Use of glycosylated proteins for diagnosis and treatment of fertility defects

Abstract

The present invention provides methods for diagnosing or predicting fertility defects, such as infertility, in a mammalian male individual by detecting RPN1. The invention also provides kits and instruments for diagnosing or predicting fertility defects, such as infertility disorders, in mammalian male individuals by detecting RPN1. The invention discovers and proves for the first time that knockout of RPN1 can cause meiosis abnormality and complete sterility of spermatoblasts of male mice due to the fact that the glycosylation level of mitochondrial related proteins is remarkably reduced due to deletion of RPN1, and then the structure and function of mitochondria are affected. The invention provides a method for diagnosing male fertility defects of mammals including human beings, and provides a new thought and method for developing new treatment for male infertility individuals influenced by gene mutation.

Description

Technical Field

[0001] The present invention relates to the field of diseases and diagnosis, and in particular to a method and a kit for diagnosing or treating male fertility defects in mammals, especially humans, using glycosylated proteins. Background Art

[0002] Meiosis is a specialized form of cell division in which the genetic material is replicated once and the cell divides twice in succession, resulting in gametes with half the number of chromosomes as in somatic cells. Meiotic prophase I involves the exchange of genetic material between homologous chromosomes and consists of five distinct substages: leptotene, zygotene, pachytene, diplotene, and diakinesis. DNA double-strand breaks (DSBs) trigger the recruitment of meiotic factors, initiating homologous recombination to repair the damaged DNA. During zygotene, a group of recombination proteins are recruited to meiotic DSBs, leading to the formation of recombination points and synapsis of homologous chromosomes. Subsequently, DMC1 and RAD51 mediate strand invasion, promoting the formation of displacement loops. When the chromosomes are fully synaptic during pachytene, the recombination points continue to mature and ultimately become the site of crossover events. Homologous chromosome pairing, synapsis, recombination, and segregation constitute the key events of meiosis, and abnormalities in any of these stages can lead to male azoospermia / oligozoospermia.

[0003] N-glycosylation marks are important post-translational modifications (PTMs) in a wide range of biological processes in eukaryotes. To catalyze this modification, high-mannose oligosaccharides are transferred as building blocks to a specific asparagine-containing protein sequence motif (Asn-X-Thr / Ser, X=Pro) in the endoplasmic reticulum (ER). Mutations that result in changes in the degree of glycosylation or the structure of the glycan chain have been found to frequently lead to the development of various diseases. For example, abnormal glycan synthesis and the formation of glycoside-linked glycoproteins can lead to congenital diseases of glycosylation (CDGs). Human CDG-Ia and mouse CDGs (Mgat2 - / -) all present with testicular atrophy, azoospermia, and infertility, suggesting that glycosylation plays a crucial role in spermatogenesis. N-glycosylation is catalyzed by oligosaccharidase transferase (OST), which consists of two catalytic subunits, STT3A and STT3B, and a group of shared non-catalytic subunits. Recently, non-catalytic subunits have also been found to play a crucial role in promoting glycosylation efficiency. Among them, ribonucleoprotein I (RPN1) was the first mammalian OST complex subunit purified from canine pancreas. Previous studies have reported that newly synthesized membrane proteins transiently associate with RPN1 after exiting the Sec61 translocation channel, suggesting that RPN1 can retain potential substrates in the vicinity of the OST catalytic subunit to enhance N-glycosylation efficiency. Furthermore, glycosylation of single-pass transmembrane proteins requires RPN1 expression to ensure efficient glycosylation, whereas RPN1 deficiency does not affect the glycosylation of secretory and multipass transmembrane proteins. Although RPN1 has been shown to promote N-glycosylation, its underlying mechanism and role in spermatogenesis remain unclear.

[0004] Spermatogenesis triggers a series of major changes in the intracellular composition and spatial arrangement of organelles. These changes include alterations in mitochondrial morphology and a shift from glycolytic to oxidative energy metabolism. Mitochondria are highly complex and dynamic organelles that serve as the primary site of energy and lipid metabolism, playing crucial roles in various processes, including ATP synthesis, reactive oxygen species (ROS) production, calcium signaling, and apoptosis. Meiosis is considered a highly energy-dependent process, requiring a continuous supply of large amounts of ATP. As spermatogonia transform into spermatocytes, their microenvironment undergoes a transformation. Spermatogonia exhibit high glycolytic activity, while spermatocytes and spermatids primarily synthesize ATP through mitochondrial-mediated oxidative phosphorylation. Recent studies have demonstrated that mitochondrial dynamics plays a crucial role in reproductive development. ClpP / ClpX deficiency reduces mitochondrial function and spermatocyte abundance, impairing energy supply during meiosis and delaying the zygotene-pachytene transition in male germ cells. Loss of MFN1 and MFN2 leads to DNA oxidation and apoptosis during spermatogonial differentiation, resulting in male infertility. MFN2 has also been shown to be involved in spermatogenesis by regulating mitochondrial and endoplasmic reticulum function. In addition, male infertility has been identified as a major symptom of mitochondrial diseases.

[0005] Despite the central role of mitochondria in reproductive biology, including spermatogenesis, their precise functional mechanisms in male reproduction remain poorly understood. A greater understanding of the regulation of spermatogenesis in mammals, including humans, is needed to detect and prevent male reproductive disorders. Summary of the Invention

[0006] The inventors have discovered and demonstrated for the first time that knocking out ribonucleoprotein 1 (RPN1) leads to abnormal meiosis and complete infertility in male mouse spermatocytes. N-glycosylation analysis showed that the loss of RPN1 led to a significant decrease in the glycosylation level of mitochondrial-related proteins, thereby affecting the structure and function of mitochondria. The applicant's discovery provides new insights into the relationship between N-glycosylation modification and meiosis. The applicant thus provides a method for diagnosing male fertility defects in mammals and humans. The inventors thus provide a method for diagnosing or treating male fertility defects in mammals and humans.

