help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2008-0447
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by García-López, A.
Right arrow Articles by Schulz, R. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by García-López, A.
Right arrow Articles by Schulz, R. W.
Endocrinology Vol. 150, No. 1 357-365
Copyright © 2009 by The Endocrine Society

Leydig Cells Express Follicle-Stimulating Hormone Receptors in African Catfish

Ángel García-López, Jan Bogerd, Joke C. M. Granneman, Wytske van Dijk, John M. Trant, Geir Lasse Taranger and Rüdiger W. Schulz

Division of Endocrinology and Metabolism (A.G.-L., J.B., J.C.M.G., W.v.D., R.W.S.), Department of Biology, Faculty of Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands; Center of Marine Biotechnology (J.M.T.), University of Maryland Biotechnology Institute, Baltimore, Maryland 21202; and Department of Physiology of Growth and Reproduction in Fish (G.L.T., R.W.S.), Institute of Marine Research, 5817 Bergen, Norway

Address all correspondence and requests for reprints to: Dr. Rüdiger W. Schulz, Utrecht University, Faculty of Sciences, Department of Biology, Division Endocrinology and Metabolism, Kruyt Building, Room W-604, Padualaan 8, 3584 CH Utrecht, The Netherlands. E-mail: r.w.schulz{at}uu.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This report aimed to establish, using African catfish, Clarias gariepinus, as model species, a basis for understanding a well-known, although not yet clarified, feature of male fish reproductive physiology: the strong steroidogenic activity of FSHs. Assays with gonadotropin receptor-expressing cell lines showed that FSH activated its cognate receptor (FSHR) with an at least 1000-fold lower EC50 than when challenging the LH receptor (LHR), whereas LH stimulated both receptors with similar EC50s. In androgen release bioassays, FSH elicited a significant response at lower concentrations than those required to cross-activate of the LHR, indicating that FSH stimulated steroid release via FSHR-dependent mechanisms. LHR/FSHR-mediated stimulation of androgen release was completely abolished by H-89, a specific protein kinase A inhibitor, pointing to the cAMP/protein kinase A pathway as the main route for both LH- and FSH-stimulated steroid release. Localization studies showed that intratubular Sertoli cells express FSHR mRNA, whereas, as reported for the first time in a vertebrate, catfish Leydig cells express both LHR and FSHR mRNA. Testicular FSHR and LHR mRNA expression increased gradually during pubertal development. FSHR, but not LHR, transcript levels continued to rise between completion of the first wave of spermatogenesis at about 7 months and full maturity at about 12 months of age, which was associated with a previously recorded approximately 3-fold increase in the steroid production capacity per unit testis weight. Taken together, our data strongly suggest that the steroidogenic potency of FSH can be explained by its direct trophic action on FSHR-expressing Leydig cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Male germ cell development is regulated by the brain pituitary axis, which has evolved in vertebrates as a hormonal master control system over spermatogenesis and reproduction in general. Within this system, the pituitary gonadotropins, LH and FSH, play pivotal roles by regulating testis functions via their respective cognate receptors, LH receptor (LHR) and FSH receptor (FSHR) (1, 2).

In mammals, the interaction between gonadotropins and their receptors is highly specific (3, 4, 5, 6), and the physiological roles of gonadotropins are generally well understood (1, 7, 8): LH regulates steroid production in LHR-expressing Leydig cells, whereas FSH regulates proliferation and the adult functioning of the FSHR-expressing Sertoli cells, such as structural germ cell support and production of paracrine factors influencing spermatogenesis (9) or Leydig cell steroidogenesis (10, 11, 12).

In teleost fish, however, the bioactivities of the two gonadotropins seem to be less specific than in mammals. For example, FSH is a potent stimulator of testicular androgen production both in vitro and in vivo, with a similar potency as LH (2, 13, 14, 15, 16, 17, 18, 19, 20). The mechanism(s) by which this action is exerted in teleosts is unknown, although LHR cross-activation by FSH seems unlikely (6). Moreover, when tested in homologous systems with recombinant or highly purified hormones the FSHR binds and/or responds to LH as well (2, 6, 21, 22, 23, 24, 25). The apparent discrepancies between mammalian and piscine gonadotropin features, and the limited information on gonadotropin physiology in male fish, contribute to the fact that the precise regulatory functions of each gonadotropin on spermatogenesis have not yet been fully understood in fish. However, several studies, mainly performed in salmonids, indicated that FSH seems to be the relevant gonadotropin in early to mid stages of seasonal testicular development, whereas LH is linked to final gamete maturation and spermiation (26, 27, 28, 29, 30, 31, 32, 33). Another question not yet clarified in teleosts is the identity of the testicular cell type(s) expressing either the LHR or the FSHR: although LH binding was localized to Leydig cells and FSH binding to Sertoli cells of coho salmon, Oncorhynchus kisutch, testis (21), the authors could not rule out FSH binding to Leydig cells. Very recently Ohta et al. (34) have reported immunocytochemical detection of FSHR protein in Japanese eel, Anguilla japonica, Leydig cells, suggesting that FSH actions on spermatogenesis are mediated (at least partly) via androgen production.

An important constraint to improve our knowledge on gonadotropin functions in fish is the limited availability of highly purified hormones, mainly FSH. To overcome this problem, considerable effort has been directed to produce bioactive recombinant fish gonadotropins. Recently an effective protocol for producing biologically active recombinant channel catfish (rCcf), Ictalurus punctatus, gonadotropins has been developed (25) allowing us to test their specific actions in established experimental systems. To develop a basis for understanding the steroidogenic activity of fish FSH and provide new insight into the contributions of FSH and LH to the development toward sexual maturity in male teleost fish, the present report addresses the following issues using African catfish (Acf), Clarias gariepinus, as model species: 1) the in vitro bioactivities of rCcfLH and rCcfFSH on testicular androgen release and activation of gonadotropin receptors transiently transfected in human embryonic kidney T 293 (HEK-T 293) cells; 2) the implication of the cAMP/protein kinase A (PKA) pathway on the AcfLHR- and AcfFSHR-mediated stimulation of androgenesis; 3) the cellular sites of gonadotropin receptor expression in catfish testis tissue; and 4) the changes of testicular gonadotropin receptor expression during puberty and the development toward full maturity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Juvenile (11, 16, and 21 wk old), adolescent (32 wk old), and fully sexually mature (51 wk old) male African catfish were used in the experiments reported here. Fish were bred and raised in the laboratory as described before (35) and were kept under constant water temperature (25 ± 2 C) and photoperiod (14 h light, 10 h dark). Animal culture and experimentation were consistent with Dutch national regulations; the Life Science Faculties Committee for Animal Care and Use approved all experimental protocols.

Gonadotropins
AcfLH was isolated from mature African catfish pituitaries as described previously (36). rCcfLH and rCcfFSH were produced in Schneider’s S2 Drosophila cell line as reported elsewhere (25).

