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Biology of Reproduction 66, 251-257 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Estradiol-17ß Triggers Luteinizing Hormone Release in the Protandrous Black Porgy (Acanthopagrus schlegeli Bleeker) Through Multiple Interactions with Gonadotropin-Releasing Hormone Control1

Fan-Pin Yena, Yan-Horn Leeb, Chun-Lin Hea, Jing-Duan Huanga, Lian-Tien Suna, Sylvie Dufourc, and Ching-Fong Chang2,a

a Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan, Republic of China b National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan, Republic of China c Laboratoire de Physiologie, UMR 8572 CNRS, Museum National d'Histoire Naturelle, Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the mechanism of estradiol-17ß (E2) action on stimulation of LH (=gonadotropin II) release in the black porgy fish (Acanthopagrus schlegeli Bleeker) using an in vivo approach and primary cultures of dispersed pituitary cells in vitro. In vivo, E2 but not androgens (testosterone [T] and 11-ketotestosterone [11-KT]) significantly stimulated plasma LH in a dose-dependent manner. Estradiol-17ß also increased brain content of seabream GnRH. GnRH antagonist prevented E2 stimulation of LH release in vivo, indicating that the effect of E2 on LH was mediated by GnRH. In vitro, sex steroids (E2, T, 11-KT) alone had no effect on basal LH release in the cultured pituitary cells, but GnRH significantly stimulated LH release. Estradiol-17ß potentiated GnRH stimulation of LH release, an effect that was inhibited by GnRH antagonist, and 11-KT, but not T, also potentiated GnRH stimulation of LH release. The potentiating effect of 11-KT on GnRH-induced LH release in vitro was stronger than that of E2. These data suggest that E2 triggers LH release in vivo by acting both on GnRH production at the hypothalamus and on GnRH action at the pituitary. In contrast, 11-KT may only stimulate GnRH action at the pituitary. The E2 induction of LH release, through multiple interactions with GnRH control, supports a possible central role of E2 in the sex change observed in the protandrous black porgy.

estradiol, gonadotropin-releasing hormone, luteinizing hormone, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Black porgy, Acanthopagrus schlegeli Bleeker, a marine protandrous hermaphrodite fish, is widely distributed in parts of Asia and is important for aquaculture [1]. These fish are functional males for the first 2 yr of life, but some begin to change sex during the third year. Only about 40% of cultured black porgy change to females; the rest remain functional males during the third spawning season [2]. Black porgy in Taiwan have an annual reproductive cycle with a pattern of multiple spawning occurring in winter and early spring.

High levels of plasma estradiol-17ß (E2) during the prespawning and spawning season are correlated with the natural sex change of 3-yr-old black porgy [2]. Vitellogenic oocytes and sex change were observed in 2-yr-old black porgy following oral administration of E2 for at least 5 mo [3, 4]. Thus, E2 plays an important role in the natural and controlled sex change in this species during the prespawning season [5]. Oral administration of E2 for more than 5 mo induced high concentrations of plasma LH (=gonadotropin II) and sex change [6]. In vivo E2 but not testosterone (T) induced high concentrations of plasma LH in black porgy, unlike in other fish species [7, 8]. The inducible release of pituitary LH by E2 in black porgy provides a model for further study of the control of LH release by sex steroids. Gonadal steroid feedback regulation of gonadotropin release is important in the neuroendocrine control of reproduction in teleosts, as in other vertebrates [9]. However, the sites of E2 action for the stimulation of LH secretion are presently unclear in black porgy.

