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Regular Article |
a Department of Marine Science, Marine Science Institute, University of Texas at Austin, Port Aransas, Texas 78373-5015
ABSTRACT
Changes in ovarian maturation-inducing steroid (MIS; 17,20ß,21-trihydroxy-4-pregnen-3-one [20ß-S]) membrane receptor concentrations during the reproductive cycle were investigated in spotted seatrout (Cynoscion nebulosus) captured at their spawning grounds. Ovarian receptor concentrations increased gradually during ovarian recrudescence and subsequently increased rapidly during oocyte maturation, reaching 3.5-fold the prematuration values by the beginning of ovulation. The significant elevation of receptor concentrations by the germinal vesicle migration stage of oocyte maturation was accompanied by increases in circulating levels of gonadotropin (LH, GTH II) and MIS (20ß-S). The regulation and physiological significance of the increase in ovarian MIS membrane receptor concentrations were investigated in a double in vitro incubation system. Incubation of fully grown, follicle-enclosed oocytes with hCG (10 IU/ml) for 6 h caused a two- to fourfold increase in oocyte and ovarian MIS receptor concentrations and the development of oocyte maturational competence (OMC; ability to complete oocyte maturation in vitro in response to exogenous 20ß-S in a second incubation). Both upregulation of the MIS receptor and development of OMC in response to gonadotropin were blocked by coincubation with actinomycin D or cycloheximide, which are inhibitors of mRNA and protein synthesis, respectively, but not by cyanoketone, which is an inhibitor of 3ß-hydroxysteroid dehydrogenase-dependent steroid synthesis. Incubation with a variety of steroids, including 20ß-S, failed to increase receptor concentrations or to induce OMC, further supporting a steroid-independent mechanism of gonadotropin action. In contrast, insulin-like growth factor I (IGF-I) mimicked the actions of gonadotropin, which suggests IGF-I may be a component of the hormone signaling pathway. A close correlation was found between the relative increase in MIS receptor concentrations and the percentage of oocytes that became maturationally competent after treatment with different concentrations of gonadotropins and drugs that elevate cAMP levels. The finding that upregulation of the MIS receptor in response to gonadotropin and other treatments is invariably associated with the development of OMC indicates that these two processes are intimately related, and it suggests that the increase in MIS receptor concentrations is a critical regulatory step in the hormonal control of oocyte maturation.
meiosis, oocyte maturation, progestogen, nongenomic steroid action
INTRODUCTION
Many actions of steroids are too rapid to be readily explained by the classic genomic mechanism of steroid action mediated by activation of nuclear steroid receptors. Rapid increases in intracellular free-calcium levels have been observed within 30 sec of treatment of PC12 cells, sperm, neurons, and bone with glucocorticoids, progestogens, estrogens, and androgens, respectively [1–4]. In addition, acute activation of a variety of signal transduction pathways and opening of ion channels have been observed in target cells within a few minutes of steroid exposure. Many of these rapid steroid actions are nongenomic and initiated at the surface of the target cell, presumably by binding to membrane receptors [5, 6]. Indeed, an extensive body of evidence has accumulated during the past 20 years supporting the existence of binding moieties on plasma membranes which have all the binding characteristics of specific steroid receptors [5–8]. These membrane-binding moieties appear to have important roles as intermediaries in a wide range of steroid actions [9–12]. However, little information is available to date regarding the physiological regulation of these binding sites and whether changes in their abundance are consistent with their purported functions, which are important criteria for the designation of these binding moieties as hormone receptors.
The induction of meiotic maturation of oocytes by maturation-inducing steroids (MIS) in teleosts and amphibians is one of the most thoroughly characterized models of membrane receptor-mediated, nongenomic steroid actions [13]. The 21-carbon MIS have been identified as progesterone in several amphibian species, 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) in amago salmon and other salmonid fishes, and 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) in several perciform fishes, including Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus) [14–17]. Further, specific receptors for these MIS have been characterized on oocyte and ovarian plasma membranes from Xenopus, spotted seatrout, rainbow trout (Oncorhynchus mykiss), and striped bass (Morone saxatilis) [7, 18–21]. Interestingly, preliminary evidence indicates that MIS receptor abundance is increased in vivo during oocyte meiotic maturation (also called final oocyte maturation [FOM]) in spotted seatrout, striped bass, and Xenopus [7, 19, 21]. Similar increases in MIS receptor concentrations can be induced in vitro by treatment of follicle-enclosed spotted seatrout and Xenopus oocytes with gonadotropin [19, 22], although the precise physiological significance of these gonadotropin-induced changes remains unclear.
Early reports demonstrating that induction of oocyte maturation by gonadotropin (LH) could be mimicked by MIS in salmonid fishes and anuran amphibians suggested that the action of LH during oocyte maturation was solely to increase MIS synthesis [23]. However, subsequent studies with full-grown oocytes from several urodele amphibians and a variety of teleost fishes at an early phase of final development showed they are unresponsive to MIS, although they can mature when treated with gonadotropin in vitro [24–29]. Oocyte responsiveness to the MIS (oocyte maturational competence [OMC]) in these species occurs at a later stage of development and can be induced by gonadotropin. Detailed investigations of gonadotropin induction of OMC in Atlantic croaker oocytes in vitro indicate that OMC does not require steroid synthesis, because it is not blocked by an inhibitor of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cyanoketone, but is dependent on new mRNA and protein synthesis [28]. These studies suggest, therefore, that gonadotropin induction of oocyte and follicular maturation in Atlantic croaker, and probably in most other teleost and amphibian species, can be divided into two stages: induction of OMC requiring synthesis of proteins but not MIS (priming phase), followed by completion of oocyte maturation depending on synthesis of MIS (germinal vesicle breakdown [GVBD] phase) [28, 30]. Further, preliminary evidence obtained in a closely related sciaenid species, spotted seatrout, indicates that the MIS membrane receptor is one of the proteins upregulated during gonadotropin induction of OMC [22].
The aims of the present study were to investigate the seasonal and hormonal regulation of the MIS membrane receptor in spotted seatrout ovaries in detail and to determine its physiological significance. Changes in receptor abundance during ovarian recrudescence and oocyte maturation were examined in a natural population of spotted seatrout sampled at their spawning sites. Hormonal upregulation of the MIS receptor and its relationship to the development of OMC were investigated in a double in vitro incubation system, because a preliminary study had indicated they were closely correlated [31]. The results of the present study demonstrate a close association between these two processes, which suggests that upregulation of the MIS membrane receptor is an essential regulatory step in gonadotropin induction of oocyte maturation in this species.
MATERIALS AND METHODS
Chemicals
1,2-3H-11-Deoxycortisol (42.0 Ci/mmol) was purchased from New England Nuclear (Boston, MA) and enzymatically converted to 20ß-S as described by Scott et al. [32]. 125Iodine was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Unlabeled steroids (Steraloids, Wilton, NH; Sigma Chemical Company, St. Louis, MO) were dissolved in absolute ethanol and stored at -20°C. Ovine LH, ovine FSH, ovine thyroid-stimulating hormone, and porcine insulin were obtained from National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. Cyanoketone was provided by Sterling Winthrop (Rensselaer, NY). Dulbecco's modified Eagle's medium with Ham's nutrient mixture F-12 (DEM), hCG, salts, enzymes, and all other chemicals were obtained from Sigma (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA).
Field Studies
Adult female spotted seatrout were collected by gill net in Redfish Bay, Aransas Pass, Texas, during the period of ovarian recrudescence (March–April) and at the beginning of the spawning season (April–June) [33]. Fish were removed from the net within 5 min of capture, weighed, measured for total length, anesthetized by placement in ice-cold water, and humanely killed by severing the spinal cord and blood supply posterior to the skull. The ovaries were excised and stored on ice for 1–3 h until the samples were brought to the laboratory, where they were weighed and stored at -80°C for up to 3 mo until they were assayed for receptor content.
Female spotted seatrout at various stages of oocyte maturation were also captured as described above at their spawning sites in Redfish Bay during various times of the day. The fish were humanely killed and their ovarian tissues removed for subsequent receptor assay as described above. Blood samples were taken from other individuals within 10 min of capture from the caudal vein with heparinized syringes and centrifuged at 1500 x g for 7 min. The plasma was then removed and stored at -20°C for subsequent measurement of gonadotropin (LH, GTH II) and MIS by RIA. The maturation stage of the largest cohort of oocytes in each ovarian sample was determined by microscopic examination as described by Brown-Peterson et al. [33].
