Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica

General and Comparative Endocrinology 154 (2007) 161–173 www.elsevier.com/locate/ygcen

Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica Shan-Ru Jeng

a,*

, Wen-Shiun Yueh a, Guan-Ru Chen b, Yan-Horn Lee c, Sylvie Dufour d, Ching-Fong Chang e,*

a

Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung 811, Taiwan Fisheries Research Institute, Freshwater Aquaculture Research Center, Lukang 505, Taiwan c Tungkang Biotechnology Research Center, Fisheries Research Institute, Council of Agriculture, Tungkang 928, Taiwan USM 0401, UMR 5178 CNRS/MNHN/UPMC Biologie des Organismes Marins et Ecosyste`mes, De´partement des Milieux et Peuplements Aquatiques, Muse´um National d’Histoire Naturelle, 7, rue Cuvier, 75231, Paris Cedex 05, France e Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan b

d

Received 13 February 2007; revised 14 May 2007; accepted 15 May 2007 Available online 25 May 2007

Abstract Eel species have a striking life cycle with a blockade of puberty until the oceanic migration. We report the first molecular data on eel gonadotropin receptors. The partial sequences cloned covered two-third of the open reading frame and included most of the extracellular and transmembrane domains. Phylogenetic analysis partitioned the two eel gonadotropin receptors into the two teleost FSHR and LHR clusters, respectively. Real-time quantitative RT-PCR was used to quantify the expression of eel gonadotropins and their receptors. Similar levels of pituitary FSH-b and LH-b transcripts were found in the immature previtellogenic female eels. In contrast, ovarian FSHR mRNA level was at 100- to 185-fold higher than that of LHR. This revealed that FSHR rather LHR would mediate gonadotropin stimulation of the early stages of ovarian growth. Chronic treatment with fish pituitary homogenates, applied to induce eel sexual maturation, stimulated pituitary LH-b but suppressed FSH-b transcripts. In the ovaries, both FSHR and LHR mRNA were significantly increased in experimentally matured eels. Treatments with sexual steroids showed a stimulatory effect of estradiol-17b (E2) on pituitary LH-b mRNA levels, while FSH-b transcripts were suppressed by E2 or testosterone (T). In contrast, neither E2 nor T-treatment had any significant effect on ovarian FSHR nor LHR transcripts. This suggests that steroid feedbacks may be responsible for the opposite regulation of pituitary gonadotropins in experimentally matured eels, but are not involved in the regulation of gonadotropin receptors. In conclusion, these are the first data on the sequence, expression and regulation of gonadotropin receptors in the eel. They provide new foundation for basic and applied research on eel reproduction.  2007 Elsevier Inc. All rights reserved. Keywords: Eel; FSH; Gonadotropin receptors; Induced maturation; LH; Sex steroids

1. Introduction Gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are secreted by pituitary and act through binding to their specific receptors (FSHR and LHR) in the gonads of vertebrates, to induce steroidogen*

Corresponding authors. Fax: +886 2 2462 1579 (C.-F. Chang). E-mail addresses: [email protected] (S.-R. Jeng), [email protected] (C.-F. Chang). 0016-6480/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2007.05.026

esis and gametogenesis (Nagahama et al., 1995; Simoni et al., 1997; Dufau, 1998). FSH, LH and thyroid-stimulating hormone (TSH) are members of the pituitary glycoprotein hormone family, they are heterodimers with two subunits, a common a-subunit and a hormone specific b-subunit. It is believed that FSH and LH in teleosts, as in mammals, have different functions and expression patterns at different stages of the reproductive cycle (Swanson et al., 1991; Prat et al., 1996; Yaron et al., 2001). FSH is involved

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in the control of puberty and gametogenesis, whereas LH mainly regulates final gonadal maturation and spawning (Prat et al., 1996; Schulz and Miura, 2002). Accordingly, the expression pattern of FSH-b was much higher than LH-b at pre-gametogenesis and early gametogenetic stage, whereas the expression level of LH-b mRNA was significantly increased at the end of reproductive cycle in rainbow trout (Oncorhynchus mykiss) (Gomez et al., 1999). However, in European sea bass (Dicentrarchus labrax), glycoprotein-a, FSH-b, LH-b mRNA levels were increased in parallel to the gonadosomatic index (GSI) during spermatogenesis (Mateos et al., 2003). This suggested important variations among teleost species, in the expression profiles and potential roles of gonadotropins during gametogenesis. Gonadotropin receptors, FSHR, LHR as well as thyrotropin receptor, TSHR are membrane-bound receptors belonging to the superfamily of G-protein-coupled receptors, which contain seven transmembrane domains (TMD). TSHR, FSHR and LHR have a very large extracellular domain (ECD); different from the other members of G-protein-coupled receptor family, and constitute a subfamily of glycoprotein hormone receptors (Kohn et al., 1995; Simoni et al., 1997; Dufau, 1998). FSHR and LHR cDNA were isolated from gonads in several teleosts (amago salmon, Oncorhynchus rhodurus: Oba et al., 1999a,b; tilapia, Oreochromis niloticus: Hirai et al., 2000; channel catfish, Ictalurus punctatus: Kumar et al., 2001a,b; African catfish, Clarias gariepinus: Bogerd et al., 2001; Vischer and Bogerd, 2003; zebrafish, Danio rerio: Kwok et al., 2005). The FSHR in coho salmon (Oncorhynchus kisutch) was shown to be localized in the thecal cells and intensively on granulosa cells in the vitellogenic ovary, in the thecal cells but not in the granulose cells in the preovulatory ovary (Miwa et al., 1994). The FSHR was mostly localized in the Sertoli cells but not in the Leydig cells of male coho salmon at all stages of spermatogenesis (Miwa et al., 1994). In contrast, LHR was highly expressed in the granulosa cells of preovulatory follicles and Leydig cells of the testis of maturing coho salmon (Miwa et al., 1994). There were few studies about the expression of FSHR or LHR during reproductive cycles. In zebrafish, FSHR expression level significantly increased with the oocytes entering vitellogenesis and fell at the full-grown or postvitellogenic stage. In contrast, the expression of LHR reached its peak level at the full-grown stage (Kwok et al., 2005). The FSHR transcripts in channel catfish showed an increase around the time of onset of ovarian recrudescence and a decrease prior to spawning; LHR transcripts remained low during most of spawning cycle and then increased around the time of spawning (Kumar et al., 2001a,b). These results were similar to the expression of FSHR and LHR during ovarian cycle in salmonids. Indeed, the FSHR and LHR profiles during oogenesis were consistent with the profiles of FSH and LH in salmonids (Prat et al., 1996; Oba et al., 1999a,b).

