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BOR - Papers in Press, published online ahead of print February 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038190
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BIOLOGY OF REPRODUCTION 72, 1370–1381 (2005)
DOI: 10.1095/biolreprod.104.038190
© 2005 by the Society for the Study of Reproduction, Inc.

Zebrafish Gonadotropins and Their Receptors: I. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Receptors— Evidence for Their Distinct Functions in Follicle Development1

Hin-Fai Kwok , Wai-Kin So , Yajun Wang , and Wei Ge 2 

Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.

FSH, Fshr, LH, Lhr, ovary, receptors, zebrafish


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In teleosts, two distinct gonadotropins, follicle-stimulating hormone (FSH, formerly termed GTH-I) and luteinizing hormone (LH, formerly termed GTH-II), have been identified and characterized in a variety of species by either purification or molecular cloning [1]; however, their functional homology to those of mammalian counterparts has not been well defined. In female salmonids whose gonadotropins are best characterized, FSH and LH are differentially expressed and secreted during the reproductive cycle, with the level of FSH being high during oocyte growth (vitellogenesis) followed by a surge of LH secretion before or during oocyte maturation and ovulation [2–4]. However, in cyprinids, the expression patterns of FSHß and LHß mRNA are somehow similar during ovarian development as demonstrated in the goldfish (Carassius auratus) [5]. This discrepancy is likely due to the different modes of ovarian development in different species. The salmonids are annual-spawning teleosts with the oocytes developing synchronously, whereas the cyprinids spawn multiple times during their spawning season and their ovaries develop asynchronously with follicles at different developmental stages, which may have different requirements for FSH and LH [6]. Although studies in salmonids have led to the hypothesis that FSH is involved mainly in controlling gonadal growth whereas LH is responsible for the late events of gonadal development such as ovulation and spermiation [7– 9], their roles in nonsalmonid species, particularly the cyprinids, remain largely unknown. Since little has been learned about the physiological relevance of FSH and LH from the studies on hormonal profiles in these fish species, studies on their receptors promise to provide alternative information on the issue.

In contrast to the studies on FSH and LH hormones in teleosts, the efforts spent on their receptors have significantly lagged behind those in mammals, which, to some extent, has hindered our understanding of the physiological roles of FSH and LH in controlling reproductive cycle. It was suggested by earlier studies that there was only one type of GTH receptor in fish [10–13]. However, with the two distinct gonadotropins purified, evidence emerged later in salmonids that there existed two classes of GTH receptors in fish gonads, termed type I (GTH-RI) and type II (GTH-RII), respectively [7], which are likely corresponding to the FSHR and LHR identified later with molecular cloning [14–19]. In the ovary of coho salmon (Oncorhynchus kistuch), GTH-RI (FSHR) has been localized to both the theca and granulosa cells by homologous ligand-binding assay and autoradiography and binds both FSH and LH, whereas GTH-RII (LHR) specifically binds LH and is located on the granulosa cells only. These results have led to the proposal of a two-receptor model for salmonid FSH and LH [20, 21]. Subsequent cloning studies in a number of teleost species have further confirmed the duality of GTH receptors in fish [14–19, 22]. However, the functionality of fish FSHR and LHR and, particularly, their spatial-temporal expression have not been as well defined as those in mammals, and the limited studies so far by expressing the receptor proteins in heterologous cell lines have generated confusing results in terms of ligand specificity. As one of the earliest efforts to clone fish GTH receptors, Oba et al. [14, 15] isolated two distinct cDNAs coding for putative FSHR and LHR in the amago salmon (Oncorhynchus rhodurus). Overexpression of these receptors in the COS cells showed that FSHR (sGTH-RI) could be specifically activated by chum salmon (Oncorhynchus keta) FSH but not LH [15], whereas LHR (sGTH-RII) was highly responsive to LH and, to a lesser extent, FSH as well [14]. In the channel catfish (Ictalurus punctatus), the cloned LHR responded specifically to hCG but not human FSH, whereas FSHR was responsive to both but with preference for human FSH [16, 17]. A similar situation was also observed in the African catfish (Clarias gariepinus) with homologous recombinant catfish FSH and LH [23]. The cloned FSHR responded to both catfish gonadotropins with slightly higher preference for FSH. In comparison, the LHR was specifically activated by LH only with little cross-reaction with FSH [18, 19, 23].

The high variation of gonadotropin-receptor interaction among the limited teleost species studied so far raises an interesting question about the situation in other fish groups. In Labeo rohita, an Indian carp, the purified FSHR-like and LHR-like receptors significantly cross-reacted with both salmon gonadotropins [24]. In the zebrafish (Danio rerio), a cDNA for Fshr has recently been cloned and its ligand specificity characterized by expressing the protein in the COS cells. The cells expressing this zebrafish Fshr responded to both carp pituitary extract and human FSH by increasing cAMP production, but it had no response to hCG [25]. Unfortunately, no information is available about Lhr in this model organism. To provide a complete picture about gonadotropin receptors and their ligand-binding specificity in cyprinids, the present study was undertaken to clone and characterize both Fshr and Lhr in the zebrafish. In addition to the functional characterization of the receptors, we also investigated their temporal expression patterns during the period of sexual maturation and their stage-dependent expression in isolated developing follicles because this information is particularly lacking in teleosts and would surely contribute to our understanding of the potential roles of the two gonadotropins and their receptors in controlling the fish reproductive cycle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Chemicals

Sexually mature or immature zebrafish, Danio rerio, were purchased from local pet stores and maintained in flow-through aquaria (36 L) at 26°C on a 14L:10D photoperiod. The fish were fed twice a day with commercial tropical fish food. All experiments were performed under license from the government of the Hong Kong SAR and endorsed by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong.

All chemicals were obtained from Sigma (St. Louis, MO) and enzymes used for molecular cloning from Promega (Madison, WI) unless otherwise specified. All culture media and fetal bovine serum (FBS) were obtained from Gibco Invitrogen (Carlsbad, CA). Human chorionic gonadotropin (hCG) was purchased from Sigma, and bovine FSH (bFSH) and LH (bLH) were obtained from Tucker Endocrine Research Institute (Atlanta, GA).

Isolation of Total RNA

Total RNA was isolated from various tissues including the ovary and isolated follicles with TRI-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol and our previous report [26].

