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Effects of the Isoquinolinesulfonamide H-8 on Fundulus heteroclitus Ovarian Follicles: Role of Cyclic Nucleotide-Dependent Protein Kinases on Steroidogenesis and Oocyte Maturation In Vitro
Comp. Biochem. Physiol. Vol. 117C, No. 1, pp. 75–81, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0742-8413/97/$17.00 PII S0742-8413(96)00236-8
Effects of the Isoquinolinesulfonamide H-8 on Fundulus heteroclitus Ovarian Follicles: Role of Cyclic Nucleotide-Dependent Protein Kinases on Steroidogenesis and Oocyte Maturation In Vitro J. Cerda`,1 T. R. Petrino,2 A. M. Landin,2 and Y-W. P. Lin2 2
1 Whitney Laboratory, 9505 Ocean Shore Boulevard, St. Augustine, Florida 32086, U.S.A. and School of Natural and Health Sciences, Barry University, Miami Shores, Florida 33161, U.S.A.
ABSTRACT. The possible role of cyclic nucleotide-dependent protein kinases in mediating the stimulatory actions of Fundulus heteroclitus pituitary extract (FPE) during ovarian steroidogenesis and oocyte maturation in vitro was investigated. Follicle-enclosed oocytes were cultured in the presence of FPE and/or N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a compound that inhibits protein kinase A (PKA) and cGMP-dependent protein kinase. H-8 alone (0.1–1 mM) promoted oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. However, the process of GVBD initiated by H-8 was much slower than that triggered by 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP), the natural inducer of oocyte maturation in F. heteroclitus. Treatment with H-8 also increased 17,20βP production by the follicles and the accumulation of this steroid in the media was much slower than that initiated by FPE. However, in contrast to the FPE action on the oocyte, which is mediated by 17,20βP, the stimulatory action of H-8 on GVBD appears to be independent of follicular steroid production, since aminoglutethimide (AGI), an inhibitor of steroidogenesis, did not-block H-8-induced GVBD while inhibiting H-8-induced 17,20βP production. Moreover, addition of H-8 to FPE-treated follicles significantly reduced 17,20βP secretion and the percentage of GVBD. These results provide further support for the involvement of PKA in the mechanism by which FPE stimulates ovarian steroidogenesis in F. heteroclitus. Furthermore, the fact that H-8 alone increased 17,20βP levels may imply that basal follicular production of this steroid could be induced by inactivation of cyclic nucleotide-dependent protein kinases. Data also indicate that inhibition of PKA and/or c-GMP-dependent protein kinase in the oocyte may be involved in the mechanism leading to resumption of meiosis in this species. comp biochem physiol 117C;1:75–81, 1997. 1997 Elsevier Science Inc. KEY WORDS. cGMP-dependent protein kinase, Fundulus heteroclitus, gonadotropin, isoquinolinesulfonamide H-8, oocyte maturation, ovarian steroidogenesis, protein kinase A, teleost
INTRODUCTION In lower vertebrates, stimulation of ovarian steroidogenesis by gonadotropic hormones (GtHs) and the subsequent induction of oocyte maturation by steroids involves the binding of the agonists hormones (GtHs to follicular cells and steroid to the oocyte) to membrane receptors, and the activation of signal transduction pathways within target cells. Cumulative evidence further indicates that some of these cellular responses are the result of the interaction between several second messengers and signal-transducing systems through a complex, multi-step mechanism not fully understood. Address reprint requests to: Joan Cerda`, Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel. 149-6221-423512; fax 149-6221-423404; E-mail: [email protected] Received 16 August 1996; accepted 19 November 1996.
In most species, ovarian steroidogenesis induced by GtHs appears to be mediated through a receptor-coupled adenylate cyclase-cAMP system (20,25,28). This transduction system involves 3′,5′-cyclic adenosine monophosphate (cAMP) as the second messenger and regulator of cAMPdependent protein kinase (PKA) activity (19,25). Although the role of cAMP in the GtHs induction of the maturational steroid by the follicular cells is well known, its involvement in mediating the steroid action on the oocyte is still controversial. Other intracellular signalling molecules, such as Ca 21-calmodulin and 1,2-diacylglycerol, that activate protein kinase C (PKC) have also been implicated in amphibian steroidogenesis, as well as in the initial response of the oocyte to agonists that induce germinal vesicle breakdown (GVBD), an easily observed event during the process of oocyte maturation in some species (5,17,18,24). In this regard, most studies have indicated that agents that increase intracellular levels of cAMP in the oocyte block
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steroid-induced GVBD (4,6,14,15,21,30). In amphibians, it has been postulated that progesterone, the maturational steroid, stimulates GVBD by decreasing cAMP levels in the oocyte, which could lead to a decrease in PKA activity, thus resulting in the dephosphorylation of a putative maturationinhibiting phosphoprotein [for review see (3,33)]. However, experimental data confirming or rejecting this hypothesis are still scarce. Much less work has been done on the signal transduction pathways operating in the ovary of teleosts (7). Previous studies in several species of fish, including F. heteroclitus, have indicated that GtHs induce steroidogenesis in the follicular cells by a cAMP-mediated pathway (16,30,38). In contrast, a fall in the oocyte cAMP level seems to be associated with steroid-induced meiotic maturation (6,11,15). Most of these studies implicating the cAMP/ PKA transduction pathway in the ovary have used exogenous cAMP or agents known to elevate intracellular levels of this second messenger, but data on the role of PKA bypassing its regulation by cAMP are missing. Previously, we have reported that while activation of PKC is inhibitory to steroid production, it has a marked stimulatory action on oocyte GVBD in F. heteroclitus (31). However, the possible modulatory role(s) of other protein kinases, particularly on oocyte maturation, are unknown in this species. In the present study, we have explored the role of cyclic nucleotide-dependent protein kinases on both F. heteroclitus ovarian steroidogenesis and oocyte maturation by using N[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a potent inhibitor of PKA and 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (9,12). MATERIAL AND METHODS Animals, Chemicals, and Hormones Female F. heteroclitus were collected from salt marshes in the vicinity of the Whitney Laboratory near St. Augustine, FL. Animals were maintained reproductively active in the laboratory for 2–3 months as described by Lin et al. (23). N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8) was obtained from Research Biochemicals International (RBI, Natick, MA, USA). H-8 was dissolved in distilled water (5 mg ml21), stored in aliquots at 220°C and added to the media in 6.7 µl water vehicle. Other biochemicals and hormones were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.), unless indicated otherwise. dl-Aminoglutethimide (AGI) was dissolved in dimethyl sulfoxide and added to the media in 10 µl vehicle. The stock solution of 17α,20β-dihydroxy-4-prenen-3-one (17,20βP), the natural maturation-inducing steroid (MIS) in F. heteroclitus (32), was prepared in 95% ethanol, stored at 220°C and added to the culture medium in 5 µl of ethanol vehicle (final concentration of ethanol was 0.5%). The effect of GtH was tested by using active F. heteroclitus pituitary extract (FPE) prepared at a concentration
of 10 pituitary equivalents (PEq) per ml and stored at 220°C until use (22). Culture of Ovarian Follicles On days prior to spawning, between 9:00 and 13:00 hr, 10– 15 females were removed from the tanks and decapitated. The ovaries were immediately removed and placed in 35 3 10 mm Petri dishes (Falcon 1008 Easy Grip) containing approximately 6 ml of 75% L-15 Leivovitz culture medium with L-glutamine and 100 µg ml21 gentamicin and adjusted to pH 7.5 with HCl. Intact ovarian follicles were obtained according to protocol previously reported (8). Full-grown prematurational follicles (1.25–1.45 mm in diameter) were manually isolated from several ovaries, pooled, and randomly distributed into various treatment groups for each experiment. Follicle culture was conducted in 24-well tissue culture trays (Falcon 3043), with 15–20 follicles in 1 ml medium per well, in a humidified, temperature-controlled incubator at 25°C. Based on previous data for F. heteroclitus (22,29), medium was collected at several times during culture and stored at 220°C prior to steroid determination by radioimmunoassay (RIA). Oocyte maturation was monitored at approximately 12-hr intervals up to 72 hr of culture by scoring germinal vesicle breakdown (GVBD), which acts as an indication of the reinitiation of meiosis (34), using an Olympus stereomicroscope. In experiments directed to assess the effect of 17,20βP and H-8 on oocyte hydration, follicles were cultured individually and the diameter of each follicle was measured to the nearest 0.02 mm at time 0 and at time of complete maturation, i.e., at 30 and 72 hr, respectively. Follicle diameter was used to calculate the volume of the follicle with the formula 4/3π r 3.
Steroid RIA Aliquots of culture medium were directly assayed for estradiol-17β (E2) and 17,20βP as previously described (22). Most radiochemicals used as tracers were obtained from New England Nuclear (Boston, MA). Antiserum against 17,20βP was a gift from Dr. Y. Nagahama (National Institute for Basic Biology, Okazaki, Japan) while that against E2 was obtained from Wien Laboratories Inc. Details for the cross-reactivities of both antisera with a variety of relevant steroids have been previously described (29).
Statistics Results are presented as mean 6 standard error (SEM) of two or three experiments performed on different batches of fish. Statistical analysis of data consisted of one- or two-way analysis of variance (ANOVA I or ANOVA II) followed by
H-8 Effects on Fundulus Ovarian Follicles
FIG. 1. Effect of various doses of H-8 on F. heteroclitus oo-
cyte maturation, as measured by percentage of GVBD during culture. Intact follicles were exposed to 5 ml 95% ethanol vehicle (Control), 0.1 mg ml21 17,20bP or various doses of H-8 (0.01, 0.1 and 1 mM) for up to 66 hr. Data are mean 6 SEM from three experiments using different batches of fish, where each treatment was performed in triplicate (15– 20 follicles per treatment).
Duncan’s multiple range test. Differences were considered significant at P # 0.05. RESULTS Effects of the Protein Kinase Inhibitor H-8 on Prematurational Ovarian Follicles In order to evaluate the role of PKA, if any, during the process of oocyte maturation, we have investigated the effect of various doses of the compound H-8, a potent synthetic inhibitor of this enzyme (12), on prematurational follicle-enclosed oocytes. The competence of the oocyte to undergo GVBD was tested using exogenously added 17,20βP, the physiological inducer of oocyte maturation in F. heteroclitus (32). Results presented in Fig. 1 indicate that H-8 (0.1 and 1 mM) alone was able to trigger GVBD. Although the percentage of follicle-enclosed oocytes that underwent maturation under H-8 treatment (95.5 6 1.3%) was similar to that initiated by 17,20βP (87.2 6 7.9%), the time course of GVBD induced by the protein kinase inhibitor (66 hr) lagged several hours behind the follicular response to the steroid (32 hr). It should be noted that during oocyte maturation H-8-treated follicles displayed cytoplasmic events, such as lipid coalescence and clearing of the ooplasm (35), similar to those stimulated by the steroid. However, the increase in oocyte volume, which is due to the hydration that normally accompanies GVBD in F. heteroclitus (35), appeared to be less than usual. To confirm this observation, further experiments were conducted in
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FIG. 2. Follicle-enclosed oocyte volume change during 17,20bP (0.1 mg ml21 0.1 mg ml21) or H-8 (0.1 mM) induction of GVBD. Bar indicates mean 6 SEM of a total of 48 replicate follicle measurements. In each experiment 24 follicles per treatment were cultured individually. Both 17,20bP and H-8-treated follicles underwent 90%–95% maturation at 30 hr and 72 hr of culture, respectively, whereas control follicles did not undergo GVBD. Different letters above the bar graph denote significant differences between treatments (P # 0.05).
which 24 intact follicles were treated with 5 µl 95% ethanol (Control), 0.1 µg ml21 17,20βP, or 0.1 mM H-8 and cultured individually. Thus, the diameter of each follicle was measured at 0 hr and at the times when complete GVBD induced by 17,20βP or H-8 was observed (approximately at 32 and 72 hr of culture, respectively). Figure 2 depicts the results from these experiments and shows that the volume increase of the oocyte in H-8 treated follicles was significantly lower when compared with that of 17,20βP-treated follicles. Effects of H-8 on FPE-Induced Oocyte Maturation and Steroid Secretion FPE stimulation of GVBD in F. heteroclitus is dependent on the synthesis of 17,20βP via an elevation of intracellular cAMP levels in the follicle (granulosa) cells (30,32). Experiments were conducted to test whether inhibition of cyclic nucleotide-dependent protein kinases by H-8 affects the follicular response to the hormonal action. Addition of FPE to follicles cultured in vitro increased media concentration of 17,20βP, and E2 (Fig. 3b,c) and promoted GVBD (Fig. 3a). As the previous experiments indicated, H-8 alone caused GVBD (Fig. 3a) and also progressively elevated 17,20βP levels (Fig. 3b), but in contrast to FPE, it did not affect E2 levels (Fig. 3c). Interestingly, when H-8 was combined with FPE, the percentage of GVBD was considerably lower at earlier times compared with the percentage of
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GVBD induced by either treatment alone (Fig. 3a). Moreover, the low percentage of GVBD appeared to correlate with lower levels of media 17,20βP found in the group treated with FPE plus H-8 at early times (24–40 hr of culture), compared to 17,20βP levels induced by FPE alone (Fig. 3b). In addition, H-8 caused total inhibition of FPE induced E2 levels (Fig. 3c). These experiments indicate that H-8 was as effective as FPE in promoting GVBD and also able to continuously increase 17,20βP concentrations in the media over a period of 71 hr of culture. Therefore, the maturation-inducing activity of H-8 could be mediated by the 17,20βP action on the oocyte rather than by a direct stimulatory effect of H8 on the oocyte. To test this hypothesis, we used AGI, an inhibitor of cholesterol side-chain cleavage (10), known to abolish FPE-induced 17,20βP accumulation and GVBD in F. heteroclitus (29). Results in Fig. 4 show that AGI inhibited FPE-induced 17,20βP secretion, although not to basal levels (Fig. 4b), and thus markedly blocked oocyte maturation (Fig. 4a). Similarly, AGI effectively prevented H-8induced 17,20βP up to levels comparable to those found in the control group (Fig. 4b), but it did not inhibit H-8-induced GVBD (Fig. 4a). These results indicate that the action of H-8 is not mediated by the steroid, and that H-8 could, in fact, act directly on the oocyte where it inhibits the nucleotide-dependent protein kinases. Inhibition of these kinases seems to induce GVBD. DISCUSSION In this study, the role of cyclic nucleotide-dependent protein kinases on FPE-induced ovarian steroidogenesis and oocyte maturation have been investigated by using H-8, the isoquinolinesulfonamide derivative that binds with high affinity to the catalytic subunit of PKA and cGMP-dependent protein kinase, thus preventing substrate phosphorylation mediated by these enzymes (9,12). This compound has proved to be a useful tool to study agonist-activated intracellular pathways in vertebrate cells (13,27). In previous studies, we have demonstrated that unstimulated follicles of F. heteroclitus possess all the enzymes necessary for the metabolism of cholesterol to 17,20βP and E2; however, unstimulated follicles cultured in vitro exhibit no
FIG. 3. Effect of FPE and/or H-8 on GVBD and follicular steroids. Intact follicles were incubated in presence of 5 ml of 95% ethanol vehicle (Control), FPE (0.5 PEq ml21), H-8 (0.1 mM) or FPE plus H-8 for up to 71 hr. Data on GVBD (a), 17,20bP (b) and E2 (c) are the mean 6 SEM from three experiments (20 follicles per treatment) using different groups of fish. Means with different letters are significantly different ( P # 0.05) for a given time point. Asterisk denotes statistical differences ( P # 0.01) between the FPE group and the other groups. nd: not detectable.
