Gonadotropins in European sea bass: Endocrine roles and biotechnological applications

General and Comparative Endocrinology xxx (2015) xxx–xxx

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General and Comparative Endocrinology

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Gonadotropins in European sea bass: Endocrine roles andbiotechnological applications

http://dx.doi.org/10.1016/j.ygcen.2015.05.0020016-6480/� 2015 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +34 964 319509.E-mail address: [email protected] (A. Gómez).

1 These authors contributed equally to this work and are listed in alphabeticalorder.

Please cite this article in press as: Mazón, M.J., et al. Gonadotropins in European sea bass: Endocrine roles and biotechnological applications. GenEndocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.05.002

María José Mazón 1, Gregorio Molés 1, Ana Rocha 1, Berta Crespo, Olivier Lan-Chow-Wing,Felipe Espigares, Iciar Muñoz, Alicia Felip, Manuel Carrillo, Silvia Zanuy, Ana Gómez ⇑Instituto de Acuicultura de Torre la Sal (CSIC), Ribera de Cabanes s/n, 12595 Torre la Sal, Castellón, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Available online xxxx

Keywords:GonadotropinsFshLhFishReproductionRecombinant hormones

Follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of thegonadal function in vertebrates. They act through specific receptors located in certain cell types found inthe gonads. In fish, the differential roles of these hormones are being progressively elucidated due to thedevelopment of suitable tools for their study. In European sea bass (Dicentrarchus labrax), isolation of thegenes coding for the gonadotropin subunits and receptors allowed in first instance to conduct expressionstudies. Later, to overcome the limitation of using native hormones, recombinant dimeric gonadotropins,which show different functional characteristics depending on the cell system and DNA construct, weregenerated. In addition, single gonadotropin beta-subunits have been produced and used as antigens forantibody production. This approach has allowed the development of detection methods for native gona-dotropins, with European sea bass being one of the few species where both gonadotropins can bedetected in their native form.

By administering recombinant gonadotropins to gonad tissues in vitro, we were able to study theireffects on steroidogenesis and intracellular pathways. Their administration in vivo has also been testedfor use in basic studies and as a biotechnological approach for hormone therapy and assisted reproduc-tion strategies. In addition to the production of recombinant hormones, gene-based therapies usingsomatic gene transfer have been offered as an alternative. This approach has been tested in sea bassfor gonadotropin delivery in vivo. The hormones produced by the genes injected were functional and haveallowed studies on the action of gonadotropins in spermatogenesis.

� 2015 Elsevier Inc. All rights reserved.

1. Reproductive cycle of European sea bass

European sea bass (Dicentrarchus labrax) belongs to the teleostorder Perciformes, family Moronidae. It is essentially aMediterranean species, and, like other fish species living at moder-ate latitudes, puberty and adult reproduction are seasonal eventsthat are highly dependent on environmental cues (e.g. photope-riod, temperature). Gonadal growth begins in September–October, with the mitotic proliferation of spermatogonia (Carrilloet al., 2009) in the testis and the beginning of vitellogenesis inthe ovaries, which have already undergone primary growth duringthe summer. Spawning typically takes place in winter (January–March). Males have a long spermiating period overlapping the win-ter reproductive cycle of females, followed by a sexual resting

period (Carrillo et al., 1995; Asturiano et al., 2000). Ovarian devel-opment in this species is defined as group-synchronous, i.e.clutches of follicles at different stages of development are simulta-neously present in the ovary and are spawned successively in up tofour batches (Mayer et al., 1990; Asturiano et al., 2000, 2002). Firstsexual maturity generally occurs during the second year of life inmales and a year later in females, but males can mature as earlyas at one year of age. Early sexual maturation is strongly influencedby the individual’s metabolic/growth status in this species.

In sea bass, as in all vertebrates, gametogenesis is completelydependent on the pituitary hormone follicle-stimulating hormone(Fsh) and steroids locally produced in response to both Fsh andluteinizing hormone (Lh). This dual control has been known forlong time, but it is unclear which parts of the gametogenic path-way are regulated by each hormone. Both gonadotropins Fsh andLh are glycosylated heterodimers formed by the non-covalentassociation of a common a-subunit (Cga) with a distinctb-subunit (Fshb or Lhb), which is what confers hormone specificity(Levavi-Sivan et al., 2010; Pierce and Parsons, 1981).

. Comp.

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2. Tools to measure gonadotropin subunits

In recent decades, different types of assays have been developedto measure gonadotropins in several species. These methods can begrouped into assays based on antigen–antibody recognition, whichmeasure the number of molecules or their mass (e.g. immunoas-says), and assays that determine a response of a biological systemto stimulation with Fsh or Lh (e.g. bioassays, both in vivo andin vitro). Traditionally, the assays used to determine gonadotropinlevels in fish have been radioimmunoassays (RIA) orenzyme-linked immune sorbent assays (ELISA) based mostly onnative gonadotropin subunits purified from fish pituitaries andtheir specific antibodies (Table 1), and more recently, also onrecombinant gonadotropin subunits (Aizen et al., 2007; Moléset al., 2012). The immunological determinations of gonadotropinsdo not necessarily reflect the biological signal perceived by theircognate receptors (Christin-Maitre and Bouchard, 1996). Pituitaryglycoproteins, including gonadotropins, are secreted as highlyheterogeneous forms that differ in carbohydrate composition (gly-cosylation), which affects many of the functional characteristics ofthese hormones, including their stability and metabolic fate, andthe interaction with their cognate receptors, i.e., their bioactivity(Ulloa-Aguirre et al., 2003). Furthermore, differential terminal gly-cosylation allows for the fine-tuning of the protein properties atthe target cell, without having to change the primary sequence(Olivares et al., 2009). More specific studies in mammals havedemonstrated that changes in the content of sialic acid affect thebioactivity of Fsh isoforms. In humans and rats, Fsh variants withmore acidic/sialylated glycosylation exhibit a longer plasmahalf-life but lower receptor binding activity and in vitro biologicalpotency than their less acidic counterparts (Ambao et al., 2009;Zambrano et al., 1996). In addition, in mammals, it has beenobserved that the molecular microheterogeneity of intrapituitaryFSH may change depending on the age, sexual development, and/orthe steroidogenic milieu, provoking a range of biological responses(Ambao et al., 2009; Rulli et al., 1999). Consequently, in vitro bioas-says constitute an ideal approach to determine some functionalaspects of gonadotropins.

