GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

81, 268-275 (1991)

Role of Calcium in the Control of Steroidogenesis Ovarian Follicles of the Goldfish

in Preovulatory

GLEN VAN DER KRAAK Department of Zoology, University of Guelph, Guelph, Ontario, Canada NlG 2Wl Accepted February 16, 1990 The possible involvement of calcium in the regulation of steroidogenesis in the goldfish was investigated using preovulatory ovarian follicles incubated in vitro. Incubation of follicles in media deficient in calcium impaired testosterone production in response to human chorionic gonadotropin (hCG) in both the presence and the absence of the phosphodiesterase inhibitor IBMX. Similarly, addition of calcium channel antagonists (verapamil, nifedipine, nicardipine, and CoCl,) caused a dose-dependent inhibition of hCG-stimulated testosterone production. TMB-8, an inhibitor of intracellular calcium mobilization, also suppressed hCG-stimulated testosterone production. Basal testosterone production was not affected by incubation in calcium-deficient media or with drugs which reduce intracellular calcium availability. In other studies, nifedipine blocked forskolin and dibutyryl cyclic AMPstimulated testosterone production suggesting that one of the major sites of calcium action is distal to cyclic AMP generation. Two inhibitors of calmodulin, W5 and W7, significantly inhibited hCG-stimulated testosterone production. These findings suggest that calcium derived from intracellular and extracellular pools participate in the expression of gonadotropin effects on steroid production in goldfish ovarian follicles and that these effects are mediated intracellularly by interaction with calmodulin. B 1991 Academic PRSS, IX.

Although cyclic AMP is viewed as the principal intracellular messenger mediating the actions of gonadotropins on ovarian steroidogenesis in tetrapods, there is evidence that calcium ions also play an important role (Veldhuis and Klase, 1982; Tsang and Carnegie, 1983, 1984; Asem and Hertelendy, 1986a; Kleiss-San Francisco and Schuetz, 1987). Specific regulatory actions of calcium ions on ovarian steroidogenesis in mammalian and avian species are exerted at several levels, including effects on LH- and FSH-stimulated CAMP production and at steps in the steroidogenic pathway distal to CAMP generation. While numerous studies have demonstrated the involvement of the adenylate cyclase-CAMP pathway in the regulation of ovarian steroidogenesis in teleosts (Nagahama, 1987; Kanamori and Nagahama, 1988), there is

surprisingly little known with respect to the role of calcium. Recently, calcium ionophore A23 187 was shown to exert facilitory and inhibitory effects on CAMP-mediated testosterone production by goldfish preovulatory ovarian follicles (Van Der Kraak, 1990). A23187 potentiates the actions of low dosages of hCG and forskolin on testosterone production but at high dosages inhibits stimulated testosterone production. The effects of calcium on basal steroid production in the goldfish are unclear. A23187 caused an increase in basal testosterone production in several studies but had no effect in others (Van Der Kraak, 1990). For the present study, alterations in extracellular calcium content as well as agents which interfere with the availability of calcium or the activity of calmodulin were used to investigate the putative role of cal268

0016~6480/91 $1.50 Copyright B 1991 by Academic Press, Inc. All tights of reproduction in any form reserved.

Ca AND STEROIDOGENESIS

cium and calmodulin in the regulation of steroid production by goldfish preovulatory ovarian follicles. MATERIALS

