Submission Number: ALL-4-9-11

Abstract Number: 129

FAILURE TO RESPOND TO CALCIUM IN PRE-OVULATORY MOUSE OOCYTES: POTENTIAL ROLE FOR CaMKII.

Allison L Abbott* ¹, Rafael A Fissore* ² and Tom Ducibella* ^1,3

Sackler School of Biomedical Sciences, Tufts University, Boston, MA ¹
Dept. Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA ²
Dept. Obstetrics and Gynecology, New England Medical Center, Boston, MA ³

Abstract:
The ability to respond to intracellular calcium ([Ca2+]i) is essential for both cell cycle resumption and cortical granule (CG) secretion at fertilization of mature ovulated mouse eggs. However, fully-grown, pre-ovulatory oocytes are not competent to respond to experimentally-induced, fertilization-like [Ca2+]i oscillations as assayed by CG secretion (Abbott et al., Dev. Biol. 207:38-48, 1999). Our objective is to identify the specific step at which CG secretion is deficient in pre-ovulatory oocytes. The hypotheses were tested that CGs are blocked at one of the following regulatory steps: translocation, docking or fusion. The identification of the specific blocked step aids in further biochemical characterization of the secretory deficiency. Our strategy was to induce [Ca2+]i oscillations in pre-ovulatory oocytes and quantify the CG distance from the plasma membrane using electron microscopy. In fertilized oocytes, the mean distance (d=0.30m, n=309 CGs) was not different from unstimulated oocytes (d=0.30m, n=358; p>0.5) or unstimulated mature eggs. Sperm factor injection also did not decrease mean CG distance (d = 0.42m, n=316). These results indicate a deficiency in the mechanism responsible for CG translocation. Because Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates secretory vesicle translocation, its role was examined in fertilization-induced CG release in mature eggs. A CaMKII antagonist, KN-93, inhibited cell cycle resumption following fertilization in 80% of eggs (vs 3% with inactive analog KN-92). At the same concentration, KN-93 inhibited CG exocytosis induced by fertilization and by thimerosal (vs KN-92, p<0.01). An increase in the amount of total CaMKII in mature eggs as compared to pre-ovulatory oocytes was found by western blot analysis. Future work will determine if there is a correlated increase in CaMKII specific activity and if these maturation-associated changes are indeed causal to the observed secretory incompetence in pre-ovulatory oocytes. These results are relevant to animal and human assisted reproductive procedures in which large numbers of oocytes (before metaphase II) are routinely collected.

Keywords: Calcium, CaMKII, Secretion, Maturation

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