Greater amberjack Fsh, Lh, and their receptors: Plasma and mRNA profiles during ovarian development

General and Comparative Endocrinology 225 (2016) 224–234

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Greater amberjack Fsh, Lh, and their receptors: Plasma and mRNA profiles during ovarian development Mitsuo Nyuji a,⇑, Yukinori Kazeto b, Daisuke Izumida c, Kosuke Tani c, Hiroshi Suzuki b, Kazuhisa Hamada d, Miyuki Mekuchi a, Koichiro Gen e, Kiyoshi Soyano c, Koichi Okuzawa b a

National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama 236-8648, Japan National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki 519-0423, Japan Institute for East China Sea Research, Nagasaki University, Nagasaki 851-2213, Japan d Komame Branch, Stock Enhancement Technology Development Center, National Research Institute of Aquaculture, Fisheries Research Agency, Otsuki 788-0315, Japan e Seikai National Fisheries Research Institute, Fisheries Research Agency, Nagasaki 851-2231, Japan b c

a r t i c l e

i n f o

Article history: Received 21 June 2015 Revised 7 October 2015 Accepted 12 October 2015 Available online 28 October 2015 Keywords: Gonadotropin Gonadotropin receptor ELISA Ovarian development Multiple spawning fish Greater amberjack

a b s t r a c t To understand the endocrine regulation of ovarian development in a multiple spawning fish, the relationship between gonadotropins (Gths; follicle-stimulating hormone [Fsh] and luteinizing hormone [Lh]) and their receptors (Gthrs; Fshr and Lhr) were investigated in greater amberjack (Seriola dumerili). cDNAs encoding the Gth subunits (Fshb, Lhb, and glycoprotein a [Gpa]) and Gthrs were cloned. The in vitro reporter gene assay using recombinant hormones revealed that greater amberjack Fshr and Lhr responded strongly to their own ligands. Competitive enzyme-linked immunosorbent assays (ELISAs) were developed for measuring greater amberjack Fsh and Lh. Anti-Fsh and anti-Lh antibodies were raised against recombinant chimeric single-chain Gths consisting of greater amberjack Fshb (or Lhb) with rabbit GPa. The validation study showed that the ELISAs were precise (intra- and inter-assay coefficient of variation, <10%) and sensitive (detection limit of 0.2 ng/ml for Fsh and 0.8 ng/ml for Lh) with low cross-reactivity. A good parallelism between the standard curve and serial dilutions of greater amberjack plasma and pituitary extract were obtained. In female greater amberjack, pituitary fshb, ovarian fshr, and plasma E2 gradually increased during ovarian development, and plasma Fsh significantly increased during the post-spawning period. This suggests that Fsh plays a role throughout ovarian development and during the post-spawning period. Pituitary lhb, ovarian lhr, and plasma Lh were high during the spawning period, suggesting that the synthesis and secretion of Lh, and Lhr expression are upregulated to induce final oocyte maturation and ovulation. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction The pituitary glycoprotein hormone, gonadotropin (GtH), plays a central role in the regulation of vertebrate reproduction. Two types of GtHs, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exist. These are heterodimeric proteins consisting of a common a-subunit (glycoprotein a, GPa), which is noncovalently bound to a hormone-specific b-subunit (FSHb and LHb) (Pierce and Parsons, 1981). Each subunit has an N-linked glycosylation site which is known to influence the stability and function of GtHs (Hearn and Gomme, 2000; Fares, 2006; Mitra et al., 2006). In the gonad, GtHs exert their actions through GtH receptors (GtHRs; FSHR and LHR), which are the member of G ⇑ Corresponding author. E-mail address: [email protected] (M. Nyuji). http://dx.doi.org/10.1016/j.ygcen.2015.10.008 0016-6480/Ó 2015 Elsevier Inc. All rights reserved.

protein-coupled receptors (Costagliola et al., 2005; Caltabiano et al., 2008). GtHRs possess a large N-terminal extracellular domain (ECD) that is responsible for selective binding of the ligands, with seven transmembrane helices and a small C-terminal intracellular tail (Vassart et al., 2004). Teleost fishes exhibit a wide variety of reproductive strategies, each accompanied by different ovarian developmental patterns (i.e., synchronous, group-synchronous or asynchronous development) (Murua and Saborido-Rey, 2003). It is of great interest to understand the endocrine mechanisms by which the Gth/Gthr system controls ovarian development in fish species. Two Gths were first purified and characterized from salmon pituitary glands, and homologous immunoassays for measuring Fsh and Lh were developed in salmonids (Suzuki et al., 1988a, 1988b, 1988c; Swanson et al., 1991). In salmonids, which spawn once per life or year and have a synchronous-type ovary, Fsh acts on vitellogenesis via

