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BOR - Papers in Press, published online ahead of print October 17, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.009662
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BIOLOGY OF REPRODUCTION 68, 516–523 (2003)
DOI: 10.1095/biolreprod.102.009662
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Role of Arachidonic Acid and Protein Kinase C During Maturation-Inducing Hormone-Dependent Meiotic Resumption and Ovulation in Ovarian Follicles of Atlantic Croaker1

Reynaldo Patiño2,a,b, Goro Yoshizakic, Digbo Bolambab, and Peter Thomasd

a U.S. Geological Survey, b Texas Cooperative Fish & Wildlife Research Unit, Texas Tech University, Lubbock, Texas 79409-2120 c Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato-ku, Tokyo 108-8477, Japan d University of Texas Marine Science Institute, Port Aransas, Texas 78373-1267


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F2{alpha} and PGE2, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 µM) and PGF2{alpha} (5 µM) did not induce maturation, and NDGA (10 µM) did not affect MIH-dependent maturation. However, IM (100 µM) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 µg/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 µM; H7, 50 µM) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 µM) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF2{alpha} restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role of COX products of AA during maturation is possible. A novel model of MIH-dependent ovulation is proposed in which 1) LOX and COX metabolites of AA are both required for ovulation, but at upstream and downstream sites of the pathway, respectively, relative to PKC, and 2) PKC is downstream of genomic activation.

follicle, meiosis, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of arachidonic acid (AA) during the meiotic resumption (maturation) of oocytes is not fully understood. Stimulation of maturation by AA or its metabolites has been reported in some starfishes [1], European sea bass [2], mouse [3–5], and sheep [6]. Conversely, inhibition of meiotic resumption by cyclooxygenase (COX) products (prostaglandins [PGs]) of AA metabolism was observed in mouse [7] and Xenopus laevis [8]. Furthermore, in other species of starfishes, AA and its metabolites appeared to have no involvement in oocyte maturation [1]. Concerning ovulation, it is generally believed that AA is required for this process in most vertebrates, including teleost fishes, but the relative importance of the COX and lipoxygenase (LOX) metabolic pathways is the subject of disagreement among species or studies [9–15].

The involvement of protein kinase C (PKC) in oocyte maturation is also unclear. For example, negative [16, 17] as well as positive [18–21] regulation of meiotic resumption by PKC has been reported in several different species. Nevertheless, general agreement is found in the literature that ovulation in most vertebrates, including teleost fishes, requires PKC-dependent pathways [10, 22–27].

The process of LH-dependent ovarian follicle maturation can be described in two distinct stages for some teleost fishes [28, 29]. The basic mechanisms of each stage have been relatively well studied in Atlantic croaker and other sciaenid species [28]. Namely, the competence of ovarian follicles to produce steroidal maturation-inducing hormone (MIH) and of the oocyte to resume meiosis in response to MIH is acquired during the first stage [30]. These events are accompanied by increases in heterologous (granulosa cell-oocyte) gap junctions (GJ) [31] and MIH-receptor activity [32–34]. Activation of protein kinase A (PKA) is sufficient and necessary for LH induction of the first stage of maturation [35]. Production of MIH marks the beginning of the second stage [30]. The MIH binds to a high-affinity receptor on the surface of the oocyte [32, 34], and this hormone-receptor complex associates with a pertussis toxin-sensitive inhibitory G protein [36]. Oocyte cAMP and PKA activity levels presumably decline and, eventually, lead to meiotic resumption in sciaenid species, as has been reported for other teleosts [37]. To our knowledge, the role of AA in follicular maturation has not been investigated for Atlantic croaker. Furthermore, although pharmacological activation of PKC does not induce meiotic maturation in Atlantic croaker [35, 38], the role of MIH-regulated PKC is also unknown for this species. It has been reported that heterologous GJ decline during the periovulatory period in croaker follicles [31, 38], but the mechanisms of this downregulation are uncertain.

We are unaware of the mechanisms of ovulation having been examined in Atlantic croaker. However, in spotted seatrout [39] and in yellow perch [40], MIH-dependent ovulation involves genomic mechanisms regulated by a nuclear receptor [39]. In addition, as noted already, AA and its metabolites as well as PKC are generally believed to be part of the transduction pathway of hormonally induced ovulation across vertebrate species. Although AA and its metabolites also are effective uncouplers of GJ channels in a variety of tissues [41–43], to our knowledge the role of AA in the periovulatory uncoupling of heterologous GJ has never been examined.