To further investigate the control of LH secretion in the black porgy, we employed an in vivo approach and an in vitro apporach with primary cultures of dispersed pituitary cells. With the in vivo study, we investigated the action of various sex steroids (E2 and androgens) on plasma LH release. We also analyzed the effect of GnRH antagonist on E2-stimulated LH release and the effect of E2 on brain GnRH content. With the in vitro study, we investigated the direct actions of sex steroids at the pituitary by analyzing their effect on LH release used either alone or in combination with GnRH or GnRH antagonist. In vitro actions were compared with in vivo effects of steroids on plasma LH release.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Fish

Two-year-old male black porgy were obtained from a pond culture in Chiayi County, Taiwan. All experimental fish were acclimated to the pond at the National Taiwan Ocean University culture station in a seawater system. The fish were fed commercial feed (Fu Sou Feed Co., Taichung, Taiwan). Water temperatures ranged from 22°C to 25°C during the experimental period. Fish were anesthetized with 2-phenoxyethanol before injections, blood collection, and death.

In Vivo Sex Steroid Effects on Plasma LH

Two-year-old male fish in the prespawing season (November) (mean ± SEM body weight = 287.6 ± 13.4 g; gonadosomatic index [GSI] = 0.27 ± 0.04) were divided into 6 groups (n = 8 fish/group): E2 (2 ng, 50 ng, 1.5 µg/g body weight [BW]), T and 11-ketotestosterone (11-KT, 1.5 µg/g BW), and controls (coconut oil vehicle alone). On Day 0, all fish were given a single i.m. injection of the respective steroid or vehicle. Blood was collected on Day 2 from the caudal vasculature with an EDTA-containing needle, and the plasma was stored at -70°C for later LH analysis. All steroids were from Sigma Chemical Co. (St. Louis, MO).

In Vivo Effect of GnRH Antagonist on E2 Action

To further examine the mechanism of E2 action on LH release, we tested the effect of a GnRH antagonist (Ac-D-Trp1, D-p-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10; Sigma). Black porgy at the end of the spawning season (March) (n = 24, BW = 306 ± 10.55 g, GSI = 3.42 ± 0.23) were divided into 3 groups: control, E2 (1 µg/g BW), and E2 (1 µg/g BW) + GnRH antagonist (0.03 µg/g BW). Plasma was collected just before (0 h) and at 4 and 24 h after E2 and GnRH antagonist injections.

In Vivo Effect of E2 on Brain Seabream GnRH

To further investigate E2 action in the hypothalamus, 2-yr-old male fish (BW = 313.7 ± 14.1 g, GSI = 0.73 ± 0.13) were divided into 3 groups (n = 8): control, 0.1 µg/g BW E2, and 0.7 µg/g BW E2. Fish were injected with respective coconut oil (as a vehicle) or E2 on Days 0 (December, spawning season), 7, and 14. After the fish were killed by decapitation on Day 21, brains were removed, put in 1.0 M HCl, and frozen at -70°C for seabream GnRH (sbGnRH) measurement.

Preparation of Dispersed Pituitary Cells

Black porgy pituitary cells were cultured using enzymatic and mechanical procedures [10]. For each culture experiment, fifteen 2-yr-old black porgy were decapitated, and the pituitaries were quickly removed and placed in ice-cold dispersion buffer (DM; Dulbecco PBS without calcium and magnesium chloride but with 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone; Gibco BRL, Rockville, MD). Pituitaries were incubated in a 0.8 mg/ml DM solution of 1-tosylamino-2-phenylchloromethyl ketone-treated bovine pancrease type III trypsin (Sigma) at 25°C for 1 h with slow shaking. This trypsin solution was then removed and replaced by a 1 mg/ml DM solution of soybean trypsin inhibitor (Sigma) and 1 µg/ml DM DNase (Sigma) for 10 min. Pituitaries were mechanically dispersed in DM by repeated passages through a plastic transfer pipette. Cells were resuspended in culture medium (CM) consisting of medium 199 with Earle salts, sodium bicarbonate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml fungizone (Gibco BRL).

