After the centrifugation, a yellowish pellet was found at the bottom of the tube. This pellet was suspended in PBS and submitted to a second high-speed centrifugation. The final pellet gave a cloudy, refracting solution in PBS, which was divided into aliquots to be processed for electron microscopy or prepared for one- and two-dimensional gel separation.
Fluids and Sperm Collection
Experiments on animals were conducted according to the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. Testis and epididymis were obtained from adult Ile de France and Romanov rams (at least 10 different animals). Testicular fluid was obtained after puncture of the rete testis. Epididymal fluids were collected by microperfusion with PBS from the 10 different zones defined in the three main regions (caput, corpus, cauda) and the caudal fluid (CEP) by retroperfusion of zones 8 and 9 from the deferent duct (see Fig. 1).
Mammalian sperm collected from the testis are immobile and unable to bind to and then fertilize the oocyte. They acquire these properties gradually while they travel through the epididymal tubule. During this transit, the sperm lose their cytoplasmic droplets and their plasma membrane undergoes profound changes, including addition, removal, or transformation of proteins and lipids. These gradual surface modifications occur in response to variations in the luminal fluid composition and are thought to be responsible for their final maturation.