H-8 Effects on Fundulus Ovarian Follicles
FIG. 4. Effect of DL-Aminoglutethimide (AGI) on FPE- and
H-8-induced GVBD (a) and 17,20bP accumulation (b). Intact follicles were incubated with or without AGI (50 mg ml21) for 1 hr prior to the addition of 5 ml of 95% ethanol vehicle (Control), FPE (0.5 PEq ml21) or H-8 (0.1 mM), and cultured for a period of 72 hr. GVBD was scored at different times during culture and media were collected at 24 and 72 hr for steroid determination. Values are mean 6 SEM from three experiments (20 follicles per treatment) using different batches of fish. Different letters indicate that treatments are significantly ( P # 0.05) different for a given time point.
detectable level of 17,20βP but low levels of E2 (basal steroidogenesis) (29). Addition of FPE stimulates 17,20βP and E2 by acting at a step prior to the conversion of cholesterol to pregnenolone (possibly cholesterol mobilization and/or transport); and FPE particularly enhances aromatase activity through a cAMP and protein synthesis dependent mechanism (29,30). The increase in cAMP in the follicle (granu-
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losa) cells caused by gonadotropins has normally been associated with activation of PKA. This study presents evidence indicating that, in effect, FPE action on steroid production is mediated through activation of PKA, since direct inhibition of this enzyme by H-8, thus bypassing any cAMP effect, considerably reduced FPE-induced 17,20βP levels and totally abolished E2 stimulation (Fig. 3). Interestingly, H-8 had a differential effect on basal steroidogenesis: it continuously increased 17,20βP while it had no effect on E2 levels (Fig. 3). Thus, it appears that activation of the transduction pathway cAMP/PKA during FPE stimulation rapidly increases 17,20βP and E2 levels, while inactivation of PKA and/or cGMP-dependent protein kinase (caused by H-8) results in a slower increase in 17,20βP levels (Fig. 3). This fact suggests that basal follicular production of 17,20βP could be induced by inactivation of cyclic nucleotide-dependent protein kinases in contrast to the FPE stimulation of this steroid that requires activation of PKA. This would explain the partial inhibition on FPE-induced 17,20βP levels caused by H-8 (Fig. 3). In addition, the increase in 17,20βP levels caused by H-8 alone appears to depend upon the activity of the cholesterol side-chain cleavage enzyme, since inhibition of this enzyme by AGI effectively blocked H-8 induction of 17,20βP (Fig. 4). On the other hand, E2 synthesis appeared to be only mediated through a cAMP/ PKA transduction pathway. These results are in agreement with our previous finding that FPE stimulates E2 synthesis not only by increasing the availability of cholesterol but by inducing de novo synthesis of the aromatase enzyme through a cAMP-dependent mechanism (30). We have also previously demonstrated in F. heteroclitus that activation of PKC has an inhibitory effect on FPE-induced ovarian steroidogenesis but a stimulatory effect on oocyte maturation, respectively (31). Results from the present study suggest an inverse scenario for cyclic mucleotidedependent protein kinases in this species. As previously discussed, activation of PKA is part of the mechanism by which FPE induces steroid production by the follicular cells, while inhibition of this kinase appears to bring along GVBD in the oocyte. In effect, H-8 alone promoted GVBD of intact follicles as efficiently as 17,20βP, although its action was much slower than that of the steroid (Fig. 1). The direct action of H-8 on the oocyte GVBD was demonstrated in experiments using AGI, which while blocking 17,20βP synthesis, did not prevent H-8-induced GVBD (Fig. 4a,b). Evidence for a decrease in PKA activity (24) and no change in PKA activity (2) during oocyte maturation has been reported in amphibians. Results from this study show that in F. heteroclitus direct inhibition of cyclic nucleotide-dependent protein kinases by H-8, can induced GVBD (Fig. 1). Although a reduction of cAMP levels after steroid-induced maturation is unknown in this species, it has been shown in other teleosts (6,11,15). Recent work by Yoshikuni and Nagahama (37), using rainbow trout oocytes, has shown that 17,20βP, probably through activation of an inhibitory
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G-protein, decreases adenylate cyclase activity leading to a fall in intracellular cAMP levels. The drop in cAMP caused by 17,20βP is believed to reduce PKA activity, which would negatively regulate meiotic maturation at several points [e.g., preventing Mos translation and the activation of the maturation promoting factor (24,26)]. Taken together, the available information suggests that in teleost, a decrease in PKA might occur during 17,20βP-induced oocyte maturation. In addition, activation of PKC in the oocyte may also serve as a signal-transducing mechanism leading to GVBD (31). These data support the view that extensive cross-talk between the PKC and PKA pathways, as postulated for amphibians (33), could take place during maturation of fish oocytes. Although H-8 alone can induce GVBD (Fig. 1), it cannot completely mimic the steroid action on the oocyte. For instance, the process was much slower than when it was triggered by 17,20βP. Another difference we have noticed is that in H-8 treated follicles, the oocyte experienced a lesser increase in volume than under 17,20βP treatment (Fig. 2). F. heteroclitus oocytes undergo a significant volume increase due to hydration during meiotic maturation, which is dependent of external K 1 (the major cause of osmotically obligated water uptake into the oocyte) that appears to be translocated to the maturing oocyte via heterologous gap junctions between the follicle cells and the oocyte (1,36). Interestingly, activators of PKC, such as 12-myristate 13acetate (PMA), which induces oocyte maturation, also decrease hydration, probably by disrupting gap junctional communication (1). Whether H-8 has a similar effect on gap junctional communication between the oocyte and the follicle cells is not yet known. In summary, this study presents evidence showing that cyclic nucleotide-dependent protein kinases serve as intracellular signaling molecules during the FPE induction of steroid synthesis by the follicle cells. In addition, it shows that inhibition of these kinases was sufficient to induce GVBD in the oocyte, thus suggesting that PKA and/or cGMP-dependent protein kinase might play a part in the mechanism, yet unknown, leading to meiotic arrest of F. heteroclitus oocytes.
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This work was supported by a National Science Foundation Grant IBN-9306123 awarded to Dr. Robin A. Wallace, NIH-NIGMS MBRS (GM45455-D5) Grant awarded to Y.-W. P. L., and MARC (GM08021-14) Grant to Barry University. Participation of JC was financed by a postdoctoral fellowship from the Ministry of Education and Science (Spain). The authors acknowledge the editorial assistance of Dr. Dorothy M. Jehle, O.P. in the preparation of this manuscript.
