Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

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Contents lists available at ScienceDirect

General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen 5 6

Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin

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François Chauvigné a,b, Sara Verdura a,1, María José Mazón c, Mónica Boj a, Silvia Zanuy c, Ana Gómez c, Joan Cerdà a,⇑ a b c

IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain Department of Biology, University of Bergen, Bergen High Technology Centre, N-5020 Bergen, Norway Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre de la Sal (IATS), CSIC, Ribera de Cabanes, 12595 Castellón, Spain

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Article history: Available online xxxx Keywords: Flatfish Senegalese sole ELISA Fsh Spermatogenesis

a b s t r a c t In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh b subunit (Fshb) and the chicken glycoprotein hormone common a subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshb-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshb subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes. Ó 2014 Published by Elsevier Inc.

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1. Introduction

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The glycoprotein gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secreted from the pituitary gland are the primary endocrine regulators of the reproductive processes in vertebrates. These heterodimeric hormones are composed of a specific b subunit (FSHb and LHb) which is non-covalently bound to the common a subunit (CGA) shared by other members of the glycoprotein hormone family (Pierce and

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⇑ Corresponding author at: IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Passeig marítim 37-49, 08003 Barcelona, Spain. Fax: +34 932 309 555. E-mail address: [email protected] (J. Cerdà). 1 Present address: Bellvitge Biomedical Research Institute (IDIBELL), Gran via de L’Hospitalet 199, L’Hospitalet, 08908 Barcelona, Spain.

Parsons, 1981). Each mature FSHb and LHb subunit is about 100 amino acids in length and has a molecular mass of 15 kDa. They bear several conserved cysteines which form intra-molecular disulphide bonds that stabilize the heterodimer and are needed for proper folding of the protein and receptor interaction (Hearn and Gomme, 2000; Fox et al., 2001; Fan and Hendrickson, 2005). Both b and a subunits are N-linked glycosylated which is necessary for the stability and biological function of the hormones (Thotakura and Blithe, 1995; Fares, 2006). The FSHb and LHb confer the specific physiological function to each gonadotropin through the binding and activation of their cognate receptors in the gonads, the FSH receptor (FSHR) and the LH/chorionic gonadotropin receptor (LHCGR) (Holdcraft and Braun, 2004). During teleost spermatogenesis, Fsh is mainly involved in the initiation and early stages of germ cell development through the production of androgens, such as testosterone (T) and

http://dx.doi.org/10.1016/j.ygcen.2014.10.009 0016-6480/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Chauvigné, F., et al. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.10.009

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11-ketotestosterone (11-KT), whereas Lh stimulates spermiation (Schulz et al., 2010). The in vivo biological functions of Fsh during teleost spermatogenesis are however poorly known in part because the circulating levels of this hormone can be determined only in a few species. Recently, cDNAs encoding the Fshb subunit have been isolated and characterized in a broad variety of teleosts (Levavi-Sivan et al., 2010; Ohkubo et al., 2010; Zhou et al., 2010; Guzmán et al., 2013), including some Pleuronectiformes (flatfishes) such as the Senegalese sole (Solea senegalensis; Cerdà et al., 2008), Japanese flounder (Paralichthys olivaceus; Kajimura et al., 2001) and Atlantic halibut (Hippoglossus hippoglossus; Weltzien et al., 2003). These studies have used the fshb transcript levels in the pituitary as an indicator of the reproductive stage or the regulation of gonadotropin function. However, the fshb mRNA levels in the pituitary may not reflect the actual plasma levels of the circulating hormone (Swanson et al., 2003) and therefore to define the biological actions of Fsh in vivo it is necessary to develop methods for its accurate measurement in plasma and pituitary. Homologous immunoassays for Fsh, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), were first developed in salmonids using native Fsh purified from the pituitary (Suzuki et al., 1988; Swanson et al., 1989; Prat et al., 1996; Govoroun et al., 1998), and more recently in the killifish Fundulus heteroclitus (Shimizu et al., 2012). However, this strategy is highly challenging because of the elevated amount of pituitary glands required and the time-consuming chromatographic procedures involved (Swanson et al., 1991; Shimizu and Yamashita, 2002). An alternative method is the production of species-specific recombinant Fsh to continuously obtain high amounts of the hormone while simultaneously precluding contamination with other related pituitary glycoproteins. Recombinant Fsh peptides of several teleosts have been produced using different heterologous expression systems (Vischer et al., 2003, 2004; Levavi-Sivan et al., 2010; García-López et al., 2010; Kobayashi et al., 2010; Yu et al., 2010; Molés et al., 2011; Chauvigné et al., 2012; Chen et al., 2012; Kim et al., 2012). However, to date, successful homologous ELISAs using recombinant Fsh have only been reported for the Nile tilapia (Oreochromis niloticus) (Aizen et al., 2007) and the European sea bass (Dicentrarchus labrax) (Molés et al., 2012). Flatfishes comprise a group of teleosts of high commercial interest worldwide, but the aquaculture of some of these species, such as the Senegalese sole, is limited due to the reproductive failure of the first generation (F1) of males raised in captivity (Howell et al., 2011). To redress this situation, a number of recent studies have begun to investigate the reproductive biology of Senegalese sole (García-López et al., 2005, 2006; Cerdà et al., 2008; Guzmán et al., 2008, 2009; Forné et al., 2009, 2011; Marín-Juez et al., 2011, 2013; Chauvigné et al., 2010, 2012), including the regulatory pathways of Fsh and Lh on gene expression during spermatogenesis (Marín-Juez et al., 2011; Chauvigné et al., 2014ab). However, due to the lack of a specific Fsh immunoassay, the actual plasma levels of circulating Fsh during the spermatogenic cycle of sole, the potential role of Fsh during puberty, and whether farmed males have defects in Fsh synthesis and/or release as compared to wild males, are questions that remain to be addressed. The objective of the present study was therefore to develop and validate a homologous competitive ELISA for Senegalese sole Fsh. To date, similar assays for teleost Fsh have been established by using antibodies against the homologous Fshb subunit alone (Aizen et al., 2007; Molés et al., 2012; Shimizu et al., 2012), since the addition of the Cga in the immunogen protein can result in a high cross-reactivity of the assay with other circulating glycoprotein hormones. However, it has been shown that antibodies raised against a dimeric gonadotropin (b plus a subunit) are more sensitive in ELISA since they may more efficiently recognize the native form of the hormone in plasma (Jiang et al., 2010). In addition,

since the tertiary structure of the gonadotropins may be more conserved than the primary structure, antibodies against the Fsh dimer can potentially cross-react more effectively with the native Fsh of related species. In the present work, we produced a recombinant chimeric single-chain gonadotropin containing the sole Fshb subunit and the chicken CGA to develop a specific ELISA for sole Fsh and to test its feasibility for Fsh measurements in other flatfishes.