In vitro gonadotropin receptors activation
In vitro bioactivities of these gonadotropins were determined via ligand-stimulated, cAMP-mediated reporter gene expression in HEK-T 293 cells transiently expressing either the AcfFSHR or the AcfLHR, as described previously (37, 38, 39). Receptor activation assays were repeated three to four times using cells from independent transfections. The gonadotropin concentrations inducing half-maximal stimulation (i.e. EC50) of cAMP-mediated reporter gene expression were calculated as reported (37) and tested for significant differences by one-way ANOVA followed by the Holm-Sidak test (P < 0.05).

Androgen secretion by African catfish testis in vitro
Testicular tissue from sexually mature African catfish was prepared for in vitro incubations essentially as described previously (35). Pooled testis tissue fragments from three to four males were used for each experiment.

For the trials with different concentrations of rCcfLH or rCcfFSH (n = 3), four or six replicates per ligand concentration (range 1.5–150 ng/ml medium) were incubated for 18 h. The experiments included a positive control with 10 µM of the adenylate cyclase activator forskolin (Sigma-Aldrich, St. Louis, MO), a concentration inducing a maximal steroid release response (40).

For a time-course experiment, eight replicates were incubated from 10 min to 18 h with 45 ng/ml of either recombinant gonadotropin.

The role of the cAMP/PKA pathway on the LHR- and FSHR-mediated stimulation of androgen release was investigated by incubating testis tissue fragments for 18 h with 150 ng/ml AcfLH (isolated from pituitaries of adult African catfish) in the presence of increasing concentrations (10, 50, and 100 µM) of the PKA inhibitor H-89 (Sigma-Aldrich). At this concentration, AcfLH fully activates both the AcfLHR and AcfFSHR (2, 37, 38 ; see Results), eliciting a maximal stimulation of steroid release from primary testis tissue cultures (37, 40, 41, 42). The inhibitor was added to the tissue fragments one h before exposure to AcfLH. Two independent experiments were done, using four replicates per condition in each one.

After incubation, the supernatants were radioimmunoassayed for 4-androsten-11β-ol-3,17-dione [11β-hydroxyandrostenedione (OHA)], the main androgen produced by African catfish testis (35, 43), as described previously (35).

For the dose-response and H-89 experiments, data were standardized to fold induction of basal OHA release (nanograms per milligram testis tissue) and then compiled for statistical analyses by one-way ANOVA followed by the Student-Newman-Keuls test (P < 0.05). To allow conversion into absolute androgen levels, basal OHA release is given in the respective figure legends. The same statistical method was used for the time-course experiment, although raw instead of standardized OHA release data were used.

Laser microdissection
To identify the testicular compartments in which the gonadotropin receptors are expressed, a PALM MicroBeam Instrument (PALM Microlaser Technologies, Bernried, Germany) was used for microdissection of sexually mature African catfish testis sections. Two microdissected fractions were analyzed in two independent biological samples for the quantification of AcfLHR and AcfFSHR mRNA abundance: interstitial tissue, excluding blood vessels, and intratubular tissue, containing spermatogenic cysts (i.e. germ/Sertoli cells units) (see Fig. 4Go, A and B). To this end, 10-µm cryosections from snap-frozen African catfish testis were mounted on PALM 1-mm MembraneSlides, dried for 1 min, and fixed in 70% ethanol for 3 min inside the cryostat chamber (set to –25 C). Then sections were transferred to room temperature, stained with Mayer’s hematoxylin for 3 min, dehydrated in ethanol, and air dried. Areas of interest were immediately selected using the PALM MicroBeam system, laser-microdissected (see Fig. 4BGo), and pressure-catapulted into 40 µl RNAlater (Ambion, Austin, TX). Given that both Leydig cells and germinal cysts at different stages of development are uniformly distributed along African catfish testis (see supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org), interstitial and intratubular samples were collected randomly throughout the whole surface of the testis sections. Total RNA was extracted from the two different microdissected fractions (RNAqueous microkit), and submitted to linear amplification (MessageAmp II aRNA amplification kit) following, in both cases, the manufacturer’s instructions (Ambion). Ninety percent of total amplified RNA (0.7–1.9 µg) was reverse transcribed to cDNA in a final volume of 45 µl, as described previously (37). Primers and fluorogenic probes to detect AcfFSHR mRNA, AcfLHR mRNA, and Acf -glyceraldehyde-3-phosphate dehydrogenase mRNA (endogenous control) were used for quantitative PCRs (qPCRs) as reported previously, including the tests for specificity and amplification efficiency on serial dilutions of testis cDNA (37, 38, 44). qPCRs were performed and cycle threshold (Ct) values determined in a 7900HT real-time PCR system (Applied Biosystems, Foster City, CA) using default settings, as described previously (37). Relative AcfLHR and AcfFSHR mRNA levels were calculated using the {Delta}{Delta}Ct method as reported (37). To allow comparison of expression levels of both receptors in both microdissected fractions and considering that all primers/probe combinations showed an identical PCR amplification efficiency (37, 38, 44), the mean {Delta}Cts of all the samples and genes analyzed was used as calibrator (reviewed in Ref. 45).


Figure 4
View larger version (66K):
[in this window]
[in a new window]

  		FIG. 4. Laser microdissection and real-time qPCR analysis of sexually mature (51 wk old) African catfish testis tissue fractions. A, Target areas for microdissection were selected and tagged as follows: interstitial (tagged with number 1) containing presumptive Leydig cells (arrow) and excluding blood vessels (BV) and intratubular (tagged with number 2) containing spermatogenic cysts (i.e. Sertoli and germ cells; asterisks). B, After selection, tissue was laser microdissected and pressure catapulted into separate tubes for interstitial and intratubular tissue. Stippled lines delineate the border between interstitial tissue and spermatogenic tubules. Scale bars, 30 µm. C, Relative African catfish gonadotropin receptor mRNA expression levels in microdissected fractions. Data correspond to values from two experiments with duplicate measurements each (mean ± SEM) and normalized to Acf-glyceraldehyde-3-phosphate dehydrogenase mRNA levels.

 
In situ hybridization
Once we defined the testicular compartments (intratubular vs. interstitial) in which each gonadotropin receptor was expressed, in situ hybridization was performed to identify the cell types expressing AcfLHR and AcfFSHR mRNA.

Specific AcfLHR and AcfFSHR PCR products (~450 bp) were generated (see supplemental Table 1), gel purified, and served as template for generating digoxigenin (DIG)-labeled cRNA probes as reported (46).

In situ hybridization was performed on 10-µm cryosections. Tissue fragments from sexually mature testis were fixed in 4% paraformaldehyde-PBS [0.1 M (pH 7.4)], cryoprotected by 25% sucrose immersion, and embedded in Neg-50 resin (Richard Allen Scientific, Kalamazoo, MI) before cryosectioning. Adjacent sections were hybridized as previously described (with modifications) with either antisense or sense probes, the latter serving as negative control (46). Modified steps included proteinase K (Sigma-Aldrich) treatment for 2 min at 37 C; hybridization at 70 C with 800 ng/ml DIG-labeled cRNA probe in hybridization buffer [containing 1 mg/ml calf thymus DNA (Invitrogen, Carlsbad, CA) instead of 0.5 mg/ml herring sperm DNA] for 18 h; posthybridization washes in 2x standard saline citrate (SSC) at room temperature, 30% deionized formamide in 2x SSC (once) and 0.2x SSC (twice) at 65 C, and 0.2x SSC at room temperature; ribonuclease A (Sigma-Aldrich) treatment (20 µg/ml) for 30 min; immunodetection of DIG by preincubation for 1 h at room temperature with buffer 1 [0.1 M Tris, 0.15 M NaCl (pH 7.5)] containing 0.5% Roche blocking reagent (Roche, Mannheim, Germany; buffer 2); and incubation for 18 h at 4 C with sheep anti-DIG, alkaline phosphatase-linked antiserum (Roche) diluted 1:2000 in buffer 2. Finally, signal visualization was achieved by incubation overnight with staining buffer [containing 2 instead of 1 mM levamisole (Sigma-Aldrich)] at room temperature.

African catfish pubertal development
Whole testis tissue samples from 11-, 16-, 21-, 32-, and 51-wk-old African catfish were processed as described previously (40) to estimate by qPCR analysis the relative AcfLHR and AcfFSHR mRNA levels throughout pubertal development and until reaching full maturation. African catfish 28S rRNA was used as endogenous control. Pubertal development in African catfish has been described in detail previously (41). Briefly, at 11 wk of age, African catfish testes are still quiescent/prepubertal; single or small groups of spermatogonia are the only germ cells observed. By 16 wk of age, spermatogonial proliferation and differentiation and meiosis have started, and spermatocytes and occasionally spermatids are found in the tubules. At 21 wk of age, spermatocytes and spermatids became more abundant, and all males showed some tubules with free spermatozoa. The 32-wk-old catfish are considered as adolescent or young adults: all tubules contained free spermatozoa but androgen release still has to reach adult levels (35). Full maturation is reached at approximately 40–45 wk of age, when fish can be used for breeding purposes. Due to the constant temperature and photoperiod conditions, male catfish stay in a prespawning stage all year round after having reached full maturity.

Due to their small size, testes from three 11-wk-old (n = 7) and from two 16-wk-old (n = 8) fish were pooled per sample, whereas testis tissue from the remaining age groups were analyzed individually (21 wk old, n = 10; 32 wk old, n = 8; 51 wk old, n = 6). To account for the dramatic increase in testis size during puberty and until full maturity is reached (total testis weight increases from ~2 mg to ~20 g) (35, 42), relative mRNA transcript amounts were corrected for the gonadosomatic index (100 x gonad weight x body weight–1). Data were expressed relative to the expression levels measured in 51-wk-old fish. Differences in expression levels among age groups were analyzed by one-way ANOVA followed by the Student-Newman-Keuls test (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro activation of African catfish gonadotropin receptors by recombinant and purified gonadotropins
rCcfFSH was highly efficient in stimulating the AcfFSHR with an EC50 of 0.1 ng/ml (Fig. 1Go). Both pituitary-purified AcfLH and rCcfLH were also able to activate the AcfFSHR, although with approximately 56- (AcfLH; EC50 5.5 ng/ml) and 220-fold (rCcfLH; EC50 21.4 ng/ml) lower efficiency than rCcfFSH (P < 0.05). Both pituitary-purified and recombinant LH-activated AcfLHR, with rCcfLH being approximately 3-fold more potent than pituitary-purified AcfLH (EC50 9.3 vs. 29.6 ng/ml; P < 0.05; Fig. 1Go) in stimulating cAMP-dependent reporter gene expression. Hence, both LHs are able to half-maximally activate both FSHR and LHR at concentrations of approximately 5–30 ng/ml. Although rCcfFSH also activated the AcfLHR, its EC50 was much higher than those of the two LHs (709 ng/ml; P < 0.05; Fig. 1Go). Thus, rCcfFSH was approximately 7000-fold more potent in stimulating the AcfFSHR than the AcfLHR. For comparison, Fig. 1BGo also shows the results (between brackets) obtained previously using recombinant Acf gonadotropins produced in the Dictyostelium discoideum expression system (2).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 1. A, Effects of pituitary-purified AcfLH, rCcfFSH, and rCcfLH on HEK-T 293 cells transiently cotransfected with either the AcfFSHR or AcfLHR and a cAMP-responsive reporter gene construct. Data are expressed as arbitrary units (AU) corresponding to the level of reporter gene expression induced by 10 µM forskolin. Data shown correspond to a single representative experiment with triplicate observations per ligand concentration. B, EC50s [nanograms per milliliter; mean (±SEM) from three or four independent assays with triplicate observations each]. For comparison, the values between brackets show the EC50s obtained previously using pituitary-purified or recombinant African catfish gonadotropins (rAcfFSH and rAcfLH) produced in the Dictyostelium discoideum expression system (2 ).

 
In vitro induction of androgen secretion in African catfish testis by recombinant gonadotropins
Incubation of testis tissue fragments from adult African catfish with either rCcfFSH or rCcfLH resulted in a concentration-dependent stimulation of androgen release (Fig. 2Go). The lowest rCcfFSH concentration eliciting a significant elevation of OHA release above control levels was 15 ng/ml (~2.4-fold), whereas for rCcfLH this concentration was 45 ng/ml (~4.2-fold). At 15 ng/ml, the steroid release induced by rCcfFSH was significantly higher than that induced by rCcfLH; for all other concentrations tested both hormones showed similar potencies (P > 0.05). At concentrations of 45 ng/ml or greater, both gonadotropins induced maximal steroidogenic stimulation in the system, i.e. not significantly different from that elicited by treatment with 10 µM forskolin (~4.4-fold).


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 2. Effects of increasing concentrations of rCcfFSH or rCcfLH and 10 µM forskolin (Forsk) on OHA secretion by sexually mature (51 wk old) African catfish testis tissue in vitro. Data are expressed as fold induction over basal OHA release (nanograms per milligram testis tissue) and represent compiled data (mean ± SEM) from three independent experiments with four to six replicates per ligand concentration each. The mean OHA level measured in the basal groups (0) was 1.38 ± 0.11 ng/mg testis tissue. *, Values are significantly different from basal OHA release (P < 0.05); #, significantly different from each other (P < 0.05).

 
In a time-course experiment (see supplemental Fig. 1), OHA secretion was significantly increased with respect to controls at 30 min after addition of either recombinant gonadotropin. Both rCcfLH and rCcfFSH showed a similar potency in stimulating OHA production at all incubation times (P > 0.05).