Laboratory Studies
Adult female spotted seatrout were collected by gill net in Redfish Bay during the spawning season in the spring and fall. Fish were maintained in circular, indoor, recirculating tanks (16 000 L) with external biofilters under conditions of constant photoperiod (14L:10D) and water temperature (25–28°C). Fish were fed a diet of dead shrimp daily at the rate of 3% body weight per day and were acclimated in the laboratory for at least 2 mo before experimentation.
Tissue sampling Fish were deeply anesthetized with quinaldine sulphate (20 mg/L; Argent Chem Labs, Redmond, WA), and a few follicle-enclosed oocytes were obtained by ovarian catheterization. All animal procedures were approved by the University of Texas at Austin Institutional Animal Care and Use Committee and were in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Fish that had a high percentage of oocytes with diameters of 350–425 µm and a central germinal vesicle were considered to be competent to undergo oocyte maturation in response to hormonal stimulation and were humanely killed by severing the spinal cord and blood supply posterior to the skull. The ovaries were rapidly excised and transferred to beakers containing ice-cold incubation medium (DEM) adjusted to pH 7.4 with sodium bicarbonate (1.2 g/L) containing streptomycin sulphate (0.1 g/L) and penicillin (0.06 g/L).
Double in vitro incubation procedure to investigate hormonal regulation of MIS receptor and development of oocyte maturational competence Ovarian fragments (3 g) were incubated in 30 ml of DEM for various periods of up to 24 h in the presence or absence of hormones and enzyme inhibitors in a Dubnoff shaking water bath at 25°C under an atmosphere of oxygen. At the end of the incubation period, the ovarian tissue was removed, washed three times with 10 ml of DEM, and divided into unequal-sized fragments for separate assays of oocyte maturational competence and 20ß-S receptor concentrations. Oocyte maturational competence was assessed using an in vitro GVBD bioassay [34] with slight modifications [29]. Three ovarian fragments, each containing approximately 60 fully grown ovarian follicles, were transferred to separate wells of a 24-well incubation plate and incubated for 12 h in 1 ml of DEM containing 290 nM 20ß-S. The incubation was stopped by replacing the medium with clearing solution (ethanol, formalin, and glacial acetic acid in a ratio of 6:3:1). The oocytes were examined under a dissecting microscope and scored for completion of GVBD according to the method described by Trant and Thomas [34]. The MIS receptor concentrations were measured in the remaining tissue after storage at -80°C for up to 1 mo.
The localization of the MIS receptor on plasma membranes and its upregulation by gonadotropin (hCG) during the acquisition of OMC were confirmed by removing the follicular cell layers surrounding the oocytes before the receptor assay. Ovarian tissues were finely minced in homogenization buffer containing 1 mM EGTA to remove divalent anions. The tissue fragments were repeatedly expelled through a syringe fitted with 18- and then 22-G hypodermic needles, and the follicular tissues and early development stage oocytes were removed by repeated washes through a 400-µ mesh screen. The purity of the oocyte preparation was determined by microscopy at x70 magnification. The final preparation consisted almost entirely of fully grown oocytes (
0.5% contamination), more than 90% of which were devoid of follicle cells, with the remainder having only small patches of cells attached.
Time course of response to gonadotropin Ovarian tissue fragments were incubated with hCG (15 IU/ml) for various periods ranging from 15 min to 24 h. At the end of the incubation period, the ovarian fragments from each treatment well were divided. A small fragment was then bioassayed for oocyte maturational competence, whereas the remaining fragments were stored at -80°C for subsequent receptor measurement. The entire experiment was repeated using ovarian tissue from different donor fish.
Effects of steroids
Ovarian tissue fragments were incubated for 12 h in media alone (control), with gonadotropin (hCG, 14 IU/ml), or with the following steroids at a concentration of 290 nM: 20ß-S, 17,20ß-P, 17
-hydroxy-4-pregnen-3,20-dione, 11-deoxycorticosterone, testosterone, estradiol-17ß, or cortisol. Ovarian fragments were subsequently bioassayed for oocyte maturational competence and assayed for receptor content. The results of the 20ß-S treatment were confirmed in five additional experiments using different donor fish.
Effects of inhibitors of transcription, translation, and 3ß-HSD on response to gonadotropin Ovarian tissue was incubated for 12 h in the presence of gonadotropin (hCG, 15 IU/ml) with or without one of the following inhibitors: actinomycin D (RNA synthesis inhibitor; 10 µg/ml), cycloheximide (protein synthesis inhibitor; 10 µg/ml), and cyanoketone (3ß-HSD inhibitor; 2 µg/ml). After the incubation, tissues were assayed for oocyte maturational competence and MIS receptor concentration. The entire experiment was repeated three times.
