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Quantitative Phosphoproteomic Profiling of Mouse Sperm Maturation in Epididymis Revealed Kinases Important for Sperm Motility.

ABSTRACT: Transcriptionally and translationally silent sperm undergo functional maturation during epididymis traverse, which provides sperm ability to move and is crucial for successful fertilization. However, the molecular mechanisms governing sperm maturation remain poorly understood, especially at the protein post-translational modification level. In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. We identified 6447 phosphorylation sites in 1407 phosphoproteins, and 345 phosphoproteins were differentially phosphorylated between caput and cauda sperm. Gene ontology and KEGG pathway analyses showed enrichment of differentially phosphorylated proteins in energy metabolism, sperm motility, and fertilization. Kinase substrate network analysis followed by inhibition assay and quantitative phosphoproteomics analysis showed that TSSK2 kinase is important for sperm motility and progressive motility. This study systemically characterized the intricate phosphorylation regulation during sperm maturation in the mouse epididymis, which can be a basis to elucidate sperm motility acquisition, and to offer potential targets for male contraception and the treatment of male infertility.

SUBMITTER: Zhang X

PROVIDER: S-EPMC11338950 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Quantitative Phosphoproteomic Profiling of Mouse Sperm Maturation in Epididymis Revealed Kinases Important for Sperm Motility.

Zhang Xiangzheng X Tu Haixia H Zhou Xin X Wang Bing B Guo Yueshuai Y Situ Chenghao C Qi Yaling Y Li Yan Y Guo Xuejiang X

Molecular & cellular proteomics : MCP 20240706 8

Transcriptionally and translationally silent sperm undergo functional maturation during epididymis traverse, which provides sperm ability to move and is crucial for successful fertilization. However, the molecular mechanisms governing sperm maturation remain poorly understood, especially at the protein post-translational modification level. In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda ...[more]

PMID: 38977202

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Project description:BackgroundOvochymase 2 (Ovch2) is an epididymis-specific gene that is required for male fertility. While a multitude of reproductive tract-specific genes required for male fertility have been identified, OVCH2 is thus far the first protein required for male fertility that contains Complement C1r/C1s, Uegf, Bmp1 (CUB) domains located in tandem in the C-terminus of the protein. Identifying the functional significance of this unique domain has implications in better understanding fertility and infertility and as a potential contraceptive target.ObjectiveThe goals of these studies were to understand the influence and requirement of OVCH2 CUB domains in the localization and functional requirement of OVCH2 in sperm maturation and function.Materials and methodsTo this end, we performed in vivo localization analysis of OVCH2 and reproductive phenotype analysis of mice containing C-terminal FLAG tag on OVCH2, with either the entire protein intact, or CUB2 or both CUB1 and CUB2 genetically ablated. All mice were generated through the CRISPR/Cas9 gene editing approach.ResultsWe found that OVCH2 is specifically expressed in the proximal caput epididymidis, and the absence of CUB2 did not affect this localization pattern. Although the absence of both CUB domains significantly reduced sperm motility and progressive motility, this effect was not manifested in a reduction in fertility over a 6-month period mating trial, which showed no significant differences between control and CUB deletant mice. Further, the absence of one or both CUB domains did not affect reproductive organ structure or sperm morphology.ConclusionsOur studies demonstrate that the CUB domains are not required for fertility in male mice, at least under the normal animal housing conditions our mice were tested in, and suggest that the enzymatic activity of the OVCH2 protease, in the absence of its CUB domains, is sufficient for normal sperm processing in the epididymis. Although our findings do not preclude the possibility that OVCH2 CUB domains are required under a yet-identified stress condition, our findings demonstrate that the most likely region for deleterious mutations in men with idiopathic infertility and the most vulnerable site for inhibition of OVCH2 protein function is in its protease domain, and not its CUB domains. Our findings have implications in the genetic screening of infertile men and the development of a novel non-hormonal male contraceptive by honing in on the more critical region of a functionally required protein.

Project description:In this study, the epididymal histology, caepididymal sperm physiological parameters, serum reproductive hormones, and antioxidant enzyme SOD levels of Tibetan sheep at a 2500 m and 3500 m altitude were compared by using a combination of transcriptome and metabolomics methods. This was conducted to investigate the effects of a high-altitude environment on spermatogenesis and the maturation of Tibetan sheep. The results showed that compared to the low-altitude group, the high-altitude group had a smaller epididymal lumen, thicker epididymal wall, significantly decreased sperm survival rate, and significantly increased sperm deformation rate, but no difference in sperm motility and sperm respiratory intensity. With increasing altitude, Tibetan sheep showed a decreasing trend in serum reproductive hormones (FSH and T), while the antioxidant enzyme SOD activity was significantly reduced. Transcriptomic analysis revealed 139 differentially expressed genes in the Tibetan sheep epididymis under high-altitude conditions. The SYCP2 gene is involved in multiple biological processes related to reproduction and plays an important role in the regulation of epididymal function and sperm quality in Tibetan sheep. Genes like ADCYAP1R1, CABP2, CALN1, and ATP6V1B1 can help maintain sperm viability and maturation by regulating the cAMP signaling pathway, calcium ion homeostasis, and cellular signaling. Metabolomic analysis found that the high-altitude group had increased adenosine content and decreased prostaglandin I2 content in the epididymis. These metabolites are involved in spermatogenesis, motility, fertilization, and early embryonic development. The integrated omics analysis suggests that Tibetan sheep adapt to the high-altitude hypoxic environment by regulating cAMP signaling pathway genes like ADCY and PRKACA, as well as metabolites like adenosine and prostaglandin I2, to maintain epididymal function and sperm motility. These genes and metabolites play an important role in maintaining normal epididymal function and sperm motility at high altitudes.

