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Am J Physiol Regul Integr Comp Physiol 278: R1572-R1578, 2000;
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Vol. 278, Issue 6, R1572-R1578, June 2000

GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish

Frank E. M. Rebers1, Peter T. Bosma2, Wytske van Dijk1, Henk J. T. Goos1, and Rüdiger W. Schulz1

1 Department of Experimental Zoology, Research Group for Comparative Endocrinology, Utrecht University, Padualaan 8, 3584 CH Utrecht; and 2 Department of Medical Oncology, Josephine Nefkens Institute, University Hospital Rotterdam, 3000 DR Rotterdam, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In African catfish, two gonadotropin-releasing hormone (GnRH) peptides have been identified: chicken GnRH (cGnRH)-II and catfish GnRH (cfGnRH). The GnRH receptors on pituitary cells producing gonadotropic hormone signal through inositol phosphate (IP) elevation followed by increases in intracellular calcium concentration ([Ca2+]i). In primary pituitary cell cultures of male African catfish, both cGnRH-II and cfGnRH dose dependently elevated IP accumulation, [Ca2+]i, and the release of the luteinizing hormone (LH)-like gonadotropin. In all cases, cGnRH-II was more potent than cfGnRH. The GnRH-stimulated LH release was not associated with elevated cAMP levels, and forskolin-induced cAMP elevation had no effect on LH release. With the use of pituitary tissue fragments, however, cAMP was elevated by GnRH, and forskolin was able to stimulate LH secretion. Incubating these fragments with antibodies against cfGnRH abolished the forskolin-induced LH release but did not compromise the forskolin-induced cAMP elevation. This suggests that cfGnRH-containing nerve terminals are present in pituitary tissue fragments and release cfGnRH via cAMP signaling on GnRH stimulation, whereas the GnRH receptors on gonadotrophs use IP/[Ca2+]i to stimulate the release of LH.

gonadotropin-releasing hormone neurons; luteinizing hormone; pituitary gonadotrophs; second messengers; hormone secretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RELEASE OF luteinizing hormone (LH) by pituitary gonadotrophs is stimulated by the neuropeptide gonadotropin-releasing hormone (GnRH). Several different GnRH decapeptides have been characterized to date, and in all vertebrates, two or three different forms of GnRH are expressed in the brain (17, 27). In the African catfish, Clarias gariepinus, catfish GnRH (cfGnRH)-producing neurons are scattered in the ventral forebrain, whereas a second group of neurons is found in the midbrain, producing chicken GnRH (cGnRH)-II (40). Nerve fibers containing cfGnRH and projecting into the proximal pars distalis of the pituitary are abundant, whereas cGnRH-II-containing fibers apparently do not project to the pituitary (40). Nevertheless, both GnRHs are found in the pituitary in relevant bioactive quantities (12), cfGnRH being 700-fold more abundant than cGnRH-II. The latter may reach the pituitary via the circulation. Both GnRHs stimulate the release of LH from the pituitary in vivo and in vitro, with cGnRH-II being the more potent form (29, 30).

Stimulation of LH release is accomplished after binding of GnRH to its membrane-bound receptor (GnRH-R) (33). Autoradiographic studies indicated that binding of radiolabeled GnRH is restricted to the gonadotrophs in primary pituitary cell cultures of male African catfish (3). In goldfish (Carassius auratus), on the other hand, GnRH-Rs are also present on the growth hormone (GH)-producing cells (8), and GnRH induced GH release (39). In African catfish, GnRH treatment had no effect on GH release (2, 20).

The GnRH-Rs are G protein-coupled receptors that mainly signal via activation of phospholipase C, resulting in the production of inositol-phosphates (IP; see Ref. 16 for review); GnRH-dependent IP formation has been shown in gonadotrophs from rat (34) and in primary cultures of goldfish gonadotrophs (5). The GnRH-R has been cloned from a number of species (31), including the African catfish (36). Elevation of IP formation has also been observed in pituitary cell lines transfected with GnRH-R cDNA constructs, such as GH3 cells stably transfected with rat GnRH-R (13). Also, in nonpituitary cells equipped with a GnRH-R, such as COS cells transfected with the human GnRH-R (24) or human embryonic kidney (HEK293) cells transfected with the catfish GnRH-R, elevation of IP levels has been observed on GnRH stimulation (36).

