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Biology of Reproduction 66, 1604-1611 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Gonadotropin-Releasing Hormone Does Not Directly Stimulate Luteinizing Hormone Biosynthesis in Male African Catfish1

F.E.M. Rebers3,a, G.A.M. Hassinga, W. van Dijka, E. van Straatena, H.J.Th. Goosa, and R.W. Schulz2,a

a Utrecht University, Faculty Biology, Research Group Endocrinology, NL-3584 CH Utrecht, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Besides gonadotropin release, GnRH stimulates gonadotropin subunit gene transcription and translation in gonadotrophs. In the African catfish, Clarias gariepinus, chicken GnRH-II (cGnRH-II: [His5,Trp7,Tyr8]-GnRH) and catfish GnRH (cfGnRH: [His5,Asn8]-GnRH) are two endogenous forms of GnRH. Studying their effects on LH subunit steady-state mRNA levels, LH de novo synthesis, and LH release in primary pituitary cell cultures of adult males, we found that cGnRH-II hardly influenced the steady-state levels of LH subunit mRNAs or LH de novo synthesis, although it stimulated LH release. Although cfGnRH stimulated LH secretion as well, high concentrations—although apparently still within the physiologic range—reduced LH transcript levels and de novo synthesis in primary pituitary cell cultures. In vivo experiments demonstrated a biphasic response of LH subunit transcript levels after a single GnRH injection: a decrease after 2 h was followed by an increase at 8 h. When the testes were removed before GnRH treatment, however, LH transcript levels remained depressed at 8 h after GnRH injection, indicating that the secondary increase in LH transcript levels depends on the presence of the testes. We conclude that the up-regulation of LH production subsequent to GnRH stimulation in adult male African catfish is mediated by factors originating from the testis. Previous work suggests that aromatizable androgens may play an important role in this context. Under the present experimental conditions, however, GnRHs had no, or an inhibitory, direct effect on LH production in catfish gonadotrophs.

gonadotropin-releasing hormone, luteinizing hormone, pituitary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Besides its namesake function, GnRH stimulates the expression of the LH subunit genes and LH de novo synthesis [1–3] in gonadotroph cells. Several different forms of GnRH have been identified in vertebrates, and usually two or three different forms are expressed in one species [4]. In the African catfish, Clarias gariepinus, catfish GnRH (cfGnRH: [His5,Asn8]-GnRH) is expressed in neurons scattered in the ventral forebrain; fibers from these neurons project to the gonadotroph cells in the pituitary [5]. Chicken GnRH-II (cGnRH-II: [His5,Trp7,Tyr8]-GnRH), the second GnRH form found in catfish, is synthesized by a small group of large neurons located in the midbrain tegmentum [6]; fibers projecting from these neurons to the pituitary have not been detected [5]. Nevertheless, both GnRHs are found in the pituitary and release LH [7], and we assume that cGnRH-II reaches the pituitary via the circulation.

Biosynthesis of LH involves glycoprotein {alpha} subunit (GP{alpha}) and LH ß subunit (LHß) gene transcription, mRNA translation, posttranslational modification, and packaging into secretory granules [8]. In mammals, experiments with primary cultures of pituitary cells [9–11] and pituitary cell lines [12–14] have shown that GnRH stimulates the expression of GP{alpha} and LHß subunit genes. De novo LH protein synthesis [2, 3, 10] and subunit glycosylation [15] are also increased by GnRH treatment, and it is generally agreed that GnRH increases LH subunit gene expression and protein synthesis by direct actions on mammalian gonadotrophs.

Also in fishes, such as goldfish [16], striped bass [17], or sockeye salmon [18], GnRH injection results in increased GP{alpha} and LHß steady-state mRNA levels. In other species, however, such as the gilthead seabream [19] or the coho salmon [20], GnRH injection does not change LHß transcript levels significantly. Information on the role of GnRH in LH de novo synthesis in fish is unavailable to our knowledge. Moreover, little information is available in vertebrates in general as to possible differences in the regulatory roles between multiple GnRH forms. We therefore studied the effects of two endogenous GnRHs in mature male African catfish on LH subunit transcript levels and de novo synthesis.