The present study examined the role of AA and PKC during MIH-dependent meiotic resumption and ovulation in Atlantic croaker. The results obtained suggest that AA and PKC are primary transducers of MIH-dependent ovulation but not of maturation. These results also suggest a novel transduction pathway for MIH-induced ovulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fish and Tissue Collection

Atlantic croaker (Micropogonias undulatus) were collected in the fall near Port Aransas, Texas, and were maintained in indoor circular tanks under standard conditions as previously described [30]. During sampling for tissue collection, fish were deeply anesthetized with quinaldine sulfate and spinally transected. Ovaries were removed into Dulbecco modified Eagle medium (DME) supplemented with streptomycin sulfate (100 mg/L) and penicillin (60 mg/L) at pH 7.6. All procedures were reviewed and approved by the animal care and use committees of Texas Tech University and the University of Texas at Austin.

Chemicals

General chemicals, DME, antibiotics, hCG, AA, PGF2{alpha}, PGE2, indomethacin (IM), nordihydroguaiaretic acid (NDGA), phorbol 12-myristate 13-acetate (PMA), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), bisindolylmaleimide GF109203X (GF), and actinomycin D were obtained from Sigma Chemical (St. Louis, MO). 17{alpha},20ß,21-Trihydroxy-4-pregnen-3-one (MIH of Atlantic croaker) was purchased from Steraloids, Inc. (Newport, RI), and Lucifer Yellow from Molecular Probes (Eugene, OR). The carrier solution was ethanol for AA, PGs, and MIH and dimethyl sulfoxide for IM, NDGA, PMA, H7, and GF. The final concentrations of carrier solutions in incubation medium never exceeded 0.1% (v/v). All other chemicals were dissolved in incubation medium.

Experimental Design

The roles of AA and PKC in MIH-dependent maturation and ovulation were investigated with in vitro bioassays using a variety of compounds that act at different sites along the AA metabolic pathway as well as drugs that alter PKC activity. Namely, the involvement of COX products of AA metabolism was examined using a nonspecific inhibitor of COX, IM, as well as two COX products, PGF2{alpha} and PGE2, whereas the role of the LOX pathway of AA metabolism was investigated using a specific inhibitor of LOX, NDGA. The role of PKC was studied using PMA, a PKC activator, as well as GF, a specific inhibitor of PKC, and H7, a nonspecific inhibitor of protein kinases. The requirement for genomic mechanisms was examined with actinomycin D, a transcription inhibitor.

Human chorionic gonadotropin was used as a homologue of LH. All incubations were carried out at 25°C under gentle agitation. Two series of experiments were conducted. The first series focused primarily on meiotic resumption, although ovulation was also recorded. For these experiments, fragments of ovarian lamella containing approximately 50 full-grown ovarian follicles were incubated in 1 ml of DME with hCG (5 IU/ml) for 12–14 h to induce maturational competence (first stage of maturation). Follicles were then incubated with MIH (290 nM) or the appropriate experimental reagent for an additional 22–24 h (total incubation time, 36 h). Although MIH-dependent germinal vesicle breakdown (GVBD) under these conditions is first observed in some follicles within 4–5 h [38], a 22- to 24-h incubation with MIH was used, because pilot observations indicated that most ovulation occurs within 36 h of total incubation time. When inhibitors were used, they were added 45–60 min before the addition of MIH (or other stimuli) to allow adequate time for their action. At the end of the incubations, ovulation was determined by counting the number of oocytes that were expelled from their follicles. Meiotic resumption was then determined by measuring the incidence of GVBD in follicles cleared with Serra solution (ethanol:formalin:glacial acetic acid, 60:30:10 [v/v]). These observations were made using a binocular stereoscope.