Relationship between MIS receptor induction and development of oocyte maturational competence after various treatments Ovarian tissue was incubated for 12 h with the following hormones and drugs: ovine LH (10, 50, and 100 ng/ml), ovine FSH (100 ng/ml), dibutyryl cyclic AMP (1, 10, 100, and 1000 µM), forskolin (1 and 10 µM), porcine insulin (0.1, 1, 10, and 100 µM), and recombinant human insulin-like growth factor I (IGF-I; 1 and 10 µM). The experimental treatments were repeated two to three times with ovarian tissues from different donors.
LH radioimmunoassay The LH (GTH II) concentrations in spotted seatrout plasma were measured by a ß-subunit-directed LH RIA validated for GTH measurement in spotted seatrout and other sciaenid fishes [35]. Spotted seatrout LH ß-subunit is used as a radioligand in the RIA with Atlantic croaker LH antiserum. Spotted seatrout plasma and pituitary extracts dilute parallel to the croaker LH standards in the RIA, which has a sensitivity of 0.1 ng/ml plasma and intra- and interassay variances of 9% and 11%, respectively [35].
MIS radioimmunoassay
Immunoreactive 20ß-S concentrations in spotted seatrout plasma were measured by RIA after solvent extraction as described previously using antisera specific for 20ß-S and its 3,5ß reduced metabolites, 3,17,20ß,21-tetrahydroxy-5ß-pregnane and 17,20ß,21-trihydroxy-5ß-pregnane [16, 36], which have been identified in seatrout plasma (unpublished observations). In a subset of the samples, these steroid metabolites were separated by LH-20 column chromatography before RIA to estimate their relative contributions to the total immunoreactive 20ß-S.
MIS membrane receptor assay Plasma membrane fractions of ovarian tissues and oocytes were prepared as described previously [7]. The MIS membrane receptor concentrations were measured using a one-point assay that was based on the procedure described by Patiño and Thomas [7]. In brief, 5 nM 3H-20ß-S in ethanol was added to each of three total and three nonspecific-binding (NSB) tubes, whereas 100-fold excess unlabeled steroid in ethanol was added to each NSB tube. The ethanol was evaporated under N2, and 500-µl aliquots of the samples were added to each assay tube. The tubes were incubated for half an hour to allow equilibrium to be reached, and then bound and free steroid were separated by filtration over glass-fiber filters as described previously [7].
Statistics
The SEMs were calculated for receptor concentrations for all in vitro experiments, and significance was determined by one-way ANOVA and Tukey HSD or by linear/curvilinear regression. Slidewrite Plus 2.0 (Advanced Graphics, Carlsbad, CA) was used to generate sigmoidal curves (time course and ovarian seasonal study).
RESULTS
Field Studies
Receptor concentrations during ovarian recrudescence Ovarian MIS receptor numbers increased significantly during the 2-mo period of ovarian recrudescence (Fig. 1) from less than 0.5 pmol/g ovary in fully regressed fish (gonadosomatic index [GSI], <1.0) to 0.5–1.5 pmol/g ovary (mean, 0.7 pmol/g) in individuals with fully grown ovaries (GSI range, 4–8). This increase was significant when it was expressed on a per gram tissue, per milligram protein, or on a whole-ovary basis (P < 0.001). The mean receptor concentrations in females with fully recrudesced ovaries were similar to those observed in control (untreated) ovarian tissues in the laboratory studies.
