Submission Number: JER-4-9-12

Abstract Number: 133

PORCINE SPERM FACTOR INITIATES [Ca²⁺]_i OSCILLATIONS BY STIMULATING THE PHOSPHOINOSITIDE PATHWAY.

JT Smyth ¹, V Luzzi ², H Wu ¹, NL Allbritton ² and Rafael A Fissore* ¹

Department of Vet and Animal Sciences, University of Massachusetts, Amherst, MA, USA ¹
Department of Physiology and Biophysics, University of California, Irvine, CA, USA ²

Abstract:
Injection of a cytosolic porcine sperm factor (SF) into mammalian metaphase II (MII) oocytes has been shown to trigger calcium ([Ca²⁺]_i) oscillations. To determine if SF activates the phosphoinositide pathway, we first tested the effects of a membrane permeable phospholipase C (PLC) inhibitor, U-73122, on SF-induced oscillations in murine MII oocytes. Incubation of oocytes in 15-20 M U-73122 (n=7/7) or injection of SF incubated in 30 M U-73122 (n=14/19) inhibited SF-induced [Ca²⁺]i oscillations (P<0.05) while similar treatments with the inactive analog, U-73343 (n=4/4 for both treatments) failed to do so. The inhibition caused by U-73122 was specific to PLC since it did not block Ca²⁺ release caused by injection of inositol 1,4,5-triphosphate (IP₃), a downstream agonist. Also, the inhibitory effects of U-73122 on SF were reversed by co-incubation with 10 mM dithiothreitol (n=4/4; P>0.05). The ability of SF to directly activate IP₃ production was monitored in Xenopus oocytes. Following SF injection, a sample of ooplasm was transferred onto a permeabilized reporter cell calibrated to measure [IP₃]_i. Ooplasm of SF injected oocytes induced a mean increase in [IP₃]_i of 1.1 0.4 M (n=6) from a baseline of about 40 nM. Injection of buffer was unable to induce IP₃ production. Finally, to test if high [IP₃]_i could sensitize Ca²⁺-induced Ca²⁺ release (CICR), a hallmark of fertilization, MII oocytes were injected with Adenophostin A (Ad; 10 M), a non-hydrolyzable analog of the IP₃ receptor. Once oscillations induced by Ad had ceased/reduced in frequency, CaCl₂ (2.0 mM) was injected. Oocytes treated with the agonist showed a mean [Ca²⁺]_i rise of 494 40 nM (n=15) following CaCl₂ injection compared to 100 70 nM in oocytes not pretreated with the agonist (n=6; P<0.05). These results support the hypothesis that SF stimulates production of IP₃ and that high [IP₃]_i sensitizes CICR, both of which may play important roles in the initiation and persistence of [Ca²⁺]_i oscillations at fertilization.

Keywords: mammalian oocytes; fertilization; Ca²⁺; IP₃; PLC inhibitor

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