GENERAL

AtiD

COMPARATIVE

ENDOCRINOLOGY

77,

221-228 (1990)

Arachidonic Acid Stimulates Steroidogenesis in Goldfish Preovulatory Ovarian Follicles GLENVANDERKRAAKANDJOHN Department

of Zoology, Zoology,

University University

P. CHANG

of Guelph, Guelph, Ontario, Canada NIG of Alberta, Edmonton, Alberta, Canada

2W1, and Department T6G 2E9

of

Accepted April 18, 1989 The possibility that arachidonic acid (AA) plays a role in the regulation of steroidogenesis in goldfish was investigated using preovulatory ovarian follicles incubated in vitro. AA was shown to act in a time- and dose-dependent manner to stimulate testosterone production. AA in the range of lo-’ to 10m4 M increased testosterone production within 2 hr and had a maximal effect by 9 hr. The magnitude of the testosterone response to AA was similar to that observed when ovarian follicles were incubated with human chorionic gonadotropin (hCG). Ovarian follicles incubated with AA and either hCG or forskolin (adenylate cyclase activator) produced more testosterone than follicles incubated with either of these compounds alone. The actions of AA on testosterone production were completely blocked by cyclooxygenase inhibitors (indomethacin or ibuprofen) and were reduced by 50% by the lipoxygenase inhibitor nordihydroguaiaretic acid. Phospholipase C was far more effective than phospholipase A, in the stimulation of testosterone production. Taken together, these results suggest that AA formed subsequent to the action of phospholipase C on membrane phospholipids has a role in the regulation of steroidogenesis in preovulatory goldfish ovarian follicles. 0 1990 Academic Press, Inc.

The involvement of cyclic AMP and cyclic AMP-dependent protein kinase in gonadal steroidogenesis in mammals has been recognized for many years (Dufau and Catt, 1978; Hseuh et al., 1984). Recent studies have demonstrated that polyphosphoinositide metabolites also regulate gonadal steroidogenesis. Hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP,) results in the formation of two second messengers: 1,2-diacylglycerol and inositol-1,4,5-trisphosphate (IP,; Nishizuka, 1984). IP3 can mobilize calcium and diacylglycerol activates protein kinase C. The diacylglycerol may also be acted upon sequentially by diacylglycerol and monoacylglycerol lipases to release arachidonic acid (AA; Chan and Tai, 1981), and AA in turn serves as a precursor to various eicosanoids. Membrane stores of AA can also be mobilized directly by the action of phospholipase A,. Studies using calcium

ionophore A23187 and either phorbol esters or synthetic diacylglycerols to mimic the actions of IP, and diacylglycerol, respectively, have demonstrated a marked stimulation of rat ovarian steroidogenesis, suggesting a role of calcium and protein kinase C in the steroidogenic responses (e.g., Kawai and Clark, 1985; Shinohara et al., 1986; Wang and Leung, 1987). AA, presumably via its lipoxygenase metabolites, also stimulates progesterone production by rat granulosa cells (Wang and Leung, 1988). As in mammals, the steroidogenic actions of gonadotropins in teleosts are mediated by cyclic AMP (Fontaine ef al., 1970; Kanamori and Nagahama, 1988). To our knowledge the involvement of polyphosphoinositides on steroidogenesis in fish has not been reported. Recently, Ranjan and Goetz (1987) showed that calcium ionophore A23187 and activators of protein kinase C induce in vitro ovulation of goldfish 221 0016~6480/90 $1.50 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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oocytes. Furthermore, the actions of these compounds were blocked by inhibitors of AA metabolism, indicating an important role for eicosanoids in the ovulation process. These results provide a strong impetus for work on the ovarian actions of membrane phospholipid metabolites in teleosts. The present studies were conducted to determine if AA stimulates steroidogenesis in preovulatory goldfish ovarian follicles in vitro. MATERIALS

