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a Department of Experimental Zoology, Research Group for Comparative Endocrinology, Utrecht University, 3584 CH Utrecht, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone (DHT), a nonaromatizable analogue of T, and to the likewise nonaromatizable 11-ketotestosterone (KT) and 11ß-hydroxyandrostenedione (OHA), physiologically relevant androgens in fish. Both T and E2 elevated glycoprotein (GP) and LHß steady-state mRNA levels (quantified by RNase protection assay), de novo synthesis (metabolic incorporation of radioactive amino acids and subsequent immune precipitation of LH), and release of preferentially newly synthesized LH, while DHT had no effect. Inhibiting the aromatase activity abolished the stimulatory effects of T. The effects of E2 on LH mRNA levels and de novo synthesis were dose dependent. Incubation with 10 ng/ml KT elevated GP and LHß mRNA levels, while other concentrations of KT or all concentrations of OHA tested had no effect. The amount of newly synthesized LH, on the other hand, was decreased dose-dependently by OHA but not by KT. Since this OHA-induced decrease did not change the specific activity (dpm immune precipitable [3H]-LH/ng immune-reactive LH) of LH, we hypothesize that OHA exerted its effect by activating a crinophagic breakdown of secretory granules in catfish gonadotrophs. Electron microscopic examination of gonadotrophs after in vitro exposure to 50 ng OHA/ml revealed that breakdown organelles had increased in size significantly. We conclude that the balanced production of aromatizable (mainly stimulatory) and 11-oxygenated androgens (mainly inhibitory) may be an important factor in regulating the amounts of LH available for secretion in male African catfish.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit (GP) transcript levels in cultures of male rat pituitary cells were reduced after incubation with testosterone (T), whereas estradiol (E2) had no effect [4]. In vitro treatment of male rat pituitary cells with T (E2 was not studied) also inhibited the de novo synthesis of LH [5, 6]. It appears that T in males can have direct inhibitory effects on the gonadotrophs' activity, at least in rodents [7]. This notion is supported by in vivo experiments in which implantation of T-containing capsules into intact male rats decreased the LHß expression level, whereas GP was unaffected [8]. Moreover, the castration-induced rise in LHß and GP mRNA levels in rats is reversed by T, 5-dihydrotestosterone (DHT) or E2 treatment [9, 10]. The effect of E2 in the latter study may be related to an indirect effect on the GnRH neurons, for example, since E2 had no effect on LHß or GP transcript levels in gonadotrophs in vitro [4]. In adult female mammals, on the other hand, positive effects of T or E2 on LH steady-state transcript levels have been observed [2, 3]. None of these studies combined determinations of de novo synthesis with measurement of mRNA levels.
In fish, in vivo treatment with T or E2 increased pituitary levels of the LH-like gonadotropin II in mixed-sex juvenile rainbow trout (Oncorhynchus mykiss) [11], castrated mature male Atlantic salmon (Salmo salar) [12], and castrated adult male African catfish [13]. This increase reflects in part a direct steroid action on the pituitary, as indicated by the following in vitro experiments. Treatment of pituitary tissue fragments from mixed-sex juvenile rainbow trout with T or E2 increased LHß transcript levels [14], and both GP and LHß steady-state mRNA levels increased after in vitro treatment of mixed-sex goldfish pituitary fragments with T [15]. It is possible that the effects of T depend on its aromatization to E2. Very high aromatase activity has been found in the pituitary of teleost fishes [16–18], and functional estrogen response elements are located in the promoter region of the salmon LHß gene [14, 19]. It therefore appears that in male mammals and male fish, LH biosynthesis is directly regulated by sex steroid hormones, but that the nature of their effects is different—in general inhibitory in mammals versus stimulatory in fish. This feature renders fish an interesting model for study of the regulation of pituitary gonadotrophs.
