CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation.
BACKGROUND
Mammalian oocytes acquire competence to be fertilized during meiotic maturation. The protein kinase CDC2 plays a pivotal role in several key maturation events, in part through controlled changes in CDC2 localization. Although CDC2 is involved in initiation of maturation, a detailed analysis of CDC2 localization at the onset of maturation is lacking. In this study, the subcellular distribution of CDC2 and its regulatory proteins cyclin B and SPDY in combination with several organelle markers at the onset of pig oocyte maturation has been investigated.


RESULTS
Our results demonstrate that CDC2 transiently associates with a single domain, identified as a cluster of endoplasmic reticulum (ER) exit sites (ERES) by the presence of SEC23, in the cortex of maturing porcine oocytes prior to germinal vesicle break down. Inhibition of meiosis resumption by forskolin treatment prevented translocation of CDC2 to this ERES cluster. Phosphorylated GM130 (P-GM130), which is a marker for fragmented Golgi, localized to ERES in almost all immature oocytes and was not affected by forskolin treatment. After removal of forskolin from the culture media, the transient translocation of CDC2 to ERES was accompanied by a transient dispersion of P-GM130 into the ER suggesting a role for CDC2 in redistributing Golgi components that have collapsed into ERES further into the ER during meiosis. Finally, we show that SPDY, rather than cyclin B, colocalizes with CDC2 at ERES, suggesting a role for the CDC2/SPDY complex in regulating the secretory pathway during oocyte maturation.


CONCLUSIONS
Our data demonstrate the presence of a novel structure in the cortex of porcine oocytes that comprises ERES and transiently accumulates CDC2 prior to germinal vesicle breakdown. In addition, we show that SPDY, but not cyclin B, localizes to this ERES cluster together with CDC2.
Hölzenspies
Jurriaan J
JJ
Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. j.j.holzenspies@uu.nl
Stoorvogel
Willem
W
Colenbrander
Ben
B
Roelen
Bernard Aj
BA
Gutknecht
Dagmar R
DR
van Haeften
Theo
T
D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D018613:Microscopy, Confocal; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D013552:Swine
D019926:Cyclin B; D005576:Colforsin; D016203:CDC2 Protein Kinase