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Receptor and hormone concentrations during oocyte maturation
Ovarian MIS receptor numbers increased three- to fourfold during the 12- to 14-h period of oocyte maturation from approximately 0.5 pmol/g ovary in fish before or at the beginning of oocyte maturation (vitellogenic and lipid coalescence, respectively) to a maximum of 2 pmol/g ovary when GVBD and hydration were complete (Fig. 2). Receptor numbers subsequently decreased to 1 pmol/g ovary during ovulation and reached prematuration levels (mean ± SEM, 0.39 ± 0.16 pmol/g ovary; n = 3) in ovaries of spawned individuals, which still contained large numbers of developing (vitellogenic) follicles. Receptor binding was barely detectable in ovulated oocytes. A significant increase in receptor concentrations was observed by the germinal vesicle migration (GVM) stage of oocyte maturation (P < 0.01). Plasma levels of LH (GTH II) were also significantly elevated in seatrout at this stage of oocyte maturation compared to those in nonmaturing individuals (P < 0.05; Table 1) and showed a further elevation to reach 3–5 ng/ml 4 h later, when GVBD was occurring. Circulating levels of 20ß-S, the MIS in this species, showed a similar pattern of stage-related changes. Plasma levels of immunoreactive 20ß-S increased to 0.37 ± 0.07 ng/ml (mean ± SEM, n = 9) at a late stage of lipid coalescence, and they continued to rise to 0.70 ± 0.07 ng/ml (n = 16) during GVM to reach maximum levels of 1.74 ± 0.09 ng/ml (n = 13) at GVBD. However, by this stage of oocyte maturation, the 5ß and 3,5ß reduced metabolites of 20ß-S composed approximately 80% of the total immunoreactive 20ß-S in the plasma.
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Laboratory Studies
Time course of response to gonadotropin Addition of hCG (15 IU/ml) to postvitellogenic ovarian fragments resulted in significant, fivefold increases in receptor concentrations (P < 0.005), peaking between 6 and 9 h (Fig. 3). The threefold increase in receptor concentrations to 1.5 pmol/g ovary after 5 h treatment with hCG was associated with the development of OMC (i.e., the ability of the oocytes to undergo GVBD in response to the MIS in a subsequent 12-h, in vitro bioassay) for 90%–100% of the oocytes.
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Analysis of purified oocyte preparations confirmed the localization of the MIS receptor on the oocyte plasma membrane and its 2.3-fold upregulation during OMC (receptor concentration: 0 h, 30.8 ± 3.3 pmol/g protein; 6 h incubation with 15 IU/ml hCG, 71.0 ± 17.3 pmol/g protein; mean ± SEM, n = 6; P < 0.002).
Receptor concentrations declined during longer-term incubations in seven separate experiments and approached control (prestimulation) values (0.5 pmol/g ovary) after 18–26 h of incubation when the oocytes had undergone GVBD and were hydrating, which is in agreement with our previous findings [22].
Effects of steroids Incubation with a variety of steroids, including the MIS, at concentrations of 290 nM did not cause significant changes in receptor concentrations (Fig. 4), although they were significantly increased (P < 0.001) after treatment with hCG (15 IU/ml). Moreover, subsequent incubation with the MIS did not induce GVBD in the control or steroid-treated samples, although GVBD was observed in the hCG-treated samples.
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Effects of inhibitors of transcription, translation, and 3ß-HSD Coincubation with actinomycin D or cycloheximide completely blocked the fivefold induction of the 20ß-S receptor by gonadotropin (hCG, 15 IU/ml), indicating a requirement for both mRNA and protein synthesis (Fig. 5). Moreover, these treatments also blocked the development of OMC in response to gonadotropin. However, coincubation with cyanoketone, which is an inhibitor of 3ß-HSD-dependent steroid synthesis, failed to block either the gonadotropin-induced receptor increase or the ability of oocytes to undergo GVBD in response to MIS.
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Relationship between MIS receptor induction and oocyte maturational competence Concentration-dependent increases in MIS receptor concentrations and the percentage of maturationally competent oocytes (oocyte priming) were observed after treatment with a variety of hormones and drugs (Fig. 6). Similar maximum levels for receptor induction (12-fold increase compared to controls) and oocyte priming (80%) were observed after treatment with high concentrations of ovine LH, hCG, seatrout pituitary extracts, forskolin, and dbc AMP.
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Ovine LH caused concentration-dependent increases in MIS receptor concentrations and oocyte priming over a range of 10–100 ng, whereas 100 ng of ovine FSH had lower activities in the receptor induction and oocyte maturational competence bioassays (Fig. 6). Although both dbc AMP and forskolin treatments caused upregulation of the MIS receptor and induction of OMC, forskolin was more effective than dbc AMP in increasing receptor concentrations at the two lower concentrations tested. Forskolin was also a more potent inducer of maturational competence. Overall, a close correlation was found between receptor upregulation and the development of OMC after these hormone and drug treatments (R = 0.88).