Japanese eel is a catadromous fish with striking life cycle and mature eels have not been found in wild or under captive condition. Before the oceanic reproductive migration or under captive condition, the eels remain at a prepubertal stage; they have immature gonads containing previtellogenic oocytes in the females (GSI < 2%) and spermatogonia in the males (GSI < 0.1%) due to a lack of gonadotropin synthesis and release (Fontaine and Dufour, 1991; Nagae et al., 1996; Dufour et al., 2003). Up to now, the larvae for eel farms must rely on catch from the wild, although glass eels could be produced in captivity recently (Tanaka et al., 2003). Since the natural eel stocks are dramatically declining (Stone, 2003), further basic and applied knowledge on regulatory mechanisms of eel reproduction are required to allow for the artificial induction of eel reproduction. Eel gonadal growth, steroidogenesis and gametogenesis, can be induced by gonadotropic treatments, using chronic injections of human chorionic gonadotropin in male and fish pituitary extracts in the female (European eel: Fontaine, 1936; Fontaine et al., 1964; Dufour et al., 1989; Pedersen, 2003, 2004; Japanese eel: Yamamoto and Yamauchi, 1974; Miura et al., 1991a,b, 2002; Ijiri et al., 1995; Tanaka et al., 1997; Jeng et al., 2002). The gonadal response to the exogenous gonadotropins is related to the abundance of gonadotropin receptors. The eels, as other teleosts, have two types of gonadotropins, and the glycoprotein-a, FSH-b and LH-b cDNA have been cloned (European eel: Que´rat et al., 1990; Degani et al., 2003; Japanese eel: Nagae et al., 1996; Yoshiura et al., 1999). The FSH-b transcripts were reported to be highly expressed in the pituitary of immature eels while LH-b transcripts could only be detected at the late vitellogenic stage and germinal vesicle migration stage (Japanese eel: Nagae et al., 1996; Yoshiura et al., 1999; European eel: Degani et al., 2003). However, recent studies from our group indicated that both gonadotropins are already expressed in the pituitary of immature previtellogenic eels (European eel: Schmitz et al., 2005; Aroua et al., 2005). Concerning the expression of gonadotropin receptors, FSHR and LHR, in the eel, no data are yet available, while they are of major importance for deciphering the regulatory mechanisms of eel ovarian development and improving the protocols for induced reproduction. The present study reports the first data on eel LH and FSH receptors. After partial cloning of Japanese eel LHR and FSHR, we measured their ovarian expression by real-time quantitative PCR and investigated the variations of their expression during induced gonadal development and after sex steroid treatments. Real-time quantitative PCR analyses were also performed in the same experimental fishes to measure the pituitary expression of the eel gonadotropin subunits (glycoprotein-a, FSH-b, LH-b) in order to compare the differential regulations of the expression of gonadotropins and their receptors.

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2. Materials and methods

2.1. Experimental fish Female eels, Anguilla japonica, were obtained from aquaculture farm in Taiwan (8-year-old eels in the Exp. 1 and 3-year-old fish in the Exps. 2–4). All the experimental fish were acclimated to a pond at the University (National Kaohsiung Marine University, Taiwan) culture station in a 2.5ton freshwater and natural light system. No clear effects of age difference (8-year-old versus 3-year-old) in the induced maturation were observed (unpublished data). Also, it is difficult to obtain enough 8-year-old eels in a same batch. Therefore, 3-year-old eels were used in the Exps. 2–4. The water temperatures ranged from 20 to 27 C. The fish were handled in an appropriate way to fulfil the ethical procedures of animal experimentation.

2.2. Tissue sampling procedures The fish were anaethesized in 2-phenoxyethanol before sacrificed. The ovary and pituitary were quickly dissected and weighed. Samples were frozen in liquid nitrogen and stored at 80 C until radioimmunoassay (RIA) or real-time quantitative PCR analysis. Total body weight (BW) and ovarian weight were measured for the calculation of gonadosomatic index (GSI) (gonad weight/BW · 100). A piece of gonad was fixed in a Bouin’s solution, embedded in paraffin, and sectioned at 6 lm. Transverse sections were stained with hematoxylin and eosin (Chang and Yueh, 1990).

2.3. Gonadotropic treatment Eels received a chronic treatment with salmon pituitary extracts, in order to induce ovarian development as previously described (Yamamoto and Yamauchi, 1974; Tanaka et al., 1997; Jeng et al., 2002). Salmon pituitary was commercially collected from wild salmon in North America during the reproductive season and sold by King Yorker (Taipei, Taiwan). Pituitaries were homogenized with a saline solution and centrifuged to obtain the supernatant (pituitary extract). Our preliminary data showed that plasma E2 levels in the control and pituitary-treated eels were 212 ± 60 pg/ml and 1064 ± 98 pg/ml, respectively, according to E2 RIA when fish plasma was collected from 3 days after the last injection. 2.3.1. Experiment 1 Eight-year-old female eels (n = 14, BW = 1586.4 ± 140.2 g, body length (BL) = 92.1 ± 1.8 cm; GSI = 1.7 ± 0.2) were divided into 2 groups: control (n = 6) and treatment (n = 8). The treated fish received weekly intraperitoneal (ip) injections of 12 mg salmon pituitary homogenates in 0.5 ml saline/fish for 14 weeks. The control fish were injected with saline alone. Fish were sacrificed 3 days after the last injection. Pituitary was collected and stored at 80 C until LH RIA. 2.3.2. Experiment 2 Three-year-old female eels (n = 22, BW = 753.2 ± 19.2 g, BL = 74.2 ± 0.9 cm; GSI = 0.77 ± 0.12) were divided into 2 groups: control (n = 8) and treatment (n = 14). The treated fish were injected ip weekly with 10 mg salmon pituitary homogenates in 0.5 ml saline/fish for 17 weeks. The control fish were injected with saline alone. Fish were sacrificed 3 days after the last injection. Pituitary and ovary were collected and stored at 80 C for the measurement of mRNA levels of gonadotropin subunits and gonadotropin receptors by real-time quantitative PCR.

