Pubertal Development of Male African Catfish (Clarias Gariepinus). Pituitary Ultrastructure and Responsiveness to Gonadotropin-Releasing Hormone

Abstract

Pubertal development was studied in male African catfish by immunocytochemical examination of pituitary gonadotrophs and by monitoring the responsiveness of gonadotropin (GTH) secretion to salmon GnRH analogue (sGnRHa) in vitro. Experiments were carried out with fish from 9 to 28 wk of age. Fish were assigned to four groups, according to the stage of spermatogenesis: I, spermatogonia alone; II, spermatogonia and spermatocytes; III, spermatogonia, spermatocytes, and spermatids; IV, all germ cell stages, including spermatozoa. Basal and sGnRHa-stimulated secretion of the LH-like GTH II increased 3- to 4-fold from stage I to II and from stage II to III, whereas a 15-fold increase was recorded from stage III to IV. The ED50 values of sGnRHa varied between 0.08 and 0.49 nM, stages II and III being less sensitive. The highest dosage of sGnRHa (100 nM) led to a reduction of GTH secretion. In the first three stages, the pituitary secreted large amounts of free alpha-subunit while free GTH II beta-subunit was not detected at any stage of development. Antisera against GTH II and its alpha- and beta-subunits were used for immunocytochemical studies. In stages I and II, two subtypes of gonadotrophs, which differed in the size and labeling intensity of their secretory granules, were present. Both types of granules were immunopositive for the two subunits of GTH II. In stages III and IV, only gonadotrophs of the subtype with the larger granules were found. Globules and irregular, membrane-bound masses (IMs), probably arising through fusion of secretory granules, appeared in the gonadotrophs in stage III and became more prominent in stage IV. Globules and IMs were immunopositive for the beta-subunit but negative for the alpha-subunit. We conclude that the two subtypes of gonadotrophs represent different developmental stages of GTH II-producing cells, as they shared immunolabeling for the alpha- and the beta-subunits of GTH II. The scarcity of GTH II beta-subunit may be rate-limiting for the amount of intact GTH II available for secretion, particularly at early stages of development. In contrast, at more advanced stages when the readily releasable pool of GTH II has greatly increased, the amount of GTH II also appears to be controlled by modification or elimination of the alpha-subunit from globules and IMs.

Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor

“…cfGP and cfLH mRNA as well as the heterodimeric hormone are present in the pituitary, and gonadotropin-releasing hormone-induced release of cfLH from the pituitary has been demonstrated, at all sexual developmental stages (Schulz et al 1995. Purified, pituitary-derived cfLH stimulates intracellular cAMP production in HEK-T 293 cells expressing either the cfLH-R or the cfFSH-R ) with comparable efficacies, as compared with the situation in mammals.…”

“…Recombinant catfish gonadotropins were quantified by RIA using antisera against intact cfLH and the cfGP -subunit, as described previously (Schulz et al 1995).…”

“…This showed that the Dictyostelium cells in a single 9 cm plate produced 18-61 ng rcfFSH and 31-80 ng rcfLH per ml of medium. These amounts are very Table 1 Amount of recombinant catfish gonadotropins/ml medium, after ∼20-fold concentration by ultrafiltration, as quantified by RIA using antisera against intact cfGP -subunit and intact cfLH, as described by Schulz et al (1995) similar to the production levels of recombinant human gonadotropins by Dictyostelium cells (Linskens et al 1999).…”

Receptor-selective determinants in catfish gonadotropin seat-belt loops

“…Hormone concentrations were quantified by radioimmunoassays using antisera against intact cfLH and the cfGP␣-subunit as described previously (Schulz et al, 1995).…”

Immunohistochemically detected ontogeny of prolactin and growth hormone cells in the African catfish Clarias gariepinus

“…The African catfish Clarias gariepinus (Burchell, 1822) (Teleostei, Siluroidei) has been used as an endocrinological model, especially for the regulation and functioning of the gonadotrophic hormone (GtH) [38]. The genes GH and PRL have been studied to a lesser degree, but they have been cloned [16], the growth hormone protein has been purified [6] and a radioimmunoassay has been developed [26].…”