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Biology of Reproduction 66, 813-822 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Involvement of Protein Kinase C and Arachidonic Acid Pathways in the Gonadotropin-Releasing Hormone Regulation of Oocyte Meiosis and Follicular Steroidogenesis in the Goldfish Ovary1

Debananda Pati3,a, and Hamid R. Habibi2,a

a Department of Biological Sciences and Endocrine Research Group, University of Calgary, Calgary, Alberta, Canada T2N 1N4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The involvement of protein kinase C (PKC) and arachidonic acid (AA) pathways were investigated in the GnRH regulation of oocyte meiosis and follicular testosterone production in the goldfish ovary. The results clearly demonstrate differences in the postreceptor mechanisms involving the stimulatory and inhibitory actions of GnRH peptides on basal and gonadotropin (GtH)-induced reinitiation of oocyte meiosis and steroidogenesis. In isolated goldfish follicles in vitro, the observed stimulatory effects of both salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) on germinal vesicle breakdown were completely blocked by addition of PKC inhibitors, suggesting the involvement of PKC, presumably through activation of phospholipase C/diacylglycerol pathways in the GnRH-induced reinitiation of oocyte meiosis. Administration of an AA metabolism inhibitor, however, only blocked the stimulatory effect of sGnRH without affecting cGnRH-II-induced meiosis. As observed previously, in the presence of GtH, sGnRH was found to inhibit GtH-induced resumption of meiosis and testosterone production, whereas cGnRH-II was without effect. The inhibitory effect of sGnRH on GtH-induced meiosis and steroidogenesis was completely reversed by addition an AA metabolism inhibitor, whereas PKC inhibitors had no effect. These findings provide functional evidence in support of the novel hypothesis that goldfish ovarian follicles contain GnRH-receptor subtypes with different ligand selectivity mediating stimulatory and inhibitory actions of sGnRH and cGnRH in the goldfish ovary.

follicular development, gonadotropin-releasing hormone, mechanisms of hormone action, meiosis, oocyte development, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clear evidence supports the presence of GnRH and GnRH-receptor subtypes in the nervous system and in nonneuronal tissues, including gonads [1–5]. In addition to its hypophysiotrophic activity, GnRH is known to function as a paracrine/autocrine regulator of gonadal function. Two or more forms of GnRH, as well as at least three classes of G protein-coupled GnRH receptors with different ligand selectivity, exist in the pituitary, gonads, and other tissues [1, 3, 6–10]. The mechanism of GnRH action involves the Gq and/or G11 protein and multiple second-messenger systems integrating phospholipid turnover, calcium mobilization, protein kinase C (PKC), arachidonic acid (AA) metabolism, phospholipase D, and activation of mitogen-activated protein kinase. Many similarities exist among the intracellular signaling cascades mediating GnRH actions in the pituitary and ovary, including involvement of phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D, as well as activation of mitogen-activated protein kinase [11–13]. The signal transduction pathways mediating GnRH-induced pituitary gonadotropin (GtH) release have been thoroughly investigated in mammals [14] and in certain nonmammalian vertebrates, such as fish [15, 16]. Like those in mammals, the main pathways for GnRH-induced GtH release in goldfish involve PKC activation, AA metabolism, and Ca2+ mobilization [15]. However, recent studies have demonstrated differences in the postreceptor mechanisms activated by the two native GnRH peptides, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), in the goldfish pituitary [15, 16]. The sGnRH stimulation of LH release involves both PKC and AA-mediated pathways, whereas cGnRH-II action is mainly dependent on the PKC pathway and does not involve the mobilization of AA in the goldfish pituitary [15, 16]. In addition, sGnRH action involves the mobilization of both intracellular and extracellular Ca2+, but cGnRH-II-induced response depends primarily on extracellular Ca2+ entry [17, 18]. To our knowledge, no information is available regarding GnRH signal transduction in the ovary of nonmammalian vertebrates.