2.4. Sex steroid treatments Chronic treatments with sex steroids were performed according to the protocols previously described (Jeng et al., 2002). Estradiol-17b (E2) or testosterone (T) treatments were administered (by injection) since the production of both estrogens and androgens by the eel ovaries has been shown to be stimulated during induced ovarian development (Ijiri et al., 1995). E2 and T were purchased from Sigma and dissolved in coconut

163

oil (Sigma–Aldrich Corp., St. Louis, MO). Our preliminary data showed that plasma E2 levels in the control and E2 treated eels were 212 ± 60 pg/ml (control), 928 ± 325 pg/ml (low E2-treated), 860 ± 136 pg/ml (high E2-treated), respectively, according to E2 RIA when fish plasma was collected from 1 week after the last injection. Plasma E2 concentrations in eels with E2-injection were similar to the levels in pituitaryinduced maturation eels (Exp. 1). 2.4.1. Experiment 3 Three-year-old female eels (n = 30, BW = 765 ± 27.5 g, BL = 76 ± 0.9 cm; GSI = 0.96 ± 0.01) were divided into 5 groups (n = 6 eels per group). Treated eels received ip injections of 0.75 mg or 3.75 mg steroid (T or E2)/kg BW. Control eels received injections of coconut oil alone. Injections were given for 3 times at a 2-week interval. Fish were sacrificed 1 week after the last injection. Pituitary was collected and stored at 80 C until LH RIA. 2.4.2. Experiment 4 Three-year-old female eels (n = 37, BW = 746.5 ± 79 g, BL = 85.6 ± 3.3 cm; GSI = 0.77 ± 0.12) were divided into 5 groups (n = 7 or 8 eels per group) and treated with steroids as described above. Injections were given for 6 times at 1-week interval. Fish were sacrificed 1 week after the last injection. Pituitary and ovary were collected and stored at 80 C for the measurement of mRNA levels of gonadotropin subunits and gonadotropin receptors by real-time quantitative PCR.

2.5. Radioimmunoassay of LH Individual pituitaries were extracted by sonication (VC 130PB, Sonics & Materials, Inc., Newtown, CT, USA) in 0.5 ml saline. Pituitary LH content was assayed by radioimmunoassay (RIA) previously established for the b-subunit of carp (Cyprinus carpio) LH and validated for the measurement of European eel LH (Dufour et al., 1983a). Iodination of carp LH-b was performed using the iodogen (Pierce) method according to Salacinski et al. (1981). Primary antiserum (antiserum against carp LH-b) was used at the final dilution of 1/75,000. Serial dilutions of Japanese eel pituitary extracts gave displacement curves parallel to the European eel LH standard curve. Results are expressed in ng LH standard/pituitary.

2.6. Cloning of eel FSHR and LHR Total RNA from ovarian tissues of eels which were induced with salmon pituitary homogenates (as described above), was extracted by homogenization in Trizol reagent (Gibco BRL, grand Island, NY). Reverse transcription (RT) was performed using Superscript II (Gibco BRL) with oligo (dT)12–18 primers under the following conditions: 42 C for 50 min, 37 C for 15 min, and 70 C for 15 min. The degenerated primers (Table 1) were based on the nucleotide sequences of tilapia, salmon and catfish gonadotropin receptors (Hirai et al., 2000; Oba et al., 1999a,b; Kumar et al., 2001a,b) and the polymerase chain reaction (PCR) assay was carried out for FSHR and LHR cloning.

2.7. Quantification of glycoprotein-a, FSH-b, LH-b, FSHR and LHR mRNA levels using real-time quantitative PCR analysis Absolute quantification with real-time quantitative PCR analysis was modified from the methods and principles previously reported (Bustin, 2000). 2.7.1. Standards Partial sequences of Glycoprotein-a (255 bp), FSH-b (315 bp) and LHb (310 bp) cDNA were cloned from total RNA of Japanese eel pituitaries by RT-PCR using specific primers designed from the published sequences of Japanese eel gonadotropins (Nagae et al., 1996; Yoshiura et al., 1999; GenBank Accession Nos.: glycorprotein-a, AB 175834; FSH-b, BAA 36546; LH-b, BAD 14302). The obtained sequences were 100% identical to the published sequences.

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Table 1 Nucleotide sequence of the primers used for gonadtropins (GtHs, FSH and LH) and gonadotropin receptors (GtHRs, FSHR and LHR) cloning and for real-time quantitative PCR (F, forward primer; R, reversed primer) Primers:

Nucleotide sequence

Primers for cloning GtHR-F1 5 0 -GCNGANABRGCRSARAANGARATVGG NGCCATRCA-3 0 GtHR-F2 5 0 -TAGTACTGGCGGCGGGAG-3 0 GtHR-R1 5 0 -GCNGAYGMBTTYAAYCCBTGYGAR G-3 0 GtHR-R2 5 0 -TGAGCATMTSCAACACKGG-3 0 R = A + G, Y = C + T, K = T + G, S = C + G, V=A+C+G B = T + C + G, M = A + C, N = A + T + G + C Primers for real time PCR Glycoprotein-a-F 5 0 -TGGTGTGTCCAGGAAAG-3 0 FSH-b-F 5 0 -GCGGTG GTGTTGAAGGTGAT-3 0 LH-b-F 5 0 -GCGTGGATCCCCATGTGA-3 0 FSHR-F 5 0 -GTCGGTCTACACACTCACCATGA-3 0 LHR-F 5 0 -GCTGATGTCTTTGCTGAGTGGAG-3 0 Glycoprotein-a-R 5 0 -TGGTAGTAGCAGGTGCT-3 0 FSH-b-R 5 0 -CAGTTGTGGTGTCGCCAACAT-3 0 LH-b-R 5 0 -ACTCTGGATGGCGCAGTCA-3 0 FSHR-R 5 0 -CTTCCGGTCCAGCTGCAT-3 0 LHR-R 5 0 -GGAGGTCTATGCTGCCGTAGG-3 0

