Meiotic arrest of sheep oocytes using roscovitine under different medium compositions

Abstract

Attempts to maintain the meiotic arrest under suitable culture conditions have been performed in order to improve the quality of oocytes in vitro matured. So, our study aimed to evaluate the potential of roscovitine, an inhibitor of cyclin-dependent kinases, to reversibly arrest the meiosis of sheep oocytes in medium supplemented with gonadotropins and/or serum. Cumulus-oocyte complexes (COCs) were treated, for 20h, with 75μM roscovitine (Rosco) in basic maturation medium (Basic) containing 10% fetal bovine serum (FBS) and 0.1IU/mL FSH plus 0.1IU/mL LH (Enriched). After this, COCs were in vitro matured (IVM) for a further 18h in the presence of gonadotropins and serum. At 20 and 38h of culture, cumulus expansion and nuclear maturation were assessed under stereomicroscope and by Hoechst 33342 staining, respectively. Our findings demonstrate that total cumulus expansion occurred only in medium containing gonadotropins and serum. In the presence of roscovitine, however, the cumulus expansion was inhibited in a not fully reversible manner, independently of medium composition. The GV rate in the Rosco FBS group (64.6%) was significantly higher than that of Rosco Enriched (51.8%); however, both did not differ from that observed in the Rosco Basic (56.2%). In all roscovitine treatments, the meiotic arrest was reversible after the additional culture for 18h in inhibitor-free medium. Besides, significant proportion of oocytes from Basic, FBS and Enriched groups reached the MII stage after 20 and 38h of culture. Therefore, we can infer that (i) gonadotropins and serum did not affect the reversible meiotic arrest promoted by roscovitine in sheep oocytes; (ii) the maximum efficiency of meiosis inhibition occurred in serum-supplemented medium; (iii) roscovitine action on cumulus cells was not fully reversible independently of medium composition.