AND METHODS

Materials. Verapamil, nifedipine, nicardipine, (8 [N,N-diethylaminol]octyl)-3,4,5-trimethoxybenzoateHCl (TMB-8), N-(6-aminohexyl)-l-naphthalenesulfonamide (W5), N-(6-aminohexyl)-5-chloro-I-naphtha lenesulfonamide (W7), human chorionic gonadotropin (hCG), 3-isobutyl-1-methylxanthine (IBMX), forskolin, and dibutyryl cyclic AMP (db CAMP) were obtained from Sigma Chemical Co. (St. Louis, MO). Powdered Medium 199 (M199) containing Hanks’ salts (without bicarbonate) was obtained from GIBCO (Burlington, Ontario). Animals. Goldfish, common or comet varietes, were obtained from Grassyfork Fisheries Co. (Martinsville, IN). Goldfish were maintained in 4-ft-diameter tanks with flow through water at 14-16” under a constant photoperiod (14 hr light:10 hr dark). Fish were fed a commercial trout diet once a day to satiation. Follicle incubations. Details of the follicle incubation procedure have been described by Van Der Kraak and Chang (1990). Briefly, preovulatory fish were killed by spinal transection and their ovaries placed in Cortland’s saline containing 0.1% bovine serum albumin, 0.1% glucose, and 0.01% streptomycin sulphate. This medium contains 1.6 mM calcium. Each experiment utilized full-grown preovulatory ovarian follicles (0.9-l. 1 mm in diameter) obtained from a single fish. Intact preovulatory follicles were separated from smaller vitellogenic follicles under a dissecting microscope. Follicles were then added to each well of a polystyrene tissue culture plate (Falcon 3047; Fisher Scientific Co., Toronto). For experiments testing calcium-free incubation conditions, Cortland’s saline was prepared without calcium and supplemented with 0.1 mM EGTA. Cortland’s saline was not appropriate for experiments testing the effects of CoCl, as high concentrations of this compound result in the formation of a precipitate. In these cases, Ml99 containing 4.0 mM sodium bicarbonate, 25 mM Hepes, 0.1% bovine serum albumin and 0.01% streptomycin sulfate, pH 7.2 (total calcium content = I .25 mM), was used. In cases where follicles were incubated in a medium other than Cortland’s, follicles were washed at least two times with the appropriate medium before the addition of test compounds. The standard incubation protocol was four replicate groups of 20 follicles each in 1 ml of medium incubated for 18 hr at 20”, after which the incubation medium was collected and stored at - 30” prior to measurement of testosterone content by radioimmunoassay (Van Der Kraak and Chang, 1990). Immediately prior to the addition of test compounds the incubation medium was

IN GOLDFISH

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269

routinely replaced with the appropriate buffer containing the phosphodiesterase inhibitor IBMX (1 mM). Human chorionic gonadotropin (hCG). db CAMP, IBMX, and CoCl, were diluted appropriately in medium before use. Forskolin, TMB-8, and calcium channel blockers (verapamil, nifedipine, and nicardipine) were dissolved in ethanol before dilution in medium. The amount of ethanol 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 (Van Der Kraak and Chang, 1990). Calmodulin antagonists W5 and W7 were dissolved in 50% propylene glycol before dilution with Cortland’s saline. Statistical analysis. Group differences were determined using analysis of variance and Duncan’s Multiple Range test or t test. The data presented are representitive of at least two experiments which gave statistically similar or identical results.

RESULTS Influence of Extracellular Calcium on Testosterone Production

The initial studies were conducted to evaluate the effects of incubation media of differing calcium content on steroidogenesis. The responsiveness of ovarian follicles to hCG stimulation was tested with follicles incubated in Cortland’s saline or calciumfree Cortland’s and in the presence or the absence of IBMX. Follicles incubated in normal Cortland’s saline produced significantly more testosterone than sister follicles incubated in calcium-free Cortland’s regardless of whether incubations were done in the presence or the absence of IBMX (Fig. 1). In contrast, basal testosterone production was not affected by extracellular calcium content. Addition of IBMX greatly potentiated the actions of hCG on testosterone production by follicles incubated in calcium-free and normal Cortland’s saline. Addition of IMBX also caused a small but significant increase in basal testosterone production. Other studies have demonstrated that the amounts of testosterone produced in response to graded dosages of hCG were reduced in calcium-free media. Stimulated testosterone production was reduced by approximately

270

GLEN

VAN

1200. T vx Y 8 2 8 F kJ

+IBYx

OCOh-tROL I hCG (10 I.U./ml)

lm.

DER

KRAAK

1600 1

00

1

-

NIFEDIPINE hCG (10 I.U.)

800.

L

600. +IBYx 400. 200. o-

-WIUM

*

m

ULCIUY

NKIFFER

NICARDIPINE

FIG. 1. Basal and hCG (10 IU/ml)-stimulated testosterone production by goldfish ovarian follicles incubated in Cortland’s saline or Ca-free Cortland’s containing 0.1 mM EGTA. Testosterone production under both test conditions was assessed in the presence or absence of IBMX (1 .O r&f). Follicles were incubated for 18 hr at 20”. Values represent the means 2 SEM of four replicate incubations. 1600,

50% in calcium-free media but there was no change in the ED,, for hCG action (Fig. 2). Effects of Calcium Channel Antagonists on Testosterone Production

1200

600

400

A series of experiments were conducted to evaluate the effects of calcium channel blockers on agonist-induced testosterone production. Addition of verapamil, nifedipine, and nicardipine caused a dosedependent inhibition of hCG-stimulated testosterone production (Fig. 3). These

0

0.1

0.5

2.5

12.5

hCG (W./ml)

FIG. 2. Effects of graded dosages of hCG on testosterone production by goldfish ovarian follicles incubated in Cortland’s saline or Ca-free Cortland’s containing 0.1 mM EGTA. Follicles were incubated for 18 hr at 20” in the presence of 1 mM IBMX. Values represent the means 2 SEM of four replicate incubations.