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ISSN 0742-8413/97/$17.00 PII S0742-8413(96)00236-8
Effects of the Isoquinolinesulfonamide H-8 on Fundulus heteroclitus Ovarian Follicles: Role of Cyclic Nucleotide-Dependent Protein Kinases on Steroidogenesis and Oocyte Maturation In Vitro J. Cerda`,1 T. R. Petrino,2 A. M. Landin,2 and Y-W. P. Lin2 2
1 Whitney Laboratory, 9505 Ocean Shore Boulevard, St. Augustine, Florida 32086, U.S.A. and School of Natural and Health Sciences, Barry University, Miami Shores, Florida 33161, U.S.A.
ABSTRACT. The possible role of cyclic nucleotide-dependent protein kinases in mediating the stimulatory actions of Fundulus heteroclitus pituitary extract (FPE) during ovarian steroidogenesis and oocyte maturation in vitro was investigated. Follicle-enclosed oocytes were cultured in the presence of FPE and/or N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a compound that inhibits protein kinase A (PKA) and cGMP-dependent protein kinase. H-8 alone (0.1–1 mM) promoted oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. However, the process of GVBD initiated by H-8 was much slower than that triggered by 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP), the natural inducer of oocyte maturation in F. heteroclitus. Treatment with H-8 also increased 17,20βP production by the follicles and the accumulation of this steroid in the media was much slower than that initiated by FPE. However, in contrast to the FPE action on the oocyte, which is mediated by 17,20βP, the stimulatory action of H-8 on GVBD appears to be independent of follicular steroid production, since aminoglutethimide (AGI), an inhibitor of steroidogenesis, did not-block H-8-induced GVBD while inhibiting H-8-induced 17,20βP production. Moreover, addition of H-8 to FPE-treated follicles significantly reduced 17,20βP secretion and the percentage of GVBD. These results provide further support for the involvement of PKA in the mechanism by which FPE stimulates ovarian steroidogenesis in F. heteroclitus. Furthermore, the fact that H-8 alone increased 17,20βP levels may imply that basal follicular production of this steroid could be induced by inactivation of cyclic nucleotide-dependent protein kinases. Data also indicate that inhibition of PKA and/or c-GMP-dependent protein kinase in the oocyte may be involved in the mechanism leading to resumption of meiosis in this species. comp biochem physiol 117C;1:75–81, 1997. 1997 Elsevier Science Inc. KEY WORDS. cGMP-dependent protein kinase, Fundulus heteroclitus, gonadotropin, isoquinolinesulfonamide H-8, oocyte maturation, ovarian steroidogenesis, protein kinase A, teleost
INTRODUCTION In lower vertebrates, stimulation of ovarian steroidogenesis by gonadotropic hormones (GtHs) and the subsequent induction of oocyte maturation by steroids involves the binding of the agonists hormones (GtHs to follicular cells and steroid to the oocyte) to membrane receptors, and the activation of signal transduction pathways within target cells. Cumulative evidence further indicates that some of these cellular responses are the result of the interaction between several second messengers and signal-transducing systems through a complex, multi-step mechanism not fully understood. Address reprint requests to: Joan Cerda`, Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel. 149-6221-423512; fax 149-6221-423404; E-mail: [email protected] Received 16 August 1996; accepted 19 November 1996.
In most species, ovarian steroidogenesis induced by GtHs appears to be mediated through a receptor-coupled adenylate cyclase-cAMP system (20,25,28). This transduction system involves 3′,5′-cyclic adenosine monophosphate (cAMP) as the second messenger and regulator of cAMPdependent protein kinase (PKA) activity (19,25). Although the role of cAMP in the GtHs induction of the maturational steroid by the follicular cells is well known, its involvement in mediating the steroid action on the oocyte is still controversial. Other intracellular signalling molecules, such as Ca 21-calmodulin and 1,2-diacylglycerol, that activate protein kinase C (PKC) have also been implicated in amphibian steroidogenesis, as well as in the initial response of the oocyte to agonists that induce germinal vesicle breakdown (GVBD), an easily observed event during the process of oocyte maturation in some species (5,17,18,24). In this regard, most studies have indicated that agents that increase intracellular levels of cAMP in the oocyte block
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steroid-induced GVBD (4,6,14,15,21,30). In amphibians, it has been postulated that progesterone, the maturational steroid, stimulates GVBD by decreasing cAMP levels in the oocyte, which could lead to a decrease in PKA activity, thus resulting in the dephosphorylation of a putative maturationinhibiting phosphoprotein [for review see (3,33)]. However, experimental data confirming or rejecting this hypothesis are still scarce. Much less work has been done on the signal transduction pathways operating in the ovary of teleosts (7). Previous studies in several species of fish, including F. heteroclitus, have indicated that GtHs induce steroidogenesis in the follicular cells by a cAMP-mediated pathway (16,30,38). In contrast, a fall in the oocyte cAMP level seems to be associated with steroid-induced meiotic maturation (6,11,15). Most of these studies implicating the cAMP/ PKA transduction pathway in the ovary have used exogenous cAMP or agents known to elevate intracellular levels of this second messenger, but data on the role of PKA bypassing its regulation by cAMP are missing. Previously, we have reported that while activation of PKC is inhibitory to steroid production, it has a marked stimulatory action on oocyte GVBD in F. heteroclitus (31). However, the possible modulatory role(s) of other protein kinases, particularly on oocyte maturation, are unknown in this species. In the present study, we have explored the role of cyclic nucleotide-dependent protein kinases on both F. heteroclitus ovarian steroidogenesis and oocyte maturation by using N[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a potent inhibitor of PKA