Inositol phosphates elevate intracellular calcium concentrations ([Ca2+]i) by release from intracellular stores (34). Primary cultures of mammalian gonadotrophs show spontaneous [Ca2+]i oscillations (35). On stimulation with GnRH, the oscillation frequency increases, and at higher GnRH concentrations, a rapid peak increase is followed by a plateau of increased [Ca2+]i (34). In goldfish gonadotrophs, a similar pattern was observed (25). A subset of the goldfish gonadotrophs reacted with a transient rise followed by a plateau phase, whereas other gonadotrophs reacted with a series of [Ca2+]i increases. In contrast to the situation in mammals, the [Ca2+]i changes in goldfish gonadotrophs depend on influx of extracellular calcium, but the two native GnRHs cause a different response (5, 14). In calcium-depleted medium, cGnRH-II-stimulated LH release is blocked, whereas salmon GnRH-induced LH secretion is only partially inhibited (14). Similarly in tilapia hybrids (Oreochromis niloticus × O. aureus) and in African catfish, gonadotropin release was reduced when pituitaries were stimulated with GnRH in the presence of a voltage-sensitive calcium channel blocker (22, 38).

Besides the calcium-signaling pathway, activation of adenylate cyclase and production of cAMP have also been implicated in the mediation of GnRH effects. However, the direct involvement of cAMP in GnRH-R signaling in gonadotrophs is a matter of debate. In gonadotrophs from goldfish (6), rat (7), or in alpha T3 cells (16), direct cAMP elevation on GnRH stimulation was not shown. Nevertheless, stimulation of goldfish gonadotrophs with cAMP induced the secretion of LH; however, this effect was additive to the GnRH-stimulated LH release (15). Stimulation of rat gonadotrophs with cAMP results in an elevation of the GnRH-stimulated LH secretion due to recruitment of gonadotrophs to the pool of releasing cells (4). Primary gonadotroph cultures of tilapia hybrids showed an increase in cAMP levels after stimulation with GnRH (21). In Sf9 cells stably transfected with the rat GnRH-R, cAMP elevation was observed with a maximum after 2 h, IP levels in this system being maximally stimulated after 30 min (9). In GH3 cells transfected with the rat GnRH-R, elevation of cAMP was observed only after 24 h (19). Also, HEK293 cells transfected with the catfish GnRH-R and a cAMP responsive reporter gene were shown to react with elevated cAMP levels after GnRH stimulation (36). These data indicate that the GnRH-R has the intrinsic capacity to activate the cAMP-generating system, whereas this capacity is not used in all cellular contexts. Moreover, cAMP may have effects in gonadotrophs that do not (directly) interfere with the GnRH signaling pathway.

Earlier studies on GnRH signal transduction in African catfish gonadotrophs indicated the involvement of cAMP (37). However, in these studies, the nonendogenous LH releasing hormone was used, and the experimental setup did not exclude juxtacrine effects between pituitary cells or a nongonadotroph origin of cAMP. In the present study, we reexamined the generation of second messengers, including besides cAMP, also IP and [Ca2+]i, on stimulation with the two endogenous GnRHs of the African catfish.


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

Fish. In this study mature male African catfish, Clarias gariepinus, 8-12 mo of age were used. The fish were bred and raised in the laboratory hatchery as described before (11), except that a catfish pituitary extract instead of human chorionic gonadotropin was used to induce ovulation.

Chemicals. All chemicals and solutions used in the cell/tissue culture procedures were from Gibco (Gaithersburg, MD) or Sigma Chemical (St. Louis, MO) unless stated otherwise. Other chemicals were from Sigma or Merck (Darmstadt, Germany).