In previous studies, the presence of GnRH and GnRH receptors have been demonstrated in the goldfish ovary [19–21]. Both sGnRH and cGnRH-II were found to individually stimulate oocyte meiosis in vitro as well as histone H1 kinase activity, which is an indicator of maturation-promoting factor activity [22, 23]. However, in the presence of GtH, sGnRH was found to inhibit GtH-induced meiosis and steroidogenesis, whereas cGnRH-II had no effect on GtH-induced responses [22]. Addition of a GnRH antagonist was found to effectively block the stimulatory effect of both sGnRH and cGnRH-II on oocyte meiosis, without affecting the inhibitory actions of sGnRH on GtH-induced response, suggesting the involvement of different pathways mediating the stimulatory and inhibitory actions of sGnRH [4, 5, 24].

In the present study, we investigated the involvement of PKC and AA pathways in sGnRH and cGnRH-II regulation of goldfish ovarian function. The findings provide a framework for better understanding the role of multiple GnRH peptides in the paracrine control of ovarian function in fish and other vertebrates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Female goldfish (Carassius auratus) of the common or comet varieties (length, 8–10 cm) were purchased from Aquatic Imports (Calgary, AB, Canada). Fish were maintained in a 1500-L, semirecirculating aquarium (60% replacement per day) at 17°C on a 16L:8D photoperiod and were fed a commercial fish diet (Nutrafin floating pellets; Rolf C. Hagen Inc., Montreal, QC, Canada).

Hormones and Drugs

The sGnRH (pGlu, His, Trp, Ser, Tyr, Gly, Trp, Leu, Pro, Gly-NH2) and cGnRH-II (pGlu, His, Trp, Ser, His, Gly, Trp, Tyr, Pro, Gly-NH2) were purchased from Peninsula Laboratories (Belmont, CA). Carp GtH, which is similar to LH, was a generous gift from Dr. J. Yu (Academia Scinica, Department of Biochemistry, Taipei, Taiwan). The compounds 5,8,11,13-eicosatetrayonic acid (ETYA) and phorbol 12-myristate 13-acetate (Sigma, St. Louis, MO) were made up as stock solutions (1 mM) in ethanol and kept in the dark under nitrogen gas at -70°C. The 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; Calbiochem, La Jolla, CA) was prepared in distilled water at a concentration of 10 mM and kept at -70°C. Bisindolylmaleimide (Calbiochem) was dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml, and aliquots were stored at -70°C. Appropriate concentrations of the hormones were prepared by diluting the stock solutions immediately before use in an experiment, and the final concentration of the vehicle was <0.5% and did not alter the basal or hormone-stimulated germinal vesicle breakdown (GVBD) and steroid release.

Follicle Incubation

Animals were killed by a blow to the head in accordance with the principles and guidelines of the Canadian Council on Animal Care, followed immediately by spinal transection. Ovaries were rapidly dissected out and transferred into the goldfish Ringer solution [25] containing 7.3 g/L of NaCl, 0.18 g/L of KCl, 0.07 g/L of MgSO4(7H2O), 0.18 g/L of MgCl(6H2O), 0.35 g/L of CaCl2(2H2O), 0.95 g/L of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 1 g/L of glucose, 1 g/L of BSA, 100 000 IU of penicillin, and 0.1 g of streptomycin sulfate (pH 7.5). Follicles were manually separated, and fully grown ovarian follicles (postvitellogenic follicle-enclosed oocyte diameter, >1 mm) were selected. These follicles were shown to have high-affinity GnRH receptors in previous studies [19].

The chosen follicles were washed with incubation medium and transferred to sterile, 24-well plates (Falcon; Becton Dickinson, Franklin Lakes, NJ), with each well containing 20 follicle-enclosed oocytes in 1 ml of incubation medium. The follicles were incubated, in the first instance, for 16–18 h (unless otherwise stated) in a CO2 water-jacketed incubator at 18°C (5% CO2, 95% air) following addition of the appropriate compounds. At the end of the initial incubation, 200 ml of media were removed and analyzed immediately for testosterone in a radioimmunoassay.

