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Pituitary |
a Department of Endocrinology, Utrecht University, 3584 CH Utrecht, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, follicle-stimulating hormone, pituitary, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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African catfish were bred and raised in the laboratory as previously described [16] except that catfish pituitary extract instead of hCG was used to induce ovulation. In our hands, sexual maturation of male African catfish started at 
3 mo of age, when the first males were found in which spermatogonial proliferation had started. At 6 mo of age, spermatozoa were found in the testes of most fish. Fully mature males can be used for breeding purposes at 9–12 mo of age. All tissues and sperm samples used in this study for the isolation of RNA and genomic DNA were collected from sexually mature (9–12-mo-old) catfish. For the studies on pubertal development (see below), pituitaries from at least 10 animals were sampled every 2 wk between 10 and 24 wk of age. Experiments involving hormone treatment and/or surgery were conducted on males between 9 and 10 mo of age. Animal culture and experimentation was consistent with the Dutch national regulations; the Life Science Faculties Committee for Animal Care and Use approved the experimental protocols.
RNA Isolation, Poly(A)+ RNA Isolation, Genomic DNA Isolation, and Genomic Library Construction
Total RNA was isolated from various tissues of mature male African catfish using the guanidium isothiocyanate method [17]. Poly(A)+-rich pituitary RNA was purified using Dynabeads-oligo dT25 (Dynal A.S., Oslo, Norway) according to the manufacturer's instructions. Pituitaries of male African catfish between 10 and 24 wk of age were collected and stored separately. The developmental stage of each male was determined by testicular histology as described previously [16]. Next, pituitaries from the same stage of development were pooled for RNA isolation with RNAzol B (Campro Scientific, Veenendaal, The Netherlands) according to the manufacturer's instructions. To this end, pools of 10 pituitaries (stage I; n = 8), 5 pituitaries (stage II; n = 5), or 4 pituitaries (stage III; n = 3) were used; from stage IV (n = 7) or adult (n = 6) males, single pituitaries were used.
Genomic DNA was extracted from sperm of an adult male African catfish according to Ausubel et al. [18]. An African catfish genomic library (in total, 3 x 106 independent clones, and amplified in 27 aliquots of 35 x 104 original clones each) was constructed in the EMBL3 SP6/T7 vector from genomic DNA extracted from liver tissue (Clontech, Palo Alto, CA).
Primers and Polymerase Chain Reaction
Primers were obtained from Life Technologies (Breda, The Netherlands) and were as follows: degFSH-S, 5'-GARWSIGAIGARTGYGGIWSITGY-ATIAC–3'; degFSH-AS, 5'-GTYTCRTAIGTCCAITCICKRAARTTRCA-3'; cfFSH1, 5'-CACAGCCTGTGCCGGCCTCT-3'; cfFSH2, 5'-CCATTGGA-CTGCGGTACGCTCT-3'; cfFSH3, 5'-ATGCGTGGCGTTGCCATGGTGT-3'; cfFSH4, 5'-GTAGTAGGCGTGTGTGTGGCAG-3'; cfFSH5, 5'-TAGT-AGGCGTGTGTGT-3'; cfFSH6, 5'-CTGAAGGCGCCGCAGTCTGTGATC-3'; cfFSH7, 5'-CTGCACAGCTCAGAGCCACAGG-3'; cfFSH8, 5'-TT-TACCTG
CGTTGCCATGGTGTTGCT-G3'; and cfFSH9, 5'-TTTACCTG
CTGA-AGGCGCCGCAGTCTG-3', in which Y = T or C, R = G or A, W = T or A, K = T or G, S = C or G, and I = inosine. Primers cfFSH8 and cfFSH9 contained a T3 and T7 RNA polymerase promoter sequence engineered at their 5' ends, respectively, which are underlined.
Polymerase chain reactions (PCRs) were carried out in 50-µl volumes containing 50 mM KCl, 10 mM Tris/HCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 200 µM each dNTP, and 50 pmol primers, with either 100 ng genomic DNA or 1 µl each of the genomic sublibraries or with 1 µl each of the pituitary cDNA sublibraries as template (see below) in a Perkin-Elmer Cetus cycler (Applied Biosystems, Foster City, CA), using 1 U SuperTaq (HT Biotechnologies Ltd., Cambridge, UK).