Studies dealing with LH synthesis have often addressed the effect of steroid hormones on gene transcription, mRNA, or protein levels. To our knowledge, studies on the regulation of LH de novo synthesis have not been done on teleost fish. Hence we investigated the effects of important steroid hormones [20, 21] in adult male African catfish on the steady-state GP and LHß mRNA levels, on the amounts of de novo-synthesized LH in primary pituitary cell cultures, and on the secretion of newly synthesized LH. Finally an electron microscopical study assessed changes in the ultrastructure of pituitary gonadotrophs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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African catfish were bred and raised in the laboratory as described previously [22], except that a catfish pituitary extract instead of hCG was used to induce ovulation. Sexually mature males between 8 and 14 mo of age were used for all experiments. At this age, the lumen of the testis lobules and of the seminal vesicles contains numerous spermatozoa, and the males are used for breeding purposes.
Cell Culture
If not stated otherwise, chemicals were from Sigma Chemical Co. (St. Louis, MO). Primary cultures of pituitary cells were prepared and cultured as described by Lescroart et al. [23]. The cells were plated in 24-well culture plates (Costar, Acton, MA) at a density of 250 000 cells per well. For analysis of steady-state mRNA levels, the cells were cultured in 800 µl of L-15 medium (15 mM Hepes buffered, pH 7.4, 26 mM sodium bicarbonate, 100 000 U/L penicillin/streptomycin; all from Gibco, Gaithersburg, MD).
To study de novo synthesis, 800 µl medium (1% [v:v] minimum essential medium vitamin solution, 100 000 U/L penicillin/streptomycin, 26 mM sodium bicarbonate, 24 mg/L L-cysteine, 292 mg/L L-glutamine, 15 mg/L L-methionine, 10 mg/L L-tryptophan, and 50 mg/L L-asparagine, in Earle's Balanced Salt Solutions, 15 mM Hepes buffered, pH 7.4; all from Gibco) was used. After the cells were allowed to attach for 1.5 h at 25°C and 5% CO2 in air, horse serum (Gibco) was added to a final concentration of 5%. Then a mixture of tritiated amino acids (containing 15 labeled amino acids; TRK 440; Amersham, Little Chalfont, UK) was added to the cultures (2 µCi/well).
The steroid hormones used in these experiments were T, E2, the nonaromatizable androgen DHT, and the likewise nonaromatizable fish androgens 11ß-hydroxyandrostenedione (OHA) and 11-ketotestosterone (KT) (from Sigma or Steraloids, Wilton, NH). The steroids were dissolved in propylene glycol (250 µg/ml), diluted with medium, and added to the cultures together with the serum and the tritiated amino acids. Control incubations received a corresponding concentration of propylene glycol. The 5% dilution of horse serum did not show specific binding of tritiated steroids using a previously described binding assay technique [24] (data not shown), suggesting that the bioavailability of the steroids was not changed by horse serum proteins. At the end of the culture period, media were collected and the cells were lysed (see below).
In a pilot experiment the steroids were used at a concentration of 50 ng/ml for a continuous exposure (24 or 48 h). After this initial study, the effects of various doses of E2, OHA, and KT were determined after continuous exposure for 48 h.
The involvement of aromatization in the effects of T was studied by coincubating primary cultures for 48 h with T and 10 µM of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). Pilot experiments showed that at this concentration the conversion of [3H]androstenedione to estrogens by pituitary fragments of African catfish was reduced to 1% of the control values (data not shown).
Determination of Transcript Levels
After removal of the medium, the plates were put on liquid nitrogen to snap-freeze the cells. They were stored at -80°C for up to 1 wk, until RNase protection analysis for the levels of GP and LHß mRNA, using the Direct Lysate Ribonuclease Protection Analysis Kit (Amersham) as described before [25]. However, the following adaptations were made to accommodate for the smaller mRNA quantities from the cell cultures: to each well 50 µl lysis solution was added, and 45 µl was used for determination of GP and LHß transcript levels, while the remainder was diluted 18 times for determination of 28S rRNA levels. Transcript levels are expressed as percentages of control incubations after normalization to the 28S rRNA levels.
