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Regular Article |
a Research Group for Comparative Endocrinology, Faculty of Biology, Department of Developmental Biology, Utrecht University, 3584 CH Utrecht, The Netherlands
ABSTRACT
In juvenile African catfish (Clarias gariepinus), the pituitary LH content strongly increased after the beginning of spermatogonial proliferation. We hypothesized that a signal of testicular origin is involved in stimulating the gonadotrophs. We investigated the effects of castration and sex steroid treatment on gonadotrophs in juvenile males by quantifying LH production and release and LH subunit transcript levels and by examining gonadotroph morphology and proliferation. Castration reduced but did not abolish the maturation-associated elevation in pituitary LH content. Treatment with testosterone but not with 11-ketotestosterone, an otherwise potent androgen in fish, reversed the castration-induced decrease of pituitary LH levels. An increased pituitary LH content was accompanied by an increased number of cytologically mature gonadotrophs. However, no evidence was found for gonadotroph proliferation, so that quiescent gonadotrophs may have become activated. Although 11-ketotestosterone treatments had no effect in castrated males, this androgen attenuated gonadotroph activation in intact males. Because androgen production in juvenile catfish is downregulated by treatment with 11-ketotestosterone, its inhibitory effects on gonadotrophs in gonad-intact males may be due to suppression of Leydig cell testosterone production, which appears to be a limiting factor for the activation of catfish gonadotrophs. Aromatizable androgens may have opposite effects on fish (stimulatory) and mammalian (inhibitory) gonadotrophs.
luteinizing hormone, pituitary, steroid hormones
INTRODUCTION
The GnRH neurons in juvenile mammals are sensitive for a sex steroid-mediated negative feedback [1]. In juvenile fish, however, sex steroids have stimulatory effects on the GnRH system. Testosterone (T) increases the GnRH content in the brain of immature salmonids [2, 3] and African catfish (Clarias gariepinus) [4]. A nonaromatizable androgen typically found in fish, 11-ketotestosterone (KT) [5], also activates the GnRH system in different species [6, 7]. Similarly, sex steroids in juvenile fish activate the pituitary gonadotrophs. In immature salmonid fish, the LH content increases after treatment with T [3, 8, 9], which may be mediated by estradiol-17ß (E2) derived from aromatization of T in gonadotrophs. Information on aromatase activity in the pituitary of juvenile fish is not available. In adult male African catfish, however, T stimulates LH ß-subunit expression only after aromatization [10], and functional estrogen response elements have been identified in regulatory regions of the salmon LH ß-subunit gene [11]. The overall stimulatory effect of sex steroid hormones on the brain-pituitary axis in juvenile fish suggests that fish represent a vertebrate model different from mammals in regard to the regulatory roles of sex steroid hormones.
The pituitary gonadotrophs are stimulated during the beginning of spermatogenesis in African catfish, the most significant changes occurring during the transition from spermatogonial proliferation to meiosis [12]. In view of the stimulatory effect of sex steroids in fish, we hypothesized that along with the beginning of spermatogenesis, the testes produce steroids that stimulate gonadotroph activity. In the present study, we tested this hypothesis by investigating the effects of castration or steroid hormone treatment on the gonadotrophs in juvenile male African catfish.
MATERIALS AND METHODS
Animals
All experimental procedures involving the use of animals were conducted in accordance with the national Dutch regulations. An internal Animal Care and Use Committee approved the protocols. African catfish were bred and raised as described previously [13], except that catfish pituitary extract instead of hCG was used to induce ovulation. The fish were kept in a copper-free recirculation system at a water temperature of 25°C ± 2°C, exposed to a photoperiod of 14L:10D, and fed with Trouvit pellets (Trouw, Putten, The Netherlands). Fish were used for steroid implantation or castration at 10 or 11 wk of age (stage I, spermatogonia but no meiotic germ cells present in testis) [14].