Static Culture

Plates were precoated with a solution of 0.1 mg/ml poly-L-lysine (Sigma) for 5 min, rinsed with sterile water, and dried. Cells were cultured in 96-well tissue culture plates (Nunc, Roskilde, Denmark) at a density of 62 000 cells/125 µl CM in each well with 1% fetal bovine serum (Gibco BRL) in a tissue culture incubator (NU4500 CO2 water-jacketed incubator; Nuaire) at 24°C under 5% CO2 and saturated humidity. Release experiments were performed on the third day of culture (Day 0). Prior to addition of treatments, cells were washed 3 times and incubated in CM (0.25 ml/well) in the presence of 1.0 % fetal bovine serum. Six wells were used for each treatment

In Vitro Treatment of Sex Steroid Alone

Six pituitary cell culture experiments were conducted using 2-yr-old black porgy at different stages of the reproductive cycle, with GSIs of 0.31 ± 0.03, 0.41 ± 0.06, and 0.43 ± 0.05 (prespawning season), 0.45 ± 0.01 (nonspawning season), and 1.08 ± 0.27 and 3.14 ± 0.45 (spawning season). Estradiol-17ß, T, and 11-KT were tested alone over 8 days at final concentrations of 10-6, 10-8, and 10-10 M. Aliquots of cell culture supernatants were removed on Days 0, 2, 4, 6, and 8 for LH measurement.

In Vitro Treatment of Sex Steroids in Combination with a GnRH Analogue

One experiment was performed during the spawning season (January) (BW = 331.5 ± 10.09 g, GSI = 3.14 ± 0.45) to further study sex steroid action. A GnRH analogue ([D-Ala6]-LHRH ethylamide; Sigma) was tested over 4 h in the presence or absence of sex steroids using GnRH analogue (10-6, 10-8, and 10-10 M) and E2, T, or 11-KT (10-8 M). Aliquots of CM were removed for LH measurement after 1, 2, and 4 h of culture.

In Vitro Effects of a GnRH Antagonist on E2 and GnRH Action

To further examine the action of E2 on LH release, the GnRH antagonist (Ac-D-Trp1, D-p-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10) was tested in vitro. Pituitaries were collected from black porgy at the prespawning season (October) (BW = 231.7 ± 10.4 g, GSI = 0.15 ± 0.015). Cells were treated with medium alone (controls) or with GnRH analogue (10-8 M), GnRH analogue (10-8 M) + E2 (10-8 M), and GnRH analogue (10-8 M) + E2 (10-8 M) + GnRH antagonist (10-8 M, 10-6 M). Aliquots of CM were removed for LH measurement after 1 h of incubation.

LH Assay

Concentrations of LH were measured using a homologous RIA employing purified black porgy LH as a standard with an anti-black porgy LHß antibody as previously described [8].

Seabream GnRH Assay

Brains were homogenized in 0.1 N HCl and neutralized with NaOH in a phosphate buffer. A specific RIA for brain sbGnRH was used. The sbGnRH antiserum was induced in a rabbit using sbGnRH-hemolycyanin of keyhole limpets (Genosys Biotechnologies, The Woodlands, TX). The sbGnRH was iodinated with chloramines T and separated with a sephadex G-25 column. The RIA employed sbGnRH (Genosys) as the standard and 125I-sbGnRH as the labeled peptide. No cross-reactivity of mammalian GnRH, salmon GnRH, chicken GnRH-I, or chicken GnRH-II was observed with sbGnRH antiserum (data not shown).

Data Analysis

An ANOVA followed by the Duncan multiple-range test were used to determine significant differences among group means. Results are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma LH Release In Vivo

Estradiol-17ß stimulated LH release in a dose-dependent manner (Fig. 1). Two days after the injection, plasma LH concentrations were significantly higher in fish injected with E2 (50 ng and 1.5 µg/g BW) than in control fish or in fish injected with a low dose (2 ng) of E2. Plasma LH concentrations in the fish treated with 1.5 µg E2 reached a mean value (152 ± 33.3 ng/ml) that was greater than that for controls (3.1 ± 0.4 ng/ml). Treatment with the high dose of T (1.5 µg/g BW) resulted in a slight but significant increase in LH concentrations as compared with Day 2 controls, but concentrations were not different from those of Day 0 controls. No difference in plasma LH concentrations was observed with 11-KT (1.5 µg/g BW).