Determination of De Novo Protein Synthesis
After collection of the medium, addition of 500 µl distilled water and one round of freezing and thawing lysed the cells. The lysate was collected and the wells were washed with 500 µl distilled water that was added to the first aliquot. After the total amount of LH present in medium and cell lysate was determined by RIA using an antiserum raised against the heterodimeric catfish LH [26], the samples were diluted to 400 ng LH/ml in 0.05 M veronal buffer (sodium-5,5-diethylbarbiturate and barbituric acid, pH 8.6, with 0.2% BSA and 0.1% sodium azide). Two aliquots of 250 µl received 250 µl of 1/100-diluted anti-LH (raised against the heterodimeric catfish LH [26]); two other aliquots received 250 µl of 1/100-diluted normal rabbit serum. After a 3-h incubation period at 37°C, all tubes received 100 µl of a 1/10-diluted goat anti-rabbit serum (Arnel, New York, NY) and were incubated for another hour at 37°C. Then 2 ml ice-cold veronal buffer was added, and the tubes were spun for 30 min at 5400 x g and 4°C. The supernatant was discarded and the pellet washed with 1 ml ice-cold veronal buffer. After centrifugation as above, the pellet was resuspended in 200 µl 2 N NaOH, which was transferred to a scintillation vial. The tubes were rinsed with 300 µl distilled water, which was added to the same scintillation vials. The radioactivity was counted in a scintillation counter using Ultima Pro scintillation liquid (Packard, Meriden, CT). The amount of newly synthesized LH was calculated by subtracting the mean dpm in the normal rabbit serum-incubated tubes from the mean in the anti-LH-incubated tubes. After accounting for the different dilutions of the samples, the mean of all wells that received the same treatment was determined. The level of de novo synthesis under control conditions was set to zero.
Conversion of [3H]T and [3H]OHA by the Primary Cell Culture
Pituitary cells were cultured at a density of 1 x 106 cells per well in 6-well culture plates (Costar) as described above. Together with the horse serum, 1 nM [3H]T (53 Ci/mmol; NEN Life Science Products, Boston, MA) or [3H]OHA (synthesized from [3H]cortisol as described previously [27], 53 Ci/mmol; NEN Life Science Products) was added to the cells, each in duplicate. After 48 h at 25°C and 5% CO2, the medium was collected and 25 µg/200 µl propanol of radioinert T and androstenedione and 50 µg/200 µl propanol of E2 and estrone were added as carriers for the [3H]T experiment; 25 µg/200 µl of OHA, KT, 11-ketoandrostenedione, and 11ß-hydroxytestosterone were added to the [3H]OHA-containing media. Subsequently, the steroids were extracted from the medium using 3 times 10 ml of dichloromethane. The dichloromethane fractions were pooled and evaporated; the steroids were redissolved in a small volume of dichloromethane-methanol (9:1) and transferred to 60F254-precoated silica plates with concentrating zone (Merck, Darmstadt, Germany). Samples from the [3H]T incubation were separated in a diisopropylether-chloroform-hexane (7:2:1) solvent system (3 times), whereas the samples from the [3H]OHA incubation were separated using a toluene-ethylacetate (3:1) solvent system (3 times). The chromatograms were analyzed with a Berthold (EG&G Walllac, Gaithersburg, MD) LB 2841 thin-layer radiochromatogram scanner. The peak surfaces were determined and taken as a quantitative measure of the yielded compounds.
