Submission Number: GLO-4-1-7

Abstract Number: 66

FURTHER STUDIES ON THE ROLE OF MITOCHONDRIA IN CONTROLLING OOCYTE APOPTOSIS.

Gloria I Perez ¹, Alexander M Trbovich ¹, Andrea Jurisicova ^1,2, Robert F Casper ² and Jonathan L Tilly ¹

Vincent Center for Reproductive Biology, Department of OB/GYN, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA. ¹
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. ²

Abstract:
Isolated mouse oocytes spontaneously undergo apoptosis in vitro (Molecular Human Reproduction 1999 5:414-420). Since mitochondria play a predominant role in the regulation of apoptosis in many cell types, herein we studied if microinjection of mouse oocytes with mitochondria isolated from cells prior to or after receipt of a pro-apoptotic stimulus exert different effects on the occurrence of oocyte apoptosis. As a source of mitochondria, healthy granulosa cells isolated from preovulatory follicles present in gonadotropin-primed prepubertal mouse ovaries were processed immediately by differential centrifugation (non-apoptotic) or were briefly UV-irradiated (125 mJ, 25 sec) to induce pro-apoptotic signaling events prior to mitochondria isolation. Mature oocytes isolated from FVB female mice (which exhibit inherently high rates of spontaneous apoptosis in vitro; Molecular Endocrinology 1999 13:841-850) were denuded of cumulus cells and microinjected with buffer (control group) or with approximately 510³ mitochondria, and were then incubated for 24 h to monitor the occurrence and extent of oocyte apoptosis by cell morphology, DNA fragmentation and caspase activation. Intracytoplasmic injection of mitochondria prepared from non-apoptotic granulosa cells into FVB oocytes decreased the rate of spontaneous apoptosis from 71% (n=139) in the control group to 36% (n=127) in the mitochondria-microinjected group. By comparison, microinjection of mitochondria isolated from granulosa cells that were exposed to UV irradiation failed to attenuate spontaneous oocyte death (n=40). Furthermore, electron microscopic analysis, FVB oocytes revealed mitochondria with electron- dense matrices containing small membrane vacuoles that ruptured during culture. These structures and events were not observed in mitochondria of B6C3F1 oocytes which undergo inherently low rates of spontaneous apoptosis in vitro (<5% in 24 hours). These findings suggest that oocyte apoptosis is influenced by changes in biochemical and ultrastructural properties of mitochondria. (Supported by NIH grant RO1-AG12279 and a Harvard Center of Excellence in Women's Health Grant). .

Keywords: Apoptosis, Oocyte, Mitochondria

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