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estradiol-17b (E2) production in ovarian follicles, while Lh induces final oocyte maturation (FOM) and ovulation via the production of 17,20b-dihydroxy-4-pregnen-3-one, the maturation-inducing hormone in this species (Planas and Swanson, 1995; Swanson et al., 2003). In contrast, most marine fish species spawn multiple batches of eggs and show group-synchronous or asynchronous patterns of ovarian development (Murua and Saborido-Rey, 2003). The physiological mechanisms regulating ovarian development in multiple spawning fishes have not been clearly defined. One reason for this is the lack of a suitable method for measuring Fsh and Lh in non-salmonids. Though two Gths have been isolated in some multiple spawning fishes, the establishment of homologous immunoassays has been hampered by difficulties with isolating large quantities of Gths, especially Fsh, and in obtaining specific antibodies against each Gth (Levavi-Sivan et al., 2010). Recently, recombinant protein production has made it possible to obtain larger quantities of hormones. Using recombinant proteins, homologous enzyme-linked immunosorbent assays (ELISAs) for the detection of both Fsh and Lh have been established in the Nile tilapia (Oreochromis niloticus) (Aizen et al., 2007). ELISAs for measuring Fsh were also developed using recombinant proteins in the Senegalese sole (Solea senegalensis) (Chauvigné et al., 2015) and the European sea bass (Dicentrarchus labrax) (Molés et al., 2012). Thus, recombinant protein production has been successfully used to establish immunoassays for teleost Gths. Additionally, in multiple spawning fish, ELISAs for two Gths have recently been developed for the mummichog (Fundulus heteloclitus) using purified native proteins (Shimizu et al., 2012). Seriola species, such as Japanese amberjack (Seriola quinqueradiata), yellowtail amberjack (Seriola lalandi), and greater amberjack (Seriola dumerili), are widely distributed in tropical and temperate waters and are commercially important fish worldwide. Greater amberjack have a group-synchronous ovary, spawn multiple times during the breeding season and is a major aquaculture species in the Mediterranean region and Japan (Marino et al., 1995; Nakada, 2002; Mylonas et al., 2004; Jerez et al., 2006). Given that the aquaculture of this species generally depends on wild captured juveniles, control of reproduction is key to successful aquaculture, and therefore the endocrine mechanisms regulating reproduction are an important area of research. Two forms of Gths have been purified and characterized from the pituitary of Mediterranean greater amberjack, and a homologous radioimmunoassay (RIA) for measuring Lh has been developed (García-Hernández et al., 1997; García Hernández et al., 2002). There are seasonal changes in the levels of sex steroids and vitellogenin (the yolk protein precursor) in the blood of greater amberjack (Takemura et al., 1999; Mandich et al., 2004). However, a homologous assay for Fsh in this species has not been established and information on Gthrs is lacking. In the present study, homologous ELISAs for measuring Fsh and Lh using recombinant protein production were developed and the relationship between the Gth/Gthr system and ovarian development in greater amberjack was evaluated. cDNAs encoding greater amberjack Gth subunits (Fshb, Lhb, and Gpa) and Gthrs were cloned and four types of recombinant proteins were produced. Recombinant greater amberjack Gths (re-Fsh and re-Lh) consisting of greater amberjack Fshb (or Lhb) and Gpa, were used as standards for the ELISAs, while recombinant chimeric Gths (re/ch-Fsh and re/ch-Lh), consisting of greater amberjack Fshb (or Lhb) with rabbit GPa, were used to produce the antisera tested using ELISA. The specificity of binding of greater amberjack Gthrs to their ligands was determined using an in vitro reporter gene assay using recombinant hormones. To characterize seasonal endocrine changes in cultured female greater amberjack, gene expression of fshb and lhb in the pituitary and fshr and lhr in the ovary were

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analyzed using TaqMan real-time PCR assays, and plasma levels of Fsh, Lh and E2 were measured using ELISAs. The development of ovaries in female greater amberjack was examined using histology. 2. Materials and methods 2.1. Fish and sample collection Juvenile greater amberjack were caught in the wild and maintained in sea pens at Komame Branch, Fisheries Research Agency, Otsuki, Kochi, Japan for 2–3 years. Fish were fed thawed mackerel and squid three times weekly. Sampling was conducted at 1– 2 month intervals from September 2011 to August 2012, when fish were aged 3 to 4 years. At the first sampling event (14 September 2011), the average size of greater amberjack was 650.3 ± 9.0 mm (fork length) and 4.6 ± 0.2 kg (body weight), while on the final day of sampling (22 August 2012) the average size was 759.3 ± 8.1 mm (fork length) and 7.5 ± 0.3 kg (body weight). At each sampling time, six females were anesthetized with 2phenoxyethanol, and blood was collected from the caudal vessel using a heparinized syringe before the fish were killed. Gonads were removed and a portion was fixed in Bouin’s solution. The remaining gonad and the pituitary were dissected and immersed in RNAlater (Ambion, Austin, TX) for 24 h at 4 °C, then stored at
30 °C for cDNA cloning. The gonadosomatic index (GSI) was calculated as follows: GSI ¼ ðgonad weight=body weightÞ  100. Blood samples were centrifuged (2000g, 10 min, 4 °C), and the plasma collected and stored at
80 °C. Pituitary extract (PE) was prepared from five additional female greater amberjack with immature gonad. The samples were collected and stored at
80 °C. Frozen pituitaries were dehydrated in cold acetone at
20 °C, dried in a vacuum and homogenized at 4 °C in 1.6 ml of phosphate-buffered saline (PBS) containing a protease inhibitor cocktail (Complete; Boehringer Mannheim, Indianapolis, IN). The homogenate was stirred for 3 h at 4 °C and centrifuged. The supernatant was then removed and filtered. Plasma samples were also obtained from cultured female Japanese amberjack in the same manner as described above. The care and use of the animals adhered to the guidelines of animal experiments set by the National Research Institute of Aquaculture, the Fisheries Research Agency. 2.2. Histology Ovary tissue samples fixed in Bouin’s solution were dehydrated through an ethanol series, embedded in paraffin, sectioned at 5 lm and stained with hematoxylin and eosin. 2.3. cDNA cloning of Gth subunits and Gthrs in greater amberjack Greater amberjack Gth subunits and Gthrs were cloned from pituitary and gonad tissue, respectively. Briefly, total RNA was extracted from tissues using an RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) or Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) with oligo-dT and random hexamer primers. The complete nucleotide sequences for Fshb, Lhb, Gpa, Fshr and Lhr were determined using basic molecular cloning techniques as previously described for other perciform species (Nyuji et al., 2012, 2013). Pairwise amino acid sequence identities of Gth subunits and ECDs of Gthrs were calculated using the BioEdit program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