Implication of the cAMP/PKA pathway on the AcfLHR- and AcfFSHR-mediated stimulation of steroidogenesis
Incubation of testis tissue fragments with 150 ng/ml AcfLH resulted in a maximal 3.8-fold elevation of OHA release above control levels (Fig. 3Go). In the presence of increasing concentrations of the PKA inhibitor H-89, however, the AcfLH-stimulated androgen release was abolished in a concentration-dependent manner. Complete inhibition of the gonadotropic stimulation (i.e. not significantly different from basal OHA release) was reached in the presence of 100 µM H-89.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 3. Effects of increasing concentrations of the PKA inhibitor H-89 on 150 ng/ml AcfLH-stimulated OHA secretion by adolescent (32 wk old) African catfish testis tissue in vitro. Data are expressed as fold induction over basal OHA release (nanograms per milligram testis tissue) and represent compiled data (mean ± SEM) from two independent experiments with four replicates per condition. The mean OHA levels measured in the basal group (0) was 0.67 ± 0.08 ng/mg testis tissue. Different letters denote significant differences among groups (P < 0.05).

 
Cellular localization of gonadotropin receptors gene expression in African catfish testis
Analysis of microdissected testicular tissue fractions by qPCR (Fig. 4CGo) revealed coexpression of both AcfFSHR and AcfLHR mRNA in the interstitial compartment. AcfFSHR mRNA was also found in the intratubular compartment, in which AcfLHR mRNA expression, on the contrary, was hardly detectable. In the interstitial fraction, average AcfFSHR transcript levels were approximately 23-fold higher than those of the AcfLHR. In addition, mean relative AcfFSHR expression in this fraction was approximately 9-fold higher than in the intratubular fraction, which could be related to the higher density of Leydig cells in the interstitial fraction compared with the density of Sertoli cells in the intratubular compartment.

In situ hybridization of African catfish testis sections with antisense probes demonstrated AcfFSHR gene expression in Sertoli cells, marking their cytoplasmic extensions lining the spermatogenic cysts within the tubules, as well as in Leydig cells in the interstitial compartment (Fig. 5AGo). AcfLHR mRNA expression was detected exclusively in interstitial Leydig cells (Fig. 5BGo). All Leydig cells present were stained with an approximately similar intensity. Also, there was no apparent distribution pattern of AcfFSHR mRNA- or AcfLHR mRNA-positive Leydig cells, which seemed to be randomly distributed in the interstitial compartment as single cells, pairs, or in small groups, in a pattern similar to 3β-hydroxysteroid dehydrogenase-positive cells (supplemental Fig. 1). Germ cells, blood cells, blood vessels, or any other interstitial cell type did not show a positive signal. No specific staining was found when incubating sections with sense probes for AcfFSHR (Fig. 5CGo) or AcfLHR mRNA (not shown).


Figure 5
View larger version (129K):
[in this window]
[in a new window]

  		FIG. 5. In situ hybridization for AcfLHR and AcfFSHR on testis sections of sexually mature (51 wk old) African catfish. A, Antisense riboprobe for AcfFSHR; note the positive staining in Leydig cells (L) in the interstitial tissue (IT) and in the cytoplasmic extensions of Sertoli cells (S) lining the spermatogenic cysts. B, Antisense riboprobe for AcfLHR; note the positive staining in Leydig cells (L) grouped within the interstitial tissue (IT) between spermatogenic tubules. In all the cases, germ cells (G) were completely devoid of staining. C, Representative sense riboprobe signal for Acf gonadotropin receptors; note the absence of a clear staining. Scale bars, 25 µm.

 
Testicular gonadotropin receptor expression during African catfish pubertal development
Both testicular AcfFSHR and AcfLHR mRNA expression levels increased in a similar manner from 11 to 21 wk of age (Fig. 6Go). At later stages, however, the pattern of transcript levels diverged. AcfLHR mRNA expression increased up to adult levels (51 wk old) already at 32 wk of age, whereas AcfFSHR mRNA levels continued to increase significantly from adolescent to fully mature males.


Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 6. Relative gonadotropin receptor mRNA expression levels in African catfish testis in juvenile fish (11 wk old), during the first wave of spermatogenesis (16 and 21 wk old), in adolescent fish (first wave of spermatogenesis completed; 32 wk old), and in sexually mature adults (51 wk old). Relative mRNA levels (mean ± SEM) were normalized to African catfish 28S rRNA, corrected for the gonadosomatic index (100 x gonad weight x body weight–1) and expressed as relative values of the expression levels measured in 51-wk-old fish testis. For each receptor, different letters denote significant differences among groups (P < 0.05). See text for sample sizes and description of the developmental stage of spermatogenesis in each age group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In search of an explanation for the strong steroidogenic activity of piscine FSH-type gonadotropins, the bioactivities of recombinant and pituitary-purified gonadotropins, the role of the cAMP/PKA pathway for LHR- and FSHR-mediated stimulation of steroid release as well as the cellular sites of LHR and FSHR expression in African catfish testis tissue was studied. In addition, to provide new insights into the contributions of FSH/FSHR and LH/LHR to the development toward sexual maturity in male African catfish, developmental changes in testicular LHR and FSHR expression were studied.

Channel and African catfish are closely related species and their gonadotropins show very high amino acid identities [e.g. 92% homology among the FSH β-subunits) (44, 47)]. Activation studies for AcfFSHR and AcfLHR using recombinant proteins from either species showed only slightly different EC50 values; with regard to stimulation of androgen release from primary tissue cultures, Channel and African catfish gonadotropins showed very similar bioactivities too (this study and Ref. 2). This suggests that rCcf gonadotropins are physiologically valid ligands for studies on African catfish gonadotropin receptors, allowing the following conclusions. Both AcfLHR and AcfFSHR were activated preferentially by their cognate ligands, LH (rCcfLH and rAcfLH) and FSH (rCcfFSH and rAcfFSH), with respective EC50s of 10–30 and 0.1–0.3 ng/ml. Second, cross-activation of each receptor required 30- to 220-fold excess of the other gonadotropin, a situation depicting certain but limited selectivity for both piscine gonadotropin receptors. This limited selectivity, which has been reported in other fish species (2, 21, 24, 25, 48) and for the chicken FSHR (49), differs from observations in mammals in which cross-reactivity of gonadotropin receptors is always less than 0.1% (3). Therefore, it is possible that the high selectivity of gonadotropin receptors has evolved in context with features specific to mammalian reproductive biology.