Steroid Treatment of Castrated and Intact Fish
All steroids and salmon GnRH analogue (sGnRHa; D-Arg6, Trp7, Leu8, Pro9-NEt GnRH) were purchased from Sigma (St. Louis, MO). 11ß-Hydroxyandrostenedione (OHA) was administered because it is the main product of testicular steroidogenesis in African catfish. However, circulating OHA is readily converted in the liver to KT [15], the main circulating androgen in African catfish and in teleost fish in general [5]. 11-Ketoandrostenedion (KA) is an intermediary product [16, 17]. Testosterone and androstenedione (A) were included because they are prominent plasma androgens in adult catfish [18]. Estradiol-17ß and the nonaromatizable 5
-dihydrotestosterone (DHT) were administered to test if treatment effects were related to the metabolization of T or A.
Before castration, fish were anesthetized with a 2% aqueous solution of phenoxyethanol (Sigma) directly applied to the gills (250 µl/gill chamber). After 20–30 sec, the body cavity was opened by a 
2-cm-long incision and the testes were removed. Before suturing, silastic pellets containing no steroid (control pellets, C) or pellets containing T or KT (both at a dose of 30 µg/g body weight) were placed in the body cavity. The pellets were prepared as described previously [19]. In sham-operated animals, testes were left in place. The four experimental groups in this experiment were sham operated (SHAM), castrated plus control pellets (CAS+C), and castrated plus pellets containing KT (CAS+KT) or T (CAS+T). A start control group (START) was sampled on the day of castration. Three weeks after surgery, all groups were sampled for the quantification of LH in pituitary and blood samples and for sex steroids in blood samples.
In a similar experiment with only two groups (SHAM and CAS), pituitaries were collected 3 wk after surgery to measure their LH protein and subunit mRNA content, and blood samples were taken for LH quantification.
At necropsy, the body cavity was inspected for testicular remnants; if remnants were encountered, the animal was excluded from analysis. In addition, the presence or absence of the urogenital papilla, an androgen-dependent secondary sexual characteristic [20], was recorded.
In three independent experiments with fish from different broods, immature intact males were implanted with silastic pellets containing T, A, KT, KA, OHA, DHT, or E2; the testes were not removed. Control animals received steroid-free pellets. On the day of implantation, a start control group was also examined for LH plasma levels, pituitary LH content, and analysis of gonadotroph ultrastructure. In the third experiment, fish received C, KT, KA, or OHA pellets. Except for pituitary mRNA levels, data from this experiment are not presented and did not differ from the results obtained with the same androgens in experiments 1 or 2.
Two weeks after implantation, blood samples were taken and plasma was stored at -20°C until use. Following decapitation, the pituitaries were removed and assayed for LH content, the LH subunit steady-state mRNA levels, and basal and GnRH-stimulated LH release in vitro. In addition, pituitaries were fixed for electron microscopy.
Quantification of Transcript and Hormone Levels
The steady-state mRNA levels of the common glycoprotein -subunit (GP) and of the LH ß-subunit were determined from 8–10 individual pituitaries per treatment group by RNase protection assay as described previously [21], except that the homogenization volume was reduced to 47.5 µl lysis buffer to account for the low mRNA amounts present in pituitaries of juvenile males.
For pituitary LH quantification, 10–15 glands per treatment group were homogenized individually. The homogenates were centrifuged, and supernatants were stored at -20°C until use. We used a previously described RIA with an antiserum against intact heterodimeric African catfish LH [22] to determine the LH concentration in plasma and pituitary samples. The androgen concentration in plasma of castrated animals was quantified by specific RIAs [23].
