Two-Dimensional Gel Electrophoretic Analysis of Vectorially Labeled Surface Proteins of Human Spermatozoa1

Abstract

The objective of this study was to identify the repertoire of proteins exposed on the surface of ejaculated human spermatozoa. High-resolution two-dimensional gel systems for separation of human sperm and seminal plasma proteins were developed using both isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) followed by polyacrylamide gel electrophoresis (IEF/PAGE, NEPHGE/PAGE). Proteins were visualized by silver staining of gels and by electroblotting followed by gold staining. The protein patterns were analyzed by computer after laser or camera scanning. One thousand three hundred ninety-seven sperm proteins with a molecular mass between 5 and 160 kDa and isoelectric points (pI) from 4 to 11 were catalogued from silver-stained gels loaded with approximately 0.25 mg of NP-40/urea extracts of sperm harvested by Percoll density gradient centrifugation, and 1191 proteins were resolved following extraction with SDS/3-[3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate/urea. Analysis of seminal plasma proteins obtained from vasectomized patients revealed over 300 silver-stained proteins, which aided the identification of sperm-coating proteins acquired from secretions of the accessory sex organs. Sperm surface proteins accessible to vectorial labeling with 125I or N-hydroxysuccinimide biotin were identified; 181 protein spots were radiolabeled with 125I, while 228 protein spots were biotinylated, including several groups of protein isoforms. Cytoskeletal and intra-acrosomal control proteins were not iodinated or biotinylated, thus verifying the surface specificity of both labeling methods. Ninety-eight sperm surface proteins were labeled by both iodine and biotin, and 22 sperm surface proteins, representing five groups of protein isoforms, were shown to contain phosphotyrosine. A composite computer image showing the position of the dually vectorially labeled sperm surface proteins was constructed, together with a table of the proteins' molecular weight, pI, and relative concentration. In addition, novel isoforms of actin, beta-tubulin, PH-20, and several phosphotyrosine-containing proteins were identified in human sperm.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

“…However, the number of biotinylated proteins comprised less than 10% of the 1754 protein spots distinguished by silver staining of the extracted proteins (Figure 1a), a fraction which is in accordance with findings from previous studies (Naaby-Hansen et al, 1997;Coonrod et al, 1999).…”

Particulate Adenylate Cyclase Plays a Key Role in Human Sperm Olfactory Receptor-mediated Chemotaxis

“…For Ca 2ϩ imaging, acrosome reaction assays, and immunocytochemistry a Percoll density gradient centrifugation was performed after liquefaction (30 min at 35.5°C) to isolate mature and motile sperm (20). In brief, liquefied semen was overlaid on a two-layer Percoll (Amersham Biosciences) density gradient consisting of 80 and 55% isotonic Percoll in Ham's F-10 medium (Invitrogen).…”

Proteomics of spermatogenesis: from protein lists to understanding the regulation of male fertility and infertility

“…Other studies have also revealed a similar number of proteins. [44][45][46] These recent global analyses of human sperm are landmark studies and provide rich resources for exploring normal sperm physiology, including sperm motility.…”