Electron Microscopy
To investigate the possibility that treatment with OHA (reduction of levels of newly synthesized LH) also affected the morphology of the gonadotrophs, we incubated pituitary fragments for 48 h in the presence or absence of 50 ng/ml OHA in L-15 medium with 5% horse serum (Gibco). The pituitary fragments were processed for electron microscopical analysis of the size of the LH-containing organelles as described previously [28]. In brief, after fixation and epoxy resin embedding, ultrathin sections were cut and labeled for GP or LHß subunits using antisera raised against these subunits, followed by a secondary antibody complexed to 10-nm gold particles. After contrasting, the sections were examined by electron microscopy, and photographs were taken and analyzed using quantitative image analysis. Special attention was given to the organelles containing GP and/or LHß-subunit immune-reactive material, the secretory granules, globules, and irregular membrane-bound masses (IMs). The latter two structures are part of a crinophagic breakdown pathway [29], in which cellular products initially destined to be secreted (secretory granules and their content) are shunted to the lysosomal pathway and degraded within the cell. The surface areas of the granules, globules, and IMs are expressed as the mean organelle surface determined in 3 pituitaries and 10 different gonadotrophs per pituitary.
Statistics
Except for Figures 1 and 5, the graphs show means ± SEM with data combined from 3 or 4 independent experiments. Differences between multiple groups were analyzed by one-way ANOVA, followed by Fisher's protected least significant difference test; a Student's t-test was used to analyze the electron microscopical data (StatView 4.5 for Windows; Abacus Concepts, Berkeley, CA). Differences were considered statistically significant when P < 0.05. Groups sharing the same underscore in the legends do not differ significantly.
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RESULTS |
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and LHß Steady-State Transcript Levels
In an initial experiment, all steroids (50 ng/ml) decreased GP and LHß mRNA levels after 24 h of continuous exposure (Fig. 1a), whereas after 48 h of continuous exposure to T, the levels were increased compared to those in the control (Fig. 1b). Incubation with T (50 ng/ml) for 48 h in the presence of the aromatase inhibitor ATD (10 µM) led to steady-state mRNA levels not significantly different from control values (Fig. 2a). Neither ATD alone nor the nonaromatizable androgen DHT (50 ng/ml) had a significant effect on the steady-state messenger levels for GP or LHß after 48 h (Fig. 2a). Incubating cells with [3H]T showed that 84% of the activity was recovered in the organic fraction, of which 80% comigrated with authentic E2 in thin-layer chromotography.
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Although the above data indicate that aromatization of T to E2 is required for T to exert its effects, incubation with 50 ng E2/ml did not have a significant effect on the steady-state messenger levels after 48 h (Fig. 1b). We therefore tested whether the effect of E2 is dose dependent. Incubations for 48 h with E2 concentrations of 0.4 and 2.0 ng/ml significantly increased LHß mRNA levels (Fig. 3a).
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Treatment of the cells for 48 h with increasing concentrations of KT resulted in elevated GP and LHß mRNA levels for 10 ng/ml KT (Fig. 4b). None of the OHA concentrations tested changed GP or LHß mRNA levels significantly (Fig. 4a).
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LH De Novo Synthesis Levels
The de novo-synthesized LH in medium and cell lysate is expressed as dpm/250 000 cells of immune precipitable [3H]LH, the total representing the sum of cell lysate and medium [3H]LH. Moreover, we calculated the "specific activity" of de novo-synthesized LH as follows: specific activity (dpm/ng) = de novo [dpm; from immune precipitation]/(de novo + preexisting) [ng; from LH RIA]. Finally, we have calculated the percentage of the total amount of newly synthesized LH that was secreted into the medium: % in medium = 100% * dpm in medium/(dpm in medium + dpm in cell lysate). With these parameters we aimed to detect possible effects of the steroids on the relation between newly synthesized LH and preexisting LH, and the level of secretion of de novo-synthesized LH, respectively.