The ability of croaker ovarian follicles to ovulate in response to MIH is LH-dependent and follows the acquisition of maturational competence (unpublished observations). Thus, the second series of experiments, focusing on ovulation, used ovarian follicles at a more advanced stage of follicular maturation to ensure adequate ovulatory responses. For this purpose, the length of the priming incubation with hCG was increased to 14–18 h, until follicles showed signs of endogenous MIH-dependent ooplasmic clarification and hydration (stages D and E according to Yoshizaki et al. [38]), which correspond to the second (and irreversible) stage of maturation [28]. Also, for experiments concerning PMA-induced ovulation, follicles were first incubated with exogenous MIH (290 nM) for 30 min immediately after the hCG incubation and before adding the appropriate inhibitors (45–60 min) or PMA. This strategy was used to ensure that the process of maturation was well underway and to prevent the atypical morphology of croaker follicles that results when PMA is added early during the maturational process (unpublished data). A similar protocol was used in a study of MIH-dependent ovulation with spotted seatrout [39]. Total incubation times in this second series of experiments were also 36 h.

All treatments within an experiment were conducted with quadruplicate incubates using ovarian fragments obtained from a single fish. Except when noted, all experiments were repeated with tissues from three or more fish.

Dye Transfer Assay for Heterologous Coupling

The methods of Yoshizaki et al. [38] were used for this procedure. Briefly, following their appropriate treatment, ovarian follicles were isolated using watchmaker forceps under a binocular stereoscope. The isolated follicles were held in grooves made on an agarose plate, and approximately 10 nl of a Lucifer Yellow (457.2 daltons) solution (0.1% in DME) were microinjected into the cytoplasm of the oocytes using a glass micropipette (outer diameter, ~10 µm). The position of the micropipette was controlled using a micromanipulator (IM-5B; Narishige, East Meadow, NY). After an equilibration period of 15 min, the follicular wall containing the thecal cell layer, basement membrane, and granulosa cell layer was manually peeled off the oocyte surface using watchmaker forceps under the binocular stereoscope and carefully rinsed. The isolated follicular cell layer was observed under a fluorescence microscope (Nikon E600; Nikon Instruments, Inc., Lewisville, TX) with B-filter set (excitation filter, 465–495 nm; emission filter, 515–555 nm). The appropriate treatment was continued during the entire procedure to eliminate the possibility of reversal of effects on coupling. The presence or absence of fluorescence in the follicle wall was interpreted as indicating the presence or absence of functional heterologous coupling, respectively.

Data Analysis

Percentage GVBD (100 x number of GVBD ÷ number of full-grown follicles) and percentage ovulation (100 x number ovulated eggs ÷ number of GVBD) were scored in all replicates. The results of all four replicates per treatment per fish were averaged to obtain an individual fish value, and sample size for statistical analyses was considered to be the number of fish. The variability in percentage ovulation among individual fish was sometimes high, but this variability was greatly reduced with data transformed relative to stimulated values. The among-fish variability in GVBD was also generally reduced when using relative values. Thus, except when noted, relative values were used for one-way ANOVA followed by, when appropriate, Duncan multiple-range tests. This data transformation has been previously used in studies of ovarian follicle maturation with Atlantic croaker [35, 44]. All analyses were conducted using the Statistica for Windows 1998 software package (StatSoft, Inc., Tulsa, OK).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preliminary experiments indicated that IM (1–100 µM), NDGA (1–50 µM), GF (0.05–5 µM), and H7 (5–50 µM) inhibited MIH-dependent ovulation in a dose-dependent manner. However, NDGA at 50 µM occasionally caused atypical follicular appearance, presumably resulting from toxic effects. Thus, in subsequent experiments, we used IM at 100 µM, NDGA at 10 µM, GF at 5 µM, and H7 at 50 µM. The effective dose-response ranges for GF and H7 observed in the present study are similar to those reported in an earlier study of maturational competence in croaker follicles [35].

Ovarian Follicles During Early Maturation

When added before the irreversible commitment to meiotic resumption in most follicles, IM (100 µM), a nonselective inhibitor of COX, partially inhibited MIH-dependent GVBD, but NDGA (10 µM), a selective inhibitor of LOX, was without effect (Fig. 1, upper panel). However, neither AA (0, 1, 10, and 100 µM; one-way ANOVA, P > 0.05, n = 4) nor PGF2{alpha} (0, 0.5, and 5 µM; one-way ANOVA, P > 0.05, n = 2) was able to induce GVBD (in both instances, negative controls [untreated medium] yielded negligible or no GVBD, whereas positive controls [MIH-induced] showed near 50% GVBD). The selective inhibitor of PKC, GF (5 µM), as well as the nonselective inhibitor of protein kinases, H7 (50 µM), did not affect MIH-dependent GVBD (Fig. 2). In contrast, inhibitors of AA metabolism (IM and NDGA) and of PKC activity (GF and H7) completely blocked the small incidence of MIH-dependent ovulation observed in these follicles (Figs. 1, upper panel, and 2).



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  		FIG. 1. Effects of IM and NDGA on MIH-dependent GVBD and ovulation in ovarian follicles of Atlantic croaker. Ovarian fragments containing approximately 50 follicles were each incubated in 1 ml of medium with hCG (5 IU) for 12–14 h (upper panel) or 14–18 h (lower panel). Following a brief rinse period, follicles were incubated with or without IM (100 µM) or NDGA (10 µM) for 45–60 min and then with or without MIH (290 nM) for the appropriate length of time for a total of 36 h of incubation (exposure to IM or NDGA continued until the end of the incubation). The incidence of ovulation and GVBD were determined. Results are expressed relative to the values obtained in the presence of MIH. Bars indicate the mean (± SEM) of three (upper panel) or eight (lower panel) separate experiments, and those associated with common letters (large, GVBD; small, ovulation) are not significantly different (one-way ANOVA and Duncan multiple-range test; P < 0.05). Follicles incubated for 14–18 h showed relatively high levels of "spontaneous" GVBD and ovulation when subsequently placed in untreated medium, indicating that considerable production of endogenous MIH occurred as result of the longer hCG incubation time. Average MIH-dependent GVBD and ovulation were 38% and 5% (upper panel), respectively, and 86% and 40% (lower panel), respectively



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  		FIG. 2. Effects of GF and H7 on MIH-dependent GVBD and ovulation in ovarian follicles of Atlantic croaker. Ovarian fragments containing approximately 50 follicles were each incubated in 1 ml of medium with hCG (5 IU) for 12–14 h. Following a brief rinse period, follicles were incubated with or without GF (5 µM) or H7 (50 µM) for 45–60 min and then with or without MIH (290 nM) for the appropriate length of time for a total of 36 h (exposure to GF or H7 continued until the end of the incubation). The incidence of ovulation and GVBD were determined. Results are expressed relative to the values obtained in the presence of MIH. Bars indicate the mean (± SEM) of three separate experiments, and those associated with common letters (large, GVBD; small, ovulation) are not significantly different (one-way ANOVA and Duncan multiple-range test; P < 0.05). Average MIH-dependent GVBD and ovulation were 82% and 17%, respectively

The PKC-stimulator PMA, but not AA, PGF2{alpha}, or PGE2, induced the uncoupling of heterologous GJ after a 6-h incubation, which is the time period necessary for MIH- and PMA-dependent uncoupling of heterologous GJ in croaker follicles (Table 1) [38]. However, MIH-dependent meiotic resumption and uncoupling of heterologous GJ were not affected by coincubation with GF (PKC inhibitor). Namely, all follicles that underwent GVBD after a 10-h incubation with MIH had completely lost their heterologous GJ coupling regardless of the presence or absence of GF (a longer incubation time was used in this second experiment to ensure complete maturation of the ovarian follicles).


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  		TABLE 1. Effects of PMA, AA, PGF2{alpha}, and PGE2 on heterologous gap-junction coupling in maturationally competent ovarian follicles of Atlantic croaker. Each treatment was applied to 12 hCG-pretreated (12–14 h) ovarian follicles per individual fish (experiment), and each experiment was repeated with ovarian follicles from three fish

Ovarian Follicles During Late Maturation

High incidences of "spontaneous" maturation were observed when follicles preincubated for 14–18 h in hCG were subsequently placed in untreated medium. This spontaneous maturation is caused by the onset of endogenous MIH production after prolonged hCG incubations [30, 45]. As noted already, the longer preincubation times were to ensure adequate development of gonadotropin-dependent ovulatory competence. In these follicles, IM (COX inhibitor) inhibited the small increase in (exogenous) MIH-dependent GVBD, but NDGA (LOX inhibitor) was without effect (Fig. 1, bottom panel). Both IM and NDGA effectively blocked MIH-dependent ovulation (Fig. 1, bottom panel).