Treatment with porcine insulin (0.1–100 µM) and IGF-I (1–10 µM) also caused concentration-dependent increases in MIS membrane receptor concentrations and induction of OMC (Fig. 6). The patterns of receptor upregulation and oocyte priming after insulin treatment differed, however, from those observed with both the various gonadotropin preparations and the agents that elevate tissue cAMP levels. At the highest concentration (100 µM), porcine insulin caused priming of approximately 70% of the oocytes, whereas it only caused a fourfold increase in receptor concentrations. In contrast, a similar rate of priming was associated with a 12-fold increase in receptor concentrations after gonadotropin treatment.
DISCUSSION
This study demonstrates that ovarian MIS receptor abundance increases several-fold during oocyte maturation in spotted seatrout collected at their spawning sites, which is consistent with the suggested role of the receptor as the intermediary in MIS induction during the later phase of oocyte maturation. The periovulatory surge of LH is the likely hormonal stimulus that initiates this increase, because similar dramatic elevations in oocyte and ovarian MIS receptor concentrations were induced by gonadotropins with LH-like actions in vitro. The physiological importance of this increase in receptor concentrations for maturation of seatrout oocytes was clearly demonstrated in the ovarian incubation experiments. The double-incubation studies showed that upregulation of the MIS receptor after treatment with gonadotropin, other hormones, and drugs was invariably associated with development of the ability of fully grown oocytes to respond to the MIS and complete oocyte maturation (OMC). In contrast, treatments that blocked gonadotropin upregulation of the MIS receptor or that did not increase receptor concentrations consistently failed to induce OMC. Therefore, these results suggest that MIS receptor abundance is one of the critical factors regulating the onset of oocyte maturation, and that gonadotropin upregulation of the MIS receptor is a prerequisite for the development of OMC and successful completion of oocyte maturation.
The demonstration of a close association among increases in MIS membrane receptor concentrations, OMC, MIS secretion, and the completion of oocyte maturation provides further substantial evidence that this receptor mediates MIS induction of oocyte maturation. Previously, a close correspondence was observed between the relative binding affinities of a wide range of steroids for the ovarian MIS membrane receptor in this species and their relative potencies as agonists and antagonists in an acute, in vitro oocyte maturation bioassay [12], whereas they showed a poor correlation with ovulation in these species [37]. In contrast, the binding affinities of steroids for a nuclear MIS receptor in seatrout ovaries did not correlate with their potencies in inducing oocyte maturation but, instead, showed a close correlation with steroid induction of ovulation in this species [37]. Taken together, these results fully satisfy the criteria of biological relevance for designating the seatrout ovarian membrane MIS binding moiety as a hormone receptor. Moreover, they clearly establish a biological role for a steroid membrane receptor in mediating a physiologically important steroid action, maturation of oocytes, but not their subsequent ovulation.
The field study results demonstrate marked changes in ovarian MIS receptor concentrations during the reproductive cycle of a natural population of spotted seatrout. The initial increase in receptor levels at the beginning of the reproductive season coincided with the appearance of vitellogenic oocytes, reaching a maximum mean value of 0.7 pmol/g ovary when growth of the ovary and the first batch of vitellogenic oocytes were complete (GSI, >4; Fig. 6) [33, 38]. These MIS receptor concentrations are similar to those observed previously for spotted seatrout with vitellogenic oocytes maintained in the laboratory (0.77 pmol/g ovary) [7]. Information is currently lacking on the mechanism regulating this gradual increase in receptor concentrations and whether it is hormonally mediated by FSH or LH. However, FSH has been identified in a closely related sciaenid species, Atlantic croaker [39], and is the principal circulating gonadotropin at this stage of the reproductive cycle in several salmonid fishes [40]. Interestingly, ovine FSH caused a moderate increase in MIS receptor concentrations in the present in vitro studies, although its activity was low (Fig. 6). In contrast, ovine LH and hCG, which has LH activity, both displayed markedly higher potencies in upregulating receptor levels. Moreover, measurable LH levels are present in Atlantic croaker plasma during the early phase of vitellogenic growth [41], and higher levels (2 ng/ml) were detected in spotted seatrout ovaries with fully recrudesced ovaries in the present study.