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

Male African catfish (9–12 mo of age) were used for experimentation. The fish were sexually mature as regards spermatogenesis (spermatozoa in many tubules) and did not differ in testis size or histology from wild fish collected from spawning grounds [21, 22]. However, the fish were in a prespawning condition; the full spawning condition is triggered by environmental stimuli (e.g., rainy season) in natural habitats.

The fish were bred and kept in the laboratory as described previously [23], except that catfish pituitary extract instead of hCG was used to induce ovulation. Animal culture and experimentation was consistent with the Dutch national regulations; the Life Science Faculties Committee for Animal Care and Use approved the experimental protocols.

In Vitro Experiments

Primary pituitary cell suspensions were prepared [24] and cultured [25] as detailed previously. In the experiments on LH de novo synthesis, the culture medium contained a mixture of 16 tritiated amino acids (TRK 440; Amersham, Little Chalfont, U.K.). The cells were incubated with GnRH for different times and at different concentrations, as detailed subsequently. The medium was collected and stored at -20°C until immunoprecipitation of LH. The cells were lysed in distilled water (1 ml), and the lysates were also stored at -20°C until immunoprecipitation of LH. De novo-synthesized LH was determined by counting the radioactivity after immunoprecipitation of LH from both medium and cell lysates as described previously [25]. Quantification of the LH content in the medium samples allowed calculating the specific activity in medium (i.e., disintegrations per minute of 3H-LH per nanogram of LH as determined by RIA). A detailed description of the catfish LH RIA using an antiserum against heterodimeric catfish LH has been given previously [26]. Intraassay and interassay variability at the EC50 concentration were 6% and 11%, respectively. Finally, we calculated what percentage of the total amount of newly synthesized 3H-LH was present in the medium.

Gene expression was measured in parallel incubations (absence of tritiated amino acids) by lysing the cells after culture in lysis buffer and determining GP{alpha} and LHß mRNA, and 28S rRNA levels by means of an RNase protection assay [27] as adapted for the lower mRNA levels in the primary cell culture system [25]. In all experiments, GP{alpha} and LHß mRNA levels were related to the level of rRNA determined in the same samples. The results are expressed as the percentage of control incubations (absence of GnRH).

In the dose-response experiments, pituitary cell cultures were exposed for 48 h continuously to 10 pM to 0.1 µM cGnRH-II or 1 nM to 10 µM cfGnRH. In the time course experiments, cell cultures were incubated in medium containing 10 nM cGnRH-II for 4–48 h or in medium containing 10 µM cfGnRH for 0.5–48 h. A large excess of cfGnRH over cGnRH-II is the physiologic situation in adult males, considering that the concentration of cfGnRH in the pituitary of mature males is 2 µM versus 3 nM for cGnRH-II [7]. We also studied whether the effects as determined 48 h after continuous exposure to GnRH differed from conditions in which GnRH was present only during the first 2 h of a 48-h incubation period. In this case, pituitary cells were rinsed with GnRH-free medium 5 times after the first 2 h and cultured for another 46 h in medium without GnRH, but still containing tritiated amino acids when appropriate.

In Vivo Experiments

Both cfGnRH and cGnRH-II were dissolved in a small volume of 1 M acetic acid and diluted to 250 µg/ml and 50 µg/ml shortly before i.p. injection in physiologic salt solution containing 0.25% BSA (Fraction V; Sigma, St. Louis, MO) sodium metabisulfite; the dose applied was 250 µg/kg cfGnRH or 50 µg/kg cGnRH-II. Two to 24 h after GnRH injection, the pituitaries were removed, frozen in liquid nitrogen, and stored at -80°C until assayed for GP{alpha} and LHß mRNA levels as described previously [27].

Mature males were sham-operated or castrated 3 days before an i.m. injection of 250 µg/kg cfGnRH or 50 µg/kg cGnRH-II, dissolved as described previously. The fish were decapitated 2 or 8 h after the injection with GnRH, and the pituitaries were collected. Blood samples were collected shortly before decapitation to verify the effect on LH plasma levels under conditions of surgery. Plasma samples were stored at -20°C until assayed for LH as described previously [26].