0 Is. 0

1.56

6.25

25

100

CALCIUM CHANNEL BLOCKER (/I,,,)

FIG. 3. Effects of nifedipene, nicardipine, and verapamil on basal and hCG-stimulated testosterone production. Ovarian follicles in Cortland’s saline containing 1 m&f IBMX were incubated with graded dosages of each of the calcium channel blockers (&lo0 l&f) alone and in combination with hCG (10 III/ml) for 18 hr at 20”. Results are the means f SEM of four replicate incubations.

drugs did not influence basal testosterone production. Similarly, addition of CoCl, caused a dose-dependent inhibition of hCGstimulated testosterone production but had no effect on basal testosterone production (Fig. 4). The effects of the calcium channel antagonist nicardipine on forskolin and db CAMP-stimulated testosterone production are shown in Fig. 5. Forskolin (1 and 10 $V) and db CAMP (3 miI4) stimulated testosterone production; a lower dosage of db

Ca

\ ic

AND

600 I wo-

STEROIDOGENESIS

00 I

g 400. is P g zwlz F

l-l

0

0

l-l

nl

0.19 COBALT

hCG (10 I.U.)

0.5 CHLORIDE

nl

1.6

5.0

(mM)

FIG. 4. Effect of CoC1, on basal and hCG-stimulated testosterone production. Ovarian follicles in Ml99 containing 1 n&f IBMX were incubated with graded dosages of CoCl, (O-5 nu%f)alone and in combination with hCG (10 NJ/ml) for 18 hr at 20”. Results are the means ? SEM of four replicate incubations.

CAMP (1 mM) was ineffective. Addition of nicardipine (25 CLM) caused a significant (P < 0.01) inhibition of forskolin and db CAMP-stimulated testosterone production but did not effect basal testosterone production. Injluence of Intracellular Calcium on Testosterone Production

Stores

The possible role of calcium derived from intracellular stores in the regulation of ovarian steroid production was investigated using TMB-8 which is a reported inhibitor

IN

GOLDFISH

271

OVARY

of intracellular calcium mobilization (Chiou and Malagodi, 1975). For these experiments, follicles incubated in calcium-free Cortland’s were pretreated with TMB-8 for 1 hr prior to the addition of hCG (10 III/ml). TMB-8 over the dose range of 25400 t~J4 caused a dose-dependent inhibition of hCGstimulated testosterone production (Fig. 6). TMB-8 did not affect basal testosterone production. Very similar results were obtained using ovarian follicles incubated in normal Cortland’s saline. In this case, preincubation for 1 hr with TMB-8 (6.25400 t&4) resulted in a significant inhibition of hCG-stimulated testosterone production (data not shown). Effects of Calmodulin Antagonists Testosterone Production

on

The effects of the calmodulin antagonists W5 and W7 on hCG (10 III/ml)-stimulated testosterone production are shown in Fig. 7. W5 and W7 act in a dose-related manner to inhibit hCG-stimulated testosterone production. W7 was more potent than W5; the IDS, for W7 action was approximately 5 @4 (data not shown) compared to 20 t& for W5 action. These calmodulin antagonists hCG 00 I IO I.lJ./ml

1600 =\

E 1400

1

g 1200. y

1000.

g

600.

P 8

600.

L

400 -

P

zoo0

-

CONTROL

l@

104

FORSKOLIN

1mY

db

3mY

CAMP

FIG. 5. Effects of nicardipine on forskolin- and db CAMP-stimulated testosterone production. Ovarian follicles in Co&and’s saline containing 1 mM IBMX were incubated with forskolin (1 and 10 +W’) or db CAMP (1 and 3 m&f) alone and in combination with nicardipine (25 uit4) for 18 hr at 20”. Values represent the means + SEM of four replicate incubations.

n CONTROL

n

n 6.25

r-l 25

TMB-8

100

nm 400

(#4)

FIG. 6. Effects of TMB-8 on hCG-stimulated testosterone production. Ovarian follicles in calcium-free Cortland’s saline containing 1 mM IBMX were preincubated for 1 hr with varying dosages of TMB-8 (6.25400 p&f) prior to the addition of hCG (10 IU/ml). The follicles were incubated for an additional 17 hr at 20”. Values represent the means + SEM of three to four replicate incubations.