[0007] Specifically, the present invention provides a method for diagnosing or predicting fertility defects such as infertility in a male mammal, which comprises detecting the gene or protein of ribonucleoprotein 1 (RPN1) in the male subject.

[0008] In the present invention, the infertility disorder of the male individual includes but is not limited to teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, asthenozoospermia and the like.

[0009] In the present invention, the mammal can be any mammal, including but not limited to rodents, canines, felines, equines, ovines, bovines, porcines and primates. Generally speaking, mammals also include humans.

[0010] Ribophorin I (RPN1) is a type I membrane protein found in the rough endoplasmic reticulum. It is part of the N-oligosaccharyltransferase complex that links high-mannose oligosaccharides to asparagine residues within the Asn-X-Ser / Thr consensus motif. RPN1 is part of the 26S proteasome regulatory subunit and mediates the association of ubiquitin-like domains with the proteasome. The Rpn1 gene, encoding RPN1, has a conserved coding sequence across mammals. The human Rpn1 gene is assigned the Gene ID 6184. The mouse Rpn1 gene is assigned the Gene ID 103963.

[0011] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention comprises the step of detecting the Rpn1 gene.

[0012] The gene detection method used in the present invention is a method commonly used for gene detection in the art and is not particularly limited. Examples thereof include mass spectrometry, microarray methods, sequencing methods, and detection methods using base sequence amplification methods such as PCR (polymerase chain reaction).

[0013] In addition, detection of PCR products obtained when PCR is performed using primers specific for each gene can be performed by any method commonly used for detecting and quantifying PCR products. For example, detection can be performed by electrophoresis, by real-time PCR using a fluorescent intercalator such as SYBR Green, or by single molecule fluorescence analysis.

[0014] 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 this gene or its complementary strand. The design of polynucleotide can adopt any one of the well-known methods in this technical field to carry out. For example, it is possible to design simply by utilizing known genome sequence data and a universal primer design tool. As this primer design tool, for example, Primer3 that can be utilized on the Internet is arranged. In addition, known genome sequence data can usually be obtained in the NCBI as an international base sequence database.

[0015] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention includes the step of detecting RPN1 protein, such as detecting the expression of the protein (including whether it exists, and whether there is a phenomenon of reduced expression relative to normal or standard) or its activity.

[0016] In yet another aspect of the present invention, the method includes the step of detecting RPN1 protein expression by immunoassay. For example, RPN1 expression can be detected by immunofluorescence or Western blotting using an antibody that specifically recognizes the protein. Another example is detecting RPN1 expression by detecting the presence or amount of mRNA encoding the protein, such as by detecting the amount of mRNA encoding RPN1 in a sample by RT-PCR.

[0017] In yet another aspect of the present invention, the method comprises the step of determining the activity of the RPN1 protein.

[0018] The present invention also provides a kit or apparatus (including a gene chip) for diagnosing or predicting fertility defects such as infertility in a mammalian individual, which includes a reagent for detecting the Rpn1 gene of the subject.

[0019] In yet another aspect of the present invention, the kit or apparatus includes reagents for detecting the expression or activity of the RPN1 protein. For example, the kit or apparatus includes reagents for an immunoassay, such as an ELISA or Western blot assay using an antibody that specifically recognizes the protein, to detect RPN1 expression. In another example, the kit or apparatus includes reagents for detecting RPN1 expression by detecting the presence or amount of mRNA encoding the protein, such as reagents for detecting the amount of mRNA encoding RPN1 in a sample by RT-PCR.

[0020] In yet another aspect of the present invention, the kit or apparatus comprises a reagent for detecting the Rpn1 gene.

[0021] In one aspect of the present invention, in the above-mentioned methods, kits or instruments of the present invention, the reagents for detecting genetic markers in samples include primers or probes for detecting Rpn1, or reagents for examining RPN1, such as specific antibodies or reagents for detecting the mRNA of the protein.

[0022] The present invention also provides the use of a reagent for detecting RPN1 in preparing a kit or apparatus for diagnosing or prognosing fertility defects, such as infertility, in a mammal, as described above. The reagent for detecting genetic markers includes primers or probes for detecting the Rpn1 gene, or reagents for detecting the RPN1 protein, such as specific antibodies or reagents for detecting the protein's mRNA.

[0023] The present invention also provides a method for treating or preventing fertility defects in a mammalian subject, comprising treating the subject having an Rpn1 defect, such as an Rpn1 mutation.