Noticeably, the EC50s for FSHs/AcfFSHR interactions were 100-fold lower than those for LHs/AcfLHR interactions (this study and Ref. 2). Thus, it seems possible that LH concentrations half-maximally cross-activating the FSHR (9–21 ng/ml) fall into the range of physiological plasma levels in spawning fish (peak levels range between 10 and 70 ng/ml) (26, 27, 28, 31). On the other hand, the FSH concentrations leading to half-maximum cross-activation of the LHR (~700 ng/ml) are far above the physiological levels measured in other fish species (27, 28); no information is available on circulating FSH levels in catfish species. This suggests that FSH-mediated cross-activation of the LHR is unlikely to occur in vivo. The absence of selectivity shown by catfish LH, which does not clearly discriminate between AcfLHR and AcfFSHR (EC50s for both receptors are 10–30 ng/ml), may be tolerable in the presence of low plasma LH levels such as during the first to the middle part of the yearly reproductive cycle in salmonid fish (27, 28, 31) or during African catfish pubertal development (50). However, LH levels are elevated during the spawning season, and thus, they might reach concentrations also activating the FSHR (see above for references). The physiological relevance of this phenomenon is not known yet and may also not be similar in all species. However, it can be speculated that LH cross-activation of the FSHR may have complementary, additive, or synergistic effects on FSH-triggered FSHR-mediated signaling mechanisms (6). Because African catfish Leydig cells coexpress both the LHR and FSHR, the physiological relevance of LH cross-activation of the FSHR might be related to LH effects on Sertoli cells, such as the opening of spermatogenic cysts to release spermatozoa into the tubular lumen (i.e. spermiation).

In accordance with previous results in a number of teleost species (13, 14, 15, 16, 19, 20), incubation of mature African catfish testis tissue with increasing concentrations of either FSH or LH (recombinant or pituitary purified) resulted in a concentration-dependent stimulation of androgen release (this study and Refs. 6, 25). The analysis of full concentration-response curves in the present report showed that the rCcfFSH concentrations inducing a first significant stimulation of steroid release (~15 ng/ml) are lower than those required to initiate cross-activation of the AcfLHR (~150 ng/ml). In addition, rCcfFSH elicited a significant androgen release response at lower concentrations than rCcfLH, which agrees with the lower FSH concentrations needed to activate the AcfFSHR than those required of LH to stimulate the AcfLHR (this study and Ref. 2). This suggests that FSH stimulated steroidogenesis in African catfish testis via a FSHR-dependent mechanism.

In mammals, androgen production by Leydig cells is mainly regulated directly by LH, in an endocrine or trophic fashion, and indirectly by FSH, through paracrine effects of Sertoli cell-derived factors (10, 11, 12, 51). The cAMP/PKA signaling pathway is the most important second messenger cascade for trophic hormone-stimulated steroid biosynthesis (i.e. for LH), whereas Sertoli cell factors, as well as other locally produced factors that modulate Leydig cell steroidogenesis, exert their effects via signal transduction pathways not involving cAMP/PKA (10, 11, 12, 51, 52, 53, 54). It is worth noting that, regardless of the nature of other locally produced factors that modulate Leydig cell steroidogenesis, the cAMP-independent induction of steroidogenesis is quite modest when compared with the cAMP/PKA-mediated response (10, 51, 52, 54, 55, 56). Taking into account our observations showing that spent medium from zebrafish Sertoli cell lines did not modulate basal or stimulated androgen release by African catfish or zebrafish primary testis tissue cultures (García-López, Á., P. P., de Waal, J. Bogerd, and R. W. Schulz, unpublished results) and that both rCcf gonadotropins had similar time-course of steroid release dynamics and potencies in the current study, it was hypothesized that the FSH-triggered steroid release by African catfish testis can be explained by a trophic, FSHR-mediated, and cAMP/PKA-transduced influence on Leydig cells.

The hypothesis with regard to the signal transduction pathway was addressed experimentally by challenging primary testis tissue cultures with an AcfLH concentration able to fully activate both the AcfLHR and the AcfFSHR. The maximal stimulation of steroid release was completely abolished in the presence of the PKA inhibitor H-89, suggesting that LHR/FSHR-mediated androgen release makes use predominantly of the cAMP/PKA signaling in African catfish Leydig cells. The implication of the cAMP/PKA signaling pathway on both LH- and FSH-stimulated androgen production has been previously suggested for teleost fish (15, 56).

A straightforward explanation for a direct action of FSH on Leydig cells is to assume FSHR expression by Leydig cells, an assumption we then examined experimentally. As we demonstrate through localizing gonadotropin receptor gene expression by laser microdissection coupled to qPCR analysis of interstitial vs. intratubular tissue and in situ hybridization, African catfish Leydig cells coexpress both the LHR and FSHR genes, whereas Sertoli cells exclusively express the FSHR gene. Coexpression of gonadotropin receptors has also been found in zebrafish Leydig cells (García-López, Á., J. Bogerd, and R. W. Schulz, unpublished results). Recently Ohta et al. (34) detected FSHR protein immunohistochemically in Japanese eel Leydig cells. These data are in line with our findings, although cross-reaction of FSHR antibodies with epitopes on other proteins, such as the LHR, was not tested. Leydig cell FSHR expression differs from the situation reported in mammals, i.e. mutually exclusive expression of LHR and FSHR on Leydig cells and Sertoli cells, respectively (1, 7, 8). Coexpression of both LHR and FSHR in African catfish Leydig cells opens possibilities for new interactions among signaling mechanisms targeted by the two gonadotropins in the regulation of steroid biosynthesis, in addition to those already described in mammalian Leydig cells (51). FSHR expression by both Leydig and Sertoli cells may allow FSH to regulate exclusively the activities of both cell types during early to mid-spermatogenesis, when plasma LH levels are very low or undetectable in salmonids (27, 28, 31 ; see below). Close to and during the spawning season, on the other hand, both plasma LH and FSH levels are at their maximum values (27, 28, 31), and thus, Leydig cells may have access to two potent steroidogenic hormones. This situation may constitute an adaptive mechanism ensuring maximal production of hormones during the prespawning and spawning period, when high amounts of steroids, both androgens and progestins (such as 17{alpha},20β dihydroxy-4-pregnen-3-one), are present in the circulation, and seem required for completing gamete maturation, or for aggressive as well as courtship behavior, and/or to be released into the water as pheromones. In addition, the notably higher expression levels of FSHR compared with those of LHR in African catfish Leydig cells may have the advantage of a more tight control of steroid release, because the FSHR, in contrast to the LHR (38), is not constitutively active.

Less than 0.5 ng rCcfFSH/ml activated the AcfFSHR, and thus, a significant steroidogenic response was expected to occur already at FSH concentrations lower than 15 ng/ml. Unfortunately, an assay to detect FSH bioactivity in fish Sertoli cells is not yet available, so it cannot be assessed at present whether FSH concentrations close to the EC50 do activate FSH-dependent processes in Sertoli cells and thus whether FSH effects on Sertoli cell activity occur at lower concentrations than on Leydig cell steroid release. In any case, this FSH-specific discrepancy between receptor-activation and steroid release bioactivity also may indicate that yet-uncharacterized mechanisms temper FSH-mediated stimulation of steroid release. Because spent medium from zebrafish Sertoli cell cultures did not modulate androgen release (see above), such a mechanism may be operating in Leydig cells. In this regard, it is interesting to refer again to the constitutive activity of the AcfLHR (38), which seems relevant also in vivo, considering the fully differentiated morphology of Leydig cells and high androgen release capacity already in juvenile males (41, 57). Accordingly, we hypothesized that the lower efficiency of rCcfFSH to stimulate steroid release in primary culture compared with that to activate the AcfFSHR transfected in HEK-T 293 cells (no LHR present) may be related to a modulatory influence of LHR-dependent signaling on FSHR-dependent pathways, such as LHR-induced down-regulation of FSHR expression in Leydig cells. Unfortunately, this hypothesis could not be tested because we did not succeed in including a suitable tag at the N terminus of AcfFSHR to be cotransfected with the AcfLHR in HEK-T 293 cells (58). In addition, the mechanisms in fish Leydig cells, whether cell autonomous or modulated by exogenous signaling molecules from testicular or extratesticular sources, regulating FSHR or LHR expression are largely unexplored.