AND METHODS

Goldfish, common or comet varieties, were purchased from Grassyfork Fisheries Co. (Martinsville, IN). Fish were maintained at the University of Guelph in 4-ft-diameter tanks with flow-through water at 1416” under a constant photoperiod (14 hr light:10 hr dark). Fish were fed a commercial trout diet once a day to satiation. Hormones and chemicals. Human chorionic gonadotropin (hCG), forskolin, 3-isobutyl-1-methylxanthine (IBMX), indomethacin (INDO), ibuprofen (IBUP), phospholipase C, and phospholipase A, were purchased from Sigma (St. Louis, MO). AA and nordihydroguaiaretic acid (NDGA) were from Calbiochem (La Jolla, CA). HCG, IBMX, and phospholipases were dissolved in incubation buffer which consisted of (g/liter): NaCl, 7.25; KCI, 0.38; CaCl, . 2H,O, 0.23; NaH,PO, . H,O, 0.41; M&l, . 6Hz0, 0.20; MgSO, . 7H,O, 0.23; NaHCO,, 1.0; bovine serum albumin, 1.0; glucose, 1.0; and streptomycin sulphate, 0.1; pH 7.6. The other drugs or hormones were dissolved in ethanol and then diluted to working strength with incubation buffer or added directly to the follicle incubation. The amount of ethanol in experimental groups and controls did not exceed 1% of the final incubation volume and did not alter basal or stimulated steroid release. Follicle incubation. Preovulatory fish were killed by spinal transection, the ovaries quickly removed and placed in incubation buffer. Intact preovulatory follicles (0.9 to 1.1 mm in diameter) were isolated under a dissecting microscope and then transferred to 24-well tissue culture plates (Falcon 3047). Immediately prior to the addition of test compounds, medium was replaced with buffer containing IBMX (1 r&). IBMX, a phosphodiesterase inhibitor, was included in the follicle incubations owing to low basal steroid secretion by goldfish ovarian follicles. Routinely, groups of 20 follicles in 1 ml of medium were incubated for 18 hr at 20”. After incubation, the medium was removed and stored at - 30” prior to hormone analysis.

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Steroid determination. Aliquots of culture medium were assayed directly for testosterone content using the protocol described by Van Der Kraak and Donaldson (1986). The antiserum used for these studies was generously provided by Dr. R. J. Etches, Department of Animal and Poultry Science, University of Guelph. This antiserum cross-reacts 100% with testosterone, 41% with So-dihydrotestosterone, 15% with androstenedione, 5% with 11-ketotestosterone, and less than 1% with 17@estradiol, 17a-hydroxyprogesterone, and 17a,20l3-dihydroxy-4-pregnen-3-one. Assay sensitivity was less than 3 pg and intraassay and interassay variabilities were 5.2 and 7.3%, respectively . Statistics. Group differences were determined using analysis of variance and Duncan’s multiple range test or t test. All experiments were repeated at least two times and similar or identical results were obtained.

RESULTS Effects of AA on Testosterone Production The effects of AA on basal and hCGstimulated testosterone production are shown in Fig. 1. AA (100 @f) increased (P < 0.01) basal testosterone production and enhanced (P < 0.01) the stimulatory effect of hCG (1 .O IU/ml) on testosterone production. Ethanol serves as the vehicle for AA and did not influence basal or hCGstimulated testosterone production when included in the incubation media at a final concentration of 1.O%. The effects of increasing amounts of AA on testosterone production were determined 18 hr after the addition of AA to ovarian follicles (Fig. 2). Maximum stimulation of testosterone production was observed at AA concentrations above 100 pJ4; a significant (P < 0.05) increase in testosterone production occurred with AA at a dosage of 11 t&f. Further studies showed that AA (100 PM) stimulates testosterone production within 2 hr, reaching maximal levels between 9 and 24 hr (Fig. 3). The time course of AA action on testosterone production was similar to that obtained with follicles incubated with hCG at a dosage of 1.0 III/ml (Fig. 3).

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STEROIDOGENESIS

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600

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100

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2. Effects of graded doses of AA on testosterone production by goldfish ovarian follicles incubated for 18 hr at 20”. Values represent the mean 2 SEM of four replicate incubations. FIG.

0IId 0

hCG

100

( I.U. /ml)

FIG, 1. Effects of AA on basal and hCG (1.0 IU/ ml)-stimulated testosterone production by goldfish ovarian follicles. For both test conditions, groups of follicles were incubated for 18 hr at 20” with buffer alone (control), EtOH (1% final concentration), or AA (100 uM). Values represent the mean * SEM of four replicate incubations.