Basal and GnRH-stimulated LH release in vitro was measured as described previously [22]. Eight to 10 pituitaries per treatment group were preincubated individually in 0.5 ml Leibovitz 15 medium (L-15; Gibco, Paisley, U.K.) supplemented with 10 mM NaHCO3, 15 mM Hepes-NaOH (pH 7.4), 100 U/ml penicillin (Gibco), and 100 U/ml streptomycin (Gibco) at 25°C for 18 h. After rinsing with L-15, pituitaries were incubated for another 3 h to measure basal LH release. The pituitaries were rinsed once and then incubated in the presence of 1 nM sGnRHa for a further 3 h to determine GnRH-stimulated LH secretion. Salmon GnRHa is a high-affinity ligand for catfish pituitary GnRH receptors [24] and readily stimulates LH secretion in vivo and in vitro [25].
Immunocytochemical Analysis of Gonadotrophs
For the ultrastructural study, four fish were selected randomly from the start and end control, and from the T- and KT-treated groups of the second experiment with intact fish. The fish were killed by decapitation, and the pituitary was fixed, cryosubstituted, and embedded in Lowicryl HM20 (Electron Microscopic Science, Fort Washington, PA) as described previously [26].
In gonadotrophs of adult African catfish, three different types of organelles containing LH subunits can be distinguished [22]: secretory granules, larger globules, and very large irregular membrane-bound masses (IM). Globules and IMs are part of a crinophagic pathway, in which secretory granules are shunted to the lysosomal pathway, and are no longer available for secretion [27].
To identify the gonadotrophs, ultrathin sections (60–90 nm) were processed for immunocytochemistry using an antiserum raised against intact catfish LH (anti-LH,ß; diluted 1:1000) and gold-labeled goat anti-rabbit serum as second antibody [22]. To distinguish between big secretory granules (available for secretion) and small globules (not available for secretion), an antiserum against the catfish GP was used (diluted 1:1000), because granules but not globules show GP immunoreactivity [22, 28]. The sections were treated with uranyl acetate and lead citrate and examined in a Zeiss electron microscope 109 (Oberkochen, Germany). Interactive imaging analysis was carried out with the IBAS system provided by Kontron & Zeiss (Eching, Germany) to determine the total gonadotroph cell and secretory granules area and the number of secretory granules, globules, and IMs per gonadotroph.
Determination of Bromodeoxyuridine Incorporation in Gonadotrophs
Immature African catfish (10 wk of age, 15–20 g body weight) were implanted with C or T silastic pellets as described above. On the day of implantation and at 3, 7, 10, and 13 days after implantation, the animals received injections with bromodeoxyuridine (BrdU; Sigma) dissolved in 0.8% w/v NaCl at a dose of 50 µg/g body weight. Cells in the S-phase of the cell cycle incorporate BrdU into their DNA. On Day 14, pituitaries were collected and cell suspensions were prepared according to the method of Lescroart et al. [29]. After dilution of the dispersed pituitary cells to 375000 cells/ml with L-15 medium, 200-µl aliquots were placed on glass coverslips (24 x 24 mm) and incubated for 90 min at 25°C in 5% CO2/95% air to allow attachment of the cells to the coverslip. Subsequently, the coverslips were washed with PBS and placed in Bouin fixative for at least 10 min. After rinsing three times in PBS, nonspecific binding was blocked with 0.5% w/v nonfat dry milk in PBS for 30 min. The coverslips were then subjected to a double-staining procedure. For BrdU detection, a kit was used according to the manufacturer's instructions (Amersham, Buckinghamshire, U.K.). The staining reaction was developed with diaminobenzidine. For the subsequent LH ß-subunit immunostaining, coverslips were incubated for 1 h with rabbit anti-catfish LH ß-subunit antiserum (40-2) [22] diluted 1:2000 in PBS containing 0.2% v/v Triton X-100 and 0.01% w/v acetylated bovine serum albumin (Aurion, Wageningen, The Netherlands). Immunostaining was revealed with fluorescein isothiocyanate-conjugated goat anti-rabbit serum (1:200 in PBS; Sigma) for 1 h. All steps were carried out at room temperature.
The LH- and BrdU-positive cells were identified in the following way. For each coverslip, the number of stained cells and the total number of cells per field of view were counted. At least 10 fields/coverslip were analysed. At least 1000 cells were analyzed per experimental condition. Double-labeled cells were recorded separately.
