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.

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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 microM 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 microM). This inhibitory effect of roscovitine was fully reversible since 89 +/- 4% of the oocytes cultured for 24 hr in the presence of 25 microM 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 microM) 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.

The aim of this study was to investigate the role of growth hormone (GH) on in vitro cumulus expansion and oocyte maturation in equine and porcine cumulus-oocyte complexes (COCs), and to approach its way of action. Equine COCs were cultured in a control medium (TCM199, 5 mg/ml BSA, 1 g/ml estradiol, and antibiotics) supplemented with either 0.5 g/ml equine GH or 5 g/ml equine LH. Porcine COCs were cultured in a basal medium (TCM199 with 570 M cysteamine) supplemented with 0, 0.1, 0.5, or 1 g/ml porcine GH or in a control medium (basal medium with 10 ng/ml epidermal growth factor and 400 ng/ml FSH) supplemented with 0 or 0.5 g/ml porcine GH. After culture, cumulus expansion and nuclear stage were assessed. The cytoplasmic maturation of porcine oocytes was evaluated by in vitro fertilization and development for 7 days. The modifications of the expression of proteins implicated in cumulus expansion were analyzed in equine COCs by SDS-PAGE with antibodies against connexins 32 and 43 and hyaluronan synthases (Has) 1, 2, and 3. The expression of GH receptor mRNA was studied in oocytes and cumulus cells of the two species using reverse transcription-polymerase chain reaction with specific primers. The addition of GH in maturation medium increased cumulus expansion in equine but not porcine COCs. It improved nuclear maturation in equine and porcine, but had no effect on porcine fertilization and embryo development. The GH receptor mRNA was detected in equine and porcine oocytes and cumulus cells. GH did not influence the expression of Has 1, Has 3, and connexin 43 in equine cumulus cells. cumulus cells, gamete biology, growth hormone, oocyte development, ovum INTRODUCTIONGonadotropins are the major regulators of ovarian function. In the last few years, however, there has been evidence that growth factors and metabolic hormones are also involved in ovarian regulation. The role of growth hormone (GH) in ovarian functions, follicular growth, and steroidogenesis is well known [1, 2 for review], and some evidence shows a positive effect of GH on oocyte maturation.