Submitted March 11, 2004
Returned for revision April 19, 2004
Accepted April 29, 2004
Ovary
Cdc2-Cyclin B-Induced G2 to M Transition in Perch Oocyte
Is Dependent on Cdc25
Dipanjan Basu ,
A.K. Navneet ,
Subrata Dasgupta ,
and
Samir Bhattacharya *
* To whom correspondence should be addressed. E-mail: samir{at}iicb.res.in.
Abstract
The G2 to M phase transition in perch oocyte is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17
, 20
-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although, a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. p30 cyclin B was present in uninduced oocyte but over expressed by MIH. MIH increased p30 cyclin B accumulation at 3h, high level of which was maintained between 9-21h, but effective increase in GVBD and H1 kinase activation could only be observed between 15 - 21h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12h, and a substantial increase occurred during 15h - 18h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocyte and its conversion to active MPF requires dephosphorylation by Cdc25, existence of which has not yet been shown in fish.
Key words:
Oocyte development
