Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro.
Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture system.
Reyes-Moreno
Carlos
C
Centre de Recherche en Biologie de la Reproduction, Départment des Sciences Animales, Université Laval, Sainte-Foy, Québec, Canada.
Boilard
Mathieu
M
Sullivan
Robert
R
Sirard
Marc-André
MA
D000818:Animals; D001685:Biological Factors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D015925:Cryopreservation; D017077:Culture Media, Conditioned / Q000737:chemistry* / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004822:Epididymis / Q000166:cytology* / Q000378:metabolism*; D006358:Hot Temperature; D008297:Male; D008970:Molecular Weight; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
D001685:Biological Factors; D017077:Culture Media, Conditioned