Ovulation was induced in a dose-dependent manner with AA and PGF2{alpha} (Fig. 3). In addition, PGE2 (5 µM) induced ovulation, but its potency seemed to be lower than that of PGF2{alpha} (data not shown). The AA-induced ovulation was inhibited by IM and the PKC inhibitor, GF, but not by NDGA (Fig. 4). Ovulation was also induced by activation of PKC with PMA (1 µg/ml), and like AA-induced ovulation, PMA-induced ovulation was suppressed by IM and GF but not by NDGA (Fig. 5).



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  		FIG. 3. Effects of AA (upper panel) and PGF2{alpha} (lower panel) on ovulation in ovarian follicles of Atlantic croaker. Ovarian fragments containing approximately 50 follicles were each incubated in 1 ml of medium with hCG (5 IU) for 14–18 h. Following a brief rinse period, follicles were incubated with or without AA (1–100 µM) or PGF2{alpha} (0.5–5 µM) for the appropriate length of time for a total of 36 h. The incidence of ovulation was determined. Results are expressed relative to the values obtained in the presence of AA at 100 µM (average ovulation, 31%) or PGF2{alpha} at 5 µM (average ovulation, 34%). Bars indicate the mean (± SEM) of three (upper panel) or two (lower panel) separate experiments, and those associated with common letters are not significantly different (one-way ANOVA and Duncan multiple-range test; P < 0.05)



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  		FIG. 4. Effects of GF, IM, and NDGA on AA-dependent ovulation in ovarian follicles of Atlantic croaker. Ovarian fragments containing approximately 50 follicles were each incubated in 1 ml of medium with hCG (5 IU) for 14–18 h. Following a brief rinse period, follicles were incubated with or without GF (5 µM), IM (100 µM), or NDGA (10 µM) for 45–60 min and then with or without AA (100 µM) for the appropriate length of time for a total of 36 h (exposure to inhibitors continued until the end of the incubation). The incidence of ovulation was determined. Results are expressed relative to the values obtained in the presence of AA (average ovulation, 31%). Bars indicate the mean (± SEM) of three separate experiments, and those associated with common letters are not significantly different (one-way ANOVA and Duncan multiple-range test; P < 0.05). This experiment was conducted in conjunction with that for AA shown in Figure 3



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  		FIG. 5. Effects of GF, IM, and NDGA on PMA-dependent ovulation in ovarian follicles of Atlantic croaker. Ovarian fragments containing approximately 50 follicles were each incubated in 1 ml of medium with hCG (5 IU) for 14–18 h. Following a brief rinse period and a 30-min incubation with MIH (290 nM), follicles were incubated with or without GF (5 µM), IM (100 µM), or NDGA (10 µM) for 45–60 min and then with or without PMA (1 µg/ml) for the appropriate length of time for a total of 36 h (exposure to inhibitors continued until the end of the incubation). The incidence of ovulation was determined. Results are expressed relative to the values obtained in the presence of PMA (average ovulation, 65%). Bars indicate the mean (± SEM) of three separate experiments, and those associated with common letters are not significantly different (one-way ANOVA and Duncan multiple-range test; P < 0.05)

The MIH-induced ovulation was inhibited by the transcription inhibitor, actinomycin D (5 µM), and restored by cotreatment with PGF2{alpha} (Fig. 6). The AA-induced ovulation, but not the PMA- or PGF2{alpha}-induced ovulation, was also inhibited by actinomycin D (Fig. 7). Finally, PMA- and AA-induced ovulation were completely inhibited by GF, but PGF2{alpha}-induced ovulation was only partially inhibited by GF (Fig. 7).



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  		FIG. 6. Blockage of MIH-dependent ovulation by actinomycin D (ActD) and rescue by PGF2{alpha} in ovarian follicles of Atlantic croaker. Ovarian fragments containing approximately 50 follicles were each incubated in 1 ml of medium with hCG (5 IU) for 14–18 h. Following a brief rinse period, follicles were incubated with or without ActD (5 µM) for 45–60 min and then with or without MIH (290 nM) or PGF2{alpha} (5 µM) for the appropriate length of time for a total of 36 h (exposure to ActD continued until the end of the incubation). The incidence of ovulation was determined. Results are expressed relative to the values obtained in the presence of MIH (average ovulation, 46%). Bars indicate the mean (± SEM) of three separate experiments, and those associated with common letters are not significantly different (one-way ANOVA and Duncan multiple-range test; P < 0.05)