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  		FIG. 1. Plasma LH concentrations in female black porgy injected with various doses of E2 (2 ng, 50 ng, or 1.5 µg/g BW), T (1.5 µg/g BW), 11-KT (1.5 µg/g BW), or vehicle. Steroid was given on Day 0, and plasma was obtained on Days 0 and 2. Values with different characters differ from the control on the same date (P < 0.05)

Effect of GnRH Antagonist on LH Release In Vivo

In agreement with the previous experiment, injection of E2 (1 µg/g BW) induced a significant increase in plasma LH concentrations (Fig. 2). The effect of E2 was significant at 24 h but not at 4 h after the injection. Coinjection of E2 and a GnRH antagonist inhibited the stimulatory effect of E2:plasma LH concentrations at 24 h in fish injected with E2 and GnRH antagonist were not different from control values.



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  		FIG. 2. In vivo effects of GnRH antagonist (0.03 µg/g BW) on the plasma concentrations of LH in black porgy after injection with E2 (1 µg/g BW) at 0 h. Plasma LH concentrations were expressed as the relative value; control LH levels at 0 h were designated as 100%. Values with different characters differ from the control of same group (P < 0.05)

Estradiol-17ß Actions on Brain sbGnRH Content In Vivo

Three injections of E2 (0.1 or 0.7 µg/g BW) induced a significant increase (2.3- and 3.2-fold, respectively) in brain sbGnRH as compared with control fish injected with vehicle alone (P < 0.05) (Fig. 3).



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  		FIG. 3. Brain sbGnRH content in male black porgy injected with various doses of E2 (0.1 µg and 0.7 µg/g BW) or vehicle. Estradiol-17ß was given on Days 0, 7, and 14, and brain was collected on Day 21. Values with different characters differ from the control (P < 0.05)

Sex Steroid (E2, T, or 11-KT) Actions on LH Release In Vitro

Treatment of cultured pituitary cells with sex steroids (E2, T, or 11-KT) at various doses (10-10 M, 10-8 M, and 10-6 M) did not significantly regulate LH release at any time point (Days 2, 4, 6, and 8) (Fig. 4).



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  		FIG. 4. Relative LH value (%, mean from 6 experiments) in the CM of pituitary cells from black porgy. The pituitary cells were treated with various doses (control, 10-10 M, 10-8 M, and 10-6 M) of E2, T, and 11-KT on Days 0, 2, 4, and 6. Aliquots of CM were collected on Days 2, 4, 6, and 8. The control value of LH concentrations in 1 of the experiments was defined as 100%, and the relative LH values were calculated. No significant difference was observed between the steroid and control groups (P > 0.05)

GnRH Actions on LH Release In Vitro

GnRH (10-10–10-6 M) significantly (P < 0.05) and dose-dependently stimulated the release of LH after 1 and 2 h of incubation compared with controls (Fig. 5A). There was no significant stimulation of LH concentrations by GnRH after the longer incubation period (4 h).



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  		FIG. 5. LH concentrations in CM of the pituitary cells of black porgy. The pituitary cells were treated with various doses (0, 10-10 M, 10-8 M, and 10-6 M) of GnRH analogue alone (A) or with a combination of various doses (0, 10-10 M, 10-8 M, and 10-6 M) of GnRH and 10-8 M E2 (B). The treatment was given at 0, 1, and 2 h, and aliquots of CM were collected after 1, 2, and 4 h of culture. Values with different characters differ from the control of same period (P < 0.05)

Estradiol-17ß Actions on GnRH and LH Release In Vitro

GnRH (10-10–10-6 M) in combination with 10-8 M E2 significantly (P < 0.05) stimulated the release of LH in a dose (GnRH)-dependent manner at 1 and 2 h of incubation (Fig. 5B). Estradiol-17ß significantly potentiated the effect of GnRH. Combining GnRH (10-6 M) with E2 (10-8 M) induced a 4.2-fold and 1.7-fold greater LH release than did GnRH (10-6 M) alone at 1 and 2 h of incubation, respectively (P < 0.05) (Fig. 5, A and B).