Interaction of AA with hCG and Forskolin on Testosterone Production

Testosterone production by ovarian follicles was increased in a dose-dependent fashion by hCG over the dose range of 0.1 to 10 III/ml (Fig. 4). AA (100 p&) alone stimulated (P < 0.01) testosterone production and significantly (P < 0.05) enhanced the actions of hCG at each dose tested. A similar effect of AA was observed when tested in combination with forskolin, a direct activator of CAMP production (Fig. 5). Forskolin at dosages of 0.01, 0.1, and 1.0 pJ4 stimulated testosterone production by ovarian follicles; coincubation of AA (100

lam with each dosage of forskolin further increased (P < 0.05) testosterone production. Influence of Inhibitors of AA Metabolism on Testosterone Production

To investigate the possible role of AA metabolites on testosterone production, ovarian follicles were incubated with AA alone and in combination with indomethatin or NDGA, inhibitors of cyclooxygenase and lipoxygenase activities, respectively. As before, AA (100 CLM) stimulates testosterone production; the addition of NDGA (40 t&f) significantly (P < 0.05) reduced AA-stimulated testosterone production (Fig. 6). Indomethacin, at the same molar concentration, caused a more pronounced reduction in testosterone production, reducing the levels to near that of control incubations. NDGA and indomethacin did not influence basal GtH secretion. In a separate study, indomethacin at 10 and 40 pM effectively reduced (P < 0.01) AAstimulated testosterone production (Fig. 7).

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VANDERKRAAKANDCHANG 600 - 0 Control . AA

600

- 0 Control .

hCG

F . -8

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Y B iii :

200

-

0’

400

200

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:/’

0 4

9

24

TIME

” 0

I

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6

4

( hour)

20

TIME

t hour)

3. Time course of action of AA (100 pLIM)and hCG (1 .O III/ml) on testosterone production by goldfish ovarian follicles at 20”. Values represent the mean * SEM of four replicate incubations. FIG.

A second cyclooxygenase inhibitor, ibuprofen, had a similar action, causing a significant reduction in AA-induced steroid production. Effects of Phospholipases on Testosterone Production

Phospholipase C at dosages of 0.5 and 2.0

mu/ml stimulated (P < 0.01) testosterone production after 4 and 18 hr of incubation with ovarian tissue (Fig. 8). In a separate study, phospholipase C at dosages of 0.1 and 0.5 mu/ml caused a dose-related stimulation of testosterone production (Fig. 9). By comparison, phospholipase A, was ineffective at the low dosage but caused a small but significant increase in testosterone production at the higher dosage.

0 Control .

AA

0 C AA

.Ol

0.1

1.0

FORSKOLIN hCG

, 1.0

0.1

10

(I.U./ml)

FIG. 4. Effects of AA (100 t&) and increasing dosages of hCG on testosterone production by goldfish ovarian follicles incubated for 18 hr at 20”. Values represent the mean +- SEM of four replicate incubations.

FIG. 5. Effects of ages of forskolin on fish ovarian follicles represent the mean tions .

CUM)

AA (100 m and increasing dostestosterone production by goldincubated for 18 hr at 20”. Values 2 SEM of four replicate incuba-

ARACHIDONIC

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ACID

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00

q

200 AA

1OOuM

ii \ -p”

400 160

z

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E \ E

z k

200

P

-

2 I-

120

2 O

C

NDGA

ts w

INDO

FIG. 6. Effects of indomethacin or NDGA (40 ufl on AA (100 l&f)-induced testosterone production by goldfish ovarian follicles incubated for 18 hr at 20”. Values represent the mean -C SEM of four replicate incubations.

&

60

P Y I40

DISCUSSION

The demonstration that full-grown ovarian follicles from goldfish secrete large quantities of testosterone in response to gonadotropin stimulation in vitro was consistent with earlier work on this species (Kagawa et al., 1984; Nagahama et al., 1986; Habibi et al., 1989). Importantly, the present results also suggest the involvement of AA in the regulation of steroido-

010 4010 40 INDO

CONTROL

IBUP

IN00

IBUP

AA

FIG. 7. Effects of graded dosages of indomethacin (INDO) or ibuprofen (IBUP) (10 and 40 pJ4) on AA (100 CLM)-induced testosterone production by goldfish ovarian follicles incubated for 18 hr at 20”. Values represent the mean -+ SEM of four replicate incubations.