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  		FIG. 7. Effects of actinomycin D (ActD) and GF on PMA-, AA-, and PGF2{alpha}-dependent ovulation in ovarian follicles of Atlantic croaker. Ovarian fragments containing approximately 50 follicles were each incubated in 1 ml of medium with hCG (5 IU) for 14–18 h. Following a brief rinse period and a 30-min incubation with MIH (290 nM), follicles were incubated with or without ActD (5 µM) and GF (5 µM) for 45–60 min and then with or without PMA (1 µg/ml), AA (100 µM), or PGF2{alpha} (5 µM) for the appropriate length of time for a total of 36 h (exposure to inhibitors continued until the end of the incubation). The incidence of ovulation was determined. Results are expressed relative to the values obtained in the presence of PMA (average ovulation, 86%). Bars indicate the mean (± SEM) of four separate experiments, and those within experimental groups (indicated by a line over the bars) associated with common letters in reference to the negative control (medium) are not significantly different (one-way ANOVA and Duncan multiple-range test on all treatments; P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Maturation

At concentrations that induce ovulation (see later discussion), AA was insufficient to induce meiotic resumption in maturationally competent ovarian follicles of Atlantic croaker. Ovulation-inducing concentrations of PGF2{alpha} (COX product) were also insufficient to induce maturation. Furthermore, ovulation-inhibiting concentrations of NDGA (LOX inhibitor) did not affect MIH-dependent maturation. These results differ from those obtained with another teleost fish, the European sea bass, in which exogenous AA as well as the COX products, PGF2{alpha} and PGE2, were sufficient to induce meiotic resumption in maturationally competent ovarian follicles [2]. The mechanisms of AA-induced oocyte maturation were not examined in European sea bass, however, and whether the action of AA was indirect, via regulation of follicular MIH production, or direct, as transducer of MIH action, is unclear [2]. The possibility of indirect effects via induction of MIH production is suggested by the results of studies with goldfish showing that exogenous AA can enhance the steroidogenic activity of ovarian follicles [46]. Nevertheless, in the present study, IM seemed to partially inhibit MIH-induced meiotic resumption. Although the latter observation allows the possibility that an intact COX pathway is necessary to facilitate MIH-induced maturation in Atlantic croaker, caution must be used when interpreting results obtained with IM, because effects of this reagent on cell functions unrelated to AA metabolism have been described [47]. Overall, the results of the present study strongly suggest that AA and its metabolites are insufficient to directly or indirectly induce meiotic resumption in ovarian follicles of Atlantic croaker, but the possibility of a permissive role of COX metabolic pathways in MIH-dependent meiotic resumption cannot be ruled out.

Studies with vertebrates other than teleosts also have yielded inconsistent observations regarding the involvement of AA and its metabolites in oocyte maturation. For example, the LOX pathway of AA metabolism was linked to hormonally induced oocyte maturation in some, but not all, species of starfishes [1]. Also, PGE1 and PGE2 inhibited progesterone-dependent meiotic resumption in Xenopus oocytes [8], whereas positive [3–6], negative [7], or no [15] involvement of AA or its metabolites was suggested in oocytes of a number of mammalian species. Although it is possible that conditions associated with specific experimental systems can lead to artifactual results, the information available also suggests that differences exist in the roles and mechanisms of AA during oocyte maturation among species.

In croaker follicles, activators of PKC (PMA) do not seem to induce meiotic resumption ([35, 38]; present study), and inhibitors of PKC (GF and H7) do not affect MIH-induced meiotic resumption at concentrations that suppress ovulation (see later discussion). Also, treatment of croaker follicles with PMA before MIH stimulation appears to be toxic to the oocytes (data not shown). These observations indicate that MIH-dependent meiotic resumption in croaker oocytes does not involve PKC and that premature PKC activation may be inhibitory or toxic to maturation. A maturation-suppressing effect of PKC was also suggested for mouse oocytes [17] and Xenopus oocytes [16]. Conversely, pharmacological activation of PKC was reported to promote oocyte maturation in species such as the rat [18], the amphibian Rana dybowskii [19], and the teleost killifish [20, 21]. However, like in Atlantic croaker, PKC activation was found to be unnecessary for MIH-dependent oocyte maturation in killifish [21]. Thus, PKC does not seem to have a physiological role in the process of MIH-dependent maturation in those teleost species examined to date. Preliminary information (unpublished data) suggests that specific inhibitors of PKA (H89) also do not affect MIH-induced meiotic resumption in croaker follicles at concentrations that inhibit hCG-dependent maturational competence (first stage of maturation) [35]. Furthermore, PKA inhibitors are sufficient to induce maturation in killifish follicles [21]. These observations are consistent with the concept that a decline in PKA activity is involved in MIH-dependent meiotic resumption of teleost oocytes [37].