GnRH Antagonist Actions on the E2-Potentiating Effect of GnRH-Induced LH Release In Vitro

Treatment with GnRH antagonist (10-6 M) significantly inhibited the stimulation of LH release in response to GnRH analogue (10-8 M) (Fig. 6). Furthermore, GnRH antagonist (10-8 M and 10-6 M) also significantly inhibited the E2 (10-8 M)-potentiating effects of the GnRH analogue (10-8 M) on LH release.



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  		FIG. 6. LH concentrations in the CM of pituitary cells of black porgy. The 3 treatment groups at 0 h were 1) control, 2) GnRH group: GnRH analogue (10-8 M), GnRH analogue (10-8 M) + GnRH antagonist (10-8 M), and GnRH analogue (10-8 M) + GnRH antagonist (10-6 M), and 3) GnRH + E2 group: GnRH analogue (10-8 M) + E2 (10-8 M), GnRH analogue (10-8 M) + E2 (10-8 M) + GnRH antagonist (10-8 M), and GnRH analogue (10-8 M) + E2 (10-8 M) + GnRH antagonist (10-6 M). The treatment lasted for 1 h. LH concentrations in the CM were expressed as the relative value (%), with the control value designated as 100%. Values with different characters differ from each other within the group (P < 0.05)

11-KT and T Actions on LH Release In Vitro

11-Ketotestosterone (10-8 M) potentiated GnRH effect on LH release in a dose (GnRH)-dependent manner (Fig. 7). Combining 11-KT (10-8 M) with GnRH (10-6 M) produced a 12-fold greater LH release than did GnRH (10-6 M) alone. The combination of 11-KT with any dose (10-10–10-6 M) of GnRH had a stronger potentiating effect on LH release as compared with combining E2 and GnRH treatments. In contrast to the potentiating effect of 11-KT, no difference in LH concentration in the medium was found between treatment with GnRH alone and treatment with GnRH + T.



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  		FIG. 7. LH concentrations in the CM of pituitary cells of black porgy. The pituitary cells were treated with various doses (0, 10-10 M, 10-8 M, and 10-6 M) of GnRH analogue alone and with a combination of 10-8 M of different sex steroids (E2, T, or 11-KT) and various doses of GnRH. The treatment was given at 0 h and lasted for 1 h. Values with different characters differ from the control of the same treated group (P < 0.05). Values with an asterisk are significantly different from the values in the rest of the GnRH group at the respective dose of GnRH (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present data indicate there are differential effects of sex steroids on LH release in vivo and in vitro in black porgy. In vitro, E2, T, and 11-KT had no effect on basal LH release from cultured pituitary cells, but E2 and 11-KT exerted a potentiating effect on GnRH-stimulated LH release. In vivo, a single injection of E2 (but not of T or 11-KT) induced a strong increase in plasma LH concentrations. An estrogen-specific stimulation of LH secretion in vivo was also found in previous studies within 24 h after injection [7, 8, 11]. Our recent data indicated that E2 stimulatory effects on LH release occur throughout the reproductive cycle, with the strongest responsiveness during the prespawning season [11]. In the present study, injection of a GnRH antagonist abolished E2 stimulation of LH release in vivo, clearly demonstrating that the E2 effect was mediated by GnRH control of LH release.

In vivo E2 stimulation of plasma LH has been largely documented in mammals [12]. In contrast, the in vivo inducible release of plasma LH by a single E2 injection, as observed in black porgy, has not been found in other teleosts. Sex steroids regulate LH secretion in various teleosts species, but these effects have been demonstrated only under conditions of long-term treatment: long-term administration of T increased plasma LH in goldfish (Carassius auratus) [13] and long-term administration of E2 stimulated plasma LH in rainbow trout (Oncorhynchus mykiss) [14]. Various effects of sex steroids have also been observed in vitro in teleost species. In the common carp (Cyprinus carpio), E2, T, and 11-KT inhibited LH secretion by cultured pituitary cells [15], whereas in the European eel (Anguilla anguilla) T but not E2 stimulated LH accumulation and release in vitro [16]. In the eel, as in the black porgy, differential effects of sex steroids were observed between in vivo and in vitro experiments [17], suggesting multiple targets of steroid action on the brain-pituitary axis. The differential in vivo and in vitro responses and the species differences in the regulation of LH secretion lead us to further investigate the mechanisms of E2-induced LH release in the black porgy, especially its possible inteactions with GnRH.