Linear plasmid DNAs containing the respective inserts of glycoprotein-a (255 bp), FSH-b (315 bp), LH-b (310 bp), FSHR (770 bp; see Section 3) and LHR (800 bp; see Section 3) in the pGEM-T Easy vector (Promega) were obtained by cutting with SalI (New England BioLabs, Beverly, MA). In vitro transcription was conducted in a mixture of rNTP mix, DTT, Sal I single-digested plasmid and T7 polymerase (Promega) to obtain standard RNA. Different concentrations of RNA (1, 0.1, 0.01, 0.001, 0.0001, 0.00001 and 0.000001 lg) were prepared and the respective cDNAs were synthesized using a random primer (hexamers) and Superscript II RT (Gibco BRL) for the preparation of standard curve. 2.7.2. Samples Total RNA from individual pituitaries or ovarian tissue samples were extracted as described above. One microgram of sample total RNA was prepared and cDNAs were synthesized using Superscript II RT (Gibco BRL) with oligo (dT)12–18 primers to analyze the mRNA expression levels. 2.7.3. Primers Specific primers were designed for the real-time quantitative PCR for Glycoprotein-a, FSH-b, LH-b, FSHR and LHR (Table 1).

2.7.4. Real- time quantitative PCR For real-time PCR quantification, gene quantification of standards and samples was simultaneously conducted by a real-time quantitative PCR (GeneAmp 5700 Sequence Detection System; Applied Biosystems, Foster City, CA) using SYBR GREEN PCR Master Mix (Applied Biosystems) which containing SYBR green I as a double-strand DNA minorgroove binding dye. The real-time quantitative PCR was evaluated according to the following criteria. The correlation of the standard curve was 0.999. The values detected from different amounts of RNA (1, 0.1, 0.01, and 0.001 lg) from the representative samples were parallel with the respective standard curve. A single and highly similar dissociation curve was obtained in standard and samples in the reaction. The obtained PCR products (with realtime quantitative PCR primers) from the standard or sample templates had the identical size as compared to the expected size according to the known cDNA sequence according to the separation by a multi-channel capillary electrophoresis system (HAD-GT12, eGene Inc., Irvine, CA). Our preliminary data showed that the changes in the expression of the house-keeping genes such as b-actin and glyceraldehydes-3-phosphate dehydrogenase were highly significant in eels with exogenous treatments. Therefore, in order to compare the quantity and changes of the transcripts among genes or various treatments, absolute values (ng standard transcripts/lg total RNA) calculated from the standard curve were chosen in these experiments.

2.8. Data analysis The Clustal W 1.83 multiple sequence alignment was used for sequence analysis and comparisons; the Phylip 3.573 was used for phylogenetic analysis. Data from RIA or from real-time quantitative PCR are expressed as means ± SEM (n = 6 to 14 eels per group, according to the experiment). The values were subjected to a one-way ANOVA to test significance (P < 0.05, 0.01) followed by a Duncan multiple-range test.

3. Results 3.1. Partial cloning of eel FSHR and LHR Putative A. japonica FSHR (GenBank Accession AY742794, 1554 bp) and LHR (GenBank Accession AY742795, 1398 bp) were partially cloned. Phylogenetical analyses of the obtained sequences in comparison with sequences of FSHR, LHR and TSHR from other teleosts and other vertebrates, indicated that these eel receptors corresponded to the respective clusters of teleost FSHR and teleost LHR (Fig. 1). Eel FSHR and LHR were clearly distinct from the teleost TSHR cluster (Fig. 1). The sequences of FSHR and LHR in Japanese eel covered 78% (133rd–655th amino acid sequence) of FSHR and

c Fig. 1. Unrooted phylogenetic tree of glycoprotein hormone receptors, FSHR, LHR and TSHR. The phylogenic tree was constructed using the Phylip 3.573.The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of 1000.00 trees. Comparisons were made with the FSHR amino acid sequences (GenBank Accession No. between brackets): chicken (NP-990410), duck (AAM34793), newt (BAB13501), lizard (CAC82173), Japanese eel (AAU90017), zebrafish (AAR84280), African catfish (CAB51907), channel catfish (AAK16067), Nile tilapia (BAB16106), European sea bass (AAV48628), gilthead seabream (AAT01413), Atlantic salmon (CAD98923), rainbow trout (AAQ04551), cattle (NP-776486), human (AAR07899), rat (AAA41175), mouse (AAC67559); the LHR amino acid sequences: zebrafish (AAR84281), African catfish (AAN75752), rat (AAB22682), mouse (NP-038610), human (NP-000224), cattle (AAC24012), chicken (NP-990267), Japanese eel (AAU90018), Turkey (AAB64409), Nile tilapia (BAB16107), channel catfish (AAK16066), gilthead seabream (AAT01412), European sea bass (AAV48629), Atlantic salmon (CAE30288), rainbow trout (AAQ04550); the TSHR amino acid sequences of human (AAR07906): monkey (AAO11782), rat (NP-037020), cat (AAK00133), dog (NP-001003285), cattle (NP-776631), sheep (NP-001009410), chicken (BAE44410), channel catfish (AAS45557), African catfish (AAN01360), striped sea bass (AAF80596), tilapia (BAB39132), amago salmon a (BAB07801), and amago salmon b (BAB07800).

