BIOLOGY
OF REPRODUCTION
42, 231-238
(1990)
The Influence of Calcium lonophore and Activators of Protein Kinase C on Steroid Production by Preovulatory Ovarian Follicles of the Goldfish1 GLEN
VAN
Department University Guelph, Canada
DER J&&J(2 of Zoology of Guelph Ontario NJG2WI
ABSTRACT Steroidogenesis in teleost fish, as in other vertebrate groups, is mediated by the activation of adenylate cyclase. For the present studies, calcium ionophore A23187 and either phorbol 12-myristate 13-acetate (PMA) or 1 -olecyl-2-acetylglycerol (OAG) were used to investigate the possible roles that changes in intracellular calcium content and protein kinase C activation play in steroid production by goldfish preovulatory ovarian follicles incubated in vitro. While ineffective alone, PMA (1.6 - 400 riM) and OAG (25 - 100 p.g/ml) exhibited classical synergism with A23187 (1.0 - 10 .iM), leading to increased testosterone production. The magnitude of these responses was at least tenfold lower than that obtained with human chorionic gonadotropin (hCG),forskolin, or dibusryl cyclic adenosine 3’, 5’-monophosphwe. Testosterone production stimulated by hCG and forskolin was blocked by addition of PMA but not OAG. Unlike PMA, the inactive phorbol ester 4a-phorbol 12, 13-dideconate did no: influence basal or stimulated testosterone production. A23187 had a biphasic effect on stimulated testosterone production: a dosage of025 or 1.0 jiM potenfiated the action of submaxunally effective dosages of hCG or forskolin on testosterone production; a higher dosage of 4 tM inhibited stimulated testosterone production by up to 50%. In conclusion, these studies suggest that, in addition to the adenylate cyclase second messenger system, changes in intracellular calcium and activation of protein kinase C may modulate steroidogenesis in goldfish
ovarian
follicles.
phore A23187 and either phorbol esters or synthetic diacyiglycerols to mimic the actions of 1P3 and diacylglycerol, respectively, suggest that phosphoinositide metabolites modulate basal and cyclic adenosine 3’, 5’-monophosphate (cAMP)-mediated steroid production in ovarian tissue from mammalian (Welsh et a!., 1984; Baum and Rosberg, 1987), avian (Tilly and Johnson, 1988), and amphibian species (Kleiss-San Francisco and Schuetz, 1988). The involvement of cAMP in regulating ovarian steroid production in teleost fish is well established (Nagahama, 1987; Kanamori and Nagahama, 1988). In contrast, the possible involvement of calcium or protein kinase C activation in controlling steroidogenesis in teleosts has not been addressed. A recent study showing that phospholipase C stimulates testosterone production in goldfish preovulatory ovarian follicles points to the possible role of phosphoinositide metabolites in steroidogenesis (Van Der Kraak and Chang, 1990). Other studies suggest that phosphoinositide metabolites may be active in goldfish since addition of calcium iono-
INTRODUCTION There is substantial evidence that the products of polyphosphoinositide turnover serve as second messengers that are important regulators of gonadal steroidogenesis in tetrapods. Phospholipase C mediates the hydrolysis of the membrane phospholipid phosphatidyl inositol 4, 5 bisphosphate, leading to the formation of two secondary messengers, 1,2 diacyiglycerol and mositol-1, 4, 5-trisphosphate (1P3; Nishizuka, 1984; Berridge, 1987). Diacyiglycerol is responsible for activating a calcium/phospholipid-dependent protein kinase (protein kinase C) and 1P3 for the mobilization of intracellular calcium stores. Studies using calcium iono-
Accepted Received
September 27. 1989. lone 15, 1989. study was supported by the
Natural
Sciences
and
Engineering
Research Council of Cknth Grant U0551. A preliminary report of some of these results was presented at the 22nd Annual Meeting of the Society for the Stu4y of Reproduction, August 1989. ‘Reprint requests.
231
VAN DER KRAAK
232
phore A23 187 together with phorbol 12-myristate 13-acetate (PMA) or synthetic diacyiglycerols induces ovulation of ovarian follicles incubated in vitro (Ranjan and Goetz, 1987). For the present study, calcium ionophore A23 187 and the phorbol ester PMA or the synthetic diacyiglycerol 1 oleoyl-2 acetyiglycerol (OAG) were used to investigate the possible roles of calcium and protein kinase C activation in steroid production by preovulatory goldfish ovarian follicles incubated in vitro. MATERIALS
AND
phore A23187 were purchased from Sigma Chemical Co. (St. Louis, MO). HCG, dbcAMP, and IBMX were dissolved directly in Cortland’s saline. Forskolin was first dissolved in ethanol, whereas phorbols, OAG, and A23 187 were dissolved in dimethylsulfoxide (DMSO); then each of these was diluted with incubation buffer before use. The amounts of ethanol of DMSO included in the follicle incubations did not exceed 0.5% of the final incubation volume and at this concentration did not influence basal or stimulated testosterone production.
METHODS Statistical
Animals
Goldfish, common or comet varieties, were purchased from Grassfork Fisheries Co., Martinsville, IN. Fish were maintained in tanks (1.22 m in diameter) with flow-through water at 14- 16#{176}C under a constant photopenod (14L:1OD). Fish were fed a commercial trout diet once a day to satiation.
Incubations
Preovulatory fish were killed by spinal transection and their ovaries were removed quickly and placed in modified Cortland’s saline containing 0.1% bovine serum albumin, 0.1% glucose, and 0.01% streptomycin sulfate (Van Der Kraak and Chang, 1990). Each experiment used full-grown preovulatory ovarian follicles (0.9 - 1.1 mm in diameter) obtained from a single fish. Intact preovulatory follicles were separated from smaller vitellogenic follicles under a dissecting microscope. Preovulatory follicles were then added in groups of 20 to each well of polystyrene tissue culture plates (Falcon 3047; Fisher Scientific Co., Toronto, ON). Immediately before addition of test compounds, the incubation medium was replaced by Cortland’s saline containing 3-isobutyl l-methylxanthine (IBMX; 1 mM). The phosphodiesterase inhibitor IBMX was included in the follicle incubations because goldfish ovarian follicles secrete low levels of basal steroid. Follicles were incubated with test compounds in air for 18 h at 20#{176}C; the fmal incubation volume was 1 ml. The medium was then removed and stored at - 30#{176}C before testosterone content was measured by radioinimunoassay (see Van Der Kraak and Chang, 1990). Human choriomc gonadotropin (hCG), dibutryl (db)cAMP, forskolin, IBMX, PMA, 4a-phorbol 12, 13-didecanoate (4aPDD), OAG, and calcium iono-
Analysis
Group differences were determined by using analysis of variance and Duncan’s Multiple Range test or t-test. The data presented represent at least two experiments that gave statistically similar or identical results. RESULTS Effects A23187
of Protein on Basal
Kinase C Activators and Testosterone Production
The effects of varying dosages of the phorbol ester PMA in combination with calcium ionophore A23187 on testosterone production are shown in Figure 1. Neither PMA (400 nM) alone nor A23187 (4000 nM) alone had any effects on testosterone production. When tested in combination with A23187, PMA at 6.25 nM caused a significant (p
OF REPRODUCTION
42, 231-238
(1990)
The Influence of Calcium lonophore and Activators of Protein Kinase C on Steroid Production by Preovulatory Ovarian Follicles of the Goldfish1 GLEN
VAN
Department University Guelph, Canada
DER J&&J(2 of Zoology of Guelph Ontario NJG2WI
ABSTRACT Steroidogenesis in teleost fish, as in other vertebrate groups, is mediated by the activation of adenylate cyclase. For the present studies, calcium ionophore A23187 and either phorbol 12-myristate 13-acetate (PMA) or 1 -olecyl-2-acetylglycerol (OAG) were used to investigate the possible roles that changes in intracellular calcium content and protein kinase C activation play in steroid production by goldfish preovulatory ovarian follicles incubated in vitro. While ineffective alone, PMA (1.6 - 400 riM) and OAG (25 - 100 p.g/ml) exhibited classical synergism with A23187 (1.0 - 10 .iM), leading to increased testosterone production. The magnitude of these responses was at least tenfold lower than that obtained with human chorionic gonadotropin (hCG),forskolin, or dibusryl cyclic adenosine 3’, 5’-monophosphwe. Testosterone production stimulated by hCG and forskolin was blocked by addition of PMA but not OAG. Unlike PMA, the inactive phorbol ester 4a-phorbol 12, 13-dideconate did no: influence basal or stimulated testosterone production. A23187 had a biphasic effect on stimulated testosterone production: a dosage of025 or 1.0 jiM potenfiated the action of submaxunally effective dosages of hCG or forskolin on testosterone production; a higher dosage of 4 tM inhibited stimulated testosterone production by up to 50%. In conclusion, these studies suggest that, in addition to the adenylate cyclase second messenger system, changes in intracellular calcium and activation of protein kinase C may modulate steroidogenesis in goldfish
ovarian
follicles.
