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Regular Article |
a Utrecht University, Faculty of Biology, Research Group Comparative Endocrinology, 3584 CH Utrecht, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone, or estradiol-17ß; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17ß further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17ß or 5-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.
luteinizing hormone, pituitary, puberty, spermatogenesis, steroid hormones, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous work in juvenile male African catfish (Clarias gariepinus) showed that treatment with T or estradiol-17ß (E2) elevated the pituitary LH content and LH ß-subunit steady-state mRNA level, whereas 5-dihydrotestosterone (DHT) had no such effect [5]. Inhibiting aromatase activity in primary pituitary cell cultures of adult catfish abolished the stimulatory effect of T on LH ß-subunit expression [6]. This enzyme activity is also present in the pituitary of immature catfish [5], so T-induced LH expression may be mediated via an estrogen-dependant process, which is a notion supported by data in other species [7]. Hence, the increase in pituitary LH levels during puberty in catfish [8] may reflect increased production by the testes of T, which is converted to E2 in the pituitary.
Stimulation of spermatogenesis, however, appears to be a specific effect of KT in catfish, because neither T, DHT, nor E2 has such an effect [9]. A direct stimulatory effect of KT on spermatogenesis has been described in Japanese eel (Anguilla japonica) testicular explants [10]. In the present study, male catfish at the beginning of spermatogenesis were subjected to a cotreatment with KT and T in an attempt to induce precocious stimulation of both pituitary and testis development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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African catfish were bred and raised as described previously [11], 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, exposed to a photoperiod of 14L:10D, and fed with Trouvit pellets (Trouw, Putten, The Netherlands). Fish in experiments A, B, and C were 10 wk of age (i.e., only spermatogonia present in the testis), with an average body weight of 12.5 ± 1.4 g at the beginning of the experiment. In experiment D, 15-wk-old fish were used, with an average body weight of 26.4 ± 4.3 g. All procedures involving experimental animals were conducted in accordance with Dutch national regulations; the Life Sciences Faculties Animal Care and Use Committee approved the protocols.
All steroids and salmon GnRH analogue ([D-Arg6, Trp7, Leu8, Pro9-NEt]GnRH, sGnRHa) were purchased from Sigma Chemical Company (St. Louis, MO). The catfish LH preparation used for the stimulation of testicular androgen secretion in vitro has been described previously [12].
Experimental Protocol
African catfish were implanted with silastic pellets containing different steroids at a dose of 30 µg/g body weight as described previously [9]. An end control group received steroid-free pellets. A start control group was sampled at the day of pellet implantation.
In experiment A, three groups of 40 fish received pellets containing KT, T, or androstenedione (A); two other groups received combined treatments (KT + T or KT + A). The sex of juvenile catfish cannot be determined externally, and at the end of experiments, the treatment groups were found to contain 15–25 males. Two weeks after implantation, blood plasma was collected and stored at -20°C until quantification of LH and androgens by RIA. The pituitaries were removed and used to quantify LH content or basal and GnRH-stimulated LH release in a tissue culture system. The presence of secondary sexual characteristics (i.e., seminal vesicles and urogenital papilla, as described previously [9]) was recorded and expressed as the percentage of animals in which the structures were developed. Testis and body weight were determined to calculate the gonadosomatic index (GSI; testis weight x 100/body weight). The stage of spermatogenesis was determined histologically.
In experiment B, groups of 10–15 males were implanted with KT, DHT, E2, KT + DHT, or KT + E2, respectively. In this experiment, only the GSI and the pituitary LH content were analyzed 2 wk after implantation of steroid-containing pellets.
In experiments C and D, groups of 11 or 14–18 males received T implants at 10 (experiment C) or 15 (experiment D) wk of age, respectively. Two weeks later, another T pellet was implanted, and after another 2 wk, blood and pituitary samples were collected and used for LH quantification. Testis samples were collected for histological analysis of spermatogenesis.
