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Biology of Reproduction 62, 731-738 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Inhibitory and Stimulatory Interactions Between Endogenous Gonadotropin-Releasing Hormones in the African Catfish (Clarias gariepinus)1

Peter T. Bosma3,a, Frank E.M. Rebers4,a, Wytske van Dijka, Peter H.G.M. Willemsb, Henk J.Th. Goosa, and Rüdiger W. Schulz2,a

a University of Utrecht, Faculty of Biology, Department of Experimental Zoology, Research Group Reproductive Endocrinology, 3508 TB Utrecht, The Netherlands b Catholic University of Nijmegen, Department of Biochemistry, 6500 HB Nijmegen, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the brain of all vertebrate classes, chicken (c) GnRH-II ([His5,Trp7,Tyr8]GnRH, cGnRH-II) is expressed in the mesencephalon. In addition, at least one other form of GnRH is expressed in the preoptical area/hypothalamus. In the human pituitary stalk and the mouse median eminence, cGnRH-II is present together with mammalian GnRH. Similarly, in the pituitary of several teleost fish (e.g., goldfish and eel, but not salmon or trout), a teleost GnRH is found together with cGnRH-II. These GnRHs are not colocalized in the same cells. Hence, these GnRH peptides may differentially regulate gonadotropin secretion and, in addition, may exert their effects simultaneously. The current study therefore investigated the effects of combinations of the two forms of GnRH present in the African catfish (Clarias gariepinus) pituitary—cGnRH-II and catfish GnRH ([His5,Asn8]GnRH, cfGnRH)—on the cytosolic free calcium concentration ([Ca2+]i) in single, Fura-2-loaded catfish gonadotrophs, as well as their effects on both in vitro and in vivo LH secretion. Both inhibitory and stimulatory effects of combinations of cfGnRH and cGnRH-II on [Ca2+]i were observed, which were mirrored by their effects on both in vitro and in vivo LH secretion. The following pattern became apparent. The effect of intermediate or maximal effective cfGnRH doses was inhibited by the simultaneous presence of subthreshold or borderline effective cGnRH-II doses. Conversely, subthreshold or borderline effective concentrations of cfGnRH enhanced the effects of intermediate and maximal concentrations of cGnRH-II. In addition, combinations of cfGnRH and cGnRH-II concentrations that were equally active when tested separately showed an additive effect. The observed interactions between the two GnRHs may be of particular physiological relevance in the control of seasonal LH levels in the African catfish, as well as in other teleost species. Moreover, the occurrence of mutual inhibitory and stimulatory interactions between endogenous GnRHs may be a widespread aspect of GnRH action in vertebrates.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To date, GnRHs form a family of twelve decapeptides [1]. In all vertebrates, two or three forms of GnRH are expressed in the brain [2–5]. In the African catfish, Clarias gariepinus, chicken GnRH-II ([His5,Trp7,Tyr8]GnRH; cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH; cfGnRH) were identified [6]. Chicken GnRH-II is found in all vertebrates [5], whereas cfGnRH has been identified to date in the Thai (Clarias macrocephalus) and the African catfish only [6, 7].

The primary function of GnRH is to regulate the synthesis and secretion of pituitary gonadotropin. Teleost fish lack a functional hypothalamo-hypophyseal portal system. Instead, the pituitary is directly innervated by neurosecretory fibers from the brain [8–10]. Regarding the African catfish, we have shown that cfGnRH-producing neurons form a prominent, hypophysiotropic fiber tract, while the cGnRH-II-producing neurons do not appear to project to the pituitary [11]. Nevertheless, both cfGnRH and cGnRH-II were identified in pituitary extracts, and both stimulated the release of LH, with cGnRH-II being more potent than cfGnRH [12, 13]. Hence, it is reasonable to assume that both endogenous GnRHs are involved in the regulation of the gonadotrophs in catfish.