[0024] Throughout this document, protein symbols are not italicized and are capitalized; gene symbols are italicized. For example, RPN1 is a protein, and the gene encoding it is written as Rpn1. Sometimes, protein symbols are not italicized throughout this document. For example, "Rpn1" is sometimes used herein to refer to the RPN1 protein. Sometimes, gene symbols are not italicized throughout this document. For example, "RPN1" or "RPN1 gene" is sometimes used herein to refer to the gene Rpn1 that encodes the RPN1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This image shows the important role of RPN1 in mouse spermatogenesis and male fertility. A. Quantitative analysis of the relative expression levels of RPN1 at different ages. The relative expression level of RPN1 protein was calculated by quantitatively calculating the grayscale value of RPN1 / β-Actin for each sample. B. Western blot (WB) analysis of RPN1 in spermatogenic cells isolated from adult WT mice by FACS (L / Z, tetany / zygotic, P / D, pachytene / diploid, RS, round spermatid). The relative expression level of RPN1 protein was calculated by quantitatively calculating the grayscale value of RPN1 / β-Actin for each sample. C. Western blot analysis of RPN1 in the nucleus and cytoplasm of 2M WT testicular spermatogenic cells. β-Actin and Lamin B1 were used to indicate the isolation efficiency of cytoplasmic and nuclear proteins, respectively. D. Spatial and temporal expression pattern of RPN1 in postnatal male germ cells. DNA was stained with DAPI. SPGs, spermatogonia. Scale bar, 5 μm. E. Schematic diagram of the Rpn1 conditional knockout strategy in germ cells. F. Western blot analysis of RPN1 knockout efficiency in testes of adult WT and Rpn1-SKO mice. β-actin was used as a control. G. Immunofluorescence staining of RPN1 in testes of WT and Rpn1-SKO mice. Spermatocytes are co-stained with γH2AX. Scale bar, 30 μm. H. A 6-month breeding experiment between Rpn1-SKO male mice and age-matched WT female mice. The number of pups born from each male mating with one female mouse. Data represent mean ± SEM. **p < 0.01 by Student's t-test. I. 2M testicular morphology of WT and Rpn1-SKO mice. J. Testicular weights of WT and Rpn1-SKO mice. Data represent mean ± SEM. **p < 0.01 by Student's t-test. K. Testis weight / body weight ratio of WT and Rpn1-SKO mice. Data represent mean ± SEM. **p < 0.01 by Student's t-test. L. Histological staining of the testes and epididymis of WT and Rpn1-SKO mice. Scale bar, 20 μm. M. Quantitative analysis of germ cells in seminiferous tubules of WT and Rpn1-SKO mice. Data represent mean ± SEM. **p < 0.01 by Student's t-test.

[0026] Figure 2 Showing defects in spermatogenesis in Rpn1-SKO mice. A. Hematoxylin staining of WT and Rpn1-SKO testes at different ages. Scale bar, 20 μm. B. Immunofluorescence (IF) co-staining of PLZF (green) and SOX9 (red) in PD12 (WT and Rpn1-SKO mice). Scale bar, 30 μm. C. PLZF per tubule in PD12 WT and Rpn1-SKO mice. +Quantification of cells. Data represent mean ± SEM. ns, not significant. D. Quantitative analysis of c-Kit+ cells per tubule in WT and Rpn1-SKO mice at PD12. Data represent mean ± SEM. ns, not significant. E. Immunofluorescence co-staining of STRA8 (green) and MVH (red) in WT and Rpn1-SKO mice at PD12. Scale bar, 30 μm. F. STRA8 per tubule in WT and Rpn1-SKO mice at PD12. + Quantification of cells. Data represent mean ± SEM. **p < 0.01 by Student's t-test. G. SYCP3 per tubule in WT and Rpn1-SKO mice. + Quantification of cells. Data represent mean ± SEM. **p < 0.01 by Student's t-test. H. Left: IF co-staining of H1t (green) and SYCP3 (red) in WT and Rpn1-SKO mice at PD18 and 2M. Scale bar, 30 μm. Right: H1t per total tubule. + Number of cells in seminiferous tubules. Data represent mean ± SEM. **p < 0.01 by Student's t-test. I. Left: TUNEL assay in testicular sections of PD18WT and Rpn1-SKO mice. Scale bar, 30 μm. Right: TUNEL per tubule. + Quantification of cells. Data represent mean ± SEM, **p < 0.01 by Student's t-test.

[0027] Figure 3Showing synapsis defects and DSB repair defects in Rpn1-deficient spermatocytes. A. SYCP3 (red) and SYCP1 (green) are co-stained in 1M spermatocyte spreads from WT and Rpn1-SKO mice. Scale bar, 5 μm. B. Percentage of meiotic spermatocytes at various stages of meiotic prophase. C. Representative images of WT and Rpn1-SKO spermatocytes at different stages of meiotic prophase, co-stained with SYCP3 (red) and γH2AX (green). Scale bar, 5 μm. D. IF co-staining of SYCP3 (red) and MLH1 (green) in WT and Rpn1-SKO mice. Scale bar, 5 μm. E. Quantification of MLH1 foci per nucleus from WT and Rpn1-SKO mice in D. Data represent mean ± SEM, **p < 0.01 by Student's t-test. F. IF co-staining of SYCP3 (red) and RPA2 (green) in WT and Rpn1-SKO mice. Scale bar, 5 μm. G. Quantification of RPA2 puncta per nucleus in WT and Rpn1-SKO mice. Data represent mean ± SEM, **p < 0.01, Student's t-test. H. IF co-staining of SYCP3 (red) and DMC1 (green) in WT and Rpn1-SKO mice. Scale bar, 5 μm. I. Quantification of DMC1 puncta per nucleus in WT and Rpn1-SKO mice. Data represent mean ± SEM, **p < 0.01, Student's t-test. J. Quantification of RAD51 puncta per nucleus in WT and Rpn1-SKO mice. Data represent mean ± SEM, **p < 0.01, Student's t-test.

[0028] Figure 4 Figure 5. Effects of Rpn1 deficiency on the expression of mitochondrial-associated glycoproteins. A. Workflow for N-glycoproteomic analysis of testes from PD12 WT and Rpn1-SKO mice. B. Venn diagram of proteins identified in the testicular N-glycoproteome versus the proteome. C, E. Venn diagram of N-glycopeptides (C), N-glycan sites (D), and N-glycoproteins (E) identified in PD12 WT and Rpn1-SKO testes. F. Percentage of NXT / S / C tripeptide sequences in WT and Rpn1-SKO testes. G. Pie chart showing the proportion of proteins containing different numbers of N-glycan sites in WT and Rpn1-SKO testes in vivo. H. Bar graph showing the number of differentially expressed glycoproteins and glycan sites in WT and Rpn1-SKO testes after normalization to the total proteome. I. Glycoproteins significantly downregulated by GO analysis. J. Downregulated N-glycoproteins were significantly enriched in pathways involving reactive oxygen species, peroxisomes, and ferroptosis.