Pituitary Tissue Culture
Determination of basal and sGnRHa-stimulated LH release was carried out essentially as described previously [13], except that only the maximally effective dose (1 nM sGnRHa) was used. The sGnRHa is a high-affinity ligand for the catfish GnRH receptor, and it effectively stimulates LH secretion in vitro [14]. Briefly, the pituitaries were preincubated for 18 h in 0.5 ml of Earle balanced salt solution (Life Technologies-Gibco, Grand Island, NY). After one rinse, 0.5 ml of fresh medium was added, and the incubation was continued for 3 h. The medium was collected for the determination of basal LH secretion, and pituitaries were rinsed once with fresh medium. Finally, 0.5 ml of medium containing 1 nM sGnRHa was added for another 3 h of incubation to determine the sGnRHa-stimulated LH release. After incubation, the medium was collected and stored at -20°C until assayed for LH.
Testis Histology
Testes were removed and weighed to calculate the GSI. The tissue was then processed for light microscopic analysis of the stage of spermatogenesis using 1-µm sections stained with 1% (v/v) methylene blue in 1% (v/v) borax as described previously [9].
In the African catfish, pubertal spermatogenesis has been subdivided into four histological stages [9]. Stage I testis contains spermatogonia only; no meiotic or postmeiotic germ cells are present. Stage II testis shows spermatogonia and spermatocytes, but no postmeiotic germ cells. Stage III testis contains spermatogonia, spermatocytes, and spermatids, but no spermatozoa are present. In stage IV testis, all germ cell stages, including spermatozoa, are present. Testis development in treatment groups is indicated as the percentage of testicular stages (number of animals in a certain stage x 100/total number of animals per treatment group).
Stages I–IV do not correspond to stages of the germinal epithelium, such as those known from mammalian testis. In fish, a group of Sertoli cells forms an intratubular space, or a cyst, housing germ cells that all belong to one germ cell clone. In this way, the descendants of a given stem cell that proceed synchronously through spermatogenesis are taken care of by "their" group of Sertoli cells. Fish Sertoli cells, therefore, contact germ cells that are all in the same stage of spermatogenesis. Hence, recurrent associations along the Sertoli cell surface of different germ cell clones at different stages of development, which constitute the stages of the germinal epithelium in mammals (and other amniotes), do not occur in fish (and other anamniotes).
Radioimmunoassay
The LH concentrations in plasma, pituitary incubation media, and pituitary homogenates were quantified by RIA according to the method described by Goos et al. [15], but with the modifications as described previously [13], using an antiserum raised against the ß subunit of LH. The limit of detection for LH is 0.25 ng/ml, with intra- and interassay variability at median effective concentrations of 6% and 11%, respectively. The plasma levels of KT and T were determined as described previously [16]. The limit of detection for the androgen RIAs is 0.3 ng/ml, with intraassay variabilities of 4% and 9% and interassay variabilities of 5% and 9% in the KT and T assays, respectively.
Statistics
Hormone levels are expressed as the mean ± SEM. The LH levels are given as nanograms per milliliter of plasma or incubation medium or as nanograms per pituitary. Steroid levels are given as nanograms per milliliter of plasma. For statistical analysis of hormone levels, data were log10 transformed and subjected to analysis of variance followed by the Fisher least significant difference test. For analysis of the stimulatory effect of sGnRHa on the release of LH, a one-tailed, paired Student t-test was used. Secondary sexual characteristics and histological data were analyzed by the Fisher exact test, followed by the Kruskal-Wallis H-test. In all cases, differences were considered to be statistically significant at P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In all groups implanted with KT pellets, plasma levels of KT increased to 7–10 ng/ml (Table 1). Such concentrations are typically found in 10- to 12-mo-old adult males [17]. The KT plasma levels remained unchanged in T- and A-treated groups, whereas elevated levels of T were found in T- and A-treated groups. Fish receiving a second implant of T after 2 wk showed higher plasma T levels than those receiving a single implant. Also, the elevated plasma levels of T found in fish carrying T or A pellets were similar to those levels recorded in adult males [17].