S.-R. Jeng et al. / General and Comparative Endocrinology 154 (2007) 161–173

66% (124th–597th amino acid sequence) of LHR as compared to the full open reading frame in zebrafish (total 668 amino acid in FSHR, total 708 amino acid in LHR). They corresponded to 61% (FSHR) and 65% (LHR) of

the extracellular domain (ECD), and 100% (FSHR) and 84% (LHR) of the transmembrane domain (TMD) compared with the full length sequences of zebrafish. Comparisons between amino acid sequences of putative eel FSHR cattle sheep

100% 95.5% 96.1% 74.7%

100%

100%

chicken duck lizard snake

100% 40.8%

33.5%

pig horse human monkey

rat mouse

100% 100%

165

100%

FSHR

newt 100% 99.9%

100% 100% 92.8%

51.3% 100%

European sea bass gilthead seabream tilapia rainbow trout amago salmon Atlantic salmon African catfish

100%

channel catfish zebrafish 51.5% Japanese eel European sea bass 98.5% gilthead seabream 100% rainbow trout 100% amago salmon 100% Atlantic salmon 100% zebrafish 70.7% 98.7% African catfish Japanese eel 57.7% 100% tilapia 100% channel catfish 100%

100%

100%

100% 100% 100%

100%

100% 27.2% 100%

chicken

100%

rat 91.6%

37.4% 100%

1000

monkey human

LHR

cattle 92.5% 98.3% pig 95.8% horse human 92.3% mouse 100% 100% rat sheep turkey 100% chicken frog

dog cat sheep cattle

tilapia striped sea bass amago salmon b amago salmon a channel catfish African catfish

TSHR

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Table 2 Amino acid identities of Japanese eels FSHR and LHR of other species Species

Overall (%)

Receptor domain

Accession No.

ECD (%)

TMD (%)

Human (Homo sapiens) House mouse (Mus musculus) Chicken (Gallus gallus) Newt (Cynops pyrrhogaster) Lizard (Podarcis sicula) African catfish (Clarias gariepinus) Channel catfish (Ictalurus punctatus) Zebrafish (Danio rerio) Salmon (Salmon salar) Rainbow trout (Oncorhynchus mykiss) Tilapia (Oreochromis niloticus) Gilthead seabream (Sparus aurata)

FSHR 59 60 61 60 56 68 70 74 64 64 66 63

47 48 45 47 45 65 66 65 56 56 54 53

74 72 75 75 70 74 75 82 74 73 77 75

AAR07899 AAC67559 NP-990410 BAB13501 CAC82173 CAB51907 AAK16067 AAR84280 CAD98923 AAQ04551 BAB16107 AAT01413

Human (Homo sapiens) House mouse (Mus musculus) Chicken (Gallus gallus) Newt (Cynops pyrrhogaster) Lizard (Podarcis sicula) African catfish (Clarias gariepinus) Channel catfish (Ictalurus punctatus) Zebrafish (Danio rerio) Salmon (Salmon salar) Rainbow trout (Oncorhynchus mykiss) Tilapia (Oreochromis niloticus) Gilthead seabream (Sparus aurata)

LHR 56 57 55 — — 62 60 62 58 58 58 60

46 47 47 — — 59 48 59 54 54 47 55

66 69 68 — — 65 76 65 64 63 70 65

NP-000224 NP-038610 NP-990267 — — AAN75752 AAK16066 AAR84281 CAE30288 AAQ04550 BAB16107 AAT01412

ECD, extracellular domain; TMD, transmembrane domain; ‘‘—’’, no data available.

and LHR with those of other vertebrates are shown in Table 2. Eel FSHR showed 56–74% identities and eel LHR 55–62%, as compared to other vertebrates (Table 2). Eel FSHR ECD shared 53–66% of identity with FSHR from other teleosts and 47–48% identity with those of mammals (Table 2). Eel FSHR TMD had 73–82% of identity with FSHR from other teleosts and 72–74% of identity with mammalian ones (Table 2). Eel LHR ECD had 47– 59% of identity to other teleosts and 46–47% of identity to mammals (Table 2). Eel LHR TMD had 63–76% identity to other teleosts and 66–69% of identity to mammals (Table 2). 3.2. Expression of gonadotropins and their receptors in control eels Measurement by real-time quantitative PCR of ovarian gonadotropin receptor transcripts revealed that the absolute amount of ovarian FSHR mRNAs in control eels (Exp. 2: 0.0962 ± 0.0169 ng/lg ovarian total RNA) was 100-fold higher than that of LHR (0.957 ± 0.189 pg/lg ovarian total RNA). Similar results were obtained in control eels from another independent experiment (Exp. 4) with mean ovarian level of FSHR mRNAs being 185-fold more elevated than that of LHR. Concerning the expression of pituitary gonadotropins, all three subunits were detectable in individual pituitaries

from all control eels. The levels of FSH-b subunit and LH-b subunit were similar (Exp. 2, FSH-b = 0.42 ± 0.11 ng/lg pituitary total RNA, LH-b = 0.53 ± 0.16 ng/ lg pituitary total RNA; Exp. 4, FSH-b = 0.46 ± 0.18 ng/ lg pituitary total RNA, LH-b = 0.55 ± 0.16 ng/lg pituitary total RNA), while the levels of a subunit were higher (Exp. 2: 11.8 ± 0.91 ng/lg pituitary total RNA; Exp. 4: 18.5 ± 3.02 ng/lg pituitary total RNA). 3.3. Effect of gonadotropic treatment Chronic treatments with fish pituitary homogenates significantly stimulated ovarian growth (Exp. 1: GSI = 20.4 ± 3.3 in treated eels versus 1.7 ± 0.2 in controls; Exp. 2: GSI = 12.8 ± 1.5 in treated eels versus 0.77 ± 0.12 in controls). Histological analysis of the ovaries showed the induction of large vitellogenic oocytes in experimentally matured eels (oocyte diameter = 0.5–1 mm; presence of numerous yolk globules in the ooplasma) as compared to the previtellogenic oocytes (no visible yolk globules; oocyte diameter = 0.1 mm) (Guraya, 1986) in the controls. Measurement of LH content in the pituitary by RIA (Exp. 1) revealed a large and significant increase in pituitary LH content (245-fold; P < 0.01) after injection with fish pituitary homogenates as compared to control eels (Fig. 2A).