Statistics

All graphs and tables show means ± SEMs. For in vitro studies, data were combined from 3 to 5 independent experiments. Overall statistical significance was determined by ANOVA. In the case of significance, individual groups were compared by contrast analysis according to Fisher; P values of less than 0.05 were considered significant. Differences between two groups were analyzed by two-sided Student t-tests (P < 0.05). Statistical analysis was performed with StatView 4.5 for Windows (Abacus Concepts, Berkley, CA). The in vivo experiments with intact fish were carried out twice with 3 fish per treatment group. Similar results were obtained, and data were pooled. The experiment involving surgery was carried out once with 5 fish per treatment group.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Experiments

Incubation of primary pituitary cell cultures with 10 nM cGnRH-II for 4–48 h did not change significantly the steady-state GP{alpha} or LHß mRNA levels (Fig. 1a). The amount of newly synthesized LH in the medium, however, increased in response to cGnRH-II, became statistically significant 8 h after addition of cGnRH-II, and increased further with prolonged treatment (Fig. 1b). The amounts of newly synthesized LH in the cell lysate did not differ from that in the control group, but an increase in the calculated total amount (medium plus cell lysate) of de novo synthesized LH was found after 48 h of incubation (Fig. 1b). Expressing the amount of newly synthesized LH in the medium as a percentage of the total amount of 3H-LH (Fig. 1c) confirmed the role of cGnRH-II as an LH secretagogue.



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  		FIG. 1. Time course of cGnRH-II (a–c) and cfGnRH (d–f) effects in primary pituitary cell cultures. Levels of the GP{alpha} (open bars) and LHß (solid bars) mRNAs expressed as percentages of control after different times of exposure to 10 nM cGnRH-II (a) or 10 µM cfGnRH (d). De novo-synthesized, immunoprecipitable 3H-LH in the incubation medium (open bars) and cell lysate (hatched bars) and the calculated total (solid bars) expressed as the difference in disintegrations per minute from control after different times of exposure to 10 nM cGnRH-II (b) or 10 µM cfGnRH (e). 3H-LH released into the medium expressed as a percentage of the total amount of de novo-synthesized 3H-LH after different times of exposure to 10 nM cGnRH-II (c) or 10 µM cfGnRH (f). * Significant difference (P < 0.05, Student t-test) from respective control

The specific activity of LH in the medium increased from 3 to 6 dpm/ng but did not differ between controls and cell cultures exposed to cGnRH-II. This finding indicates that for secretion under basal and cGnRH-II-stimulated conditions, newly synthesized and preexisting LH are recruited in a similar way.

Exposure of primary pituitary cell cultures to 10 µM cfGnRH resulted in decreased GP{alpha} mRNA levels after 48 h; LHß mRNA levels were decreased after 24 and 48 h of exposure (Fig. 1d). During the first hour, there was no effect on the levels of newly synthesized LH (Fig. 1e). After 2 and 4 h, however, the level of newly synthesized LH in the medium was increased, yet accompanied by a decrease in newly synthesized LH in the cell lysate. After culture periods longer than 4 h, the level of newly synthesized LH in the medium no longer differed from that in controls, with a trend toward a reduction. In cell lysates, however, the amount of 3H-LH decreased progressively. Similar to the findings for cGnRH-II, the specific activity of LH in the medium increased with time (from 2 to 5 dpm/ng) but did not differ between controls and cell cultures exposed to cfGnRH, indicating a similar recruitment for secretion of preexisting and newly synthesized LH. Expressing the amount of newly synthesized LH in the medium as a percentage of the total amount of 3H-LH (Fig. 1f) therefore reflects the activity of cfGnRH as an LH secretagogue.

Primary pituitary cell cultures exposed for 48 h to cGnRH-II concentrations between 10 pM and 0.1 µM did not show a significant change in LHß mRNA steady-state levels, whereas the GP{alpha} mRNA levels were moderately elevated in the presence of 0.1 µM cGnRH-II (Fig. 2a). Exposure for 48 h to 10 µM cfGnRH, on the other hand, significantly reduced GP{alpha} and LHß transcript levels (Fig. 2d).