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Testicular Histology, GSI, and Secondary Sexual Characteristics
An increase in GSI was observed when comparing the start and end control groups in experiments A and B (Fig. 1, a and c), reflecting the onset of testis development during the experimental period of 2 wk. Treatment with KT increased the GSI above end control levels and promoted spermatogenesis, as indicated by the higher proportion of males with spermatocytes (Fig. 1b). Treatment with T, A, DHT, or E2 did not affect the GSI and/or spermatogenesis compared to the end control group. However, T slightly promoted seminal vesicle, but not urogenital papilla, development. Unexpectedly, the stimulatory effect of KT on testis development was abolished by cotreatment with T or A, which also attenuated the stimulatory effect of KT on seminal vesicle, but not on urogenital papilla, development (Fig. 1b). Cotreatment with DHT or E2, on the other hand, failed to inhibit the stimulatory effect of KT on testis growth (Fig. 1c).
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In experiment C (4 wk of T treatment beginning at 10 wk of age), the end control group presented immature (n = 4) and maturing (n = 5) subgroups, reflecting individual differences in the timing of maturation. The data obtained from these two subgroups were distributed in two combinations: low GSI, absence of postmeiotic germ cells, and low pituitary LH content; or high GSI, presence of postmeiotic germ cells, and high pituitary LH content. The end control group, therefore, was split in two subgroups, which showed statistically significant differences in all parameters analyzed. The GSI values and stages of spermatogenesis (Fig. 1, d and e) were similar in the T-treated fish and in the immature end control group, whereas the maturing controls were clearly advanced in spermatogenesis.
In experiment D (conducted as experiment C, except that T treatment started at 15 wk of age), the end control group was well advanced as regards testis growth and germ cell development (Fig. 1, f and g) compared to the start control group. Treatment with T clearly attenuated testis growth. Because in this experiment the treatment started at a more advanced stage, the histological analysis also provided evidence for a fallback of spermatogenesis behind the start control group levels, indicating that more advanced germ cell stages that had been present, as in the start control group, were lost during the 4 wk of exposure to T.
Pituitary LH Content, Basal and sGnRHa-Stimulated LH Secretion In Vitro, and Plasma LH Levels
The pituitary LH content had increased significantly when the start and the end control groups were compared (Fig. 2, a, d, and e), reflecting normal development during the experimental period of 2 wk. Treatment of 10-wk-old fish with aromatizable androgens (T and A) or E2 elevated the pituitary LH content four- to sixfold above the end control levels, and cotreatment with KT had no effect in this regard. However, after treatment with KT alone, pituitary LH contents were lower than in the end control groups. Implantation of DHT alone had no effect on pituitary LH levels (Fig. 2d), but it abolished the inhibitory effect of KT in the respective cotreatment study (experiment B).
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Also with regard to pituitary LH levels, two subgroups (<70 ng/pituitary [immature] and > 1110 ng/pituitary [maturing]) were found in the end control group of experiment C (Fig. 2e). Different from the shorter treatment of 10-wk-old fish, in experiments involving longer treatment with T, this androgen did not elevate pituitary LH levels above those found in the maturing end control fish in experiment C (Fig. 2e) or in experiment D, in which similar pituitary LH amounts were found for end control (3.3 ± 0.8 µg/pituitary) and T-treated fish (3.1 ± 0.5 µg/pituitary). Elevated plasma T levels (Table 1) and the inhibitory effects on testis growth and spermatogenesis (Fig. 1, d–g) indicate, however, that T treatment in these fish was effective.
The pituitary release of LH was significantly stimulated by 1 nM sGnRHa in all groups (Fig. 2b, experiment A). Whereas basal LH release was similar in most groups (except for the KT + T treatment), the amount of LH released in response to sGnRHa was higher in those groups showing a higher pituitary LH content than in the end control group. The reduced LH content in the KT-treated group, however, was not reflected in a reduced LH release in response to sGnRHa compared to that in the end control group (Fig. 2b).
