Japanese Eel Follicle-Stimulating Hormone (Fsh) and Luteinizing Hormone (Lh): Production of Biologically Active Recombinant Fsh and Lh by Drosophila S2 Cells and Their Differential Actions on the Reproductive Biology1

Abstract

Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.

Fsh and Lh have common and distinct effects on gene expression in rainbow trout testis

“…Indeed, we found a stimulation of 11KT production by both gonadotropins, together with a positive regulation of mRNA abundance of several key actors involved in steroid metabolism (star, hsd3b1, cyp17a1, and cyp11b2-2). This latter observation is in agreement with previous data obtained in vitro in eel (Kazeto et al 2008), zebrafish (Garcia-Lopez et al 2010, or female coho salmon (Luckenbach et al 2011). In fact, several genes regulated by both Fsh and Lh were also found to be similarly regulated by androgens in vivo (A D Rolland, A Lardenois, A-S Goupil, J-J Lareyre, F Chalmel, R Houlgatte and F Le Gac, unpublished observations).…”

Two states of active spermatogenesis switch between reproductive and non‐reproductive seasons in the testes of the medaka, Oryzias latipes

“…5). Gonadotropins induce all steps of spermatogenesis in immature testis of eels both in vivo and in vitro (Ohta et al 2007;Hayakawa et al 2008;Kazeto et al 2008) and FSH stimulates proliferation of A-gonia and early B-gonia through (Ohta et al 2007). Thus, the result of the present study suggests that the gonadotropins keep inducing spermatogenesis of O. latipes in vivo and its effect was still present in the cultured testes for the initial 5 days.…”

Sertoli cell proliferation in the adult testis is induced by unilateral gonadectomy in African catfish

“…but not Lh was able to up-regulate the expression of steroidogenesis related genes in juvenile eel 25 [24] and in adult zebrafish testis [18]. Therefore, we propose that Fsh, rather than Lh, mediated the 26 up-regulation of the steroidogenic capacity of the testis remaining after ULG.…”