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2.4. Production of recombinant Gths Four different DNA constructs were generated and cloned into the pCAGGS expression vector using In-Fusion cloning technology (Clontech, Palo Alto, CA). The constructs pCAGGS-Fshb (or Lhb)-GSH6-Gpa, which is referred to as re-Fsh or re-Lh, consisted of the entire open reading frames (ORFs) of greater amberjack Fshb (or Lhb) without stop codons and the spacer sequence (GGGSGGGSGGGSGGG) with the six-His tag, followed by the ORF of greater amberjack Gpa without a signal peptide. In the constructs of recombinant chimeric Gths (re/ch-Fsh and re/ch-Lh), the ORF of rabbit GPa without a signal peptide was used in place of the greater amberjack Gpa. Four pairs of primers with a Kozak translation initiation sequence, start and stop codons, were used in PCR (see Supplementary Table S1). The expression vectors were transformed separately into Escherichia coli (E. coli) DH5a (Toyobo, Tokyo, Japan), plated onto a Luria broth plate containing ampicillin, and incubated overnight at 37 °C. Plasmid DNA was obtained from the culture medium of transformed cells and sequenced to confirm the proper reading frame of constructs. For transfection in mammalian cells, endotoxin-free plasmids were purified from large-volume E. coli cultures using NucleoBond Xtra Maxi EF (Macherey–Nagel, Düren, Germany). Recombinant Gths were produced by transient transfection of suspension-growing mammalian cells using a FreeStyle Max expression system (Invitrogen) according to the manufacturer’s recommendations. Briefly, FreeStyle 293-F cells were maintained as a suspension culture in FreeStyle 293 expression medium. The culture was produced in an Erlenmeyer flask with a vented cap at 37 °C, in a humidified atmosphere containing 8% CO2 with orbital shaking (135 rpm/min). For transient expression of recombinant protein, the cells were grown in 300 ml of medium (1.0  106 cells/ml). The transfection reagent FreeStyle MAX (375 ll), 300 lg of each expression plasmid vector, and 75 lg of pAdVAntage (Promega, Madison, WI), which enhances the yield of protein production, were mixed in OptiPRO Serum Free Medium, incubated for 15 min, and added to the culture medium. After incubation for 5–7 days, the cells were removed by centrifugation and proteinase inhibitor was added into the medium. The medium (1600–2400 ml) was concentrated by tangential flow filtration using a 10-kDa filter Viva Flow 200 system (Sartorius, Goettingen, Germany), added to a 1 ml bed volume of Ni–NTA agarose beads (Qiagen), and then the His-tagged recombinant proteins were eluted with 250 mM imidazole, 0.3 M NaCl, 50 mM NaH2SO4, pH 8.0. The extracts were dialyzed against 1.5 M ammonium sulfate, and the supernatant was re-dialyzed against PBS. The purified recombinant proteins were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) using 15% gel (SuperSep; Wako Pure Chemical Industries, Osaka, Japan) and stained with Coomassie brilliant blue (CBB) to confirm purity. The protein concentration was determined using the BCA reagent (Pierce, Rockford, IL) and bovine serum albumin as a standard.

2.5. Antibody production Polyclonal antibodies against the re/ch-Fsh and re/ch-Lh (referred to as anti-Fsh and anti-Lh) were raised in New Zealand white rabbits. Approximately 1 mg of each recombinant chimeric protein in 1 ml of PBS was mixed with the same volume of TiterMax Gold (CytRx, Norcross, GA) and injected subcutaneously into a rabbit. The rabbit was injected four times at two-week intervals by using about 250 lg of each protein in TiterMax Gold. The antiserum was collected twice a week starting after 19 days of after the first injection. The antiserum collected from 19 to 40 days after the

first injection was mixed and IgGs were affinity-purified using nProteinA Sepharose (GE Healthcare Biosciences, Piscataway, NJ). The specificity of anti-Fsh and anti-Lh antibodies was analyzed using Western blotting. Recombinant Fsh and Lh, and greater amberjack PE were resolved by SDS- or native-PAGE using 15% gel (SuperSep; Wako), transferred to nitrocellulose membranes, immunoblotted with anti-Fsh and anti-Lh antibodies, both of which were diluted 1:4000 in immunoreaction enhancer solution (Can Get Signal Solution 1; Toyobo). Primary antibodies were detected using poly-HRP-labeled anti-rabbit secondary antibodies (Envision+; DakoCytomation, Carpinteria, CA) diluted 1:15 in Can Get Signal Solution 2 (Toyobo), and 3, 30 -diaminobenzidine (ImmPact DAB; Vector Laboratories, Burlingame, CA). 2.6. ELISAs for measuring greater amberjack Fsh and Lh Ninety-six well polystyrene plates (Corning, Corning, NY) were coated with 50 ll per well of re-Fsh (30 ng/ml) or re-Lh (40 ng/ml) solution, both of which were diluted in 50 mM carbonate buffer (pH 9.6) and incubated overnight at 4 °C. After washing four times with PBS containing 0.05% Tween20 (TPBS), plates were blocked with 100 ll per well of Superblock in PBS (Pierce) for 1 h at room temperature (RT). Samples and standards were diluted with TPBS; plasma samples were diluted 1:4, while greater amberjack PE and standards (reGths) were serially diluted. The primary antibodies (anti-Fsh or anti-Lh) were diluted 1:80,000 (for Fsh ELISA) or 1:40,000 (for Lh ELISA) in TPBS containing 5% normal goat serum (NGS). Samples and standards were mixed with an equal amount of primary antibody solution in a 1.5 ml microtube, and incubated overnight at 4 °C. The antigen-coated plate was washed and 50 ll of reacted mixture of either sample or standard and antibody were dispensed in triplicate (samples) or duplicate (standards) and incubated overnight at 4 °C. Subsequently, the wells were incubated with 50 ll per well of biotinylated antibody to rabbit immunoglobulins (Zymed, South San Francisco, CA) diluted 1:10 (for Fsh ELISA) or 1:100 (for Lh ELISA) in 5% NGS-TPBS for 1 h at RT. After washing, the wells were incubated with 50 ll per well of streptavidin-polyHRP80 (Stereospecific Detection Technology [SDT], Baesweiler, Germany) diluted into 200 ng/ml with Universal Casein Diluent/Blocker (SDT) for 1 h at RT. After washing, the plates were developed with 100 ll of 1-Step Ultra TMB-ELISA solution (Thermo Scientific, Rockford, IL). After incubation for 30–60 min at RT, the reaction was stopped by the addition of 100 ll of 2 M sulfuric acid and the absorbance was read at 450 nm. 2.7. In vitro reporter gene assay Two different DNA constructs were generated and cloned into the pcDNA3.1 expression vector using In-Fusion cloning technology (Clontech), pcDNA3.1-L21-Fshr (or Lhr), which includes the ORFs of greater amberjack Fshr or Lhr. A lobster L21 promoter sequence was inserted before the start codon to improve translation efficiency. The procedure for the reporter gene assay was performed as previously described (Nyuji et al., 2013). In brief, CHO cells were maintained under 5% CO2 in Ham’s F12 medium supplemented with 10% fetal bovine serum, 1% HT supplement (Life Technologies), and penicillin/streptomycin solution (Nakarai Tesque, Kyoto, Japan). Plasmid pcDNA3.1-L21-Fshr (or Lhr) was co-transfected with reporter plasmid pCRE-Luc (Agilent, Palo Alto, CA) and pRLTK plasmid (Promega) using X-treme GENE HP DNA transfection reagent (Roche) according to the manufacture’s protocol. After 24 h, the cells were split into 96-well plates and incubated for 1 day. The cells were stimulated with increasing concentrations

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of re-Fsh and re-Lh, and a serial dilution of greater amberjack PE. After incubation for 3 h, the medium was removed. The ligandinduced cyclic adenosine monophosphate (cAMP) production was assessed by measuring luciferase activity of the cells using reagents from a Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s protocol in a Tecan Infinite F200 plate reader (Tecan, Switzerland). Firefly luciferase activity was normalized to the Renilla luciferase activity. Three replicates were conducted for each experiment.

at 0.2 lM, 0.4 lM and 30 nM, respectively, in a final volume of 10 ll containing template cDNA corresponding to 45 ng of total RNA and 5 ll of 2 master mix. For each PCR, a standard curve from serial dilutions of plasmid containing full-length fshr or lhr was constructed. The thermocycling conditions were 3 min at 95 °C and 40 cycles of 10 s at 95 °C and 20 s at 60 °C. All samples and standards were run in duplicate.

2.8. Quantitative real-time PCR

The plasma steroids were extracted into diethyl ether, which was then evaporated, and the remaining steroids were dissolved in an assay buffer supplied with the Enzyme Immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). The levels of E2 extracted were measured using the EIA kit according to the manufacturer’s instructions.

Gene specific primers for greater amberjack fshb, lhb, fshr, and lhr were designed for TaqMan real-time PCR assays (see Supplementary Table S2). The real-time PCR for measuring greater amberjack Gth subunit transcripts was performed using the Light Cycler instrument (Roche) with FastStart Essential DNA Probes Master (Roche) according to the manufacturer’s protocol. Total RNA was extracted from each fish pituitary using RNeasy Lipid Tissue Mini Kit (Qiagen) and treated with RNase-Free DNase Set (Qiagen). Firststrand cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche). The TaqMan probes were labeled at the 50 end with the reporter dye 6-carboxyfluorescein (FAM), double quencher of the internal ZEN, and at the 30 end with the quencher dye Iowa Black FQ. The concentrations of primers and probe were optimized at 0.5 lM and 0.1 lM, respectively, in a final volume of 20 ll containing template cDNA corresponding to 1.25 ng of total RNA and 10 ll of 2 master mix. For each PCR, a standard curve from serial dilutions of plasmid containing fshb or lhb was constructed. The thermocycling conditions were 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. All samples and standards were run in duplicate. The real-time PCR for measuring greater amberjack Gthr transcripts was performed using the MX3005P (Stratagene, La Jolla, CA) device with the reagents from the Brilliant III Ultra-Fast QPCR master mix (Agilent) according to the manufacturer’s protocol. Total RNA was extracted from a piece of ovary from each fish using the same methods as used for the pituitary. First-strand cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). The TaqMan probes were labeled at the 50 end with the reporter dye FAM and at the 30 end with the Eclipse Dark Quencher. The concentrations of primers, probe and reference dye were optimized

2.9. Measurement of plasma E2 levels

2.10. Calculations and statistics In the ELISA, sigmoid curves for standards and samples were made linear by log–logit transformation to estimate the linearity between standards and samples as follows: logitðB=B0 Þ ¼ ln½ðB=B0 Þ=1
ðB=B0 Þ. The detection limit was calculated as B0-2 standard deviations. Data are represented as means ± standard errors of the mean (SEM). The data for annual changes were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Gth subunits and Gthrs in greater amberjack In the present study, cDNA clones for greater amberjack fshb, lhb, gpa, fshr, and lhr were obtained and full-length nucleotide sequences were confirmed (Table 1). For greater amberjack Gth subunits, there are one (Lhb) or two (Fshb and Gpa) amino acid residue replacements as compared with those previously identified by amino acid sequences of mature proteins (García-Hernández et al., 1997; Supplementary Fig. S1). The deduced amino acid sequences of greater amberjack and Japanese amberjack Gth subunits share high sequence identity (98–100%). In Fshb, amino acid residues differed between the two Seriola species in two positions,

Table 1 Sequence information of greater amberjack (Seriola dumerili) Gth subunits and Gthrs, and amino acid identities between greater amberjack Gth subunits or extracellular domains of Gthrs and those of other vertebrate species. GtH subunits

Sequence information GenBank accession number Nucleotide sequences Full-length (bp) ORF (bp) Amino acid sequences Amino acid identity (%) Japanese amberjack European sea bass Zebrafish Human Rabbit Chicken

GtHRs

FSHb

LHb

GPa

FSHR

LHR

LC019038

LC019039

LC019040

LC015526

LC015527

528 363 121

584 447 149

585 345 115

3339 2064 688

3255 2169 723

98 71 32 29 27 27

100 85 52 39 36 40

99 83 57 43 55 50

–

– 69 45 34 35 31

87 59 43 43 45

Full-length nucleotide sequences without a poly(A) tail are shown. GenBank accession numbers of FSHb, LHb, GPa, FSHR, and LHR are as follows: Japanese amberjack (Seriola quinqueradiata), AHC70790, AHC70791, AHC70792; European sea bass (Dicentrarchus labrax), AAN40506, AAN40507, AAK49431, AAV48628, AAV48629; Zebrafish (Danio rerio), NP_991187, NP_991185, AAS01761, NP_001001812, NP_991188; Human (Homo sapiens), NP_000501, NP_000885, NP_001239312, NP_000136, AAA59515; Rabbit (Oryctolagus cuniculus), NP_001075640, NP_001076164, NP_001076193, XP_002709764, XP_002709921; Chicken (Gallus gallus), NP_989588, ADY03193, NP_001264950, NP_990410, NP_990267.

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Fig. 1. Analysis of the purity of recombinant chimeric Gths (re/ch-Fsh and re/ch-Lh) and non-chimeric greater amberjack Gths (re-Fsh and re-Lh), and the reactivity of antiFsh and anti-Lh antibodies against recombinant Gths and greater amberjack pituitary extract (PE). (A) SDS–PAGE followed by CBB staining. (B) SDS–PAGE followed by Western blotting with anti-Fsh and anti-Lh antibodies (diluted 1:4000). (C) Native-PAGE followed by Western blotting with anti-Fsh and anti-Lh (diluted 1:4000). Molecular mass marker are shown on the right (A) or left (B, C).

while those of Gpa differed in one position at the signal peptide sequence. The amino acids of Lhb matched completely (Supplementary Fig. S2). The amino acid sequences of greater amberjack Gth subunits are 71–85%, 32–57% and 27–55% identical with the Gth subunits of European sea bass, zebrafish (Danio rerio), and non-fish vertebrate species (human, rabbit and chicken), respectively. The deduced amino acid sequences of greater amberjack Gthr ECDs are 45–87% identical with European sea bass and zebrafish Gthr ECDs, whereas they are 31–45% identical with the Gthr ECDs of non-fish vertebrate species. 3.2. Production of recombinant Gths and antibody characterization Four recombinant Gths were produced in mammalian cells. Each His-tagged protein was affinity-purified, and then the purity estimated using SDS–PAGE and CBB staining. Wide bands around 31 kDa were found in re/ch-Fsh and re-Fsh, while those in re/ch-Lh and re-Lh were around 35 kDa (Fig. 1A). The re/ch-Fsh and re/ch-Lh were used to immunize rabbit. After preparation of affinity-purified IgGs, the reactivity of these antibodies against re-Gths and greater amberjack PE were tested using SDS–PAGE and native-PAGE with Western blot analysis. Anti-Fsh antibodies recognized a protein band around 31 kDa on SDS–PAGE, corresponding to re-Fsh, and anti-Lh antibodies recognized a protein band around 35 kDa, corresponding to re-Lh (Fig. 1A and B). Anti-Fsh antibodies recognized a protein band around 15 kDa in the greater amberjack PE fraction on SDS–PAGE, while anti-Lh antibodies recognized a protein band around 16 kDa (Fig. 1B). Using native-PAGE and Western blotting, anti-Fsh antibodies strongly reacted with re-Fsh and hardly reacted with re-Lh, whereas antiLh antibodies strongly reacted with re-Lh and had a weak reaction with re-Fsh (Fig. 1C). 3.3. Validation of ELISAs for greater amberjack Fsh and Lh In the Fsh ELISA, the standard curve ranged from 80 pg/ml to 400 ng/ml and cross-reactivity with re-Lh was less than 1% (Fig. 2A). The assay sensitivity was 7.0 ng/ml in 50% inhibition (IC50) with a detection limit of 0.2 ng/ml (5 pg/well). On log–logit transformed data, linear responses were found in the serial dilutions of greater amberjack plasma and PE, and Japanese amberjack plasma, corresponding to a linear re-Fsh response (160 pg/ml to

40 ng/ml) (Fig. 2B). The intra-assay coefficients of variation (CV) for standard of 10 ng/ml in the same plate were 3.0% (n = 10). The inter-assay CV for the same plasma sample on different plates were 9.6% (n = 7). The Fsh levels measured from the diluted samples in this study ranged from 1.0 to 10.5 ng/ml, which is positioned at the linear part of the standard curve. In the Lh ELISA, the standard curve ranged from 0.4 ng/ml to 1.6 lg/ml and cross-reactivity with re-Fsh was 4% (Fig. 2C). The assay sensitivity was 21.1 ng/ml in IC50 and the detection limit was 0.8 ng/ml (20 pg/well). On log–logit transformed data, linear responses were found in the serial dilutions of greater amberjack plasma and PE, and Japanese amberjack plasma corresponding to a linear re-Lh response (2.6–100 ng/ml) (Fig. 2D). The intra- and inter-assay CVs were 3.9% (n = 10) and 8.1% (n = 7), respectively. The Lh levels measured from the diluted samples in this study ranged from 2.1 to 27.6 ng/ml, which is positioned at the linear part of the standard curve. 3.4. Binding specificity of greater amberjack Gthrs to recombinant Gths The biological activity of re-Gths and pharmacological characterization of greater amberjack Gthrs were analyzed using an in vitro reporter gene assay with a cAMP-responsive reporter. Both Fshr and Lhr were activated following serial dilutions with greater amberjack PE (Fig. 3A and B). The re-Fsh activated Fshr at concentrations of 100–30,000 ng/ml (EC50, 632.5 ng/ml), but re-Lh did not enhance reporter gene expression (Fig. 3A). The re-Lh activated Lhr at concentrations of 100–10,000 ng/ml (EC50, 217.9 ng/ml), but re-Fsh did not enhance reporter gene expression (Fig. 3B). 3.5. Seasonal changes in ovarian development The water temperature during the sampling period ranged between 15 °C in February and 29 °C in August, while daylight hours were shortest in December and longest in June (Fig. 4A). The GSI significantly increased in June, then decreased in August (Fig. 4B). The developmental stages of female greater amberjack were classified into six stages based on the histology: immature (IM), cortical alveolus (CA), early vitellogenesis (EV), middle vitellogenesis (MV), late vitellogenesis (LV) and atresia (AT). In fish at the IM stage, perinucleolus stage primary growth oocytes occupied the

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Fig. 2. Validation of competitive ELISAs for measuring greater amberjack Fsh and Lh. (A) Standard curve for Fsh ELISA and cross-reactivity of re-Lh. (B) Standard curve for Lh ELISA and cross-reactivity of re-Fsh. (C) Parallelism between standard curve for Fsh ELISA and serial dilutions of plasma and pituitary extract (PE). (D) Parallelism between standard curve for Lh ELISA and serial dilutions of plasma and PE.

