Plasma levels of follicle-stimulating and luteinizing hormones during the reproductive cycle of wild and cultured Senegalese sole ( Solea senegalensis )

Toward developing recombinant gonadotropin-based hormone therapies for increasing fertility in the flatfish Senegalese sole

“…The glycosylation of the rFsh and rLh produced in CHO cells was confirmed by SDS-PAGE and Western blotting using specific antibodies against Senegalese sole Fshβ and Lhβ previously developed [21, 22] (Fig 2). As expected, each sole Fsh and Lh antiserum was able to detect one thick band, corresponding to rFsh (Fig 2A) and rLh (Fig 2B), respectively.…”

“…The Alexa Fluor 647-conjugated wheat germ agglutinin (WGA) was obtained from Life Technologies Corp. (W32466). The specific antibodies against Senegalese sole Fsh β and Lh β subunits have been previously characterized [21, 22]. The secondary antibodies employed included Alexa Fluor 488-coupled goat anti-rabbit and anti-mouse IgG (A-11008 and A-11029, respectively; Life Technologies Corp.), and horseradish peroxidase-coupled goat anti rabbit IgG (sc-2004; Santa Cruz Biotechnology, Inc.).…”

“…The cells were cultured in suspension for 120 h, and the secreted recombinant hormones were subsequently purified from the culture medium by ion exchange chromatography. The hormones were concentrated, and quantified by semiquantitative Western blot, using an antibody against European seabass ( Dicentrarchus labrax ) Cga (gently provided by A. Gómez, CSIC, Spain), as well as by specific enzyme-linked immunosorbent assay (ELISA) as described previously [21, 22]. Hormones were stored at -80°C and diluted just before use with saline (0.9% NaCl) to various concentrations (2.5–10 μg/ml) which enabled all treatment groups to be injected with the same volume containing the appropriate hormone dose.…”

“…Levels of 11-KT in plasma were determined by commercial enzyme immunosorbent assay (EIA; Cayman Chemical Company) as described previously [18, 22]. Free steroids were extracted from plasma (3.5 μl) in methanol and the resulting pellet was diluted 1:150 in EIA buffer (0.1 M K 2 HPO 4 /KH 2 PO 4 , 1.54 mM sodium azide, 0.4 M NaCl, 1 mM EDTA, and 0.1% BSA, pH 7.4).…”

“…Studies in wild and F1 sole males, using newly developed enzyme-linked immunosorbent assays (ELISAs) specific for Fsh and Lh [21, 22], have revealed lower levels of plasma Lh in F1 males than in wild males, while the levels of Fsh and androgens are comparable, suggesting that the low fertilization capability of F1 males may be related to low circulating levels of Lh [22]. Based on these observations it seems plausible that hormone therapies using the administration of the hypothalamic gonadotropin-releasing hormone analogue (GnRHa), which can stimulate the pituitary release of endogenous gonadotropins, might stimulate sperm production in sole as in other farmed teleosts [3].…”