Continuous exposure to E2 or T (50 ng/ml) in the initial experiment increased the levels of newly synthesized LH (Fig. 1, c and d) after 24 and 48 h in medium and total (i.e., medium + cells). Moreover, the percentage of de novo-synthesized LH present in the medium (Table 1; % [3H]LH secreted) and the specific activity of the newly synthesized LH in medium and cell lysates increased in response to T and E2. These data indicate that E2 and T stimulated the de novo synthesis of LH and preferentially induced secretion of newly synthesized LH. Treatment with KT for 24 or 48 h did not affect any of the parameters studied (Fig. 1, c and d; Table 1), except that after 48 h of continuous exposure the cellular level of de novo-synthesized LH was decreased (Fig. 1d).
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A 48-h coincubation of T (50 ng/ml) with the aromatase inhibitor ATD (10 µM), as well as incubations with ATD or DHT alone (50 ng/ml), did not result in changes of de novo-synthesized LH levels in cells or medium (Fig. 2b) or other parameters studied (Table 1). Consistent with the results of the pilot study (Fig. 1, b and d), incubation with T alone led to increased LH de novo synthesis and increased secretion of newly synthesized LH (Fig. 2b; Table 1). The results using the aromatase inhibitor indicate also that the effect of T on the stimulation of de novo LH synthesis, and on the increased release of de novo-synthesized LH, is dependent on aromatization.
Exposure for 48 h to increasing concentrations of E2 resulted in a complex dose-response curve (Fig. 3b). Low concentrations of E2 (1 and 4 pg/ml) significantly reduced the de novo-synthesized LH in the cells and in cells plus medium (i.e., total), while the specific activities of de novo-synthesized LH in the cell lysates were not altered significantly at these concentrations (Table 2). The specific activity of newly synthesized LH in the medium was significantly elevated at a concentration of 0.02 ng/ml and above (Table 2). Increasing the E2 concentrations above 0.1 ng/ml elevated the amount of newly synthesized LH in the medium and the total amount, reaching a maximum at 2 ng/ml (Fig. 3b). Moreover, at E2 concentrations of 0.4 ng/ml and higher, the percentage of newly synthesized LH found in the medium increased above control levels (Table 2).
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In the initial experiment, OHA (50 ng/ml) treatment decreased the amount of newly synthesized LH after 24- or 48-h exposure (Fig. 1, c and d), especially in the cell lysate. Surprisingly, this decrease was not accompanied by a change in the specific activity of the newly synthesized LH (Table 1). Considering the equation used to calculate the specific activity, it can only remain constant, despite a decrease in the amounts of de novo-synthesized LH, when one is assuming a corresponding decrease of preexisting LH. In contrast to the extensive conversion of [3H]T to mainly [3H]E2, incubations with [3H]OHA indicated that the primary pituitary cell culture is unable to convert [3H]OHA to a noteworthy extent. We recovered 96% of the activity in the organic fraction, 87% of which comigrated with authentic OHA. The remaining 13% did not migrate in a concise peak and was not analyzed further. This indicates that the effects observed following exposure to OHA are specific for this androgen.
The decrease in the amounts of newly synthesized LH in the cells during a 48-h exposure to OHA was dose dependent (Fig. 4c), while no change was observed in the medium. There was no effect of KT on the de novo-synthesized LH amounts (Fig. 4d). After 48 h of continuous exposure to the various doses of OHA and KT, no significant changes were observed with respect to the percentage of newly synthesized LH secreted, the absolute amounts of LH, or its specific activity in cells or medium (data not shown).
