Immunohistochemical Localization of Sperm‐Preserving Proteins in the Ram Reproductive Tract

Abstract

Previously, we reported that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury, and that 2 proteins of approximately 14 (P14) and 20 (P20) kDa, the main components of fraction 6 isolated by exclusion chromatography, are responsible for this protective effect. The objective of the present study was to localize P14 and P20 in tissues of the ram reproductive tract to determine their origin. Antiserum generated against purified P14 and P20 reacted with proteins in seminal vesicles and vas deferens by Western blot analyses of protein tissue extracts. However, these antisera failed to detect P14 and P20 in testis, prostate, efferent ductules, bulbourethral glands, and epididymis (caput, corpus, and cauda). Immunohistochemical analyses by both indirect immunofluorescence and the avidin-biotin complex technique confirmed that only seminal vesicles showed reactivity, restricted to the secretory cells, with both antibodies. Obtained results indicate that P14 and P20 are secreted specifically in the seminal vesicles. To further confirm that P14 and P20 are specifically expressed in seminal vesicles, we used Northern blot analyses to investigate the expression of both proteins in seminal vesicles and vas deferens. These assays corroborated again that P14 and P20 were specifically expressed in seminal vesicles. Consequently, we suggest referring to these 2 proteins as RSVP14 and RSVP20, respectively, according to their origin and molecular weight.

Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction

“…Our data are consistent with those in a previous report in which we identified the presence of a seminal plasma protein in the ram sperm membrane (RSVP14, Fernandez-Juan et al 2006), phosphorylated at serine and threonine residues, which undergoes a partial loss and redistribution during capacitation and acrosome reaction towards the post-acrosomal and equatorial regions (Barrios et al 2005). The results of the present study support our previous suggestion that serine/ threonine phosphorylation may play an important role in sperm capacitation, since consolidation of phosphoproteins to specific domains of the head and tail may facilitate the acrosome reaction and egg penetration (Naz 1999).…”

The Positive Effects of Seminal Plasma During the Freezing Process on Cryosurvival of Sperm with Poor Freezability in the Rhesus Macaque (Macaca mulatta)

“…The complex mixture of a wide variety of components in seminal plasma affects the survival of sperm. Certain seminal plasma protein bands have been identified in boar semen that could prevent cold shock injury [19]. In the present study, individual responses to the presence and absence of seminal plasma were observed after cold shock, and semen volumes and sperm concentrations varied among the five rhesus macaque males, which indicated that the components of seminal plasma and the proteins that could prevent cold shock might be genetically inherited rather than being random.…”

Remodelling of Lipid Rafts during In vitro Capacitation and Acrosome Reaction of Ram Spermatozoa

“…We have already shown that RSVP14 and RSVP20 (ram seminal plasma proteins) are adsorbed onto the ejaculated ram sperm surface [75], and that they are redistributed and partially lost from the plasma membrane during in vitro capacitation and the acrosome reaction (only approximately 35% of them remained on the spermatozoa after both processes, [76]. Thus, we can speculate that there is both a loss and a redistribution of proteins in discrete plasma membrane domains.…”