Submission Number: MAS-4-22-7

Abstract Number: 166

ARTIFICIAL ACTIVATION BY TREATMENT WITH EDTA OF PORCINE OOCYTES AT THE GV STAGE.

Tohru Azuma, Shuntaro Ikeda and Masayasu Yamada*

Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan ¹

Abstract:
In vitro culture of mammalian oocytes arrested at the germinal vesicle (GV) stage induces their resumption of the first meiosis and progression to metaphase of the second meiosis (MII), where they undergo arrest again. After fertilization or artificial activation, the oocytes exit from the arrest followed by emission of the second polar body and formation of pronuclei. In the present study, we found that treatment with a cell membrane-impermeable chelator, EDTA of porcine oocytes at the GV stage collected from follicles (4-6 mm in diameter) induced artificial activation of the oocytes followed by formation of a pronucleus. When the oocytes were cultured for 48 hrs in 1 mM EDTA containing TCM199 medium supplemented with 0.9 mM pyruvate, 3 mM glucose, 10 IU/ml hCG, 10 IU/ml eCG and 0.1 % PVA, 63 % of the oocytes (n=68) were activated and more than 90 % of the activated oocytes formed one pronucleus without emission of the first and the second polar bodies. This result suggests that EDTA may force the oocytes to form an interphase nucleus without sister chromatid segregation during the first meiosis. When the activated oocytes (n=160) were further cultured in NCSU medium with 0.4 % BSA for 6 days, blastocysts that appeared to be morphologically normal were formed at the rate of 10 %. Next, we examined the effects of metal ion-saturated EDTA and a Ca²⁺ specific chelator, EGTA at a concentration of 1 mM. As a result, Ca-EDTA but not Zn-EDTA and EGTA had the ability to activate the oocytes. Moreover, it was found that activation of the oocytes with Ca-EDTA was significantly inhibited by addition of Zn²⁺ but not Cu²⁺, Ni²⁺ or Co²⁺ at concentrations of more than 15 &muM. Most of the oocytes treated with 15 &muM Zn²⁺ alone for 48 hrs appeared to normally develop to the MII stage. From these results, we propose that extracellular chelation of Zn²⁺ with EDTA of porcine oocytes at the GV stage results in activation of the oocytes with pronuclear formation. .

Keywords: EDTA, Zn²⁺, activation, porcine oocyte

This abstract is being presented at: 8:00 AM in session:
Oocyte Development