Statistics
All data are expressed as the mean ± SEM. Levels of LH are expressed in nanograms per milliliter of plasma or incubation medium or in nanograms per pituitary. Levels of mRNA for GP and LHß were corrected for the 28S rRNA and expressed as a percentage of end control values or SHAM values. Data, if necessary, were log10 transformed prior to analysis. The overall significance of any differences was determined by analysis of variance (ANOVA). In the case of significance (P < 0.05), individual groups were compared by contrast analysis according to Fisher. P values of <0.05 were considered significant. A two-tailed unpaired Student t-test was carried out for analysis of the effects of sGnRHa on LH release and of the effect of T on gonadotroph proliferation.
Steroid levels are given as nanograms per milliliter of plasma (Table 1). Because several samples were below the limit of detection (0.16 ng/ml), a nonparametric analysis was applied, in which the limit of detection was used to represent nondetectable plasma levels. The overall significance of any differences was determined by the Kruskal-Wallis H-test. In the case of significance (P < 0.05), pairs of groups were compared using the Mann-Whitney U-test.
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RESULTS
Experiments with Castrated Fish
Androgen levels in castrated animals were low, and in many fish levels were below the limit of detection of 0.16 ng/ml plasma (Table 1). Implantation of pellets containing KT or T increased the respective circulating hormone levels two- to fourfold above the levels found in sham-operated controls. A male-type urogenital papilla, an androgen-dependent secondary sexual characteristic, was observed in all sham-operated animals but was absent in nearly all castrated fish. Development of the urogenital papilla was retained in the CAS+KT but not in the CAS+T group. The sham-operated control animals displayed a higher gonadosomatic index (GSI) after 3 wk than did the start control groups, reflecting testicular development during the experimental period of 10–13 wk of age. The plasma androgen levels in sham-operated males also were increased above start control levels, where many samples were below the limit of detection.
The plasma LH level in sham-operated animals was higher than that in the start control group (Fig. 1A). Neither castration nor subsequent treatment with KT led to a change of LH plasma levels as compared with sham-operated controls. Treatment of castrated fish with T, however, lowered plasma LH levels significantly, although they remained above start control levels. The pituitary LH content increased strongly in sham-operated controls within 3 wk (Fig. 1B). Castration impaired this increase, and similar data were obtained in a repeated castration experiment (start control, 16 ± 5; sham-operated, 852 ± 192; CAS, 143 ± 31 ng LH/pituitary). Treatment with T increased the LH content to a value similar to that recorded in pituitaries of sham-operated animals. Treatment of castrated males with KT, however, led to pituitary LH levels that were similar to those found in castrated males receiving no steroid treatment.
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RNase protection assay analysis revealed that 3wk after castration the steady-state levels of GP and LHß mRNA in pituitary were 75% ± 7% and 30% ± 4% of the levels in the sham-operated controls, respectively, the decrease being significant (P < 0.05) in the case of the LH ß-subunit. The amount of GP mRNA in the sham-operated group exceeded that of the LH ß-subunit mRNA 12- ± 2-fold, whereas in pituitaries of castrated fish a 94- ± 9-fold excess of GP over LHß was recorded (P < 0.05).
Experiments with Intact Fish
Plasma LH levels were significantly higher in the C pellet-implanted end control groups than in the start control groups, which were sampled 2 wk earlier (Fig. 2, A and D). Such an elevation of circulating LH was not seen in intact fish treated with T, A, or E2. After treatment with the 11-oxygenated androgens KT, OHA, or KA or with the likewise nonaromatizable DHT, plasma LH levels did not differ significantly from those of the end control group.
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The pituitary LH content in the end control groups was significantly higher than that in the start control groups (Fig. 2, B and E). Treatment with E2 or with the aromatizable androgens T and A further elevated the pituitary LH content, but DHT had no effect. Although KT did not decrease the pituitary LH content in castrated fish (Fig. 1B), treatment of intact fish with KT, OHA, or KA resulted in reduced pituitary LH contents in comparison with the end control group. We reported previously that OHA and KA are converted to KT in vivo and that elevated KT levels are found in OHA- or KA-treated fish [15, 20].