We have previously shown that gonadotrophs were the only cell type in an African catfish pituitary cell suspension displaying GnRH binding activity [14]. Furthermore, we demonstrated that the effects of both cfGnRH and cGnRH-II on the cytosolic free calcium concentration ([Ca2+]i) in gonadotrophs in vitro are correlated with their potency to induce LH secretion, both in vitro and in vivo [12, 14]. Recently, a GnRH receptor was cloned from African catfish pituitary [15]. In a cell line transfected with this receptor, the relative efficacies of cfGnRH and cGnRH-II to stimulate second messenger systems corresponded with the effects of GnRHs on in vivo and in vitro release of LH [12, 14, 15]. In addition, when the cloned catfish GnRH receptor was challenged with both endogenous GnRHs simultaneously, mutual inhibitory effects were observed on the second messenger response of the transfected cells [15]. The present study addresses possible mutual modulating effects of both GnRHs through the catfish GnRH receptor in its natural place by investigating the [Ca2+]i response of gonadotrophs upon simultaneous challenges of cfGnRH and cGnRH-II. Further, we studied the possibility that the two peptides modulate each other's LH release activity, both in vivo and in vitro.


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

African catfish were bred and raised in the laboratory hatchery as described previously [16], except that catfish pituitary extract instead of hCG was used to induce ovulation. Mature male fish (8–10 mo old) were used in all experiments.

GnRH Peptides

Synthetic cfGnRH (American Peptide Co., Sunnyvale, CA) and cGnRH-II (Peninsula Laboratories, Belmont, CA) were used in all experiments. An analogue of salmon GnRH ([D-Arg7,Leu8,Pro9-NEt]GnRH, sGnRHa; Syndel Laboratories Ltd., Vancouver, BC, Canada) was used to determine maximally stimulated LH plasma levels [17].

In Vitro Experiments with Dispersed Pituitary Cells or Enriched Gonadotrophs

The preparation of a pituitary cell suspension and the culture conditions have been described previously [14, 18, 19]. Briefly, fish were killed by decapitation, and pituitaries were removed under sterile conditions. Pituitary glands were minced with a scalpel, and tissue fragments were transferred to 5 ml PBS-Dulbecco, pH 7.4 (Gibco/BRL, Paisley, UK), containing 0.3% (w:v) BSA (fraction V; Sigma, St. Louis, MO), 0.1% collagenase (type I; Sigma), and 0.125% (w:v) dispase-II (Boehringer, Mannheim, Germany). Enzymatic dispersion was performed in a water bath (25°C) for 1 h with shaking (60 cycles/min). Tissue fragments were collected by centrifugation (5 min, 200 x g) and washed in 5 ml Ca2+- and Mg2+-free PBS-Dulbecco, pH 7.4, containing 0.3% (w:v) BSA (CMF-PBS) and 0.04% (w:v) EDTA (Sigma). After centrifugation (5 min, 200 x g), the pituitary fragments were resuspended in 5 ml CMF-PBS containing 0.125% dispase-II and returned to the water bath (25°C; 60 cycles/min) for 30 min. Fragments were allowed to settle, and subsequently the supernatant containing single cells was transferred to a centrifuge tube. Remaining tissue fragments were treated again with CMF-PBS containing 0.04% (w:v) EDTA, and then by CMF-PBS containing 0.125% dispase-II as described above, after which the single cell suspensions were pooled and centrifuged (10 min, 200 x g). Cells were washed with 5 ml CMF-PBS containing 0.08% (w:v) EDTA, centrifuged (5 min, 200 x g), and resuspended in 5 ml PBS-Dulbecco, pH 7.4, containing 0.3% (w:v) BSA. The cell suspension was filtered through nylon gauze (50-µm mesh), centrifuged (5 min, 200 x g), and resuspended in sodium bicarbonate-buffered (26 mM) L-15 medium containing 1% (v:v) penicillin-streptomycin mixture (Gibco/BRL).

For static culture experiments, dispersed cells were plated at a density of 125 000 cells/well in 48-well tissue culture plates (Costar, Cambridge, MA) [14]. Cells were incubated for 2 days in 500 µl of sodium bicarbonate-buffered (26 mM) L-15 medium containing 5% (v:v) horse serum (Gibco/BRL) before GnRH treatment was begun. Before an experiment, cells were washed three times with medium, with 350 µl exchanged each time, thus leaving 150 µl of medium on the cells to prevent their exposure to air. Cells were allowed to rest for 30 min at 25°C. Then, 350 µl was exchanged with medium containing freshly diluted cfGnRH and cGnRH-II, either alone or in combination (6 wells per treatment). After 30 min of incubation, medium was collected and centrifuged (10 min, 200 x g, 4°C), and the supernatant was stored at -20°C until assayed for LH by RIA [20].