Recently both macrophage-derived factors, such as TNF{alpha} or IL-1β (53), and nitric oxide (59) have been shown to modulate teleost Leydig cell androgen biosynthesis. However, whether these compounds exert their actions on Leydig cell steroidogenesis by modulating LHR or FSHR expression, influencing LHR- or FSHR-dependent mechanisms, and/or acting through LHR- and FSHR-independent mechanisms is not known to date. In vivo androgen treatments (40) as well as unilateral orchidectomy (García-López, Á., W. van Dijk, and R. W. Schulz, unpublished results) have been recently shown to up-regulate testicular FSHR mRNA expression in African catfish. It is not clear, however, whether the increased FSHR expression levels corresponded to elevated expression in Leydig, Sertoli, or both testicular cell types.

Studying testicular AcfLHR and AcfFSHR mRNA expression during puberty and until reaching full maturity showed that up until 21 wk of age, expression of both receptors increased in a similar manner. At 21 wk of age, all males had started the first wave of spermatogenesis, had progressed until the spermatid stage, and free spermatozoa were observed in the lumen of some tubules (41). At this stage, pituitary AcfFSH β-subunit mRNA levels had increased steeply reaching similar levels to those measured in adult males (44), whereas LH protein levels in the pituitary kept increasing until completion of the first wave of spermatogenesis in adolescent fish at 32 wk of age and further until full maturation is reached (60, 61). Circulating LH levels, however, remained less than 1 ng/ml during puberty up until 49 wk of age (50); no information on circulating FSH levels is available in catfish. Altogether, these data suggest a relatively higher relevance of the FSH/FSHR system (acting on both Sertoli and Leydig cells) for driving early to mid-stages of pubertal testis development in African catfish, as previously suggested for other teleost species (27, 28, 29, 30, 31, 32, 33). From 21 to 32 wk of age, spermiogenesis and spermiation continued and free spermatozoa appeared in the lumen of all spermatogenic tubules (41), whereas testicular androgen release capacity still has to reach adult levels (35). Hence, males at this age are considered as adolescent or young adults. Testicular AcfLHR mRNA expression had increased to adult levels until wk 32, although circulating LH remained at low levels (<1 ng/ml) (50) until entering the spawning season. Attaining adult levels of testicular steroid release capacity from 32 to 51 wk of age was accompanied by a further increase in AcFSHR mRNA levels, whereas those of AcfLHR stayed constant. We therefore speculate that also at this more advanced stage of development, AcFSHR-mediated signaling may be responsible for the up-regulation of the steroid release capacity (35) (see basal release levels in legends of Figs. 2Go and 3Go).

In summary, this study showed that in African catfish, FSH stimulates steroid release directly via its trophic action on FSHR-expressing Leydig cells. This expression pattern, which would explain the strong steroidogenic activity that has been associated with several piscine FSHs, has been found to date in species belonging to orders Siluriformes (this study), Cypriniformes (García-López, Á., J. Bogerd, and R. W. Schulz, unpublished results), and Anguilliformes (34). Future comparative studies on species belonging to other major fish taxonomic groups and on species representing other vertebrate classes will show whether Leydig cell FSHR expression is a feature typical for the reproductive physiology of fish and whether the situation in mammals with FSHR expression being restricted to Sertoli cells (8) is the phylogenetically conserved setting.


   Acknowledgments
 
The authors thank DSM Food Specialties (Delft, The Netherlands) and SenterNovem (The Netherlands) for using their PALM MicroBeam Instrument at the Department of Cell Architecture and Dynamics (Utrecht University). The technical assistance of H. C. Schriek and J. van Rootselaar (Utrecht University aquarium facility) is highly appreciated.


   Footnotes
 
This work was supported by Norwegian Research Council Grant 159645/S40 (to G.L.T., J.B., and R.W.S.), and National Institutes of Health Grant DK69711 (to J.B.). Á.G.-L. was supported by a postdoctoral fellowship from the Fundación Ramón Areces (Spain).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 28, 2008

Abbreviations: Acf, African catfish; Ct, cycle threshold; DIG, digoxigenin; FSHR, FSH receptor; LHR, LH receptor; OHA, 11β-hydroxyandrostenedione; PKA, protein kinase A; qPCR, quantitative PCR; rCcf, recombinant channel catfish; SSC, standard saline citrate.

Received April 1, 2008.