[0029] Figure 5Rpn1-deficient spermatocytes demonstrate mitochondrial dysfunction and impaired energy metabolism. A. TEM imaging of mitochondrial structure in testicular sections from 2MWT and Rpn1-SKO mice. N, nucleus. Scale bar, 1 μm. B. Representative images of WT and Rpn1-SKO spermatocytes, immunostained for TOM20 (red), and quantified for fluorescence intensity. Data represent mean ± SEM, **p < 0.01, Student's t-test. Scale bar, 5 μm. C. RT-qPCR analysis of mitochondrial copy number expression in WT and Rpn1-SKO testes. Hk2 was used as an internal control. Data represent mean ± SEM, **p < 0.01, Student's t-test. D. Mitochondrial membrane potential in WT and Rpn1-SKO spermatocytes assessed by JC-1 staining. JC-1 aggregates, red; JC-1 monomers, green. Data represent mean ± SEM, **p < 0.01, Student's t-test. Scale bar, 2 μm. EF. Detection of CM-H2DCFDA fluorescence in L / Z and P / D spermatocytes from WT and Rpn1-SKO mice. Data represent mean ± SEM, **p < 0.01, Student's t-test. G. ATP content in WT and Rpn1-SKO spermatocytes. Data represent mean ± SEM, **p < 0.01, Student's t-test. H. Detection of Fluo-2 AM fluorescence in L / Z and P / D spermatocytes from WT and Rpn1-SKO mice by flow cytometry.

[0030] Figure 6 Schematic model of RPN1 function in spermatogenesis. Rpn1 deficiency suppresses mitochondrial function and abundance and reduces energy availability during meiosis. This deficiency leads to a significant inhibition of meiotic progression. Consequently, aberrantly regulated spermatocytes undergo apoptosis, resulting in a decrease in spermatocyte number and testicular size. Created with BioRender.com. DETAILED DESCRIPTION

[0031] The essential content and beneficial effects of the present invention will be further illustrated below with reference to examples, which are only used to illustrate the present invention rather than to limit the present invention.

[0032] Example 1 Experimental methods and reagents

[0033] mice

[0034] In this study, C57BL / 6J mice were used to establish an Rpn1 conditional knockout model. flox / floxMice with germline Rpn1 knockout were obtained by crossing Vasa-Cre and Stra8-GFPCre mice, respectively. Stra8-GFPCre mice were obtained from the laboratory of Dr. Tong Minghan at the Chinese Academy of Sciences. All experimental protocols were approved by the Animal Ethics Committee of Shandong University School of Medicine (Jinan, Shandong, China). All mouse experiments were performed in accordance with the protocols of the Institutional Animal Care and Use Committee of Shandong University (IACUC; #2018-77). All mice were housed under specific pathogen-free (SPF) conditions with access to water and food and a 12-h dark / 12-h light cycle.

[0035] Generation and genotyping of conditional knockout mice

[0036] Rpn1 flox / flox Mice were generated by Cyagen Biosciences (Guangzhou, China) using the CRISPR-Cas9 system for gene editing. Based on the structure of the Rpn1 gene, exon 2 of the Rpn1-201 (ENSMUST00000032143.7) transcript was selected as the deletion target. Briefly, Cas9 mRNA and gRNA were transcribed in vitro. Vectors encoding Cas9, sgRNA, and the donor were microinjected into fertilized eggs of C57BL / 6J mice. The fertilized eggs were transplanted into female mice to obtain F0 positive mice, which were confirmed by PCR and Sanger sequencing. Stable F1 generation mice were obtained by mating F0 positive mice with wild-type C57BL / 6J mice. Genomic DNA was extracted from the mouse toes for mouse genotyping and PCR amplification. The floc and wild-type bands were 407 base pairs (bp) and 306 bp, respectively. The Rpn1 gene knockout band was 443 bp.

[0037] Spermocyte spreading and immunofluorescence staining

[0038] Testicular cells were prepared from 1- to 2-month-old mice for meiotic chromosome spreading and subsequent immunofluorescence staining. Briefly, after washing the testes with phosphate-buffered saline (PBS), the tunica albuginea was removed. Then, 1.5- to 2-cm seminiferous tubules were immersed in hypotonic extraction buffer (30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM ethylenediaminetetraacetic acid [EDTA], 0.5 mM DTT, and 0.5 mM PMSF, pH 8.2) for 30-40 min at room temperature. Subsequently, the tubules were mechanically disaggregated, and spermatocytes were released into a solution containing 100 mM sucrose (pH 8.2) and spread onto clean glass slides containing 1% paraformaldehyde (PFA; pH 9.2) containing 0.15% Triton X-100. The slides were air-dried in a humidity chamber for at least 2 h before immunofluorescence staining.

[0039] For immunostaining, slides were placed in 1× PBS containing 5% bovine serum albumin (BSA) for 10 minutes and then blocked with 5% BSA for at least 1 hour at room temperature. Next, slides were incubated with primary antibodies (Table S2) overnight at 4°C. After incubation, slides were washed and incubated with secondary antibodies. Nuclei were stained with DAPI (Vector Laboratories) and then sealed using a Vector Laboratory H-1000 (Vector, Shanghai, China).

[0040] Histology, immunostaining, and TUNEL analysis

[0041] Mouse testes and ovaries were fixed with 4% PFA (Solarbio, Beijing, P1110) and Bouin's solution at 4°C overnight for histological analysis and immunostaining. The samples were dehydrated, paraffin-embedded, and cut into 5 μm sections. The sections were stained with hematoxylin for histological analysis. For immunofluorescence analysis, the sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for 20 min, soaked in 1× PBS containing 5% BSA for 10 min, and then blocked with 5% BSA in a humidity chamber for 1 hour. The sections were incubated with primary and secondary antibodies. After a brief rinse with PBS, the sections were stained with DAPI.