Cloning of Zebrafish fshr and lhr cDNA Fragments from the Ovary

Two pairs of degenerate primers were designed based on the consensus amino acid sequences of vertebrate FSHR and LHR to amplify zebrafish fshr and lhr cDNA fragments (Table 1). The total RNA (3 µg) from the ovary was reverse transcribed into single-stranded cDNA with SuperScript II (Gibco) under the following condition: 70°C for 10 min without the enzyme followed by 2-h incubation at 42°C after addition of the enzyme. Polymerase chain reaction (PCR) was carried out using the degenerate primers listed in Table 1. The synthesized cDNA was subjected to 35 cycles of PCR with cycle profile of 30 sec at 94°C, 30 sec at 55°C, and 1 min at 72°C. The reaction was finished with a final extension at 72°C for 10 min. The amplified PCR products of expected sizes (fshr: ~280 bp; lhr: ~380 bp) were resolved by agarose gel electrophoresis, isolated, purified by phenol/chloroform extraction, and cloned into pBluescript II KS (+) (Stratagene, La Jolla, CA) by T/A cloning. The cloned plasmids were prepared with the NucleoSpin Plasmid Isolation Kit (Macherey-Nagel, Duren, Germany) for sequence analysis.


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  		TABLE 1. Primers used.a

Rapid Amplification of 5' cDNA Ends (5'-RACE) and Full-Length cDNAs

Gene-specific antisense primers for fshr and lhr were designed based on the sequenced fshr and lhr cDNA fragments for 5'-RACE (Table 1). The reaction was carried out using the SMART RACE cDNA Amplication Kit (Clontech, Palo Alto, CA), and the amplification products were cloned into pBluescript II KS (+) and sequenced. Based on the sequences of the 5'-RACE products, new gene-specific sense primers were then designed near the 5' ends for 3'-RACE to amplify the full-length cDNAs of fshr and lhr (Table 1). PCR was performed with the following reaction profile: 5 cycles at 94°C for 5 sec and 72°C for 3 min; 5 cycles at 94°C for 5 sec, 70°C for 10 sec, and 72°C for 3 min; and 31 cycles at 94°C for 5 sec, 68°C for 10 sec, and 72°C for 3 min. The amplified cDNAs were cloned into pBluescript II KS (+) and subjected to sequencing after generating a series of overlapping subclones by exonuclease III and mung bean nuclease deletion. Both strands of fshr and lhr cDNAs were sequenced with the BigDye Terminator Cycle Sequencing Kit v3.1 and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assays

The total RNA extracted from the ovary or ovarian follicles were reverse transcribed into cDNA at 42°C for 2 h in a total volume of 10 µl reaction solution consisting of 1x M-MLV RT buffer, 0.5 mM each dNTP, 0.5 µg oligo(dT), and 80 U M-MLV reverse transcriptase. PCR amplification was then carried out on 1 µl RT mixture using gene-specific primers (Table 1). The PCR reaction mixture (30 µl) consisted of 1x PCR buffer, 0.2 mM each dNTP, 2.5 mM MgCl2, 0.2 µM each primer, and 0.5 U Taq polymerase. PCR profiles were 30 sec at 94°C, 30 sec at specific annealing temperature for each gene (62, 58, 56, and 56°C for zebrafish fshr, lhr, bactin [ß-actin] and gapd [glyceraldehyde-3-phosphate dehydrogenase], respectively), and 1 min at 72°C. For semiquantitative RT-PCR analysis, we validated the assays by first obtaining the cycle number that generated half maximal amplification for each gene followed by testing it on serially diluted cDNA template. The cycle numbers used were 31, 32, 21, and 23 for fshr, lhr, bactin, and gapd, respectively.

Isolation of Ovarian Follicles

Fifteen to 20 female zebrafish were anaesthetized in each experiment by immersion in ethyl 3-aminobenzoate methanesulfonate (MS-222) and decapitated before dissection for the ovaries. The ovaries removed were placed in a 35-mm culture dish containing 60% medium Leibovitz L-15, and the follicles of different stages were manually isolated.

Sampling of Zebrafish Ovaries During Sexual Maturation

About 200 sexually immature zebrafish were used for studying the developmental profiles of fshr and lhr expression in the ovary during sexual maturation. The sexual immaturity of the fish was first confirmed by histological examination of the ovary before the experiment started. The day of the first sampling was designated Day 0, and subsequent sampling was performed on Days 3, 6, 9, 12, 15, and 18. At each time point, both ovaries of each zebrafish were quickly removed. One of the ovaries was fixed with Bouin solution for histological sectioning and microscopic observation of follicle development, and the other one was subjected to total RNA extraction with TRI-Reagent for semiquantitative RT-PCR analysis.

Construction of Expression Plasmids

For functional analysis of the cloned zebrafish Fshr and Lhr, we directly amplified the open reading frame (ORF) of each receptor with Pfu polymerase from the cDNA generated from the zebrafish ovary to ensure high sequence fidelity. The primers (Table 1) flanking the ORF were designed from the cloned cDNAs with appropriate restriction sites added for subsequent cloning into the expression vector pBK-CMV (Stratagene) with the lac promoter removed as suggested by the manufacturer. The sense primers were also modified before the translation initiation codon ATG to conform to the Kozak consensus sequence GCCGCC[A/ G]CCATGG to enhance the efficiency of translation [27]. The ORF of fshr was cloned at EcoRI/XbaI sites (pBK-CMV/fshr) and that of lhr at BamHI/ XbaI sites (pBK-CMV/lhr). The clones were sequenced to confirm their identity and sequence accuracy.

Transient Transfection and Reporter Gene Assay

The Chinese hamster ovary (CHO K-1) cells were cultured under 5% CO2 in Ham F-12 medium supplemented with 10% FBS. The expression plasmid pBK-CMV/fshr or pBK-CMV/lhr was cotransfected into the CHO cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) together with the reporter plasmid pCRE-SEAP (Clontech), which encodes the secreted human placental alkaline phosphatase (SEAP) reporter driven by a promoter containing multiple cAMP-responsive elements. The expression vector pBK-CMV was used as the control. Twelve hours after transfection, the cells were subcultured into 24-well plates at the density of 105 cells/ well. The medium was changed to serum-free medium after 24-h incubation, and the incubation continued for 24 h before drug treatment. The cells were treated with different concentrations of goldfish pituitary extract or hCG for 18 h before sampling the medium for SEAP assay. We also used COS-1 cells to confirm the results using similar transfection and assay protocols. The COS-1 cells were cultured under 5% CO2 in DMEM (Dulbecco Modified Eagle Medium) supplemented with 10% FBS.

The level of SEAP protein in the medium was assayed with the Chemiluminescent SEAP Reporter Gene Assay Kit (Roche, Mannheim, Germany) according to the manufacturer's protocol and our recent report [28]. The signals were detected and analyzed with the Lumi-Imager F1 Workstation and the software LumiAnalyst 3.1 (Roche).