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2. Materials and methods

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2.1. Fish and sample collection

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Senegalese sole adult F1 breeders (937 ± 72 g; mean ± SEM) were obtained from the Spanish Institute of Oceanography (IEO, Santander, Spain) and the Institut de Recerca i Tecnologia Agroalimentàries (IRTA, Tarragona, Spain), whereas juvenile F1 males (15 ± 1 g) were obtained from Base Viva S.L. (Girona, Spain). Fish were transported to the laboratory and maintained under natural photoperiod and temperature as described by Agulleiro et al. (2006). At each sampling time, fish were anesthetized with 500 ppm phenoxyethanol, weighed and sacrificed by decapitation. Blood was collected from the caudal vein or the heart, centrifuged at 10,000g for 15 min at 4 °C, and the plasma stored at 80 °C until analysis. Testis and pituitary samples were removed, frozen in liquid nitrogen, and stored at 80 °C. The gonadosomatic index (GSI) was determined according to the formula: gonad weight (g)/fish weight (g) 100. Finally, pieces of testis were fixed in modified Bouin solution (75% picric acid and 25% formalin) and processed for histology as previously described (Agulleiro et al., 2007), or fixed in 4% paraformaldehyde (PFA) in PBS for immunohistochemistry and in situ hybridization (ISH) as previously described (Chauvigné et al., 2010). Biological samples (plasma and pituitary) were also obtained from wild common sole (Solea solea), and cultured Atlantic halibut, turbot (Scophthalmus maximus), Atlantic salmon (Salmo salar), gilthead seabream (Sparus aurata), and zebrafish (Danio rerio). The procedures employed relating to the care and sacrifice of animals followed local legislation and ethics committees in accordance with the European Union Council Guidelines (86/609/EU).

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2.2. Histological analysis

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Sections (7 lm) from the testicular cortex were stained with hematoxylin and eosin. The proportion of the various germ cell types was determined by light microscopy as previously described (Chauvigné et al., 2014b).

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2.3. Expression constructs

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The methylotrophic yeast Pichia pastoris was employed to produce the recombinant constructs by using the Pichia Expression kit (Life Technologies Corp.). The recombinant chimeric singlechain Fsh (rFsh-C) cDNA was synthesized in vitro (Life Technologies Corp.) following the same strategy as described previously (Chauvigné et al., 2012), and was subcloned into the pPIC9K expression vector, which contains the yeast methanol-inducible AOX1 promoter followed by the a-Factor signal sequence that directs the recombinant protein to the secretory pathway. The expression construct comprised the coding sequence of Senegalese sole mature Fshb subunit (GenBank accession No. ABW81403; Q2 from amino acid 19 to amino acid 115), followed by a 6 His-Tag, the carboxyl-terminal peptide (CTP) sequence of the human chorionic gonadotropin (hCG) b subunit as a linker, and the mature sequence of the chicken (Gallus gallus) CGA (GgCGA;

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GenBank accession No. XM_429886; from amino acid 25 to amino acid 120). Another construct containing only the mature GgCGA sequence bearing a 6 His-Tag in the C-terminus was also subcloned in the pPIC9 K vector (GgrCGA). Recombinant homologous single-chain Senegalese sole Fsh and Lh (SsrFsh and SsrLh, respectively) were produced in Chinese hamster ovary (CHO) cells as previously described (Chauvigné et al., 2012).

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2.4. Transformation of yeast and selection of positive clones

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The P. pastoris GS115 host strain (Mut+ His) was transformed by electroporation (ECM 830 Electroporation system, BTX) with the two constructs described above previously linearized with BglII (Roche). Different volumes (50–200 ll) of the electroporated cells were plated on minimal dextrose plates (2% dextrose, 1.34% yeast nitrogen base, 4 105% biotin, 2% agar) without His and incubated at 29 °C for 3–10 days. Colonies were replicated on different media, MM (0.5% methanol, 1.34% yeast nitrogen base, 4 105% biotin) with 2% agar, or YPD (1% yeast extract, 2% peptone, 2% dextrose) with 2% agar, supplemented with different concentrations (0.5–2 mg/ml) of Geneticin antibiotic (G418; Sigma–Aldrich) which enabled the initial selection of the positive transformants. Selected clones were confirmed as positive by PCR using an AOX1 forward primer (50 -GACTGGTTCCAATTGACAAGC-30 ) and a GgCGA reverse primer (50 -TGCAGTGACAGTCTGTGTGG-30 ). For the initial production tests, three different pairs of media, for the growth and production phases, respectively, were used: minimal glycerol/minimal methanol (MGY/MM; MM media with 1% glycerol or 0.5% methanol, respectively), buffered minimal glycerol/ buffered minimal methanol (BMG/BMM; same composition as MGY/MM but supplemented with 100 mM potassium phosphate, pH 6), and buffered minimal glycerol yeast/buffered minimal methanol yeast (BMGY/BMMY; same composition as BMG/BMM but supplemented with 1% yeast extract and 2% peptone). The growth phase was carried out in 4 ml of MGY, BMG or BMGY under shaking for 21–28 h at 29 °C until OD600 reached 2. The cells were then harvested by centrifugation at 2000g for 5 min at room temperature and resuspended at OD600 = 1–2 in 4 ml of MM, BMM or BMMY production media. Every 24 h, methanol was added at a final concentration of 0.5% and production was maintained up to 96 h. Samples were collected every 24 h, centrifuged at 14,000g for 5 min, and the supernatant and pellet frozen separately at 80 °C. After concentration of the supernatant by 100-fold using Amicon Ultra-15 3 K centrifugal filter devices (Millipore), the timecourse production for each clone was characterized by Western blot (see below) using an anti-6 His-Tag monoclonal antibodyHRP conjugate (Clontech Laboratories Inc.). As a negative control, GS115 cells transformed with the empty expression vector were used.

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2.5. Production and purification of recombinant proteins

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The final selected clone for each construct was grown in 500 ml of BMGY, and the production in BMMY at OD600 = 1 was carried out in 2 l during 72 h. The culture medium was recovered after centrifugation at 2000g for 5 min, and concentrated 50-fold using Centricon Plus-70 Biomax 3 centrifugal filter devices (Millipore). The concentrated supernatant was diluted 1:5 in phosphate buffer (PB; 20 mM Na3PO4, 500 mM NaCl, pH 7.4) containing 20 mM imidazole and purified by immobilized metal affinity chromatography using His GraviTrap columns (GE Healthcare). The columns were pre-equilibrated with PB plus 20 mM imidazole, according to the manufacturer instructions. After the diluted sample (60 ml/column) was run, columns were washed twice with PB containing 20 mM imidazole, and bound proteins were eluted

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with PB containing 500 mM imidazole. The eluate was further concentrated to 1 ml using Amicon Ultra-15 3 K centrifugal filter units (Millipore), and washed twice with standard PBS to reduce the final concentration of imidazole to 0.2 mM. Protein concentration was determined by the Bradford method (Biorad), and the purity of the recombinant hormones was estimated by densitometry analysis of SDS–PAGE gels stained with silver nitrate solution (Sigma–Aldrich) using the Quantity-One software (Bio-Rad Laboratories), as well as by Western blotting.

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2.6. Antibody production

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Polyclonal antibodies against rFsh-C were generated in rabbits by a commercial company (Agrisera, Sweden). Two rabbits were first immunized with 200 lg of rFsh-C each. Three subsequent immunizations (II–IV) were carried out every subsequent month with 100 lg of rFsh-C. Blood samples were collected 2 weeks before immunization (pre-immune serum) and 2 weeks after immunization III, in order to perform the titration test. The final bleeding was performed 2 weeks after the immunization IV. The antiserum was further purified by affinity chromatography on GgrCGA-bound columns to deplete the serum of anti-GgrCGA antibodies.

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2.7. Development of the ELISA for Fsh

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A competitive ELISA for Senegalese sole Fsh was developed using the specific antiserum generated against rFsh-C, before and after purification, as well as the rFsh-C to coat the plates and prepare the standards. The protocol was based on previously described ELISAs for Nile tilapia (Aizen et al., 2007) and European sea bass (Molés et al., 2012) Fsh with some modifications as described below. All steps were carried out at room temperature and without shaking except when specified.

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2.7.1. Coating of the plates and blocking Polystyrene ELISA microtiter plates (F96 Maxisorp NuncImmuno Plates; Nunc) were coated with 100 ll/well of 20 ng rFsh-C/ml carbonate buffer (50 mM sodium carbonate, pH 9.6), and incubated overnight at 4 °C. Two wells for the blank were not coated and two wells for the non-specific binding were coated with 100 ll/well of 20 ng BSA/ml carbonate buffer. The following day, coated plates were washed 3 times for 3 min each with 200 ll/well of PBST (PBS plus 0.05% Tween 20) and, to reduce the potential background, blocked with 200 ll/well of PBST buffer containing 2% normal goat serum (NGS; Sigma–Aldrich) for 1 h at 37 °C. The wells were washed as above prior to the addition of the samples and standards.

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2.7.2. Preincubation of standard and samples Plasma samples were used directly, whereas pituitaries were weighed and homogenized in 100 volumes of PBST, centrifuged at 5000g for 15 min at 4 °C, and the supernatant used for the ELISA or frozen at 80 °C. The standard (0.01–50 ng rFsh-C) and experimental samples (plasma and pituitary) were diluted in PBST with 0.5% NGS containing the primary antibodies diluted at 1:20,000 and pre-incubated overnight at 4 °C. To confirm the parallelism of the curves, plasma samples were diluted 1:2 to 1:200, while pituitary samples were diluted 1:103 to 1:105. To measure Fsh levels during the Senegalese sole reproductive cycle, plasma samples were diluted 1:10 and 1:20. After pre-incubation, 100 ll of each sample/standard was loaded into the wells of the previously blocked rFsh-C-coated plate and incubated overnight at 4 °C. All samples were processed in duplicate.

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2.7.3. Incubation with secondary antibodies and color development The next day, the plates were washed as described above and antigen–antibody complexes were detected by incubation with 100 ll/well of EIA grade affinity purified goat anti-rabbit IgG (H + L) HRP conjugated (Bio-rad Laboratories) diluted 1:5000 in PBST-0.5% NGS for 2 h. The plates were washed again and enzymatic color development was carried out by the addition of 100 ll/well of tetramethylbenzidine (TMB) peroxidase EIA substrate kit (Bio-rad Laboratories). The reaction was completed in darkness and stopped after 30 min with 100 ll/well 1 N sulfuric acid (Sigma–Aldrich). Absorbance was read at 450 nm with an Infinite M200 PRO plate reader (Tecan).

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2.7.4. Validation of ELISA for Senegalese sole Fsh The ELISA was validated for the determination of Fsh in plasma and pituitary of Senegalese sole and was tested for cross-reactivity with various teleost species. Displacement curves for plasma and pituitary samples were obtained by serial dilutions of the samples as indicated above and compared with the ELISA standard curve. The intra-assay coefficient of variation (CV) was calculated by assaying twelve replicates of the 0.5 ng/ml standard concentration at 50% and 80% of maximum binding on the same plate. The interassay CV was determined by measuring the same standard concentration (0.5 ng/ml) on ten different plates.

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2.8. Steroid determination

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Levels of 11-KT in plasma were determined in duplicate by enzyme immunosorbent assay (EIA; Cayman Chemical Company) as previously described (Chauvigné et al., 2012).

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2.9. Immunohistochemistry, ISH and immunofluorescence

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Immunohistochemistry and combined ISH and immunofluorescence were carried out as previously described (Cerdà et al., 2008; Chauvigné et al., 2012). ISH was carried out on pituitary sections using specific digoxigenin (DIG)-labeled riboprobes for Senegalese sole cga and fshb (Cerdà et al., 2008), and on testicular sections using sole gonadal soma derived factor (gsdf) and 3b-hydroxysteroid dehydrogenase (hsd3b) specific probes (Chauvigné et al., 2014b). Immunostaining of Fshb and Fshra in pituitary and testis, respectively, was carried out using the purified rFsh-C antiserum (1:250) and a Senegalese sole affinity-purified Fshra antibody (1:400) previously characterized (Chauvigné et al., 2012). For immunofluorescence, sections were counterstained with 40 ,6diamidino-2-phenylindole dihydrochloride (DAPI; 1:5000; Sigma–Aldrich) for 3 min before mounting. Control sections for ISH and immunostaining were respectively probed with sense probes or with the antibodies preadsorbed with rFsh-C or the peptide used for immunization as previously described (Chauvigné et al., 2012).

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2.10. Native-PAGE, SDS–PAGE and Western blotting

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For native-PAGE, samples were homogenized in Laemmli sample buffer without SDS and DTT, loaded into non-denaturing 12% gels and electrophoresed at 200 V for 4–5 h at 4 °C. For SDS–PAGE, samples were homogenized in Laemmli buffer containing SDS and DTT, denaturated at 95 °C, and separated in 12% gels. Testis samples of juvenile fish were homogenized in 150 mM NaCl, 50 mM Tris pH 7.4, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and a cocktail of protease inhibitors (Roche) and the supernatant mixed with Laemmli. In some cases, samples were deglycosylated by incubation with 500

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units of N-glycosidase F (PNGase F; New England Biolabs Inc.) for 3 h at 37 °C prior to SDS–PAGE. Proteins were blotted into nitrocellulose membranes, which after blocking in 5% nonfat dry milk in TBST (20 mM Tris, 140 mM NaCl, 0.1% Tween, pH 8) were incubated overnight at 4 °C with the rFsh-C antiserum diluted (1:2000) or a Senegalese sole-specific anti-Fshra antibody (1:600) (Chauvigné et al., 2012) in TBST with 1% nonfat dry milk. Bound antibodies were detected with horseradish peroxidase-coupled goat anti rabbit IgG antibody (1:5000; Santa Cruz Biotechnology Sc-2004) and Western HRP substrate (Millipore). Control membranes were incubated with preadsorbed antibodies as above.

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2.11. Statistics

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Data are presented as mean ± SEM and were statistically analyzed by the Student’s t-test, or one-way ANOVA, followed by the Duncan’s multiple range test. P value < 0.05 was considered statistically significant. To calculate the data in the ELISA and define the linearity of the serial dilutions of the standards and experimental samples, linearization of the sigmoid curves was carried out by using the logit transformation, logit (B/B0) = log [r/(1 r)], where r = B/B0, B represents the binding for each data point, and B0 the maximum binding. The slope of the curves and the coefficient of correlation r2 were calculated from regression lines from each serial dilution of standard and samples.

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3. Results

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3.1. Production of recombinants and antibody characterization

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Two recombinants were produced in the methylotrophic yeast P. pastoris, the chimeric single-chain rFsh-C containing the mature Senegalese sole Fsh b subunit in the N-terminal region and the mature GgCGA in the C-terminus, and the mature GgrCGA alone. The rFsh-C was used to generate an antiserum in rabbits, which was subsequently depleted of GgCGA antibodies by affinity chromatography on GgrCGA-bound columns. Both non-purified and purified rFsh-C antisera were then characterized by Western blot on recombinant single-chain SsrFsh and SsrLh produced in CHO cells (Chauvigné et al., 2012), as well as on rFsh-C, GgrCGA, and pituitary extracts. The non-purified antiserum was able to recognize the SsrFsh as one major band at 45 kDa in 12% SDS–PAGE, which was reduced to 36 kDa after PNGase F treatment, whereas it did not recognize the SsrLh (Fig. 1A). As expected, this antibody reacted with the rFsh-C and the GgrCGA revealing a smear ranging from 25 kDa to 70 kDa and 30 to 50 kDa, respectively, probably due to a high glycosylation rate since the size of some bands was reduced after PNGase F treatment (Fig. 1A). After purification of the rFsh-C antiserum, immunoblotting indicated that the antibody recognized only the SsrFsh and rFshC recombinants, but not the GgrCGA or SsrLh (Fig. 1B). In pituitary extracts subjected to SDS–PAGE major immunoreactive bands were detected at 20–27 kDa, 33 kDa and 100 kDa, which were no longer detected when the antiserum was preadsorbed with the rFsh-C recombinant. After PNGase F treatment, the 25-kDa and 100-kDa bands disappeared and novel bands at 15 kDa and 50–64 kDa were visible, indicating that sole Fshb is highly N-glycosylated in vivo (Fig. 1B). The 33-kDa, 27-kDa and 20-kDa bands were not affected by PNGase F, suggesting that Fshb is also posttranslationally modified in the pituitary by other mechanisms (Fig. 1B). Native-PAGE using the purified rFsh-C antiserum confirmed the results obtained with SDS–PAGE since the rFsh-C was revealed as a band at 35–40 kDa and a smear, whereas two major

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Fig. 1. Characterization of the Senegalese sole Fsh antisera by SDS–PAGE, native-PAGE and Western blot. (A) Representative immunoblots of sole homologous recombinant single-chain Fsh and Lh (SsrFsh and SsrLh, respectively) produced in CHO cells, and recombinant chimeric single-chain Fsh (rFsh-C) and chicken CGA (GgrCGA) produced in yeast, using the non-purified rFsh-C antiserum. (B) Representative immunoblots of recombinant hormones as above and pituitary extracts using the rFsh-C antiserum after depletion of anti-GgrCGA antibodies. Pituitary blots preabsorbed with rFsh-C (P) gave no signal. In A and B, plus or minus indicates preincubation of protein extracts with PNGase F prior to SDS–PAGE and Western blot. Molecular mass markers (kDa) are on the left.

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immunoreactive bands at 30 and 100 kDa were detected in the pituitary extract (Fig. 1B). The specificity of the purified rFsh-C antisera was also tested by immunohistochemistry and ISH on serial histological sections of the Senegalese sole pituitary gland. Cells located mostly in the periphery of the proximal pars distalis strongly expressed the cga (Fig. 2A and D) and to a lesser extent the fshb (Fig. 2B and E). The rFsh-C antisera labeled the native Fshb subunit in the cells specifically expressing the fshb transcripts (Fig. 2C and F), with most of them coexpressing the cga (Fig. 2D and E, arrows). However, some other cga positive cells did not express either fshb mRNA or Fshb protein product (Fig. 2D and E, arrowheads).

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3.2. Development and validation of an ELISA for Senegalese sole Fsh

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Competitive ELISA was developed for sole Fsh determination in plasma and pituitary using rFsh-C as standard and for coating the plates, and the rFsh-C antiserum, before and after purification, for detection (Fig. 3). Dilution tests were carried out to determine the appropriate dilutions of antigen in the wells (coating) and the

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antisera. The optimal concentration of rFsh-C in the wells to reach an optical density close to 1.0 by using a 1:20,000 dilution of antisera was 20 ng/ml, whereas the rFsh-C used to generate the standard curves ranged from 1 pg to 500 ng (Fig. 3A and B). The sensitivity of the assay, defined as the amount of rFsh-C sufficient to reduce the optical density determined in the absence of rFsh-C by 2 standard deviations, using the rFsh-C antisera before purification was 50 pg/ml (B/B0 > 93%) with the optical density decreasing as a linear function of rFsh-C concentration (r2 = 0.9892). The intra- and inter-assay CVs were 6.2% and 4.9% respectively. After purification of the antisera, the sensitivity of the assay was 10 pg/ml (B/B0 > 95%; r2 = 0.9932), and the intra- and inter-assay CVs were 5.8% and 5.4%, respectively. The cross-reactivity of the assay with GgrCGA, SsrFsh and SsrLh was also evaluated. Before purification, the cross-reactivity for GgrCGA was 25.0% and that of SsrLh was 11.2%. Surprisingly, however, the cross-reactivity of the assay for SsrFsh was only of 16.7%. (Fig. 3A). After purification of the antisera, the cross-reactivity for GgrCga and SsrLh was reduced to 0.7% and 1.0%, respectively, whereas that for SsrFsh increased to 40.2% (Fig. 3B). Therefore,

Fig. 2. Expression of cga and fshb, and Fshb immunolocalization, in Senegalese sole serial sections of the pituitary. (A–B) ISH of cga (A) and fshb (B) transcripts using DIGlabeled antisense riboprobes. (C) Immunostaining of Fshb using the purified rFsh-C antiserum. Sense probes or preadsorbed antibody gave no signal (insets in A, B and C). (D–F) Higher magnification on the regions indicated in A, B and C showing coexpression of cga, fshb and Fshb in some cells (arrow), whereas other cells were only positive for cga (arrowheads). Scale bars, 100 lm (A–C), 25 lm (D–F).


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Fig. 3. Competitive binding curves for the rFsh-C standard, and cross-reactivity with serial dilutions of SsrFsh, SsrLh and GgrCGA, using non-purified (A) and purified (B) rFsh-C antiserum. Data are the mean of 2–5 replicates.

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subsequent assays were carried out using only the purified rFsh-C antisera. Validation of the assay for the detection of Senegalese sole Fsh was evaluated by testing the parallelism between the standard curves for rFsh-C and the displacement curves obtained with serial dilutions of sole plasma (Fig. 4A) and pituitary extracts (Fig. 4B). The slope of the curve obtained for the standard rFsh-C (slope ± SEM, 0.72 ± 0.04) was not significantly different from that obtained with native plasma (slope ± SEM, 0.74 ± 0.05) or pituitary extracts (slope ± SEM, 0.67 ± 0.06) from two different males and females. The circulating levels of Fsh in these particular fish ranged from 9 to 40 ng/ml, with a working detection limit of 10 pg/ml plasma, whereas in the pituitary the total Fsh amount was of 6–13 lg/fresh pituitary tissue and the detection limit of 50 pg/ml.

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3.3. Validation of the Fsh ELISA for other pleuronectiform teleosts

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In order to test whether the Senegalese sole Fsh ELISA could be used to determine the levels of Fsh in other teleosts, displacement curves obtained from plasma and pituitaries from different pleuronectiform, perciform, salmoniform and cypriniform species were compared to that obtained with the recombinant rFsh-C. For pleuronectiforms, such as Atlantic halibut, common sole and turbot, the slope of the displacement curves (slope ± SEM) obtained with serially diluted plasma (Fig. 5A) and pituitary extracts (Fig. 5B) was not significantly different from that of rFsh-C (0.72 ± 0.04), being 0.74 ± 0.05 and 0.70 ± 0.03, 0.73 ± 0.08 and 0.68 ± 0.03, and 0.71 ± 0.08 and 0.75 ± 0.04, respectively. The plasma levels of Fsh were 4.68 ± 0.44 and 7.79 ng/ml in Atlantic halibut mature males and females, respectively, 3.95 ± 0.29 and 21.12 ± 2.94 ng/ ml in common sole spermiating males and immature females, respectively, and 4.06 ± 0.02 and 2.41 ± 0.51 ng/ml in turbot

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Fig. 4. Parallelism between the standard curve of ELISA using rFsh-C for the measurement of Senegalese sole Fsh and displacement curves obtained with serial dilutions of plasma (A) and pituitary (B) from two males and two females. Each point is the mean of a duplicate of two representative males and females.

females and males, respectively. The total amount of Fsh in the pituitary of Atlantic halibut, common sole and turbot was 4.3, 3.2, and 7.5 lg/fresh pituitary tissue, respectively. In more phylogenetically distant teleosts, such as the Atlantic salmon (Salmoniformes), the zebrafish (Cypriniformes), or the gilthead seabream (Perciformes), the slope of the curves obtained from plasma (Fig. 5C) and pituitary extracts (Fig. 5D) did not parallel that obtained with rFsh-C.

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The plasma levels of Fsh and 11-KT were assessed in Senegalese sole F1 juvenile fish showing differentiated but immature testes (Viñas et al., 2013) as well as in F1 adult males during the course of spermatogenesis (Fig. 6). The fish investigated were classified into five groups based on the GSI and the progression of spermatogenesis, which was evaluated by the proportion of the different germ cell types, spermatogonia, spermatocytes, spermatids and spermatozoa, in the seminiferous lobules of the cortical area of the testis (Fig. 6A–F), where more active spermatogenesis is found (Agulleiro et al., 2007). The testis of the juvenile males was very small (GSI < 0.01) and contained only spermatogonia and primordial Sertoli cells (Fig. 6A, F and G). In the four groups of adult males, the increase of GSI (0.048 ± 0.009, 0.101 ± 0.001, 0.118 ± 0.003, and 0.175 ± 0.017, in groups A1–A4, respectively) correlated with a drop in the proportion of spermatogonia and spermatocytes in the cortical testis and the concomitant increase in the number of spermatids and spermatozoa (Fig. 6B–E, F and G). In the juvenile fish, the Fsh plasma levels were 12.01 ± 0.98 ng/ml, while those of 11-KT were of only 0.07 ± 0.004 ng/ml (Fig. 6H). In adult males,

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Fig. 5. Parallelism between the standard curve of ELISA using rFsh-C for the measurement of Senegalese sole Fsh and displacement curves obtained with serial dilutions of plasma (A–C) and pituitary (B–D) from males and females of teleost species belonging to different orders: Pleuronectiformes (Atlantic halibut, common sole and turbot), Salmoniformes (Atlantic salmon), Perciformes (gilthead seabream) and Cypriniformes (zebrafish). Each point is the mean of a duplicate of a representative male and female.

Fig. 6. Plasma levels of Fsh and 11-KT in Senegalese sole males at different spermatogenic stages. (A–E) Representative photomicrographs of testis histological sections stained with hematoxylin and eosin of juvenile fish (A; group J1) and four groups of adults males showing increasing GSI (B–E, groups A1–A4). Scale bars, 20 lm (A–E), 10 lm (A inset). (F) GSI of the five groups of males studied. (G) Percentage of the different types of germ cells in the cortical region of the testis. (H) Fsh and 11-KT plasma levels in the different groups. In F–H, data (mean ± SEM; n indicated in parenthesis in panel F) with different superscript are significantly different (P < 0.05).

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the plasma levels of Fsh were the highest in adult fish at early spermatogenesis (35.82 ± 1.06 and 38.22 ± 1.60 ng/ml, in the A1 and A2 groups, respectively), showing a higher occurrence of spermatogonia and spermatocytes in the testis (Fig. 6G and H). When the testicular number of spermatocytes decreased, the Fsh plasma levels

dropped significantly (30.89 ± 3.51 ng/ml in the A3 group), and the levels decreased further during the period of maximum spermiation (14.52 ± 2.25 ng/ml in the A4 group) to the levels observed in juvenile fish (Fig. 6G and H). The circulating levels of 11-KT increased only in the males at early spermatogenesis with the


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lower GSI (15.02 ± 2.99 ng/ml in group A1), whereas androgen levels dropped as spermatogenesis progressed (7.04 ± 2.54 ng/ml and 4.37 ± 1.24 ng/ml, in groups A2 and A3). However, during spermiation the plasma levels of 11-KT increased again (19.83 ± 5.02 ng/ ml in group A4). 3.5. Expression pattern of the Fshra in the Senegalese sole juvenile testis The above findings were unexpected, since they indicated that the Fsh plasma levels in juvenile males were as high as in adults at the maximum spermiation stage. We therefore decided to investigate the Fsh pathway in juvenile testes, by characterizing the cell type-specific expression of the Fshra in the testis using a specific Senegalese sole Fshra antibody (Chauvigné et al., 2012). Immunoblotting for Fshra in whole testicular extracts showed two major reactive bands of 80 and 100 kDa which were also noted in the adult testis (Fig. 7A). Both reactive bands were no longer detected after preadsorption of the Fshra antisera with large amounts of the corresponding immunizing peptides (Fig. 7A, right panel). Immunolocalization of the Fshra in the juvenile testis revealed that the receptor was expressed in interstitial cells showing long protrusions, but surprisingly, not in the primordial Sertoli cells of the forming seminiferous lobules (Fig. 7B). The labelling of the interstitial cells disappeared when using the preadsorbed antibody suggesting that these cells expressed the Fshra.

To further characterize the Fshra-positive cells in the juvenile testis, we performed ISH with markers specific for Leydig and Sertoli cells (hsd3b and gsdf, respectively). The hsd3b staining was found in the interstitial cells corresponding to the expected location of Leydig cells (Fig. 7E), whereas gsdf specifically localized to primordial Sertoli cells delimitating the seminiferous lobules and enclosing spermatogonia (Fig. 7G). Hybridizations using hsd3b and gsdf sense probes were negative (Fig. 7, F and H). Combined Fshra immunofluorescence and ISH for hsd3b revealed a precise colocalization in the interstitial cells, indicating that the Fshraexpressing cells were indeed Leydig cells (Fig. 7I–L). However, when combined immunostaining and ISH was carried out using the Sertoli cell specific marker gsdf, the Fshra did not colocalize, suggesting that the primordial Sertoli cells in the juvenile testis did not express the Fshra (Fig. 7M–P).

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4. Discussion

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Previously reported immunoassays for teleost Fsh have been established using native or recombinant Fshb alone as antigens (Suzuki et al., 1988; Swanson et al., 1989; Prat et al., 1996; Govoroun et al., 1998; Aizen et al., 2007; Molés et al., 2012). In this study, a specific ELISA for Senegalese sole Fsh was developed using a novel approach based on the production of rFsh-C as a singlechain molecule. The N- and C-termini of the rFsh-C monomer were specific for the mature sole Fshb and the GgCGA, respectively, and

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Fig. 7. Pattern of expression of the Fshra in the testis of Senegalese sole juvenile fish. (A) Representative Fshra immunoblot of testicular extracts from juvenile males (J1) and adult males at early spermatogenesis (A1) using intact (left) or preabsorbed (right) Fshra-specific antibody. (B and C) General (B) and higher magnification (C) view of the immunolocalization of Fshra in the juvenile testis. (D) Preadsorbed antibody gave no signal. (E–H) Localization of hsd3b (E) and gsdf (G) expression by ISH. Control sections (F and H) probed with the respective sense probes were negative. (I–P) Bright-field (I and M) and epifluorescence (J–K and N–O) photomicrographs showing combined hsd3b (J) or gsdf (N) ISH and Fshra immunofluorescence (K and O) on the testis. L and P show the merged images. In J–L and N–P, cell nuclei were stained with DAPI (blue). Scale bars, 20 lm (B–H), 10 lm (I–P). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)


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produced by the yeast P. pastoris for subsequent purification of Fshb specific antibodies. The use of a dimeric gonadotropin in single-chain form can increase its immunogenicity (Jiang et al., 2010), as well as the specificity of the generated antibodies, since the dimer may expose more accurate epitopes of native Fsh than the b subunit alone. We also inserted the CTP of hCG as a linker between both subunits, which confers four additional O-linked glycosylation sites that may further increase the stability of the protein (Furuhashi et al., 1995). The rFsh-C produced in this study showed a molecular mass between 25 and 50 kDa, which was higher than the in silico predicted size of the recombinant (23.5 kDa) probably due to the high level of N-linked and O-linked glycosylation occurring in yeast cells (Aizen et al., 2007; Molés et al., 2012). As expected, initial ELISA assays using the crude rFsh-C antiserum showed a cross-reactivity with SsrLh produced in CHO cells of around 11%, which was higher than that reported in previous RIA (0.001–6%) (Swanson et al., 1989; Van der Kraak et al., 1992; Prat et al., 1996) or ELISA (0.001–2.3%) (Aizen et al., 2007; Molés et al., 2012; Shimizu et al., 2012) developed for teleost Fsh. Since such high cross-reactivity with Lh was most likely caused by the presence in the rFsh-C antiserum of anti-GgCGA antibodies that cross-reacted with the Senegalese sole Cga subunit, the antiserum was further depleted on GgrCGA-bound columns to recover anti-sole Fshb specific antibodies. Both Western blot and ELISA using the purified antiserum showed very low cross-reactivity with either SsrLh or GgrCGA (1% and 0.6% respectively), thus confirming the high specificity of the Fshb antibodies recovered. However, the ELISA only crossreacted with SsrFsh by 40%, which could be the result of a different glycosylation pattern of the protein in the two different hosts as reported for European sea bass Fsh produced in baculovirus or yeast (Molés et al., 2011). The specificity of the purified rFsh-C antiserum was also confirmed by Western blot and immunocytochemistry on the Senegalese sole pituitary. Immunoblots for Fsh in pituitary extracts under reducing conditions showed a reactive band at the expected size of the Fshb subunit (15 kDa) although its intensity was lower when compared to the upper bands suggesting that the dimer in SDS– PAGE was not completely reduced and dissociated, as observed for the Fsh from other pleuronectiform teleosts (Weltzien et al., 2004). However, in NATIVE-PAGE the major bands found in the pituitary were comparable to those detected for rFsh-C, which suggests that the pattern of post-translational modifications in yeast cells and sole may be conserved. Immunocytochemistry of Fsh in the pituitary showed that a subpopulation of cells of the proximal pars distalis coexpressed fshb mRNA and Fsh protein, as well as cga transcripts, as expected (Cerdà et al., 2008), thus corroborating the specificity of the purified rFsh-C antiserum. However, the signal intensity in the positive gonadotrope cells was low which could be caused by epitope masking when the dimer is intracellular. The ELISA developed for Senegalese sole Fsh showed a precise reproducibility, as the inter- and intra assay CVs were 5%, and a sensitivity of 10 pg/ml plasma, which is higher than previous assays for killifish (0.125 ng/ml plasma; Shimizu et al., 2012) and European sea bass (0.5 ng/ml; Molés et al., 2012) Fsh, with the exception of that developed for Nile tilapia Fsh (0.24 pg/ml; Aizen et al., 2007). The Fsh levels measured in the Senegalese sole plasma (9–42 ng/ml) were within a similar range to that found in the killifish (0.3–6 ng/ml), European sea bass (15–50 ng/ml) and Nile tilapia (2–10 ng/ml). In addition, good parallelism was observed for plasma and pituitary samples from other pleuronectiform species, including the Atlantic halibut, common sole and turbot (where the amino acid sequence of mature Fshb of halibut and common sole show 65.7% and 94.0% identity, respectively, with that of sole), with a detection limit of 50 pg/ml plasma, thus allowing the use of this assay for Fsh measurements in these species. In

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contrast, little or no parallelism was found with serial dilutions of plasma and pituitaries from Salmoniformes, Perciformes or Cypriniformes, such as the Atlantic salmon, gilthead seabream or zebrafish. In teleosts, plasma levels of Fsh are very low in immature males, peak at the onset of spermatogenesis, and decline at spermiation, which is in agreement with the role of Fsh during early germ cell development (Levavi-Sivan et al., 2010; Molés et al., 2011). However, most of the species investigated to date show cystic and cyclic spermatogenesis, whereas nothing is known on the changes in the circulating levels of Fsh in teleosts with semicystic spermatogenesis, such as the Senegalese sole. In this species, spermatids are released into the seminiferous lumen where they complete spermiogenesis without direct contact with the Sertoli cells (García-López et al., 2005; Chauvigné et al., 2014a). Nevertheless, previous studies in Senegalese sole have shown, as it occurs in teleosts with cystic spermatogenesis (Levavi-Sivan et al., 2010), that an increase of the fshb mRNA levels in the pituitary during winter, when meiosis of germ cells in the testis is increased, precedes the period of maximum spermiation in spring, during which the pituitary fshb expression levels can remain high (Cerdà et al., 2008). Accordingly, T and 11-KT plasma levels peak in winter, whereas during spring the androgen levels are reduced although maintained higher than during the summer (García-López et al., 2006; Cerdà et al., 2008). In this study, the Fsh plasma levels measured in Senegalese sole F1 males at different spermatogenic stages are in accordance with previous studies on fshb mRNA expression, although the samples collected did not cover the complete reproductive cycle. Thus, adult males with low GSI and the testis containing higher occurrence of early germ cells (spermatogonia and spermatocytes) showed the highest plasma levels of Fsh, whereas the plasma levels of Fsh dropped significantly at the stage when more spermatozoa are found in the testis. Since high mRNA levels of fshb in the pituitary are apparently maintained during this period (Cerdà et al., 2008), the reduction of plasma Fsh during spermiogenesis, suggests that fshb translation and Fsh accumulation and/or secretion in the pituitary, and/or clearance of the hormone in plasma, can change during the reproductive cycle. As expected, the high plasma levels of Fsh during early spermatogenesis in sole were associated with an increase of the 11-KT concentrations, which may result from the direct action of Fsh on Leydig cells expressing the Fshra (Chauvigné et al., 2012), as reported for other teleosts (Ohta et al., 2007; García-López et al., 2009, 2010; Alam et al., 2010). At the spermiation stage, however, low Fsh plasma levels were associated with a second rise of plasma 11-KT. This second peak of androgen is likely related to a Lh surge at this stage since Lh is more potent than Fsh at inducing the secretion of 11-KT in vitro in spermiating testis explants (Chauvigné et al., 2014b), although this conclusion should be corroborated in future studies. Nevertheless, our findings suggest that in Senegalese sole Fsh is involved in spermatogonia mitosis and entrance into meiosis during early spermatogenesis as has been proposed for teleosts with cystic spermatogenesis (Schulz et al., 2010). The role of Fsh during Senegalese sole larval development and puberty is largely unknown, although an early expression of fshb and cga transcripts in larvae at 1 day posthatching (dph), which peaks at mid metamorphosis (15 dph), has been reported (Guzmán et al., 2009). An interesting observation of the present study was the detection of Fsh in the plasma of juvenile males with differentiated but immature testes (i.e. containing only spermatogonia) at levels as high as in adult males at the maximum spermiation stage. Interestingly, by using specific markers of Leydig and Sertoli cells, hsd3b and gsdf, respectively, and a Fshra affinity purified antibody, we found that at this juvenile stage the Fshra was expressed in Leydig cells but not in the primordial Sertoli cells


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forming the tubules. This observation suggests that the action of Fsh at this early testicular stage likely relies mainly on steroids produced by Leydig cells, and that a direct action of Fsh on Sertoli cells must occur later in development. This hypothesis would be supported by the finding that during early and mid-spermatogenesis in adult sole Fsh promotes the expression of gsdf in vitro, which is involved in spermatogonial proliferation and differentiation (Sawatari et al., 2007; Chen et al., 2013), through steroid production (Chauvigné et al., 2014b). However, the 11-KT plasma levels in juvenile males were about 100-times less than in adults, and therefore whether a small local Fsh-mediated production of androgen in the testis is sufficient to support spermatogonia proliferation, or if there is also a Fsh-mediated production of estradiol or progestins at this stage (Schulz et al., 2010; Chen et al., 2013), remains unknown. In addition, a role of Lh at this stage cannot be discarded because Leydig cells from the juvenile testis also express the Lhcgrba (data not shown), and the sole Fshra can be cross-activated by Lh (Chauvigné et al., 2012). In the European sea bass, Lh plasma levels increase towards the end of the testicular differentiation and growth period in juvenile males, suggesting a potential synergic role of Lh with that of Fsh at this stage (Rodríguez et al., 2004; Carrillo et al., 2010). Therefore, future studies will be necessary to establish the role of Fsh, and perhaps also of Lh, during puberty in Senegalese sole. In summary, the results of this report show that recombinant chimeric Fsh produced in yeast can be used to generate specific antibodies against the Fshb subunit and develop a highly sensitive and specific ELISA for flatfish Fsh. This assay allowed for the first time the determination of the Fsh plasma levels during the semicystic spermatogenesis of Senegalese sole and provided indirect evidence for the role of Fsh during puberty and early spermatogenesis in this species. The ELISA developed here will also be a useful tool to decipher the role of this gonadotropin during gonadogenesis in other pleuronectiform species.

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Acknowledgments

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We thank P. Swanson (National Oceanic and Atmospheric Administration, USA) for valuable suggestions that inspired the methodological approach used in this work. We also thank S. Ibañez (IATS, CSIC, Spain) for excellent technical assistance during the production of the recombinants. Finally, we are indebted to A. Mateos (Base Viva S.L., Spain), N. Duncan (IRTA, Spain), O. Chereguini (IEO Santander, Spain), R. Cal (IEO Vigo, Spain), M.-L. Bégout (IFREMER, France), and B. Norberg and P.G. Fjelldal (Institute of Marine Research, Norway), for providing animals and biological samples used in this study. This work was Q3 supported by the Spanish Fundación Ramón Areces (to J.C.), and partially funded by the Spanish Ministry of Science and Innovation Q4 (MICINN) Aquagenomics project (CSD2007-00002 to S.Z.). M.B. was supported by a predoctoral fellowship (FPI) from MICINN. References

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Carrillo, M., Begtashi, I., Rodríguez, L., Marin, M.C., Zanuy, S., 2010. Long photoperiod on sea cages delays timing of first spermiation and enhances growth in male European sea bass (Dicentrarchus labrax). Aquaculture 299, 157–164. Cerdà, J., Chauvigné, F., Agulleiro, M.J., Marin, E., Halm, S., Martínez-Rodríguez, G., Prat, F., 2008. Molecular cloning of Senegalese sole (Solea senegalensis) folliclestimulating hormone and luteinizing hormone subunits and expression pattern during spermatogenesis. Gen. Comp. Endocrinol. 156, 470–481. Chauvigné, F., Tingaud-Sequeira, A., Agulleiro, M.J., Calusinska, M., Gómez, A., Finn, R.N., Cerdà, J., 2010. Functional and evolutionary analysis of flatfish gonadotropin receptors reveals cladal- and lineage-level divergence of the teleost glycoprotein receptor family. Biol. Reprod. 82, 1088–1102. Chauvigné, F., Verdura, S., Mazón, M.J., Duncan, N., Zanuy, S., Gómez, A., Cerdà, J., 2012. 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Development of a highly sensitive enzyme linked immunosorbent assay for human serum progesterone using penicillinase.

Detection of Leptospira-specific antibodies using a recombinant antigen-based enzyme-linked immunosorbent assay.

Development of concanavalin A-enzyme immunosorbent assay for glycated haptoglobin using polyclonal and monoclonal antibodies.

Development of an enzyme-linked immunosorbent assay for caffeine.

Development of Fluorescence-Linked Immunosorbent Assay for Paeoniflorin.

Immunodiagnosis of human dirofilariasis by enzyme-linked immunosorbent assay using recombinant DNA-derived fusion protein.

A serodiagnostic assay by microenzyme-linked immunosorbent assay for human anisakiasis using a monoclonal antibody specific for Anisakis larvae antigen.

A highly sensitive enzyme-linked immunosorbent assay for etoposide using beta-D-galactosidase as a label.

Development of a competitive radioimmunoassay and a sandwich enzyme-linked immunosorbent assay for recombinant human granulocyte colony-stimulating factor. Application to a pharmacokinetic study in rats.

Development a monoclonal antibody-based enzyme-linked immunosorbent assay for screening carotenoids in eggs.

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay.

Development of a keratinase activity assay using recombinant chicken feather keratin substrates.

Development and application of an indirect enzyme-linked immunosorbent assay using recombinant truncated Cap protein for the diagnosis of porcine circovirus-like virus P1.

Human Menopausal Gonadotropin versus Recombinant FSH in Polycystic Ovary Syndrome Patients Undergoing In Vitro Fertilization.

Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A.

Enzyme-linked immunosorbent assay for the phytotoxin cleistanthin A.

A two-site enzyme-linked immunosorbent assay for cytokeratin 8.

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay.

Comparison of a latex agglutination assay and an enzyme-linked immunosorbent assay for detecting cholera toxin.

recombinant-FSH versus standard protocol with recombinant-FSH in women of advanced age undergoing IVF.

Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay.

Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine.

Detection of cytomegalovirus antibodies by an enzyme-linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150.