[0042] TUNEL assays were performed strictly according to the manufacturer's instructions and were accompanied by a one-step TUNEL apoptosis detection kit (Merlen Bio, MA0223). Images were acquired using an LSM 780 / 710 microscope (Zeiss) or an SP8 microscope (Leica).

[0043] Western blotting

[0044] Testes were dissected and immediately suspended in NP40 buffer containing a protease inhibitor cocktail (Roche, 04693116001). After sufficient shaking and lysis, the supernatant protein extract was incubated with 1× SDS loading buffer (Beyotime, P0015L) at 95°C for 5 min. Equal concentrations of protein were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then immunoblotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated secondary antibodies and then photographed on a Bio-Rad ChemiDoc MP 453 imaging system and image analysis was performed using Image Lab software (Bio-Rad, USA). β-actin was used as a loading control.

[0045] Flow cytometry analysis of spermatogenic cells

[0046] Flow cytometric analysis of spermatogenic cells was performed as described previously. Briefly, after removing the tunica albuginea, testes were gently dispersed with forceps, and seminiferous tubules were incubated in 1× collagenase type I (120 U / ml, Thermo Fisher Scientific, 17100017) at 35°C with gentle rotation for 10 min. After digestion in 5 mL of 0.25% trypsin containing 5 mg / mL DNase I at 35°C for 7 min with rotation, tubule fragments were pipetted up and down to disperse germ cells. Trypsin was inactivated by the addition of 0.5 mL of fetal bovine serum (FBS), and the suspension was filtered through a 70 μm honeycomb filter. The suspension was centrifuged at 500 g for 5 min, and the supernatant was discarded. Microspheres were resuspended in DMEM supplemented with 10% fetal bovine serum. After staining with Hoechst 33342 (1 x 106 / ml, 3 μg / μL) for 40 min, cells were sorted using a flow cytometer (BD Biosciences, FACS Aria II, USA) based on different fluorescent channels. Germ cell samples were then used for ROS detection, mitochondrial membrane potential detection, and Ca 2+ Detection.

[0047] Quantification of ATP levels

[0048] ATP levels were measured using an ATP assay kit (Beyotime Biotech, S0026) according to the manufacturer's protocol. Different types of spermatogenic cells separated by flow cytometry were suspended in lysis buffer for 10 min. After the cells were completely lysed, they were centrifuged at 12,000 g for 5 min, and the supernatant was collected and the ATP content was determined. 20 μl of each supernatant or ATP standard control was added to the ATP assay buffer in a 96-well plate. Relative brightness (RLU) was calculated using an infinite number of ions. TMThe concentrations were measured using an M200 microplate reader (Thermo Fisher Scientific). A standard curve was generated using concentrations ranging from 0.01 mM to 1 mM and used to estimate the total ATP content in the samples.

[0049] ROS level detection

[0050] ROS levels in the testis were assessed using a ROS detection kit (Beyotime, S0033S). The DCFH-DA probe was diluted in DMEM medium to prepare culture droplets, which were then incubated at 37°C for 30 minutes to reach equilibrium. Spermatogonia at different stages were obtained by flow cytometry and placed in droplets containing 10 μM DCFH-DA. The droplets were incubated in the dark at 37°C for 30 minutes. After washing, analysis was performed using a FACS Aria II flow cytometer (BD Biosciences, USA).

[0051] Proteomic and N-glycosylation analysis

[0052] Testicular samples from WT and Rpn1-SKO mice were subjected to proteomic and N-glycosylation profiling at PD12. After three rinses with phosphate-buffered saline (PBS), the samples were placed in microcentrifuge tubes and snap-frozen in liquid nitrogen. Samples were shipped on dry ice to Hangzhou Jingjie Biotechnology Co., Ltd. for proteomics, N-glycoproteomics, and corresponding data analysis. A threshold of p < 0.05 and a |fold change| > 1.5 was used to identify significantly differentially regulated proteins between WT and Rpn1-SKO samples.

[0053] Gene Ontology (GO) functional annotation analysis and Fisher's exact test were performed to identify significantly enriched terms among the total identified proteins (p < 0.05). Pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, with Fisher's exact test (p < 0.05) used to assess significant, differentially enriched pathways among the total identified proteins. The R packages cluster analyzer and metascape (http: / / metascape.org) were used for GO and KEGG pathway enrichment analysis.

[0054] Statistical analysis

[0055] Unless otherwise stated, all experimental data were statistically analyzed using at least three independent replicates. Significant differences between groups were determined using a two-tailed unpaired Student's t-test (*p < 0.05, significant difference; **p < 0.01, very significant difference). Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA).

[0056] Example 2 RPN1 is highly expressed in mouse pachytene / diplotene spermatocytes

[0057] To investigate the role of RPN1 in spermatogenesis, we first used immunoblotting to detect RPN1 in extracts from the reproductive organs of adult (2-month-old, 2M) wild-type (WT) male mice of the C57BL / 6J genetic background. High expression levels of RPN1 protein were observed in both the testes and ovaries. Further monitoring of RPN1 levels by Western blot (WB) from postnatal day 6 (PD6) to 3M revealed that its levels remained consistently high ( Figure 1 A). Germ cells at different developmental stages, including leptotene / zygotene (L / Z), pachytene / diplotene (P / D) spermatocytes, and round spermatids (RS), were then isolated from adult WT male mice by fluorescence-activated cell sorting (FACS) for further Western blot analysis of RPN1 levels. RPN1 is expressed in spermatocytes entering meiotic prophase I (L / Z), increases in P / D spermatocytes, and then decreases to a relatively low level in RS ( Figure 1 B).