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  		FIG. 2. Dose-response effects of cGnRH-II (a–c) and cfGnRH (d–f) in primary pituitary cell cultures after 48 h of continuous exposure to the indicated GnRH concentrations. Levels of the GP{alpha} (open columns) and LHß (solid columns) mRNAs after exposure to cGnRH-II (a) or cfGnRH (d). De novo-synthesized, immunoprecipitable 3H-LH in the incubation medium (open bars) and cell lysate (hatched bars) and the calculated total (solid bars) expressed as the difference in disintegrations per minute from control after exposure to cGnRH-II (b) or cfGnRH (e). 3H-LH released into the medium after exposure to cGnRH-II (c) or cfGnRH (f) expressed as a percentage of the total amount of de novo-synthesized 3H-LH. * Significant difference from control (ANOVA, followed by Fisher least significance difference test, P < 0.05)

Incubation with cGnRH-II did not change significantly the total or cellular level of newly synthesized LH after 48 h (Fig. 2b), whereas levels of newly synthesized LH in the medium increased dose-dependently in response to cGnRH-II. The specific activity in the medium did not change significantly in response to cGnRH-II (from 2.5 to 3.2 dpm/ng LH), whereas the increase in the percentage of de novo-synthesized LH in the medium (Fig. 2c) confirmed the LH-releasing activity of cGnRH-II. Incubation with cfGnRH resulted in a different pattern, as decreased total and cellular levels of de novo synthesized LH were recorded at 1 and/or 10 µM cfGnRH (Fig. 2e). Elevated percentages of de novo-synthesized LH in the medium (Fig. 2f), with the specific activity remaining unchanged (from 2.6 to 3.3 dpm/ng LH), indicate the LH-releasing activity of cfGnRH.

Long- and Short-Term Exposure

Primary pituitary cell cultures were exposed for 2 h to 10 µM cfGnRH or 0.1 µM cGnRH-II, after which GnRH was removed, and incubation of the cells was continued for another 46 h. The results under this condition were compared with those obtained with continuous exposure to GnRH for 48 h. In general, the effects were similar under both conditions but more pronounced after the long-term exposure. As reported previously, cfGnRH but not cGnRH-II decreased LHß transcript levels (Fig. 3a) and LH de novo synthesis (Fig. 3b). However, the long exposure period was required to increase the amount of 3H-LH present in the medium in response to cGnRH-II (Fig. 3b).



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  		FIG. 3. Comparison between effects after short-term and continuous exposure to GnRH of primary pituitary cell cultures. Transcript levels (a) of GP{alpha} (open columns) and LHß (solid columns) and levels of de novo-synthesized 3H-LH (b) from cultures exposed during the first 2 h of a 48-h incubation period, to 10 µM cfGnRH or 0.1 µM cGnRH-II (short), or from cultures exposed for 48 h continuously (cont.) to the same GnRH concentrations. * Significant difference from control (Student t-test, P < 0.05)

In Vivo Experiments

Transcript levels of GP{alpha} (Fig. 4a) and LHß (Fig. 4b) were significantly decreased 2 h after injection of 250 µg/kg cfGnRH or 50 µg/kg cGnRH-II. After 4 h, LHß mRNA levels were still decreased in the cfGnRH-injected fish. Eight hours after injection of either GnRH, both GP{alpha} and LHß steady-state transcript levels were increased above control levels, although LHß transcript levels were not significantly elevated after cGnRH-II injection.



