Activation of pig and cattle oocytes by butyrolactone I: morphological and biochemical study

Abstract

In this study a specific inhibitor of cyclin-dependent kinases (cdks), butyrolactone I (BL I), was used for activation of pig and cattle metaphase II (MII) oocytes. BL I at a concentration of 100 μM was able to induce activation of both pig and cattle MII oocytes in a manner dependent on exposure time; however, precise timing of BL I exposure was required for the best activation results. The optimum activation rates were obtained when cattle MII oocytes were treated for 5 h with BL I and subsequently for 3-11 h in control medium, and pig MII oocytes for 8 h in BL I and then for 8-16 h in control medium; the percentage of activated oocytes after such treatment varied between 55% and 74% and between 53% and 81% for cattle and pig oocytes, respectively. Shorter exposures to BL I led to re-entry of the oocytes to the metaphase state in 35-50% of oocytes, the remaining oocytes forming a pronuclear stage; longer exposure to BL I led to increased numbers of oocytes being abnormal or degenerated. The behaviour of histone H1 kinase and mitogen activated protein (MAP) kinase, also measured during the experiment, reflected the morphological changes in the oocytes: both were inactivated after BL I treatment, though the inactivation of histone H1 kinase occurred 2 h ahead of that of MAP kinase. However, in the oocytes treated for a shorter time with BL I, with the reoccurrence of condensed chromatin in proportion of the oocytes cultured in control medium after BL I treatment, both kinases became reactivated. Taken together, these results suggest the possibility of using BL I for activation and cloning experiments in both species.

Parthenogenetic Activation and Subsequent Development of Porcine Oocytes Activated by a Combined Electric Pulse and Butyrolactone I Treatment

“…Similar results were obtained by using a combination of EP and protein synthesis inhibitor, cycloheximide (CHX), in pigs [16], cattle [31]. BL I, which acts as a competitive inhibitor of ATP, is a potent and specific inhibitor of cyclin-dependent kinases (cdks) and has few inhibitory effects on other protein kinases such as MAP kinases [32,33]. Kubelka et al (2002) showed that the timing of exposure of oocytes to BL I impaired the time course of cdc2 and MAP kinase activities, cdc2 kinase became inactivated after 3 h of BL I exposure and MAP kinase became inactivated after about 5 h and 8 h of BL I treatment in cattle and pig oocytes.…”

“…BL I, which acts as a competitive inhibitor of ATP, is a potent and specific inhibitor of cyclin-dependent kinases (cdks) and has few inhibitory effects on other protein kinases such as MAP kinases [32,33]. Kubelka et al (2002) showed that the timing of exposure of oocytes to BL I impaired the time course of cdc2 and MAP kinase activities, cdc2 kinase became inactivated after 3 h of BL I exposure and MAP kinase became inactivated after about 5 h and 8 h of BL I treatment in cattle and pig oocytes. It was suggested that the inactivation of MAP kinase might not be induced by BL I but triggered by the inactivation of cdc2 kinase.…”

Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer

“…Chromosome condensation is correlated with histone H1 and H3 phosphorylation, and histone H1 is often used as a substrate for MPF in vitro (Kubelka et al 2002). However, the overall significance of H1 phosphorylation is not clear.…”

Effects of Different Oocyte Activation Procedures on Development and Gene Expression of Porcine Pre-Implantation Embryos

“…Therefore, additional treatments are required to stabilize the MPF and MAP kinases at low levels. To do so, treatments such as repetitive electrical pulses, protein synthesis inhibitors [cycloheximide (CHX) (Soloy et al 1997;Liu et al 1998)] or protein phosphorylation inhibitors [6-DMAP, butyrolactone I or bohemine (Liu et al 1998;Loi et al 1998;Alberio et al 2000;Kubelka et al 2002)] are currently used.…”