and 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (9,12). MATERIAL AND METHODS Animals, Chemicals, and Hormones Female F. heteroclitus were collected from salt marshes in the vicinity of the Whitney Laboratory near St. Augustine, FL. Animals were maintained reproductively active in the laboratory for 2–3 months as described by Lin et al. (23). N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8) was obtained from Research Biochemicals International (RBI, Natick, MA, USA). H-8 was dissolved in distilled water (5 mg ml21), stored in aliquots at 220°C and added to the media in 6.7 µl water vehicle. Other biochemicals and hormones were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.), unless indicated otherwise. dl-Aminoglutethimide (AGI) was dissolved in dimethyl sulfoxide and added to the media in 10 µl vehicle. The stock solution of 17α,20β-dihydroxy-4-prenen-3-one (17,20βP), the natural maturation-inducing steroid (MIS) in F. heteroclitus (32), was prepared in 95% ethanol, stored at 220°C and added to the culture medium in 5 µl of ethanol vehicle (final concentration of ethanol was 0.5%). The effect of GtH was tested by using active F. heteroclitus pituitary extract (FPE) prepared at a concentration
of 10 pituitary equivalents (PEq) per ml and stored at 220°C until use (22). Culture of Ovarian Follicles On days prior to spawning, between 9:00 and 13:00 hr, 10– 15 females were removed from the tanks and decapitated. The ovaries were immediately removed and placed in 35 3 10 mm Petri dishes (Falcon 1008 Easy Grip) containing approximately 6 ml of 75% L-15 Leivovitz culture medium with L-glutamine and 100 µg ml21 gentamicin and adjusted to pH 7.5 with HCl. Intact ovarian follicles were obtained according to protocol previously reported (8). Full-grown prematurational follicles (1.25–1.45 mm in diameter) were manually isolated from several ovaries, pooled, and randomly distributed into various treatment groups for each experiment. Follicle culture was conducted in 24-well tissue culture trays (Falcon 3043), with 15–20 follicles in 1 ml medium per well, in a humidified, temperature-controlled incubator at 25°C. Based on previous data for F. heteroclitus (22,29), medium was collected at several times during culture and stored at 220°C prior to steroid determination by radioimmunoassay (RIA). Oocyte maturation was monitored at approximately 12-hr intervals up to 72 hr of culture by scoring germinal vesicle breakdown (GVBD), which acts as an indication of the reinitiation of meiosis (34), using an Olympus stereomicroscope. In experiments directed to assess the effect of 17,20βP and H-8 on oocyte hydration, follicles were cultured individually and the diameter of each follicle was measured to the nearest 0.02 mm at time 0 and at time of complete maturation, i.e., at 30 and 72 hr, respectively. Follicle diameter was used to calculate the volume of the follicle with the formula 4/3π r 3.
Steroid RIA Aliquots of culture medium were directly assayed for estradiol-17β (E2) and 17,20βP as previously described (22). Most radiochemicals used as tracers were obtained from New England Nuclear (Boston, MA). Antiserum against 17,20βP was a gift from Dr. Y. Nagahama (National Institute for Basic Biology, Okazaki, Japan) while that against E2 was obtained from Wien Laboratories Inc. Details for the cross-reactivities of both antisera with a variety of relevant steroids have been previously described (29).
Statistics Results are presented as mean 6 standard error (SEM) of two or three experiments performed on different batches of fish. Statistical analysis of data consisted of one- or two-way analysis of variance (ANOVA I or ANOVA II) followed by
H-8 Effects on Fundulus Ovarian Follicles
FIG. 1. Effect of various doses of H-8 on F. heteroclitus oo-
cyte maturation, as measured by percentage of GVBD during culture. Intact follicles were exposed to 5 ml 95% ethanol vehicle (Control), 0.1 mg ml21 17,20bP or various doses of H-8 (0.01, 0.1 and 1 mM) for up to 66 hr. Data are mean 6 SEM from three experiments using different batches of fish, where each treatment was performed in triplicate (15– 20 follicles per treatment).
Duncan’s multiple range test. Differences were considered significant at P # 0.05. RESULTS Effects of the Protein Kinase Inhibitor H-8 on Prematurational Ovarian Follicles In order to evaluate the role of PKA, if any, during the process of oocyte maturation, we have investigated the effect of various doses of the compound H-8, a potent synthetic inhibitor of this enzyme (12), on prematurational follicle-enclosed oocytes. The competence of the oocyte to undergo GVBD was tested using exogenously added 17,20βP, the physiological inducer of oocyte maturation in F. heteroclitus (32). Results presented in Fig. 1 indicate that H-8 (0.1 and 1 mM) alone was able to trigger GVBD. Although the percentage of follicle-enclosed oocytes that underwent maturation under H-8 treatment (95.5 6 1.3%) was similar to that initiated by 17,20βP (87.2 6 7.9%), the time course of GVBD induced by the protein kinase inhibitor (66 hr) lagged several hours behind the follicular response to the steroid (32 hr). It should be noted that during oocyte maturation H-8-treated follicles displayed cytoplasmic events, such as lipid coalescence and clearing of the ooplasm (35), similar to those stimulated by the steroid. However, the increase in oocyte volume, which is due to the hydration that normally accompanies GVBD in F. heteroclitus (35), appeared to be less than usual. To confirm this observation, further experiments were conducted in
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FIG. 2. Follicle-enclosed oocyte volume change during 17,20bP (0.1 mg ml21 0.1 mg ml21) or H-8 (0.1 mM) induction of GVBD. Bar indicates mean 6 SEM of a total of 48 replicate follicle measurements. In each experiment 24 follicles per treatment were cultured individually. Both 17,20bP and H-8-treated follicles underwent 90%–95% maturation at 30 hr and 72 hr of culture, respectively, whereas control follicles did not undergo GVBD. Different letters above the bar graph denote significant differences between treatments (P # 0.05).