S.-R. Jeng et al. / General and Comparative Endocrinology 154 (2007) 161–173 72400

∗∗

A

50

72200

20

10

Pituitary Extracts

Control

0

B

Control E2

d

2500

T 2000 1500 1000 500

c a

Control

b

b

0.75mg/kg

3.75mg/kg

Steroid treatment Fig. 2. Effect of experimental maturation (A) and steroid treatments (B) on LH pituitary content in female Japanese eels, as measured by radioimmunoassay. (A) Eight-year-old cultivated female eels received weekly injections of salmon pituitary homogenates for 14 weeks for inducing maturation. Control eels were injected with saline alone (Exp. 1). Results are expressed as ng LH standard/pituitary. Means are given ±SEM (n = 6 eels per group). *Significant difference between the two groups (P < 0.01). (B) Three-year-old cultivated female eels received two injections per week of estradiol-17b (E2) or testosterone (T) (0.75 or 3.75 mg in coconut oil/kg BW) for 3 weeks (Exp. 3). Control eels were injected with coconut oil alone. Results are expressed as ng LH standard/ pituitary. Means are given ±SEM (n = 6 eels per group). Different letters represent significant differences among groups (P < 0.05).

mRNA levels (ng/ug pituitary total RNA)

LH contents (ng/pituitary)

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400 300 200 100 0

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B FSH-β

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50

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1

Measurement of gonadotropin subunit gene expression in the pituitary by real-time quantitative PCR (Exp. 2), showed that treatment with fish pituitary homogenates significantly stimulated LH-b transcript levels (80-fold; P < 0.01; Fig. 3C). Glycoprotein-a transcripts were also slightly but significantly increased in treated eels (2-fold; P < 0.05; Fig. 3A). In contrast, FSH-b transcripts were decreased (0.73-fold; P < 0.05; Fig. 3B). Measurement by real-time quantitative PCR of ovarian gonadotropin receptor transcripts indicated that both FSHR and LHR mRNA were significantly increased (6fold for FSHR P < 0.01 and 2.2-fold for LHR; P < 0.05) in eels treated with fish pituitary homogenates (Fig. 4A and B). 3.4. Effect of sex steroid treatments No or moderate increase in GSI was observed after treatment with sex steroids (E2 and T) with GSI varying from 0.96 to 1.7 in Exp. 1 and from 0.6 to 1.0 in Exp. 3.

0

Control

Pituitary Extracts

Fig. 3. Effect of experimental maturation on pituitary glycoprotein-a (A) FSH-b (B) and LH-b mRNA levels (C) in female Japanese eels, as measured by real-time quantitative PCR. Three-year-old cultivated female eels were weekly treated with salmon pituitary homogenates for 17 weeks (Exp. 2). Control eels were injected with saline alone (Exp. 2). Results (absolute mRNA levels) are expressed as ng gonadotropin subunit mRNA/lg pituitary total RNA). Means are given ±SEM (n = 6 eels per group). Significant difference between the two groups: *P < 0.05 and **P < 0.01.

Gonadal histology showed that oocytes remained at the previtellogenic lipid droplet stage in steroid-treated eels as in controls. No induction of vitellogenin incorporation in the oocyte could be observed (no yolk stage) in the gonad of steroid-treated eels. Pituitary LH content, as measured by RIA (Exp. 3) was significantly stimulated by E2 injection in a dose-dependent

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A FSHR

**

0.8

mRNA levels (ng/ug ovarian total RNA)

0.6

0.4

high doses of T also significantly suppressed (0.9-fold, P < 0.05) FSH-b mRNA levels (Fig. 5B). Measurement by real-time quantitative PCR of ovarian gonadotropin receptor transcripts indicated that neither E2 nor T-treatments had any significant effect on ovarian FSHR (Fig. 6A) or LHR (Fig. 6B) transcripts as compared to the control eels. However, high dose of E2-treatment had slightly higher (2-fold, P = 0.05) LHR transcripts than the control (Fig. 6B).

0.2

0.0 0.005

40 35

B LHR

E2

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25

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20 0.003

15

a

a

10 0.002 5 0

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Pituitary Extracts

Fig. 4. Effect of experimental maturation on ovarian FSHR (A) and LHR (B) mRNA levels in female Japanese eels, as measured by real-time quantitative PCR. For treatment see legend of Fig. 3 (Exp. 2). Results (absolute mRNA levels) are expressed as ng gonadotropin receptor mRNA/lg ovarian total RNA). Means are given ±SEM (n = 6 eels per group). Significant difference between the two groups: *P < 0.05 and **P < 0.01.

manner (Fig. 2B), low dose of E2 inducing a 13-fold increase (P < 0.01) and high dose a 108-fold increase (P < 0.01), as compared to the controls (Fig. 2B). Low and high doses of T induced a significant increase in pituitary LH content (2.7-fold increase P < 0.05 and 4.5-fold increase P < 0.05 as compared to controls, respectively) but the maximal effect was much lower than that of E2 (P < 0.01) (Fig. 2B). Pituitary LH-b mRNA transcripts, as measured by realtime quantitative PCR (Exp. 4), were also significantly increased by E2 treatment in a dose-dependent manner 7fold in low dose (P > 0.05) and 26-fold (P < 0.05) in high dose of E2 as compared to controls (Fig. 5C). In contrast, LH-b transcripts remained at a similar level after low or high doses of T treatment as compared to the controls (Fig. 5C). Glycoprotein-a transcripts were not significantly changed by any dose of E2-treatment, while they were slightly but significantly decreased by low and high doses of T (0.56-fold, P < 0.05) (Fig. 5A). FSH-b transcripts were strongly and significantly decreased (0.9-fold, P < 0.05) by low and high doses of E2 treatment (Fig. 5B). Low and

mRNA levels (ng/ug pituitary total RNA)

0.001

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B FSH-β b

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16 12 8

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a

a

0.75mg/kg

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Steroid treatment Fig. 5. Effect of steroid treatments on on pituitary glycoprotein-a (A), FSH-b (B), and LH-b mRNA levels (C) in female Japanese eels, as measured by real-time quantitative PCR. Three-year-old cultivated female eels received weekly injections of estradiol-17b (E2) or testosterone (T) (0.75 or 3.75 mg in coconut oil/kg BW) for 6 weeks (Exp. 4). Control eels were injected with coconut oil alone. Results (absolute mRNA levels) are expressed as ng gonadotropin subunit mRNA/pituitary total RNA). Means are given ±SEM (n = 7 or 8 eels per group). Different letters represent significant differences among groups (P < 0.05).