ovary (Fig. 5A). Early secondary growth oocytes appeared during the CA stage, but vitellogenic oocytes were not observed (Fig. 5B). During vitellogenesis, the developmental stage was determined by the diameter of the most advanced vitellogenic oocytes present: EV (<300 lm) (Fig. 5C), MV (300–500 lm) (Fig. 5D) and LV (<500 lm) (Fig. 5E). In fish at the AT stage, most vitellogenic oocytes underwent atresia (Fig. 5F). The frequency distribution was calculated by number of fish at each developmental stage (Fig. 5G). From 14 September to 12 December 2011, all female fish had immature ovaries (IM stage). By 25 January, a few fish were at the CA stage, but most remained IM. On 14 March some females were at EV and MV stages, and by 25 April all females were MV or LV. During the spawning season, on 13 June, most fish were LV but some fish were undergoing atresia (AT stage). By 22 August, post-spawning, all females had immature gonads. 3.6. Seasonal changes in fshb, lhb, fshr, and lhr The expression of fshb in the pituitary gradually increased from the lowest level in November, to high levels between April and June, then decreased significantly in August (Fig. 6A). The expression of lhb was low between September and January, then gradually increased to reach a peak in June, before significantly declining in August (Fig. 6B). In the ovary, the expression of fshr gradually increased from January, and remained high between April and August, with a peak

in June (Fig. 6C). The expression of lhr was low between September and April, markedly increased in June, and then decreased to the lowest level in August (Fig. 6D). 3.7. Seasonal changes in plasma Fsh, Lh, and E2 Although not statistically significant, levels of plasma Fsh fluctuated between September and June. Levels of plasma Fsh increased significantly from June to August (Fig. 7A). Levels of plasma Lh gradually increased from January and remained high between April and August (Fig. 7B). Plasma E2 levels gradually increased from January and reached a peak in June, but declined significantly in August (Fig. 7C). 4. Discussion Evaluating the role of the Gth/Gthr system during ovarian development is key to understanding the physiological mechanisms regulating reproduction in multiple spawning fish. In the present study, the complete nucleotide sequences of cDNAs encoding greater amberjack Gth subunits (Fshb, Lhb, and Gpa) and Gthrs (Fshr and Lhr) were determined using molecular cloning. The Gth subunits of greater amberjack showed high sequence identity with those of perciform fishes, but not with other vertebrate species including non-perciform fishes. The Gth subunits also showed very high homology between two Seriola species, greater amberjack and

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used as an antigen, although chicken GPa was used, and antichicken GPa antibodies were removed (Chauvigné et al., 2015). Likewise, recombinant chimeric single-chain Gths (re/ch-Fsh and re/ch-Lh) were constructed as antigens in this study. Here, rabbit GPa was connected to a greater amberjack b-subunit to test the idea that the homogeneous GPa would not be recognized as a foreign antigen in the rabbit host. After immunization, IgGs were purified from antisera against re/ch-Fsh and re/ch-Lh (anti-Fsh and anti-Lh) by protein A-agarose and used for the immunoassays. Recombinant non-chimeric single-chain Gths consisting of a greater amberjack b-subunit and its Gpa (re-Fsh and re-Lh) were also produced. Western blot and SDS–PAGE analysis showed that anti-Fsh and anti-Lh antibodies recognized re-Fsh and re-Lh, respectively (Fig. 1A and B). The reactivity of antibodies to native hormones was also analyzed by SDS–PAGE using greater amberjack PE. Studies on teleost pituitary Gths have consistently shown that Fshb resolves as a low molecular weight band compared with Lhb in SDS–PAGE, and that Gpa has a low molecular weight compared with b-subunits (Copeland and Thomas, 1993; Mañanós et al., 1997; Mateos et al., 2006; Kim et al., 2011; Ohga et al., 2012). In the present study, the molecular weight of protein detected by anti-Fsh antibodies was slightly lower than those detected by anti-Lh antibodies (Fig. 1B), indicating that these bands may correspond to Fshb and Lhb, respectively. Also, greater amberjack Gpa was not detected, as no other bands were found. Reactions of anti-Fsh and anti-Lh antibodies against re-Lh and reFsh were weak compared with those against re-Fsh and re-Lh using native-PAGE and Western blotting, although both re-Fsh and re-Lh contain greater amberjack Gpa (Fig. 1C). The immunoassay results also exhibited low cross-reactivity. Therefore, it seems likely that both antibodies strongly react with each b-subunit, but hardly react with greater amberjack Gpa. Immunization with recombinant chimeric single-chain Gths, which are constructed with b-subunits of the target species and a-subunits of the host animal, was successful in obtaining an antibody specific for one Gth. This

A Fig. 3. Analysis of binding specificity of greater amberjack Fshr (A) and Lhr (B) to reFsh, re-Lh, and PE by in vitro luciferase reporter gene assay.

Japanese amberjack, where mature Lhb and Gpa peptides matched completely and FSHb differed in only two amino acids. The Gthr sequences showed high sequence identity with perciform fishes, but not with other vertebrates. Sequences and structures of Lhr are more conserved than those of Fshr among vertebrate species (Levavi-Sivan et al., 2010). Indeed, amino acid identities of the ECD of greater amberjack Lhr were higher than that of Fshr when compared with other vertebrates. To establish immunoassays for greater amberjack Fsh and Lh, recombinant Gths were produced in mammalian cells. Compared with the baculovirus-insect cell system, oligosaccharides of glycoproteins attached by the mammalian cell line are more similar to native vertebrate hormones because they are sialylated in the N-glycosylation pathway (Kost et al., 2005; Hossler et al., 2009; Molés et al., 2011). Immunization with the b-subunit alone is generally more effective than the dimer in producing specific antibodies against one Gth, because Fsh and Lh share a common a-subunit (Govoroun et al., 1998; Aizen et al., 2007; Shimizu et al., 2012). The single-chain form enhances the immunogenicity of recombinant human chorionic gonadotropin (Jiang et al., 2010). In the Senegalese sole Fsh ELISA, a recombinant single-chain protein consisting of an N-terminal Fshb and C-terminal Gpa were successfully

B

Fig. 4. Seasonal changes in water temperature, day length and gonadosomatic index (GSI) of female greater amberjack.

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Fig. 5. Histological sections of greater amberjack ovaries at various stages of development (A–F) and the frequency distribution of females at each developmental stage (G). Ovarian stages are as follows: immature, IM (A); cortical alveolus, CA (B); early vitellogenesis, EV (C); middle vitellogenesis, MV (D); late vitellogenesis, (E); atresia, AT (F). Bars = 100 lm.

may be due to high binding specificity against the b-subunit, resulting in low cross-reactivity with another Gth. Using Fsh and Lh assays to detect Gths in fish helps to better understand the relationship between the timing of Gth secretion and reproductive status. In greater amberjack, an RIA for measuring Lh was developed using purified native hormones (García Hernández et al., 2002). The use of RIA is limited because of the need for special facilities and expertise with handling radioactive materials. The detection of specific proteins using ELISA is preferred as it does not require special equipment or the use of radioisotopes. In multiple spawning fishes, ELISAs for measuring

both Fsh and Lh have been established in Nile tilapia (Aizen et al., 2007), European sea bass (Mateos et al., 2006, Molés et al., 2012) and mummichog (Shimizu et al., 2012). The ELISA assays for greater amberjack Fsh and Lh had inter- and intra-assay CVs (3.0–9.6%) that were within the range estimated for other fish species. The cross-reactivity of the greater amberjack Fsh ELISA with Lh was very low (1%), indicating high specificity, while that of Lh ELISA with Fsh was 4%. The IC50 values indicated that the sensitivity of the greater amberjack Fsh ELISA (IC50, 7.0 ng/ml) was 3-fold higher than that of the Lh ELISA (IC50, 21.1 ng/ml). The parallelism between the standard curve and the serial dilutions of greater

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Fig. 7. Seasonal changes in plasma Fsh (A), Lh (B), and E2 (C) in female greater amberjack.

Fig. 6. Seasonal changes in pituitary fshb (A) and lhb (B), and ovary fshr (C) and lhr (D) in female greater amberjack.

amberjack plasma and pituitary samples was observed for both Fsh and Lh ELISAs (Fig. 2B and D). The serial dilutions of Japanese amberjack plasma also showed good correspondence with the standard curve. The ELISA assays used in our study are useful for measuring Fsh and Lh in the blood and pituitary of greater amberjack, and are likely to be applicable for other Seriola species. The in vitro reporter gene assay showed that Fshr and Lhr were activated by greater amberjack PE (Fig. 3), demonstrating that Gthrs isolated in this study are functional receptors for native hormones derived from the pituitary. The ligand-binding specificity of greater amberjack Gthrs to Gths was investigated

using recombinant hormones. In mammals, Fshr and Lhr bind their own ligands with high specificity (Vassart et al., 2004). Similar specificity has been obtained for some fish species such as rainbow trout (Oncorhynchus mykiss) (Sambroni et al., 2007), mummichog (Ohkubo et al., 2013), and chub mackerel (Scomber japonicus) (Nyuji et al., 2013). On the other hand, Fshr is activated not only by Fsh, but also by Lh in some species, including African catfish (Clarias gariepinus) (Vischer et al., 2003), Japanese eel (Anguilla japonica) (Kazeto et al., 2008) and Atlantic salmon (Salmo salar) (Andersson et al., 2009). Thus, the ligand-binding properties of teleost Gthrs display species specificity. In the present study, greater amberjack Fshr and Lhr were activated by their corresponding ligands but did not show cross-activation (Fig. 3). Seasonal changes in oocyte development and hormone levels were evaluated in female greater amberjack. Histological observations demonstrated that oocytes during the cortical alveolus stage first appeared in January. Vitellogenesis started in March and was completed between April and June during the spawning period, which corresponded with a significant increase in the GSI. Atretic oocytes were found in some fish in June, but by August the ovaries of all females sampled had completely regressed (immature) (Figs. 4 and 5). In the pituitary, fshb expression increased from

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November to April as ovarian development progressed, and remained at high levels between April and June when females completed vitellogenesis (Fig. 6A). Similar results have been described for other multiple spawning fishes including goldfish (Carassius auratus) (Sohn et al., 1999), European sea bass (Mateos et al., 2003) and chub mackerel (Nyuji et al., 2012), where elevated fshb expression was accompanied by ovarian development. In the ovarian follicles of European sea bass and chub mackerel, Fsh has been found to stimulate the production of E2, which induces hepatic vitellogenin production (Molés et al., 2008; Ohga et al., 2012). In the current study, the levels of plasma E2 gradually increased during vitellogenesis (Fig. 7C). It has been reported that in greater amberjack, serum vitellogenin gradually increased from the prespawning period to the spawning period (Takemura et al., 1999). Similar changes in plasma E2 and vitellogenin were also observed in wild Mediterranean greater amberjack (Mandich et al., 2004). This suggests that Fsh may act on vitellogenesis in greater amberjack via E2 production, resulting in a synchronized increase in pituitary fshb, plasma E2 and vitellogenin, and ovarian development. In mummichog, plasma Fsh increased during vitellogenesis (Shimizu et al., 2012); however, no significant changes in the levels of plasma Fsh occurred in greater amberjack (Fig. 7A). Similar results have been reported in European sea bass, which showed no significant changes in plasma Fsh between pre- and post-vitellogenesis (Molés et al., 2012). In the present study, levels of fshr in the ovary exhibited a similar profile to pituitary fshb and plasma E2. In multiple spawning fishes, high levels of fshr expression in ovarian follicles have been associated with vitellogenesis (García-López et al., 2011; Kitano et al., 2011; Nyuji et al., 2013). This suggests that in greater amberjack, increase of fshr expression is more important for regulating vitellogenesis than elevation of plasma Fsh content. Further studies are necessary to determine this observation. On the other hand, a significant increase in plasma Fsh was found during the post-spawning period (Fig. 7A). Similar results were obtained for rainbow trout, where increased levels of plasma Fsh postspawning is considered important for the start of the new gametogenic cycle (Breton et al., 1998; Gomez et al., 1999). Our results also showed high fshr expression in the greater amberjack ovary during the post-spawning period (Fig. 6C). As for salmonids, Lh induces final oocyte maturation (FOM) and ovulation in multiple spawning fishes (Van Der Kraak et al., 1992; Kagawa et al., 1998; Ohga et al., 2012). In Mediterranean greater amberjack, spawning was initiated by implantation of agonist gonadotropin-releasing hormone (GnRH), which was demonstrated to induce Lh secretion in the pituitary (García Hernández et al., 2002; Mylonas et al., 2004). This indicated that the release of GnRH-induced Lh initiated FOM, followed by ovulation and spawning. In the present study, pituitary lhb significantly increased in April and peaked in June (Fig. 6B), while levels of ovarian lhr were only high in June (Fig. 6C). High levels of lhr have been found in ovarian follicles with fully-grown vitellogenic oocytes (GarcíaLópez et al., 2011; Kitano et al., 2011; Nyuji et al., 2013). The high levels of greater amberjack lhr found during the spawning period may be associated with the presence of fully-grown vitellogenic oocytes. Levels of plasma Lh gradually increased during ovarian development and remained at high levels during the spawning period (Fig. 7B). This suggests that the synthesis and secretion of Lh and the expression of Lhr are upregulated at the completion of vitellogenesis. It has been reported that a surge of plasma Lh occurs during FOM and ovulation in goldfish (Kobayashi et al., 1987), striped bass (Morone saxatilis; Mylonas et al., 1998) and salmonids (Gomez et al., 1999). Further investigation is required to assess whether a surge of Lh occurs in association with FOM and ovulation in greater amberjack. Peak levels of plasma Lh were found during the post-spawning period in this study (Fig. 7B). High levels of plasma Lh during the post-spawning period were also