Currently, several methods are available for measuring plasmaand pituitary gonadotropin levels in European sea bass: oneELISA for Lh derived from the purified native Lhb subunit(Mateos et al., 2006); two immunoassays for Fsh, a dot-blot(Molés et al., 2011b) and an ELISA (Molés et al., 2012), both basedon recombinant Fshb subunits generated in insect cells and Pichiapastoris, respectively; and one in vitro bioassay for Fsh based on aHEK-293 cell clone containing the sea bass fshr cDNA and a lucifer-ase reporter gene (Molés et al., 2011b). This bioassay, which wasthe first to be developed and validated in fish to measure Fshbioactivity in pituitary and plasma samples (Table 2), has provideda more complete vision of sea bass Fsh activity. The combination ofboth immuno- and bioassay methods makes it possible to evaluatethe relative bioactivity of defined amounts of Fsh according to thebiological stage of the animal.

3. Gonadotropins in the first year of life of sea bass: gonaddifferentiation period and first tentative maturation ofprecocious males

The timing of the appearance of Fsh and Lh-expressing cells inthe pituitary during ontogeny appears to be species-specific(Chen and Ge, 2012). The expression profiles of gonadotropin sub-unit genes in the pituitary gland of sea bass during early develop-ment, including the period of gonad differentiation (from 50 to300 days post hatching (dph)), have been studied using female-and male-dominant populations produced by size-grading (Molés

Please cite this article in press as: Mazón, M.J., et al. Gonadotropins in EuropeaEndocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.05.002

et al., 2007). In addition, isolated pituitaries and plasma from thosepopulations – available from 150 dph onwards – were used to ana-lyze gonadotropin content by means of a combination of bio- andimmunoassays (Molés et al., 2011b). The expression profile of cgawas equivalent for both populations. At 150 dph, fshb expressionwas similar in males and females and corresponded to a low con-tent of Fsh in the pituitary, but high plasma levels. At 200 dph fshbwas up-regulated in males, while it remained constant in females,and in both cases expression stayed high until the final samplingpoint at 300 dph. Throughout this period, the Fsh content in thepituitary increased without being secreted, whereas Fsh bioactivityin plasma remained high until 200 dph and then graduallydecreased in males and females. Additionally, plasma Fsh bioactiv-ity levels were higher in females than in males, and the Fsh biopo-tency in pituitary (measured as Fsh bioactivity (B):Fsh quantity (I)ratio, B:I) was also higher in females than in males. All these datasuggest an important role for Fsh at the time of gonad differentia-tion in this species, and a possible sexual dimorphism in the syn-thesis and potency of Fsh at this stage (Molés et al., 2011b). Onthe other hand, the gonadal expression of fshr peaked at 250 daysin both males and females (Felip, Zanuy, Gómez, unpublished).

During this period of gonad differentiation lhb expression andpituitary and plasma Lh levels were low until 150 dph in bothmales and females, after which expression levels increased andremained high from 200 dph onwards. From that moment a con-tinuous increase in Lh content in the pituitary was also observed,which reached the highest levels at 300 dph (the end of this exper-iment). Lh plasma levels in males followed the same trend while infemales Lh in plasma dropped at 300 dph (Molés et al., 2007). Theexpression of lhcgr increased at 250 days in male gonads, while nosignificant differences were found during the period analyzed infemales. The increase in Lh content and lhb/lhcgr expression couldbe related to the significant number of precocious males thatappeared in this population (Papadaki et al., 2005; Felip, Zanuy,Gómez, unpublished).

The expression of the gonadotropin subunit genes and levels ofgonadotropins in pituitary and plasma of juvenile male sea bassduring their first year of life have also been reported in other stud-ies related with research on precocious puberty. It was found thatall three subunits have similar expression profiles, peaking duringthe tentative gonadal maturation period (December–February)(Rodríguez et al., 2005), but lhb and cga levels were much higherthan those of fshb, a peculiarity that was not seen in adult males.The pituitary content of Fsh and Lh was higher in juveniles thatentered precocious puberty than in those that remained immatureat the same age. And in plasma, precocious animals showed higherFsh levels than non-precocious specimens during testiculargrowth, including spermiogenesis. This was also observed for Lh,which reached the highest values in precocious males in the sper-miation phase (Espigares, Carrillo, Rocha, Gómez, Ameur, Zanuy,unpublished results). On the other hand, the transcription of thesegenes was completely blocked when fish were exposed to a long(12-month) continuous light treatment, which is known to inhibitthe onset of precocious puberty in juvenile sea bass (Felip et al.,2008; Rodríguez et al., 2005). Other light regimes consisting ofshorter (4-month) continuous light periods administered before(June–September) or during (October–March) spermatogenesisinduced alternative expression profiles, but only for fshb (Felipet al., 2008).

4. Gonadotropins in the first maturation of male and female seabass and in adults

In adult male sea bass, all three pituitary gonadotropin subunitsare transcribed throughout the entire reproductive cycle, including

n sea bass: Endocrine roles and biotechnological applications. Gen. Comp.


Table 1Teleost species with immunoassays for gonadotropins.

Species Assay GTH Sensitivity (ng/ml) Intra-CV (%) Inter-CV (%) References

Hypophthalmichthys molitrix RIA Gth 0.58 6.8 8.6 Kobayashi et al. (1985)Oncorhynchus tschawytscha RIA Gth 0.44 6.0 7.9 Kobayashi et al. (1987)Oncorhynchus keta RIA Fsh <2 3.6 10.4 Suzuki et al. (1988)

Lh <2 2.5 9.8Micropogonias undulatus RIA Gth 0.05 – 14.5 Copeland and Thomas (1989)Oncorhynchus kisutch RIA Fsh �0.28 – – Swanson et al. (1989, 1991)

Lh �0.28 – –Carassius auratus ELISA* Gth 0.125 5.0 9.0 Kah et al. (1989)Oncorhynchus mykiss ELISA* Lh 0.070 4.2 6.3 Salbert et al. (1990)Sparus aurata RIA Gth 0.3 10–11 – Zohar et al. (1990)Pagrus major RIA Lh 0.78 6–7 11 Tanaka et al. (1993)Oncorhynchus mykiss RIA* Fsh 2.34 – – Prat et al. (1996)

Lh 0.26 – –M. saxatilis x M. chrysops ELISA Lh 0.156 7–8 8–15 Mañanós et al. (1997)Oncorhynchus mykiss RIA Fsh 0.87–1.42 4.6 9.8 Govoroun et al. (1998)

Lh 0.1–0.2 5.9 8.3Oncorhynchus mykiss RIA Fsh 1 – – Santos et al. (2001)Seriola dumerilii RIA Lh 0.25 – <15 García Hernández et al. (2002)Dicentrarchus labrax ELISA Lh 0.65 11.7 11 Mateos et al. (2006)Oreochromis niloticus ELISA Fsh 0.00024 8 12.5 Aizen et al. (2007)

Lh 0.0158 7.2 14.8Dicentrarchus labrax Dot-blot Fsh 162.8 9.8 11.5 Molés et al. (2011b)Dicentrarchus labrax ELISA Fsh 0.50 2.12 5.44 Molés et al. (2012)

* Heterologous assay adapted from Molés (2011).

Table 2Species with bioassays for gonadotropins.

Species Cell line Reporter gene GTHR Sensitivity Intra-CV (%) Inter-CV (%) References

Homo sapiens HEK 293 Luciferase FSHR – – – Tilly et al. (1992)Homo sapiens Y-1 – FSHR – – – Kelton et al. (1992)Homo sapiens HEK 293 Luciferase LHR 0.3 ng/ml 18 13 Jia et al. (1993)Rattus norvegicus Ltk� – FSHR 0.3 UI/l 5.2 16.2 Gudermann et al. (1994)Homo sapiens CHO Luciferase FSHR <3 UI/l (1.1 ng/ml) – – Albanese et al. (1994)Homo sapiens CHO – FSHR 6.2 UI/l 7.3 10.3 Tano et al. (1995)Homo sapiens CHO Luciferase FSHR <4 UI/l 8 16 Christin-Maitre et al. (1996)Homo sapiens CHO Luciferase FSHR 0.010 UI/l – – Kajitani et al. (2008)Dicentrarchus labrax HEK 293 Luciferase Fshr 0.104 ng/ml 9.3 10.9 Molés et al. (2011b)Anguilla anguilla HEK 293 Luciferase Fshr – – – Minegishi et al. (2012)Anguilla japonica HEK 293 Luciferase Lhr – – – Minegishi et al. (2012)

Adapted from Molés (2011).

M.J. Mazón et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx 3

the period of sexual resting (August) (Mateos et al., 2002, 2003),and show similar profiles. The expression of fshb, lhb and cga isup-regulated from October (initiation of gametogenesis, stage II)to February (spermiation, stage V), concomitantly with the gona-dosomatic index (GSI) and the accumulation of Lh protein in thepituitary, with all genes being sharply down-regulatedpost-spermiation (Mateos et al., 2003). This profile of fshb expres-sion found in male sea bass is similar to that observed in othermultiple spawners such as goldfish, red sea bream or Atlantic hal-ibut (Levavi-Sivan et al., 2010) and also in Senegalese sole (Cerdàet al., 2008), but different from salmonids and Atlantic cod, whereonly fshb is up-regulated at the onset of pubertal testis growth,partially down-regulated with the completion of spermatogenesis,and fully down-regulated in spent fish; while lhb only increases inthe more advanced stages of development (spermiogenesis, sper-miation), reaching peak values at the time of spawning(Campbell et al., 2003; de Almeida et al., 2011; Gomez et al.,1999; Maugars and Schmitz, 2008).

The analysis of the gonadotropin hormones in plasma revealedthat Fsh levels increase during testicular growth (stages II–IV) anddecline before spermiogenesis-spermiation (stage V) (Molés et al.,2012) (Table 3). However, plasma Lh levels increased gradually asspermatogenesis progressed, with a significant elevation in late


recrudescence (stage IV), which was maintained in full spermiation(stage V) (Rocha et al., 2009).

In female sea bass, the expression of the gonadotropin subunitsduring the reproductive cycle is not known, but the pituitary con-tent of the hormones showed overlapping profiles for Fsh and Lh,as was the case for the expression of the subunits in adult males,with high levels during late-/post-vitellogenesis andmaturation-ovulation (Molés et al., 2011b). However, the plas-matic profiles were quite different for both gonadotropins. Themaximum plasma Fsh levels were detected from early vitellogene-sis to post-vitellogenesis, whereas a significant decrease wasobserved in maturation-ovulation (Molés et al., 2012). These datapoint to the active synthesis and secretion of Fsh during the courseof vitellogenesis, whereas during maturation–ovulation Fsh secre-tion would decrease, while accumulating in the pituitary.Conversely, plasma Lh levels peaked at maturation–ovulation(Rocha et al., 2009), the time when plasma Fsh reached minimumvalues.

Altogether, data on the pituitary/plasma content of gonadotro-pins in adult females suggest a differential control of Fsh/Lh releaseto the bloodstream in sea bass and an important role for Fsh in theregulation of early-mid phases of spermatogenesis and vitellogen-esis, as is the case in salmonids (Breton et al., 1998; Prat et al.,



Table 3Approximate plasma levels of gonadotropins in some teleost species.

Species GTH Males (�ng/ml) Females (�ng/ml) References

Immat. Test. Rec. Sperm. Immat. Vtg Mat. Ovul. Postovul.

Oncorhynchus keta Fsh – – – – – – 40 – Suzuki et al. (1988)Lh – – – – – – 70 –

Oncorhynchus rhodurus Fsh – – – – – – 2 – Suzuki et al. (1988)Lh – – – – – – 18 –

Oncorhynchus mykiss Fsh <2 5 – <2 8 – – – Suzuki et al. (1988)Lh <2 1.5 – <2 2 – – –

Oncorhynchus mykiss Fsh <2.5 5–6 3–4 <2.5 17 <7 34 – Prat et al. (1996)Lh <0.3 <0.3 3 <0.3 <0.3 2 70 –

Oncorhynchus mykiss Fsh – – – 8 17 10 8 >25 Breton et al. (1998)Lh – – – <0.5 1.5 21 13 4.5

Oncorhynchus mykiss Fsh 2 9 4 3.5 15 3–8 – 15 Gomez et al. (1999)Lh N.D. <0.5 0.8 N.D. <0.1 2.5–6 – 15

Oncorhynchus mykiss Fsh – – – 3 11 – 32 – Santos et al. (2001)Oncorhynchus kisutch Fsh <2 50 20 <2 30 – 10–15 – Swanson (1991)

Lh <1 <1 5–12 <1 <1 – 15–40 –Morone saxatilis Lh – – 5 – 1 4.5 – 3 Mylonas and Zohar (2001)Oreochromis niloticus Fsh – – – – 5–6 – 5–6 – Aizen et al. (2007)

Lh – – – – 5–6 – 8–10 –Dicentrarchus labrax Lh 1.2 2–3 2.6 <0.5 1 2.7 2.7 2.4 Rocha et al. (2009)Dicentrarchus labrax Fsh 18 29–42 15 24 32 16 16 22 Molés et al. (2012)Solea senegalensis Fsh 12 30–38 14 Chauvigné et al. (2014)

Immature (Immat.); Testicular Recrudescence (Test. Rec.); Spermiation (Sperm.); Vitellogenesis (Vtg); Maturation (Mat.); Ovulation (Ovul.); Post-ovulation (Postovul.); Nodetectable (N.D.). Adapted from Molés (2011).


1996; Swanson, 1991), whereas Lh would be involved in spermio-genesis, spermiation and maturation-ovulation.

When Fsh bioactivity was assessed in the pituitary of sexuallymature females, the highest values were observed duringlate-/post-vitellogenesis and maturation–ovulation, while they fellduring atresia at the end of the reproductive season. In plasma, Fshbioactivity peaked at vitellogenesis and declined gradually there-after (Molés et al., 2011b). These trends are similar to thoseobserved when measuring the amount of Fsh by immunoassay,as described above. However, Fsh biopotency (B:I ratio) in pituitarywas higher during pre-vitellogenesis and then fell as the reproduc-tive cycle progressed to reach lower values than those obtainedduring gonad differentiation. These data suggest the presence ofFsh isoforms in the pituitary with different biopotencies that mightbe associated with the reproductive status, indicating that a lessbiopotent, but probably longer-lived Fsh, would be necessary dur-ing ovarian growth in mature females (Molés et al., 2011b).

As regards the gonadotropin receptors, the expression of seabass fshr is restricted to the gonads in adult males and females(Rocha et al., 2007). The specific cell type expressing fshr in seabass testis has not yet been determined. In mammals, this geneis exclusively expressed in Sertoli cells, but in some fish speciesit has been found that fshr is also expressed in Leydig cells(Chauvigné et al., 2012; García-López et al., 2009, 2010; Ohtaet al., 2007). By contrast, lhcgr expression in sea bass is not limitedto the gonads and its mRNA can be found in different tissues.Whether these mRNAs are translated into protein in these tissuesis not yet known.