Catfish genomic DNA was used as template for PCR amplification with two degenerate primers (degFSH-S and degFSH-AS) corresponding to conserved FSHß-specific amino acid sequences in goldfish [12] and channel catfish [14]. Denaturation was at 94°C for 5 min, followed by the addition of Taq polymerase at 75°C. The PCR amplification was performed using 35 cycles at 94°C for 45 sec, at 60°C for 30 sec, at 72°C for 1.5 min, and a final extension at 72°C for 7 min. A PCR product of 1.2 kb was amplified and subcloned in pGEM-T (Promega, Madison, WI) for sequence analysis.
Isolation of cfFSHß Genomic and cDNA Clones
The cloned 1.2-kb PCR product contained parts of exons 2 and 3, interrupted by intron 2, cfFSHß sequences. Next, two sets of specific primers (cfFSH1 and cfFSH2, and cfFSH3 and cfFSH4) were designed to isolate the cfFSHß gene and cDNA from catfish genomic and cDNA [19] sublibraries, respectively, by a PCR-based filter screening method [20]. Positive clones were isolated by rescreening at lower plaque densities. DNA from two positive genomic 
DNA clones was isolated using a Lambda DNA isolation kit according to the manufacturer's instructions (Roche, Almere, The Netherlands), while five positive cfFSHß cDNA clones were excised in vivo as pBluescript SK(-) phagemids. Sequence analysis showed that all cDNA clones were identical but were missing sequences at the 5' end. In order to obtain additional cDNA sequence information at the 5' end, African catfish poly(A)+-rich pituitary RNA was reverse transcribed to 5'-RACE-ready first-strand cDNA with a cfFSH-specific reverse primer (cfFSH5) using the SMART RACE cDNA amplification kit according to manufacturer's instructions (Clontech). Next, 5'-RACE amplification was carried out on this cDNA by performing a touchdown PCR using the Advantage 2 PCR kit (Clontech) with primer cfFSH6 in combination with the universal primer mix, supplied with the SMART RACE cDNA amplification kit. The PCR was diluted 50-fold in Tricine-EDTA buffer (10 mM Tricine-KOH, pH 8.5, and 1 mM EDTA), and a nested PCR was performed using primer cfFSH7 and the nested universal primer, supplied with the SMART RACE cDNA amplification kit. Both PCRs were carried out in a Perkin-Elmer 2400 cycler (Applied Biosystems) according to the manufacturer's instructions (Clontech). The PCR products were cloned in pGEM-T (Promega) and sequenced.
Sequence and Phylogenetic Analyses
DNA sequence analyses were performed on automated ABI PRISM 310 and 377 DNA sequencers (Applied Biosystems) using Dye Terminator cycle sequencing chemistry (Applied Biosystems). Nucleotide and deduced amino acid sequences were analyzed with Lasergene software (DNASTAR, Madison, WI). Multiple sequence alignment was performed by means of the Megalign program of the Lasergene software using the clustal method (PAM250). The putative signal peptide cleavage site was predicted using SignalP V1.1 software (Center for Biological Sequence Analysis, Lyngby, Denmark; http://www.cbs.dtu.dk/services/SignalP/).
Real-Time, Quantitative PCR
The relative cfFSHß mRNA levels in different tissues of mature catfish and in the pituitary during male pubertal development and induced by in vivo experimentation (see below) were determined as described previously [7]. Specific primers and fluorogenic probes for the cfFSHß mRNA and the endogenous control RNAs (i.e., catfish 28S rRNA and catfish GAPDH mRNA) are shown in Table 1. Optimization and validation were performed according to the manufacturer's guidelines (Applied Biosystems) and as previously described [7].
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In Situ Hybridization
In situ hybridization was performed on sections of adult catfish pituitary as described previously [10]. An 347-base pair (bp) DNA template (comprising nucleotides 33–380 of the cfFSHß cDNA sequence; Fig. 1B) was generated by PCR amplification using primers cfFSH8 and cfFSH9. Next, sense and antisense digoxigen (DIG)-labeled cfFSHß cRNA probes were synthesized from this template by in vitro transcription using a DIG-RNA labeling mix (Roche) according to the manufacturer's instructions. DIG-labeled cfLHß probes were synthesized as described previously [10].