[0058] Immunofluorescence (IF) staining with antibodies against RPN1 and γH2AX (indicating different spermatocyte types) was used to assess the subcellular localization of RPN1 in WT adult testes. The results showed that RPN1 could be detected in spermatogonia, leptotene, and zygotene, but the level of RPN1 was much higher in pachytene and diplotene spermatocytes ( Figure 1 C) In addition, testicular extracts from adult mice were separated and subjected to Western blotting analysis. Co-detection with the cytoplasmic marker β-Actin and the nuclear marker Lamin B1 indicated that RPN1 was present in the cytoplasmic fraction.

[0059] These findings demonstrate that RPN1 plays a role in mouse spermatogenesis.

[0060] Example 3 Germ cell-specific Rpn1 deletion leads to male infertility

[0061] By CRISPR Cas9 genome editing, two LoxP sites were introduced on both sides of exon 2 of the Rpn1 gene to generate Rpn1 flox / flox Then, Vasa-Cre transgenic mice were crossed with Rpn1 flox / flox Crossbreeding mice to obtain Rpn1 flox / - Vasa-Cre (hereinafter referred to as Rpn1-VKO) mice, in which Rpn1 was specifically knocked out at embryonic day 15.5. Morphological observation showed that the Rpn1 flox / + and Rpn1 flox / floxCompared with male littermates, the testes of Rpn1-VKO mice were significantly smaller, and the testis-to-body weight ratio of Rpn1-VKO mice was significantly reduced. Histological examination using hematoxylin and IF staining revealed an almost complete absence of spermatocytes in the seminiferous tubules of Rpn1-VKO mice, suggesting that Rpn1 is required for spermatogenesis in mice.

[0062] Then, by flox / flox The mice were crossed with Stra8-GFPCre transgenic mice expressing CRE recombinase in PD3 male germ cells to further study the role of Rpn1 in meiosis and obtain Rpn1 flox / - ;Stra8-GFPCre mice (Rpn1-SKO; Figure 1 D). Immunoblotting and IF staining of RPN1 and γH2AX confirmed that RPN1 levels were indeed reduced in Rpn1-SKO testes compared with WT ( Figure 1 E, F). A 6-month fertility test showed that Rpn1-SKO male mice were completely infertile ( Figure 1 G), although the body weights were comparable between Rpn1-SKO and WT mice. The testes of Rpn1-SKO male mice were much smaller than those of WT male mice (Figure H), and the testis weight and testis-to-body weight ratio were significantly reduced ( Figure 1 I, J). Histological analysis showed that spermatocytes in Rpn1-SKO testes were very few, contained apoptotic cells or were almost empty, while seminiferous tubules in WT mice contained abundant spermatocytes at different stages ( Figure 1 K) Subsequent IF staining with the germ cell marker MVH showed that Rpn1-SKO mice carried MVH compared to wild-type male mice. + Significantly fewer cells ( Figure 1 L). In addition, no sperm were found in the epididymis of adult Rpn1-SKO mice ( Figure 1 K).

[0063] Taken together, these results demonstrate that germ cell knockout of Rpn1 leads to germ cell loss in mice and that RPN1 is required for spermatogenesis and male fertility.

[0064] Example 4 RPN1 deficiency leads to severe defects in the meiotic process of mouse spermatogenesis

[0065] Given the fertility and spermatogenesis defects observed in Rpn1-SKO mice, we investigated the developmental stage at which germ cell number initially decreases by collecting testes from WT and Rpn1-SKO mice for histological and IF analysis at PD12, PD15, PD18, PD23, and PD30. Although no significant differences were observed between Rpn1-SKO and WT testes at PD12, germ cell number decreased in Rpn1-SKO at PD15 and further decreased dramatically from PD18 to PD30 ( Figure 2 A).

[0066] To verify whether the loss of RPN1 affects spermatogonial differentiation, IF antibodies against PLZF, a marker of undifferentiated spermatogonial cells, and SOX9, a marker of Sertoli cells, were co-stained. Image analysis showed that there was no difference in the abundance of staining between Rpn1-SKO and WT mice ( Figure 2 B, C). Next, we used the differentiated spermatogonial marker c-KIT to focus on the stage of spermatogonial differentiation. Image analysis again showed no significant differences between Rpn1-SKO and WT testes at PD12 ( Figure 2 D) shows that knocking out Rpn1 in germ cells does not affect spermatogonia differentiation. Spermatogonia develop into primary spermatocytes and then enter meiosis.

[0067] Next, we used MVH fluorescent-labeled antibodies and co-stained the meiotic initiation marker STRA8 or the spermatocyte meiotic marker SYCP3 on paraffin sections to investigate whether Rpn1 deficiency affects meiotic entry. Figure 2 E) Compared with WT testes, Rpn1-SKO testes contained more STRA8 per seminiferous tubule. + and SYCP3 + There are significantly fewer cells ( Figure 2 F, G), suggesting that RPN1 may be involved in the entry of spermatocytes into meiosis. We then examined the expression of the testis-specific histone marker H1t at the pachytene stage, which is upregulated in spermatocytes at the mid-late pachytene and diplotene stages, to investigate the fate of Rpn1-SKO spermatocytes. H1t staining analysis showed that Rpn1-SKO spermatocytes could at least reach the mid-late pachytene stage (PD18), while the first wave of spermatogenesis in WT mice could reach the late pachytene stage (PD18). Figure 2 H). In addition, it was observed that H1t +The number of cells was significantly reduced, indicating that the progression of meiotic prophase in Rpn1-SKO spermatocytes was delayed or blocked. In the absence of RPN1, most spermatocytes were lost during spermatogenesis, and TUNEL assays showed that TUNEL-positive cells were abundant in Rpn1-SKO tubules at PD18 when the first wave of meiotic prophase reached late pachytene. Figure 2 I).

[0068] These cumulative results indicate that loss of Rpn1 leads to meiotic defects during spermatogenesis in mice.