In vitro, GnRH stimulated the release of LH with a dose-response profile after 1 and 2 h of incubation. The stimulatory effect of GnRH was inhibited in the presence of a GnRH antagonist. GnRH also stimulated in vitro LH release by cultured pituitary cells in several teleosts such as the European eel [10], rainbow trout [18, 19], and goldfish [20]. The stimulation of LH release by GnRH in the present experiment assessed the successful development of static primary cultures of pituitary cells in black porgy. The stimulatory effects of GnRH on LH release from black porgy pituitary cells were no longer observed after 4 h of incubation, indicating a desensitization phenomenon. In vitro, desensitization of LH release to GnRH stimulation also has been observed in mammals [21], chickens [22], turtles [23], and goldfish [24].

In vitro, E2 alone did not stimulate basal LH release from black porgy pituitary cells. In contrast, E2 (10-8 M) significantly potentiated GnRH action on the release of LH by pituitary cells. This potentiating effect of E2 on GnRH-induced LH release during the spawning season was also found in black porgy during the nonspawning season (unpublished data). Treatment with GnRH antagonist inhibited the effects of combined GnRH analogue and E2 on the stimulation of LH release in vitro.

These in vitro data indicate that the pituitary is one of the target organs responsive to the E2 stimulation of LH release, with a potentiating effect of E2 on GnRH-induced LH release. In mammals, several possible pathways for E2-potentiating effects on GnRH-stimulated LH release have been proposed. In vivo, E2 increased both plasma LH and the number of GnRH receptors in the ovine pituitary [25]. Implantation with E2 increased the amount of mRNA for GnRH receptor in sheep [26]. In vitro, E2, but not cortisol and progesterone, increased the number of GnRH receptors in cultured ovine pituitary cells [27, 28]. Similar E2 effects on the increase of GnRH binding were found in rats [29, 30]. Estradiol-17ß also induced the hyperglycosylation of GnRH receptors in ewes [31]. Estradiol-17ß regulation of the responsiveness of gonadotropins to GnRH action can also act at postreceptor sites to modulate Ca2+-mediated and protein kinase C-mediated signal transducing pathways in rats [32]. These data indicate that E2 potentiates GnRH releasing effects by acting on GnRH receptor number and posttranslational processes and on GnRH postreceptor signaling. Such mechanisms could also mediate E2 potentiating effects in the black porgy pituitary.

A potentiating effect of 11-KT (10-8 M) on GnRH action was also found in the present experiment. The potentiating effect of 11-KT was even greater than that of E2. In contrast, T (10-8 M) did not potentiate GnRH effects on LH release. These data provide the first evidence that a nonaromatizable androgen (11-KT), but not an aromatizable one (T), significantly potentiated GnRH effect on LH release in teleosts. 11-Ketotestosterone is a physiological androgen in black porgy [3] as in other teleosts. This androgen is more potent than T in inducing androgen-dependent transactivation of nuclear androgen receptor {alpha} and ß types in the Japanese eel (Anguilla japonica) [33]. In addition, steroid-binding sites specific to 11-KT were found on the cellular membrane in the kidney of the three-spine stickleback (Gasterosteus aculeatus) [34]. Therefore, specific (11-KT) androgen receptors may be involved in the 11-KT potentiating effect in the black porgy pituitary.