The second major increase in MIS receptor concentrations to 2.0 pmol/g ovary coincided with oocyte maturation and subsequently declined during ovulation, which is in agreement with the results of our earlier, preliminary field and laboratory studies on this species [7, 22]. Elevated MIS receptor concentrations during oocyte maturation have also been reported in another perciform species, striped bass [21]. The rapid elevation in receptor concentrations during oocyte maturation and results of the in vitro experiments suggest that the periovulatory surge of LH, which triggers oocyte maturation, is the likely mediator of the increase in MIS receptor levels. However, direct evidence in support of this hypothesis is not available, because field data on plasma LH levels could not be collected before the increase in MIS receptor concentrations at GVM. Moreover, by this stage of oocyte maturation, plasma concentrations of another potential hormonal mediator, the MIS, were also elevated, and they continued to increase in parallel with the further increase in receptor concentrations during the later stages of FOM. Thus, although the field study results clearly demonstrated that ovarian MIS receptor concentrations increased during oocyte maturation, the hormonal regulation of this increase remained unclear.
Convincing evidence was obtained from the in vitro studies that the increase in MIS receptor concentrations on seatrout oocytes during oocyte maturation is regulated by gonadotropin. The experiments with inhibitors of transcription and translation, actinomycin D and cycloheximide, showed that this action of gonadotropin was dependent on new mRNA and protein synthesis. In contrast, none of the results indicated that this action of gonadotropin was dependent on steroid hormone synthesis. Coincubation with cyanoketone, an inhibitor of 3ß-HSD-dependent steroid synthesis, did not block the gonadotropin response. Moreover, incubations with a variety of steroids failed to significantly upregulate receptor concentrations. Interestingly, the modulatory influences of mRNA and protein synthesis inhibitors on gonadotropin upregulation of the MIS receptor paralleled their effects on OMC. A similar requirement for mRNA and protein synthesis, but not for steroidogenesis, for the development of OMC has been demonstrated previously in Atlantic croaker [29]. In addition to greater activity in upregulating the MIS receptor, gonadotropins with LH-like activity were more potent than ovine FSH in inducing OMC, which is in agreement with results using homologous gonadotropins in red seabream [42, 43]. The finding that induction of the MIS receptor and OMC by gonadotropins was mimicked by incubation with drugs that increase intracellular cAMP levels (dbc AMP and forskolin) suggests that this gonadotropin-activated signal transduction pathway involves activation of adenylate cyclase. Similarly, Jalabert and Finet [44] showed that elevation of cAMP levels in rainbow trout oocytes was associated with increased sensitivity to 17,20ß-P, the MIS in that species.
The in vitro studies also demonstrated a close association between gonadotropin-induced upregulation of the receptor and the development of OMC. The present results extend our earlier, preliminary observations of a likely relationship between these two events during oocyte maturation in spotted seatrout [22]. Unpublished results discussed in a recent review by Nagahama [45] suggest that a similar relationship between these processes also occurs in flounder during oocyte maturation. Maturational competence was only induced in most of the fully grown seatrout oocytes in vitro when MIS receptor concentrations reached 2 pmol/g ovary, which is similar to the maximum receptor levels recorded in seatrout undergoing natural oocyte maturation on their spawning grounds. The time-course data showed that widespread OMC developed shortly after receptor levels reached this point. In addition, a close correlation (R = 0.88) was found between the percentage of oocytes that became maturationally competent and the relative increase in receptor concentrations after treatment with graded concentrations of various gonadotropins with LH-like activities or with drugs that elevate the cAMP level (Fig. 6). Moreover, as discussed previously, several blockers of gonadotropin-induced intracellular processes inhibited both the increase in receptor concentrations and the development of OMC. The gonadotropin-induced increase in MIS receptor concentrations during oocyte maturation in spotted seatrout is intimately related, therefore, with development of OMC in this species.