Establishment and Characterization of Stable Fshr- or Lhr-Expressing Cell Lines

CHO cells cotransfected with pBK-CMV/fshr or pBK-CMV/lhr together with pCRE-SEAP were subcultured into a six-well plate 12 h after transfection and cultured in the presence of 500 µg/ml G418 sulfate (Gibco) for 21 days. The expression of zebrafish fshr or lhr mRNA by the selected CHO cells was verified by RT-PCR. The stable transfectants were trypsinized and serially diluted into a 96-well plate for selection of single G418-resistant clones. All single clones were screened for SEAP response to gonadotropin(s) by treating the cells with 30 µg/ml goldfish pituitary extract (for Fshr cells) or 10 IU/ml hCG (for Lhr cells) for 18 h. The responsive CHO-Fshr and CHO-Lhr clones were expanded, and the expression of fshr or lhr mRNA was further verified by RT-PCR.

To investigate the functionality of the cloned zebrafish Fshr and Lhr, the stable CHO-Fshr or CHO-Lhr clones were subcultured into 24-well plates at the density of 105 cells/well for 24 h followed by 24-h serum starvation before the treatment with the goldfish pituitary extract or mammalian gonadotropins for 18 h. The assay for SEAP activity was performed as described previously.

Data Analysis

The sequences were analyzed with MacVector 7.2.2 (Accelrys, San Diego, CA). For semiquantitative RT-PCR assays, the mRNA level of each gene was presented as the percentage of the control or reference group after being normalized to that of bactin or gapd, which were amplified as the internal controls. For the SEAP reporter assay, the SEAP activity in each sample was expressed as the percentage of the control. All values were expressed as the mean + SEM, and the data were analyzed by one-way ANOVA followed by the Dunnett test (for comparing treatment groups with the control group) or the Newman-Keuls test (for comparing all pairs of groups) using the software Prism 4.0b for Macintosh (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of fshr and lhr cDNA from the Zebrafish Ovary

The full-length cDNAs for zebrafish fshr and lhr were cloned by 3'-RACE using the gene-specific primers designed from the 5'-RACE products. The cloned fshr cDNA is 2543 bp in length, containing an ORF of 2007 bp that encodes a protein of 668 amino acids (Fig. 1). A putative N-terminal signal peptide of 16 amino acids was predicted by the software SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP) [29]. Zebrafish Fshr shows the highest homology with that of channel catfish (72% amino acid identity) [16] and African catfish (71% identity) [19] (Fig. 3). Characterization of zebrafish Fshr with the software TMHMM v2 (http://www.cbs.dtu.dk/services/TMHMM) [30] reveals the presence of three major regions: a long N-terminal extracellular region of 347 amino acids, a 263-amino-acid region consisting of seven transmembrane domains, and a C-terminal intracellular tail of 58 amino acids.



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  		FIG. 1. Nucleotide and deduced amino acid sequences of zebrafish Fshr (GenBank accession no. AY424301). The predicted signal peptide at the N-terminal and a potential polyadenylation signal (ATAAA) in the 3'-untranslated region are underlined. The boxed regions are the seven predicted transmembrane domains



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  		FIG. 3. Phylogenetic relationship of zebrafish Fshr and Lhr with the gonadotropin receptors from other teleosts as well as model mammals and chicken as demonstrated by bootstrapping analysis using neighbor joining method. The numbers at the forks are the bootstrap proportions

The lhr cDNA is 2965 bp in length with a 2127-bp ORF that encodes a 708-amino-acid protein. As expected for a transmembrane receptor, there is also a typical hydrophobic signal peptide of 21 amino acids at the N-terminal (Fig. 2). The deduced amino acid sequence of zebrafish Lhr shares the highest homology with that of African catfish (73% identity) [18] (Fig. 3). Similar to Fshr, zebrafish Lhr also includes three regions: an N-terminal extracellular region of 374 amino acids, a transmembrane region of 266 amino acids including seven transmembrane domains, and a 68-amino-acid intracellular tail.



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  		FIG. 2. Nucleotide and deduced amino acid sequences of zebrafish Lhr (GenBank accession no. AY424302). The predicted signal peptide at the N-terminal and two potential polyadenylation signals (ATTAAA) in the 3'-untranslated region are underlined. The boxed regions are the seven predicted transmembrane domains

Expression of fshr and lhr in the Gonads and Extragonadal Tissues

Using gene-specific primers, we examined a variety of tissues including the brain, gill, kidney, liver, muscle, pituitary, ovary, and testis for the expression of zebrafish fshr and lhr. As expected, both fshr and lhr were expressed abundantly in the ovary and testis. Interestingly, fshr and lhr also seemed to be expressed in certain extragonadal tissues. The expression of fshr could be easily detected in the kidney, whereas lhr was obviously expressed in the liver (Fig. 4). We repeated the experiment twice using up to 40 individuals in each experiment depending on the size of organs (40 for the pituitary), and the results were fully reproducible.



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  		FIG. 4. Tissue distribution of zebrafish fshr and lhr expression. Total RNA from each tissue (1.6 µg for pituitary and 3.0 µg for others) was subjected to RT-PCR analysis with gapd as the internal control. B, brain; G, gill; K, kidney; L, liver; M, muscle; P, pituitary; O, ovary; T, testis. +, RT with reverse transcriptase; –, RT without reverse transcriptase

Functional Characterization of Zebrafish Fshr and Lhr

To test the functionality of the cloned Fshr and Lhr, we overexpressed each receptor in both CHO K-1 and COS-1 cells by transiently transfecting the cells with pBK-CMV/ fshr or pBK-CMV/lhr together with the cAMP-responsive reporter construct pCRE-SEAP. The goldfish pituitary extract that is supposed to contain both FSH and LH significantly stimulated SEAP expression in both Fshr- and Lhr-expressing cells. However, hCG, which is commonly used in teleosts to induce spawning, significantly increased reporter expression in the Lhr-expressing cells only, and it had no effect on Fshr-expressing cells. A slight effect of the pituitary extract was also noticed on the control cells that were transfected with the expression vector pBK-CMV only. This might be due to certain unknown factors in the extract that may influence the intracellular cAMP level in the CHO cells; however, the level of response in the control cells was much lower than those expressing zebrafish Fshr or Lhr, and hCG had no effect on the control cells (Fig. 5). The results were reproducible with both CHO and COS cells. Consistent with the reports in the channel catfish and African catfish [17, 18], we also observed a significantly high level of basal expression of SEAP in the cells that expressed zebrafish Lhr but not Fshr (data not shown).