For [Ca2+]i measurements, enrichment of gonadotrophs by density gradient centrifugation of dispersed pituitary cells was carried out as described previously [19]. The enriched population contained 73 ± 3% gonadotrophs, n = 6, as determined immunocytochemically [14]. Cells were cultured overnight on glass coverslips in 6-well tissue culture plates (Costar) at a density of 30 000 cells/coverslip in 5 ml sodium bicarbonate-buffered (26 mM) L-15 medium containing 5% (v:v) horse serum. Before an experiment, cells were washed four times with 4 ml medium, leaving 1 ml medium in the wells to cover the cells between wash steps. The cells were loaded with 10 µM Fura-2/AM (Molecular Probes, Eugene, OR) in 1 ml L-15 medium in the presence of 0.02% (w:v) Pluronic (Molecular Probes) [21] for 1 h at room temperature. The cells were subsequently washed four times with 5 ml PBS containing 1 mM CaCl2, pH 7.4, placed in a Leiden perifusion chamber (volume 400 µl) [22], and superfused with the same buffer for 5 min at a flow rate of 1 ml/min. [Ca2+]i measurements on single cells were carried out by dynamic video imaging using the MagiCal hardware and TARDIS software provided by Joyce Loebl (Dukesway, Team Valley, Gateshead, Tyne and Wear, UK) as described previously [14, 23]. In brief, the relative changes in [Ca2+]i were measured as the fluorescence emission ratio at 492 nm after excitation at 340 and 380 nm. The cells were challenged by adding GnRH to the superfusion buffer. On average, 28 ± 2 (SEM, n = 15) single cells were monitored simultaneously. Responding and nonresponding cells were counted during an accelerated replay of the consecutive emission frames. The [Ca2+]i response to GnRH was quantified in each reacting cell, by taking the four ratio frames with the highest fluorescence intensity (peak [Ca2+]i response) and calculating the mean of these four frames.

LH Plasma Levels

Groups of eight fish received i.p. injections of different dosages of cfGnRH and cGnRH-II, either alone or in combination. Blood samples were taken just before and 60 min after injection, when peak levels of circulating gonadotropic hormone (GTH) II are observed [12]. To determine the maximum LH response, 5 µg sGnRHa/kg BW was injected [17]. Plasma samples were frozen and stored at -20°C until assayed for LH by RIA [20].

Data Analysis

All data on the effects of combinations of cfGnRH and cGnRH-II on the [Ca2+]i in gonadotrophs and the release of LH were subjected to ANOVA, followed by Fisher's least-significant-difference test ({alpha} = 0.05). Two-group comparisons were done by a two-tailed Student's t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Combined GnRH Treatment on [Ca2+]i In Vitro

Both cfGnRH and cGnRH-II transiently increased [Ca2+]i in 68 ± 5% of the cells (mean ± SEM; 15 coverslips; 413 cells) in a primary culture of enriched gonadotrophs (Fig. 1). Figure 2 (insert) shows that the peak increase in [Ca2+]i was a function of the GnRH concentration. The maximal value for this peak increase was reached with 1 µM cfGnRH and 10 nM cGnRH-II, while both GnRHs were equally effective at these concentrations. Figure 2 shows that a subthreshold (1 pM) cGnRH-II concentration reduced the effect of an intermediate (0.1 µM) cfGnRH concentration (Fig. 2A), but did not affect the stimulatory action of a maximal (1 µM) cfGnRH concentration (Fig. 2B). Conversely, a subthreshold (0.1 nM) cfGnRH concentration potentiated the effect of both an intermediate (1 nM; Fig. 2C) and a maximal (10 nM; Fig. 2D) cGnRH-II concentration. Also, the combined action of maximal concentrations of 1 µM cfGnRH and 10 nM cGnRH-II was higher than the effect of each GnRH alone (Fig. 2E). Finally, no mutual effect was observed when both GnRHs were used at intermediate (0.1 µM cfGnRH and 1 nM cGnRH-II; Fig. 2F) concentrations.



