High developmental competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture by specific inhibition of MPF kinase activity

Abstract

Roscovitine, a potent inhibitor of M‐phase Promoting Factor (MPF) kinase activity, was used to maintain cattle oocytes at the germinal vesicle stage for a 24‐hr culture period. A concentration of 25 μM of roscovitine was sufficient to reach the maximum level of meiotic resumption inhibition with 83 ± 6% of the oocytes remaining at the germinal vesicle stage after the 24 hr of culture. The histone H1 kinase activity was maintained at a basal level after culture under roscovitine inhibition at any of the concentrations tested (12.5, 25, 50, and 100 μM). This inhibitory effect of roscovitine was fully reversible since 89 ± 4% of the oocytes cultured for 24 hr in the presence of 25 μM of roscovitine reached the metaphase II stage after a further culture of 24 hr in permissive medium (TCM199 supplemented with 10 ng/ml EGF). The cleavage rate as well as the development to the blastocyst stage was not different for oocytes cultured for 24 hr under roscovitine (25 μM) inhibition and then matured for 24 hr in the presence of EGF as compared to oocytes not submitted to prematuration culture (82 ± 8% cleavage and 41 ± 4% blastocysts at 8 days post insemination for control oocytes compared to 90 ± 7% and 36 ± 7% respectively for roscovitine‐treated oocytes). Roscovitine meiotic inhibition was also effective in the presence of EGF, and the final developmental potential as well as the kinetics of blastocyst formation were not affected after such prematuration treatment. The EGF induced cumulus expansion was also inhibited by roscovitine. These results indicate for the first time the feasibility of culturing cattle oocytes under meiotic inhibition without decreasing their resulting developmental potential. Mol. Reprod. Dev. 55:89–95, 2000. © 2000 Wiley‐Liss, Inc.

Retinoid‐dependent mRNA expression and poly‐(A) contents in bovine oocytes meiotically arrested and/or matured in vitro

“…The presence of meiotic inhibitors is likely to allow neo-transcription or polyadenylation of pre-existing mRNAs or both. The implications of these findings in subsequent oocyte developmental ability are unknown, although roscovitine alone does not reduce the potential of oocyte development (Mermillod et al, 2000;Lattanzi et al, 2003). However, the differences we observed in the inhibited oocyte at the transcription level, as well as ultrastructural anomalies (Lonergan et al, 2003b), coexist with normal or improved embryonic development.…”

“…Those COCs assigned to groups undergoing meiotic inhibition were washed twice in basic medium (BM: TCM199 þ PVA 0.5 mg ml À1 ) containing 25 mM roscovitine previously dissolved in 50 mM DMSO and stored in aliquots at À208 C. Once thawed, the stock solution was diluted to 10 mM in DMSO before use, as described by Mermillod et al (2000). Meiotic inhibition was performed by culturing COCs in BM with 25 mM roscovitine for 24 hr.…”

“…Most transcriptional activity in the oocyte must occur before maturation, but transcription is not fully abolished within the first hours after meiotic resumption (Hyttel et al, 1997;Pavlok et al, 2000;Tomek et al, 2002). Maintenance of reversible meiotic arrest in vitro at the germinal vesicle stage is feasible in cattle by using a variety of chemicals including roscovitine (Mermillod et al, 2000;Lattanzi et al, 2003), a selective inhibitor of cyclin-dependent kinases (Cdks). Roscovitine enables bovine oocytes to be cultured under meiotic inhibition without decreasing their capacity to develop.…”

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

“…Those COCs assigned to groups undergoing meiotic inhibition were washed twice in basic medium (BM: TCM199 + PVA 0.5 mg/mL) containing roscovitine 25 mM, which was previously dissolved in DMSO and stored in aliquots at À20 8C until use, as described by Mermillod and co-workers [4]. Meiotic arrest was accomplished by culturing COCs in BM with roscovitine 25 mM during 24 h. Those COCs cultured under permissive, non-meiotically inhibited maturation conditions were washed three times in HM and twice in maturation medium (MM).…”

“…Inhibition of meiosis is thought to reproduce this period necessary for the oocyte to synthesize and store mRNA and protein. The use of meiotic inhibitors, such as roscovitine, allowed oocytes to improve developmental competence without reducing blastocyist rates [4].…”