Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common α-subunit (α-glycoprotein hormone (α-GP)), β-FSH, and β-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the α-subunit without the His-tag with each of the His-tagged β-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and α-GP in abundance; however, expression of the individual β-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose–response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

Abstract

The sensitivity of the pituitary to gonadotrophin-releasing hormone (GnRH) and that of the testis to gonadotrophin (GTH) was monitored in African catfish in vivo at different stages of pubertal development (20, 21, 24, 31, 39, 42 and 49 weeks of age). The fish were injected i.p. with chicken GnRH-II (cGnRH-II) or catfish GnRH (cfGnRH), their two endogenous GnRHs. Blood samples were collected to quantify LH-like GTH-II and three androgens 11-ketotestosterone (11-KT), testosterone and 11β-hydroxyandrostenedione (OHA). The testes of 20- and 21-week-old fish contained spermatogonia alone, or spermatogonia and spermatocytes, or – in a limited number of specimens – some spermatids as well. Spermatozoa were first observed in the testes of 24-week-old fish and became predominant as the fish attained full maturity (49 weeks of age). In 20- to 24-week-old fish, significantly elevated plasma GTH-II levels were only recorded after treatment with cGnRH-II. In 31- to 49-week-old fish, injection of both GnRHs led to increased plasma GTH-II levels, but cGnRH-II was always more effective than cfGnRH. Whereas basal GTH-II plasma levels hardly changed throughout the study, GnRH-stimulated levels increased with the age of the fish. Plasma concentrations of 11-KT were not different from controls in 20- and 21-week-old males despite their elevated GTH-II levels following injection of cGnRH-II. The first significant increase in levels of 11-KT after cGnRH-II treatment was observed in 24-week-old fish and, after cfGnRH treatment, in 39-week-old fish. Basal and GnRH-stimulated 11-KT plasma levels increased with the age of the fish. Basal and cGnRH-II-stimulated plasma levels of OHA and testosterone also increased with the age of the fish. However, the levels of OHA and testosterone were five- to tenfold lower than those of 11-KT and, except for OHA in the 49-week-old fish, no increases were recorded in the cfGnRH-injected fish. Our data show that at the beginning of spermatogenesis the pituitary gland is already sensitive to GnRH stimuli. However, sensitivity of the testicular steroidogenic system to GTH-II, sufficient to be reflected in consistently elevated androgen plasma levels, was not observed until 3–4 weeks later. The restricted testicular GTH-II responsiveness at the beginning of spermatogenesis may represent a limiting factor for further pubertal development.

Journal of Endocrinology (1994) 140, 265–273

The Atlantic salmon shows substantial life cycle plasticity, which also applies to the timing of puberty. While it is characterized by the activation of the brain–pituitary–gonad axis, many morphophysiological aspects of puberty and the influence of environmental conditions, such as water salinity, are not well understood in fish. Here, 12-month-old Atlantic salmon coming from an out-of-season smoltification regime in December were exposed to freshwater (FW) or seawater (SW) at 16 °C to stimulate puberty under a 24-h constant light (LL) or 12 h light:12 h darkness (LD) photoperiod. These four treatment groups (FWLL, SWLL, FWLD, and SWLD) were studied from January to March. Next to 11-ketotestosterone (11-KT) plasma levels, the expression of pituitary genes (gnrhr4, fshb, and lhb) and spermatogenesis was quantified. When spermatogonial proliferation started, fshb mRNA levels increased steeply and began to decrease when spermatogonial mitosis approached completion and most germ cells had reached meiotic or post-meiotic stages. Conversely, lhb mRNA levels increased progressively during spermatogenesis. Most males in all treatment groups matured, but exposure to SW resulted in the strongest stimulation of the onset of spermatogenesis and elevation of pituitary gnrhr4 and fshb mRNA levels. Later on, the LD photoperiod accelerated, irrespective of the salinity, the completion of spermatogenesis, associated with higher lhb mRNA and 11-KT plasma levels than in the LL groups. We find that both salinity and photoperiod modulated different aspects of spermatogenesis, and resulted in a differential activation of pituitary and testis functions; SW stimulating the onset and the shorter photoperiod the completion of spermatogenesis.

Gonadotrophs are the primary target cells for GnRH in the pituitary. However, during a limited period of neonatal life in the rat, lactotrophs and somatotrophs respond to GnRH as well. Also, in the adults of a number of teleost fishes (e.g. carp, goldfish, and tilapia but not trout), GnRH is a potent GH secretagogue. In studying hypophysiotrophic actions of the two forms of GnRH present in the African catfish (Clarias gariepinus), chicken GnRH-II ([His⁵,Trp⁷,Tyr⁸]GnRH; cGnRH-II) and catfish GnRH ([His⁵,Asn⁸]GnRH; cfGnRH), we have investigated the effects of GnRH on catfish gonadotrophs and somatotrophs. GnRH binding was examined by incubating dispersed pituitary cells attached to coverslips with ¹²⁵I-labelled [d-Arg⁶,Trp⁷,Leu⁸,Pro⁹-Net]GnRH (sGnRHa), a salmon GnRH analogue with high affinity for the GnRH receptor. Following fixation and immunohistochemistry using antisera against catfish LH and GH, ¹²⁵I-labelled sGnRHa was localised autoradiographically and silver grains were quantified on gonadotrophs and somatotrophs. Specific binding of ¹²⁵I-labelled sGnRHa was restricted to gonadotrophs. Both cfGnRH and cGnRH-II dose-dependently inhibited ¹²⁵I-labelled sGnRHa binding to gonadotrophs. To substantiate the localisation of functional GnRH receptors, the effects of cfGnRH and cGnRH-II on the cytosolic free calcium concentration ([Ca²⁺]_i) were examined in Fura-2-loaded somatotrophs and gonadotrophs. GnRH-induced increases in [Ca²⁺]_i appeared to be confined to gonadotrophs, in which both endogenous GnRHs caused a single and transient increase in [Ca²⁺]_i. The amplitude of this [Ca²⁺]_i transient depended on the GnRH dose and correlated well with the GnRHs' effect on LH release. In vivo experiments demonstrated that GnRH treatments which markedly elevated plasma LH levels had no effect on plasma GH levels, while a dopamine agonist (apomorphine) significantly elevated plasma GH levels. We conclude that the two endogenous forms of GnRH in the African catfish are not directly involved in the regulation of the release of GH, suggesting that GnRHs cannot be considered as GH secretagogues in teleosts in general.

Journal of Endocrinology (1997) 152, 437–446