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  		FIG. 5. Functional test of recombinant zebrafish Fshr and Lhr in the CHO-K1 (A) and COS-1 (B) cells. The cells were transiently cotransfected with pBK-CMV/fshr or pBK-CMV/lhr and the reporter plasmid pCRE-SEAP with the vector pBK-CMV as the control. The SEAP activity was assayed after 18-h treatment with goldfish pituitary extract (PE) or hCG. The dose dependence was better demonstrated in the COS cells because the doses used were slightly different from those used in the CHO cells. Each data point represents the mean ± SEM of three replicates. **, P < 0.001 vs. untreated cells in each group

To further assess the identity and functionality of zebrafish Fshr and Lhr and also to establish a specific and reliable system for assaying and monitoring recombinant production of zebrafish FSH and LH in the future, we went on to establish two stable CHO cell lines that express zebrafish Fshr and Lhr, respectively, together with the reporter gene SEAP. We chose CHO cells because they are more commonly used to generate stable cell lines. Consistent with the results shown in Figure 5 with the transiently transfected cells, the goldfish pituitary extract significantly increased SEAP levels in both Fshr and Lhr cell lines in a clear dose-dependent manner; however, hCG stimulated only Lhr cells with no effect on Fshr cells (Fig. 6). Interestingly, when tested with bFSH and bLH, the Fshr cells responded to bFSH dose dependently only at high concentrations (1–4 µg/ml), and they showed no response to bLH at all doses tested (0–5 µg/ml); however, the Lhr cells responded to both bFSH and bLH, although the effective doses of bFSH were obviously higher than those of bLH (Fig. 7). The effect of bLH was similar to that of hCG in that both hormones were specific to zebrafish Lhr without any effect on Fshr, and high concentrations appeared to cause reduced response (Figs. 6 and 7).



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  		FIG. 6. Functional test of the stable CHO-Fshr (A) and CHO-Lhr (B) cell lines. The SEAP activity was assayed after 18-h treatment with goldfish pituitary extract or hCG. Each data point represents the mean ± SEM of three replicates. *, P < 0.05; **, P < 0.001 vs. untreated control cells



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  		FIG. 7. Effects of bovine FSH and LH on SEAP expression in the stable CHO-Fshr (A and B) and CHO-Lhr (C and D) cell lines. The media were assayed for SEAP activity after 18-h treatment with the hormones. Two ranges of concentrations were tested (0.005–5 and 1–4 µg/ml). Bovine FSH stimulated both zebrafish Fshr and Lhr over the high concentration range (1–4 µg/ml), whereas bovine LH was effective in stimulating Lhr at low concentrations (0.005–5 µg/ml). Each data point represents the mean ± SEM of three replicates. *, P < 0.05; **, P < 0.001 vs. control

Expression of fshr and lhr at the Ovary Level During Sexual Maturation

As receptors of gonadotropins, which are the most important regulators of gonadal development and function, FSHR and LHR themselves are also subject to tight control by endocrine and paracrine factors in the gonads [31–47]. Understanding the expression profiles of FSHR and LHR will provide important clues to the specific roles of FSH and LH in controlling ovarian development and function, an issue that remains poorly understood in fish, particularly the cyprinids. In this experiment, we analyzed the temporal patterns of fshr and lhr expression in the zebrafish ovary during sexual maturation using semiquantitative RT-PCR. The assays were validated as described in Materials and Methods (Fig. 8). The experiment was started with sexually immature fish of about 2 mo old, and the ovaries were collected every 3 days over an 18-day period. Nine to 15 female zebrafish were sampled each time. Since the fish used in the experiment showed evident individual variation, we fixed one ovary from each sampled fish for histological examination of follicle development and staging. The total RNA was extracted from the other ovary for RT-PCR analysis. The average level of fshr mRNA gradually increased from Day 0 to Day 18 and peaked on Day 12 and afterward. In contrast, the level of lhr mRNA was generally low during the period of the first 9 days despite a slight trend of increase, but it dramatically increased in the late stage of development from Day 12 through Day 18 (data not shown). To demonstrate the correlation between the stage of the first wave of developing follicles and the expression of fshr and lhr, we reanalyzed the data according to the histological examination of the ovary from each fish. Representative histological sections of the whole ovary in each group are shown in Figure 9C. The staging of the follicles was according to a recent report from our laboratory [48] and that by Selman et al. [49]. The expression of fshr increased significantly during vitellogenesis with the peak level reached when the first cohort of developing follicles developed to the midvitellogenic stage. However, with the appearance of the full-grown follicles in the ovary, the overall fshr expression at the ovary level decreased (Fig. 9A). In contrast to fshr, although the expression of lhr also showed a slight but steady increase during follicle growth, a phenomenal increase was observed when the first wave of follicles reached the full-grown stage (Fig. 9B).



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  		FIG. 8. Validation of the semiquantitative RT-PCR assay for zebrafish fshr and lhr expression. RT-PCR was performed on the RNA isolated from the whole ovary for different cycle numbers (A and C), and the cycle number that generated half maximal reaction was used to analyze the expression of the gene. The assays were validated by testing these cycle numbers on the serially diluted cDNA (B and D). The assays for gapd and bactin were also validated in the same way (data not shown)



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  		FIG. 9. Expression profiles of zebrafish fshr (A) and lhr (B) at the ovary level during sexual maturation. The ovaries were collected over an 18-day period at a 3-day interval with 9–15 female zebrafish sampled at each time point. The expression level was normalized to gapd and expressed as the percentage of that at PV (for fshr) or EV (for lhr) stage. All individuals sampled were subjected to histological examination for staging the ovarian development, and the representative histological images with the leading wave of developing follicles at different stages (labeled by *) are shown on the right (C). The table at the bottom shows the stages of leading follicles in the fish sampled at each time point. PG, primary growth stage; PV, previtellogenic or cortical alveolus stage; EV, early vitellogenic stage; MV, midvitellogenic stage; FG, full-grown stage. Each data point represents the mean ± SEM (n = 8–27). Bars with different letters are different with statistical significance (P < 0.05)

Stage-Dependent Expression of fshr and lhr at the Follicle Level in Sexually Mature Zebrafish

To further confirm the stage dependence of fshr and lhr expression observed in the last experiment at the ovary level, we carried out another experiment to examine the expression of the two receptors at the follicle level using different stages of follicles isolated from mature gravid zebrafish. The results demonstrated a similar but clearer stage dependence of fshr and lhr expression as compared with that observed at the ovary level (Fig. 10). This is because the follicles in the zebrafish ovary exhibit asynchronous development and the ovary contains multiple stages of follicles at any time after puberty; therefore, the expression level of any gene measured at the ovary level reflects the average of the follicles present. At the primary growth stage (PG or stage I, <0.20 mm), both fshr and lhr had extremely low expression. The expression of fshr dramatically increased at the previtellogenic or cortical alveolus stage (PV or stage II, 0.20–0.30 mm) when the cortical vesicles started to appear and accumulate in the oocyte, which is an important morphological marker for the recruitment and activation of the follicle. However, this increase of fshr expression was not accompanied by that of lhr. The expression of fshr steadily increased throughout vitellogenesis (stage III) and reached its peak level at the midvitellogenic stage (MV, 0.45–0.55 mm). However, after the follicles reached full-grown or postvitellogenic stage (FG, >0.65 mm), there was a slight but significant drop in fshr expression (Fig. 10A). In contrast, the expression of lhr was obviously delayed as compared with that of fshr. Its expression became noticeable at the early vitellogenic stage (EV, 0.35–0.40 mm) but significantly increased at the midvitellogenic stage. While fshr expression decreased at the full-grown stage, the expression of lhr reached its peak level at this stage (Fig. 10B).