[0069] Example 5 Rpn1 deletion leads to defects during synapsis and crossover recombination

[0070] To better understand the relationship between RPN1 deficiency and meiotic defects in Rpn1-SKO spermatocytes, IF staining of the central element (SYCP1) and the axial / lateral element (SYCP3) of the synaptonemal complex in chromosome spreads of 1M mouse spermatocytes was performed. All meiotic prophase I stages (leptoterene to diakinesis, Figure 3 A). However, Rpn1-SKO pachytene and pachytene spermatocytes contain many cells with abnormal chromosome synapsis, such as multiple partners synapsing to different regions of a single chromosome, resulting in frequent pairing between non-homologous chromosomes. In addition, many unsynapsed homologous chromosomes are present in Rpn1-SKO pachytene spermatocytes. Statistical analysis of meiotic prophase populations showed that Rpn1-SKO mice had significantly fewer pachytene and diptene cells, while significantly more pachytene cells, compared with WT controls ( Figure 3 B), which is also reflected in the evaluation of spermatocytes by FACS.

[0071] These results suggest that in the absence of RPN1, meiosis may be arrested or delayed during the zygotene-pachytene transition, resulting in a small fraction of spermatocytes with fewer defects that can progress to pachytene.

[0072] The completion of pachytene synapsis coincides with the completion of DSB repair in autosomes. Given the synapsis defects observed in Rpn1-SKO testes, it was hypothesized that DSB repair might also be impaired. To test this possibility, DSB formation and repair events were detected by co-staining for the DNA DSB markers γH2AX and SYCP3 ( Figure 3C). It was found that the γH2AX signal had comparable intensity between Rpn1-SKO and WT at the leptotene stage, indicating that DSBs formed normally in Rpn1-SKO spermatocytes. However, strong γH2AX signals persisted on the autosomes in Rpn1-SKO pachytene spermatocytes, while they were confined to the XY body in WT spermatocytes at the pachytene stage. These results indicate that DSB repair is indeed defective in Rpn1-SKO spermatocytes. By co-staining for SYCP3 and the crossover marker MLH1, we further determined whether these pachytene spermatocytes were able to complete homologous recombination and form crossovers, and found that MLH1 foci were significantly reduced in Rpn1-SKO pachytene spermatocytes ( Figure 3 D, E). These results indicate that loss of RPN1 leads to severe defects in chiasma formation, suggesting that RPN1 plays a critical role in processes related to DSB repair and chiasma formation in mouse spermatocytes.

[0073] To further investigate the function of RPN1 in DSB repair and recombination during meiosis, we performed staining for RPA2, a component of the RPA complex (which binds single-stranded 3' DNA overhangs in DSBs), as well as DMC1 and RAD51, key recombinases recruited after the RPA complex that mediate sister chromatid single-strand invasion. We found that Rpn1-SKO and WT spermatocytes had comparable numbers of RPA2 dots at leptotene. As recombination progressed during meiosis, RPA2 dots decreased in WT spermatocytes at zygotene and pachytene. However, RPA2 dots were found to persist in zygotene-like and pachytene-like spermatocytes of Rpn1-SKO mice, indicating that RPA2 was not effectively cleared ( Figure 3 F, G). DMC1 and RAD51 foci were then examined to assess the effect of Rpn1 depletion on early homologous recombination. The results showed that in WT spermatocytes, the foci of both recombinases decreased at the transition from leptotene to pachytene, indicating successful DSB repair. However, these foci remained at relatively high levels in Rpn1-SKO spermatocytes ( Figure 3 HJ).

[0074] Collectively, these results indicate that RPN1 loss impairs DSB repair.

[0075] Example 6: Changes in mitochondrial-related protein glycosylation in N-glycosylation analysis of Rpn1 knockout spermatocytes

[0076] Considering the known role of RPN1 in promoting N-glycosylation, we performed TMT-based quantitative proteomics and N-glycoproteomics analyses to quantify the differences in protein expression and N-glycosylation between the testes of PD12 WT and Rpn1-SKO mice ( Figure 4 A).

[0077] In total, N-glycosylation analysis identified 6721 total proteins (including 505 N-glycoproteins), 667 N-glycans, and 653 N-glycopeptides in mouse testis. Figure 4 BE). Of the identified proteins, 109 were detected solely by N-glycoproteomics and 396 were also quantified by proteomics ( Figure 4 B).

[0078] N-glycoproteomic analysis revealed 505 and 487 glycopeptide spots in WT and Rpn1-SKO testes, which were located on 493 and 481 glycopeptides and 391 and 385 glycoproteins, respectively. Figure 4 CE). The typical N-glycosylation motif NXS / T was present in 95.5% of the identified N-glycosylation sites, with no significant difference in its distribution in Rpn1-SKO samples ( Figure 4 F). In WT testes, approximately 80.8% of N-glycoproteins carried one N-glycosylation site, 14.1% had two N-glycosylation sites, and 4.7% had three or more N-glycosylation sites. In contrast, in Rpn1-SKO testes, approximately 82.3% of glycoproteins contained one N-glycoside, while the percentage of proteins containing two N-glycosides decreased to 13% ( Figure 4 G). Differential glycosylation analysis showed that 22 glycopeptides from 20 glycoproteins were upregulated, while 38 glycopeptides from 34 glycoproteins were downregulated in Rpn1-SKO mice (|FC|>1.5; p<0.05) ( Figure 4 H). Gene ontology (GO) analysis showed that the down-regulated glycoproteins were enriched in “release mitochondria”, “apoptotic mitochondrial changes”, and “regulate mitochondrial organization” ( Figure 4 ), while KEGG pathway analysis showed that the down-regulated glycoproteins were enriched in the “reactive oxygen species”, “peroxisome” and “ferroptosis” pathways ( Figure 4 J).

[0079] Taken together, these results suggest that RPN1 deficiency may impair DSB repair and transition to pachytene through abnormal glycosylation of mitochondrial-associated proteins.