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  		FIG. 4. Time course of GnRH effects (vehicle, open columns; cfGnRH, hatched columns; cGnRH-II, solid columns) in intact males. At the indicated times after injection into the body cavity of 250 µg/kg cfGnRH or 50 µg/kg cGnRH-II, pituitary samples were taken to determine GP{alpha} (a) and LHß (b) mRNA levels. The data were corrected for 28S rRNA and expressed as a percentage of that present vehicle-injected controls for each time point. * Significant difference from vehicle-injected control (Student t-test, P < 0.05)

Similar to the results noted in the previous experiment with intact fish, sham-operated fish showed reduced GP{alpha} (Fig. 5a) and LHß (Fig. 5b) transcript levels 2 h after i.m. injection of 250 µg/kg cfGnRH or 50 µg/kg cGnRH-II. In castrated fish that received a vehicle injection, LHß transcript levels were lower than those in sham-operated fish, which is in agreement with previous observations [27]. Treatment of castrated fish with GnRH did not further change the low transcript levels after 2 h. In sham-operated fish, GP{alpha} and LHß transcript levels had recovered to control levels 8 h after GnRH injection. Remarkably, such a recovery was not observed in castrated fish 8 h after GnRH injection; in these fish, the transcript levels were significantly lower than those in vehicle-injected, gonadectomized fish (Fig. 5, a and b). Plasma LH levels were elevated in response to GnRH treatment (Fig. 5c). The reduced LH transcript levels in castrated fish were not reflected in reduced circulating hormone levels.



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  		FIG. 5. Effects of GnRH treatment (vehicle, open columns; 250 µg/kg cfGnRH, hatched columns; 50 µg/kg cGnRH-II, solid columns) in sham-operated or castrated males 2 or 8 h after i.m. injection of GnRH. The mRNA levels of GP{alpha} (a) and LHß (b) were corrected for 28S rRNA and expressed as a percentage of that present in sham-operated, vehicle-injected fish for both time points. Plasma LH levels (c) are given in nanograms per milliliter. * Significantly different from vehicle-injected control; # Significantly different from sham-operated control (Student t-test, P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Two endogenous GnRHs failed to increase LH subunit transcript levels or de novo synthesis in primary pituitary cell cultures of male African catfish; high doses of cfGnRH were even inhibitory. Yet, both GnRHs functioned as LH secretagogues. This is not in line with the stimulatory role of GnRH on LH production in mammals [1–3, 8–15].

An important point to discuss is the continuous exposure to GnRH in the present study, which can have inhibitory effects in mammalian gonadotrophs [28, 29]. However, LH de novo synthesis [2, 10] and subunit glycosylation [15] also increases in response to continuous GnRH treatment of up to 8 h in mammalian gonadotrophs, so that a pulsatile mode is required for some (e.g., LH secretion) but apparently not for all (e.g., LH de novo synthesis or glycosylation) aspects of GnRH action. In fish, there are no indications for a regular, pulsatile mode of GnRH release, although LH secretion bursts can occur [30]. In African catfish, LH release in vitro is maintained at elevated levels under continuous stimulation with GnRH for 3.5 h [31]. In commercial fish farming, long-term continuous GnRH-releasing devices are widely used to promote gonadal maturation and to enhance reproductive success [32]. In the present study, GnRH effects were qualitatively similar after short-term (2-h) and long-term (48-h) exposures, and LH release kept increasing with time. The molecular basis of the catfish gonadotrophs' responsiveness even after prolonged agonist exposure may be related to the fact that agonist-induced internalization of the catfish GnRH receptor levels off after ~30 min, leaving about 50% of the receptors at the cell surface [33]. It seems that prolonged exposure to physiologic concentrations of GnRH in fish has little, if any, inhibitory effect on the functioning of gonadotrophs.