Changes in gonadal expression of the gonadotropin receptorgenes fshr and lhcgr in male and female European sea bass havebeen analyzed throughout a complete gonadal cycle (Rocha et al.,2009). In males, the fshr expression profile was seen to be bimodal.Levels were gradually up-regulated from the immature to earlymaturation stage, followed by a progressive and significantdown-regulation during mid and late maturation, only to beup-regulated once again in fully spermiating males. Regardinglhcgr expression, a small, non-significant up-regulation was firstobserved during early maturation. Expression was thendown-regulated during mid and late maturation, peaking in full


spermiation and reaching its lowest levels in spent fish (Rochaet al., 2009). In the ovary, fshr levels were very low inpre-vitellogenic females, progressively up-regulated in vitellogenicfish, peaking during maturation/ovulation and fullydown-regulated after ovulation. Lhcgr transcript levels remainedconsistently low during pre- and early vitellogenesis, wereup-regulated during late and post-vitellogenesis and reached theirhighest values during maturation/ovulation. Expression wasdown-regulated in post-ovulating females (Rocha et al., 2009).The profiles described above should be contextualized in light ofthe European sea bass mode of gonadal development. During theannual cycle, fish gonads undergo dramatic changes in cellularcomposition. In males, these changes result in a dilution ofSertoli cell-derived mRNAs by germ cell mRNAs during germ cellproliferation, which diminishes towards spawning, onceRNA-poor haploid cells gradually become prevalent (Schulz et al.,2005). Due to the group synchronous type of ovarian development,the ovaries in female European sea bass contain heterogeneousoocyte populations that develop at different rates, which couldobscure stage-specific transcripts. However, in a gene expressionsurvey performed on isolated European sea bass ovarian follicles,primary oocytes showed low transcript amounts of gonadotropinreceptors. Follicles at the beginning of the secondary growth phaseshowed elevated mRNA amounts of fshr. Early-to-mid vitellogenicfollicles showed high levels of fshr, while mid-to-late vitellogenicfollicles expressed increasing amounts of lhcgr (García-López et al.,2011). Overall, the analysis of these expression profiles in relationto changes in plasma levels of important reproductive hormones,steroidogenesis-related transcripts and histological data, allows usto conclude that the expression of fshr is connected with the earlystages of gonadal development and with the spermiation/maturation–ovulation periods. The lhcgr expression profile seen in bothsexes supports the involvement of Lh in the regulation of the finalstages of gamete maturation and spermiation/ovulation.

5. Regulation of the gonadotropin system in sea bass

The expression of gonadotropins is closely controlled by a feed-back from sexual steroids and Gnrh. The implantation of estradiol




(E2), testosterone (T) or a non-aromatizable androgen in adult seabass during the sexual resting period almost suppressed the basalexpression of fshb, while slightly up-regulating that of lhb. Gnrhhad no effect on fshb levels, but up-regulated pituitary lhb andcga expression (Mateos et al., 2002). Moreover, treatment withE2 inhibited the expression of the fshb gene in pituitary cells cul-tured in vitro (Muriach et al., 2014), showing that the negativefeedback of E2 observed in vivo is acting, at least in part, directlyat pituitary level. Regarding the transcriptional control of sea bassgonadotropin receptor genes, some studies performed on the pro-moter region of fshr have shown the existence of functional tan-dem binding sites for the transcription factors Sp1/Sp3 (Crespoet al., 2012) and the presence of an E-box where Usf2 can bind toactivate gene expression (Lan-Chow-Wing et al., 2014). Althoughthere is very limited information about fish fshr promoters(Hayashi et al., 2010), the existence of an E-box has also beendescribed in some mammalian fshr promoters (Goetz et al., 1996;Putowski et al., 2004; Xing and Sairam, 2001). Nevertheless, noneof these factors explains how the spatial expression is restrictedto gonad level.

As regards the regulation of gonadotropin secretion, in sea bass,as in other fish species (Levavi-Sivan et al., 2010), different Gnrhanalogs are able to stimulate Lh release from gonadotrophsin vitro (Forniés et al., 2003) and in vivo (Mañanós et al., 2002).However, less information exists for sea bass on the stimulationof Fsh release by Gnrh, but preliminary information provided bythe recently available Fsh assay indicates that low doses of Gnrh,which can efficiently induce Lh release from pituitary cellsin vitro, are nevertheless incapable of stimulating Fsh release(Espigares, Zanuy, Gómez, unpublished). The in vivo effects ofKiss-10 forms on gonadotropin release have also been studied insea bass. Gonadotropin levels were measured in blood after sys-temic administration of the peptides, and it was found thatKiss2-10 was more potent than Kiss1-10 in inducing Lh secretionin pre-pubertal and pubertal males, while Fsh release could onlybe observed in pre-pubertal males injected with Kiss2-10 (Felipet al., 2009). In a recent study longer forms of kisspeptins(Kiss1-15 and Kiss2-12) were injected directly into the brain(intracerebroventricular administration) of mature male sea bass.The injections provoked an increase of Lh in plasma, Kiss2-12 beingthe most potent peptide, whereas none of the peptides had aneffect on Fsh release (Espigares et al., 2015). This same Kiss2-12peptide was able to induce Lh release, but not Fsh release, fromcultured pituitaries of adult males during the spermiating period,when both hormones are present in the pituitary (Espigares,Zanuy, Gómez, unpublished).

6. Sea bass recombinant gonadotropins: functionality andactions

6.1. Expression systems and DNA constructs

Although both native gonadotropins have been purified in seabass (Mateos et al., 2006; Molés et al., 2008), the availability ofcDNA coding for their subunits allowed the production of sea bassrecombinant gonadotropins, which provides a continuous sourceof these hormones with no cross-contamination. We used two dif-ferent approaches in both expression systems and DNA constructs.The insect cell line Sf9 infected with recombinant baculovirus andstable clones of mammalian CHO cells containing standard recom-binant expression plasmids were used as cellular expression sys-tems. As DNA constructs, the single subunits were used indifferent vectors, resulting, after co-infection/transfection, in theproduction of non-covalently bound dimers that resemble thenative heterodimers. Alternatively, we used a fused open reading


frame coding for both subunits, which generated covalently boundsingle-chain (sc) gonadotropins, with the carboxy-terminal peptideof the human chorionic gonadotropin (CTP) acting as a linkerbetween the subunits. All these sea bass recombinant gonadotro-pins are bioactive, but the insect cell based system producedhigher amounts of extracellular hormone. The gonadotropins pro-duced in different systems showed the same behavior in terms ofreceptor activation, but displayed some different characteristics,as explained below (Molés et al., 2011a).

6.2. Receptor activation and specificity

Fshr and Lhcgr are members of the G-protein coupled receptor(GPCR) superfamily. Hormone binding to their extracellulardomain induces conformational changes, initiating the transduc-tion of extracellular signals to the inside of the target cell by acti-vating Gs-proteins (Pierce et al., 2002). This activation increases theintracellular levels of cAMP, guiding the translocation of the cat-alytic subunit of protein kinase A (PKA) to the nucleus, and laterthe phosphorylation of cAMP-response element binding protein(CREB), which bind to CRE sites in specific gene promoters(Mukherjee et al., 1996; Salvador et al., 2001). In mammals, thespecificity barriers between each gonadotropin receptor coupleare such that no cross-signaling occurs under physiological condi-tions in which hormone concentrations are low (Braun et al., 1991;Moyle et al., 1994). Evidence indicates that the specificity of thepiscine gonadotropin receptors is less obvious (Bogerd et al.,2005). Human Embryonic Kidney (HEK) 293 cell lines stablyexpressing sea bass Fshr or Lhcgr and the firefly luciferase geneunder the control of CRE-binding sites have been used to evaluatesea bass gonadotropin receptor ligand specificity (Rocha et al.,2007). Both native and different homologous recombinant sea bassgonadotropins were used (Molés et al., 2008, 2011a). None of thegonadotropins showed cross-receptor binding at any of the con-centrations tested, as they were only able to stimulate their cog-nate receptors. These specific interactions have been found inother fish species, such as tilapia (Aizen et al., 2012), rainbow trout(Sambroni et al., 2007), Manchurian trout (Ko et al., 2007), andchub mackerel (Nyuji et al., 2013); while amago salmon (Obaet al., 1999), Atlantic salmon (Andersson et al., 2009), African cat-fish (Vischer et al., 2003), Senegalese sole (Chauvigné et al., 2012)and zebrafish (So et al., 2005) have shown promiscuous activationof Fshr by Lh.

Different results were obtained when heterologous mammalianhormones (human FSH and CG; bovine Fsh and Lh) were used toactivate sea bass gonadotropin receptors. Bovine Fsh was able toactivate sea bass Fshr, but human FSH was only effective whenhigh doses (8–40 IU/ml) were applied. Both human and bovineFSHs were able to stimulate sea bass Lhcgr in a dose-dependentmanner. Human CG only activated Lhcgr, but not the Fshr in seabass (Rocha et al., 2007; Molés et al., 2011a).

6.3. Biopotency of the recombinant gonadotropins

The combined use of specific immuno- and bio-assays for seabass Fsh allowed us to determine the B:I ratio of particularamounts of the different recombinant Fshs produced. The dataobtained showed that the insect-derived Sf9-Fsh was more biopo-tent than CHO-Fsh, but less so than the single-chain CHO-scFsh(Molés et al., 2011a). These differences could be due to a differenttype and degree of glycosylation depending on the expression sys-tem used (Ambao et al., 2009; Grossmann et al., 1997; Olivareset al., 2009). It is known that insect cells, unlike mammalian cells,are not able to add sialic acid residues, producing instead glycopro-teins that contain high mannose-type oligosaccharides (Kost et al.,




2005). The gonadotropin isoforms that contain more sialylatedoligosaccharides are long-lived but less biopotent. This supportsour findings that Sf9-Fsh is more potent than CHO-Fsh. In the par-ticular case of CHO-scFsh, the higher B:I ratio might be influencedby other aspects, such as the fact of being a fusion protein and not adimer, or the presence of the CTP (Ben-Menahem and Boime, 1996;Fares et al., 1992).

6.4. In vitro activity and steroidogenesis: steroid production and geneexpression

In vitro stimulation of sea bass gonad explants with recombi-nant or native gonadotropins has demonstrated the steroidogeniccapacity of both Fsh and Lh (Molés et al., 2008, 2011a; Mazónet al., unpublished, Muñoz et al., unpublished). E2 productionwas equally stimulated by Fsh and Lh in cultured ovarian explants.In in vitro cultures of sea bass testis, Lh stimulated 11KT synthesismore potently than Fsh (Molés et al., 2011a), as it has also beendescribed in other teleost species (Kagawa et al., 1998; Planasand Swanson, 1995). However, in channel catfish and zebrafish,Fsh has been demonstrated to be more potent than Lh in stimulat-ing androgen release (García-López et al., 2010; Zmora et al., 2007).These differences in 11KT levels could be due to the differentdevelopmental stages of the gonadal tissue used in all these exper-iments, and further research is needed to help elucidate the role ofeach hormone at each spermatogenic stage.

Steroid hormones result from the conversion of cholesterol, as aresult of the activity of different steroidogenic enzymes. The gate-keeper of this process is the steroidogenic acute regulatory protein(StAR) (Stocco, 2000), and the expression of its coding gene can bemodulated by the action of Fsh as has been shown in several spe-cies (Balasubramanian et al., 2008; García-López et al., 2010;Pescador et al., 1997), including sea bass (Mazón et al., unpub-lished). The ability of gonadotropins to modulate the expressionof steroidogenic enzymes has been widely described in the litera-ture for mammals (Gyles et al., 2001; Zhang and Mellon, 1996)and fish species (García-López et al., 2010; Paul et al., 2010;Sambroni et al., 2013; Skaar et al., 2011; Tong and Chung, 2003),and therefore the steroid production differences among speciesmight also be a consequence of their ability to modulate the geneexpression or the activity of the steroidogenic enzymes.

6.5. Intracellular pathways in the gonads

As mentioned above, the cAMP/PKA pathway is the main intra-cellular route used by gonadotropin receptors to transduce theirsignal. This pathway, which is well defined in mammals, inducessteroid synthesis by regulating the expression and activity of ster-oid enzymes, such as StAR (Ariyoshi et al., 1998; Manna et al.,2003; Stocco, 2000) and Cyp19a (Young and Mcphaul, 1998), viathe activation of CRE sites localized at promoter level. Similarlyto findings in mammals, an up-regulation of star and cyp19a1expression was observed in sea bass gonads following Fsh orcAMP analog stimulation (Mazón et al., unpublished). BesidescAMP/PKA, Fsh signals can be transduced through other pathways,such as PKC or MAPK (Hunzicker-Dunn and Maizels, 2006;Ulloa-Aguirre et al., 2007), which have been less well studied infish. In Atlantic croaker (Benninghoff and Thomas, 2006) and tila-pia (Paul et al., 2010), blocking of the MAPK route has been shownto affect hCG-induced steroid levels. In a similar manner, blockingMAPK activity in sea bass inhibited Fsh-stimulated E2 and T pro-duction, an inhibition that was accompanied by down-regulationof the expression of some steroidogenic enzymes, includingcyp17a1 and star (Mazón et al., unpublished).


6.6. In vivo stability

The stability or half-life of recombinant sea bass gonadotropinshas been evaluated to increase knowledge of the pharmacokineticsinvolved before undertaking any in vivo functional experimenta-tion. The results showed that CHO-produced single-chain gonado-tropins are more stable in plasma than Sf9-derived gonadotropins(Molés et al., 2011a). This behavior could be due to glycosylationdifferences, since the content in terminal sialic acid residues deter-mines the rate at which glycoproteins are cleared from circulation(Ambao et al., 2009; Olivares et al., 2009; Ulloa-Aguirre et al.,2001). Moreover, the fusion of the two subunits and the presenceof O-linked oligosaccharides in CTP may additionally contributeto prolonging the circulating half-life of single-chain gonadotro-pins (Ben-Menahem and Boime, 1996; Fares et al., 1992; Kleinet al., 2003). In a comparison of the hormones, Fsh showed greaterstability than Lh within each production system (Molés et al.,2011a). A relationship between the stability and the physiologicalrole of the hormones could be hypothesized. Maintained highlevels of Fsh in the bloodstream would be necessary forlong-term stimulation of gonadal growth, whereas short-termincreases in plasma Lh would have a more specific effect in a cer-tain phase of the reproductive cycle and would probably be clearedmore quickly.

7. Somatic gene transfer in muscle: a delivery route forgonadotropins

In 1990, Wolff et al. (1990) demonstrated that naked DNA wasinternalized and expressed when injected into skeletal muscle.This technology, referred to as somatic gene transfer, allows a for-eign gene to be introduced into adult tissue to achieve long-lastinggene expression, without it being permanently incorporated in thegenome or altering the immune system (Tonheim et al., 2008).Numerous target tissues have been tested for DNA injection(Escoffre et al., 2010), with muscle being one of the most attractivedue to its abundance, accessibility and rich blood supply (Miyazakiand Miyazaki, 2008). In fish, the use of this methodology has beenscarcely developed and any undertaking in this respect has focusedon immunoprophylaxis (Kurath, 2008). In mammalian models,naked DNA-based gene therapies have been tested to treat defi-ciencies in hormones or other circulating proteins (Fewell et al.,2001; Ratanamart and Shaw, 2006; Rizzuto et al., 2000; Shawet al., 2002; Xie et al., 2004). Successful somatic gene transfer isconditioned by three major variables: the promoter selected todrive the gene expression, the fish metabolic conditions and theamount of plasmid injected. To date, several promoters have beentested in fish, the most prominent of which has been the viral pro-moter CMV. Despite the high expression levels achieved by viralpromoters, they are a cause for concern in the approval of biotech-nological applications (Chico et al., 2009), making it important tofind alternatives. Experiments injecting reporter gene plasmids insea bass have established that for successful plasmid uptake andexpression to take place, fish should be reared at a warm temper-ature, indicating that a metabolically active muscle is required toapply this technique (Muñoz et al., 2013). The optimal amount ofplasmid to be delivered can also vary in distinct fish species withmarked differences in size. In addition, variations in the expressionlevels and time of response may also be found between juvenilesand adults (Mazón et al., 2013). In fact, a saturation limit forDNA uptake might exist, whereby the doses necessary might notincrease proportionally to the weight of the fish (Heppell andDavis, 2000). Moreover, some authors have detected low plasmidexpression when excessively high doses of DNA are used(Gómez-Chiarri et al., 1996; Schulte et al., 1998). Several works



Fig. 1. Schematic representation of the somatic gene transfer technique applied in systemic treatments of Lh and Fsh to male sea bass, as described in this review.


suggest that electrotransfer improves the efficiency of plasmidinjection by increasing the period and level of gene expression(Draghia-Akli and Fiorotto, 2004) and DNA uptake into the cells(Bettan et al., 2000; Vicat et al., 2000). When we applied this tech-nique using sea bass muscle as the target tissue and plasmids con-taining the sequences of Lh and Fsh as the coding genes injected(Mazón et al., 2013, 2014), we found that after plasmid uptake,muscle cells were able to express the introduced genes and secretethe proteins produced into the bloodstream (Fig. 1). This demon-strates that somatic gene transfer may be a powerful tool forin vivo research of gonadotropin functions and a hormonal therapyin fish. Plasmids were injected in the skeletal muscle at the dorsalfin area and this was followed by square electroporation pulsesusing needle-array electrodes applied in the area surrounding theinjection site. In the case of the Lh expression plasmid, it was foundthat injection followed by electroporation favored a more uniformresponse than injection alone, with 100% of the injected fish pre-senting increased plasma levels of Lh. One of the most outstandingdifferences when comparing the injection of the plasmid con-structs with direct administration of the recombinant proteinwas the persistence of the hormone in the bloodstream.Secretion of the hormone by the plasmid-injected muscle cellscould be detected up to 30 days later, in contrast to the 13 daysafter direct administration of CHO-scLh recombinant hormone,emphasizing the advantage of gene therapy for long-lasting treat-ments. In agreement with these observations in sea bass, treat-ments with plasmids encoding erythropoietin (Klinman et al.,1999) or human-alpha-1-antitrypsin (Levy et al., 1996) in miceresulted in a peak of recombinant protein in serum 7 days afterthe first injection, with the levels remaining low but detectableduring at least 3 weeks. The bioactivity of the plasmid-derived Lhproduced by the muscle cells was confirmed after detecting a sig-nificantly higher amount of sperm in the fish treated withCHO-scLh or its coding plasmid. To study the role of Fsh in sea bassspermatogenesis, similar experiments were carried out, in whichimmature juvenile sea bass were injected with an scFsh coding


plasmid or recombinant CHO-scFsh (Mazón et al., 2014).Regardless of the administration system used, Fsh plasma levelssignificantly increased in scFsh-injected fish, and were accompa-nied by an increase in plasma 11KT levels. Fsh-treated fish alsopresented changes in testis functioning, with a remarkableincrease in Sertoli and spermatogonia cell proliferation, whichfinally led them to enter meiosis and resulted in the appearanceof cysts of spermatocytes and spermatids. These morphologicalchanges were also accompanied by changes in gene expression,showing that Fsh treatment suppressed amh expression, whileinducing lhcgr expression. These results strongly support a rolefor Fsh in the onset of spermatogenesis in sea bass, as suggestedfor rainbow trout (Loir, 1999) and other vertebrate species (Abelet al., 2009; Mendez et al., 2003; Méndez et al., 1998; Singh andHandelsman, 1996).

8. Conclusions and future perspectives

In sea bass, the gonad expression patterns of gonadotropinreceptors and the profiles of gonadotropin plasma levels point toa role for gonadotropins similar to that described in mammalsand salmonids, where Fsh would control the first phases of sper-matogenesis in testis and vitellogenesis in the ovary, and Lh wouldhave a major role in the later phases of spermiation and matura-tion/ovulation. Whereas experimental verification of gonadotropinactions in sea bass ovaries in vivo awaits future research, in malesthe proposed roles of gonadotropins are supported by functionalexperiments in which Lh applied in vivo induced sperm productionin mature animals, while Fsh triggered spermatogenesis in imma-ture juveniles. However, Fsh and its receptor are also present dur-ing the end phases of gametogenesis in adults, which could berelated to the group synchronous type of gonad development,where different clutches of germ cells are consecutively recruitedfor maturation. Functional studies with recombinant sea bassgonadotropins have shown a very specific interaction betweeneach hormone–receptor couple, with no cross reactivity between




Lh and Fshr, as has been described in other fish species. In addition,both gonadotropins possess steroidogenic activity, but they showdifferent levels of potency in androgen production in the testis.In vivo studies have revealed that the recombinant single-chaingonadotropins produced in mammalian systems provide the moststable forms for long-term availability in vivo. Moreover, intramus-cular injection of the plasmids coding for these hormones resultedin long-lasting high levels of circulating functional gonadotropins,demonstrating that somatic gene transfer may be an approach forhormone therapy in fish species, in order to solve reproductivedysfunctions associated with low hormone levels. The return topre-treatment hormone levels 3 weeks after cessation of therapysuggests that repeated delivery of a gonadotropin plasmid couldbe used for long-term treatment. In addition, future improvementof this technique could involve the exogenous control of the timingof hormone production, in a similar way as we have alreadydemonstrated, to control the expression of injected reporter genesby means of the tetracycline-regulated system (Muñoz et al.,2013). Taken as a whole, this technology could be of great interestand extremely useful for research into reproductive endocrinology,as it offers a low-cost alternative to the production of recombinantproteins.

Bearing in mind all the tools and acquired knowledge describedabove, European sea bass has become a realistic model for studiesof gonadotropin action. However, some major knowledge gaps stillexist, such as the cellular localization of gonadotropin receptorsand the precise actions of Fsh and Lh, both steroid-dependentand steroid-independent.

Acknowledgments

We thank Alexandra Willis for drawing a figure for this article,and Amparo Gil for help with the bibliographic references. Theresearch summarized in this article was supported by Grants ofthe Spanish Government (AGL2008-02937, AGL2011-28890,CSD2007-00002), Generalitat Valenciana (PROMETEO/2010/003,PROMETEOII/2014/051) and the EU (LIFECYCLE FP7-222719-1).

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Gonadotropins in European sea bass: Endocrine roles and biotechnological applications

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