Variations in the interactions between sex steroids and GnRH have been observed among vertebrate species. In teleosts, in vivo implantation with E2 or T could potentiate in vitro GnRH action on LH release in goldfish [35, 36], whereas nonaromatizable androgens 11-KT and 5{alpha}-dihydrotestosterone had no effect on LH response to GnRH [35]. In vitro, T (10-7 M) did not affect basal LH release but increased LH response to GnRH stimulation in goldfish pituitary cells [37, 38]. Implantation with E2 or T significantly inhibited in vivo GnRH-induced LH secretion in the Atlantic croaker (Micropogonias undulatus), whereas 5{alpha}-dihydrotestosterone was ineffective [39]. In chickens, E2, T, and progesterone all inhibited GnRH-induced LH secretion from cultured pituitary cells [40]. In contrast, in juvenile female bullfrogs (Rana catesbeiana) in vivo or in vitro pretreatment with 5{alpha}-dihydrotestosterone elevated the GnRH-induced secretion of FSH and LH in vitro [41]. In sheep and cattle, E2 enhances the release of LH from pituitary cells in response to GnRH in vitro [42, 43] but does not affect unstimulated release of LH [44].

Our data showed that E2 and 11-KT, but not T, significantly potentiated GnRH action on LH release in vitro in black porgy and suggest that actions of these steroids are mediated by specific E2 and 11-KT receptors, respectively. As for E2, the potentiating effect of 11-KT and E2 on GnRH action could involve genomic action on GnRH receptor expression, posttranslational modulatory effects on GnRH receptors, or interactions with GnRH receptor intracellular signaling pathways. Data for goldfish suggest that T potentiates GnRH-stimulated LH release by enhancing the sensitivity of protein kinase C-dependent LH release but not through an increase in GnRH receptors [38]. Possible actions of E2 and 11-KT via membrane receptors should also not be excluded; E2 and androgens rapidly increase cytosolic calcium concentrations through respective membrane receptors on mammalian cells [45, 46].

Estradiol-17ß and 11-KT are the major plasma sex steroids in female (or changing sex) and spermatogenesis-active male black porgy, respectively [3, 5]. Oral administration of E2 resulted in increased gonadal aromatase activity, elevated plasma LH, and sex changes in black porgy [2–4, 6]. The evidence of a close link between E2 and LH release in these experiments and the induction of sex change by exogenous E2 [2–4, 6] suggest that LH is probably involved in the process of E2-controlled sex changes in black porgy.

The present study revealed different mechanisms of action of E2 and 11-KT on LH release. Although 11-KT had an even greater potentiating effect than E2 on pituitary cell response to GnRH in vitro, only E2 was effective in inducing LH release in vivo. The in vivo effect of E2, which was abolished by GnRH antagonist, should therefore include an additional hypothalamic target, leading to the stimulation of GnRH release. The lack of effect of 11-KT and T in vivo indicated that this positive control of GnRH release in the black porgy is E2 specific. Estradiol-17ß stimulatory effects on the surge of GnRH release have been documented in mammals [47, 48]. Our data also indicated that E2 had a stimulatory effect on brain sbGnRH, the major hypophysiotropic GnRH form in perciforms [49], including the black porgy (data not shown). Sex steroids exert positive feedback on the hypophysiotropic GnRH form in various teleost species, e.g., E2 on mammalian GnRH in the European eel [50].

The present data provide in vitro and in vivo evidence to elucidate the possible mechanism of E2-stimulatory effects on LH release in black porgy. Estradiol-17ß may act at both the hypothalamus and pituitary to stimulate GnRH release and to modulate GnRH action, respectively. 11-Ketotestosterone may mainly act at the pituitary, possibly to increase GnRH receptor number or action, but does not modulate GnRH release at the hypothalamus. The E2 induction of LH release, through multiple interactions with GnRH control, supports a possible central role of E2 in the sex change in the protandrous black porgy.


   FOOTNOTES
 
First decision: 15 May 2001.

1 This work was supported in part by the National Science Council (NSC 91-2313-B019-058), Taiwan. Back

2 Correspondence. FAX: 886 2 2462 1579; b0044{at}mail.ntou.edu.tw Back

Accepted: August 30, 2001.

Received: April 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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