We hypothesize that treatment with OHA decreased the amounts of immune precipitable de novo-synthesized LH by activating LH breakdown via the crinophagic route, which directs secretory granules to the lysosomal pathway, rather than by inhibiting LH synthesis. In fish gonadotrophs this pathway gives rise to two LHß subunit-containing organelles [29], globules and IMs. After treatment of pituitary fragments with 50 ng OHA/ml for 48 h, we determined the surface area of the various organelles in gonadotrophs containing the LH subunits. The treatment did not result in changes in the surface area of the secretory granules or the globules, while the surface area of the IMs increased significantly (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These results are opposite to what has been observed in male mammals. Using pituitary cells from castrated rats it was shown that T inhibited the de novo synthesis of LH subunits [5]. In vitro exposure of pituitary cells from juvenile and adult male rat to T decreased the LHß transcript levels, while exposure to E2 was without effect [4, 7]. This indicates that in mammals a direct, inhibitory effect is mediated by androgens. In the African catfish, on the other hand, T has stimulatory effects following conversion to E2. Stimulatory effects of T and E2 have been recorded in other teleost fishes. For example, LHß and GP transcript levels increased after incubation of pituitary fragments from mixed-sex goldfish with T [15]. Also, the LHß mRNA levels were elevated after treatment of pituitary cells from immature rainbow trout with T or E2 for at least 10 h [14], or after treatment of pituitary cells from mature male tilapia with T for at least 12 h [31]. The functional estrogen response elements found in the promoter of the salmon LHß gene offer an explanation for direct stimulatory effects of E2 [14, 19].
The E2 dose-response data indicate that E2 has differential effects on gene transcription and de novo LH synthesis, and that the type of effect (stimulatory or inhibitory) changes with the dose of E2. In mature male African catfish, E2 amounts of 148 pg/pituitary were found [32]. With an average wet weight per pituitary of 5 mg (~5 µl), this amount is similar to a concentration of 30 ng E2/ml. The plasma E2 levels in mature male catfish are usually below the detection limit (0.1 ng/ml); however, the gonadotrophs exhibit aromatase activity [33]. Hence the pituitary E2 level would reflect the local production, depending on the supply of substrate. We therefore conclude that E2 has specific and discrete effects on both LH gene transcription and LH de novo synthesis. The nature of these separate effects and of the change between inhibitory and stimulatory effects requires further investigation.
In the present study, T/E2 not only elevated the absolute levels of de novo LH synthesis, but also increased the specific activity of newly synthesized LH, indicating a true stimulation of LH biosynthesis. Besides the effects on de novo synthesis of LH, these steroids furthermore increased the secretion of de novo-synthesized LH. E2 has been shown to facilitate the GnRH-stimulated release of gonadotropin in mammals [34] and goldfish [35]. In the present study, however, the basal secretion of LH is elevated by E2. Similar effects were observed in female rats [36], while in immature rainbow trout [11] and eel, [37] T/DHT, but not E2, elevated LH secretion. In ovariectomized rhesus monkeys with hypothalamic lesions abolishing the endogenous GnRH release, an injection of estradiol benzoate induced LH release from the pituitary, indicating that E2 may be a releasing factor in its own right [38]. Basal secretion could be increased by E2 affecting the availability of secretory granules for secretion or up-regulating the machinery of the regulated secretory pathway, as suggested by Thomas and Waring [39]. Interestingly, in the present study, T or E2 preferentially stimulated the release of de novo-synthesized LH, indicating that the gonadotrophs are able to differentiate between preexisting and newly synthesized LH, the latter apparently being recruited preferentially for T/E2-induced secretion.
The direct stimulatory effect of T/E2 on LH secretion in vitro is not in line with the inhibitory effect of these steroids in vivo. Castration of adult male catfish increased (doubled) plasma LH levels, and this increase was reversed by replacement of aromatizable androgens or estrogens [13]. This discrepancy suggests that an inhibitory effect of T/E2 on GnRH secretion may dominate a direct stimulatory effect on the pituitary, akin to what has been proposed for various mammalian systems (e.g., [7]). In mature male African catfish and Atlantic croaker, however, testicular control of LH secretion appears to be less prominent than in mammals, as castration either only doubles [13], or has no effect at all [30], on circulating LH levels in the absence of exogenous GnRH, while testicular control of LH synthesis is prominent also in fish.