Cell and tissue cultures. Primary pituitary cell cultures were prepared as described before (20), and 250,000 cells were plated out into 24-well culture plates (Costar; Acton, MA) in 800 µl L-15 medium [15 mM HEPES buffered, pH 7.4, 26 mM sodium bicarbonate, 1% (vol/vol) penicillin/streptomycin]. After 1.5 h, when the cells were attached to the plate, 200 µl horse serum (HS) were added to a final concentration of 5% (vol/vol). The GnRH-stimulated LH release was studied as described previously (2). All cultures were performed at 25°C in 5% CO2 in air at saturated humidity.

To study changes in [Ca2+]i or cAMP, gonadotrophs were enriched to ~75% (2) by Percoll density gradient centrifugation (10). Cells were rinsed four times with PBS (10 mM with 15 mM NaCl, pH 7.4) to remove the Percoll. For [Ca2+]i studies, 30,000 cells in 50 µl L-15 were put on glass coverslips in six-well culture plates (Costar) and cultured at 25°C, 5% CO2 for 1.5 h, after which 3 ml L-15 with 5% HS were added. For cAMP measurements, 125,000 cells were plated out in 24-well culture plates and processed as described above.

Pituitary tissue fragment cultures were performed by cutting the pituitaries into quarters and incubating each quarter in 500 µl L-15 with 5% HS overnight at 25°C and 5% CO2 in air. Thereafter, the fragments were incubated under the conditions described in cAMP measurements.

IP measurements. For the study of IP production, the cells were prepared and cultured as described in Cell and tissue cultures, except that dialyzed calf serum was used instead of HS. After overnight culture, 700 µl medium were removed and 0.3 µCi tritiated inositol (TRK 911, Amersham; Little Chalfont, UK) in 10 µl medium were added per well. After overnight incubation, the cells were washed three times by exchanging 700 µl medium with 700 µl L-15 without bicarbonate [L-15(-)]. After the third wash, 700 µl medium were removed, 150 µl 25 mM LiCl in L-15(-) were added, and the cells were incubated for another 10 min at 25°C. Next, 50 µl 10× concentrated GnRH solution in 25 mM LiCl in L-15(-) were added. After 1 h at 25°C, the medium was removed and the cells were lysed by incubation for 10 min on ice in 500 µl ice-cold chloroform and methanol (2:1). The chloroform-methanol mixture was transferred to 2.5-ml glass tubes, and 500 µl distilled water and 500 µl chloroform were added. After centrifugation (5 min at 2,000 g, 4°C), 500 µl of the top phase were transferred to 10-ml conic glass tubes containing 500 µl of a 1:1 Dowex slurry (1 × 8, 100-200 mesh; Fluka, Buchs, Switzerland) in distilled water. The Dowex was washed three times with 3 ml of water, after which IP was eluted by washing twice with 500 µl formic buffer (1.2 M ammonium formate, 0.1 M formic acid) and collecting the eluate. After addition of 3 ml Ultima Pro scintillation liquid (Packard; Meriden, CT) to the eluate, the vials were counted. The results are presented as percent differences after GnRH treatment compared with the control value.

Calcium determination. Relative [Ca2+]i determination was performed as described before (2). In short, enriched gonadotrophs attached to glass coverslips were loaded with 10 µM fura 2-AM (Molecular Probes; Eugene, OR) and 0.02% Pluronic (Molecular Probes) for 1 h at room temperature and washed four times with PBS (10 mM, 0.8% NaCl, 1 mM CaCl2, pH 7.4). The coverslip was transferred to a Leiden tissue culture dish and superfused with PBS (1 ml/min) for at least 5 min to allow the cells to equilibrate. The cells were incubated with GnRH at the desired concentration for 2 min via the superfusion medium. Changes in [Ca2+]i were determined by dynamic video imaging using the MagiCal hardware and TARDIS software from Joyce Loebl (Dukesway, Team Valley, Gateshead, Tyne and Wear, UK). Because catfish gonadotrophs do not show [Ca2+]i oscillations (2), the response to GnRH was quantified by averaging the four ratio frames around the fluorescence ratio maximum, i.e., the peak [Ca2+]i response.

