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.

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In this work, we describe the participation of the adenylate cyclase/3'-5'-cyclic adenonsine monophosphate (cAMP) pathway in the seasonal follicular secretion of progesterone (P ) and testosterone (T), and its relationship with the maturation of Rhinella arenarum oocytes. Under gonadotropin stimulation, P secretion was the dominant steroid produced during the reproductive period, resulting in 100% germinal vesicle breakdown (GVBD) in oocytes in vitro; in contrast, T and estradiol (E ) secretion increased (∼16 nM/20 follicles and ∼80 pM/20 follicles, respectively) during the non-reproductive period, but only yielded 50% GVBD. Treatment of the follicles with dibutyryl-cAMP or forskolin induced a significant increase in T secretion during both periods, but P secretion did not significantly change and GVBD did not occur. These results suggest that high cAMP levels in the oocyte maintain meiotic arrest and prevent the induction effect of follicular steroids. An increase in cAMP levels in denuded oocytes, however, negatively regulated T-induced maturation since treatment with increasing db-cAMP or forskolin inhibited their maturation. Therefore, we hypothesize that an elevation in T during the non-reproductive period favors its aromatization to E , leading to follicle growth. During the reproductive period, P production might promote oocyte maturation when environmental conditions are favorable for reproduction. Together, the results indicate that steroidogenesis is seasonal and depends on gonadotropic activity in R. arenarum.