In primary pituitary cell cultures of male African catfish, neither LHß subunit transcript levels nor LH de novo synthesis was clearly affected by cGnRH-II treatment. However, cGnRH-II functioned as an LH secretagogue, indicating that GnRH receptors were activated. This suggests that a direct effect on LH subunit transcript levels or de novo synthesis is not a prominent part of the spectrum of this peptide's biologic activity, or that gonadotrophs under the present experimental conditions are unable to respond to cGnRH-II by up-regulating LH subunit transcript levels. Since LH synthesis is strongly activated developmentally already in 3-mo-old catfish [34], and since gonadotrophs from 9- to 12-mo-old males incubated under identical conditions respond to aromatizable androgens and estrogens by up-regulating LHß mRNA levels and de novo synthesis [25], we conclude that catfish gonadotrophs have the physiologic capacity to activate LHß transcription but fail to do so in response to cGnRH-II. For cfGnRH concentrations below 1 µM, a similar conclusion can be drawn, whereas concentrations of 1 to 10 µM of cfGnRH induced decreases in LH de novo synthesis and transcript levels. We believe that such concentrations are within the physiologic range. Considering that the pituitary of mature male African catfish contains micromolar concentrations of cfGnRH [7] and that the peptide is stored in axon endings close to the gonadotrophs, it is reasonable to assume that micromolar concentrations of cfGnRH can be attained in the intercellular space between axon endings and gonadotrophs. Moreover, when studying GnRH binding to cells transfected with the catfish GnRH receptor, the IC50 concentration of cfGnRH is 7 µM [33]. Similarly, in primary catfish gonadotroph cultures, the EC50 concentration of cfGnRH is 2 µM with regard to inositol phosphate production [35]. Also in other fish species, the GnRH form produced in the ventral forebrain and reaching the pituitary by axonal transport is a comparatively weak LH secretagogue and is present in high concentrations in the pituitary [36]. It therefore appears possible that the inhibitory effect of high concentrations of cfGnRH is of physiologic significance.

Previous studies using African catfish primary pituitary cell cultures have shown that LH release or second messenger signaling induced by high concentrations of cfGnRH can be inhibited by subthreshold or borderline concentrations of cGnRH-II [37]. In the present study, however, neither a low (10 pM) nor a high (0.1 µM) concentration of cGnRH-II modulated the inhibitory effect of 10 µM cfGnRH on LH transcript levels or de novo synthesis (data not shown). Since cGnRH-II attenuates cfGnRH-induced LH release [37] but not cfGnRH-induced inhibition of LH synthesis (present study), and since the cfGnRH effects on LH release are uncoupled from those on LH synthesis (present study), it is possible that cfGnRH-mediated inhibition of LH synthesis makes use of a signaling pathway distinct and independent from the one involved in LH release.

The decreased GP{alpha} and LHß transcript levels 2 h after cfGnRH injection provide in vivo evidence for the significance of the inhibitory effect of cfGnRH. Treatment with cGnRH-II had a similar effect in vivo. This might seem surprising considering that cGnRH-II had no such effects in our in vitro studies. On the other hand, the release of cfGnRH from nerve terminals in the pituitary is sensitive to GnRH stimulation [35], so that cGnRH-II may have triggered the release of endogenous cfGnRH that in turn decreased LH transcript levels.

Eight hours after GnRH treatment of intact fish, GP{alpha} and LHß transcript levels were restored to or increased above control levels, subsequent to the initial decrease 2 h postinjection. The present in vitro results indicated, however, that both cGnRH-II and cfGnRH were unable to increase LH subunit transcript levels. We therefore speculated that the rise in GP{alpha} and LHß mRNA 8 h after GnRH injection might reflect indirect effects of the GnRH treatment, such as LH-stimulated sex steroid production that may in turn affect LH subunit transcript levels. Indeed transcript levels remained suppressed after GnRH injection when the testes were removed before GnRH treatment. One explanation for this observation is that the secondary increase in LH transcript levels after injection of GnRH into intact male African catfish is due to testicular factors. Among the growth factors and hormones produced by the testis, an interesting candidate to fulfill this feedback role in catfish is testosterone, which is converted to 17ß-estradiol in catfish gonadotrophs and then increases LHß subunit transcript levels [25]. The relevance of the ligand-activated estrogen receptor and its interaction with other transcription factors in the regulation of LHß gene expression has been demonstrated in a piscine model (e.g., [38]) and might be involved in mediating the up-regulating effect of aromatizable androgens in intact catfish.

