Protein-leakage during heparin – induced in-vitro capacitation of bull sperm
Freshly ejaculated washed spermatozoa of six cross-bred bulls were capacitated by incubating in TALP medium supplemented with BSA, heparin and hepes buffer at a sperm concentration of 6 × 10
9 s/ml at 37ο C for 6 h. A control was also run in 0.85% saline. Percentage motility was observed at two h interval. Sperm smears were stained with giemsa and observed under oil emulsion to study different stages of acrosome reaction. After six h of incubation, sperm membrane proteins were extracted from capacitated- and uncapacitated- spermatozoa using deoxycholate in Tris-HCl (0.02M). Spermatozoa, incubation media (IM) and sperm membrane extract (SME) were analyzed for total proteins which were further fractionated by SDS-PAGE. Antisera raised against SME of bull # 1, 2 and 3 were used for identification of antigens in SME of all the bulls using immunodiffusion technique.
After incubation of spermatozoa for 6 h, there was 15–21% and 55–62% decline in motility of capacitated- and uncapacitated- spermatozoa respectively. A total of 62–87% spermatozoa showed acrosome reaction (swelling, vesiculation and shedding of acrosome) in all the bulls. A statistically significant (P<0.05) decrease in protein content of spermatozoa was observed during capacitation. The percentage extraction of protein and percentage leakage of proteins into medium showed a significant correlation with percentage acrosome reaction in respective bulls. A higher and positive correlation of rate of acrosome reaction with protein content of capacitated- and uncapacitated- spermatozoa and IM indicates that percentage capacitation in correlation to fertility can be predicted from the protein content of spermatozoa and proteins released into the medium. Protein profile revealed that from 12.5 to 70 kDa proteins get exposed during capacitation. There was leakage of 4–7 proteins, mainly of medium (70–130 kDa) and low mol wt (<12.5–35 kDa) from capacitated spermatozoa, whereas only 2–5 proteins of high (160–>240 kDa) and low (<12.5–25 kDa) mol wt proteins get leaked out from uncapacitated spermatozoa. Immunodiffusion reaction of SME of bull # 1, 2 and 3 also suggested the removal/exposure of antigens from sperm surface during capacitation and acrosome reaction.