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  		FIG. 1. GnRH-induced increases in the Fura-2 ratios in individual gonadotrophs challenged with cfGnRH (top) or cGnRH-II (bottom). The concentration and duration of GnRH in the medium is represented on the abscissae. Note the absence of spontaneous or GnRH-induced oscillations



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  		FIG. 2. Peak [Ca2+]i responses (Fura-2 ratios) of single gonadotrophs (1-min GnRH challenge) to cfGnRH, cGnRH-II, and combinations of these GnRHs. Bars represent the mean ± SEM of 8–30 gonadotrophs. An asterisk indicates a significant difference between basal and GnRH-challenged Fura-2 ratios, tested by a two-tailed Student's t-test (P < 0.05). Groups sharing the same lowercase letter are not significantly different (Fisher's least-significant-difference test, {alpha} = 0.05). The insert shows control and dose-response curves of Fura-2 ratios to cfGnRH and cGnRH-II

Effects of Combined GnRH Treatment on LH Release In Vitro

Both cfGnRH and cGnRH-II stimulated the release of LH from a primary culture of enzymatically dispersed, unfractionated catfish pituitary cells in a dose-dependent fashion (Fig. 3; insert). Maximal secretion was reached with 1 µM cfGnRH and 1 nM cGnRH-II, and both GnRHs were equally effective at these concentrations. Figure 3 shows that a subthreshold (1 pM) cGnRH-II concentration completely inhibited the stimulatory effect of an intermediate (0.1 µM) cfGnRH concentration (Fig. 3A). Inhibition was also observed when either a subthreshold (1 pM; Fig. 3B) or a borderline effective (10 pM; Fig. 3C) cGnRH-II concentration was added in combination with a maximal (1 µM) cfGnRH concentration. Conversely, a subthreshold (1 nM) cfGnRH concentration did not interfere with the stimulatory action of an intermediate (0.1 nM) cGnRH-II concentration (Fig. 3D), whereas a borderline effective (5 nM) cfGnRH concentration potentiated the stimulatory effect of a maximal (1 nM) cGnRH-II concentration (Fig. 3E). Finally, neither inhibition nor additivity was observed when maximal concentrations of both GnRHs were used (1 µM cfGnRH and 1 nM cGnRH-II; Fig. 3F).



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  		FIG. 3. LH levels (ng/125 000 cells) in the incubation medium of a primary culture of enzymatically dispersed, unfractionated catfish pituitary cells, after 30-min incubations with cfGnRH, cGnRH-II, and combinations of these GnRHs. Bars represent the mean ± SEM (n = 6 wells/group). An asterisk indicates a significant difference between control and GnRH-challenged LH levels, tested by a two-tailed Student's t-test (P < 0.05). Groups sharing the same lowercase letter are not significantly different (Fisher's least-significant-difference test, {alpha} = 0.05). The insert shows control and dose-response LH levels to cfGnRH and cGnRH-II.

Effects of Combined GnRH Treatment on LH Release In Vivo

Both GnRHs dose-dependently increased the plasma LH level when injected i.p. (Fig. 4; insert). Maximal effects were reached with 250 µg/kg BW cfGnRH and 5 µg/kg BW cGnRH-II. Figure 4 shows that both a subthreshold (0.025 µg/kg BW; Fig. 4A) and a borderline effective (0.5 µg/kg BW; Fig. 4B) cGnRH-II dose markedly inhibited the stimulatory effect of a maximal (250 µg/kg BW) cfGnRH dose. By contrast, a subthreshold (2.5 µg/kg BW) cfGnRH dose potentiated the effect of an intermediate (2.5 µg/kg BW) cGnRH-II dose (Fig. 4C), while a borderline effective (5 µg/kg BW) cfGnRH dose did not affect the stimulatory action of a maximal (5 µg/kg BW) cGnRH-II dose (Fig. 4D). Potentiation was also observed when fish received coinjections of either borderline effective (5 µg/kg BW cfGnRH and 0.1 µg/kg BW cGnRH-II; Fig. 4E) or maximal (250 µg/kg BW cfGnRH and 5 µg/kg BW cGnRH-II; Fig. 4F) concentrations of both GnRHs.



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  		FIG. 4. LH blood levels (ng/ml plasma) in mature male catfish, 1 h after i.p. injection of cfGnRH, cGnRH-II, and combinations of these GnRHs (µg/kg BW). Bars represent the mean ± SEM (n = 8 animals/GnRH treatment). An asterisk indicates a significant difference between pre- and postinjection LH levels, tested by a two-tailed Student's t-test (P < 0.05). Groups sharing the same lowercase letter are not significantly different (Fisher's least-significant-difference test, {alpha} = 0.05). The insert shows control and dose-response LH levels to cfGnRH and cGnRH-II.

