Abstract
Macaque sperm require treatment with dibutyryl cAMP (dbcAMP) and caffeine (termed activators; ACT) to fully capacitate in vitro. Previous studies have shown that treatment with ACT also increases sperm binding to the macaque zona pellucida and enhances the induction of acrosome reactions of the bound sperm. This study investigated whether phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol (DOG), which stimulate protein kinase C (PKC), can also increase sperm binding to the zona pellucida and/or enhance induction of the acrosome reaction of bound sperm. Cyanomolgus macaque sperm were centrifuged through 60%. Percoll and then were washed with modified Biggers, Whitten and Whittingham (BWW) medium and incubated for 2.5 h at 37°C with 5% CO2. ACT, PMA, or DOG was added to sperm during the last 15- 30 min of incubation. Sperm were evaluated immediately after incubation for motility and acrosomal status. Macaque oocytes were coincubate with stimulated sperm suspensions in BWW medium for 30 sec (pulse). Half of the oocytes were removed to sperm-free medium and further incubated for 1 h (chase). When assessed after the pulse period, sperm-zona binding increased significantly after ACT treatment compared to that in untreated controls. DOG but not PMA treatment had a similar effect on sperm binding. PMA, DOG, and ACT treatment increased the percentage of acrosome reactions in sperm bound to the zona following the 30-sec pulse compared to the percentage in controls. Inactive analogs of PMA and DOG had no effect on sperm-zona binding or the percentage of acrosome reactions (% AR) of bound sperm. The results suggest that both the production of cAMP and that of diacylglycerol second messengers are involved in mediation of the zona-induced acrosome reaction of macaque sperm. Treatment with PMA and DOG also increased the % AR in sperm suspensions incubated without oocytes. To test the hypothesis that the increase % AR of zona-bound sperm was due to preferential binding of acrosome-reacted sperm, the sperm-zona binding experiments were also performed in supplemented media with minimal Ca2+ (Ca2+-free media). The % AR of zona-bound sperm in the Ca2+ free media was significantly less than in media with Ca2+. Because Ca2+ is required for the zona-induced acrosome reaction, this results suggests that the increase in acrosome reactions of zona-bound sperm resulted after zona binding.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1418-1425 |
| Number of pages | 8 |
| Journal | Biology of Reproduction |
| Volume | 52 |
| Issue number | 6 |
| DOIs | |
| State | Published - 1995 |
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ASJC Scopus subject areas
- Cell Biology
- Developmental Biology
- Embryology
Cite this
Effect of protein kinase C stimulators on zona pellucida binding and the acrosome reaction of macaque sperm. / Tollner, Theodore L; Overstreet, J. W.; Vandevoort, C. A.
In: Biology of Reproduction, Vol. 52, No. 6, 1995, p. 1418-1425.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Effect of protein kinase C stimulators on zona pellucida binding and the acrosome reaction of macaque sperm
AU - Tollner, Theodore L
AU - Overstreet, J. W.
AU - Vandevoort, C. A.
PY - 1995
Y1 - 1995
N2 - Macaque sperm require treatment with dibutyryl cAMP (dbcAMP) and caffeine (termed activators; ACT) to fully capacitate in vitro. Previous studies have shown that treatment with ACT also increases sperm binding to the macaque zona pellucida and enhances the induction of acrosome reactions of the bound sperm. This study investigated whether phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol (DOG), which stimulate protein kinase C (PKC), can also increase sperm binding to the zona pellucida and/or enhance induction of the acrosome reaction of bound sperm. Cyanomolgus macaque sperm were centrifuged through 60%. Percoll and then were washed with modified Biggers, Whitten and Whittingham (BWW) medium and incubated for 2.5 h at 37°C with 5% CO2. ACT, PMA, or DOG was added to sperm during the last 15- 30 min of incubation. Sperm were evaluated immediately after incubation for motility and acrosomal status. Macaque oocytes were coincubate with stimulated sperm suspensions in BWW medium for 30 sec (pulse). Half of the oocytes were removed to sperm-free medium and further incubated for 1 h (chase). When assessed after the pulse period, sperm-zona binding increased significantly after ACT treatment compared to that in untreated controls. DOG but not PMA treatment had a similar effect on sperm binding. PMA, DOG, and ACT treatment increased the percentage of acrosome reactions in sperm bound to the zona following the 30-sec pulse compared to the percentage in controls. Inactive analogs of PMA and DOG had no effect on sperm-zona binding or the percentage of acrosome reactions (% AR) of bound sperm. The results suggest that both the production of cAMP and that of diacylglycerol second messengers are involved in mediation of the zona-induced acrosome reaction of macaque sperm. Treatment with PMA and DOG also increased the % AR in sperm suspensions incubated without oocytes. To test the hypothesis that the increase % AR of zona-bound sperm was due to preferential binding of acrosome-reacted sperm, the sperm-zona binding experiments were also performed in supplemented media with minimal Ca2+ (Ca2+-free media). The % AR of zona-bound sperm in the Ca2+ free media was significantly less than in media with Ca2+. Because Ca2+ is required for the zona-induced acrosome reaction, this results suggests that the increase in acrosome reactions of zona-bound sperm resulted after zona binding.
AB - Macaque sperm require treatment with dibutyryl cAMP (dbcAMP) and caffeine (termed activators; ACT) to fully capacitate in vitro. Previous studies have shown that treatment with ACT also increases sperm binding to the macaque zona pellucida and enhances the induction of acrosome reactions of the bound sperm. This study investigated whether phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol (DOG), which stimulate protein kinase C (PKC), can also increase sperm binding to the zona pellucida and/or enhance induction of the acrosome reaction of bound sperm. Cyanomolgus macaque sperm were centrifuged through 60%. Percoll and then were washed with modified Biggers, Whitten and Whittingham (BWW) medium and incubated for 2.5 h at 37°C with 5% CO2. ACT, PMA, or DOG was added to sperm during the last 15- 30 min of incubation. Sperm were evaluated immediately after incubation for motility and acrosomal status. Macaque oocytes were coincubate with stimulated sperm suspensions in BWW medium for 30 sec (pulse). Half of the oocytes were removed to sperm-free medium and further incubated for 1 h (chase). When assessed after the pulse period, sperm-zona binding increased significantly after ACT treatment compared to that in untreated controls. DOG but not PMA treatment had a similar effect on sperm binding. PMA, DOG, and ACT treatment increased the percentage of acrosome reactions in sperm bound to the zona following the 30-sec pulse compared to the percentage in controls. Inactive analogs of PMA and DOG had no effect on sperm-zona binding or the percentage of acrosome reactions (% AR) of bound sperm. The results suggest that both the production of cAMP and that of diacylglycerol second messengers are involved in mediation of the zona-induced acrosome reaction of macaque sperm. Treatment with PMA and DOG also increased the % AR in sperm suspensions incubated without oocytes. To test the hypothesis that the increase % AR of zona-bound sperm was due to preferential binding of acrosome-reacted sperm, the sperm-zona binding experiments were also performed in supplemented media with minimal Ca2+ (Ca2+-free media). The % AR of zona-bound sperm in the Ca2+ free media was significantly less than in media with Ca2+. Because Ca2+ is required for the zona-induced acrosome reaction, this results suggests that the increase in acrosome reactions of zona-bound sperm resulted after zona binding.
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U2 - 10.1095/biolreprod52.6.1418
DO - 10.1095/biolreprod52.6.1418
M3 - Article
VL - 52
SP - 1418
EP - 1425
JO - Biology of Reproduction
JF - Biology of Reproduction
SN - 0006-3363
IS - 6
ER -