Ejaculated ram spermatozoa, freed from seminal plasma by a dextran/swim-up procedure and exposed to cold shock, were incubated with ram seminal plasma proteins and analyzed by fluorescence markers and scanning electron microscopy. Seminal plasma proteins bound to the sperm plasma membrane modified the functional characteristics of damaged spermatozoa, reproducing those of live cells. Scanning electron microscopy showed that the dramatic structural damage induced by cooling reverted after incubation with seminal plasma proteins. Assessment of membrane integrity by fluorescence markers also indicated a restoration of intact-membrane cells. This protein adsorption is a concentration-dependent process that induces cell surface restoration in relation to the amount of protein in the incubation medium. Fractionation of ram seminal plasma proteins by exclusion chromatography provided three fractions able to reverse the cold shock effect. Scanning electron microscopy also confirmed the high activity of one fraction, because approximately 50% of cold-shocked sperm plasma membrane surface was restored to its original appearance after incubation. Differences in composition between the three separated fractions mainly resulted from one major band of approximately 20 kDa, which must be responsible for recovering the sperm membrane permeability characteristic of a live cell.
ABSTRACT:We have already shown that seminal plasma proteins in the ram can repair cold-shock sperm membrane damage and that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury. In this study, we prove that 2 protein bands of approximately 14 (P14) and 20 (P20) kd isolated from seminal plasma are responsible for this protective effect. In vitro capacitation (CA) and acrosome reaction (AR) modified the content of both proteins on the sperm surface. P20 release began at the beginning of CA, and the induction of the AR accounted for an additional release of both proteins; not more than 35% of these proteins remained on acrosome-reacted sperm. Cytochemical analysis detected that there are several binding sites for P14 and P20 on the sperm surface and that membrane alterations induced by CA and the AR accounted for the loss and redistribution of both proteins to the equatorial and postequatorial regions. The P14-sequenced fragment showed a high homology with several seminal plasma proteins of different species and contained the FN 2 domain like bovine PDC-109. However, the sequence of the P20 fragment was not homologous with any reported protein. By immunochemical analysis, we obtained evidence that P14 is phosphorylated in serine and threonine residues and that P20 is a glycosylated protein. These results suggest that both proteins are involved in sperm CA and gamete interaction, first by stabilizing the sperm membrane and then by participating in CA in the female reproductive tract. The protective effect of P14 and P20 could be related to their decapacitating role.Key words: Immunocytochemistry, capacitation, acrosome reaction.J Androl 2005;26:539-549 M ammalian sperm leaving the testis cannot fertilize eggs. They must undergo a series of modifications in the epididymis and then in the female reproductive tract (capacitation [CA]) to become fully competent to fertilize an ovum (Austin, 1951(Austin, , 1952Chang, 1951). Among the most frequently studied phenomena during this maturation process are the sperm surface changes and the segregation of certain proteins and lipids to specialized domains of the sperm plasma membrane (for a review, see Jones, 1999).The seminal plasma of mammals is a complex biological mixture of various fluids in the male reproductive tract. It contains several proteins (Mann and LutwakMann, 1981), some of which are adsorbed onto the surface of ejaculated sperm (Leeuw de et al, 1990;Metz et al, 1990;Desnoyers and Manjunath, 1992;Amann et al, 1999). Many of these proteins are secretory products of the seminal vesicle (Huarte et al, 1987;Aumuller et al, 1988;Chandonnet et al, 1990), an accessory reproductive gland in most male mammals, where they accumulate in the lumen after puberty. Some of the adsorbed proteins maintain the stability of the membrane until the process of CA in the female genital tract (Cross, 1996), when their removal is a prerequisite for fertilization (Desnoyers and Manjunath, 1992). Seminal plasma plays an important role in impr...
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.
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