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  		FIG. 10. Stage-dependent expression of fshr (A) and lhr (B) at the follicle level in sexually mature gravid zebrafish. Follicles of different stages were isolated from sexually mature zebrafish and the total RNA isolated from each stage for RT-PCR analysis. The staging of the follicles is the same as that described in Figure 9. The expression level was normalized to bactin and expressed as the percentage of that at PV (for fshr) or EV (for lhr) stage. Each data point represents the mean ± SEM of three to four replicates. Bars with different letters are different with statistical significance (P < 0.05). The image of PCR amplification is shown at the bottom of each graph


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In teleosts, although FSH and LH have been extensively reported in a large number of species [1], their receptors have not been as well documented in different fish models, and in particular, there has been lack of information about FSHR and LHR in cyprinids, one of the largest teleost families. In the present study, we cloned the full-length cDNAs for both fshr and lhr in the zebrafish, which represents the first cyprinid species with both receptors cloned and characterized. Recently, Laan et al. also reported the cloning of a zebrafish fshr cDNA encoding a protein of 668 amino acid residues [25]. However, sequence analysis and comparison reveal 34 nucleotide differences between this zebrafish fshr (fshra) cDNA and the one cloned in the present study (fshrb). Interestingly, these different nucleotides do not show random distribution with most (19 out of 34) concentrated in the 3'-untranslated region (396 bp; data not shown). Whether these two forms of zebrafish fshr cDNA are due to different genetic background or they represent different isoforms of the receptor remains unknown. Both zebrafish Fshr and Lhr exhibit high sequence homology with their counterparts in the African catfish, channel catfish, and amago salmon [14–19] with structural characteristics of glycoprotein hormone receptor family, including a large N-terminal extracellular domain, seven-transmembrane domains, and a short C-terminal intracellular domain [50–52].

Using gene-specific primers, we analyzed the expression of zebrafish fshr and lhr in different tissues with RT-PCR. As expected, both receptors were abundantly expressed in the ovary and testis. However, we could also easily detect the expression of fshr and lhr in the kidney and liver, respectively. This observation agrees with the reports in other vertebrates, including fish and humans. In the African catfish and channel catfish, LHR was detected in the kidney or head kidney at high levels and a variety of other tissues at lower levels [17, 18]. As for FSHR, a low level of expression was reported in the spleen of the channel catfish [16]. In the bullfrog, the existence of both FSHR and LHR in the liver was demonstrated by receptor binding assays, and in agreement with this, bullfrog FSH and LH significantly increased cAMP production in cultured hepatic cells with LH being more potent than FSH [53]. The extragonadal expression of LHR has also been documented in humans in both fetal [54] and adult stage [55, 56]. Our results in the present study, together with the reports in different vertebrate models, suggest that gonadotropins may exert actions at nongonadal tissues. Although we have little information about the nongonadal effects of gonadotropins and the physiological relevance of such effects, this is surely an interesting issue to address in the future.

We further investigated the functionality and ligand specificity of the cloned zebrafish Fshr and Lhr by expressing the recombinant receptors together with the cAMP-responsive reporter gene SEAP in mammalian cells (CHO K-1 and COS-1) followed by measurement of SEAP levels in the medium after treatment with gonadotropins. Both cell lines generated consistent results, suggesting that the activities of the expressed receptors were independent of the host cells used. As expected, both zebrafish Fshr and Lhr could be activated by goldfish pituitary extract that is supposed to contain FSH and LH. However, as also reported by Laan et al. [25], zebrafish Fshr showed no response to hCG, the most commonly used mammalian gonadotropin in fish. In contrast, Lhr exhibited significant response to hCG treatment. Since all our previous studies in the zebrafish used hCG to examine the roles of gonadotropin(s) in controlling ovarian functions [26, 57–66], the information about its receptor specificity from the present study is important because it clearly shows that all the previously reported results from our laboratory and others [67, 68] using hCG involved activation of Lhr only without coactivation of Fshr. This immediately raises an interesting question about the effects of activating Fshr alone or coactivating Fshr and Lhr, which will be an exciting issue to investigate in the future.

Using the two stable Fshr- and Lhr-expressing cell lines, we also examined the effects of purified bFSH and bLH on the two zebrafish gonadotropin receptors. Similar to hCG, bLH specifically stimulated the expression of reporter gene SEAP in the Lhr-expressing but not Fshr-expressing cells. However, bFSH could activate both zebrafish Fshr and Lhr at high concentrations (1–4 µg/ml). This is similar to the situation reported in the African catfish that human FSH stimulated both FSHR and LHR, whereas human LH and hCG activated only LHR [18, 19]. However, also using heterologous human hormones, the studies in the channel catfish showed that human FSH preferentially stimulated FSHR with little effect on LHR, but hCG can activate both receptors [16, 17]. Because of the high variability of gonadotropin-receptor specificity in fish, especially when heterologous hormones are used, the effects of bFSH and bLH on zebrafish Fshr and Lhr should be interpreted with extra caution. Whether they reflect the specificity of native zebrafish FSH and LH would have to be confirmed with recombinant zebrafish FSH and LH. The discrepancy between heterologous human gonadotropins and the homologous hormones from the same species has already been demonstrated recently in the African catfish, where recombinant catfish FSH specifically stimulated FSHR with much less preference for LHR, whereas catfish LH could stimulate both receptors with more or less equal potency [23], which is different from the effects of human FSH and LH/ hCG in the same species [18, 19, 23]. Since fish gonadotropins are not easily available, hormones from mammalian sources have been commonly used as the alternatives in various studies in fish. The results from the present study, together with those from other models, should bring it to the attention of researchers who use heterologous gonadotropins that there exists potential complexity arising from the cross activation of the two gonadotropin receptors by such hormones in various fish models. Although the physiological relevance of the coactivation of zebrafish Fshr and Lhr by bFSH is unknown, this hormone could be a useful ligand to study the interactive effects of coactivating Fshr and Lhr in this model.