Accepted for publication August 20, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McLachlan RI, Wreford NG, O'Donnell L, De Kretser DM, Robertson DM 1996 The endocrine regulation of spermatogenesis: independent roles for testosterone and FSH. J Endocrinol 14:1–9[CrossRef]
  2. Vischer HF, Granneman JCM, Linskens MHK, Schulz RW, Bogerd J 2003 Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor. J Mol Endocrinol 31:133–140[Abstract]
  3. Pierce JG, Parsons TF 1981 Glycoprotein hormones: structure and function. Ann Rev Biochem 50:465–495[CrossRef][Medline]
  4. Jia XC, Oikawa M, Bo M, Tanaka T, Ny T, Boime I, Hsueh AJ 1991 Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species. Mol Endocrinol 5:759–768[Abstract/Free Full Text]
  5. Tilly JL, Aihara T, Nishimori K, Jia XC, Billig H, Kowalsky KI, Perlas EA, Hsueh AJ 1992 Expression of human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts. Endocrinology 131:799–806[Abstract/Free Full Text]
  6. Bogerd J, Granneman JCM, Schulz RW, Vischer HF 2005 Fish FSH receptors bind LH: how to make the human FSH receptor to be more fishy? Gen Comp Endocrinol 142:34–43[CrossRef][Medline]
  7. Themmen APN, Huhtaniemi IT 2000 Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonad function. Endocr Rev 21:551–583[Abstract/Free Full Text]
  8. Petersen C, Söder O 2006 The Sertoli cell—a hormonal target and ‘super’ nurse for germ cells that determines testicular size. Horm Res 66:153–161[CrossRef][Medline]
  9. Meng X, Lindahl M, Hyvönen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H 2000 Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287:1489–1493[Abstract/Free Full Text]
  10. Verhoeven G, Cailleau J 1987 A Leydig cell stimulatory factor produced by human testicular tubules. Mol Cell Endocrinol 49:137–147[CrossRef][Medline]
  11. Saez JM 1994 Leydig cells: endocrine, paracrine, and autocrine regulation. Endocr Rev 15:574–626[Abstract/Free Full Text]
  12. Boujrad N, Ogwuegbu SO, Garnier M, Lee CH, Martin BM, Papadopoulos V 1995 Identification of a stimulator of steroid hormone synthesis isolated from testis. Science 5217:1609–1612
  13. Kagawa H, Tanaka H, Okuzaga K, Kobayashi M 1998 GTH II but not GTH I induces final maturation and development of maturational competence of oocytes of red seabream in vitro. Gen Comp Endocrinol 112:80–88[CrossRef][Medline]
  14. Weltzien FA, Norberg B, Swanson P 2003 Isolation and characterization of FSH and LH from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.). Gen Comp Endocrinol 131:97–105[CrossRef][Medline]
  15. Planas JV, Swanson P, Dickhoff WW 1993 Regulation of testicular steroid production in vitro by gonadotropins (GTH I and GTH II) and cyclic AMP in cocho salmon (Oncorhynchus kisutch). Gen Comp Endocrinol 91:8–24[CrossRef][Medline]
  16. Planas JV, Swanson P 1995 Maturation-associated changes in the response of the salmon testis to the steroidogenic actions of gonadotropins (GTH I and GTH II) in vitro. Biol Reprod 52:697–704[Abstract]
  17. Kamei H, Ohira T, Yoshiura Y, Uchida N, Nagasawa H, Aida K 2003 Expression of a biologically active recombinant follicle stimulating hormone of Japanese eel Anguilla japonica using methylotropic yeast, Pichia pastoris. Gen Comp Endocrinol 134:244–254[CrossRef][Medline]
  18. Kamei H, Kaneko T, Aida K 2006 In vivo gonadotropic effects of recombinant Japanese eel follicle-stimulating hormone. Aquaculture 261:771–775[CrossRef]
  19. Aizen J, Kasuto H, Golan M, Zakay H, Levavi-Sivan B 2007 Tilapia follicle-stimulating hormone (FSH): immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion. Biol Reprod 76:692–700[Abstract/Free Full Text]
  20. Molés G, Gómez A, Rocha A, Carrillo M, Zanuy S 2008 Purification and characterization of follicle-stimulating hormone from pituitary glands of sea bass (Dicentrarchus labrax). Gen Comp Endocrinol 158:68–76[CrossRef][Medline]
  21. Miwa S, Yan L, Swanson P 1994 Localization of two gonadotropin receptors in the salmon gonad by in vitro ligand autoradiography. Biol Reprod 59:629–642
  22. Yan L, Swanson P, Dickhoff WW 1992 A two-receptor model for salmon gonadotropins (GTH I and GTH II). Biol Reprod 47:418–427[Abstract]
  23. Nijenhuis WAJ, Male R, Swanson P, Andersson E, Bogerd J, Schulz RW The gonadotropin receptors of Atlantic salmon (Salmo salar). Proc Fifth International Symposium on Fish Endocrinology, Castellón de la Plana, Spain, 2004, p 101 (Abstract P67)
  24. So WK, Kwok HF, Ge W 2005 Zebrafish gonadotropins and their receptors: II. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone subunits—their spatial-temporal expression patterns and receptor specificity. Biol Reprod 72:1382–1396[Abstract/Free Full Text]
  25. Zmora N, Kazeto Y, Kumar RS, Schulz RW, Trant JM 2007 Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions. J Endocrinol 194:407–416[Abstract/Free Full Text]
  26. Kobayashi M, Aida K, Hanyu I 1986 Gonadotropin surge during spawning in male goldfish. Gen Comp Endocrinol 62:70–79[CrossRef][Medline]
  27. Prat F, Sumpter JP, Tyler CR 1996 Validation of radioimmunoassays for two salmon gonadotropins (GTH I and GTH II) and their plasma concentrations throughout the reproductive cycle in male and female rainbow trout (Oncorhynchus mykiss). Biol Reprod 54:1375–1382[Abstract]
  28. Gomez JM, Weil C, Ollitrault M, Le Bail P-Y, Breton B, Le Gac F 1999 Growth hormone (GH) and gonadotropin subunit gene expression and pituitary and plasma changes during spermatogenesis and oogenesis in rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 113:413–428[CrossRef][Medline]
  29. Loir M 1999 Spermatogonia of rainbow trout: II. In vitro study of the influence of pituitary hormones, growth factors and steroids on mitotic activity. Mol Reprod Dev 53:434–442[CrossRef][Medline]
  30. Campbell B, Dickey JT, Swanson P 2003 Endocrine changes during onset of puberty in male spring Chinook salmon, Oncorhynchus tshawytscha. Biol Reprod 69:2109–2117[Abstract/Free Full Text]
  31. Sohn YC, Yoshiura Y, Kobayashi M, Aida K 1999 Seasonal changes in mRNA levels of gonadotropin and thyrotropin subunits in the goldfish, Carassius auratus. Gen Comp Endocrinol 113:436–444[CrossRef][Medline]
  32. Schulz RW, van Dijk W, Bogerd J 2003 Sertoli cell proliferation and FSH signaling in African catfish, Clarias gariepinus. Fish Physiol Biochem 28:223–224[CrossRef]
  33. Kusakabe M, Nakamura I, Evans J, Swanson P, Young G 2006 Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes. J Endocrinol 189:541–554[Abstract/Free Full Text]
  34. Ohta T, Miyake H, Miura C, Kamei H, Aida K, Miura T 2007 Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica. Biol Reprod 77:970–977[Abstract/Free Full Text]
  35. Schulz RW, van der Corput L, Janssen-Dommerholt C, Goos HJTh 1994 Sexual steroids during puberty in male African catfish (Clarias gariepinus): serum levels and gonadotropin-stimulated testicular secretion in vitro. J Comp Physiol B 164:195–205[CrossRef]