Project description:PurposeDuring epididymal sperm maturation, spermatozoa acquire progressive motility through dynamic protein modifications. However, the relationship between sequential protein modifications during epididymal sperm maturation and sperm motility and fertility has not yet been investigated. This study investigated whether sequential changes in fertility-related protein expression including that of enolase 1 (ENO1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and UQCRC2), and voltage-dependent anion channel 2 (VDAC2) in spermatozoa during epididymal maturation are related to bovine sperm motility. Moreover, we found that mitochondrial metabolism is closely related to fertility-related proteins. Therefore, we investigated how the sequential modification of mitochondrial proteins during epididymal maturation regulates sperm motility.Materials and methodsTo determine the differential protein expression in caput and cauda epididymal spermatozoa from low and high motility bulls, western blot analysis was performed. Moreover, signaling pathways were identified to understand the mechanisms of regulation of sperm motility through the differential protein expression associated with fertility-related proteins.ResultsWe found that ENO1 was substantially higher in the caput spermatozoa from low motility bulls than the caput and cauda spermatozoa from high motility bulls. However, ENO1 expression in low motility bull spermatozoa was downregulated to a level comparable to that in the high motility bull spermatozoa during epididymal maturation. Moreover, there was a lack of modification of mitochondrial proteins, including glutathione peroxidase 4 and NADH:Ubiquinone Oxidoreductase Core Subunit S8, in low motility bull spermatozoa during epididymal maturation, whereas active changes were detected in high motility bull spermatozoa.ConclusionsIrregular modifications of mitochondrial proteins during epididymal sperm maturation may increase excessive ROS production and premature activation of spermatozoa during epididymal maturation. Consequently, spermatozoa may lose their motility by the earlier consumption of their energy source and may be damaged by ROS during epididymal maturation, resulting in a decline in sperm motility and bull fertility.

Project description:Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Within the molecular scope of NCSLC, a complex landscape of dysregulated cellular signaling has emerged, defined largely by mutations in select mediators of signal transduction, including the epidermal growth factor receptor (EGFR) and anaplastic lymphoma (ALK) kinases. Consequently, these mutant kinases become constitutively activated and targets for chemotherapeutic intervention. Encouragingly, small molecule inhibitors of these pathways have shown promise in clinical trials or are approved for clinical use. However, many protein kinases are dysregulated in NSCLC without genetic mutations. To quantify differences in tumor cell signaling that are transparent to genomic methods, we established a super-SILAC internal standard derived from NSCLC cell lines grown in vitro and labeled with heavy lysine and arginine, and deployed them in a phosphoproteomic workflow. We identified 9019 and 8753 phosphorylation sites in two separate tumors. Relative quantification of phosphopeptide abundance between tumor samples allowed for the determination of specific hubs and pathways differing between each tumor. Sites downstream of Ras showed decreased inhibitory phosphorylation (Raf/Mek) and increased activating phosphorylation (Erk1/2) in one tumor versus another. In this way, we were able to quantitatively access oncogenic kinase signaling in primary human tumors.Through the use of quantitative proteomics, we demonstrated the feasibility and coverage that large scale mass spectrometry can leverage for understanding kinase networks in cancer. By incorporating Super-SILAC based quantitation into a typical pathology workflow, we were able to access and compare tumors from multiple patients in this analysis with high accuracy and dynamic range. We analyzed tumors from patients diagnosed with non-small cell lung cancer and were able to detect comprehensive phosphorylation networks relaying through known hubs of oncogenesis in lung cancer. We hereby show that it is possible to track changes to phosphorylation networks across multiple tumors, opening up the possibility that drug susceptibility and patient-specific stratification can be implemented downstream of classical pathology.

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Quantitative Phosphoproteomic Profiling of Mouse Sperm Maturation in Epididymis Revealed Kinases Important for Sperm Motility.

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Quantitative Phosphoproteomic Profiling of Mouse Sperm Maturation in Epididymis Revealed Kinases Important for Sperm Motility.

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