The follicles were then incubated for an additional 24 h and assayed for meiotic maturation by monitoring for the presence or absence of germinal vesicles as described previously [26]. In brief, 100 ml of acetic acid (50% [v/v]) were added to each well and allowed to stand for 5 min to clear the opaque ooplasm. The cleared follicles were transferred into a Petri dish, and the developmental stage for each oocyte was determined under a dissecting microscope. The absence of an oocyte nucleus (i.e., germinal vesicle) indicated dissolution of the nuclear envelope (i.e., GVBD) and reinitiation of meiosis. The results are expressed in terms of percentage GVBD for a given number of oocytes. In the present experiments, interanimal variations were minimized, because each animal acted as its own control by contributing an equal number of follicles to all treatment groups in any particular experiment.

Steroid Determination

Testosterone level was determined by RIA in the media using a method described previously [27]. Anti-testosterone-BSA serum (a gift from Dr. H.R. Behrman, Yale University, New Haven, CT) was stored lyophilized, reconstituted before the experiment, and characterized for titer and cross-reactivity using [1,2,6,7-3H]testosterone from Amersham Canada (Mississauga, ON, Canada). An antiserum dilution of 1:60 000 was found to be optimal, with a sensitivity of 5 pg per 100 ml. The ED20, ED50, and ED80 values for the assays were 65.748 ± 3.2, 23.3 ± 2.5, and 8.25 ± 1.3 pg per 100 ml (w/v), respectively (n = 15). This antiserum cross-reacts with testosterone (100%), 4-androsten-17ß-ol-3-11-dione (65%), dihydroxytestosterone (54.9%), estrone (0.002%), 17ß-estradiol (no displacement), 17{alpha}-estradiol (no displacement), estriol (no displacement), progesterone (0.069%), 17{alpha},20ß-dihydroxy progesterone (no displacement), cortisol (0.004%), and pregnenolone (0.001%). Because of the cross-reactivity of the antiserum, the measured testosterone levels also may include other androgen metabolites, such as 4-androsten-17ß-ol-3-11-dione and dihydroxytestosterone.

Statistical Analysis

The differences between GVBD levels were tested using a paired test of proportions based on binomials [28] as previously described [26]. The results of the testosterone levels were analyzed using Student t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PKC-Inhibitor H7 on GnRH-Induced Meiosis

To investigate the possible role of PKC on the GnRH-stimulated reinitiation of oocyte meiosis, goldfish follicles were incubated with GnRH alone and in combination with 5 µM H7, a potent inhibitor of PKC. Treatment with increasing concentrations of sGnRH and cGnRH-II significantly stimulated GVBD in a dose-related manner (Fig. 1). The stimulatory effect of both GnRH peptides was significantly inhibited (P < 0.05) in the presence of H7. Treatment with H7 alone (5 µM) had no effect on basal GVBD response (Fig. 1).



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  		FIG. 1. Effect of PKC-inhibitor H7 (5 µM) on sGnRH and cGnRH-II-induced GVBD response in follicle-enclosed goldfish oocytes in vitro. Each value represents the percentage GVBD determined using 240 follicles at each concentration (6 experiments carried out in duplicate using ovaries from 6 fish, with each fish contributing follicles to all treatment groups, which were incubated in groups of 20 follicles/well). Individual values were compared using the binomial test of proportions; values with dissimilar superscripts are significantly different (P < 0.05).

Effect of AA Metabolism-Inhibitor ETYA on GnRH-Induced Meiosis

We used ETYA, which is known to block cyclooxygenase, lipoxygenase, and epoxygenase pathways, to investigate the possible role of AA metabolites in the GnRH-induced actions on oocyte meiosis. As before, sGnRH and cGnRH-II stimulated the GVBD in a dose-related fashion. Treatment with ETYA (5 µM) completely inhibited the stimulatory effect of sGnRH on oocyte meiosis (P < 0.05) without affecting the cGnRH-II-induced response (Fig. 2). Five micromoles of ETYA alone did not influence basal GVBD response.