Similar to the tetrapods, teleosts also produce two gonadotropins (FSH and LH) in their pituitary glands [7, 8, 69]. However, the physiological roles of FSH and LH have not been well defined in different models. In salmonids, it has been proposed based on the in vivo hormone profiles that FSH is likely important in promoting follicle growth in the ovary, whereas LH is responsible mainly for final oocyte maturation and ovulation [7, 8]. However, in nonsalmonid teleosts such as cyprinids, studies on seasonal variation of FSH and LH expression did not seem to support differential roles for the two gonadotropins during the reproductive cycle. For example, FSHß and LHß in the goldfish showed similar expression patterns during the cycle [5]. This, together with the relatively low expression level of FSHß in the pituitary compared with that of LHß, has led to the speculation that FSH may not be physiologically relevant in some teleosts, including cyprinids. To provide clues to this issue, we performed experiments to investigate the temporal expression patterns of fshr and lhr at the ovary level during sexual maturation and the stage dependence of their expression at the follicle level in sexually mature gravid zebrafish. This kind of information is particularly lacking in fish, and only channel catfish FSHR and LHR have hitherto been studied for their seasonal variation in the ovary during the reproductive cycle [16, 17]. Our experiments showed that unlike the reported expression patterns of FSHß and LHß in the pituitary of cyprinids, the two gonadotropin receptors were differentially expressed in the zebrafish at both the ovary and the follicle level. The expression of fshr was closely associated with vitellogenesis because its expression level increased significantly when the follicles were recruited from the primary growth stage and continued to rise throughout vitellogenesis. However, with the completion of vitellogenesis, its expression level fell at the full-grown or postvitellogenic stage. In contrast, the expression of lhr became evident much later than that of fshr, and it reached its peak level at the full-grown stage, strongly implicating it in the induction of oocyte maturation and/or ovulation. Although we are still lacking the information about the profiles of FSH and LH in the zebrafish, the results from the present study on fshr and lhr seem to support the concept proposed in salmonids for the roles of FSH and LH during the ovarian cycle [7, 8]. What remains to be addressed in the future is how these two receptors are activated by the two gonadotropins in vivo and the specificity of the ligand-receptor interaction. In a companion study, we have recently cloned all the subunits of zebrafish FSH and LH, which has allowed us to establish stable cell lines that produce recombinant zebrafish FSH and LH (So et al., published separately). The availability of recombinant zebrafish gonadotropins will allow for studies on their receptor specificity and signaling mechanisms as well as the effects or interactive effects of the activation or coactivation of Fshr and Lhr. These would surely provide important information on the physiological relevance of the two gonadotropins and their roles in zebrafish ovarian development and function.

In summary, full-length cDNAs coding for Fshr and Lhr were cloned from the zebrafish ovary. Both zebrafish Fshr and Lhr could be activated by the goldfish pituitary extract. Interestingly, hCG and bLH specifically stimulated zebrafish Lhr without any effect on Fshr; however, bFSH could activate both receptors. Zebrafish fshr and lhr exhibited distinct patterns of expression at the ovary level during sexual maturation and at the follicle level in sexually mature fish. The expression of fshr was strongly associated with the recruitment and growth of the follicles, whereas lhr was more likely involved in the final oocyte maturation and ovulation (Fig. 11). These results strongly suggest that the two gonadotropins, FSH and LH, may play differential roles in controlling zebrafish ovarian and follicle development, although this may not be reflected by their expression and/or secretion at the pituitary level because of the asynchronous nature of zebrafish follicle development in the ovary.



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  		FIG. 11. Schematic representation of the temporal expression profiles of zebrafish fshr and lhr during follicle development. Both receptors are expressed at extremely low levels at the PG stage. The expression of fshr is strongly correlated with the growth or vitellogenesis of the follicle, and its level drops on the completion of vitellogenesis. However, the expression of lhr is obviously involved in the final stage of follicle development, such as oocyte maturation and/or ovulation. Its expression level increases in the late phase of follicle growth and reaches the peak at the FG stage. The staging of the follicles is the same as that described in Figure 9 (LV, late vitellogenic stage). The small open and solid circles in the follicle represent the cortical alveoli and yolk granules, respectively


   FOOTNOTES
 
1 Supported by grants (CUHK4176/99M, CUHK4150/01M, and CUHK4258/02M) from the Research Grants Council of the Hong Kong Special Administrative Region to W.G. Back

2 Correspondence: Wei Ge, Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. FAX: 852 2603 5646; weige{at}cuhk.edu.hk Back

Received: 17 November 2004.

First decision: 23 December 2004.