272

GLEN

lOOO-

VAN

eZa W5 with hCG I W7 with hCG

c

ANTAGONIST

(/LM)

plus

hCG (10

I.U.)

7. Effects of the calmodulin antagonists W5 and W7 on hCG-stimulated testosterone production. Ovarian follicles in Cortland’s saline containing 1 m&f IBMX were incubated with hCG (10 IU/ml) alone or in combination with varying dosages of W5 or W7 (20-80 pA4) for 18 hr at 20”. Values represent the means 2 SEM of four replicate incubations. FIG.

did not affect basal testosterone production. In other studies, the reported calmodulin antagonists trifluoperazine and chlorpromazine at 100 l.& were found to significantly (P < 0.01) inhibit hCG-stimulated testosterone production (data not shown). DISCUSSION

The present study suggests that calcium plays an important role in GtH-induced steroidogenesis in preovulatory ovarian follicles from the goldfish. Specifically, these studies suggest that calcium derived from extracellular and intracellular pools may contribute to the full expression of GtH effects on steroid production. The importance of extracellular calcium was shown by a reduction of hCG-stimulated testosterone production by follicles incubated in calcium free media or with calcium channel blockers (verapamil, nifedipine, nicardipine, CoCl,). The possible involvement of calcium derived from intracellular stores was indicated by the reduction of stimulated testosterone production by TMB-8 which is a putative blocker of intracellular calcium mobilization. These results parallel the responses seen in mammalian and avian species where both intracellular and extracellular calcium pools participate in the reg-

DER

KRAAK

ulation of GtH-stimulated steroid production (Veldhuis and Klase, 1982; Tsang and Carnegie, 1983, 1984; Veldhuis er al., 1984; Asem and Hertelendy, 1986a; Schwartz et al., 1989). It is well established that the adenylate cyclase-CAMP pathway mediates the actions of GtH on ovarian steroidogenesis in teleosts (Nagahama, 1987; Kanamori and Nagahama, 1988). Consistent with my earlier studies (Van Der Kraak, 1990; Van Der Kraak and Chang, 1990), forskolin (a direct activator of adenylate cyclase) and db CAMP were shown to stimulate testosterone production by goldfish ovarian follicles. The observation that the calcium channel blocker nifedipine inhibits forskolin and db CAMP-stimulated testosterone production indicates that calcium has a regulatory role in the steroidogenic pathway at steps beyond the formation of CAMP. This is similar to the calcium involvement in stimulated steroid production in ovarian tissue from mammalian (Veldhuis and Klase, 1982; Tsang and Carnegie, 1983; Veldhuis et al., 1984), avian (Asem and Hertelendy, 1986a), and amphibian (Kleiss-San Francisco and Scheutz, 1987) species. Depletion of extracellular calcium has also been shown to impair GtH-stimulated CAMP formation in the rat and chicken granulosa cells indicating a calcium requirement prior to CAMP formation (Veldhuis and Klase, 1982; Tsang and Carnegie, 1983; Asem and Hertelendy, 1986a; Eckstein et al., 1986). Future work will need to consider whether calcium has a similar action in fish ovarian follicles. In the present studies, removal of extracellular calcium did not affect basal testosterone production (Fig. 1). However, owing to the very low basal testosterone production, goldfish ovarian follicles were routinely incubated in the presence of IBMX which greatly enhances the actions of activators of the protein kinase A pathway but also causes a slight but significant stimulation of testosterone production. Removal of

Ca AND

STEROIDOGENESIS

extracellular calcium or addition of calcium channel antagonists did not affect IBMXstimulated testosterone production. These lindings are in agreement with the actions of calcium on basal steroid production by swine granulosa cells (Veldhuis et al., 1984). In contrast, studies on rat and avian granulosa have clearly implicated calcium in the regulation of basal steroid production (Tsang and Carnegie, 1983; Asem and Hertelendy, 1986a). Perhaps resolution of this issue in goldfish will necessitate further tests at times when basal steroid production is elevated. The precise manner in which GtH regulates the intracellular calcium levels in goldfish ovarian follicles is unknown. It is clear that one main pathway involves the entry of calcium through membrane channels. Recently, two kinds of calcium channels (L and T) were identified in chicken granulosa cells (Schwartz et al., 1989). The activity of one of these channels was completely blocked by the addition of nifedipine (16.7 @4). In the present studies, two dihydropyridine calcium channel blockers (nifedipine and nicardipine) which exhibit a high specificity to L channels were effective in blocking hCG-stimulated testosterone production by goldfish ovarian follicles; the ID,, for these drugs was