Correlations between meiotic resumption and uncoupling of granulosa cell-oocyte GJ have been observed in all vertebrate species examined, including Atlantic croaker [31, 38]. Given the recently proposed involvement of AA in oocyte maturation of some teleosts [32] and the inhibitory effect of AA on GJ coupling in a number of tissues [41–43], the present study examined the hypothesis that AA is part of the signaling pathway for MIH-dependent uncoupling of heterologous GJ. However, the present results showed that neither AA nor its COX metabolites, PGE2 and PGF2{alpha}, are able to uncouple heterologous GJ in maturationally competent croaker follicles within the time frame necessary for MIH-dependent uncoupling and maturation [38]. Furthermore, although pharmacological activation of PKC (by PMA) caused heterologous GJ to uncouple in croaker follicles ([38]; present study), specific inhibitors of PKC (GF) had no effect on MIH-dependent GJ uncoupling. Thus, AA and PKC do not seem to participate in the MIH-dependent periovulatory uncoupling of heterologous GJ.

Ovulation

In contrast to maturation, MIH-dependent ovulation in croaker follicles seems to require functional PKC and intact COX and LOX pathways of AA metabolism. Namely, inhibitors of PKC (GF and H7), COX (IM), and LOX (NDGA) all blocked MIH-dependent ovulation, whereas PMA, AA, PGF2{alpha}, and PGE2 were all sufficient to induce ovulation. Furthermore, actinomycin D blocked MIH-dependent ovulation, and coincubation with PGF2{alpha} restored ovulation. (Transcription is not required for MIH-dependent meiotic resumption in croaker follicles [30].) These various findings are generally consistent with those obtained in other vertebrates. For example, a requirement for de novo transcription during MIH-dependent ovulation has been previously reported in spotted seatrout [39] and yellow perch [40]. This transcription requirement suggests that MIH-dependent ovulation in teleost fishes occurs via interaction with a nuclear (classical) steroid receptor recently characterized in teleost ovaries [39]. Similar conclusions were made regarding progesterone-dependent ovulation in rats [48]. Furthermore, PKC has been previously linked to ovulation in other teleosts [10, 22–24], amphibians [27], and mammals [10, 25, 26]. Finally, as observed in the present study with Atlantic croaker, AA seems to be involved in the ovulation process of most other vertebrates that have been studied to date [9–15].

The PMA-dependent ovulation was completely blocked by a specific PKC inhibitor (GF), thus confirming that PMA acts via activation of PKC. Furthermore, PMA-dependent ovulation was inhibited by IM but not by actinomycin D or NDGA. These latter observations suggest that the requirements for transcription and LOX during MIH-dependent ovulation are both upstream of PKC activation, whereas the requirement for COX is downstream of PKC (Fig. 8). This proposed sequence of events implies that AA-dependent ovulation should not be markedly affected by the LOX inhibitor, NDGA (because AA-derived PGs produced by COX would bypass the upstream requirement for LOX), or by actinomycin D. The present results are consistent with the first condition, but the inhibitory effect of actinomycin D on AA-dependent ovulation is seemingly inconsistent with the proposed model. However, because PKC inhibitor (GF) completely blocked AA-induced ovulation, it can be postulated that MIH-induced PKC activity not only serves to increase the activity of existing COX enzymes but also to slow down their turnover rate or that of their mRNAs. In such a scenario, the combination of a transcription blockage (as when in the presence of actinomycin D) and conditions of basal PKC activity would lead to a gradual decline in COX protein and activity and, thus, to a reduced follicular capacity to metabolize AA into the ovulation-active products, PGs. The overall outcome would be a reduced ability of AA to induce ovulation, thus explaining the observation of the present study. Alternatively, transcription-mediated induction of COX by AA could also explain the inhibition of AA-dependent ovulation by actinomycin D, but this scenario would be difficult to reconcile with the findings that the IM-sensitive site of ovulation (COX) is downstream of PKC and that PKC-dependent ovulation is insensitive to actinomycin D.