0

4 hr. PHOSPHOLIPASE (units/ml)

18 hr. c

FIG. 8. Time course of action of phospholipase C (0.5 or 2.0 mu/ml) on testosterone production by goldfish ovarian follicles at 20”. Values represent the mean k SEM of four replicate incubations.

genesis in preovulatory goldfish. AA stimulates testosterone production by ovarian follicles incubated in vitro and the magnitude of this response was similar to that obtained with a high dosage of hCG. The actions of AA were time and dose dependent. AA and hCG have a similar time course of action on testosterone production (Fig. 3). In the present study, AA was effective in the dose range of 10P5-lop4 M (Fig. 2), which is comparable to the actions of AA on progesterone production by rat granulosa cells (Wang and Leung, 1988) and with the ability of AA to stimulate hormone secretion in a number of endocrine tissues in mammals (Kolesnick et al., 1984;

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PLC

/

PHOSPHOLI (units/ml)

PASE

FIG. 9. Effects of phospholipases C and A, (0.15 and 0.5 mu/ml) on testosterone production by goldfish ovarian follicles incubated for 18 hr at 20”. Values represent the mean 2 SEM of four replicate incubations.

Chang et al., 1986; Abou-Samra et al., 1986). The actions of gonadotropin on steroidogenesis in teleosts are mediated by CAMPdependent mechanisms (Salmon et al., 1984; Tan et al., 1986; Kanamori and Nagahama, 1988). In support of these findings, forskolin stimulates testosterone production by preovulatory follicles from goldfish (Fig. 4). However, it appears that the effects of AA on steroidogenesis are mediated intracellularly by mechanisms which do not involve CAMP. This is suggested by the finding that AA potentiates the actions of hCG and forskolin on testosterone production (Fig. 1, 4, and 5). In mammals, ovarian steroidogenesis is controlled by CAMP and calcium-mediated mechanisms (Dufau and Catt, 1978; Tsang and Carnegie, 1984; Shinohara et al., 1986). Agents known to activate protein kinase C, including phorbol esters and diacylglycerol, stimulate steroidogenesis in rat granulosa cells independently of changes in CAMP production (Kawai and Clark, 1985; Shinohara et al., 1986). The steroidogenic actions of AA appear to be a consequence of its conversion to

AND

CHANG

other eicosanoids. Treatment of ovarian follicles with the cyclooxygenase inhibitors indomethacin or ibuprofen effectively blocked AA-stimulated testosterone production (Figs. 6 and 7). Similarly, the lipoxygenase inhibitor NDGA reduced the amount of testosterone secreted in response to AA by 50%. These data indicate that both cyclooxygenase and lipoxygenase metabolites are involved in the regulation of ovarian steroidogenesis. Studies showing that [14C]AA is metabolized to prostaglandins and hydroxyeicosatetraenoic acids by ovarian tissue from brook trout (Goetz et al., 1987) and goldfish (unpublished work cited in Ranjan and Goetz, 1987) provide direct evidence for the presence of cyclooxygenase and lipoxygenase pathways in the teleost ovary. It will be of interest in future studies to identify the AA metabolites which exhibit steroidogenic actions. It seems that active metabolites in goldfish may differ from those in the rat. In rat granulosa cells, NDGA, but not indomethacin, blocks luteinizing hormone-releasing hormone (LHRH) and AA-induced progesterone production (Wang and Leung, 1988). Ranjan and Goetz (1987) reported that lipoxygenase products of AA metabolism mediate ovulation in goldfish. In these studies, NDGA, but not indomethacin, blocked ovulation induced by calcium ionophore and phorbol ester. Unlike what occurs at ovulation, it appears that cyclooxygenase metabolites have a dominant effect on steroidogenesis. Together, these results highlight the need to examine eicosanoids throughout the periovulatory period and suggest that changes in AA metabolism may be of prime importance at this time. AA may be formed from polyphosphoinositides through sequential reactions catalyzed by phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase or alternatively, released from the SN 2-acyl position of phospholipids by the action of phospholipase A, (see Introduction and

ARACHIDONIC

ACID AND STEROIDOGENESIS

Nishizuka, 1984; Rubin, 1986). The former pathway may be the major source of AA in goldfish ovarian follicles as phospholipase C was far more effective in stimulating testosterone production than phospholipase A, (Fig. 9). However, a major question that remains is the identity of the trophic signal which activates phospholipase C and enhances AA mobilization and metabolism in goldfish. In rat granulosa cells, LHRH promotes the hydrolysis of membrane phospholipids, including phosphoinositides, leading to the formation of diacylglycerol, inositol phosphates, and AA (Leung et al., 1983; Davis et al., 1986b; Wang and Leung, 1988). Increased intracellular calcium, activation of protein kinase C and AA metabolites contribute to the steroidogenic effects of LHRH (Shinohara et al., 1986; Wang and Leung, 1987, 1988). It is not clear whether gonadotropin-releasing hormone (GnRH) or GnRH-like peptides have similar actions in teleosts. GnRH peptides stimulate a small but significant increase in the number of oocytes undergoing final maturation but had no effect on testosterone production in goldfish (Habibi et al., 1988, 1989). Luteinizing hormone also stimulates phosphoinositide turnover in mammalian granulosa and luteal cells (Davis et al., 1986a, 1987; Dimino et al., 1987). This suggests that gonadotropin actions may be expressed through activation of the phosphoinositide cycles as well as the more recognized adenylate cyclase system. However, the actions of gonadotropins may not always be stimulatory as follicle-stimulating hormone attenuates phosphoinositide hydrolysis in immature rat Sertoli cells (Monaco et al., 1988). These alternate signal transduction pathways for gonadotropin action in fish have not been investigated to the best of our knowledge. In summary, we have demonstrated that lipoxygenate and cyclooxygenate metabolites of AA stimulate testosterone production by goldfish preovulatory ovarian folli-

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cles. It appears that AA is formed secondarily to phospholipase C hydrolysis of membrane phospholipids. This suggests that steroidogenesis is regulated by two intracellular pathways, one involving CAMP and the other metabolites of phospholipid hydrolysis. Further work will be required to identify the trophic stimulus which activates phospholipase C and to consider the role of IP, and diacylglycerol which are key secondary messengers of phosphoinositide turnover. ACKNOWLEDGMENTS This work was supported by NSERC operating Grants UO554 and UO552 to G.V.D.K. and J.P.C., respectively. We thank Mrs. S. Mahaney for her excellent technical assistance and Mrs. L. Ferguson for typing the manuscript.

REFERENCES Abou-Samra, A. B., Catt, K. J., and Aguilera, G. (1986). Role of arachidonic acid in the regulation of adenocorticotropin release from rat anterior pituitary cell cultures. Endocrinology 119, 14271431. Chart, L.-Y., and Tai, H.-H. (1981). Release of arachidonate from diglyceride in human platelets requires the sequential action of diglyceride lipase and monoglyceride lipase. Biochem. Biophys. Res. Commun. 100, 1688-1695. Chang, J. P., Graeter, J., and Catt, K. J. (1986). Coordinate actions of arachidonic acid and protein kinase C in gonadotropin-releasing hormonestimulated secretion of luteinizing hormone. Bio&em. Biophys. Res. Comrnun. 134, 134-139. Davis, J. S., Weakland, L. L.., Farese, R. V., and West, L. A. (1987). Luteinizing hormones increase inositol trisphosphate and cytosolic free Ca++ in isolated bovine luteal cells. J. Biol. Chem. 262, 8515-8521. Davis, J. S., Weakland, L. L., West, L. A., and Farese, R. V. (1986a). Luteinizing hormone stimulates the formation of inositoltrisphosphate and cyclic AMP in rat granulosa cells. Biochem. J. 238, 597-604. Davis, J. S., West, L. A., and Farese, R. V. (1986b). Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacylglycerol in rat granulosa cells: Further evidence for the involvement of Ca*+ and protein kinase C in the action of GnRH. Endocrinology 118, 2561-2571.

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Dimino, M. J., Snitzer, J., and Brown, K. M. (1987). Inositol phosphates accumulation in ovarian granulosa after stimulation by luteinizing hormone. Biol. Reprod. 37, 1129-1134. Dufau, M. L., and Catt, K. J. (1978). Gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. Vitam. Horm. 36, 461-592. Fontaine, Y. A., Burzawa-Gerard, E., and DelerueLe Belle, N. (1970). Stimulation hormonale de l’activite adenyl-cyclasique de l’ovaire d’un poisson teleosteen, le cyprin (Carassius aurutus L.). C.R.

Acad.

Sci.

Paris

Ser.

D 271, 780-783.