The data obtained in the in vitro and in vivo studies are summarized in Figure 5. Results from combinations tested, but not shown in Figures 2–4, are also included to provide a complete record of our observations. From this presentation, it appears that the mutual modulatory effects of cfGnRH and cGnRH-II on Ca2+ mobilization were mirrored by their effects on LH release. Furthermore, it appears that the effects of the different GnRH combinations fall into two groups, as indicated by the diagonal line in Figure 5. Combinations below this line were either inhibitory or did not exert mutual effects, but were never stimulatory. These are combinations in which the cfGnRH dose was more effective than the cGnRH-II dose (e.g., an intermediate cfGnRH dose combined with a borderline effective cGnRH-II dose). Conversely, combinations above this line were either stimulatory or did not exert mutual effects, but were never inhibitory. In these combinations, cfGnRH and cGnRH-II doses were either equally active (e.g., maximal doses of both peptides), or the cGnRH-II dose was more effective than the cfGnRH dose (e.g., an intermediate dose of cGnRH-II combined with a subthreshold dose of cfGnRH).



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  		FIG. 5. Summary of the effects of combinations of cfGnRH and cGnRH-II on the Ca2+ mobilization in single, Fura-2-loaded catfish gonadotrophs, as well as their effects on both in vitro and in vivo LH secretion. Bars along the axes indicate effectiveness levels of GnRH concentrations/doses. Arrows indicate an inhibited (down arrow), enhanced (up arrow), or unchanged (no arrow) effect of the combination compared to the effects of either of the two GnRHs alone


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study provides evidence for mutual modulatory effects of the two endogenous GnRHs in catfish on their activities as LH secretagogues. Both the inhibitory and stimulatory interactions of combinations of cfGnRH and cGnRH-II on LH release were paralleled by their effects on Ca2+ mobilization.

To our knowledge, this is the first study showing an inhibitory action of one GnRH on the stimulated release of gonadotropin by another GnRH. Subthreshold or borderline effective doses of cGnRH-II consistently impaired intermediate and maximal cfGnRH-induced LH secretion. The characteristics of this cGnRH-II inhibitory action on the cfGnRH-stimulated release of LH may provide some insight into the presently unknown mechanism of action underlying this phenomenon. First, cGnRH-II inhibited only when the dose of cfGnRH alone was more effective than cGnRH-II alone. Furthermore, the combined response was never below the response to cGnRH-II alone. In rats, GnRH can decrease blood gonadotropin levels by blocking GnRH release via a negative ultrashort feedback loop [24–27]. However, the inhibition of cfGnRH-stimulated LH secretion by cGnRH-II was observed in vivo as well as in vitro, suggesting that the inhibitory effect was exerted directly on the pituitary gonadotrophs. This implies a receptor-mediated mechanism of action for cGnRH-II. Indeed, a cGnRH-II concentration that was ineffective by itself also impaired the cfGnRH-induced stimulation of second messenger pathways in a cell line transfected with the catfish GnRH receptor [15]. Although the mechanism of the inhibiting action of cGnRH-II on the cfGnRH effect remains unknown, our results indicate that cGnRH-II is a partial antagonist of LH secretagogues.

Conversely, combining subthreshold or borderline effective doses of cfGnRH with intermediate and high doses of cGnRH-II resulted in an enhanced LH and [Ca2+]i response compared to cGnRH alone (Fig. 5). Moreover, combinations of equally active doses of both GnRHs were more effective than the effect of each GnRH alone (Fig. 5). As in the case of the inhibitory effects of cGnRH-II, the mechanism of action underlying this phenomenon is presently unknown. However, the enhanced action of these combinations occurred in vivo and in vitro, suggesting an effect directly on the pituitary gonadotrophs through a receptor-mediated mechanism of action. Surprisingly, this enhanced action is in contrast with the second messenger response of the cloned catfish GnRH receptor, which showed an inhibition when challenged with these combinations of both peptides [15]. The different response of the cloned catfish GnRH receptor may result from a difference between the activation of second messenger systems by the cloned receptor in HEK 293 cells [15] and the second messenger activation by the native receptor in the catfish gonadotroph. However, the present results may alternatively indicate that a second GnRH receptor type is present in the catfish gonadotroph, through which the action of stimulatory GnRH combinations is regulated. Recently, two GnRH receptors were cloned from goldfish brain and pituitary [28]. Also in African catfish, two different GnRH receptor subtypes are present, showing rather similar characteristics in activating second messenger signalling systems in transfected HEK 293 cells (unpublished results). This opens the possibility of mutual interactions of signalling cascades. Moreover, heterodimerization of the G-protein-coupled {delta}- and K-opioid receptors has been reported to result in a new receptor that exhibits functional properties distinct from those of either receptor [29]. Hence, it is possible that, via homo- and heterodimerization, three receptor entities with distinct interactions of signalling cascades are present in the catfish gonadotroph, through which the interactions between cfGnRH and cGnRH-II are modulated.