which 24 intact follicles were treated with 5 µl 95% ethanol (Control), 0.1 µg ml21 17,20βP, or 0.1 mM H-8 and cultured individually. Thus, the diameter of each follicle was measured at 0 hr and at the times when complete GVBD induced by 17,20βP or H-8 was observed (approximately at 32 and 72 hr of culture, respectively). Figure 2 depicts the results from these experiments and shows that the volume increase of the oocyte in H-8 treated follicles was significantly lower when compared with that of 17,20βP-treated follicles. Effects of H-8 on FPE-Induced Oocyte Maturation and Steroid Secretion FPE stimulation of GVBD in F. heteroclitus is dependent on the synthesis of 17,20βP via an elevation of intracellular cAMP levels in the follicle (granulosa) cells (30,32). Experiments were conducted to test whether inhibition of cyclic nucleotide-dependent protein kinases by H-8 affects the follicular response to the hormonal action. Addition of FPE to follicles cultured in vitro increased media concentration of 17,20βP, and E2 (Fig. 3b,c) and promoted GVBD (Fig. 3a). As the previous experiments indicated, H-8 alone caused GVBD (Fig. 3a) and also progressively elevated 17,20βP levels (Fig. 3b), but in contrast to FPE, it did not affect E2 levels (Fig. 3c). Interestingly, when H-8 was combined with FPE, the percentage of GVBD was considerably lower at earlier times compared with the percentage of
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GVBD induced by either treatment alone (Fig. 3a). Moreover, the low percentage of GVBD appeared to correlate with lower levels of media 17,20βP found in the group treated with FPE plus H-8 at early times (24–40 hr of culture), compared to 17,20βP levels induced by FPE alone (Fig. 3b). In addition, H-8 caused total inhibition of FPE induced E2 levels (Fig. 3c). These experiments indicate that H-8 was as effective as FPE in promoting GVBD and also able to continuously increase 17,20βP concentrations in the media over a period of 71 hr of culture. Therefore, the maturation-inducing activity of H-8 could be mediated by the 17,20βP action on the oocyte rather than by a direct stimulatory effect of H8 on the oocyte. To test this hypothesis, we used AGI, an inhibitor of cholesterol side-chain cleavage (10), known to abolish FPE-induced 17,20βP accumulation and GVBD in F. heteroclitus (29). Results in Fig. 4 show that AGI inhibited FPE-induced 17,20βP secretion, although not to basal levels (Fig. 4b), and thus markedly blocked oocyte maturation (Fig. 4a). Similarly, AGI effectively prevented H-8induced 17,20βP up to levels comparable to those found in the control group (Fig. 4b), but it did not inhibit H-8-induced GVBD (Fig. 4a). These results indicate that the action of H-8 is not mediated by the steroid, and that H-8 could, in fact, act directly on the oocyte where it inhibits the nucleotide-dependent protein kinases. Inhibition of these kinases seems to induce GVBD. DISCUSSION In this study, the role of cyclic nucleotide-dependent protein kinases on FPE-induced ovarian steroidogenesis and oocyte maturation have been investigated by using H-8, the isoquinolinesulfonamide derivative that binds with high affinity to the catalytic subunit of PKA and cGMP-dependent protein kinase, thus preventing substrate phosphorylation mediated by these enzymes (9,12). This compound has proved to be a useful tool to study agonist-activated intracellular pathways in vertebrate cells (13,27). In previous studies, we have demonstrated that unstimulated follicles of F. heteroclitus possess all the enzymes necessary for the metabolism of cholesterol to 17,20βP and E2; however, unstimulated follicles cultured in vitro exhibit no
FIG. 3. Effect of FPE and/or H-8 on GVBD and follicular steroids. Intact follicles were incubated in presence of 5 ml of 95% ethanol vehicle (Control), FPE (0.5 PEq ml21), H-8 (0.1 mM) or FPE plus H-8 for up to 71 hr. Data on GVBD (a), 17,20bP (b) and E2 (c) are the mean 6 SEM from three experiments (20 follicles per treatment) using different groups of fish. Means with different letters are significantly different ( P # 0.05) for a given time point. Asterisk denotes statistical differences ( P # 0.01) between the FPE group and the other groups. nd: not detectable.
H-8 Effects on Fundulus Ovarian Follicles
FIG. 4. Effect of DL-Aminoglutethimide (AGI) on FPE- and
H-8-induced GVBD (a) and 17,20bP accumulation (b). Intact follicles were incubated with or without AGI (50 mg ml21) for 1 hr prior to the addition of 5 ml of 95% ethanol vehicle (Control), FPE (0.5 PEq ml21) or H-8 (0.1 mM), and cultured for a period of 72 hr. GVBD was scored at different times during culture and media were collected at 24 and 72 hr for steroid determination. Values are mean 6 SEM from three experiments (20 follicles per treatment) using different batches of fish. Different letters indicate that treatments are significantly ( P # 0.05) different for a given time point.
