Cloning and sequencing of the FSH-β and LH β-subunit in the three-spined stickleback, Gasterosteus aculeatus, and effects of photoperiod and temperature on LH-β and FSH-β mRNA expression

Abstract

The aim of this study was to characterize the FSH-β and LH-β subunit in the three-spined stickleback, a fish used extensively in experimental studies, and to use the cloned cDNAs as probes for measuring FSH-β and LH-β mRNA expression in sticklebacks treated with different photoperiods and temperatures. A first strand cDNA was prepared from 10 pituitaries from male sticklebacks, and cDNA fragments were amplified by PCR using degenerated primers based on highly conserved regions of known teleost FSH-β and LH-β cDNA sequences. To obtain full-length cDNAs of FSH-β and LH-β, RACE amplifications were performed. The cDNA of FSH-β was 540 bp long, encoding a protein of 122 amino acids and LH-β cDNA was 568 bp long, encoding a protein of 148 amino acids. Of gonadotropin (GTH) β-subunits published so far, those most similar to stickleback GTHs are found among other percomorph fishes, with amino acid similarities of 46–55% for FSH-β and 75–77% for LH-β subunits.The cloned cDNAs were used as probes to analyze LH and FSH mRNA expression in pituitaries from sticklebacks exposed to different photoperiods and temperatures. Two hundred males were divided into four groups and kept under different photoperiods and temperatures; light:dark (LD) 16:8, 20 °C; LD 16:8, 7 °C; LD 8:16, 20 °C, and LD 8:16, 7 °C. Red breeding colors and a marked kidney hypertrophy, androgen-dependent male secondary sexual characters in the stickleback, appeared in the group kept under LD 16:8 at 20 °C, but not in the other groups. The kidney epithelium height (KEH) was significantly lower in the LD 8:16, 20 °C group than in all other groups, and this was also the only group with active spermatogenesis. The LD 8:16 20 °C had significantly lower expression of both FSH-β and LH-β than all other groups. Thus, both GTHs displayed their lowest expression when spermatogenesis was active and when low KEH indicated that androgens levels were at their lowest.