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The sections were documented using a digital camera (Nikon DXM 1200 using Nikon ACT-1 version 2.00 software; Inca, Haarlem, The Netherlands) attached to an axioscope microscope (Zeiss, Weesp, The Netherlands). In some cases, adjacent sections incubated with cfLHß and cfFSHß probes were color edited and overlayed in Photoshop 6.0 (Adobe, San Jose, CA).
In Vivo Experiment
In order to determine presumed feedback effects of gonadal hormones on cfFSH expression levels in the pituitary, mature male African catfish were castrated or sham operated. Fish were kept individually 1 wk before and during the experiment. One week after surgery, fish were injected with estradiol-17ß (E2) or vehicle. To this end, E2 was dissolved in cacao butter and injected intraperitoneally (15 mg/kg body weight). Once inside the body cavity, the butter solidifies, resulting in the sustained release of E2 [21]. Pilot experiments revealed that E2 plasma levels stabilized at about 16 ng/ml from Day 3 to Day 7 after the E2 cacao butter injection [22]. The fish were decapitated 3 d after E2 injection and the pituitaries were collected. Next, relative cfFSHß transcript levels were determined using real-time, quantitative PCR (see above). Blood samples were taken before surgery, E2 injection, and decapitation in order to determine steroid plasma levels (testosterone [T], 11-ketotestosterone [11kT], and E2) by radioimmunoassay as described previously [16].
Statistical Analysis
The data on the cfFSHß mRNA levels during the course of puberty were grouped according to the stage of spermatogenesis (see above). All measurements were log transformed and group means were compared by analysis of variance followed by a Fisher least significant difference test using the StatView software package (Abacus Concepts, Berkeley, CA). P values <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison of the cfFSHß DNA sequences at the genomic and the cDNA levels revealed that the cfFSHß gene consisted of three exons, interrupted by two introns (cf., Fig. 1A). Inspection of the 5'-flanking region (1684 bp) of the cfFSHß gene revealed the presence of TATA-box consensus sequences located at 32, 1129, 1225, and 1622 bp upstream from the first nucleotide of exon 1. In addition, the following potential cis-acting elements have been identified in the 5'-flanking region: half androgen responsive elements (
ARE; 5'-TGTYCT-3'), half estrogen responsive elements (ERE; 5'-TGACC-3'), activator protein-1 binding elements (AP1; 5'-TGABTCA-3'), gonadotroph-specific elements (GSE; 5'-NCAAGGYCA-3') (all reviewed by [23]), pituitary homeobox 1 protein binding elements (Pitx1; 5'-YHAKCY-3') [24], and cAMP-responsive elements (CRE; 5'-TTACGTAA-3') [25]. The location of these motifs is depicted in Figure 1B.
The combined sequences of the 5'-RACE and isolated cDNA clones revealed that the cfFSHß mRNA consists of 749 nucleotides, containing an open reading frame (ORF) of 402 bp (Fig. 1B). The first nucleotide of the presumed first translation inition codon starts at position 19, taking into account the results from multiple sequence alignment analysis of the deduced amino acid sequences with other vertebrate FSH ß subunits. The cfFSHß ORF is preceded by a 5'-UTR of 18 nucleotides and is followed by a 3'-UTR of 329 nucleotides.
Deduced cfFSHß Protein
Conceptual translation of the coding region following the proposed initiation codon (starting at nucleotide position 19) predicted a polypeptide of 133 amino acids, including a signal peptide of 18 amino acids (Fig. 1B). The 12 cysteine residues that are completely conserved in mature ß subunits of vertebrate pituitary glycoprotein hormones with respect to number and position were also observed in the cfFSHß. One putative N-linked glycosylation consensus sequence (Asn-X-[Thr/Ser]) was identified at amino acid positions 47–49 of the cfFSHß subunit protein (signal peptide is included in the amino acid numbering).
Amino acid sequence alignment of a representative set of glycoprotein hormone ß subunits was performed using the Clustal algorithm. CfFSH ß subunit showed the highest amino acid identity with channel catfish FSHß (92%), followed by FSHß's of cyprinids and anguilliformes (47–70%). The percentage identities of cfFSHß were 30–43% to FSHß's of perciform and salmonid species, 38–44% to tetrapod FSHß's, and 30–40% to vertebrate LHß's and TSHß's. A phylogenetic tree was constructed from the aligned sequences (Fig. 2) using the neighbor-joining method. The glycoprotein hormone ß subunits were divided into two major branches: one consists of all vertebrate LHß's, while the other branch is further subdivided into two additional branches, where all vertebrate FSHß's form one branch and all vertebrate TSHß's form another branch.