cAMP measurements. Before stimulation, enriched gonadotrophs were rinsed three times, each time by exchanging 700 µl medium with 700 µl fresh L-15(-). After the third wash, 700 µl L-15(-), containing IBMX (final concentration of 0.3 mM) as well as GnRH or forskolin at the desired concentrations, were added. After 30 min of incubation at 25°C, the medium was removed and centrifuged for 10 min at 200 g and 4°C. The supernatant was stored at -20°C until assayed for LH content as described before (30). The cells were then lysed in 100 µl ice-cold 0.1 M HCl and stored at -20°C until assayed for cAMP.

Pituitary fragments were washed with L-15(-) and incubated for 3 h in 500 µl L-15(-) with 0.3 mM IBMX at 25°C. The medium was then collected and processed for LH quantification (basal secretion) as described before (30). The pituitary fragments were cultured for another 3 h in 500 µl L-15(-) with 0.3 mM IBMX and GnRH or forskolin in the presence or absence of 5% (vol/vol) rabbit preimmune serum (Arnell; New York) or 5% (vol/vol) rabbit antiserum raised against cfGnRH (29). The medium was collected for LH quantification (stimulated secretion). From the LH levels, the stimulation factor was calculated by dividing stimulated by basal LH secretion, thus accounting for possible differences in gonadotroph number between tissue fragments. Finally, 100 µl of ice-cold 0.1 M HCl were added to the pituitary fragments, which were stored in their incubation wells under 0.1 M HCl at -20°C until assayed for cAMP content.

Determination of cAMP content was performed according to Norstedt and Fredholm (26) with some adaptations. The cell lysate was thawed and 250 µl neutralization solution [NS; in mM: 85 Tris, 214 NaCl, 8.6 EDTA (pH 7.4 at 4°C), 40 NaOH, and 50 HEPES] was added. Pituitary fragments were transferred to Eppendorf vials, after which the wells were washed with 250 µl NS that were added to the same vials. The fragments were homogenized using a pestle, centrifuged for 10 min at 200 g and 4°C, and the supernatant collected. An aliquot of the sample (cell lysate or pituitary fragment homogenate) or cAMP standard (15-8,000 fmol), 25,000 counts/min tritiated cAMP (TRK 498, Amersham), and 200 µl protein kinase A (16 µg/ml) were incubated at 4°C for 3 h. Then, 300 µl dextrane-coated charcoal [1% Norit A (wt/vol), 0.1% dextran T-70 (wt/vol) in 30 mM sodium bisphosphate/60 mM disodium phosphate, pH 7.0 with 0.05% sodium azide, 0.9% NaCl, and 0.1% gelatin (all wt/vol)] were added, incubated for 5 min, and centrifuged at 5,400 g for 5 min at 4°C. The supernatant (500 µl) was counted.

Statistics. In all graphs, the values are given as means ± SE. Experiments were repeated at least three times, and representative data are presented. Multiple groups were compared by one-way ANOVA followed by Fisher's protected least-significant difference test. For comparing two groups unpaired, double-sided Student's t-tests were used. Both tests were calculated with StatView 4.5 for Windows (Abacus concepts, Berkely, CA). For the nonlinear fits of concentration curves, sigmoidal dose-response curves and the EC50 were calculated using GraphPad Prism 2.01 (GraphPad Software, San Diego, CA). Differences were considered statistically significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IP, calcium, and LH release. Stimulation of a primary pituitary cell culture for 1 h with cGnRH-II resulted in a dose-dependent increase in IP production (Fig. 1A). Similarly, cfGnRH induced dose-dependent increases in IP production, which reached a slightly higher maximum level than found after incubation with cGnRH-II. Curve fitting resulted in a EC50 of 0.82 nM for cGnRH-II and 1.74 µM for cfGnRH.