The presently observed inhibitory and stimulatory interactions between both GnRH peptides may be physiologically relevant. In the catfish pituitary, cfGnRH is present in a 700-fold excess over cGnRH-II [13]. However, cGnRH-II is much more potent than cfGnRH ([12–14], present study), which may well compensate for the excess of cfGnRH over cGnRH-II in the pituitary. There is a direct projection of cfGnRH-producing neurons from the ventral forebrain into the pituitary, while the cGnRH-II-producing neurons in the midbrain tegmentum do not appear to project to the pituitary [11]. Instead, cGnRH-II may reach the pituitary via the general circulation, by binding to serum GnRH binding proteins as in goldfish [30, 31] and dogfish [32], or via the cerebrospinal fluid [10, 33]. The fish used for these experiments were raised and kept in the laboratory hatchery. These fish mature sexually. However, they do not spawn spontaneously but remain in a condition comparable to the prespawning phase under natural conditions. In these fish, cGnRH-II may be one of the factors that keep circulating LH levels relatively low throughout the year [34]. During the annual reproduction cycle under natural conditions, LH plasma levels likewise are fairly low, with LH surges occurring only at the time of spawning [35]. During the prespawning period, one of the factors helping to maintain low circulating LH levels may be cGnRH-II, by its partially inhibition of the cfGnRH-stimulated GTH II secretion. At the time of spawning, high plasma LH levels are required; these may be generated by increased cGnRH-II amounts in the pituitary which, together with cfGnRH, and possibly in combination with a reduced dopaminergic inhibition [16], result in the spawning-associated LH surges, as proposed previously [12]. A first indication whether this hypothesis is valid could be obtained by measuring pituitary concentrations of cfGnRH and cGnRH-II during the natural spawning cycle of African catfish.

This study shows that, even in a situation with great differences between the GnRHs regarding their LH release activity and their pituitary content, the GnRHs can instantly modulate each other's effects as LH secretagogues. As the presence of multiple forms of GnRH has also been shown in the pituitary of a number of other teleost species [2, 36, 37], as well as in the hypothalamus of humans and rodents [4], mutual modulatory effects on the LH release activities of endogenous GnRHs may not be restricted to African catfish.

In conclusion, the GnRHs in the African catfish can, in addition to their direct stimulatory action on LH release, modulate each other's LH release activity. This mutual modulation can be both stimulatory and inhibitory, the final result depending on the relative concentrations of cfGnRH and cGnRH-II. The expression of multiple GnRHs in the brains of all vertebrates suggests that similar interactions between different endogenous GnRHs may also occur in humans and other vertebrates.


   FOOTNOTES
 
First decision: 29 March 1999.

1 This study was financially supported by the Netherlands Organization for Research (NWO). Preliminary results have been presented (abstract 152) at the 17th Conference of European Comparative Endocrinologist, Córdoba (Spain), 5-10 September 1994. Back

2 Correspondence: R.W. Schulz, University of Utrecht, Faculty of Biology, Department of Experimental Zoology, Research Group Reproductive Endocrinology, P.O. Box 80058, 3508 TB Utrecht, The Netherlands. FAX: 31 30 2532837; r.w.schulz{at}bio.uu.nl Back

3 Current address: Department of Medical Oncology, Josephine Nefkens Institute, University Hospital Rotterdam, Rotterdam, The Netherlands. Back

4 Current address: Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Experimental Immunohematology, Amsterdam, The Netherlands. Back

Accepted: November 4, 1999.

Received: March 4, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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