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Comparative Proteomic Analysis of Seminal Plasma Exosomes in Buffalo with High and Low Sperm Motility

ABSTRACT: Exosomes are nanosized membranous vesicles secreted by variety types of cells and regulate intercellular communication through deliver bioactive substances. They are found in abundance in biological fluids including semen. Although protein composition and potential fertility biomarkers of seminal plasma exosomes (SPE) have been identified in human, but they remains unclear in buffalo. Here, we aim to discover the protein correlation between spermatozoa, seminal plasma (SP) and SPE, then further compare and analyze the protein differences between high and low motility SPEs in buffalo. SPEs were concentrated and purified by ultracentrifugation combined by sucrose density gradient centrifugation, followed by verification by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPE, and protein difference in high and low motility SPEs were identified by LC-MS/MS proteomic analysis and functional analyzed by a comprehensive bioinformatic analysis.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Bubalus Bubalis

TISSUE(S): Testis, Sperm

SUBMITTER: qiang Fu

LAB HEAD: Qiang Fu

PROVIDER: PXD033442 | Pride | 2022-12-05

REPOSITORIES: Pride

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Proteomic profiles of buffalo spermatozoa and seminal plasma.

Fu Qiang Q Pan Limei L Huang Delun D Wang Zhiqiang Z Hou Zhen Z Zhang Ming M

Theriogenology 20190515

A comprehensive identification of the proteins of spermatozoa and seminal plasma enables the full characterization of sperm biology and is essential to understand how seminal plasma influences sperm fertility. In this study, proteomics of buffalo spermatozoa and seminal plasma were analyzed using a bottom-up approach. A total of 2147 and 864 proteins were identified from mature spermatozoa and seminal plasma, respectively. Of these, 371 proteins-42.9% in the seminal plasma set-were common to bot ...[more]

PMID: 31146187

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Project description:Male factors account for approximately 40% of all infertility. Conventional semen analysis focus on seminal physicochemical property and spermatozoa morphology. They yield no information concerning the functional competence of the spermatozoa(Petrunkina et al., 2007). Owning to the essential role of proteins in the reproductive process, comprehensive and systematic identification of proteome is critical to gain new insights into spermatogenesis and infertility. Recent advances in mass spectrometry have allowed the identification of hundreds to thousands of protein in spermatozoa(Oliva et al., 2008; Amaral et al., 2014; Codina et al., 2015). In major domestic mammalian species, global spermatozoa proteomic profiles have been described in rodents(Baker et al., 2008a; Baker et al., 2008b) and bovine(Peddinti et al., 2008). Meanwhile, lacking of new protein synthesis in spermatozoa supports the current view that the regulation of sperm maturation is controlled by exogenous proteins(O'Rand et al., 2011) (e.g., during epididymal transit). Among the seminal plasma proteins, The human seminal plasma has been comprehensively described (Pilch and Mann, 2006; Batruch et al., 2011; Milardi et al., 2012; Milardi et al., 2013; Sharma et al., 2013). In domestic animal species, some studies performing a systematic analysis on using high throughput proteomics have been performed(Kelly et al., 2006; Moura et al., 2007; Souza et al., 2012; Druart et al., 2013; Soleilhavoup et al., 2014). Buffalos (Bubalus bubalis) are adapted to hot–humid tropical climatic conditions, but have low reproductive efficiency. The low sperm motility maybe contributed to protein components variation of spermatozoa and seminal plasma. The composition of buffalo spermatozoa and buffalo seminal plasma (BSP) remains unknown. Proteomic study would be beneficial to the elucidation of the roles of sperm and BSP proteins in regulation of maturation, motility and fertilization. As such, the aim of the current study was to extensively characterize the differentially expressed protein of buffalo spermatozoa and seminal plasma using a comparative proteomics.

Project description:Abstract Buffalo bulls are the backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of buffalo dairy farms. Seminal plasma (SP) of bulls harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility associated proteins which contribute to the fertilizing potential of spermatozoa. In this study, we explored various parameters of EVs such as size, concentration and the complete proteome profiles of SP EVs from two distinct fertility groups in order to unravel the differentially abundant proteins that potentially affect the overall fertility of bulls. Through Dynamic Light Scattering (DLS) it was found that the purified EVs were present in 7 to 14 size exclusion chromatographic (SEC) fractions with sizes ranging from 145 to 256 nm in the high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively across all the fractions. No significant difference was observed in the size and concentration of seminal EVs between the HF and LF groups. We identified a total of 1862 and 1807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1754 proteins were common in both the groups and about 87 proteins were highly abundant in HF group while 1292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that the highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. In addition, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins of HF EVs, namely protein disulfide-isomerase A4 (PDIA4) and gelsolin (GSN), were successfully immunolocalized on spermatozoa, indicating that these proteins might be transferred to spermatozoa through EVs. The proteins; PDIA4 and GSN are intricately associated with sperm-oocyte fusion and acrosome reaction, respectively, thus they are vital for regulating the fertilizing capacity of sperm. Our evidences clearly support that the protein repertoires in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, the current investigation clearly indicates that SPEVs possess crucial protein repertoires which are essential for enhancing the sperm fertilizing capacity.

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