Accepted: 8 February 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Swanson P, Dickey JT, Campbell B. Biochemistry and physiology of fish gonadotropins. Fish Physiol Biochem 2003 28:53-59[CrossRef]
  2. Weil C, Bougoussa-Houadec M, Gallais C, Itoh S, Sekine S, Valotaire Y. Preliminary evidence suggesting variations of GtH 1 and GtH 2 mRNA levels at different stages of gonadal development in rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 1995 100:327-333[CrossRef][Medline]
  3. Prat F, Sumpter JP, Tyler CR. 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 1996 54:1375-1382[Abstract]
  4. Swanson P. Salmon gonadotropins: reconciling old and new ideas. In: Scott AP, Sumpter JP, Kime DE, Rolfe MS (eds.), Reproductive Physiology of Fish. Sheffield, UK: FishSymp91; 1991:2–7
  5. Yoshiura Y, Kobayashi M, Kato Y, Aida K. Molecular cloning of the cDNAs encoding two gonadotropin ß subunits (GTH-Iß and -IIß) from the goldfish, Carassius auratus. Gen Comp Endocrinol 1997 105:379-389[CrossRef][Medline]
  6. Kobayashi M, Aida K, Hanyu I. Hormone changes during the ovulatory cycle in goldfish. Gen Comp Endocrinol 1988 69:301-307[Medline]
  7. Swanson P, Dittman A. Pituitary gonadotropins and their receptors in fish. In: Kawshima S, Kikuyama S (eds.), Advances in Comparative Endocrinology. Bologna, Italy: Monduzzi Editore; 1997:841–856
  8. Swanson P, Suzuki K, Kawauchi H, Dickhoff WW. Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II. Biol Reprod 1991 44:29-38[Abstract]
  9. Nagahama Y. Endocrine regulation of gametogenesis in fish. Int J Dev Biol 1994 38:217-229[Medline]
  10. Breton B, Le Gac F, Sambroni E. Gonadotropin hormone (GtH) receptors in the ovary of the brown trout Salmo trutta L. in vitro studies. Gen Comp Endocrinol 1986 64:163-171[CrossRef][Medline]
  11. Kanamori A, Kagawa H, Nagahama Y. Gonadotropin receptors in the postovulatory ovary of amago salmon (Oncorhynchus rhodurus). Gen Comp Endocrinol 1987 66:210-217[CrossRef][Medline]
  12. Kanamori A, Nagahama Y. Developmental changes in the properties of gonadotropin receptors in the ovarian follicles of amago salmon (Oncorhynchus rhodurus) during oogenesis. Gen Comp Endocrinol 1988 72:25-38[Medline]
  13. Le Gac F, Breton B, Bougoussa M. Gonadotropic hormone (GtH) receptors in the testis of the trout Salmo gairdneri: in vitro studies. Fish Phys Biochem 1988 5:209-217
  14. Oba Y, Hirai T, Yoshiura Y, Yoshikuni M, Kawauchi H, Nagahama Y. Cloning, functional characterization, and expression of a gonadotropin receptor cDNA in the ovary and testis of amago salmon (Oncorhynchus rhodurus). Biochem Biophys Res Commun 1999 263:584-590[CrossRef][Medline]
  15. Oba Y, Hirai T, Yoshiura Y, Yoshikuni M, Kawauchi H, Nagahama Y. The duality of fish gonadotropin receptors: cloning and functional characterization of a second gonadotropin receptor cDNA expressed in the ovary and testis of amago salmon (Oncorhynchus rhodurus). Biochem Biophys Res Commun 1999 265:366-371[CrossRef][Medline]
  16. Kumar RS, Ijiri S, Trant JM. Molecular biology of the channel catfish gonadotropin receptors: 2. Complementary DNA cloning, functional expression, and seasonal gene expression of the follicle-stimulating hormone receptor. Biol Reprod 2001 65:710-717[Abstract/Free Full Text]
  17. Kumar RS, Ijiri S, Trant JM. Molecular biology of channel catfish gonadotropin receptors: 1. Cloning of a functional luteinizing hormone receptor and preovulatory induction of gene expression. Biol Reprod 2001 64:1010-1018[Abstract/Free Full Text]
  18. Vischer HF, Bogerd J. Cloning and functional characterization of a gonadal luteinizing hormone receptor complementary DNA from the African catfish (Clarias gariepinus). Biol Reprod 2003 68:262-271[Abstract/Free Full Text]
  19. Bogerd J, Blomenrohr M, Andersson E, van der Putten HH, Tensen CP, Vischer HF, Granneman JC, Janssen-Dommerholt C, Goos HJ, Schulz RW. Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus). Biol Reprod 2001 64:1633-1643[Abstract/Free Full Text]
  20. Yan L, Swanson P, Dickhoff WW. A two-receptor model for salmon gonadotropins (GTH I and GTH II). Biol Reprod 1992 47:418-427[Abstract]
  21. Miwa S, Yan L, Swanson P. Localization of two gonadotropin receptors in the salmon gonad by in vitro ligand autoradiography. Biol Reprod 1994 50:629-642[Abstract]
  22. Oba Y, Hirai T, Yoshiura Y, Kobayashi T, Nagahama Y. Fish gonadotropin and thyrotropin receptors: the evolution of glycoprotein hormone receptors in vertebrates. Comp Biochem Physiol B Biochem Mol Biol 2001 129:441-448[CrossRef][Medline]
  23. Vischer HF, Granneman JC, Linskens MH, Schulz RW, Bogerd J. Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor. J Mol Endocrinol 2003 31:133-140[Abstract]
  24. Basu D, Bhattacharya S. Purification of two types of gonadotropin receptors from carp ovarian follicles: overlapping recognition by two different ligands. Gen Comp Endocrinol 2002 129:152-162[CrossRef][Medline]
  25. Laan M, Richmond H, He C, Campbell RK. Zebrafish as a model for vertebrate reproduction: characterization of the first functional zebrafish (Danio rerio) gonadotropin receptor. Gen Comp Endocrinol 2002 125:349-364[Medline]
  26. Pang Y, Ge W. Gonadotropin regulation of activin ßA and activin type IIA receptor expression in the ovarian follicle cells of the zebrafish, Danio rerio. Mol Cell Endocrinol 2002 188:195-205[CrossRef][Medline]
  27. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 1987 196:947-950[CrossRef][Medline]
  28. Lau MT, Ge W. Cloning of Smad2, Smad3, Smad4 and Smad7 from the goldfish pituitary and evidence for their involvement in activin regulation of goldfish FSHß promoter activity. Gen Comp Endocrinol 2005 141:22-28[CrossRef][Medline]
  29. Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997 10:1-6[Abstract/Free Full Text]
  30. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001 305:567-580[CrossRef][Medline]
  31. Camp TA, Rahal JO, Mayo KE. Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary. Mol Endocrinol 1991 5:1405-1417[Abstract]
  32. LaPolt PS, Tilly JL, Aihara T, Nishimori K, Hsueh AJ. Gonadotropin-induced up- and downregulation of ovarian follicle-stimulating hormone (FSH) receptor gene expression in immature rats: effects of pregnant mare's serum gonadotropin, human chorionic gonadotropin, and recombinant FSH. Endocrinology 1992 130:1289-1295[Abstract]
  33. Sites CK, Patterson K, Jamison CS, Degen SJ, LaBarbera AR. Follicle-stimulating hormone (FSH) increases FSH receptor messenger ribonucleic acid while decreasing FSH binding in cultured porcine granulosa cells. Endocrinology 1994 134:411-417[Abstract]
  34. Tano M, Minegishi T, Kishi H, Kameda T, Abe Y, Miyamoto K. The effect of follicle-stimulating hormone (FSH) on the expression of FSH receptor in cultured rat granulosa cells. Life Sci 1999 64:1063-1069[CrossRef][Medline]
  35. Nakamura M, Minegishi T, Hasegawa Y, Nakamura K, Igarashi S, Ito I, Shinozaki H, Miyamoto K, Eto Y, Ibuki Y. Effect of an activin A on follicle-stimulating hormone (FSH) receptor messenger ribonucleic acid levels and FSH receptor expressions in cultured rat granulosa cells. Endocrinology 1993 133:538-544[Abstract]