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  		Fig. 1.   Dose-response relationships after stimulation of African catfish primary cells in culture with chicken gonadotropin-releasing hormone (cGnRH)-II () or catfish gonadotropin-releasing hormone (cfGnRH; ) with respect to inositol phosphate (IP) accumulation (A) or intracellular Ca2+ concentration ([Ca2+]i) changes (B) and luteinizing hormone (LH) secretion (C). Bars in B represent fura 2 ratio in response to 1 µM cGnRH-II or 10 µM cfGnRH, respectively, in absence of extracellular calcium. All graphs represent means ± SE, n = 3-10 replicate experiments in A, n = 6-7 in B, and n = 6 (replicates in representative experiment) in C. * Significantly different plateau for cfGnRH compared with cGnRH-II (P < 0.05).

Primary cultures of enriched gonadotrophs were incubated for 2 min with different concentrations of cGnRH-II and cfGnRH to induce changes in [Ca2+]i (Fig. 1B). Fitting analysis of these data resulted in EC50 of 0.34 and 89 nM for cGnRH-II and cfGnRH, respectively. No difference in the plateau maximum between the two GnRHs was observed. We also tested the requirement of the presence of 1 mM extracellular calcium on 1 µM cGnRH-II- and 10 µM cfGnRH-induced changes of [Ca2+]i. There was no significant influence of the lack of extracellular calcium on the response to both GnRHs (bars in Fig. 1B), and both GnRHs significantly elevated [Ca2+]i to the same extent as in the presence of 1 mM extracellular calcium.

Both cGnRH-II and cfGnRH induced dose-dependent increases in the amount of LH secreted over a 30-min incubation period (Fig. 1C). Both GnRHs reached the same maximal plateau of secretion. Fitting analysis of these data resulted in EC50 of 0.8 and 262 nM for cGnRH-II and cfGnRH, respectively.

Gonadotropin release and cAMP-cell cultures versus tissue fragments. Stimulation of enriched gonadotrophs for 30 min with 1 µM forskolin greatly increased the intracellular level of cAMP, which, under control conditions or after GnRH stimulation, did not surpass the assay detection limit (15.6 fmol cAMP; Fig. 2A). As in previous experiments, stimulation of enriched gonadotroph cells with 0.1 µM cGnRH-II or 10 µM cfGnRH resulted in a stimulation of LH release, whereas incubation with 1 µM forskolin did not significantly elevate LH secretion (Fig. 2C).


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  		Fig. 2.   cAMP levels in gonadotroph cells (A) or pituitary tissue fragments (B) and LH release from gonadotroph cells (C) or pituitary tissue fragments (D). pi, Incubated with 5% preimmune serum; im, incubated with 5% anti-cfGnRH antiserum. All graphs show means ± SE from a representative experiment with n = 4 (A, C) or n = 11-18 (B, D). nd, Not detectable; bars sharing same letter do not differ significantly (ANOVA, followed by Fisher's protected least-significant difference test, alpha  = 0.05).

Incubation of pituitary tissue fragments, however, with 1 µM forskolin for 3 h (in the presence of preimmune serum), resulted in an increased LH release into the medium (Fig. 2D). As expected, also 10 µM cfGnRH and 10 nM cGnRH-II (data not shown) elevated the LH secretion from the pituitary tissue fragments. Forskolin induced a large increase in the cAMP content of the pituitary tissue fragments (Fig. 2B). Unexpectedly, cfGnRH (Fig. 2B) and cGnRH-II (data not shown) also increased the cAMP level, although to a lower extent than forskolin. Replacing the preimmune serum by an antiserum raised against cfGnRH abolished the release of LH in response to forskolin (Fig. 2D). Preimmune serum had no influence on basal LH release, whereas antiserum against cfGnRH attenuated basal LH secretion (Fig. 2D).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows that both cfGnRH and cGnRH-II stimulate secretion of LH from a primary pituitary cell culture in a dose-dependent manner and that elevations of IP levels and [Ca2+]i participate in GnRH signal transduction. Both GnRHs share a simple dose-response relationship in regards to LH release, cGnRH-II being 325-fold more potent than cfGnRH; this difference may be attributed to the difference in affinity of these two endogenous ligands to the GnRH-R (29, 30).