The plasma LH levels increased between the start and end control groups (Fig. 2, c and f). Treatment with KT had no effect on circulating LH levels, whereas the T- and A-treated groups showed levels similar to those in the start control group. Cotreatment with KT had no effect on the T- or A-induced reduction of plasma LH levels. In experiment C, similar plasma LH values were found in T-treated and maturing end control fish, which had lower levels than those found in immature controls (Fig. 2f). In experiment D, the plasma LH levels did not differ significantly between the end control (0.7 ± 0.1 ng/ml) and T-treated groups (0.8 ± 0.2 ng/ml).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of single-hormone treatments are in agreement with those of previous studies [5], confirming that aromatizable androgens and E2 can elevate pituitary LH levels and sGnRHa-stimulated LH secretion while reducing plasma LH levels. Treatment with KT, on the other hand, suppressed pituitary LH levels compared to those in the end control group. This effect of KT depends on the presence of the testis [5]. Because KT treatment inhibits Leydig cell activity [18], we propose that the resulting, largely reduced testicular production of T limited the increase in pituitary LH among KT-treated fish. This notion is supported by the observations that cotreatment with an aromatizable androgen (or E2) compensates for the inhibitory effect of KT on pituitary LH levels. That pituitary LH levels in these cotreatment groups were indistinguishable from those in treatments with T or A alone suggests that KT has no major, direct effect on LH production. Aromatizable androgens, however, play a key role in stimulating gonadotroph activity in immature male catfish. Also, DHT abolished the inhibitory effect of KT on pituitary LH levels. This may relate to the slight (i.e., twofold) stimulatory effect of DHT on the steady-state mRNA levels of both LH subunits [5]. However, because DHT has no effect on primary gonadotroph cultures, at least in adult males [6], DHT may not have a direct effect on the pituitary level.
No difference was found in pituitary LH levels between end control and T-treated fish during experiment D, in which the fish were 5 wk older than those used for the other experiments. The decreased impact of exogenous T in older fish might reflect increased endogenous stimulation, so that treatment of juvenile males with T might induce precocious attainment of a predetermined level of LH in the pituitary.
Unexpectedly, aromatizable androgens inhibited the stimulatory effect of KT on catfish spermatogenesis. The initial period of spermatogenesis appears to consist of at least two phases, and the inhibitory effect of T or A seems to be restricted to the second phase. This assumption is based on the observation that neither T nor A inhibited the initial increase in testis growth from the start to the end control group, which mainly reflects spermatogonial proliferation [9]. Experiments in the Japanese eel also suggested that the first five (of ten) mitotic cell cycles of the spermatogonia are regulated differently from the second five cell cycles [19]. Similarly, in the medaka (Oryzias latipes), the spermatogonial generations appear to be subdivided into at least two subgroups, with the first one being characterized by a bisexual potential that is lost after the fourth mitotic cell cycle [20]. In rodents, spermatogonial generations are subdivided in two subgroups (i.e., undifferentiated and differentiating) that differ in their morphological and physiological aspects [21]. We assume that the initial phase of spermatogonial proliferation in catfish has a low sensitivity toward inhibition by exogenous T or A. However, T appears to prevent the development, and/or to induce the loss, of further advanced stages of germ cell development.