[0080] Example 7: Rpn1 deficiency leads to mitochondrial dysfunction and impairs energy metabolism

[0081] Given the above findings, Rpn1 deficiency leads to a decrease in the glycosylation of mitochondrial-related proteins. We directly observed the mitochondrial defects that may be caused by Rpn1 deficiency. To this end, we examined the changes in mitochondrial morphology and structure in spermatocytes using transmission electron microscopy (TEM). The results showed that a subpopulation of mitochondria in Rpn1-SKO testicular spermatocytes was swollen or enlarged, with fewer cristae ( Figure 5A). Subsequently, the mitochondrial outer membrane marker TOM20 was used to evaluate the mitochondrial abundance in WT and Rpn1-SKO spermatocytes, indicating that the TOM20 signal intensity in Rpn1-SKO spermatocytes was significantly lower than that in WT ( Figure 5 B).

[0082] Furthermore, mitochondrial DNA (mtDNA) copy number was quantified by RT-qPCR analysis of 16S and ND1 mitochondrial markers and it was found that the mtDNA copy number in Rpn1-SKO spermatocytes was significantly reduced, consistent with the observed reduction in mitochondrial abundance ( Figure 5 C). Then, the effect of Rpn1 deficiency on the mitochondrial membrane potential (Δψm) of spermatocytes from WT and Rpn1-SKO mice was investigated using JC-1 staining assay for mitochondrial function. Compared with mitochondria from WT spermatocytes, the JC-1 aggregate / monomer ratio in Rpn1-SKO spermatocytes was significantly reduced, indicating that the mitochondrial membrane potential in Rpn1-deficient spermatocytes was weakened ( Figure 5 D).

[0083] Since mitochondria play a crucial role in ATP production and scavenging reactive oxygen species (ROS), we also measured ROS and ATP levels in L / Z and P / D spermatocytes of WT and Rpn1-SKO mice. As expected, Rpn1-deficient L / Z and P / D spermatocytes had lower ATP levels but higher ROS levels compared to WT ( Figure 5 EG). Increased mitochondrial ROS levels can induce oxidative damage, leading to mitochondrial Ca 2+ Excessive accumulation of Ca2+ opens the mitochondrial membrane permeability transition pore, releasing mitochondrial contents, especially cytochrome c, and thus triggering cell apoptosis. 2+ Mitochondrial Ca2+ expression in spermatocytes was detected by flow cytometry using Rhod-2 AM as an indicator. 2+ This analysis showed that mitochondrial Ca levels in L / Z and P / D spermatocytes of Rpn1-SKO mice were significantly increased compared with those in WT controls. 2+ Levels did increase significantly ( Figure 5 G).

[0084] These observations collectively suggest that Rpn1 deficiency suppresses mitochondrial function and energy metabolism, thereby increasing spermatocyte apoptosis and ultimately leading to male infertility ( Figure 6 ).

[0085] In the male reproductive system, some key proteins are known to undergo N-glycosylation modification. However, the specific roles and mechanisms of these modifications in mammalian spermatogenesis and the entire male reproductive system are still unclear. The applicant discovered and demonstrated for the first time that knocking out the N-glycosylation-related protein ribonucleoprotein I (RPN1) would lead to abnormal meiosis and complete infertility in male mouse spermatocytes. N-glycosylation genomics analysis showed that the absence of RPN1 led to a significant decrease in the glycosylation level of mitochondrial-related proteins, thereby affecting the structure and function of mitochondria. The applicant's discovery provides new insights into the relationship between N-glycosylation modification and meiosis, and further research on this relationship will help to advance the development of treatments for male infertility. The applicant has thus provided a method for diagnosing male fertility defects in mammals, including humans, and has provided new ideas and methods for developing new treatments for male infertile individuals affected by gene mutations.

[0086] The above is an explanation of the present invention and should not be regarded as limiting 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 in addition to being particularly described in the above description and embodiments, the present invention can also be realized in other ways. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art. 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 RPN1 in a male subject for preparing a kit or instrument for diagnosing or predicting reproductive defects such as infertility in mammals and humans.

2. The method of claim 1, wherein the male individual's infertility disorder is selected from teratozoospermia, azoospermia, oligospermia, asthenozoospermia, and asthenozoospermia.

3. The use according to claim 1 or 2, wherein the reagent for detecting RPN1 in the male subject is a reagent for detecting the Rpn1 gene.

4. The use according to claim 1 or 2, wherein the reagent for detecting RPN1 in the male subject is a reagent for detecting RPN1 protein, and preferably, further comprises a reagent for measuring the activity of RPN1.

5. The use according to claim 4, which comprises a reagent for detecting the expression of RPN1 by immunoassay, such as a reagent for detecting the expression of RPN1 by ELISA or Western blotting using an antibody that specifically recognizes the protein; or These include reagents for detecting RPN1 by detecting the presence or amount of RPN1 mRNA, such as reagents for detecting the amount of RPN1 encoding mRNA in a sample by RT-PCR.

6. A kit for diagnosing reproductive defects such as infertility in mammals and humans, comprising a reagent for detecting RPN1 in a test male individual.

7. The kit of claim 6, wherein the infertility disorder of the male individual is selected from teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, and asthenozoospermia.

8. The kit of claim 6, comprising a reagent for detecting the Rpn1 gene of the test male individual.

9. The kit of claim 6, comprising a reagent for detecting RPN1 protein in a male subject, and preferably, further comprising a reagent for measuring RPN1 activity.

10. The kit of claim 9, comprising a reagent for detecting RPN1 by immunoassay, such as an antibody that specifically recognizes RPN1; or These include reagents for detecting the presence or amount of RPN1 mRNA to detect the expression of RPN1, such as reagents for detecting RPN1 mRNA in a sample by RT-PCR.

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