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Release of enzymes from rabbit sperm during capacitation in vitro
THERIOGENOLOGY
RELEASEOF ENZYMESFROM RABBITSPERM DURINGCAPACITATIONIN VITRO C.E. McBride, R.A. Fayrer-Hosken. B.G. Brackett Department of Physiology and Pharmacology College of Veterinary Medicine The University of Georgia, Athens, GA., 30602. The release of acrosomal enzymes reported during in vitro capacitation of rabbit sperm include P-N-acetylglucosaminidase (P-NAG), arylsulfatase (Aryl), acrosin. phospholipase C (PhosCI. acid phosphatase (AcidPI, alkaline phosphatase (AlkPI. hyaluronidase and neuraminidase (Akruk et al, 1979, Gam Res 21-l 3). Also, P-NAG inhibitors were found to prevent hamster sperm capacitation (Gwatkin and Anderson, 1973, J Reprod Fert 35565567). Present research was carried out to characterize sequential enzyme release from rabbit sperm (undergoing capacitation in vitro) that might be useful for indicating maximal fertilizing ability for a given sperm population. The relation between certain enzymatic changes in the medium and the earliest achievement of rabbit sperm capacitation were evaluated. Capacitation was determined by the ability of sperm cells to bind to and penetrate salt-stored zonae (SSZI. Enzymatic profiles were demonstrated for sperm populations sampled every 120 min during in vitro incubation. Preliminary experiments revealed sequential enzyme peaks of P-NAG, AlkP, AcidP. and PhosC between 240 and 480 min. However, no evidence of penetration into SSZ was seen. In further experiments enzyme peaks for P-NAG commencing at 240 min, Aryl after 240 min and PhosC after 480 min were associated with sperm capacitation as evidenced by penetration into the perivitelline space (PVS) of SSZ starting at 330 min with 25% (2/8 SSZ), and rising to 100% (I l/l 1 SSZ) by 570 min. In one experiment, penetration occurred as previously found but mass enzymatic effects of presumably acrosome reacted sperm led to complete digestion of zonae - 100% (1 O/l 0 SSZ) at 570 min. decreasing to no digestion of zonae but 70% penetration (7/l 0 SSZ). at 930 min. In three experiments in which similar enzymatic profiles were not found no penetration of SSZ occurred. Results indicate that /?-NAG and Aryl peak prior to initial penetration with the /?-NAG peak being much more pronounced. Also, a peak in PhosC was associated with increased penetration. Data point to the release of P-N-acetylglucosaminidase. arylsulfatase and phospholipase C as predictors of in vitro capacitation of rabbit sperm resulting in their ability to penetrate SSZ. (This work supported by UGA VMES Project 86225, and USDAgrant 85CRCR-l-1707.)
JANUARY
1988 VOL.
29 NO.
1
277
RELEASEOF ENZYMESFROM RABBITSPERM DURINGCAPACITATIONIN VITRO C.E. McBride, R.A. Fayrer-Hosken. B.G. Brackett Department of Physiology and Pharmacology College of Veterinary Medicine The University of Georgia, Athens, GA., 30602. The release of acrosomal enzymes reported during in vitro capacitation of rabbit sperm include P-N-acetylglucosaminidase (P-NAG), arylsulfatase (Aryl), acrosin. phospholipase C (PhosCI. acid phosphatase (AcidPI, alkaline phosphatase (AlkPI. hyaluronidase and neuraminidase (Akruk et al, 1979, Gam Res 21-l 3). Also, P-NAG inhibitors were found to prevent hamster sperm capacitation (Gwatkin and Anderson, 1973, J Reprod Fert 35565567). Present research was carried out to characterize sequential enzyme release from rabbit sperm (undergoing capacitation in vitro) that might be useful for indicating maximal fertilizing ability for a given sperm population. The relation between certain enzymatic changes in the medium and the earliest achievement of rabbit sperm capacitation were evaluated. Capacitation was determined by the ability of sperm cells to bind to and penetrate salt-stored zonae (SSZI. Enzymatic profiles were demonstrated for sperm populations sampled every 120 min during in vitro incubation. Preliminary experiments revealed sequential enzyme peaks of P-NAG, AlkP, AcidP. and PhosC between 240 and 480 min. However, no evidence of penetration into SSZ was seen. In further experiments enzyme peaks for P-NAG commencing at 240 min, Aryl after 240 min and PhosC after 480 min were associated with sperm capacitation as evidenced by penetration into the perivitelline space (PVS) of SSZ starting at 330 min with 25% (2/8 SSZ), and rising to 100% (I l/l 1 SSZ) by 570 min. In one experiment, penetration occurred as previously found but mass enzymatic effects of presumably acrosome reacted sperm led to complete digestion of zonae - 100% (1 O/l 0 SSZ) at 570 min. decreasing to no digestion of zonae but 70% penetration (7/l 0 SSZ). at 930 min. In three experiments in which similar enzymatic profiles were not found no penetration of SSZ occurred. Results indicate that /?-NAG and Aryl peak prior to initial penetration with the /?-NAG peak being much more pronounced. Also, a peak in PhosC was associated with increased penetration. Data point to the release of P-N-acetylglucosaminidase. arylsulfatase and phospholipase C as predictors of in vitro capacitation of rabbit sperm resulting in their ability to penetrate SSZ. (This work supported by UGA VMES Project 86225, and USDAgrant 85CRCR-l-1707.)
JANUARY
1988 VOL.
29 NO.
1
277