Follicular steroids cooperate with NPPC to delay nuclear maturation and to increase oocyte-cumulus communication in cattle

Oocyte maturation is precociously induced when the cumulus oocyte complex (COC) is removed from the follicle for IVM/IVF, which accounts for the limited efficiency of IVM. Natriuretic peptide type C (NPPC) delays GVBD and increases oocyte-cumulus communication. In rodents, estradiol appears to cooperate with NPPC by increasing the expression of its receptor NPR2. In this study we first tested the hypothesis that follicular steroids (estradiol, progesterone and androstenedione) at approximately physiological levels cooperate with NPPC to delay GVBD (Experiments 1) and to maintain oocyte-cumulus communication (Experiment 2). Then, we assessed the effects of follicular steroids, NPPC and the combination of both in a pre-IVM culture on embryo production (Experiment 3). Experiments 1 and 3 were performed at UNESP-Botucatu, with ovaries from Bos indicus (predominantly Nellore), and experiment 2 was conducted at the University of Milan with ovaries from Bos taurus (Holstein). COC were aspirated from 3-8mm follicles from abattoir ovaries and cultured in groups of 20. Base medium was TCM199 supplemented with BSA (0,4%), amicacin (75µg/mL) and pyruvate (22µg/mL). In Experiments 1 and 2, COCs were cultured for 9 hours in base medium only (B) or base medium supplemented with NPPC (NPPC), base medium supplemented with follicular steroids (FE) or follicular steroids plus NPPC (NPPC+FE; concentrations were within ranges described in the patent PCT 201690005). At the end of culture, the proportion of oocytes at the germinal vesicle (GV) stage (Experiments 1), and the proportion of COCs with opened gap junctions communications (GJC) were assessed (Experiment 2). To identify GV stage, oocytes were denuded and stained with Hoechst 33342 and GJC were assessed by examining the transfer of the dye injected (Lucifer Yellow) from the oocyte to cumulus cells. Effects of treatments were analyzed by ANOVA followed by Tukeyu0027s test. Experiment 3 tested the effects of the treatments above as pre-IVM culture step on embryo production. For IVM, COCs were cultured in 400µl of base medium supplemented with 100ng/ml amphiregulin (AREG) at 38.5oC and 5% CO2 in humidified air for 24h. In Experiment 1, FE+NPPC maintained oocyte GV arrested as at the time of collection (96.1 vs. 97.5, P u003e 0.05). Moreover, NPPC increased the percentage of COC with open GJC in B+NPPC group compared to B group (47.2 vs. 24.2, P 0.05). Blastocyst production and the percentage of hatched and expanded blastocysts did not differ between treatments. In conclusion, the data suggest that follicular steroids enhance the ability of NPPC to maintain meiotic arrest and to increase oocyte-cumulus communication. Further studies are in progress to ascertain the effect of delayed nuclear maturation with FE+NPPC on embryo development after transfer.