  36. Minegishi T, Tano M, Nakamura K, Nakamura M, Igarashi S, Ito I, Shinozaki H, Karino S, Ibuki Y, Miyamoto K. Regulation of follicle-stimulating hormone receptor. Horm Res 1996 46:suppl_137-44
  37. Inoue K, Nakamura K, Abe K, Hirakawa T, Tsuchiya M, Oomori Y, Matsuda H, Miyamoto K, Minegishi T. Mechanisms of action of transforming growth factor ß on the expression of follicle-stimulating hormone receptor messenger ribonucleic acid levels in rat granulosa cells. Biol Reprod 2003 69:1238-1244[Abstract/Free Full Text]
  38. Inoue K, Nakamura K, Abe K, Hirakawa T, Tsuchiya M, Matsuda H, Miyamoto K, Minegishi T. Effect of transforming growth factor ß on the expression of luteinizing hormone receptor in cultured rat granulosa cells. Biol Reprod 2002 67:610-615[Abstract/Free Full Text]
  39. Hirakawa T, Minegishi T, Abe K, Kishi H, Ibuki Y, Miyamoto K. A role of insulin-like growth factor I in luteinizing hormone receptor expression in granulosa cells. Endocrinology 1999 140:4965-4971[Abstract/Free Full Text]
  40. Nakamura K, Nakamura M, Igarashi S, Miyamoto K, Eto Y, Ibuki Y, Minegishi T. Effect of activin on luteinizing hormone-human chorionic gonadotropin receptor messenger ribonucleic acid in granulosa cells. Endocrinology 1994 134:2329-2335[Abstract]
  41. Richards JS, Hedin L. Molecular aspects of hormone action in ovarian follicular development, ovulation, and luteinization. Annu Rev Physiol 1988 50:441-463[CrossRef][Medline]
  42. Shi H, Segaloff DL. A role for increased lutropin/choriogonadotropin receptor (LHR) gene transcription in the follitropin-stimulated induction of the LHR in granulosa cells. Mol Endocrinol 1995 9:734-744[Abstract]
  43. Yaron Y, Schwartz D, Evans MI, Lessing JB, Rotter V. Alternatively spliced mRNA transcripts encoding the extracellular domain of the FSH receptor gene: expression in the mouse ovary during the ovulatory cycle. J Reprod Med 1998 43:435-438[Medline]
  44. Segaloff DL, Wang HY, Richards JS. Hormonal regulation of luteinizing hormone/chorionic gonadotropin receptor mRNA in rat ovarian cells during follicular development and luteinization. Mol Endocrinol 1990 4:1856-1865[Abstract]
  45. Menon KM, Munshi UM, Clouser CL, Nair AK. Regulation of luteinizing hormone/human chorionic gonadotropin receptor expression: a perspective. Biol Reprod 2004 70:861-866[Abstract/Free Full Text]
  46. Tsuchiya M, Minegishi T, Kishi H, Tano M, Kameda T, Hirakawa T, Ibuki Y, Mizutani T, Miyamoto K. Control of the expression of luteinizing hormone receptor by local factors in rat granulosa cells. Arch Biochem Biophys 1999 367:185-192[CrossRef][Medline]
  47. Minegishi T, Hirakawa T, Kishi H, Abe K, Abe Y, Mizutani T, Miyamoto K. A role of insulin-like growth factor I for follicle-stimulating hormone receptor expression in rat granulosa cells. Biol Reprod 2000 62:325-333[Abstract/Free Full Text]
  48. Wang Y, Ge W. Developmental profiles of activin ßA, ßB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle. Biol Reprod 2004 71:2056-2064[Abstract/Free Full Text]
  49. Selman K, Wallace RA, Sarka A, Qi X. Stages of oocyte development in the zebrafish, Brachydanio rerio. J Morphol 1993 218:203-224[CrossRef]
  50. Dufau ML. The luteinizing hormone receptor. Annu Rev Physiol 1998 60:461-496[CrossRef][Medline]
  51. Ascoli M, Fanelli F, Segaloff DL. The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr Rev 2002 23:141-174[Abstract/Free Full Text]
  52. Simoni M, Gromoll J, Nieschlag E. The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 1997 18:739-773[Abstract/Free Full Text]
  53. Kubokawa K, Ishii S. Receptors for native gonadotropins in amphibian liver. Gen Comp Endocrinol 1987 68:260-270[Medline]
  54. Abdallah MA, Lei ZM, Li X, Greenwold N, Nakajima ST, Jauniaux E, Rao Ch V. Human fetal nongonadal tissues contain human chorionic gonadotropin/luteinizing hormone receptors. J Clin Endocrinol Metab 2004 89:952-956[Abstract/Free Full Text]
  55. Lei ZM, Rao CV, Kornyei JL, Licht P, Hiatt ES. Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain. Endocrinology 1993 132:2262-2270[Abstract]
  56. Pabon JE, Li X, Lei ZM, Sanfilippo JS, Yussman MA, Rao CV. Novel presence of luteinizing hormone/chorionic gonadotropin receptors in human adrenal glands. J Clin Endocrinol Metab 1996 81:2397-2400[Abstract]
  57. Pang Y, Ge W. Activin stimulation of zebrafish oocyte maturation in vitro and its potential role in mediating gonadotropin-induced oocyte maturation. Biol Reprod 1999 61:987-992[Abstract/Free Full Text]
  58. Pang Y, Ge W. Gonadotropin and activin enhance maturational competence of oocytes in the zebrafish (Danio rerio). Biol Reprod 2002 66:259-265[Abstract/Free Full Text]
  59. Pang Y, Ge W. Epidermal growth factor and TGF{alpha} promote zebrafish oocyte maturation in vitro: potential role of the ovarian activin regulatory system. Endocrinology 2002 143:47-54[Abstract/Free Full Text]
  60. Wang Y, Ge W. Cloning of zebrafish ovarian carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase and characterization of its spatial and temporal expression. Gen Comp Endocrinol 2002 127:209-216[CrossRef][Medline]
  61. Wang Y, Ge W. Gonadotropin regulation of follistatin expression in the cultured ovarian follicle cells of zebrafish, Danio rerio. Gen Comp Endocrinol 2003 134:308-315[CrossRef][Medline]
  62. Wang Y, Ge W. Involvement of cyclic adenosine 3',5'-monophosphate in the differential regulation of activin ßA and ßB expression by gonadotropin in the zebrafish ovarian follicle cells. Endocrinology 2003 144:491-499[Abstract/Free Full Text]
  63. Wang Y, Ge W. Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes. Biol Reprod 2003 69:1998-2006[Abstract/Free Full Text]
  64. Wang Y, Ge W. Cloning of epidermal growth factor (EGF) and EGF receptor from the zebrafish ovary: evidence for EGF as a potential paracrine factor from the oocyte to regulate activin/follistatin system in the follicle cells. Biol Reprod 2004 71:749-760[Abstract/Free Full Text]
  65. Wang Y, Ge W. Cloning of zebrafish ovarian P450c17 (CYP17, 17{alpha}-hydroxylase/17, 20-lyase) and characterization of its expression in gonadal and extra-gonadal tissues. Gen Comp Endocrinol 2004 135:241-249[CrossRef][Medline]
  66. Wang Y, Wong AO, Ge W. Cloning, regulation of messenger ribonucleic acid expression, and function of a new isoform of pituitary adenylate cyclase-activating polypeptide in the zebrafish ovary. Endocrinology 2003 144:4799-4810[Abstract/Free Full Text]
  67. Kohli G, Hu S, Clelland E, Di Muccio T, Rothenstein J, Peng C. Cloning of transforming growth factor-ß1 (TGF-ß1) and its type II receptor from zebrafish ovary and role of TGF-ß1 in oocyte maturation. Endocrinology 2003 144:1931-1941[Abstract/Free Full Text]
  68. Wu T, Patel H, Mukai S, Melino C, Garg R, Ni X, Chang J, Peng C. Activin, inhibin, and follistatin in zebrafish ovary: expression and role in oocyte maturation. Biol Reprod 2000 62:1585-1592[Abstract/Free Full Text]
  69. Li MD, Ford JJ. A comprehensive evolutionary analysis based on nucleotide and amino acid sequences of the {alpha}- and ß-subunits of glycoprotein hormone gene family. J Endocrinol 1998 156:529-542[Abstract]



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