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CfFSHß Subunit mRNA Expression
Real-time, quantitative PCR that allows the specific and sensitive detection of transcripts in different tissues revealed that cfFSHß mRNA is exclusively expressed in catfish pituitary: the relative cfFSHß mRNA levels in other tissues (i.e., testis, cerebellum and other brain areas, muscle, stomach, intestine, head-kidney, kidney, seminal vesicles, liver, ovary) were <0.01% of the observed expression level in the pituitary (data not shown).
The levels of cfFSHß mRNA in the pituitary increased during the course of pubertal development. The appearance of meiotic (transition from stage I to II) as well as postmeiotic germ cells (transition from stage II to III) was accompanied by significant increases in the FSHß mRNA levels, which remained at a high level from stage III through to the adult stage (Fig. 3A).
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Castration of adult male catfish resulted in a significant 2.1-fold increase of the relative cfFSHß mRNA levels in the pituitary compared with the sham-operated group (Fig. 3B). The castration-induced increase in cfFSHß mRNA levels was reversed by subsequent E2 treatment. T and 11KT plasma levels were determined in castrated and sham-operated fish in order to validate the surgical procedure. Castration resulted in a significant 10-fold decrease in both T and 11KT plasma levels 7 days after surgery, resulting in levels of 0.5 ± 0.19 and 1.9 ± 0.71 ng/ml, respectively. Detectable E2 plasma levels of 9.82 ± 2.06 ng/ml were only observed in the E2-treated groups (i.e., the SHAM + E2 and castrated + E2 groups).
Localization of cfFSHß mRNA in the Pituitary
Comparison of cfFSHß and cfLHß expression patterns by in situ hybridization, using consecutive, frontal sections from a pituitary of an adult male, suggests that both subunits are expressed in the same area, namely the proximal pars distalis (PPD) (Fig. 4, A and B); the observed in situ hybridization signal for the cfLHß seems to be stronger than for cfFSHß. Higher magnification (Fig. 4, C and D) confirmed overlapping expression patterns because nonstained areas in the PPD, probably occupied by nongonadotroph cells, were of similar shapes and in similar positions for the two ß subunits. The cfFSHß transcript expressing gonadotropes occur in two different shapes (Fig. 4C), namely round to polygonal with a slightly excentric nucleus or, less frequently, cells with the appearance of unipolar neurons. These two cellular shapes were also observed among the cfLHß-expressing cells, although the neuronlike shape appeared to be less prominent than among the cfFSHß-expressing cells (Fig. 4D). Next to differences in shape, also differences in expression levels were found for both subunits. This is exemplified by intensely and weakly stained cells in the PPD, the latter still clearly distinct from nonstained cells, which probably represent nongonadotropic cells. Overlaying two adjacent sections that were incubated with cfLHß and cfFSHß probes, respectively, suggests that the two ß subunits are coexpressed in a limited fraction of the gonadotroph cells, while the majority expressed only one of the two ß subunits (Fig. 4E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we report the cloning and sequencing of both the cfFSHß gene and the cfFSHß cDNA, confirming the gonadotropin duality also for the African catfish. The cfFSHß gene consists of three exons interrupted by two introns. The position of the first intron (between nucleotides 18 and 19; cf., Fig. 1B) is conserved in all vertebrate FSHß genes (reviewed by Sohn et al. [23]) and is located immediately upstream of the translation initiation codon. Hence, the mature cfFSHß protein is encoded by exons 2 and 3. The first intron of the cfFSHß gene has about the same length (i.e., 327 bp) as found in the goldfish FSHß gene (300 bp), which is considerably smaller than the first intron of various mammalian FSHß genes (0.62–1.1 kb). The second intron is located between nucleotides 192 and 193 (cf., Fig. 1B) and is strictly conserved in all known pituitary glycoprotein hormone ß subunit genes. The length of the second intron of the cfFSHß gene is approximately 1.5 kb, which is about the same size as found in most mammalian FSHß genes (reviewed by Sohn et al. [23]). Considerably smaller introns, however, were observed in goldfish (0.75 kb [23]) and tilapia (0.95 kb [26]). Similar to the 3'-UTRs of goldfish [23] and tilapia [26] FSHß mRNAs, the 3'-UTR of the cfFSHß mRNA is considerably shorter than those found in mammalian FSHß genes (1 kb [23]). A nonconsensus polyadenylation signal (ATTAAA), the most common variant of the AATAAA consensus polyadenylation signal [27], was recognized 19 bp upstream of the poly(A) tail.