Steroid effects on the steady-state mRNA level of the LH ß-subunit were much more pronounced than those on GP (Fig. 3). Treatment with A, T, and E2 elevated 15- to 18-fold the steady-state levels of LH ß-subunit mRNA. Treatment with 11-oxygenated androgens, on the contrary, resulted in LH ß-subunit mRNA levels that were reduced to one third of the control levels. In the DHT-treated group, both GP and LHß mRNA levels were slightly elevated, whereas GP mRNA levels were reduced after treatment with T. Presenting the data in a normalized way, as in Figure 3, masks the excess of GP over the LH ß-subunit mRNA amounts (on average 54-fold in the end control groups). After treatment with T, A, or E2, this excess was significantly reduced to only 2.5-fold (P < 0.05, as compared with end control), whereas in KT-, OHA-, and KA-treated groups a 101-fold excess of GP over LHß mRNA was recorded (P < 0.05; as compared with both end controls and with T/A/E2-treated groups).
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To investigate whether the hormone-induced changes in the pituitary LH transcript and protein levels affect the releasable pool of LH, pituitaries were isolated and incubated first in the absence and then in the presence of 1 nMsGnRHa. In all cases 1 nM sGnRHa significantly stimulated the release of LH (Fig. 2, C and F). The stimulatory effect of aromatizable androgens and E2 and the inhibitory effect of 11-oxygenated androgens on the pituitary LH content were reflected in increased and decreased sGnRHa-evoked LH release, respectively. Basal LH release, however, showed a different pattern. An increase in pituitary LH content (T, A, or E2 treatment) was not paralleled by an increased basal LH release. The lower basal release after treatment with 11-oxygenated androgens, however, may be related to the reduced pituitary LH load. Secretion of LH from pituitaries of DHT-treated fish did not differ from that of the control group. The increase in sGnRHa-evoked LH secretion over basal secretion was similar for control and androgen-treated animals (range, 4.4- to 9.7-fold) but was significantly (P < 0.05) enhanced from 9.6-fold in controls to 20.4-fold (Fig. 2C) and from 4.0-fold in controls to 16.7-fold (Fig. 2F) in E2-treated fish, respectively.
Structural and Quantitative Analysis of LH-Immunoreactive Granules
Gonadotrophs were scarce and small in pituitaries of the start control group and contained small secretory granules that labeled weakly with anti-LH,ß (Table 2). Globules and IMs, organelles that form part of a crinophagic breakdown pathway, were not observed. In the end control group, gonadotrophs were more abundant and larger and contained more and larger secretory granules that labeled more intensely with anti-LH,ß (Table 2). In all sections examined, one globule but no IMs were observed. After treatment with KT, the ultrastructure of the gonadotrophs appeared to be similar to that in the end control group. Quantitative analysis revealed, however, a significantly lower number of secretory granules than in the end control group (Table 2); globules were seen (0.7 ± 0.3 per section). Treatment with T, however, led to the precocious appearance of adult-type gonadotrophs with large secretory granules, several globules (5.6 ± 1.6 per section), and IMs (0.7 ± 0.2 per section). In addition, the immunolabeling intensity of the secretory granules was enhanced. However, this greater intensity may be due in part to the increased size of the granules.