Goetz, F. W., Ranjan, M., Bemdtson, A. K., and Duman, P. (1987). The mechanism and hormonal regulation of ovulation: The role of prostaglandins in teleost ovulation. In “Proceedings, Third Intemational Symposium on Reproductive Physiology of Fish (D. R. Idler, L. W. Crim, and J. M. Walsh, Eds.), pp. 235-238. Memorial Univ. Press, St. John’s, Habibi, H. R., Van Der Kraak, G., Bulanski, E., and Peter, R. E. (1988). Effect of teleost GnRH on reinitiation of oocyte meiosis in goldfish, in vitro. Amer. .I. Physiol. 255, R268-R273. Habibi, H. R., Van Der Kraak, G., Fraser, R., and Peter, R. E. (1989). Effect of a teleost GnRH analog on steroidogenesis by the follicle-enclosed goldfish oocytes, in vitro. Gen. Comp. Endocrinol. 76, 95-105. Hseuh, A. J. W., Adashi, E. Y., Jones, P. B. C., and Welsh, T. H., Jr. (1984). Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5, 76-127. Kagawa, H., Young, G., and Nagahama, Y. (1984). In vitro estradiol-17B and testosterone production by ovarian follicles of the goldfish, Cnrassius auratus. Gen. Comp. Endocrinol. 54, 139-143. Kanamori, A., and Nagahama, Y. (1988). Involvement of 3’5’-cyclic adenosine monophosphate in the control of follicular steroidogenesis of the amago salmon (Oncorhynchus rhodurus). Gen. Camp. Endocrinol.

72, 39-53.

Kawai, Y., and Clark, M. R. (1985). Phorbol ester regulation of rat granulosa cell prostaglandin and progesterone accumulation. Endocrinology 116, 2320-2326. Kolesnick, R. N., Musacchio, I., Thaw, C., and Gershengom, M. C. (1984). Arachidonic acid mobilizes calcium and stimulates prolactin secretion from GH, cells. Amer. J. Physiol. 246, E458E462. Leung, P. C. K., Raymond, V., and Labrie, F. (1983). Stimulation of phosphatidic acid and phosphatidylinositol labelling in luteal cells by luteinizing hormone releasing hormone. Endocrinology 112, 1138-1140.

AND

CHANG

Monaco, L., Adamo, S., and Conti, M. (1988). Follicle-stimulating hormone modulation of phosphoinositide turnover in the immature rat Sertoli cell in culture. Endocrinology 123, 2032-2039. Nagahama, Y., Goetz, F. W., and Tan, J. D. (1986). Shift in steroidogenesis in the ovarian follicles of the goldfish (Carassius auratus) during gonadotropin-induced oocyte maturation. Dev. Growth Differ.

28, 555-561.

Nishizuka, Y. (1984). The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature (London) 308, 693-698. Ranjan, M., and Goetz, F. W. (1987). Protein kinase C as a possible mediator of goldfish (Carassius auratus) ovulation. J. Exp. Zool. 242, 355-361. Rubin, R. P. (1986). Inositol lipids and cell secretion. In “Phosphoinositides and Receptor Mechanisms” (J. W. Putney, Ed.), pp. 149-162. A. R. Liss, New York. Salmon, C., Marchelidon, J., Fontaine-Bertrand, E., and Fontaine, Y. A. (1984). Human chorionic gonadotropin and immature fish ovary: Characterization and mechanism of the in vitro stimulation of cyclic adenosine monophosphate accumulation. Gen. Camp. Endocrinol. 58, 101-108. Shinohara, O., Knecht, M., Feng, P., and Catt, K. J. (1986). Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells. .I. Steroid Biochem. 24, 161-168. Tan, D. J., Adachi, S., and Nagahama, Y. (1986). The in vitro effects of cyclic nucleotides, cyanoketone, and cyclohexamide on the production of estradiol-17B by vitellogenic ovarian follicles of goldfish (Carussius auratus). Cert. Camp. Endocrinol. 63, 110-116. Tsang, B. K., and Carnegie, J. A. (1984). Calciumdependent regulation of progesterone production by isolated rat granulosa cells: Effects of the calcium ionophore A23187, prostaglandin F,a, dlisoproterenol and cholera toxin. Biol. Reprod. 30, 787-794. Van Der Kraak, G., and Donaldson, E. M. (1986). Steroidogenic capacity of coho salmon ovarian follicles throughout the periovulatory period. Fish Physiol.

Biochem.

1, 179-186.

Wang, J., and Leung, P. C. K. (1987). Role of protein kinase C in luteinizing hormone-releasing hormone (LHRH)-stimulated progesterone production in rat granulosa cells. Biochem. Biophys. Res.

Commun.

146,93%944.

Wang, J., and Leung, P. C. K. (1988). Role of arachidonic acid in luteinizing hormone-releasing hormone action: Stimulation of progesterone production in rat granulosa cells. Endocrinology 122, 906-911.

Arachidonic acid stimulates steroidogenesis in goldfish preovulatory ovarian follicles.

The possibility that arachidonic acid (AA) plays a role in the regulation of steroidogenesis in goldfish was investigated using preovulatory ovarian f...
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