phore A23187 and either phorbol esters or synthetic diacyiglycerols to mimic the actions of 1P3 and diacylglycerol, respectively, suggest that phosphoinositide metabolites modulate basal and cyclic adenosine 3’, 5’-monophosphate (cAMP)-mediated steroid production in ovarian tissue from mammalian (Welsh et a!., 1984; Baum and Rosberg, 1987), avian (Tilly and Johnson, 1988), and amphibian species (Kleiss-San Francisco and Schuetz, 1988). The involvement of cAMP in regulating ovarian steroid production in teleost fish is well established (Nagahama, 1987; Kanamori and Nagahama, 1988). In contrast, the possible involvement of calcium or protein kinase C activation in controlling steroidogenesis in teleosts has not been addressed. A recent study showing that phospholipase C stimulates testosterone production in goldfish preovulatory ovarian follicles points to the possible role of phosphoinositide metabolites in steroidogenesis (Van Der Kraak and Chang, 1990). Other studies suggest that phosphoinositide metabolites may be active in goldfish since addition of calcium iono-
INTRODUCTION There is substantial evidence that the products of polyphosphoinositide turnover serve as second messengers that are important regulators of gonadal steroidogenesis in tetrapods. Phospholipase C mediates the hydrolysis of the membrane phospholipid phosphatidyl inositol 4, 5 bisphosphate, leading to the formation of two secondary messengers, 1,2 diacyiglycerol and mositol-1, 4, 5-trisphosphate (1P3; Nishizuka, 1984; Berridge, 1987). Diacyiglycerol is responsible for activating a calcium/phospholipid-dependent protein kinase (protein kinase C) and 1P3 for the mobilization of intracellular calcium stores. Studies using calcium iono-
Accepted Received
September 27. 1989. lone 15, 1989. study was supported by the
Natural
Sciences
and
Engineering
Research Council of Cknth Grant U0551. A preliminary report of some of these results was presented at the 22nd Annual Meeting of the Society for the Stu4y of Reproduction, August 1989. ‘Reprint requests.
231
VAN DER KRAAK
232
phore A23 187 together with phorbol 12-myristate 13-acetate (PMA) or synthetic diacyiglycerols induces ovulation of ovarian follicles incubated in vitro (Ranjan and Goetz, 1987). For the present study, calcium ionophore A23 187 and the phorbol ester PMA or the synthetic diacyiglycerol 1 oleoyl-2 acetyiglycerol (OAG) were used to investigate the possible roles of calcium and protein kinase C activation in steroid production by preovulatory goldfish ovarian follicles incubated in vitro. MATERIALS
AND
phore A23187 were purchased from Sigma Chemical Co. (St. Louis, MO). HCG, dbcAMP, and IBMX were dissolved directly in Cortland’s saline. Forskolin was first dissolved in ethanol, whereas phorbols, OAG, and A23 187 were dissolved in dimethylsulfoxide (DMSO); then each of these was diluted with incubation buffer before use. The amounts of ethanol of DMSO included in the follicle incubations did not exceed 0.5% of the final incubation volume and at this concentration did not influence basal or stimulated testosterone production.
METHODS Statistical
Animals
Goldfish, common or comet varieties, were purchased from Grassfork Fisheries Co., Martinsville, IN. Fish were maintained in tanks (1.22 m in diameter) with flow-through water at 14- 16#{176}C under a constant photopenod (14L:1OD). Fish were fed a commercial trout diet once a day to satiation.
Incubations
Preovulatory fish were killed by spinal transection and their ovaries were removed quickly and placed in modified Cortland’s saline containing 0.1% bovine serum albumin, 0.1% glucose, and 0.01% streptomycin sulfate (Van Der Kraak and Chang, 1990). Each experiment used full-grown preovulatory ovarian follicles (0.9 - 1.1 mm in diameter) obtained from a single fish. Intact preovulatory follicles were separated from smaller vitellogenic follicles under a dissecting microscope. Preovulatory follicles were then added in groups of 20 to each well of polystyrene tissue culture plates (Falcon 3047; Fisher Scientific Co., Toronto, ON). Immediately before addition of test compounds, the incubation medium was replaced by Cortland’s saline containing 3-isobutyl l-methylxanthine (IBMX; 1 mM). The phosphodiesterase inhibitor IBMX was included in the follicle incubations because goldfish ovarian follicles secrete low levels of basal steroid. Follicles were incubated with test compounds in air for 18 h at 20#{176}C; the fmal incubation volume was 1 ml. The medium was then removed and stored at - 30#{176}C before testosterone content was measured by radioinimunoassay (see Van Der Kraak and Chang, 1990). Human choriomc gonadotropin (hCG), dibutryl (db)cAMP, forskolin, IBMX, PMA, 4a-phorbol 12, 13-didecanoate (4aPDD), OAG, and calcium iono-
Analysis
Group differences were determined by using analysis of variance and Duncan’s Multiple Range test or t-test. The data presented represent at least two experiments that gave statistically similar or identical results. RESULTS Effects A23187
of Protein on Basal
Kinase C Activators and Testosterone Production
The effects of varying dosages of the phorbol ester PMA in combination with calcium ionophore A23187 on testosterone production are shown in Figure 1. Neither PMA (400 nM) alone nor A23187 (4000 nM) alone had any effects on testosterone production. When tested in combination with A23187, PMA at 6.25 nM caused a significant (p