  36. Schulz RW, Vischer HF, Cavaco JEB, Santos EM, Tyler CR, Goos HJT, Bogerd J 2001 Gonadotropins, their receptors, and the regulation of testicular functions in fish. Comp Biochem Physiol B 129:407–417[CrossRef][Medline]
  37. Bogerd J, Blomenröhr M, Andersson E, van der Putten HHAGM, Tensen CP, Vischer HF, Granneman JCM, Janssen-Dommerholt C, Goos HJT, Schulz RW 2001 Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus). Biol Reprod 64:1633–1643[Abstract/Free Full Text]
  38. Vischer HF, Bogerd J 2003 Cloning and functional characterization of a gonadal luteinizing hormone receptor complementary DNA from the African catfish (Clarias gariepinus). Biol Reprod 68:262–271[Abstract/Free Full Text]
  39. Chen W, Shields TS, Stork PJS, Cone RD 1995 A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal Biochem 226:349–354[CrossRef][Medline]
  40. Schulz RW, Liemburg M, García-López Á, van Dijk W, Bogerd J 2008 Androgens modulate testicular androgen production in African catfish (Clarias gariepinus) depending on the stage of maturity and type of androgen. Gen Comp Endocrinol 156:154–163[CrossRef][Medline]
  41. Schulz RW, Lubberink K, Zandbergen MA, Janssen-Dommerholt C, Peute J, Goos HJT 1996 Testicular responsiveness to gonadotropic hormone in vitro and Leydig and Sertoli cell ultrastructure during pubertal development of male African catfish (Clarias gariepinus). Fish Physiol Biochem 15:243–254[CrossRef]
  42. Schulz RW, van der Wind F, Janssen-Dommerholt C, Peute J, Mylonas CC, Zohar Y, Swanson P, Goos HJT 1997 Modulation of testicular androgen production in adolescent African catfish (Clarias gariepinus). Gen Comp Endocrinol 108:56–66[CrossRef][Medline]
  43. Vermeulen GJ, Lambert JGD, Lenczowski MJP, Goos HJT 1993 Steroid hormone secretion by testicular tissue from African catfish, Clarias gariepinus, in primary culture: identification and quantification by gas chromatography-mass spectrometry. Fish Physiol Biochem 12:21–30[CrossRef]
  44. Vischer HF, Teves ACC, Ackermans JCM, van Dijk W, Schulz RW, Bogerd J 2003 Cloning and spatiotemporal expression of the follicle-stimulating hormone β subunit complementary DNA in the African catfish (Clarias gariepinus). Biol Reprod 68:1324–1332[Abstract/Free Full Text]
  45. Bustin SA 2000 Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193[Abstract]
  46. Weltzien FA, Norberg B, Helvik JV, Andersen Ø, Swanson P, Andersson E 2003 Identification and localization of eight distinct hormone-producing cell types in the pituitary of male Atlantic halibut (Hippoglossus hippoglossus L.). Comp Biochem Physiol A 134:315–327[CrossRef][Medline]
  47. Liu Z, Kim S, Karsi A 2001 Channel catfish follicle-stimulating hormone and luteinizing hormone: complementary DNA cloning and expression during ovulation. Mar Biotechnol 3:590–599[CrossRef][Medline]
  48. Sambroni E, Le Gac F, Breton B, Lareyre JJ 2007 Functional specificity of the rainbow trout (Oncorhynchus mykiss) gonadotropin receptors as assayed in a mammalian cell line. J Endocrinol 195:213–228[Abstract/Free Full Text]
  49. Wakabayashi N, Suzuki A, Hoshino H, Nishimori K, Mizuno S 1997 The cDNA cloning and transient expression of a chicken gene encoding a follicle-stimulating hormone receptor. Gene 197:121–127[CrossRef][Medline]
  50. Schulz RW, van der Sanden MCA, Bosma PT, Goos HJT 1994 Effects of gonadotrophin-releasing hormone during the pubertal development of the male African catfish (Clarias gariepinus): gonadotrophin and androgen levels in plasma. J Endocrinol 140:265–273[Abstract/Free Full Text]
  51. Stocco DM, Wang XJ, Jo Y, Manna PR 2005 Multiple signaling pathways regulating steroidogenesis and steroidogenic acute regulatory protein expression: more complicated than we thought. Mol Endocrinol 19:2647–2659[Abstract/Free Full Text]
  52. Verhoeven G, Cailleau J 1985 A factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells. Mol Cell Endocrinol 40:57–68[CrossRef][Medline]
  53. Lister A, van der Kraak G 2002 Modulation of goldfish testicular testosterone production in vitro by tumor necrosis factor {alpha}, interleukin-1β, and macrophage conditioned media. J Exp Zool 292:477–486[CrossRef][Medline]
  54. Manna PR, Chandrala SP, Jo Y, Stocco DM 2006 cAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation. J Mol Endocrinol 37:81–95[Abstract/Free Full Text]
  55. Verhoeven G, Cailleau J 1986 Specificity and partial purification of a factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells. J Steroid Biochem 25:393–402[CrossRef][Medline]
  56. Wade MG, van der Kraak G 1991 The control of testicular androgen production in the goldfish: effects of activators of different intracellular signaling pathways. Gen Comp Endocrinol 83:337–344[CrossRef][Medline]
  57. Cavaco JEB, van Blijswijk B, Leatherland JF, Goos HJT, Schulz RW 1999 Androgen-induced changes in Leydig cell ultrastructure and steroidogenesis in juvenile African catfish, Clarias gariepinus. Cell Tissue Res 297:291–299[CrossRef][Medline]
  58. Vischer HF, Granneman JCM, Koelink PJ, Marques RB, Bogerd J 2008 Identification of a luteinizing hormone-selective determinant in the exodomain of a follicle-stimulating hormone receptor. Gen Comp Endocrinol 156:490–498[CrossRef][Medline]
  59. nee Pathak ND, Lal B 2008 Nitric oxide: an autocrine regulator of Leydig cell steroidogenesis in the Asian catfish, Clarias batrachus. Gen Comp Endocrinol 158:161–167[CrossRef][Medline]
  60. Schulz RW, Zandbergen MA, Peute J, Bogerd J, van Dijk W, Goos HJT 1997 Pituitary gonadotrophs are strongly activated at the beginning of spermatogenesis in African catfish, Clarias gariepinus. Biol Reprod 57:139–147[Abstract]
  61. de Leeuw R, Wurth YA, Zandbergen MA, Peute J, Goos HJT 1986 The effects of aromatizable androgens, nonaromatizable androgens, and estrogens on gonadotropin release in castrated African catfish, Clarias gariepinus (Burchell). A physiological and ultrastructural study. Cell Tissue Res 243:587–594



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
S. X. Chen, J. Bogerd, A. Garcia-Lopez, H. de Jonge, P. P. de Waal, W. S. Hong, and R. W. Schulz
Molecular Cloning and Functional Characterization of a Zebrafish Nuclear Progesterone Receptor
Biol Reprod, January 1, 2010; 82(1): 171 - 181.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
F. F.L. Almeida, G. L. Taranger, B. Norberg, O. Karlsen, J. Bogerd, and R. W. Schulz
Photoperiod-Modulated Testis Maturation in Atlantic Cod (Gadus morhua, L.)
Biol Reprod, April 1, 2009; 80(4): 631 - 640.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
E. R. d. Alvarenga and L. R. d. Franca
Effects of Different Temperatures on Testis Structure and Function, with Emphasis on Somatic Cells, in Sexually Mature Nile Tilapias (Oreochromis niloticus)
Biol Reprod, March 1, 2009; 80(3): 537 - 544.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by García-López, A.
Right arrow Articles by Schulz, R. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by García-López, A.
Right arrow Articles by Schulz, R. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals