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Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro

Department of Veterinary Physiology, Faculty of Veterinary Science, University of Murcia, 30071 Murcia, Spain and ¹ Institute of Agriculture, Tennessee Agricultural Experiment Station, Department of Animal Science, University of Tennessee, Knoxville, Tennessee 37996-4574, USA

Correspondence should be addressed to J L Edwards, Department of Animal Science, 206 Brehm Animal Science Building, 2505 River Drive, Knoxville, Tennessee 37996-4574, USA; Email: jedwards{at}utk.edu

The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34^cdc2/cyclin B kinase) to maintain bovine cumulus–oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 µmol/l S-roscovitine for 24 h. Hoechst staining showed that 50 µmol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 µmol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin–fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 µmol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8–16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.

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