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  		FIG. 2. Effect of ETYA, an inhibitor of all known pathways of AA metabolism (5 µM) on sGnRH and cGnRH-II-induced GVBD response in follicle-enclosed goldfish oocytes in vitro. Other details are as described in Figure 1

Dose-Related Effect of H7 and ETYA on sGnRH- and cGnRH-II-Induced Reinitiation of Meiosis in Follicle-Enclosed Goldfish Oocytes In Vitro

Treatment with increasing concentrations of the PKC-inhibitor H7 significantly attenuated the effects of both sGnRH- and cGnRH-II-induced (100 nM) GVBD response in a dose-related manner (Fig. 3). Treatment with ETYA significantly blocked the stimulatory effect of 100 nM sGnRH in a dose-related manner without affecting the cGnRH-II-induced GVBD response (Fig. 3). Treatment with H7 and ETYA alone did not significantly influence the basal level of GVBD (Fig. 3).



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  		FIG. 3. Dose-related effect of the PKC-inhibitor H7 and the AA metabolism-inhibitor ETYA on basal as well as sGnRH- and cGnRH-II (100 nM)-stimulated GVBD response in follicle-enclosed goldfish oocytes in vitro. Each value represents the percentage GVBD determined using 120 follicles at each concentration (3 experiments carried out in duplicate using ovaries from 3 fish, with each contributing 40–50 follicles incubated in groups of 20 follicles/well). Individual values were compared using the binomial test of proportions; values with dissimilar superscripts are significantly different (P < 0.05)

Effects of PKC and AA Inhibitors and PKC Stimulator on GtH-Induced Reinitiation of Meiosis in Follicle-Enclosed Goldfish Oocytes In Vitro

Incubation of follicles with increasing concentrations of PKC inhibitors (H7 or bisindolylmaleimide) or an AA metabolism inhibitor (ETYA) did not influence basal GVBD response. Treatment with GtH (250, 500, or 100 ng/ml) significantly (P < 0.05) increased the GVBD response in the goldfish oocytes (Fig. 4). Concomitant treatment of GtH with H7, bisindolylmaleimide, and ETYA did not significantly affect the GtH-induced GVBD response (Fig. 4).



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  		FIG. 4. Dose-related effect of PKC-inhibitors H7 and bisindolylmaleimide, AA metabolism-inhibitor ETYA, and PKC-agonist PMA on basal as well as GtH-stimulated GVBD response by follicle-enclosed goldfish oocytes in vitro. Each value represents the percentage GVBD determined using 160 follicles at each concentration (4 experiments carried out in duplicate using ovaries from four fish, with each fish contributing 40–50 follicles that were incubated in groups of 20 follicles/well). An asterisk indicates a significant difference (P < 0.05) with respect to appropriate controls according to the binomial test of proportions.

In a further set of experiments, the effect of a PKC stimulator, phorbol 12-myristate 13-acetate (PMA), on the GtH-induced GVBD response was tested. Increasing concentrations of PMA alone significantly stimulated the basal GVBD response (Fig. 4). In contrast, concomitant treatment with PMA significantly attenuated the GtH-induced GVBD response in the follicle-enclosed goldfish oocytes (Fig. 4).

Effects of PKC and AA Metabolism Inhibitors and Stimulators on GtH-Induced Testosterone Production in Follicle-Enclosed Goldfish Oocytes In Vitro

In this experiment, we investigated the effects of various inhibitors and stimulators of PKC and AA metabolism on follicular testosterone production. Because the antiserum cross-reacts with other androgens, however, it should be noted that these values also may include concentrations of 4-androsten-17ß-ol-3-11-dione and dihydroxytestosterone.