detectable level of 17,20βP but low levels of E2 (basal steroidogenesis) (29). Addition of FPE stimulates 17,20βP and E2 by acting at a step prior to the conversion of cholesterol to pregnenolone (possibly cholesterol mobilization and/or transport); and FPE particularly enhances aromatase activity through a cAMP and protein synthesis dependent mechanism (29,30). The increase in cAMP in the follicle (granu-
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losa) cells caused by gonadotropins has normally been associated with activation of PKA. This study presents evidence indicating that, in effect, FPE action on steroid production is mediated through activation of PKA, since direct inhibition of this enzyme by H-8, thus bypassing any cAMP effect, considerably reduced FPE-induced 17,20βP levels and totally abolished E2 stimulation (Fig. 3). Interestingly, H-8 had a differential effect on basal steroidogenesis: it continuously increased 17,20βP while it had no effect on E2 levels (Fig. 3). Thus, it appears that activation of the transduction pathway cAMP/PKA during FPE stimulation rapidly increases 17,20βP and E2 levels, while inactivation of PKA and/or cGMP-dependent protein kinase (caused by H-8) results in a slower increase in 17,20βP levels (Fig. 3). This fact suggests that basal follicular production of 17,20βP could be induced by inactivation of cyclic nucleotide-dependent protein kinases in contrast to the FPE stimulation of this steroid that requires activation of PKA. This would explain the partial inhibition on FPE-induced 17,20βP levels caused by H-8 (Fig. 3). In addition, the increase in 17,20βP levels caused by H-8 alone appears to depend upon the activity of the cholesterol side-chain cleavage enzyme, since inhibition of this enzyme by AGI effectively blocked H-8 induction of 17,20βP (Fig. 4). On the other hand, E2 synthesis appeared to be only mediated through a cAMP/ PKA transduction pathway. These results are in agreement with our previous finding that FPE stimulates E2 synthesis not only by increasing the availability of cholesterol but by inducing de novo synthesis of the aromatase enzyme through a cAMP-dependent mechanism (30). We have also previously demonstrated in F. heteroclitus that activation of PKC has an inhibitory effect on FPE-induced ovarian steroidogenesis but a stimulatory effect on oocyte maturation, respectively (31). Results from the present study suggest an inverse scenario for cyclic mucleotidedependent protein kinases in this species. As previously discussed, activation of PKA is part of the mechanism by which FPE induces steroid production by the follicular cells, while inhibition of this kinase appears to bring along GVBD in the oocyte. In effect, H-8 alone promoted GVBD of intact follicles as efficiently as 17,20βP, although its action was much slower than that of the steroid (Fig. 1). The direct action of H-8 on the oocyte GVBD was demonstrated in experiments using AGI, which while blocking 17,20βP synthesis, did not prevent H-8-induced GVBD (Fig. 4a,b). Evidence for a decrease in PKA activity (24) and no change in PKA activity (2) during oocyte maturation has been reported in amphibians. Results from this study show that in F. heteroclitus direct inhibition of cyclic nucleotide-dependent protein kinases by H-8, can induced GVBD (Fig. 1). Although a reduction of cAMP levels after steroid-induced maturation is unknown in this species, it has been shown in other teleosts (6,11,15). Recent work by Yoshikuni and Nagahama (37), using rainbow trout oocytes, has shown that 17,20βP, probably through activation of an inhibitory
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G-protein, decreases adenylate cyclase activity leading to a fall in intracellular cAMP levels. The drop in cAMP caused by 17,20βP is believed to reduce PKA activity, which would negatively regulate meiotic maturation at several points [e.g., preventing Mos translation and the activation of the maturation promoting factor (24,26)]. Taken together, the available information suggests that in teleost, a decrease in PKA might occur during 17,20βP-induced oocyte maturation. In addition, activation of PKC in the oocyte may also serve as a signal-transducing mechanism leading to GVBD (31). These data support the view that extensive cross-talk between the PKC and PKA pathways, as postulated for amphibians (33), could take place during maturation of fish oocytes. Although H-8 alone can induce GVBD (Fig. 1), it cannot completely mimic the steroid action on the oocyte. For instance, the process was much slower than when it was triggered by 17,20βP. Another difference we have noticed is that in H-8 treated follicles, the oocyte experienced a lesser increase in volume than under 17,20βP treatment (Fig. 2). F. heteroclitus oocytes undergo a significant volume increase due to hydration during meiotic maturation, which is dependent of external K 1 (the major cause of osmotically obligated water uptake into the oocyte) that appears to be translocated to the maturing oocyte via heterologous gap junctions between the follicle cells and the oocyte (1,36). Interestingly, activators of PKC, such as 12-myristate 13acetate (PMA), which induces oocyte maturation, also decrease hydration, probably by disrupting gap junctional communication (1). Whether H-8 has a similar effect on gap junctional communication between the oocyte and the follicle cells is not yet known. In summary, this study presents evidence showing that cyclic nucleotide-dependent protein kinases serve as intracellular signaling molecules during the FPE induction of steroid synthesis by the follicle cells. In addition, it shows that inhibition of these kinases was sufficient to induce GVBD in the oocyte, thus suggesting that PKA and/or cGMP-dependent protein kinase might play a part in the mechanism, yet unknown, leading to meiotic arrest of F. heteroclitus oocytes.
3. 4.
5. 6.
7.
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9.
10. 11. 12.
13.
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15.
This work was supported by a National Science Foundation Grant IBN-9306123 awarded to Dr. Robin A. Wallace, NIH-NIGMS MBRS (GM45455-D5) Grant awarded to Y.-W. P. L., and MARC (GM08021-14) Grant to Barry University. Participation of JC was financed by a postdoctoral fellowship from the Ministry of Education and Science (Spain). The authors acknowledge the editorial assistance of Dr. Dorothy M. Jehle, O.P. in the preparation of this manuscript.
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