S.-R. Jeng et al. / General and Comparative Endocrinology 154 (2007) 161–173 0.20

A FSHR

a

0.16

Control E2 T

a

mRNA levels (ng/ug ovarian total RNA)

0.12

a

a a

0.08 0.04 0.00 0.004

B LHR a

0.003

0.001

a

a

0.002

a

a

0.000

Control

0.75mg/kg

3.75mg/kg

Steroid treatment Fig. 6. Effect of steroid treatments on ovarian FSHR (A) and LHR (B) mRNA levels in female Japanese eels, as measured by real-time quantitative PCR. For treatments see legend of Fig. 5 (Exp. 4). Results (absolute mRNA levels) are expressed as ng gonadotropin receptor mRNA/lg ovarian total RNA). Means are given ±SEM (n = 7 or 8 eels per group. No difference was found between the groups (P > 0.05).

4. Discussion 4.1. Partial cloning of eel LH and FSH receptors We report here, for the first time, molecular data on eel gonadotropin receptors. Surprisingly, despite the increasing interest in eel reproduction, the ancient and increasing concern on eel reproduction, only a few studies, using binding of radio-labeled heterologous hormones, had been performed up to now to investigate eel ovarian receptors (European eel: Salmon et al., 1987). The partial sequences cloned covered 66–78% of the open reading frame and included most of the ECD and TMD, which are those involved in the recognition and binding of the corresponding hormones. The phylogenetic analysis based on these sequences partitioned the two Japanese eel gonadotropin receptors into the two monophyletic groups of teleost FSHR and LHR clusters, respectively. These phylogenic patterns were consistent with the previous analysis based on the amino acid sequence of a part of ECD (Maugars and Schmitz, 2006). As eels are representative species of one of the most ancient groups among teleosts (Elopomorphs), the present phylogenetic analysis also indicates an early differentiation between teleost LHR or FSHR clusters and their respective counterparts in Tetrapods.

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4.2. Differential expression of gonadotropin subunits and gonadotropin receptors in immature eels Evaluation of absolute amounts of eel gonadotropin subunit transcripts by real-time quantitative PCR revealed that similar levels of FSH-b and LH-b transcripts were expressed in the pituitary of immature previtellogenic female eels. Glycoprotein-a transcripts were at least 20-fold higher than gonadotropin-b subunit ones. This likely reflects the fact that glycoprotein-a is the common subunit of all pituitary glycoprotein hormones including not only FSH and LH but also TSH, which is more abundant than gonadotropins in immature eels (Salmon et al., 1993). The similar levels of FSH-b and LH-b transcripts, as measured by real-time quantitative PCR in immature female Japanese eels, are consistent with our previous studies in female European eels at the silver stage using dot blot assays and in situ hybridization (Schmitz et al., 2005; Aroua et al., 2005). In contrast, other previous studies, using Northern blot, reported that FSH-b was highly expressed in immature previtellogenic stage while LH-b could only be detected at the late vitellogenic and maturation stage of Japanese eel (Nagae et al., 1996; Yoshiura et al., 1999) and European eel (Degani et al., 2003). These discrepancies may come from the sensitivity of the methods, the degree of immaturity (Aroua et al., 2005) and also reflect individual variations in gonadotropin expression (Schmitz et al., 2005; Aroua et al., 2005). In any case, the expression of not only FSH but also LH in previtellogenic Japanese eels is further supported by the detection of the LH protein itself by RIA in each individual pituitary. This is also in agreement with our previous studies in the European eel (Aroua et al., 2005). The expression of both gonadotropins suggests that both FSH and LH may have potential roles during the early stage of gonadal growth. In contrast to pituitary gonadotropins, which appeared to be expressed at the same level, real-time quantitative PCR revealed that the ovary of immature eels contained much more (about 40-fold) transcripts for FSHR than for LHR. These are the first direct data on eel ovarian receptivity. More abundant transcripts for FSHR than LHR have also been indicated in the ovary of other immature teleosts such as salmon (Miwa et al., 1994), tilapia (Hirai et al., 2000), channel catfish (Kumar et al., 2001a,b). These data suggest that in the eel as in other teleosts, FSHR rather than LHR would play a major role in the mediation of gonadotropin stimulation of the early stages of ovarian growth. Very low expression of LHR may explain the difficulty of the successful induction in eel ovarian maturation. A few studies in some other teleosts have investigated the specificity of gonadotropin receptors, using homologous recombinant hormones and receptors. They suggested that LHR would have a stronger specificity than FSHR which shows a similar affinity to both LH and FSH (coho salmon: Yan et al., 1992; African catfish: Bogerd et al., 2001; Vischer and Bogerd, 2003; Vischer et al., 2003; zebra-

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fish: So et al., 2005). In both African catfish and zebrafish, it has also been shown that human chorionic gonadotropin (hCG), a LH-like hormone, specifically activated LHR but not FSHR but the homologous or recombinant LH can recognize both receptors (Bogerd et al., 2001; Vischer and Bogerd, 2003; Kwok et al., 2005). However, many experimental studies have shown that hCG can induce whole spermatogenesis in the male (Miura et al., 1991a,b, 2002) but is unable to stimulate vitellogenesis in the female eels. This suggests that FSHR, which is the predominant gonadotropin receptor in the immature eel ovary, probably could not recognize hCG. Further investigation should aim at characterizing the specificity of eel recombinant receptors towards homologous and heterologous hormones. Such data are necessary to further decipher eel reproductive endocrinology and develop new and relevant hormonal treatments. 4.3. Differential regulation of the expression of gonadotropin subunits and gonadotropin receptors after gonadotropic treatment Repeated injections of fish pituitary homogenates were used to induce ovarian growth, steroidogenesis and vitellogenesis, according to the classical protocol in the eel (Fontaine et al., 1964; Ohta et al., 1997). RIA of pituitary LH content showed a large increase in treated eel, which is consistent with our previous studies in Japanese eels (Jeng et al., 2002) and European eels (Dufour et al., 1989). Measurement of gonadotropin subunits transcripts by real-time quantitative PCR also demonstrated a large increase in pituitary LH-b mRNA levels. Conversely, FSH-b mRNA levels were significantly decreased in treated eels. The opposite regulation of LH and FSH expression in experimentally matured eels is in good agreement with the previous studies in the Japanese eel (Nagae et al., 1996; Yoshiura et al., 1999; Suetake et al., 2002) as well as in the European eel (Schmitz et al., 2005). We also investigated (by real-time quantitative PCR) the variations in the expression of ovarian gonadotropin receptors transcripts in experimentally matured eels. Both FSHR and LHR mRNA levels were significantly increased in treated eels. This is the first report on the regulation of gonadotropin receptors expression in eels. These results are in agreement with data in other teleosts. In channel catfish (Kumar et al., 2001a,b) and zebrafish (Kwok et al., 2005), the expression levels of FSHR and LHR were both increased during ovarian growth. 4.4. Role of sex steroids in the regulation of the expression of pituitary gonadotropins Experimental maturation in the eel, as induced by gonadotropic treatments, involves an increase in endogenous sexual steroid production, including both estrogens and androgens in the female (Leloup-Hatey et al., 1987; Ijiri et al., 1995). In order to decipher whether steroids

