BUTYROLACTONE I AND INDIRUBIN PREVENT
GVBD AND MICROTUBULAR
POLYMERIZATION IN PORCINE OOCYTES
J. Ju1, Y. Weng1, P. Tang1 and S. Sha1.
1
Department of Animal Science, National Chung Hsing University, Taichung,Taiwan,
ROC
Abstract- Pig oocytes undergo spontaneous germinal vesicle breakdown (GVBD)
after being released from follicular environment which potentially hinders GV-related
manipulation. In this study, we investigated the effects of specific cdc2 kinase
inhibitors, butyrolactone I (BL I) and/or indirubin oxime (IO), on the prevention of
GVBD, changes of microtubular structures (MTs), and maturation rates in porcine
oocytes after removal of the inhibitors. Experiment 1: Cumulus-oocyte-complexes
(COCs) were collected from slaughterhouse ovaries and IVM cultured (44 h) in
NCSU#23 medium containing different concentrations of BL I (0, 10, 20, 40, or 80
M). Results showed that percentages of oocytes remained in GV stage were 0, 0, 32,
80, 84 %, and the maturation rates were 86, 63, 30, 0, and 0 % for the oocytes treated
with 0, 10, 20, 40 and 80 M of BL I, respectively. Experiment 2: Thirty-two to forty
oocytes in each group were fixed at 44 h after IVM for immunocytochemical labeling
of the chromatin and the cytoskeleton. Similar proportions (61-82%) of oocytes were
prevented from GVBD as in Experiment 1. Of these, 77-88% oocytes remaining in
the GV stage showed fragmentation or pulverization of pericytoplasmic MTs after BL
I co-incubation. In contrast, the oocytes without BL I treatment (control) had normal
pericytoplasmic MT patterns. When oocytes were released from BL I (Experiment 3)
or IO (Experiment 4) treatment and IVM cultured for an additional 44 h, 81, 82, and
77% of oocytes resumed meiosis, but only 52, 36 and 5% of oocytes reached MII
stage in the 20, 40 and 80 M of BL I-treated groups, respectively. A similar pattern
(38-51%) of reversibility was also observed in the IO-treated oocytes. In conclusion,
treatment with 40-80 M of BL I effectively prevented the resumption of meiosis
and polymerization of pericytoplasmic MTs. However, the reversibilities of the
oocytes after treatment with the drugs were not satisfactory, which might be due to
the lengthened duration of coincubation with the drugs.
Key words- Germinal vesicle, Pig, Oocyte, Microtubule