Identification and validation of mouse sperm proteins correlated with epididymal maturation

Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6080, USA.
Proteomics (Impact Factor: 3.81). 10/2011; 11(20):4047-62. DOI: 10.1002/pmic.201100075
Source: PubMed


Sperm need to mature in the epididymis to become capable of fertilization. To understand the molecular mechanisms of mouse sperm maturation, we conducted a proteomic analysis using saturation dye labeling to identify proteins of caput and cauda epididymal sperm that exhibited differences in amounts or positions on two-dimensional gels. Of eight caput epididymal sperm-differential proteins, three were molecular chaperones and three were structural proteins. Of nine cauda epididymal sperm-differential proteins, six were enzymes of energy metabolism. To validate these proteins as markers of epididymal maturation, immunoblotting and immunofluorescence analyses were performed. During epididymal transit, heat shock protein 2 was eliminated with the cytoplasmic droplet and smooth muscle γ-actin exhibited reduced fluorescence from the anterior acrosome while the signal intensity of aldolase A increased, especially in the principal piece. Besides these changes, we observed protein spots, such as glutathione S-transferase mu 5 and the E2 component of pyruvate dehydrogenase complex, shifting to more basic isoelectric points, suggesting post-translational changes such dephosphorylation occur during epididymal maturation. We conclude that most caput epididymal sperm-differential proteins contribute to the functional modification of sperm structures and that many cauda epididymal sperm-differential proteins are involved in ATP production that promotes sperm functions such as motility.

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    • "The role of the BAG6/HSPA2 interaction that we observed in both the epididymal epithelium and in maturing spermatozoa within the lumen of this organ remains less certain. Interestingly, the localization of HSPA2 to epididymal spermatozoa in our study diverges from observations of the mouse epididymis where the protein is purportedly shed along with the cytoplasmic droplet during sperm transit from the caput to caudal regions of the duct (Ijiri et al., 2011). However, our detection of HSPA2 in the epithelial cells of the epididymis reflects that of previous localization studies (Legare et al., 2004). "
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    ABSTRACT: While a large cohort of sperm surface receptors underpin sperm-oocyte adhesion processes, our recent work has revealed that the molecular chaperone Heat Shock Protein A2 (HSPA2) is a key regulator of zona pellucida-receptor complex assembly in our own species. Indeed, in the infertile population, spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. While the mechanisms behind this protein deficiency are under consideration, BCL2-associated athanogene 6 (BAG6) has been identified as a key regulator of HSPA2 stability in mouse germ cells. However, in the human, the presence of BAG family proteins remains completely uncharacterized. Consequently, this study aimed to determine the presence of BAG6 in human sperm cells and to characterize its putative interaction with HSPA2 throughout sperm cell development. BAG6 was shown to co-localize with HSPA2 in human testicular germ cells and epididymal spermatozoa. Similarly, BAG6 was identified in the equatorial region of non-capacitated spermatozoa but underwent a marked relocation to the anterior region of the head upon the induction of capacitation in these cells. Protein-protein interaction assays revealed the stable interaction of BAG6 and HSPA2 proteins in mature spermatozoa. Furthermore, examination of the spermatozoa of infertile men with zona pellucida binding defects, related to a lack of HSPA2, revealed a concomitant deficiency in BAG6 protein expression. In view of the findings described in this study, we propose that BAG6 is likely a key regulator of HSPA2 stability/function in human germ cells. Moreover, its under-representation in spermatozoa with zona pellucida binding deficiency suggests that BAG6 may be an important candidate to study for a further understanding of male idiopathic infertility.
    Molecular Human Reproduction 07/2015; 21(10). DOI:10.1093/molehr/gav041 · 3.75 Impact Factor
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    • "The basic techniques used in proteomics research largely focus on illuminating the structure and conformation, purifying, and measuring the concentration of sperm proteins. The proteomic techniques that are used for the structural analysis of sperm proteins include MS and electrophoresis [26, 27], nuclear magnetic resonance spectroscopy [29, 30], and X-ray crystallography [31, 32]. However, liquid chromatography (LC) [33, 34] and the recently developed microfluidic separation technique [35, 36] are widely used for the purification of proteins from spermatozoa or other samples. "
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    ABSTRACT: Spermatozoa are highly specialized cells that can be easily obtained and purified. Mature spermatozoa are transcriptionally and translationally inactive and incapable of protein synthesis. In addition, spermatozoa contain relatively higher amounts of membrane proteins compared to other cells; therefore, they are very suitable for proteomic studies. Recently, the application of proteomic approaches such as the two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in-gel electrophoresis has identified several sperm-specific proteins. These findings have provided a further understanding of protein functions involved in different sperm processes as well as of the differentiation of normal state from an abnormal one. In addition, studies on the sperm proteome have demonstrated the importance of spermatozoal posttranslational modifications and their ability to induce physiological changes responsible for fertilization. Large-scale proteomic studies to identify hundreds to thousands of sperm proteins will ultimately result in the development of novel biomarkers that may help to detect fertility, the state of complete contraception, and beyond. Eventually, these protein biomarkers will allow for a better diagnosis of sperm dysfunctions and aid in drug development. This paper reviews the recent scientific publications available from the PubMed database to address sperm proteomics and its potential application to characterize male fertility and contraception.
    International Journal of Endocrinology 12/2013; 2013(8):360986. DOI:10.1155/2013/360986 · 1.95 Impact Factor
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    • "Although some information is available concerning TPI1 in sperm from a limited number of species, an extensive analysis of the isoforms present and the localization of the protein in sperm have not been published. We previously reported that mouse TPI1 was present in the sperm flagellar accessory structures and was identified as a cauda epididymal sperm-differential protein by proteomic analysis (Cao et al., 2006; Ijiri et al., 2011). Here, we have shown that TPI1 is restricted to the principal piece of the sperm flagellum, and is tethered to the fibrous sheath. "
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    Molecular Reproduction and Development 10/2013; 80(10). DOI:10.1002/mrd.22217 · 2.53 Impact Factor
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