Abstract: 418

ASCORBIC ACID REGULATION IN EGGS AND ZYGOTES OF THE HAMSTER.

John Pepperell^{1 *}, M. Porterfield¹ , L. Liu¹ , P.J.S. Smith¹ , D.L. Keefe¹
Dept. Ob/Gyn, Women & Infants Hospital, Brown University Medical School, Providence, RI. ¹

Cells accumulate AA by direct uptake via Na+-dependent transport, or by uptake of dehydroascorbic acid (DHA), via a glucose-dependent transporter, and intracellular reduction to AA. Specific transporters for AA or DHA have not been identified, although the glucose transporters (GLUT)-1 and GLUT-3 also transport DHA. Anti-oxidant defense is key for cell survival, but in eggs and early embryos these mechanisms are virtually unknown. Our objective is to study ascorbic acid regulation in eggs and zygotes. Cycling hamsters were used to obtain preovulatory ovaries, while PMSG (25 IU)-treated and mated females were sacrificed to harvest zygotes. Immunofluorescent confocal microscopy was used to identify GLUTs-1 and -3 by specific antibodies. AA regulation in individual zygotes was measured by means of an AA-selective, self-referencing microelectrode fixed to a computer-controlled micromanipulator, allowing precise 3D movement. Sequential measurement of AA concentration ([AA]) within 1 µm of the zona and 30 µm away allows real-time data accumulation, and net AA flux is calculated from the difference in [AA] at the two points by application of Fick's equation. We saw strong immunolabelling for GLUTs-1 and -3 in the oolemma of oocytes of preovulatory and antral follicles, but little or no labeling in oocytes of earlier follicles. Zygotes exhibited a net efflux of AA, which showed transitory oscillatory increases when treated with ionomycin (10 µM). We suggest, preovulatory eggs may accumulate AA by uptake of DHA through GLUTs-1 and -3, while conditions of oxidative stress in zygotes leads to AA efflux.

    This abstract is being presented on Tuesday, August 3 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.