Insulin-like growth factor I signaling has been clearly implicated in the induction of oocyte maturation in a broad range of vertebrate species, including mammals, amphibians, and teleosts [42, 46, 47]. The IGF-I receptor has been characterized on Xenopus oocytes and recently cloned from an oocyte cDNA library in this species [48, 49]. Moreover, several postreceptor-mediated IGF-I signaling events have been identified in Xenopus oocytes, including mitogen-activated kinases and cyclin-dependent kinases [50]. Equivalent information is currently lacking for teleosts. However, IGF-I and its mRNA have been detected in teleost ovaries and oocytes [51, 52], and IGF-I has been shown to stimulate ovarian steroidogenesis and oocyte maturation in teleosts [42, 53]. Insulin-like growth factor I mimics the action of gonadotropin in red seabream ovaries, inducing OMC as well as the completion of oocyte maturation (GVBD) [42]. The present results confirm our previous, preliminary findings [31] that IGF-I also induces OMC in spotted seatrout, and that this process is associated with upregulation of the MIS membrane receptor. Recently, IGF-I has been demonstrated to induce another critical event during the development of OMC in seabream: an increase in the concentrations of homologous and heterologous gap junctions [54]. Thus, IGF-I can mimic two important actions of gonadotropin during the development of OMC: upregulation of the MIS receptor and increases in gap junction concentrations in the ovarian follicle. The present results further support a role for IGF-I in the development of OMC in teleosts, although direct evidence of increases in ovarian IGF-I concentrations during oocyte maturation and information on the mechanism of IGF-I action are lacking. Ovarian IGF-I synthesis does not appear to be controlled by growth hormone [52], so the possibility exists that IGF-I may be a component of the gonadotropin signaling pathway, as discussed previously by Kagawa et al. [42]. In contrast, a preliminary report on oocyte maturation in striped bass indicates that IGF-I does not mimic the actions of gonadotropin in this species [55]. Interestingly, in the present study, the relationship between induction of the MIS receptor and the development of OMC differed after treatment with 100 µM porcine insulin and the gonadotropins, with insulin showing a proportionally greater induction of OMC (Fig. 6). Although these results suggest qualitative differences in the responses to insulin (or IGF-I) and gonadotropins in spotted seatrout, their significance remains unclear.
Recent studies indicate that several other processes in addition to upregulation of the MIS receptor occur during gonadotropin induction of OMC in teleosts. Increases in the number of heterologous and homologous gap junctions have been reported in the ovarian follicles of Atlantic croaker and red seabream during induction of OMC [54, 56]. In addition, increases in mRNA levels of connexin, the proteins that form gap junctions, have been observed in Atlantic croaker ovaries during OMC [57, 58]. However, the broad applicability of these results across teleost species is unclear, because decreases in gap junctions during oocyte maturation were observed in Fundulus [59]. Upregulation of 20ß-hydroxysteroid hydrogenase, which is a critical enzyme for the synthesis of teleost MIS, is also induced by gonadotropin in salmonid fishes at an early stage of maturation in preparation for the steroid-dependent GVBD phase of oocyte maturation [45].
Alterations in steroid membrane receptor concentrations under different physiological conditions have recently been described in several vertebrate/cell models [21, 60]. However, the mechanisms regulating steroid membrane receptor concentrations have received scant attention and remain poorly understood. Previous studies have shown that gonadotropin can upregulate MIS receptor concentrations in fully grown ovaries and oocytes of Xenopus and spotted seatrout [19, 22] and in testes and sperm of spotted seatrout and Atlantic croaker [8, 61]. The more extensive studies reported here show that gonadotropin upregulation of the MIS receptor involves both mRNA and protein synthesis but is not mediated by increases in steroid synthesis. Moreover, the physiological significance of the increase in receptor concentrations for the development of OMC is clearly demonstrated in the seatrout model. The present results suggest that fish and amphibian models of FOM can provide valuable insights into the hormonal regulation of steroid membrane receptors as well as the modulation of nongenomic steroid actions and their physiological significance.
ACKNOWLEDGMENTS
The authors wish to thank Sterling Winthrop (Rensselaer, New York) for the generous donation of cyanoketone and Charles Laidley for assistance in the collection of the fish for the seasonal study.
FOOTNOTES
1 Supported by PHS grant ES04214 to P.T. 
2 Correspondence: Peter Thomas, Marine Science Institute, University of Texas at Austin, 750 Channel View Dr., Port Aransas, TX 78373-5015. FAX: 361 749 6777; thomas{at}utmsi.utexas.edu
3 Current address: Department of Fisheries and Oceans, 4160 Marine Dr., West Vancouver, BC, V7V 1N6 Canada.
4 Current address: Department of Internal Medicine, Yale University, School of Medicine, VA Medical Center, West Haven, CT 06516.
Accepted: July 7, 2000.
Received: May 17, 2000.
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) dbc AMP (
), forskolin (
), seatrout pituitary extract (+), insulin ([bu1002]
), and IGF-I (
). Maturational competence was assessed by scoring GVBD after a 12-h incubation with 290 nM 20ß-S. Each point represents the mean of a minimum of three observations. Numbers refer to the concentrations of test substances (units: LH and FSH, ng; hCG, IU; pituitary extract, pituitary equivalents; dbc AMP, forskolin, insulin, and IGF-I, µM)