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.