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Effect of Testosterone on Gonadotroph Proliferation
The data obtained so far indicate that aromatizable androgens stimulate gonadotrophs in juvenile male African catfish. The previous observation that the strong increase in pituitary LH expression during puberty was paralleled by an increase in the number of immunoreactive gonadotrophs [12] raised the question of whether these steroids would induce proliferation of the gonadotrophs. Following implantion in three independent experiments with C pellets or with pellets containing T, the percentage of immunoreactive gonadotrophs doubled among enzymatically dispersed pituitary cells (control, 1.1% ± 0.4%; T, 2.1% ± 0.3%; P < 0.05) in T-treated males. However, the percentage of BrdU-labeled cells did not change significantly (control, 7.8% ± 1.8%; T, 9.1% ± 2.1%), whereas the initially already low percentage of BrdU and LHß double-labeled cells (0.13% ± 0.09%) tended to decrease after treatment with T (0.06% ± 0.03%, P < 0.07). Similar results were obtained with pituitary cells isolated from females (data not shown).
DISCUSSION
The findings of the present study are consistent with the idea that the androgens T and KT play important and distinct stimulatory roles in the initiation of puberty in male African catfish.
Evidence for the involvement of T of testicular origin in triggering gonadotroph activation comes from the following observations: 1) castration markedly reduced the elevation in the pituitary LH content, an effect reversed by T replacement; 2) T increased the number of LH-immunoreactive pituitary cells and led to a precocious development of adult-type gonadotrophs; and 3) an increased amount of LHß-subunit mRNA was found in the pituitary after T treatment. The stimulatory effects of T on the gonadotrophs seem to depend on the conversion of T to estrogens, considering that the stimulatory action of T was mimicked by E2 and that treatment with the nonaromatizable T analogue, DHT, had no (LH content) or very weak (LH mRNA levels) effects. Aromatase activity has been reported in adult catfish gonadotrophs [30] and in the pituitary of other teleost species [31, 32]. More recently, we demonstrated that T was only effective in stimulating LH ß-subunit mRNA levels in vitro after its aromatization to E2 in adult male African catfish [10]. Stimulatory effects of aromatizable androgens or E2 on the LH ß-subunit expression or pituitary LH content have been described in other fish species [33;nd36]. The aromatase activity in the pituitary of juvenile male African catfish does not appear to be a limiting factor; its quantification by analyzing the conversion of [7-3H]androstenedione to estrogens [37] showed similar activity per microgram of pituitary protein in 10- and 40-wk-old males (unpublished observations). Collectively, these data indicate that in immature male catfish, LH expression may be stimulated by a direct estrogen-dependent effect on the gonadotrophs, fueled by the testicular production of aromatizable androgens.
Castration did not completely prevent the increase in LH expression. Although additional endocrine signals, such as GnRH, may be involved in gonadotroph stimulation, it is also possible that the residual increase in pituitary LH reflects the activity of the residual T found in the blood plasma after castration. The activity of GnRH neurons, however, is unlikely to play a leading role in stimulating LH expression at the onset of sexual maturation, considering that the LH content in an additional pituitary grafted into the epaxial trunk muscles, and hence disconnected from GnRH input from the brain, was activated by treatment with T in a similar way as the recipient's own pituitary in juvenile rainbow trout (Oncorhynchus mykiss) [38]. Increased availability of T can largely explain the activation of gonadotrophs during puberty in fish.
RNAse protection assay analysis revealed that the steady-state LH ß-subunit mRNA level was strongly increased by aromatizable androgens, whereas the GP -subunit mRNA level remained largely unaffected. It was also found that the GP -subunit mRNA level exceeds that of the LH ß-subunit mRNA. Together with the observation that the amount of GP protein in juvenile males exceeds by 100-fold the amount of LHß and that in mature individuals there is still a 3-fold excess [12], these data suggest that the availability of heterodimeric LH is limited by the expression of the hormone-specific ß-subunit. An excess of GP over the pituitary glycoprotein ß-subunits is a common phenomenon in other vertebrates [39, 40].
The increase in LH production after treatment with T was accompanied by an increase in the number of immunoreactive gonadotrophs and a precocious cytological development of these cells. Previous studies with male African catfish have shown that along with an increased number of gonadotrophs the LH content per cell also rose [12]. In the present study, we found no evidence for a T-stimulated gonadotroph proliferation. Rather, it appeared that T recruited terminally differentiated but quiescent gonadotrophs into a state of increased activity. The tendency to further decrease the already low percentage of BrdU-incorporating gonadotrophs even may indicate that T finalized the terminal differentiation of the gonadotroph population.
