Identification of prostasomes in male dog seminal plasma.

Aim of this study was to determine if membrane vesicles exist and could be extracted from dog seminal plasma as it as been realized in other species. In order to obtain the membrane vesicles, semen samles were collected by digital manipulation from 8 male dogs of good fertility. The membrane vesicles were isolated following the method described by Ronquist and Brody (1985) and modified. The freshly collected prostatic fluid was submitted to 2 sequential centrifugation. The supernatant was then submitted to ultracentrifugation for 2h at + 4 °C. The final pellets were re-dissolved in 1 ml. of Tris Buffer Saline (TBS) and stored at –20C°. To identify membrane vesicles by electronic microscopy the pellets were prefixed in 2.5% glutaraldehyde for 12 h. After fixation, the samples were rinsed with phosphate buffer 0.1 M pH 7.4, post fixed in 1% osmium tetroxide for 1h, dehydrated in an increasing series of ethanol, impregnated with propylene oxide and embedded in Epon-Araldite. The ultrathin sections made with a Reichert-Jung Ultracut E and diamond blade, the slides were stained with uranyl acetate and lead citrate before examination at the Transmission Electron Microscope (EM 208, EM 400 T Philips). The ultra microscopy analysis of the pellets showed the presence of small vesicular-shaped particles with a medium diameter of 200 nm. They were coated with bilaminar membranes, some of which contained a uniformly distributed electron-dense material.