In goldfish and striped bass, GnRH treatment elevates LH transcript levels [16, 17]. Since these studies were conducted with intact fish and samples were collected 6–24 h after injection, indirect effects via gonadal steroids cannot be excluded. More recent studies in goldfish have found a direct stimulatory effect of GnRH on LHß transcript levels using primary tissue/cell culture approaches [39]. However, GnRH receptors are also present on goldfish somatotrophs [40], which are often juxtaposed to gonadotrophs, so that a paracrine effect on gonadotrophs involving activin appears possible, as discussed previously [41]. In catfish, GnRH receptors are confined to gonadotrophs [42]. Also, in primary pituitary cell cultures of tilapia, GnRH treatment increases LH transcript levels [43, 44]. In this fish, however, GnRH action on pituitary cells appears to also involve increased cAMP levels [43]. In catfish, GnRH does not increase pituitary cAMP production [35]. Analyzing GnRH effects on the salmon LHß subunit gene promoter in the context of an immortalized murine gonadotroph cell line supported the notion of a direct stimulatory effect of the peptide [45]. It should be noted, however, that mammalian, but not submammalian, GnRH receptors lack the intracellular C-terminal domain. Since this structural difference is functionally significant [33, 46], it is possible that one or more components of the signaling pathway, from the GnRH receptor down to the transcription factors regulating LH subunit promoter activities, are used differently in a murine cell line versus a piscine gonadotroph. In vivo treatment of salmon with GnRH does not result in elevated LHß transcript levels at the beginning of the reproductive cycle [20], in contrast to the pattern noted in more mature fish [18]. This might indicate that the gonadotroph's responsiveness to GnRH is a feature developing during maturation, or that additional factors related to the stage of gonadal maturity modulate the responsiveness to GnRH, such as a more prominent steroidogenic response in more mature fish to GnRH-induced gonadotropin release.

The adult male fish used in the present study—although mature with regard to spermatogenesis—were not in spawning condition because of the absence of environmental cues (e.g., rainy season). It cannot be excluded that GnRH directly stimulates LH gene expression in gonadotrophs of fish in full spawning condition. However, the amount of LH in the pituitary of the males used in the present study has increased at least 2000-fold since the beginning of puberty at 3 mo of age [34], suggesting that the LH production capacity can be considered as fully activated in the fish used in the present study. Considering furthermore that the amount of LH in the pituitary of adult males exceeds the total circulating amount of LH by ~1000-fold under basal conditions [47], we presume that the moderate increase in circulating LH levels associated with spawning from 0.5 to 4 ng/ml [48] can be covered without assuming mechanisms that remained quiescent until the spawning period. Moreover, an immediate LH synthesis response after a GnRH challenge may not be required to maintain the LH release capacity in view of the large reservoir of pituitary LH, and considering that the elevated plasma steroid levels after GnRH-stimulated LH release will stimulate LH synthesis after a lag period of only a few hours.

In conclusion, two endogenous GnRHs did not increase directly LH production in adult male catfish, whereas high levels of cfGnRH inhibited LH production. This inhibitory effect might be transient in nature in vivo, however, since testicular products released in response to GnRH-induced LH secretion mediate an increase in LH transcript levels after a lag period of ~8 h. In the adult male rat (e.g., [49]), on the other hand, castration as well as GnRH treatment increases LH transcript levels, which is representative of the response seen in many mammals. It appears that in adult male African catfish, it is the sex steroids, rather than GnRH, that stimulate LH subunit gene expression. Similar to the situation in mammals, however, GnRH functions as an LH secretagogue in fish. In a situation in which the regulation of LH synthesis after GnRH stimulation seems to depend on gonadal steroid production, changes in the steroidogenic response to gonadotropin become critical to the long-term changes in the activity of the reproductive system in fish that show a reproductive seasonality, such as African catfish.


   FOOTNOTES
 
First decision: 18 September 2001.

1 This study was supported by grant 980526165 from the Dutch National Science Foundation to R.W.S. Back

2 Correspondence: R.W. Schulz, Utrecht University, Faculty Biology, Research Group Endocrinology, Kruyt building, Room Z-211, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. FAX: 31 30 2532837; r.w.schulz{at}bio.uu.nl Back

3 Current address: Department of Experimental Hematology, Central Laboratory for Blood Transfusion, Amsterdam, The Netherlands Back

Accepted: December 19, 2001.

Received: August 15, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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