In the catfish gonadotrophs, both cfGnRH and cGnRH-II give dose-dependent increases in the IP accumulation. This is in line with previous reports on responses to GnRH by gonadotrophs from goldfish (5) and rat (34). Similarly in the gonadotroph-derived cell line alpha T3 (1), the lactotroph-derived cell line GH3 expressing the rat GnRH-R (13), or COS-1 cells transfected with the human GnRH-R (24), elevation of IP was observed after GnRH stimulation. The present study indicates that in the African catfish also, both IP and [Ca2+]i are elevated after GnRH stimulation.

Besides IP and [Ca2+]i, cAMP has also been implicated in the release of LH in goldfish (6), African catfish (37), or tilapia (21). The present study shows that GnRH does not increase cAMP levels in primary cultures of catfish pituitary cells (enriched gonadotrophs), nor does forskolin stimulate LH release from these cells. Thus apparently cAMP is not directly involved in GnRH-R signal transduction in catfish gonadotrophs. A similar conclusion was drawn for primary cultures of goldfish pituitary cells in which GnRH did not elevate cAMP levels (6) and in which inhibition of the cAMP pathway did not influence GnRH-stimulated LH secretion (15). Nevertheless, forskolin slightly elevated LH release from perifused goldfish pituitary cells, which was attributed to an indirect effect of cAMP (15). The fact that stimulation of static cultures of catfish gonadotrophs with forskolin did not change the LH release may be based on the different incubation conditions (static vs. perifusion). On the other hand, in HEK293 cells transfected with the catfish GnRH-R, addition of GnRH stimulated the expression of a cAMP-dependent reporter gene (36). Similarly, the rat GnRH-R activates adenylate cyclase in heterologous environments (9, 32, 33). It appears that the GnRH-R is able to elevate cAMP levels but that this pathway is not a prominent one regarding the GnRH signaling in gonadotrophs in most (e.g., catfish, goldfish, or rat), but not all (e.g., tilapia) species; compounds other than GnRH, for example, pituitary adenylate cyclase-activating polypeptide (PACAP) may be more important in regulating cAMP levels in gonadotrophs (23, 28). Moreover, the increases of cAMP after GnRH stimulation are relatively slow (19) and follow the faster reaction of IP and [Ca2+]i, which correspond better to the time scale of LH release.

As regards the role of cAMP in GnRH-stimulated LH secretion, strikingly different results were obtained in the present study with catfish pituitary fragments versus primary cell cultures. In tissue fragments, but not in primary cell cultures, cAMP is elevated after the addition of GnRH, and the addition of forskolin stimulated LH release. This is in contrast to the situation in tilapia in which the results did not depend on using pituitary fragments or cell cultures (21), as GnRH always elevated cAMP levels. The results from the experiments with catfish pituitary cell cultures suggested that cAMP has no direct effect on LH secretion in catfish. To reconcile this with the observations made with catfish pituitary tissue fragments, we sought evidence for a cAMP-mediated but indirect effect on LH secretion. In this context, it is important to note that in teleost fish, the GnRH neurons directly contact pituitary gonadotrophs via their axons, whereas a portal blood vessel system connecting the median eminence and the adenohypophysis is missing (40). The pituitary fragments, as used in the present study, thus contain cfGnRH-containing nerve fibers. Indeed, we have shown that 95% of all cfGnRH present in the brain and pituitary of adult catfish is found in the pituitary (12).

We hypothesize that the nerve terminals present in the catfish pituitary fragments (40) may release their stored cfGnRH on elevation of the cAMP level. Furthermore, because GnRH-Rs are expressed by GnRH neuronal cells (GT1 cells) and because GnRH analogs were shown to have autocrine effects on GnRH release in these cells (18), GnRH may induce secretion of cfGnRH via a cAMP-involving pathway. Within such a model, a primary release of cfGnRH in response to electrical stimulation would result in the binding of cfGnRH to GnRH-R on its own nerve terminals. This would elevate cAMP levels in these terminals leading to a further release of cfGnRH; such a mechanism may enable a cfGnRH release burst. Catfish GnRH will also bind to GnRH-R on gonadotrophs to stimulate LH secretion via the IP/ [Ca2+]i-dependent pathway.