Although the testis does not produce DHT and DHT is not present in the blood of African catfish [20, 21], it was used to trace possible effects of T that do not depend on aromatization. However, no evidence for such effects on mRNA levels or de novo LH synthesis was observed in the present study; all effects of T were abolished by the addition of an aromatase inhibitor, and the DHT-treated groups were similar to control groups. The latter finding is possibly also related to the fact that in other fishes [40–42], DHT is a 5- to 700-fold weaker competitor than T for androgen receptor-like binding sites.
Previous studies showed that long-term in vivo treatment (7 wk) of immature masu salmon parr (Oncorhynchus masou) with KT [43] resulted in increased LH pituitary levels. We have also shown that the transcript levels of GP and LHß are markedly increased in mature male catfish in vivo 5 days after a single injection of KT [25]. The present study indicates, however, that the direct effects of KT on GP and LHß transcript levels in pituitary cells in vitro are relatively weak compared to the effects in vivo. This suggests that in vivo KT may also use an indirect mechanism of action. In this context it is interesting to note that in immature platyfish (Xiphophorus maculatus) [44] and in bluehead wrasse (Thalassoma bifasciatum) [45], KT treatment increased the number of GnRH neurons. GnRH in turn may stimulate the expression of LH [2, 46–49]. Taking these findings together, we conclude that androgens, whether aromatizable or 11-oxygenated, apparently are of limited importance for the direct regulation of GP or LHß transcript levels in gonadotroph cells of the African catfish.
A surprising finding was that treatment with OHA decreased the amounts of newly synthesized LH while the specific activity was unchanged. Considering the equation used to calculate the specific activity of de novo-synthesized LH, there are two possibilities to explain a constant specific activity (i.e., the dpm/ng ratio of [3H]LH immune precipitated over ng of immunoreactive LH) despite decreased amounts of de novo-synthesized LH: 1) OHA stimulated the breakdown of preexisting LH to exactly the same extent that OHA inhibited the de novo synthesis of LH or 2) OHA stimulated the breakdown of LH by a process not differentiating between de novo-synthesized and preexisting LH. The presently available data do not allow exclusion of the first possibility. It is unlikely, however, that the inhibitory effect of OHA on the de novo synthesis occurs at the level of LH gene transcription, considering that OHA had no effect on the steady-state LH mRNA levels after 48 h of incubation. Secretory granules in African catfish gonadotrophs can enter a crinophagic breakdown route for LH, leading to the formation of globules and eventually IMs; both globules and IMs are no longer available for the secretory pathway [29, 50]. In the crinophagic pathway, secretory granules fuse with lysosomes, and also the resulting larger lysosomes (referred to as globules) can fuse with secretory granules and/or with other globules, eventually forming IMs. Such structures were also described in gonadotrophs of other fish species [51]. In African catfish gonadotrophs, globules and IMs appear during puberty [26] and are especially abundant in the post-spawning period [52]. We therefore consider it likely that the reduced levels of de novo-synthesized LH after OHA treatment are related to an effect on the breakdown of LH; the effect appears to be specific for OHA as the structurally related androgen KT is ineffective in this respect. Indeed, exposure of pituitary fragments to 50 ng/ml OHA for 48 h increased the surface area of the IMs compared to gonadotrophs from control incubated pituitary fragments, supporting our hypothesis that OHA stimulated the crinophagic breakdown route.
In African catfish, OHA is the main product of the testis [53] but present in relatively low concentrations in the blood plasma (2 ng/ml basal to 8 ng/ml after stimulation [54]). KT, on the other hand, is a minor product of the testis [53] but is the quantitatively dominant androgen in the plasma of male catfish (basal 5–10 ng/ml to 50–60 ng/ml after stimulation [54]) and most other male teleost fish [55]. This apparent mismatch between the patterns of testicular and circulating androgens is related to the hepatic conversion of bloodborne OHA to KT [53]. In the context of the present results, it appears that a strong stimulation by LH will increase the OHA levels, leading to a decrease of the amount of LH available for secretion through activation of the crinophagic breakdown route. At the same time, however, LH will have stimulated testicular T output, so that de novo synthesis of LH is stimulated. The overall effect of this rather complex feedback system may be to prevent the intracellular LH stocks in the secretory granules from "aging." It is possible that the high incidence of globules and IMs recorded in catfish gonadotrophs following the LH surges during spawning [52] reflect an activation of this feedback system.
In summary, in the African catfish, the testis may regulate the expression and de novo synthesis of LH by a balanced production of T and OHA. After aromatization to E2, T stimulates LH steady-state transcript levels and de novo LH synthesis, whereas we propose that OHA stimulates the breakdown of LH. However, since OHA is rapidly converted into KT, the probably indirect effects of KT may dominate in vivo, except when the steroidogenic system is stimulated strongly. Then OHA plasma levels may increase as well, for example during spawning-related LH surges, and may stimulate the crinophagic breakdown of LH.
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FOOTNOTES |
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1 This project was supported by grant no. 805–26.165 from the Netherlands Organization for Research (NWO). 
2 Correspondence: R.W. Schulz, Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 31 30 2532837; r.w.schulz{at}bio.uu.nl
Accepted: November 8, 1999.
Received: April 13, 1998.
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- and lutropin (LH) ß-subunit but not release of stored LH in cultured rat pituitary cells. Biochim Biophys Acta Mol Cell Res 1996; 1310:348–354.[Medline]-reductase in the teleost brain, spinal cord and pituitary gland. Gen Comp Endocrinol 1985; 60:244–251.[CrossRef][Medline]- and gonadotropin IIß-subunit mRNA levels in the pituitary of mature male African catfish, Clarias gariepinus. Fish Physiol Biochem 1997; 17:99–108.[CrossRef]This article has been cited by other articles:
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  Y Kazeto and J M Trant Molecular biology of channel catfish brain cytochrome P450 aromatase (CYP19A2): cloning, preovulatory induction of gene expression, hormonal gene regulation and analysis of promoter region J. Mol. Endocrinol., December 1, 2005; 35(3): 571 - 583. [Abstract] [Full Text] [PDF]
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 D. Consten, E.D. Keuning, J. Bogerd, M.A. Zandbergen, J.G.D. Lambert, J. Komen, and H.J.Th. Goos Sex Steroids and Their Involvement in the Cortisol-Induced Inhibition of Pubertal Development in Male Common Carp, Cyprinus carpio L Biol Reprod, August 1, 2002; 67(2): 465 - 472. [Abstract] [Full Text] [PDF]
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F.E.M. Rebers, G.A.M. Hassing, W. van Dijk, E. van Straaten, H.J.Th. Goos, and R.W. Schulz Gonadotropin-Releasing Hormone Does Not Directly Stimulate Luteinizing Hormone Biosynthesis in Male African Catfish Biol Reprod, June 1, 2002; 66(6): 1604 - 1611. [Abstract] [Full Text] [PDF]
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J. E. B. Cavaco, J. Bogerd, H. Goos, and R. W. Schulz Testosterone Inhibits 11-Ketotestosterone-Induced Spermatogenesis in African Catfish (Clarias gariepinus) Biol Reprod, December 1, 2001; 65(6): 1807 - 1812. [Abstract] [Full Text] [PDF]
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J.E.B. Cavaco, J. van Baal, W. van Dijk, G.A.M. Hassing, H.J. Th. Goos, and R.W. Schulz Steroid Hormones Stimulate Gonadotrophs in Juvenile Male African Catfish (Clarias gariepinus) Biol Reprod, May 1, 2001; 64(5): 1358 - 1365. [Abstract] [Full Text]
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D. Consten, J. Bogerd, J. Komen, J.G.D. Lambert, and H.J.Th. Goos Long-Term Cortisol Treatment Inhibits Pubertal Development in Male Common Carp, Cyprinus carpio L Biol Reprod, April 1, 2001; 64(4): 1063 - 1071. [Abstract] [Full Text]
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