Treatment with T stimulated the appearance of globules and IMs, which are otherwise typical for gonadotrophs in more mature catfish [41]. Globules and IMs are part of a crinophagic breakdown route for the elimination of LH, and granules are part of the secretory route [27]. Only the secretory granules contain GP- and LHß-immunoreactive material, whereas GP, but not LHß, is lost in globules and IMs in catfish [22, 28] or rainbow trout [42]. The increase in pituitary LH content may reflect in part the presence of endocrinologically inert LH ß-subunit in globules and IMs after treatment with T. The precocious appearance of globules and IMs after T treatment indicates, however, that upregulation of LH expression is associated with an activation of the crinophagic breakdown of LH.
Treatment with aromatizable androgens decreased LH blood levels while increasing LH expression, an observation also made in adult male African catfish [25]. However, castration did not result in increased plasma LH level in the present study. The amounts of T produced by the immature testis, although sufficient to stimulate LH expression, may have been insufficient to exert a negative feedback on LH release, and the stronger signal by exogenous T may be necessary to reduce the circulating LH level in immature males.
Steroid treatments led to circulating hormone levels two- to fourfold above those found in sham-operated fish. The experimentally elevated androgen concentrations in the 3-mo-old fish were similar to the levels found in 4-mo-old males and were about 10-fold lower than the levels found in 12-mo-old adults [43]; we consider these levels, albeit elevated, as still being in the physiological range. The present data on secondary sexual characteristics and pituitary LH levels also seem to indicate that physiological changes have been induced precociously without leading to pharmacological effects.
The pituitary incubation experiments show that steroid treatments resulting in increased pituitary LH levels are reflected in a more pronounced GnRH-induced LH secretion, without having an effect on basal LH release. This finding suggests that the increased pituitary LH content is available for the regulated secretory pathway and forms part of the readily releasable pool of LH. Only treatments with E2 increased the GnRH-evoked LH release. Recent experiments with castrated males indicate an increase in pituitary GnRH receptor transcript levels 3 days after E2 treatment (unpublished results). An upregulation of GnRH receptor transcript levels by E2 also has been reported for an immortalized gonadotroph cell line [44].
In contrast to aromatizable androgens and E2, the 11-oxygenated androgens KT, OHA, and KA reduced pituitary LH ß-subunit mRNA steady-state levels, LH content, and sGnRHa-stimulated LH release in vitro but had no effect on circulating LH levels. The similarity of the effects after treatment with OHA, KA, and KT seen in the present study and in previous studies on spermatogenesis [20] suggests that these androgens exert their effects via a common mechanism of action or that KT is the biologically active androgen after bioconversion of OHA and/or KA. We favor the latter possibility because previous studies have shown that treatment of adult and juvenile male catfish with KA or OHA led to increased plasma levels of KT [15, 20, 25], reflecting the rapid conversion of both OHA and KA to KT in the liver [15].
There was no effect of KT on the pituitary LH content in castrated animals. In intact fish, however, KT treatment reduced the pituitary LH content, suggesting that the inhibitory effect of KT depends on the presence of the testis. Because a similar androgen treatment of juvenile males reduced Leydig cell androgen production [14], the inhibition of pituitary LH production induced by KT and other 11-oxygenated androgens may result from an indirect effect via a reduced Leydig cell T production, leading to a weakened stimulation of LH expression. However, the testicular signal activating pituitary LH production may be more complex, involving other factors besides T. The decreased pituitary LH levels after KT treatment of intact fish also could be related to an effect via FSH, such that possibly reduced FSH levels after KT treatment would reduce the cross-reaction of FSH in the LH RIA. Because catfish FSH protein is not available yet, we cannot directly test its cross-reactivity. However, the KT-induced decrease in pituitary LH was accompanied by similarly reduced LH ß-subunit transcript levels, so that the possible contribution of decreasing FSH levels (if occurring) may be of minor relevance quantitatively.