This model predicts that forskolin-induced LH secretion by catfish pituitary tissue fragments does not reflect a direct effect of cAMP on gonadotrophs, but rather a cAMP-dependent cfGnRH secretion from cfGnRH nerve terminals. The latter should be sensitive to immunoneutralization by an antiserum against cfGnRH, which, however, should not modulate the forskolin-induced elevation of cAMP levels. Moreover, cfGnRH should elevate cAMP levels in the tissue fragments (i.e., GnRH-R-equipped, cfGnRH-containing nerve terminals). Indeed, incubating pituitary tissue fragments with forskolin in the presence of antibodies against cfGnRH abolished the effect of forskolin on LH secretion but not on cAMP accumulation. Moreover, cfGnRH elevated cAMP levels in the tissue fragments. This small but significant response may reflect the contribution of the cfGnRH-containing nerve terminals to the total cAMP response evoked by forskolin, which is probably composed of contributions by several cell types in the pituitary tissue fragment. The fact that immunoneutralization of extracellular cfGnRH is able to dissociate the two effects forskolin has on tissue fragments (increased cAMP and increased LH secretion) is evidence to support the existence of a cAMP-mediated release of an LH release-inducing factor from pituitary tissue fragments of adult male African catfish exists. Because primary cultures of the pituitary gonadotrophs do not show this phenomenon, we attribute the forskolin effect to a cAMP-induced release of cfGnRH from the nerve terminals.

We cannot exclude that GnRH elevated cAMP in the pituitary fragments in cells other than the gonadotrophs. Previous work has shown, however, that catfish gonadotrophs are the only cells in a primary pituitary cell culture showing GnRH binding activity (3). We also cannot exclude that forskolin induced the secretion of a synergistic factor acting on LH release, for instance PACAP. Such a mechanism, if present, is probably of minor relevance, because antiserum against cfGnRH completely abolished the forskolin effect on LH release in tissue-fragment experiments. Finally, it is interesting to note that incubation with cfGnRH antiserum decreased "basal" LH secretion, indicating that when working with tissue fragments, LH secretion in the absence of exogenous GnRH comprises the real, non-GnRH-stimulated basal LH secretion plus the LH secretion induced by the endogenous release of cfGnRH.

In summary, in the gonadotrophs in the African catfish pituitary, both cfGnRH and cGnRH-II regulate LH release by directly influencing the gonadotroph cells via IP/[Ca2+]i signaling. Moreover, we propose that both GnRHs have the potential to stimulate cfGnRH secretion in the vicinity of the gonadotrophs through a cAMP-dependent mechanisms in the cfGnRH-containing, GnRH-R-expressing nerve terminals.


    ACKNOWLEDGEMENTS

The authors thank Dr. P. H. G. M. Willems (Dept. of Biochemistry, Catholic Univ. of Nijmegen, Nijmegen, The Netherlands) for help with the calcium determinations.


    FOOTNOTES

This research was funded by a grant from the Netherlands Organization for Research.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. W. Schulz, Dept. of Experimental Zoology, Research Group for Comparative Endocrinology, Utrecht Univ., Padualaan 8, 3584 CH Utrecht, The Netherlands (E-mail: R.W.Schulz{at}bio.uu.nl).

Received 4 October 1999; accepted in final form 5 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, L, Mcgregor A, Cook JV, Chilvers E, and Eidne KA. Rapid desensitization of GnRH-stimulated intracellular signaling events in alpha T3-1 and HEK-293 cells expressing the GnRH receptor. Endocrinology 136: 5228-5231, 1995[Abstract].

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4.   Cassina, P, Sellers J, and Neill JD. Effect of cAMP on GnRH-stimulated LH secretion from individual pituitary gonadotropes. Mol Cell Endocrinol 114: 127-135, 1995[Web of Science][Medline].

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Am J Physiol Regul Integr Comp Physiol 278(6):R1572-R1578
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