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  		FIG. 8. Major steps of the proposed transduction pathway for MIH-induced ovulation in ovarian follicles of Atlantic croaker. The MIH binds to a nuclear receptor (nMIHR), possibly in granulosa or thecal cells, and activates genomic mechanisms. Enzymes associated with the LOX pathway of AA metabolism are consequently synthesized or activated, and an undefined product or products (X) of LOX directly or indirectly induce the activity of preexisting PKC. Increased PKC activity in turn induces COX and increases the synthesis of prostaglandins (PGF2{alpha}) from AA. The PGF2{alpha} causes rupture of the follicle and consequent expulsion of the oocyte (ovulation). Lines ending with solid circles point toward steps along the pathway that are blocked by the inhibitors used in the present study (see text). The PMA directly activates PKC and bypasses the actinomycin D (ActD)- and NDGA-sensitive steps of ovulation

The proposed transduction pathway (Fig. 8) also indicates that PG-dependent ovulation should not be affected by transcription inhibitors. This condition was met by the present results. Interestingly, PGF2{alpha}-induced ovulation was partially diminished in the presence of a concentration of GF that completely blocked PMA-induced ovulation. Thus, it appears that PKC activity facilitates, but is not strictly required for, PG-induced ovulation. Although endogenous PGs were not measured in the present study, ovarian PGEs and PGFs have been observed in all vertebrate species for which measurements have been taken [10]. In teleost fishes, PGF2{alpha} has consistently induced ovulation, whereas the effects of PGEs have been variable [10]. In Atlantic croaker, exogenous PGF2{alpha} and PGE2 both induced ovulation, but PGF2{alpha} seemed to be slightly more potent.

The transduction pathway for MIH-induced ovulation proposed here for Atlantic croaker (Fig. 8) is remarkably consistent with the following observations in the rat: 1) LH-induced PG production and ovulation seem to be mediated by progesterone production and interaction with a classical progesterone receptor [48], 2) LOX metabolites (leukotrienes) are produced earlier than COX products (PGs) during hCG-induced ovulation [49], 3) treatment of perfused ovaries with LOX inhibitor (NDGA) inhibits LH-dependent PG production and ovulation [12], and 4) certain LOX metabolites can induce PG production by ovarian granulosa cells [50]. These observations suggest that LH (progesterone)-dependent ovulation in rat follicles is mediated by LOX product-dependent synthesis of PGs. Furthermore, although its relative position in the transduction pathway is unknown, PKC is also thought to be required for LH-dependent ovulation in rat follicles [26, 51]. In yellow perch, the COX and LOX pathways of AA metabolism have been implicated in ovulation as well [52], but unlike in Atlantic croaker, PMA-induced ovulation is inhibited only by NDGA and not by IM [22]. Thus, the requirements for LOX and COX during perch ovulation seem to be downstream and upstream of PKC, respectively, which is the reverse situation relative to the proposed croaker ovulation pathway (Fig. 8).

Conclusion

The present observations suggest that AA and PKC are key elements of the transduction pathway for MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in ovarian follicles of Atlantic croaker. However, these results are inconclusive regarding the possibility of a permissive role of COX products during MIH-dependent maturation. A novel model of MIH-dependent ovulation is proposed for Atlantic croaker in which LOX and COX metabolites of AA are both required for ovulation but at upstream and downstream sites of the pathway, respectively, relative to PKC and in which PKC is downstream of genomic activation. This heuristic model is amenable to experimental testing and, if verified, would allow further studies focusing on specific steps of the pathway and their respective mechanisms.


   ACKNOWLEDGMENTS
 
Dr. Frederick W. Goetz and Mr. Naresh Pandey provided critical comments on a draft version of this manuscript.


   FOOTNOTES
 
1 Funding for this work was provided by the U.S. Department of Agriculture (NRICGP Animal Reproduction, 00-35203-9135) and Japan Society for the Promotion of Science's Research for the Future program (97L00902). The U.S. Geological Survey, Texas Tech University, Texas Parks and Wildlife Department, and the Wildlife Management Institute jointly sponsor the Texas Cooperative Fish and Wildlife Research Unit. Back

2 Correspondence. FAX: 806 742 2946; r.patino{at}ttu.edu Back

Received: 22 July 2002.

First decision: 14 August 2002.

Accepted: 28 August 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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