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CSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit.

ABSTRACT: Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.

SUBMITTER: Krapf D

PROVIDER: S-EPMC3424100 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit.

Krapf Dario D Ruan Ye Chun YC Wertheimer Eva V EV Battistone Maria A MA Pawlak John B JB Sanjay Archana A Pilder Stephen H SH Cuasnicu Patricia P Breton Sylvie S Visconti Pablo E PE

Developmental biology 20120630 1

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associa ...[more]

PMID: 22750823

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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:BackgroundPaternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit.ResultsUsing proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation.ConclusionsThese data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Project description:AbstractIn laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits.Lay summaryIn mice, sperm quality is usually assessed in sperm collected from the epididymis (organ where ripe sperm is stored) of euthanized males. However, there is one non-terminal and minimal invasive alternative to collect sperm, called percutaneous epididymal sperm aspiration (PESA), which allows repeated sample collections from the same individual. Given that individual sperm quality is variable and can change according to various factors, PESA could allow to track sperm quality over time and would be highly appreciated in different research fields. Here, we tested the suitability of PESA to determine sperm quality by comparing sperm samples collected by PESA vs the commonly applied terminal epididymis dissection. We used computer-assisted sperm analysis to determine various sperm quality traits. Surprisingly, we found that sperm collected by PESA showed significantly reduced motility, swimming velocity and more morphological abnormalities compared to sperm samples collected by epididymis dissection. Thus, we cannot recommend PESA as a suitable method to determine sperm quality traits as the procedure itself seems to affect collected sperm cells.

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CSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit.

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cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit.

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