Development of specific enzyme-linked immunosorbent assay for determining LH and FSH levels in tilapia, using recombinant gonadotropins.

We recently produced Oreochromis niloticus recombinant LH and FSH as single-chain polypeptides in the methylotrophic yeast Pichia pastoris. Glycoprotein subunit alpha was joined with tilapia (t) LHbeta or tFSHbeta mature protein-coding sequences to form a fusion gene that encodes a ;;tethered" polypeptide, in which the gonadotropin beta-subunit forms the N-terminal part and the alpha-subunit forms the C-terminal part. Recombinant (r) gonadotropins were used to develop specific and homologous competitive ELISAs for measurements of FSH and LH in the plasma and pituitary of tilapia, using primary antibodies against rtLHbeta or rtFSHbeta, respectively, and rtLHbetaalpha or rtFSHbetaalpha for the standard curves. The wells were coated with either rtLHbeta (2ng/ml) or rtFSHbeta (0.5ng/well), and the final concentrations of the antisera were 1:5000 (for tLH) or 1:50,000 (for tFSH). The sensitivity of the assay was 15.84pg/ml for tLH and 0.24pg/ml for tFSH measurements in the plasma, whereas for the measurements in the pituitary, the sensitivity was 2.43ng/ml and 1.52ng/ml for tLH and tFSH, respectively. The standard curves for tFSH and tLH paralleled those of serially diluted pituitary extracts of other cichlids, as well as of serially diluted pituitary extract of seabream, European seabass and hybrid bass. We examined plasma tFSH and tLH levels in the course of one reproductive cycle, between two successive spawnings, in three individual tilapia females. Plasma levels of both FSH and LH increased during the second day after the eggs had been removed, probably related to the vitellogenic phase. LH levels increased toward spawning, which occurred on the 11th day. FSH levels also increased on day of cycle, probably due to recruitment of a new generation of follicles for the successive spawning. The development of specific ELISAs using recombinant gonadotropins is expected to advance the study of the distinct functions of each of these important hormones.

Tilapia follicle-stimulating hormone (FSH): immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion.

In fish, FSH is generally important for early gonadal development and vitellogenesis. As in mammals, FSH is a heterodimer composed of an alpha subunit that is noncovalently associated with the hormone-specific beta subunit. The objective of the present study was to express glycosylated, properly folded, and biologically active tilapia FSH (tFSH) using the Pichia pastoris expression system. Using this material, we aimed to develop a specific ELISA and to enable the study of FSH response to GnRH. The methylotrophic yeast P. pastoris was used to coexpress recombinant genes formed by fusion of mating factor alpha leader and tilapia fshb and cga coding sequences. Western blot analysis of tilapia pituitary FSH, resolved by SDS-PAGE, yielded a band of 15 kDa, while recombinant tFSH beta (rtFSH beta) and rtFSH beta alpha had molecular masses of 17-18 kDa and 26-30 kDa, respectively. Recombinant tFSH beta alpha was found to bear only N-linked carbohydrates. Recombinant tFSH beta alpha significantly enhanced 11-ketotestosterone (11-KT) and estradiol secretion from tilapia testes and ovaries, respectively, in a dose-dependent manner (similar to tilapia pituitary extract, affinity-purified pituitary FSH, and porcine FSH). Using antibodies raised against rtFSH beta, FSH-containing cells were localized adjacent to hypothalamic nerve fibers ramifying in the proximal pars distalis (PPD), while LH cells were localized in a more peripheral region of the PPD. Moreover, FSH is under the control of hypothalamic decapeptide GnRH, an effect that was abolished through the use of specific bioneutralizing antisera, anti-rtFSH beta. It also reduced basal secretion of 11-KT.

Production of biologically active tethered tilapia LHbetaalpha by the methylotrophic yeast Pichia pastoris.

In fish, luteinizing hormone (LH) stimulates processes leading to final oocyte maturation and ovulation in females, and spermiation in males. The hormone is a heterodimeric glycoprotein composed of two non-covalently associated subunits. In this study, we describe the expression of tilapia LH (tLH) as a biologically active, single-chain polypeptide using the methylotrophic yeast Pichia pastoris. The tLHbeta and alpha mature protein-coding sequences were joined to form a fusion gene that encodes a "tethered" polypeptide in which the tLHbeta-chain forms the N-terminal part and the alpha-chain forms the C-terminal part. A "linker" sequence of six amino acids (three Gly-Ser pairs) was placed between the beta- and alpha-chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the beta-subunit, to enable purification of the recombinant protein. Western blot analysis of the pituitary LH resolved by SDS-PAGE yielded a band of 35 kDa, while the recombinant tLHbetaalpha had a molecular mass of 45 kDa, and was found to possess only N-linked carbohydrates. Recombinant tLHbetaalpha stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced.