PARENT SESSION
Oocyte Development

DEVELOPMENT OF BOVINE EMBRYOS DERIVED FROM OOCYTES CULTURED IN ROSCOVITINE (INHIBITOR OF p34^cdc2/CYCLIN B KINASE) FOR 24 OR 48 HOURS BEFORE OOCYTE MATURATION.

McCann, Lisa¹, Miller, Jennifer¹, Edwards, J Lannett¹, ¹

ABSTRACT- Inhibiting resumption of meiosis, without compromising further development, will be important for development of culture systems allowing the study of immature oocytes after removal from follicles. Objective of this study was to evaluate development of bovine embryos derived from oocytes cultured with roscovitine (a cell cycle inhibitor of p34^cdc2/cyclin B kinase preventing resumption of meiosis) for 0, 24 or 48 h before oocyte maturation. Cumulus oocyte complexes were cultured in 0 (control medium: M199 with 10% fetal bovine serum, 1 X nucleosides, 2 mM L-glutamine, 1 X nonessential amino acids, 0.1 mM -mercaptoethanol, 50 U/ml penicillin, 50 g/ml streptomycin, 0.3 g/ml LH, and 5.0 g/ml FSH) or 50 M roscovitine (control medium without gonadotropins) at 38.5°C in 5.5% CO₂. After 24 or 48 h, oocytes were washed of roscovitine and cultured for an additional 24 h in control medium containing gonadotropins. Oocytes, presumed matured, were divided within treatment; half were fertilized while remaining served as parthenogenetic controls. Data were arranged in a randomized block design and analyzed using mixed models of SAS. Culture of oocytes with roscovitine for 24 or 48 h did not compromise ability to cleave after maturation and fertilization. In fact, proportion of putative zygotes cleaving by day 3 post-fertilization, was higher for oocytes cultured with roscovitine for 24 h (81.1%) compared to 48 h (61.0%) or control (52.2%; SEM=6.2; P<0.02). Ability of cleaved embryos to develop to 8-16 cell was similar (88.6, 91.9 and 91.5% for 0, 24 and 48 h with roscovitine, respectively; SEM=2.7); however, proportion developing to blastocyst, by day 9 post-fertilization, was higher for oocytes cultured with roscovitine for 24 h (30.9%) compared to 48 h (17.6%) or control (16.6%; SEM=3.1; P<0.01). Number of nuclei in blastocysts was similar regardless of initial treatment of oocytes (95.6; SEM=5.7). Moreover, culture of oocytes with 50 M roscovitine did not increase parthenogenetic activation (6.1, 2.4 and 6.5% cleaved for 0, 24 and 48 h with roscovitine, respectively; SEM=2.1). Culture of bovine oocytes with roscovitine for 48 h after follicle removal did not compromise subsequent development of embryos resulting after maturation and fertilization.

KEY WORDS: oocyte maturation, roscovitine, bovine, embryo