Consensus sequences for potential CCAAT- and TATA-box proximal promoter elements were identified in the 5'-flanking region of the cfFSHß gene. The putative TATA-box is located at a conserved position, as found in goldfish and mammalian FSHß genes (reviewed by Sohn et al. [23]), at 32 bp upstream of the transcription initiation site. Three other TATA-box sequences are located further upstream. Multiple putative cis-acting elements with near or identical homology to the consensus sequences were identified. This indicates that transcription of the cfFSHß gene can be regulated via multiple pathways and that the effects mediated by steroid hormone receptors might be integrated with other signaling systems. As discussed below, E2 reduced cfFSHß mRNA levels in the catfish pituitary, whereas GnRH has been shown to stimulate FSHß gene expression in cultured tilapia pituitary cells [28]. Promoter truncation studies followed by mutational analyses of the putative cis-acting elements are required to evaluate the significance of transcription factor-binding sites in regulating cfFSHß gene expression.
Comparison of the deduced amino acid sequence of cfFSHß with those of other glycoprotein hormone ß subunits revealed two obvious peculiarities. Like in most other teleost FSHß's, an additional cysteine residue (Cys22) is present next to the 12 conserved disulfide-bound half-cystines and is located four amino acids upstream from the first conserved half-cystine (i.e., Cys26). In addition, cfFSHß contains only a single putative N-linked glycosylation site (Asn47), which is situated at a similar position as the second putative N-linked glycosylation site in the FSHß subunits of Japanese eel and mammals. Hence, the otherwise conserved first glycosylation site of all gonadotropin ß subunits is absent in cfFSHß.
Phylogenetic analysis of the amino acid sequence of a representative set of vertebrate glycoprotein hormone ß subunits showed that cfFSHß is clustered into the teleost FSHß branch. Typically, sequence alignment revealed a lower degree of amino acid identity between the FSHß subunits for every given teleost species (33–92%) as compared with the amino acid identity between the fish LHß subunits (50–90%). This may indicate a more rapid diversification of FSHß, compared with LHß, during teleost evolution.
Expression of the cfFSHß mRNA was restricted to the pituitary and was observed in all developmental stages. The cfFSHß mRNA levels increased during pubertal development but remained at a constantly high level after the appearance of spermatids. In contrast, cfLHß mRNA levels increased significantly during the successive transitions through all spermatogenic stages (I–IV) and reached peak levels in the adult stage [22]. Similar gonadotropin ß subunit gene expression patterns have been reported during testicular maturation for striped bass [29] and tilapia [30], although in these species, the FSHß transcript levels decreased at final maturation. The absence of such a decrease in the present study might be related to the absence, in captive catfish, of the natural triggers inducing the spawning conditions, such as the spring rainy season and consequently flooded river meadows, which serve as spawning grounds. In rainbow trout, a salmonid species, a different pattern was found. Relatively high FSHß mRNA levels were already present in immature fish, which then increased only gradually during testicular maturation but increased dramatically at final maturation; LHß mRNA levels on the other hand, remained low during an extended period, to increase abruptly when approaching the spawning season [2].
In contrast with the situation in salmonids [1] and eel [13], cfLHß mRNA as well as the hormone is detectable in the catfish already in juvenile fish [10], suggesting that cfLH may fulfill gonadotrophic functions during early stages of testis development. Moreover, cfLH is able to activate the cfFSH receptor [7], opening the possibility that cfLH can replace cfFSH. The observed cfFSHß mRNA expression patterns suggest, however, that also cfFSH may act at an early stage of testicular development to regulate testicular functions. However, further discussions about potential physiological function(s) of cfFSH must await confirmation of the presence of the hormone in the circulation and experiments using recombinant cfFSH.