The available data indicate that an increased testicular output of T is an important factor in the maturation-associated increase in gonadotroph activity. How is this increased androgen output regulated? The testicular capacity to secrete T and the LH concentration inducing the half-maximal secretion of T were similar in male catfish before and after the appearance of spermatocytes [22], which indicates the start of the maturation-associated increase in LH production [12]. However, in the present study plasma LH levels were significantly higher in the end than in the start control groups. Because LH is detectable in pituitary and blood and is available for secretion already in immature fish [22], an increased LH release and, consequently, a stimulation of Leydig cells may result in an increased availability of T. It is not clear how the increase in LH levels is regulated. Higher activities of the GnRH system or an increased pituitary sensitivity to GnRH are obvious possibilities currently under investigation. This increase, however, may initiate the cascade that, via aromatizable androgens of testicular origin, leads to the puberty-associated increase in gonadotroph activity. Because 11-oxygenated androgens also are important products of the teleost Leydig cell, the same gonadotropic stimulation may explain the increased KT plasma levels found in the end control groups. Because KT stimulates spermatogenesis [20, 45], the supra-allometrical growth of the testes may be indirectly related to increased LH levels. A role for FSH in spermatogenesis has not yet been defined in fish. After all, KT triggers complete spermatogenesis in eel testis tissue culture [45], for which gonadotropins are not required. However, growth factors are required [46], which may be a field of FSH action.
The data obtained in the present study suggest that aromatizable androgens of testicular origin play an important role in the dramatic increase in LH expression in pubertal male African catfish. In mammalian models, the regulation of gonadotroph functions requires aromatization of T and the presence of a functional estrogen receptor-. However, the steroid effects are opposite of those seen in the African catfish and in other fishes, considering that castration of adult male mice leads to increased levels of LH ß-subunit mRNA levels, which are reversed by aromatizable androgens or estrogens [47]. In prepubertal male rats, androgen deprivation by castration or treatment with nonsteroidal antiandrogens led to a precocious gonadotroph activation, and androgen replacement suppressed the castration-induced activation [48]. In catfish, on the contrary, aromatizable androgens/estrogens seem to be major stimulators of LH ß-subunit gene expression; castration [21] or treatment with a nonsteroidal aromatase inhibitor (unpublished observation) results in depressed LHß mRNA levels. In both fish and mammals, however, the transition from immature to mature males is associated with increased circulating levels of sex steroids and an increased activity of the pituitary gonadotrophs, and a negative steroid-mediated feedback on LH secretion seems to operate in both fish and mammals.
ACKNOWLEDGMENTS
The authors thank Drs. F.E.M. Rebers and M.A. Zandbergen (Department Developmental Biology, University of Utrecht) for their help with the RNAse protection assays and the morphology, respectively. The help of the undergraduate students Astrid van Bemmel, Joanneke Huck, and Rachel Veal and of the staff of the Department for Image Processing and Design (Utrecht University) is gratefully acknowledged.
FOOTNOTES
First decision: 18 October 2000.
1 This work was supported by grant BD/2603/93 from PRAXIS XXI J.N.I.C.T.–Portugal and by grant ENV4-CT97-0567 from the European Union. A preliminary report on part of the data was presented as a poster at the 18th European Conference for Comparative Endocrinologists, 10–14 September 1996, Rouen, France. 
2 Correspondence: R.W. Schulz, Research Group Comparative Endocrinology, Faculty of Biology, Department of Developmental Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 31 30 2532837; r.w.schulz{at}bio.uu.nl
3 Current address: Molecular and Comparative Endocrinology, Universidade do Algarve-UCTRA, Centro de Ciências do Mar, Campus de Gambelas, P-8000-810 Faro, Portugal.
Accepted: December 12, 2000.
Received: September 19, 2000.
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