Validation of Electrochemiluminiscence Immunoassay for Ovarian Steroid Determination in Rhinella arenarum

Abstract

In this work, we describe the validation of an electrochemiluminescence immunoassay (ECLIA) that allowed us for the first time to determine the levels of progesterone (P4 ) and testosterone (T) secreted by Rhinella arenarum follicles during the preovulatory (POP) and reproductive (RP) periods. We also verified the relation between P4 and T levels and oocyte maturation. Moreover, we demonstrated that the extraction protocol developed for the determinations of P4 and T by ECLIA proved to be efficient and reproducible since the efficacy of the extraction was above 95% in all assays conducted. The results indicate that in the validation process the variation coefficient (CV) between assays is compatible with the analytical procedures based on automated immunoassays (CV < 8%) and that the adaptation proposed for the samples allows the determination of T and P4 with the Cobas e-411 analyzer. Our results indicate that in basal conditions the levels of T released by R. arenarum follicles were higher than those of P4 during POP and RP. In these conditions, steroid secretion failed to induce germinal vesicle break down (GVBD) in the follicles. Under gonadotropin stimulation, steroidogenesis showed a remarkable increase in both periods, especially during POP. This increase was correlated with a high maturation percentage in the follicles incubated in vitro (GVBD = 72 ± 16%) during POP. During RP, human Chorionic Gonadotropin (hCG) induced 81.75 ± 9.1% GVBD. This study is the first report of the seasonal steroidogenic activity in the ovary of R. arenarum in situ using an ECLIA-modified protocol developed in our laboratory.

Oocyte maturation in the toad Rhinella arenarum (Amphibia, Anura): Evidence of cAMP involvement in steroid production and action

“…Although the absolute concentration of T was great than P 4 , gonadotropin stimulation during the reproductive period caused a remarkably greater increase in P 4 (∼sixfold) compared to T secretion (∼twofold) over basal ovarian secretion. Such differential follicular response in T versus P 4 production support our previous work demonstrating that R. arenarum oocytes mature under the action of different steroids, wherein androgens can induce oocyte maturation (Arias Torres, Bühler, et al, ) but P 4 proved to be the most effective (Arias Torres, Páez, et al, ). Therefore, oocyte maturation in this species might be more sensitive to fold‐change increases in P 4 secretion rather than to absolute concentrations.…”

“…Several publications describe the influence of hypophyseal hormones in the secretion of ovarian steroids in amphibians (Norris & Lopez, ), but few specifically address this for R. arenarum . Steroid concentrations in the plasma of adult females are known (Medina, Ramos, Crespo, Gonzalez Calvar, & Fernandez, ), as well as in the ovary in situ (Arias Torres, Páez, et al, ). Here, we extended this knowledge base by tracking the seasonal secretion of sex steroids from full‐grown R. arenarum follicles.…”

“…Steroids were extracted from the incubation medium, and quantified using ECLIA with the method validated previously by our group (Arias Torres, Páez, et al, ). In this analytical system, the reporting range for P 4 is 0.095–191 nM, for T is 0.087–52.0 nM, and for E 2 is 18.4–15781.0 pM.…”

“…Using an electrochemiluminescence immunoassay (ECLIA), we determined that R. arenarum follicles in basal conditions release more T than P 4 during the preovulatory and reproductive periods, but these steroids failed to induce GVBD. Under gonadotropin stimulation, however, steroidogenesis remarkably increased in both periods, and resulted in a high in vitro maturation percentage (72 ± 16% GVBD in follicles from the preovulatory period, 81.75 ± 9.1% GVBD from the reproductive period) (Arias Torres, Páez, & Zelarayán, ). Given that the downstream signaling involved in this androgen‐induced GVBD have not yet been described for toads, we aimed to study the participation of the AC/cAMP signaling in T‐induced R. arenarum oocyte maturation as well as follicular steroid secretion, and its relationship with oocyte maturation.…”

Data collection in support of the Endocrine Disruption (ED) assessment for non‐target vertebrates

“…The literature search found no eligible studies describing original and/or detailed methods or report sufficient methodological data for the quantification of estrogens or androgens in amphibian samples using ELISA methods. Arias Torres et al (2016) (reference 15412 in Annex B -) validated a competitive chemiluminescence enzyme immunoassay (ECLIA) for ovarian steroid in Rhinella arenarum using a method previously developed for human samples. Korte et al (2011) Chemical analysis Chang et al (2010) quantified the estrogens (E1, 17β-E2, 17α-E2) in African clawed frogs (Xenopus laevis), a standard test species (see reference 1407 in Annex B -) using liquid chromatographyelectrospray tandem mass spectrometry (LC-ESI-MS/MS).…”