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seen in rainbow trout, but its role is unclear (Breton et al., 1998; Gomez et al., 1999). Unlike fshr expression, lhr expression was at the lowest levels during the post-spawning period in greater amberjack (Fig. 6D), suggesting that Lh does not play a role in initiating gametogenesis. In conclusion, competitive ELISAs for measuring Fsh and Lh in greater amberjack were developed using recombinant protein production. Analysis of the Gth/Gthr system in female greater amberjack suggests that Fsh plays a role throughout ovarian development and post-spawning. The synthesis and secretion of Lh, and Lhr expression are upregulated at the completion of vitellogenesis to activate FOM and ovulation. The application of ELISA has extended our understanding of endocrine regulation in fish reproduction. Information obtained in the present study provides detailed insight into the role of the Gth/Gthr system during ovarian development in multiple spawning fishes. Acknowledgments The authors thank the students in the Nagasaki University, Dr. A. Fujiwara of the National Research Institute of Fisheries Science, and Dr. T. Yamaguchi of the National Research Institute of Aquaculture, for sampling support. This study was supported by a Grant for Technological Development for Selection and Secure Stock of Bloodstock for Culture of Bluefin Tuna from the Fisheries Agency of Japan. The work of M.N. was partly supported by the Japan Society for the Promotion of Science (JSPS) Research Fellowship. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ygcen.2015.10. 008. References Aizen, J., Kasuto, H., Levavi-Sivan, B., 2007. Development of specific enzyme-linked immunosorbent assay for determining LH and FSH levels in tilapia, using recombinant gonadotropins. Gen. Comp. Endocrinol. 153, 323–332. Andersson, E., Nijenhuis, W., Male, R., Swanson, P., Bogerd, J., Taranger, G.L., Schulz, R.W., 2009. Pharmacological characterization, localization and quantification of expression of gonadotropin receptors in Atlantic salmon (Salmo salar L.) ovaries. Gen. Comp. Endocrinol. 163, 329–339. Breton, B., Govoroun, M., Mikolajczyk, T., 1998. GTH I and GTH II secretion profiles during the reproductive cycle in female rainbow trout: relationship with pituitary responsiveness to GnRH-a stimulation. Gen. Comp. Endocrinol. 111, 38–50. Caltabiano, G., Campillo, M., De Leener, A., Smits, G., Vassart, G., Costagliola, S., Pardo, L., 2008. The specificity of binding of glycoprotein hormones to their receptors. Cell. Mol. Life Sci. 65, 2484–2492. Chauvigné, F., Verdura, S., Mazón, M.J., Boj, M., Zanuy, S., Gómez, A., Cerda, J., 2015. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin. Gen. Comp. Endocrinol. 221, 75–85. Copeland, P.A., Thomas, P., 1993. Isolation of gonadotropin subunits and evidence for two distinct gonadotropins in Atlantic croaker (Micropogonias undulatus). Gen. Comp. Endocrinol. 91, 115–125. Costagliola, S., Urizar, E., Mendive, F., Vassart, G., 2005. Specificity and promiscuity of gonadotropin receptors. Reproduction 130, 275–281. Fares, F., 2006. The role of O-linked and N-linked oligosaccharides on the structure– function of glycoprotein hormones: Development of agonists and antagonists. BBA-Gen. Subjects 1760, 560–567. García Hernández, M.P., García Ayala, A., Agulleiro, B., García, A., van Dijk, W., Schulz, R.W., 2002. Development of a homologous radioimmunoassay for Mediterranean yellowtail (Seriola dumerilii, Risso 1810) LH. Aquaculture 210, 203–218. García-Hernández, M.P., Koide, Y., Díaz, M.V., Kawauchi, H., 1997. Isolation and characterization of two distinct gonadotropins from the pituitary gland of Mediterranean yellowtail, Seriola dumerilii (Risso, 1810). Gen. Comp. Endocrinol. 106, 389–399. García-López, Á., Sánchez-Amaya, M.I., Prat, F., 2011. Targeted gene expression profiling in European sea bass (Dicentreachus labrax, L.) follicles from primary growth to late vitellogenesis. Comp. Biochem. Physiol. A 160, 374–380. Gomez, J.M., Weil, C., Ollitrault, M., Le Bail, P.Y., Breton, B., Le Gac, F., 1999. Growth hormone (GH) and gonadotropin subunit gene expression and pituitary and

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