323 EXPRESSION OF PSP-I AND PSP-II IN THE REPRODUCTIVE TRACT OF THE BOAR BY IMMUNOHISTOCHEMISTRY, WESTERN BLOTTING, AND RT-PCR

Abstract

In boar seminal plasma, PSP-I and PSP-II form a noncovalent heterodimer, which is known to have a beneficial effect on highly extended boar spermatozoa. These proteins are mainly produced by the seminal vesicles of the male reproductive tract and are mixed with the spermatozoa during ejaculation. This study assessed the epithelial localization and expression of spermadhesin PSP-I and PSP-II subunits using immunohistochemical, western blotting, and RT-PCR methods in porcine testis, epididymis (caput, corpus, and caudal), seminal vesicles, and bulbourethral glands. Tissues were collected from 10 mature boars (Swedish Yorkshire) of proven fertility, frozen or fixed, and embedded in paraffin. Sections of 5 µm were mounted on poly-l-lysine-coated glass slides for immunohistochemistry, and 50 mg from the frozen counterparts were homogenized to isolate proteins (Western blotting study) or RNA (RT-PCR study). Polyclonal antibodies (antiPSP-I and antiPSP-II) against proteins (PSP-I and PSP-II) were used. The immunohistochemistry showed positive labelling for both antibodies in the epithelium of seminal vesicles in all males. Positive immunolabelling, but of variable intensity, was present in the epididymal epithelium (caput, corpus, and caudal), with variation among segments and boars. No labelling was found in the seminiferous epithelium or bulbourethral glands of any boar. After SDS-PAGE and Western blotting, immunoreactive bands were obtained in the extracts of all tissues for both PSP proteins. Among tissues, the highest intensity corresponded to the seminal vesicle lane for both proteins. Epididymis (caput, corpus, and caudal), bulbourethral gland, and testis showed more feeble bands, yet present. The intensity of the bands varied among boars. Amplification products from mRNA were obtained in all tissues explored by RT-PCR, using specific primers for PSP-I and PSP-II. The same trend was obtained regarding intensity bands, corresponding with the intensity obtained by Western blotting. The results indicate that PSP-I and PSP-II are mainly expressed in seminal vesicles and epididymal segments and, in lower amount, in the testis and bulbourethral gland.

This work was supported by Formas, Stockholm, and Fundación Séneca, Murcia, Spain.