Dynamics of steroid-induced oocyte maturation in three amphibian species: Mathematical modeling and simulation.

Oocyte maturation in vertebrates involves morphological, biochemical, and functional changes through which meiosis, previously arrested in Profase I, is resumed. Within these changes, germinal vesicle breakdown (GVBD) constitutes morphological evidence of the resumption of the cellular cycle. Sex steroids (progestins and androgens) play a key role in oocyte maturation and are considered inducers of this process. The aims of this study were to analyze the effect of sex steroids on the oocyte maturation of amphibians and to model and simulate the dynamics of this process through the experimental data obtained. The oocytes of sexually mature females of Scinax fuscovarius, Pleurodema borellii, and Physalaemus biligonigerus were treated with different concentrations (0.001-1 µM) of sex steroids progesterone (P 4 ) and testosterone (T) for 24 hr. Dose- and time-response curves were performed with the results achieved. Sex steroids induced oocyte maturation in a dose-dependent manner in all three species. The dose at which the highest maturation percentage was found was 1 µM. No significant differences were observed between GVBD percentages of P 4 and T. However, time-response curves show that oocytes responded earlier and achieved higher GVBD percentages when treated with P 4 . Gompertz mathematical model was proposed in this study to simulate GVBD dynamics. This model properly fits the corresponding experimental data and allows the analysis of the behavior of oocyte maturation in amphibians and the quantification of parameters with biological meaning that are indicative of sex steroids efficiency in this process.

Validation of Electrochemiluminiscence Immunoassay for Ovarian Steroid Determination in Rhinella arenarum.

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.

In vitro steroid-induced meiosis in Rhinella arenarum oocytes: role of pre-MPF activation.

In this work we showed the relationship between seasonal periods and the response of R. arenarum follicles and oocytes to different steroids. Using in vitro germinal vesicle breakdown (GVBD) assays, we demonstrated that P4 is the main steroid capable of inducing maturation in R. arenarum oocytes and follicles. In the second part of this work we showed that androgens can activate pre-maturation promoting factors (pre-MPFs) such as P4, by cytoplasm microinjection experiments. The results indicated that the steroids assayed induced oocyte and follicle maturation in a dose- and time-dependent manner. In oocytes, P4 was the most efficient steroid as a maturation inducer (EC50 of the reproductive period, 6 nM, EC50 of the non-reproductive period ≅ 30 nM). Androgens (DHEA, dehydroepiandrosterone; T, testosterone; and AD, androstenedione) were less efficient maturation inducers than P4 (EC50 reproductive period ≅ 50, 120 and 600 nM respectively). Similar results were obtained with intact follicles in both seasonal periods. Although the response of follicles to the different androgens was variable, in no case was it above the above the response induced by P4. Independently of the season, oocytes and follicles incubated in P4, P5 and T underwent GVBD after 6-10 h while oocytes and follicles incubated in DHEA and AD matured more slowly. Furthermore, we demonstrated that microinjection of mature cytoplasm from androgen-treated oocytes is sufficient to promote GVBD in immature recipient oocytes (DHEA, 57 ± 12%; AD, 60 ± 8%; T, 56 ± 13%). Thus, androgens such as DHEA, T and AD are as competent as P4 to activate pre-MPF.

Role of arachidonic acid cascade in Rhinella arenarum oocyte maturation.

There are no studies that document the production of prostaglandins (PGs) or their role in Rhinella arenarum oocyte maturation. In this study, we analysed the effect of arachidonic acid (AA) and prostaglandins (PGs) on maturation, activation and pronuclear formation in R. arenarum oocytes. Our results demonstrated that AA was capable of inducing maturation in time-dependent and dose-dependent manner. Arachidonic acid-induced maturation was inhibited by indomethacin. PGs from AA hydrolysis, such as prostaglandin F2α (PGF2α) and, to a lesser extent, PGE2, induced meiosis resumption. Oocyte maturation in response to PGF2α was similar to that produced by progesterone (P4). Oocyte response to PGE1 was scarce. Rhinella arenarum oocyte PGF2α-induced maturation showed seasonal variation. From February to June, oocytes presented low sensitivity to PGF2α. In following periods, this response increased until a maximum was reached during October to January, a close temporal correlation with oocyte response to P4 being observed. The effect of PGF2α on maturation was verified by analysing the capacity of oocytes to activate and form pronuclei after being injected with homologous sperm. The cytological analysis of activated oocytes demonstrated the absence of cortical granules in oocytes, suggesting that PGF2α induces germinal vesicle breakdown (GVBD) and meiosis resumption up to metaphase II. In turn, oocytes matured by the action of PGF2α were able to form pronuclei after fertilization in a similar way to oocyte maturated by P4. In microinjection of mature cytoplasm experiments, the transformation of pre-maturation promoting factor (pre-MPF) to MPF was observed when oocytes were treated with PGF2α. In summary, our results illustrated the participation of the AA cascade and its metabolites in maturation, activation and pronuclei formation in R. arenarum.

Involvement of PLA2, COX and LOX in Rhinella arenarum oocyte maturation.

In Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism - phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 µM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation.