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ABSTRACT
The gonadotrophic function of the European eel (Anguilla anguilla L.) at the silver stage is very weak: gonadotrophin-releasing hormone (GnRH) secretion is deficient and, moreover, dopamine inhibition overrides GnRH action. At the silver stage, œstradiol stimulates the biosynthesis of the type-II gonadotrophin (GTH-II). To study the molecular mechanism of this activation further, we examined the effect of testosterone and œstradiol administration on pituitary levels of mRNA encoding GTH-II α and β subunits. Corresponding eel cDNA probes and Northern blot analysis were used. After 2 weeks, testosterone and œstradiol implantation resulted in a strong increase in mRNA encoding the GTH-II β subunit (7-fold and 25-fold, respectively) and in a slight, but non-significant, rise in the a subunit mRNA level (1.8-fold and 1.5-fold, respectively). Co-implantation of these two steroids suggested a potentiation of their effects on the β subunit (104-fold) while an additive effect was indicated on the α mRNA level (2.9-fold). Effects were detectable within 4 days and were maximal 4 weeks after implantation. These results indicate that in the European eel at the silver stage, gonadal steroids stimulate differentially the expression of GTH-II subunit genes at a pretranslational level.
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ABSTRACT
Oestradiol treatment enhances type-II gonadotrophin (GTH-II) synthesis in the European eel (Anguilla anguilla) at the silver stage. As a first step in studying the molecular mechanisms involved in this stimulation, we cloned and characterized the cDNA encoding the β subunit of eel GTH-II.
A cDNA library was constituted in λgt10 from oestradiol-treated eels. It was screened using an oligodeoxyribonucleotide mixed probe designed to be complementary to a highly conserved region of cDNAs from several LH-related β subunits. Several clones were obtained and four were subcloned in pUC13 and sequenced. The longest clones comprised a 420 bp coding sequence, plus 5′ and 3′ untranslated regions of 36 and 172 bp respectively. Comparison with GTH-II from other teleost fish permitted the localization of the putative cleavage site of a 24 amino acid signal peptide. The resulting 116 amino acid apopeptide had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 70–80% with GTH–II from other fish, 45% with LH from mammals and birds, 38% with mammalian FSH and only 35% with fish GTH-I. Preliminary results indicated a strong positive effect of oestradiol treatment on the level of the putative GTH-II β-subunit mRNA. This supports our proposal that the European eel provides a suitable model for studying the positive regulation of gonadotrophin synthesis by gonadal steroids.