Ovarian follicles were incubated with PKC inhibitors (H7 or bisindolylmaleimide), a PKC agonist (PMA), or an AA metabolism inhibitor (ETYA) alone or in combinations with three different doses of GtH (250, 500, and 1000 ng/ml). Treatment with H7, bisindolylmaleimide, PMA, or ETYA had no effect on basal testosterone production by follicle-enclosed goldfish oocytes (Fig. 5). Treatment with GtH increased the testosterone production in a dose-related fashion (Fig. 5). Treatments with both PKC inhibitors (H7 and bisindolylmaleimide) significantly inhibited the GtH-induced testosterone production at the higher doses tested (Fig. 5). At the lower doses (H7, 0.1–5.0 µM; bisindolylmaleimide, 0.1–1.0 nM), the two inhibitors did not influence GtH-induced testosterone production. Increasing concentrations of PMA had no effect on the basal testosterone levels but significantly attenuated the GtH-induced testosterone production in a dose-related manner (Fig. 5); GtH-induced steroid production was completely abolished in the presence of 0.1–1 µM PMA. Concomitant treatment with ETYA had no significant effect on the dose-related effect of GtH-induced testosterone production (Fig. 5).



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  		FIG. 5. Dose-related effect of PKC-inhibitors H7 and bisindolylmaleimide, AA metabolism-inhibitor ETYA, and PKC-agonist PMA on basal as well as increasing doses of GtH-induced testosterone production by follicle-enclosed goldfish oocytes in vitro. Because of the cross-reactivity of the antiserum used, values also may include other androgen metabolites, such as 4-androsten-17ß-ol-3-11-dione and dihydroxytestosterone. Testosterone concentration in the incubation media was measured after 16 h of incubation at 18°C. Each value represents the mean ± SEM of 8 observations (4 fish, with each fish contributing 40–50 follicles that were incubated in duplicate groups of 20 follicles/well). The results of the testosterone production were analyzed using Student t-test. An asterisk indicates a significant difference (P < 0.05) with respect to appropriate controls.

Based on the above results, noninhibitory doses of H7 (5 µM), bisindolylmaleimide (1 nM), ETYA (5 µM), and PMA (1 nM) were selected to further investigate the action of GnRH peptides on GtH-induced testosterone production and reinitiation of oocyte meiosis.

Involvement of PKC Pathway and AA Metabolism in sGnRH-Mediated Inhibition of GtH-Induced GVBD Response in Follicle-Enclosed Goldfish Oocytes In Vitro

As demonstrated previously [22], sGnRH inhibited the GtH-stimulated GVBD response. Administration of the PKC blockers (H7 and bisindolylmaleimide) did not affect the inhibitory action of sGnRH on GtH-induced GVBD response (Fig. 6). Treatment with the PKC-agonist PMA also was without effect on sGnRH inhibition of GtH-induced GVBD response (Fig. 6), indicating a lack of PKC involvement in the sGnRH inhibition of the GtH-induced response. Treatment with ETYA, however, completely reversed the sGnRH inhibition of the GtH-induced GVBD response, indicating the involvement of AA metabolites in the inhibitory effect of sGnRH (Fig. 6). It should be noted that ETYA, H7, and bisindolylmaleimide significantly attenuated the stimulatory effect of sGnRH alone, as demonstrated before.



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  		FIG. 6. Effect of PKC-inhibitors H7 (5 µM) and bisindolylmaleimide (1 nM), AA metabolism-inhibitor ETYA (5 µM), and PKC-agonist PMA (1 nM) on sGnRH-mediated inhibition of GtH-induced GVBD response in follicle-enclosed goldfish oocytes in vitro. Follicles were incubated with increasing doses of GtH in combination with 100 nM sGnRH, 100 nM sGnRH + drug, or drug alone. Each value represents the percentage GVBD determined using 120–200 follicles at each concentration (3–5 experiments, as indicated in the figure, were carried out in duplicate using ovaries from 3 to 5 fish, with each fish contributing 40–50 follicles that were incubated in groups of 20 follicles/well). Individual values were compared using the binomial test of proportions; values with dissimilar superscripts are significantly different (P < 0.05).

In a set of control studies, the above experiments were repeated using cGnRH-II instead of sGnRH. As demonstrated previously, cGnRH-II had no effect on GtH-induced meiosis. Concomitant treatment of the follicles with increasing concentrations of GtH and cGnRH-II in the presence of either PKC inhibitors (H7 and bisindolylmaleimide), PKC agonist (PMA), or AA metabolism inhibitor (ETYA) had no effect (Fig. 7). However, treatment with H7 and bisindolylmaleimide significantly attenuated the stimulatory effect of cGnRH-II alone, whereas treatment with ETYA had no effect (Fig. 7). These results confirm earlier findings indicating a lack of effect from cGnRH-II on GtH-induced meiosis.



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  		FIG. 7. Effect of PKC-inhibitors H7 (5 µM) and bisindolylmaleimide (1 nM), AA metabolism-inhibitor ETYA (5 µM), and PKC-agonist PMA (1 nM) on GtH-induced GVBD response in the presence or absence of 100 nM cGnRH-II in follicle-enclosed goldfish oocytes in vitro. Follicles were incubated with increasing doses of GtH in combination with 100 nM cGnRH-II, 100 nM cGnRH-II + appropriate drug, or drug alone. Other details are as described in Figure 6

Effects of PKC Agonist and Antagonists and AA Metabolism Inhibitor on sGnRH-Mediated Inhibition of GtH-Induced Testosterone Production in Follicle-Enclosed Goldfish Oocytes In Vitro

Similar to the results of previous experiments [22], sGnRH significantly attenuated the GtH-stimulated production of testosterone (Fig. 8). The sGnRH-induced inhibition was completely reversed in the presence of ETYA (Fig. 8), confirming earlier observations that AA metabolites mediate the inhibitory effect of sGnRH on GtH-induced steroidogenesis. The inhibitory effect of sGnRH on GtH-induced testosterone production remained unaffected by concomitant treatment with PKC inhibitors (H7 and bisindolylmaleimide) (Fig. 8). However, treatment with a PKC agonist, PMA, completely reversed the sGnRH-induced inhibition (Fig. 8). Treatments with ETYA (5 µM), H7 (5 µM), bisindolylmaleimide (1 nM), or PMA (1 nM) had no effect on GtH-induced testosterone production.



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  		FIG. 8. Effect of PKC-inhibitors H7 (5 µM) and bisindolylmaleimide (1 nM), AA metabolism-inhibitor ETYA (5 µM), and PKC-agonist PMA (1 nM) on sGnRH-mediated inhibition of GtH-induced testosterone production in follicle-enclosed goldfish oocytes in vitro. Because of the cross-reactivity of the antiserum used, values also may include other androgen metabolites, such as 4-androsten-17ß-ol-3-11-dione and dihydroxytestosterone. Follicles were incubated with increasing doses of GtH or in combination with 100 nM sGnRH, 100 nM sGnRH + appropriate drug, or drug alone. Testosterone concentration in the incubation media was measured after 16 h of incubation at 18°C. Each value represents the mean ± SEM of 6–10 observations, as indicated in the figure (3–5 fish, with each fish contributing 40–50 follicles that were incubated in groups of 20 follicles/well; i.e., in duplicate). The results of the testosterone production were analyzed using Student t-test; values with dissimilar superscripts are significantly different (P < 0.05)

In a set of control studies, the above experiments were repeated using cGnRH-II instead of sGnRH. As shown before, cGnRH-II exerted no effect on GtH-induced steroidogenesis (Fig. 9). Concomitant treatment of the follicles with increasing concentrations of GtH and cGnRH-II in the presence of PKC inhibitors (H7 and bisindolylmaleimide), PKC agonist (PMA), or AA metabolism inhibitor (ETYA) had no effect. These findings confirm earlier observations indicating a lack of effect from cGnRH-II on the GtH-induced steroidogenesis.



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  		FIG. 9. Effect of PKC-inhibitors H7 (5 µM) and bisindolylmaleimide (1 nM), AA metabolism-inhibitor ETYA (5 µM), and PKC-agonist PMA (1 nM) on GtH-induced testosterone production in the presence or absence of 100 nM cGnRH-II in follicle-enclosed goldfish oocytes in vitro. Because of the cross-reactivity of the antiserum used, values also may include other androgen metabolites, such as 4-androsten-17ß-ol-3-11-dione and dihydroxytestosterone. Follicles were incubated with increasing doses of GtH in combination with 100 nM cGnRH-II, 100 nM cGnRH-II + appropriate drug, or drug alone. Other details are as described in Figure 8


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study investigated the involvement of the PKC pathway and AA metabolism in the GnRH-mediated regulation of oocyte meiosis and steroidogenesis in follicle-enclosed goldfish oocytes in vitro. The results clearly demonstrate a difference in the postreceptor mechanisms involving the stimulatory and inhibitory actions of sGnRH on reinitiation of oocyte meiosis and steroidogenesis. The stimulatory effect of both sGnRH and cGnRH-II on the reinitiation of oocyte meiosis was completely blocked by the PKC inhibitors (H7 and bisindolylmaleimide), suggesting the involvement of a PLC/diacylglycerol (DAG) pathway in the mechanisms of GnRH-induced meiosis. Administration of ETYA, an inhibitor of all known pathways of AA metabolism, including cyclooxygenase, lipoxygenase, and epoxygenase, only inhibited the stimulatory effect of sGnRH and had no effect on cGnRH-II-induced meiosis. Furthermore, the results demonstrate that sGnRH inhibition of GtH-induced meiosis and steroidogenesis could only be reversed by ETYA, an inhibitor of AA metabolism, and not by PKC blockers. These findings provide functional evidence in support of the hypothesis that goldfish ovarian follicles contain GnRH-receptor subtypes with different ligand selectivity that mediate the stimulatory and inhibitory actions of sGnRH in the goldfish ovary. Further support for this hypothesis was provided by a recent report that addition of a GnRH antagonist effectively blocked the stimulatory effect of both sGnRH and cGnRH-II on oocyte meiosis but was without effect on the inhibitory actions of sGnRH on GtH-induced meiosis [22], also suggesting the involvement of different pathways mediating the stimulatory and inhibitory actions of sGnRH. Similarly, evidence supports the presence of different GnRH receptors, coupling independently to PLC and PLA2, in rat luteal cells [29]. In this context, goldfish pituitary, ovary, and testis were found to contain two GnRH-receptor subtypes (A and B) with different ligand selectivity but sharing 71% identity [8, 30]. To our knowledge, however, no information is available regarding the functional significance of GnRH-receptor subtypes.

In the present study, administration of the PKC-agonist PMA was found to reverse the sGnRH inhibition of GtH-induced steroidogenesis. This finding appears to be contrary to the idea that the PKC pathway may not be involved in the inhibitory actions of sGnRH. However, a possible explanation for the observed effect of PMA would be the production of AA through the sequential breakdown of phosphoinositides by PLC, DAG lipase, and monoacylglycerol lipase resulting from a cross-communication between the PKC and AA metabolism pathways. In this regard, an earlier study [31] demonstrated a stimulatory effect of AA alone on the production of testosterone in the goldfish ovary and suggested the production of AA subsequent to the action of PLC, possibly through the PLC/DAG lipase pathway. There appear to be differences between the mechanisms of GnRH stimulation and the inhibition of gonadal functions in goldfish and rat [11]. In the rat ovary, the PKC pathway and AA metabolism have been suggested to mediate the inhibitory and stimulatory effects of GnRH, respectively. Furthermore, nordihydroguaiaretic acid (lipoxygenase inhibitor), but not indomethacin (cyclooxygenase inhibitor), was found to block GnRH- and AA-induced progesterone production in the rat ovary [32]. In contrast to the rat, cyclooxygenase metabolites have been reported to be important for the AA-induced testosterone production in the goldfish ovary [31]. The use of a general inhibitor of AA metabolism, ETYA, reduces the products of cyclooxygenase and lipoxygenase metabolites and could potentially increase the concentration of AA itself. Therefore, the possibility that AA might have played a direct role in reversing the inhibitory actions of sGnRH cannot be ruled out. However, in view of the studies by Van Der Kraak and Chang [31], the sGnRH-mediated inhibitory effect may involve cyclooxygenase metabolites, such as prostaglandins and thromboxanes. The contribution of the PLC/DAG pathway to AA production compared to PLA2 activation is not clear, at present, in the goldfish ovary. However, the observed effect of PMA in reversing the sGnRH-induced inhibition in steroidogenesis may suggest an involvement of PLC/DAG stimulation. Such interactions have been reported previously in the rat ovary [32] and other systems [33].

It is now evident that the dominant role played by GtHs in regulating steroidogenesis in mammals is mediated by more than one intracellular signaling pathway involving cAMP and other intracellular messengers, including those derived from the PLC pathway [11]. Studies in goldfish indicate that, in addition to cAMP pathways [34], PLC and AA metabolism are also involved in the steroidogenic action of GtH [31, 35]. Therefore, the antigonadotropic actions of sGnRH may be due to an interference in the GtH receptor-induced signaling cascade. However, the exact nature of this interference remains to be elucidated. In the present study, PMA was found to significantly inhibit the GtH-induced testosterone production. This is consistent with previous observations in rats [36], chickens [37, 38] and goldfish ovarian follicles [39, 40], in which PMA was found to block the steroid production stimulated by GtH and cAMP agonists without affecting basal steroid production. However, it should be noted that the PKC agonist also resulted in a paradoxical effect on steroidogenesis. In fact, PMA has been shown to stimulate cAMP and steroid production in rats [41], pigs [42], and cows [43]. This finding suggests a modulation of adenylate cyclase activity by PKC, which may occur through one or more sites in the steroidal cascade distal to cAMP generation. In goldfish, PKC acts before the cholesterol side-chain cleavage, because 25-hydroxycholesterol metabolism was not affected by PMA, suggesting a role for PKC in inhibiting cholesterol esterase activity or transport to the side-chain cleavage enzyme [35]. On the other hand, in goldfish, PLC has been reported to be far more effective than PLA2 in the stimulation of testosterone production [31]. Therefore, the inhibitory tone of sGnRH on GtH-induced testosterone production may be through AA metabolism and may play a role as a control mechanism in preovulatory goldfish follicles. It is becoming apparent that such a "check and balance" in reproductive processes is important in seasonal breeders such as goldfish, which possess a relatively homogeneous ovary and undergo ovulation during a narrow window of time in response to environmental cues. Such fine-tuning mechanisms may be possible by cross-communication between multiple signaling pathways by GtH, GnRH, and other gonadal peptides and growth factors [5].

In summary, the present study provides evidence for the involvement of both PKC and AA metabolism in the actions of GnRH in the goldfish ovary. An important finding is that the inhibitory action of sGnRH on oocyte meiosis and steroidogenesis is mediated by AA metabolism, whereas the stimulatory effects of sGnRH and cGnRH-II on oocyte meiosis may be through a PKC pathway.


   FOOTNOTES
 
First decision: 18 September 2001.

1 Supported by a Natural Sciences and Engineering Research Council Research Grant to H.R.H. Back

2 Correspondence: H.R. Habibi, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4. FAX: 403 289 9311; habibi{at}ucalgary.ca Back

3 Current address: Texas Children's Cancer Center, Baylor College of Medicine, 6621 Fannin Street MC 3-3320, Houston, TX 77030-2399 Back

Accepted: October 30, 2001.

Received: August 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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