FSH and LH are produced by different gonadotropic cells in the pituitaries of a number of teleosts (e.g., tilapia [31], Atlantic salmon, and rainbow trout [32]). In the pituitary of African catfish, three cell types were found that expressed the 
subunit [33], which were situated in the PPD. Based on morphological criteria, one of them was identified as thyrotropic cells. The other two cell types were identified as two developmental stages of cfLH-producing gonadotropic cells [9]. The gonadotropes predominating during early stages of spermatogenesis are characterized by small secretory granules being weakly anti-cfLHß antigenic, whereas most gonadotropes during advanced stages of spermatogenesis are characterized by large secretory granules that are strongly labeled with anti-cfLHß.
In situ hybridization detection revealed the presence of cfFSHß mRNA-positive cells in the PPD. Analysis of adjacent sections hybridized with either cfFSHß or cfLHß DIG-labeled probes seemed to indicate that cfFSHß and cfLHß are coexpressed in at least part of the gonadotropic cells. Coexpression is regularly found in pituitaries of tetrapod vertebrates [34]. Coexpression, however, is difficult to reconcile with the previous observation that all subunit-positive cells were identified as either cfTSH- or cfLH-producing cells. Because cfFSH is unavailable, cross reactivity of anti-cfLH with cfFSH cannot be ruled out [9], so that the weak anti-cfLHß antigenicity in the small secretory granules of the gonadotropes predominating at early stages of spermatogenesis might represent a cross reaction with cfFSHß. Combined immunocytochemical and in situ hybridization studies during pubertal development will provide conclusive evidence for the ontogeny and identity of the catfish gonadotropic cells.
In order to provide evidence for the physiological relevance of the cloned cfFSHß cDNA, the influence of testicular hormones on cfFSHß mRNA expression was studied. Our results suggested a negative feedback regulation of cfFSHß gene expression by aromatizable androgens in mature male catfish and indicated that cfFSHß is of functional relevance in the brain-pituitary-gonad axis. A similar negative regulation of FSHß by gonadal hormones was observed 3–14 days after surgery in mature male coho salmon [35], while an opposite effect (i.e., positive feedback) was found in mature male Atlantic salmon parr after long-term (3–7 mo) castration [36]. No net effect was recorded in mature male goldfish [37], while in fish at the beginning of the rapid testicular growth phase, T and E2 decreased FSHß transcript levels. The mechanism by which aromatizable androgens regulate cfFSHß gene expression and the involvement of other gonadal factors (e.g., activin and inhibin) remains to be elucidated. In addition, FSHß gene expression may be regulated by GnRH derived from the brain, as reported for cyprinids [38], perciforms [39], and salmonids [40]. Future studies using primary pituitary cell culture will be undertaken to investigate the direct effects of E2 as well as GnRH or their combination on cfFSHß gene expression.
In conclusion, we cloned the gene as well as the cDNA, coding for the cfFSH ß subunit from the African catfish. The presence of the mature protein in the pituitary and circulation remains to be shown and is currently under investigation. Together with the recent cloning of the cfFSH-R, this study provides tools to shed light on the identification and physiological significance of FSH-dependent processes in fish reproduction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: R.W. Schulz, Utrecht University, Faculty of Biology, Department of Endocrinology, Kruyt Building, Room Z-203, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. FAX: 31 30 2532837; r.w.schulz{at}bio.uu.nl
Received: 7 August 2002.
First decision: 31 August 2002.
Accepted: 25 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and gonadotropin IIß-subunit mRNA levels in the pituitary gland of mature male African catfish, Clarias gariepinus. Fish Physiol Biochem 1997 17:99-108[CrossRef]This article has been cited by other articles:
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  A. Garcia-Lopez, J. Bogerd, J. C. M. Granneman, W. van Dijk, J. M. Trant, G. L. Taranger, and R. W. Schulz Leydig Cells Express Follicle-Stimulating Hormone Receptors in African Catfish Endocrinology, January 1, 2009; 150(1): 357 - 365. [Abstract] [Full Text] [PDF]
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W.-K. So, H.-F. Kwok, and W. Ge 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, June 1, 2005; 72(6): 1382 - 1396. [Abstract] [Full Text] [PDF]
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B. E. West, G. E. Parker, J. J. Savage, P. Kiratipranon, K. S. Toomey, L. R. Beach, S. C. Colvin, K. W. Sloop, and S. J. Rhodes Regulation of the Follicle-Stimulating Hormone {beta} Gene by the LHX3 LIM-Homeodomain Transcription Factor Endocrinology, November 1, 2004; 145(11): 4866 - 4879. [Abstract] [Full Text] [PDF]
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