Effects of cell cycle dependent kinases inhibitor on nuclear and cytoplasmic maturation of porcine oocytes

Abstract

The aims of this study were to assess the effectiveness of roscovitine, a potent inhibitor of cell cyclin kinases, to prevent meiotic resumption in porcine oocytes, and to test the subsequent fertilisability and developmental competence of these oocytes. Roscovitine blocked porcine oocytes at the GV stage during 22-44 hr of culture. This effect was dose-dependent, and a concentration of 25 microM was sufficient to prevent meiotic resumption in 92+/-5% of the oocytes after 22 hr in the presence of EGF and FSH. Cumulus expansion was also inhibited under these conditions. The histone H1 kinase activity in oocytes was inhibited in a dose-dependent way, and maintained at a basal level with 25 microM of roscovitine. Synthesis of proteins of 29, 47 and 79 kDa, normally synthesized during maturation, was inhibited too. All these effects were fully reversible. However, the kinetics of maturation were accelerated after roscovitine removal, and the acceleration was more pronounced after 44 hr of inhibition than after 22 hr. Fertilization of oocytes blocked for 22 hr before a 44 hr maturation was decreased compared to control, but was not different from that of oocytes matured for 66 hr. The developmental competence was decreased for the oocytes cultured for 66 hr, including or not an inhibition period, but it was less reduced for oocytes maintained under inhibition for 22 hr. Roscovitine may thus protect oocytes against the aging mechanisms responsible for developmental competence loss, but not against loss of fertilisability. In conclusion, roscovitine provides a useful tool to study the morphological and biochemical basis of porcine oocyte terminal differentiation.

Human oocyte maturation in vitro

“…In fact, GV oocytes treated transiently with roscovitin, an inhibitor of MPF, display a delay in GVBD and cytoskeletal characteristics more similar to in vivo matured oocytes (Sanfins et al, 2004). Consistently, it has been described in the pig (Marchal et al, 2001), cow (Ponderato et al, 2001) and horse (Franz et al, 2003) that temporary arrest at the GV stage with roscovitin at the beginning of the maturation period increases the developmental potential of IVM oocytes. The implications of these findings are least two-fold.…”

Regulation of Maternal Gene Expression by MEK/MAPK and MPF Signaling in Porcine Oocytes During In Vitro Meiotic Maturation

“…MPF is required for oocyte GVBD. The effect of roscovitine on the maintenance of oocyte meiotic arrest and oocyte developmental competence has been previously assessed in pigs using different concentrations, inhibitory periods and sources of oocytes [8][9][10][11][12][13]20]. However, the molecular basis of MPF action is still unclear.…”

“…However, the data for the interplay of these two kinases in oocytes are still controversial. Previous studies using pig oocytes have focused primarily on the dynamic changes in MAPK and MPF activities and on the effects of inactivation of these two kinases on porcine oocyte maturation in terms of nuclear maturation, microtubule organization and oocyte developmental competence [5][6][7][8][9][10][11][12][13]. The underlying molecular mechanisms, such as the cytoplasmic maturation and regulation of maternal gene expression, remain to be reported.…”

Application of In Vitro Maturation to Assisted Reproductive Technology

“…In fact, we observed different expression patterns of mRNA between meiotic arrested oocytes and non-treated oocytes (unpublished data). In addition, Marchal et al [88] have also shown that immature porcine oocytes synthesize some proteins during meiotic arrest by roscovitine, a potent inhibitor of cyclindependent kinase.…”