Image_3_Development of an enzyme-linked immunosorbent assay for determining FSH plasma concentrations in green sea turtle (Chelonia mydas), using recombinant gonadotropins.tif

Follicle-stimulating hormone (FSH) is involved in the regulation of essential reproductive processes such as gametogenesis and follicular growth. There are presently no immunoassays for measuring FSH in turtles. Recently we produced green sea turtle (Chelonia mydas) recombinant (r) FSH as a single-chain polypeptide using the methylotrophic yeast Pichia pastoris expression system, and polyclonal antibodies for the recombinant FSH. In this work we developed a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of FSH concentrations in plasma samples from green sea turtles. We used the rFSHβα for standard, rFSHβ for coating and a cmFSHβ antibody. The sensitivity of the assay was 0.13 ng/ml and the intra-assay and inter-assay coefficients of variation were 5.54% and 13.52% respectively. Parallelism was observed between the linearized FSH standard curves and the corresponding serial dilutions of green sea turtle plasma samples. We also observed parallelism between the linearized standard and serial dilutions of plasma samples from the loggerhead sea turtle (Caretta caretta), hawksbill sea turtle (Eretmochelys imbricate), and African softshell turtle (Trionyx triunguis). The ELISA was used to study the FSH plasma concentrations during the reproductive cycles and was compared to hormonal steroid concentrations (Testosterone, Estradiol and Progesterone). This revealed a positive correlation between FSH and estradiol concentrations in females; estradiol concentrations were increased immediately after FSH elevation. In addition, nested females presented an increase in FSH concentrations prior to progesterone elevation in January to April, slightly before egg laying. This ELISA will increase our understanding of gonadotropin functions, and their effects on reproduction in the green sea turtle.</p

Effect of combined GnRH and testosterone treatment on gonadal staging.

<p>Effect of combined GnRH and testosterone treatment on gonadal staging.</p

Competitive binding curves for Russian sturgeon FSH and LH standards.

<p>Typical standard curve for the measurement of Russian sturgeon FSH (A) and LH (B).</p

Parallelism between the standard curves of ELISA for the measurements of Russian sturgeon gonadotropins.

<p>Russian sturgeon FSH (A) or LH (B) and displacement curves obtained with serial dilutions of plasma from a male (Fish 1) and female (Fish 2). (C) Displacement curves obtained with serial dilutions of pituitary extract of Russian sturgeon FSH and (D) Pituitary extract of Russian sturgeon LH.</p

Secretion of estradiol from sturgeon ovaries.

<p>Sturgeon ovaries containing white (A), yellow (B), gray (C) or black (D) oocytes from a mature female at 10-year age, in response to various doses of recombinant sturgeon FSHβα, rsLHβα, adenylate cyclase activator (Forskolin, FSK;E) or carp pituitary extract (CPE; F).</p

Effect of hormonal treatments on E2, 11-KT, FSH and LH plasma level in 6-year-old Russian sturgeon females.

<p>The control group was injected with 0.2 mL/kg saline. The first treatment group was injected (IM) with salmon GnRH analog ([D-Arg⁶,Pro⁹- NEt]-salmon GnRH; sGnRHa) in slow-release implants (Evac) up to a final dose of 10 μg/kg. Fish from the second group received testosterone (T) as food supplements (60 mg/kg food; 0.4% BW food per day). The third group received both GnRH implants and T as food supplements, at the above concentrations. Each bar represents the mean ± SEM. Different letters indicate that treatments have significantly different effects (ANOVA followed by Newman–Keuls test, (p<0.05).</p

Characterization of Pichia-expressed recombinant stLHβ, stLHβα, stFSHβ and stFSHβα by Western-blot analysis and deglycosilation.

<p>Supernatants of transformed Pichia cultures were separated by 15% semi-native PAGE and immunoreacted with antibodies raised against anti-His (A) recombinant stLHβ (B) or recombinant stFSHβ (C). For deglycosylation analysis, denatured and reduced proteins were incubated with (lanes 2, 4, 6, 8 and 10) or without (lanes 1, 3, 5, 7 and 9) N-glycosidase F. stLHβ: lanes 1,2; stLHβα: lanes 3,4; stFSHβ: lanes 5,6; stFSHβα: lanes 7,8; sturgeon pituitary extract (diluted 1:100): lanes 9,10; PM-protein marker; HM-His-tag protein marker.</p

Secretion of 11-KT from sturgeon testes.

<p>Testes from pre-mature (A) or mature (B) sturgeon, in response to various doses of recombinant sturgeon FSHβα, rsLHβα, adenylate cyclase activator (Forskolin, FSK; C) or carp pituitary extract (CPE; D).</p

Russian sturgeon gonadotropin models.

<p>A. stFSH model. B. stLH model. Red colored: Subunit α, blue colored: Subunit β, αC: subunit α C-termini, αN: subunit α N- termini, βC: Subunit β C- termini, βN: subunit β N- termini.</p