proteomics analysis of mouse and control mouse testes was performed to obtain Ube2j 2- / - Quantification of protein expression and ubiquitination modification in the testes of mice and control mice.

[0091] Example 6: Ube2j2 gene knockout affects spermatogenesis and steroid synthesis pathway protein expression

[0092] In order to explore the effects of Ube2j2 knockout on protein expression and ubiquitination modification in mouse testis, 2- / - Differentially expressed proteins in the testis of mice and control mice, Ube2j 2- / - Testis-specific expression proteins, GSEA and Ube2j in mice and control mice 2- / - Four aspects of changes in ubiquitination-modified proteins in mouse testis were analyzed.

[0093] First, differentially expressed proteins were analyzed using Ube2j 2- / - The results of GO enrichment analysis of differentially expressed down-regulated proteins in mouse testis showed that biological process (BP) was mainly enriched in spermatogenesis, DNA repair, chromatin remodeling, protein-DNA complex assembly and other processes, cellular component (CC) was mainly enriched in chromatin, centrosome and other components, and molecular function (MF) was mainly enriched in transcription factor binding, ubiquitin binding function ( Fig.11 A).

[0094] Then, we analyzed the specific expression proteins. Among the 5791 proteins identified by proteomics, there were 168 WT-specific expression proteins and 53 KO-specific expression proteins. WT-specific expression proteins were mainly enriched in biological processes such as ubiquitination modification, spermatogenesis, and chromatin structure ( Fig.11 B), KO-specific expressed proteins were mainly enriched in the steroid anabolic pathway ( Fig.11 C).

[0095] GSEA analysis of slightly differentially expressed proteins showed that the pathways significantly enriched in the WT group were related to spermatogenesis, meiotic cell cycle, and chromosome dissociation (see Fig.11 B), pathways significantly enriched in the KO group were related to steroid metabolism (see Fig.11 C).

[0096] The results of the ubiquitination group analysis showed that among the 1,372 ubiquitination sites identified, 166 ubiquitination sites were specific to the WT group and 140 ubiquitination sites were specific to the KO group. The pathways in which ubiquitination-downregulated proteins were significantly enriched in the KO group were mainly related to ubiquitination-dependent protein degradation processes, while the pathways in which ubiquitination-specific proteins were enriched in the WT group were related to ubiquitination-dependent protein degradation, spermatogenesis, cell cycle and other processes.

[0097] Summarizing the bioinformatics analysis results of ubiquitination proteomics, it was found that UBE2J2 was associated with steroidogenesis and chromosome dissociation in addition to ubiquitin modification and spermatogenesis.

[0098] Example 7 Ube2j2 gene knockout affects synaptonemal complex disassembly

[0099] Experiments showed that UBE2J2 did not affect androgen synthesis (data not provided). In order to find the pathway through which UBE2J2 exerts its effects, we next explored the role of UBE2J2 in chromosome dissociation. In the late pachytene stage, dissociation of the synaptonemal complex between homologous chromosomes occurs. The first key step in SC dissociation is the recruitment of PLK1 (Polo-like kinase1) to the SC, which disassembles the central region of the SC by phosphorylating the SC central axis molecules SYCP1, TEX12, and SYCE1. Cyclin-dependent kinases (CDKs) and Aurora kinases (AURKs) are required for the disassembly of SC lateral axis elements. The CDK1-Cyclin B complex is involved in the dissociation of the SC, but before CDK1 binds to Cyclin B, CDK1 needs to be activated by HSPA2. HSPA2 is localized in the SC during the pachytene stage and may recruit CDK1 to the SC, causing SC components containing CDK1 phosphorylation sites (such as SYCP1) to be phosphorylated by CDK1, thereby leading to SC disassembly.

[0100] The SC localization of kinases PLK1 and CDK1, which play a role in SC disassembly, revealed that Ube2j 2- / - Increased expression of PLK1 on mouse spermatocyte SC (see Fig.12 A), but the expression of CDK1 was greatly reduced (see Fig.12 B) This shows that Ube2j 2- / - Abnormal SC disaggregation in mouse spermatocytes.

[0101] CDK1 needs to be activated by HSPA2, so the chromosome spread was stained with HSPA2 fluorescence, and the results showed that Ube2j 2- / - HSPA2 is not localized on chromosomes or around chromosomes in mouse spermatocytes (see Fig.13 ). The above results show that Ube2j 2- / - Abnormal SC disaggregation in mouse spermatocytes.

[0102] Example 8 UBE2J2 promotes meiosis by interacting with HSPA2

[0103] To further investigate the role of UBE2J2 in SC disassembly, we identified the interacting proteins of UBE2J2 and compared the control mice with those of UBE2J2. 2- / - Mouse testes were subjected to IP-MS analysis.

[0104] Mass spectrometry analysis showed that Ube2j was present in control mice. 2- / - Of the 16 UBE2J2-interacting proteins not detected in mice, only one was associated with reproduction, HSPA2.

[0105] First, the overall expression level of HSPA2 in testis was detected by Western Blot, and it was found that HSPA2 was expressed in Ube2j 2- / - Expression was significantly reduced in mouse testis (see Fig.14 A). The results of subsequent cytoplasmic-nuclear separation experiments showed that UBE2J2 was mostly localized in the cytoplasm, and also partially localized in the nucleus (see Fig.14 B), suggesting that in addition to the known ERAD, UBE2J2 may also play a role in the nucleus.

[0106] Summarizing the above results, Ube2j 2- / - The overall ubiquitination level in mouse spermatocytes did not change significantly, but chromosome disassembly was abnormal. UBE2J2 could interact with HSPA2 to recruit HSPA2 to the nucleus and SC, and HSPA2 subsequently recruited CDK1 to promote SC disassembly.

[0107] Example 9 The phenotype of Ube2j2 germ cell conditional knockout mice is consistent with that of whole body knockout mice

[0108] Because UBE2J2 is not a protein specifically expressed in germ cells, in order to exclude the possibility that UBE2J2 affects the reproductive system by affecting other tissues and organs, a conditional knockout of Ube2j2 in germ cells was constructed. fl / - ; Stra8-Cre mice (hereafter referred to as cKO mice) to confirm the role of UBE2J2 in spermatocytes.

[0109] Hematoxylin staining of testicular paraffin sections showed that there were no sperm in the epididymis of cKO mice, no cells in the late meiotic phase in the testes, and a large number of spermatocytes apoptotic during the first meiotic phase, which was consistent with the results of KO mice. Fluorescence staining of synaptic molecules on chromosome spreads showed that the first meiotic phase of cKO mice was blocked at the mid-pachytene phase, and the synaptic complex could synapse normally at the stage that spermatocytes of cKO mice could progress to.

[0110] Subsequently, the marker molecules of each recombination event were stained to observe the recombination of cKO mice. The staining results of the γH2AX molecule that marks DSBs showed that there was no abnormality in the generation of DSBs at the stage that the spermatocytes of cKO mice could progress to. Subsequently, the molecules ATR and pATM that initiate DSBs repair were stained, and it was found that there was no abnormality in the initiation of DSBs repair in spermatocytes of cKO mice. Staining of the single-strand protection protein RPA2 showed that there was no abnormality in the single-strand protection of spermatocytes of cKO mice. Subsequently, staining of the single-strand invasion molecule RAD51 showed that there was no abnormality in the single-strand invasion of spermatocytes of cKO mice. Staining of MSH4, which stabilizes homologous recombination intermediates, showed that the localization of MSH4 on the SC in spermatocytes of cKO mice was reduced, and the homologous recombination intermediates were unstable, and thus the recombination could not be completed to form COs, and there was no MLH1 signal.

[0111] The phenotype of Ube2j2 cKO mice is consistent with that of KO mice, which excludes the possibility that UBE2J2 affects the reproductive system by affecting other tissues and organs, and further proves that UBE2J2 plays an important role in the prophase of the first meiotic division of spermatocytes.

[0112] The inventors of the present application discovered for the first time that UBE2J2 plays an important role in the first meiotic division of spermatocytes by regulating the recombination and disaggregation of homologous chromosomes. Ube2j2 gene knockout leads to azoospermia in male mice, and spermatocyte meiosis is blocked at the mid-pachytene stage. Ube2j2 gene knockout does not affect the generation and initiation of DSBs repair, nor does it affect the synapsis and MSCI of SC, but it causes instability of homologous recombination intermediates and the inability to form COs. Ube2j2 gene knockout affects spermatogenesis, steroid synthesis, cell cycle and chromosome disaggregation pathways, but does not affect the synthesis of sex hormones. The inventors of the present application found that UBE2J2 can activate CDK1 by interacting with HSPA2, thereby playing a role in the process of homologous chromosome SC disaggregation.

[0113] The inventors of this application built Ube2j 2- / - The role of UBE2J2 in spermatogenesis was studied in mice, and it was found that Ube2j2 gene knockout caused azoospermia in male mice, and spermatocytes were arrested in the pachytene stage of the first prophase of meiosis. In order to exclude the possibility that UBE2J2 affects the reproductive system by affecting other tissues and organs, the inventors of the present application constructed a conditional knockout of Ube2j2 in germ cells. 2fl / - ; Stra8-Cre mice, whose phenotypes are consistent with those of whole-body knockout mice, further demonstrated that UBE2J2 plays an important role in the prophase of the first meiotic division of spermatocytes.

[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 UBE2J2 in a subject in the preparation of a kit for diagnosing fertility defects such as infertility in mammals.

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 method according to any one of claims 1 to 3, wherein the detection reagent is a reagent for detecting the Ube2j2 gene.

5. The use according to any one of claims 1-3, comprising a reagent for detecting UBE2J2 protein, preferably, further comprising a reagent for determining the activity of UBE2J2 protein.

6. The use according to claim 5, which comprises a reagent for detecting the expression of the UBE2J2 protein by immunoassay, such as a reagent for detecting the expression by ELISA or Western blotting using an antibody that specifically recognizes the protein; or These include reagents for detecting the presence or amount of mRNA of the UBE2J2 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 disorders in mammals of different sexes, comprising a reagent for detecting UBE2J2 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 UBE2J2 of the subject is a reagent for detecting the Ube2j2 gene.

10. The kit according to claim 7, wherein the reagent for detecting UBE2J2 in the subject is a reagent for detecting UBE2J2 protein, and preferably, further comprises a reagent for detecting the activity of UBE2J2 protein.

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