Germ cell proliferation and development and, hence, growth of the spermatogenic cyst are associated with proliferation of the cyst-forming Sertoli cells in the adult fish testis [22]. In mammals, Sertoli cell proliferation is a prominent regulatory function of FSH, affecting the spermatogenic capacity of the testis [23]. Further experiments will show if the inhibitory effects of T target, for example, Sertoli cell FSH-receptor expression and/or FSH blood levels. The latter decreased in response to treatment with T in juvenile salmon [24]. With regard to FSH-receptor expression, the inhibitory effect of T on KT-stimulated seminal vesicle development is interesting, because the seminal vesicles in catfish develop from the caudal part of the testis anlage. The epithelium of the vesicles is derived from Sertoli cells [25], and we recently showed a high level of FSH-receptor expression in seminal vesicle tissue [26]. One possible explanation for the inhibitory effect of T on KT-stimulated development of both testis and seminal vesicles, therefore, might be a shared dependency on regulatory processes involving FSH or its receptor. We are presently examining if FSH-receptor expression in testis and seminal vesicle tissue behaves similarly under different experimental conditions.
Although reduced plasma LH levels were found after treatment with aromatizable androgens or E2, indicating that these steroids inhibited LH secretion, reduced plasma LH levels are unlikely to have been responsible for the detrimental effect of T and A on KT-stimulated spermatogenesis, considering that treatment with E2 also reduced circulating LH levels [5] but did not impair KT-stimulated spermatogenesis. Another possible explanation of the inhibitory effect of T on KT-stimulated spermatogenesis relates to the observation that the inhibitory effect of aromatizable androgens on the testis was not mimicked by E2 or DHT, and that the latter steroids were also ineffective in compromising Leydig cell function and morphology [18]. This opens the possibility that the inhibitory effect of T on spermatogenesis might be mediated via suppression of Leydig cell functions that are relevant for spermatogenesis but distinct from supplying 11-oxygenated androgens. Experiments involving long-term testis tissue cultures (e.g., [10, 27]) should be able to address some of these questions.
Regardless of the exact mode of action of T, its effects are clearly distinct from those of KT. The recent detection of two androgen receptors in fish [2–4] with different ligand-binding characteristics opens the possibility that T and KT use different receptors [28], making fish a unique model system for studying the role of androgens in the regulation of spermatogenesis.
In a gonadotropin-deficient mice model, T always had stimulatory effects on spermatogenesis [29], with the extent depending on the androgen dose administered. Under certain conditions in the rat, however, such as after interruption of spermatogenesis following radiation or a cytotoxic treatment, a transient decrease of intratesticular T levels promoted the proliferation and differentiation of A spermatogonia [30]. Experimental models based on fish spermatogenesis may provide interesting aspects for future research in this regard, considering that the stimulatory effects on spermatogenesis appear to be linked to KT, with T and A showing inhibitory effects.
Testosterone is needed to fully stimulate gonadotroph activity in juvenile fish [5]. Although it can have detrimental effects on spermatogenesis under experimental conditions, such effects are not apparent during normal puberty in catfish, when stimulation of gonadotrophs provides evidence for testicular production and biological activity of aromatizable androgens [8] at the time when testis growth and spermatogenesis progress swiftly. We therefore speculate that the biological activity of endogenous T is limited by binding to a hepatic sex steroid-binding globulin and/or a testicular androgen-binding protein. It is interesting to note that catfish sex steroid-binding globulin shows high binding affinities for T and A, but not for KT [31].
In summary, the present study showed that cotreatment of juvenile male African catfish with T and KT did not induce further stimulation of pubertal maturation compared to treatments with single hormones. Instead, T inhibited the stimulatory effect of KT on spermatogenesis. It appears that a balanced production of T and KT and/or a control of their biological activities, possibly involving two types of androgen receptors as well as nonreceptor androgen-binding proteins, enable these androgens to regulate testis and gonadotroph development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 J.E.B.C. was supported by Project "PRAXIS XXI" J.N.I.C.T.-Portugal, grant BD/2603/93. 
2 Correspondence: R.W. Schulz, Utrecht University, Faculty of Biology, Research Group Comparative Endocrinology, 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, Faculdade de Ciências do Mar e Ambiente-CCMar, Campus de Gambelas, P-8000-810 Faro, Portugal.
Accepted: July 31, 2001.
Received: April 13, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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