could be involved in the regulation of the expression of gonadotropins and their receptors as observed in experimentally matured eels, we investigated the effect of in vivo treatments with E2 or T. It has been largely evidenced that T or E2 could regulate the mRNA expression levels, the synthesis or secretion of gonadotropins in teleosts through brain or act at pituitary directly (Trudeau et al., 1993; Borg et al., 1998; Dickey and Swanson, 1998). We demonstrated here that pituitary LH protein content and LH-b mRNA levels were strongly increased in a dose-dependent manner after E2 treatment. In contrast, T could only slightly stimulate pituitary LH content and the extent of the stimulation by T was much less than by E2. We did not observe any significant effect of T on pituitary LH-b transcript levels. These results are in good agreement with previous studies in female European silver eels showing a dramatic stimulatory effect of E2 in vivo on pituitary LH mRNA and protein levels, while T had no or only a moderate effect (Dufour et al., 1983b; Que´rat et al., 1991; Schmitz et al., 2005). The differential effects of T and E2 on the regulation of LH expression in the eel contrasts with their similar effects in many other teleosts (salmonids: Breton et al., 1997; Dickey and Swanson 1998; Borg et al., 1998; goldfish: Huggard et al., 1996). This is likely related to the low aromatase activity in the eel, as compared to other teleost species (Jeng et al., 2005). Both T and E2 decreased FSH-b transcripts in immature female Japanese eels in the present study. In female European silver eels, a significantly negative effect by T treatment on FSH-b mRNA levels was also found but E2 had no significant effect (Schmitz et al., 2005). The different doses of steroid treatment, physiological stage of eels, duration of treatment and the method of application may result in this difference. All together these data indicate that sex steroids exert opposite regulatory controls on the expression of LH and FSH in the Japanese and European eels, with a stimulatory effect on LH and an inhibitory one on FSH. Endogenous sex steroid feedbacks may therefore be responsible for the opposite variations in the expression of pituitary LH and FSH observed in experimentally matured eels. 4.5. Lack of role of sex steroids in the regulation of the expression of ovarian gonadotropin receptors These are the first data concerning the potential role of sex steroids in the regulation of FSHR and LHR in the eels and any other teleosts. In contrast to the effects of sex steroids on the expression of pituitary gonadotropins, E2 or T-treatment did not induce any significant variation in the expression of ovarian gonadotropin receptors, FSHR and LHR, in the Japanese eels. This indicates that sex steroids could not directly act on the ovaries to regulate the expression of FSHR or LHR. In contrast, FSHR and LHR transcripts were significantly stimulated by the injection of pituitary homogenates. Our data suggest that endogenous sex steroids,

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differently from their significant role in the differential regulation of pituitary gonadotropins, are only slightly involved in the regulation of gonadotropin receptors during experimental maturation. There is very limited information about the regulation of FSHR and LHR transcripts in teleosts. The likely hypothesis is that gonadotropins contained in exogenous pituitary homogenates, upregulated the expression of FSHR and LHR in the eel ovary. Or, the increased expression of gonadotropin receptors was a concomitant of the ovarian growth and maturation induced by exogenous pituitary homogenates. In mammals, both up and down regulation of their receptors by gonadotropins have been evidenced (i.e. in mice: Piquette et al., 1991; Takase and Tsutsui, 1997). Other hormones, in addition to gonadotropins, from pituitary homogenates may also have regulatory effects on the gonadotropin receptors. For instance, FSH reduced FSHR but prolactin had a stimulatory role on FSHR in mice (Takase and Tsutsui, 1997) and porcine (Porter et al., 2000). Further studies, using in vivo and in vitro approaches, should aim at investigating the regulatory factors involved in eel gonadotropin receptor expression. In conclusion, these are the first data on the sequence, expression and regulation of gonadotropin receptors, FSHR and LHR in the eel. This study demonstrated that gonadotropins and their receptors are differentially regulated during experimental maturation and steroid treatments. Our findings provide new foundation for basic and applied research on eel reproduction. Acknowledgment This work was supported in part by the Fisheries Agency, Council of Agriculture (Taiwan). References Aroua, S., Schmitz, M., Baloche, S., Vidal, B., Rousseau, K., Dufour, S., 2005. Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event. Neuroendocrinology 82, 221–232. Bogerd, J., Blomenrohr, M., Andersson, E., van der Putten, H.H., Tensen, C.P., Vischer, H.F., Granneman, J.C., Janssen-Dommerholt, C., Goos, H.J., Schulz, R.W., 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. Borg, B., Antonopoulou, E., Mayer, I., Andersson, E., Berglund, I., Swanson, P., 1998. Effects of gonadectomy and androgen treatments on pituitary and plasma levels of gonadotropins in mature male Atlantic salmon, Salmo salar, parr–positive feedback control of both gonadotropins. Biol. Reprod. 58, 814–820. Breton, B., Sambroni, E., Govoroun, M., Weil, C., 1997. Effects of steroids on GTH I and GTH II secretion and pituitary concentration in the immature rainbow trout Oncorhynchus mykiss. C. R. Acad. Sci. III, Sci. Vie 320, 783–789. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193. Chang, C.F., Yueh, W.S., 1990. Annual cycle of gonadal histology and steroid profiles in the juvenile males and adult females of the

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