OVARY - C
Wednesday, August 4, 2004
10:30 AM–12:30 PM
(784) CASPASE-3 EXPRESSION IN SMALL OVARIAN FOLLICLES.
Fenwick, Mark1, Hurst, Peter1, 1 Department of Anatomy & Structural Biology, Dunedin, New Zealand
ABSTRACT- Small single-layered (primordial) follicles must remain viable in the ovary until they are periodically selected to develop and mature for ovulation. However, once they are selected, the default developmental pathway is to degenerate as a consequence of follicle cell apoptosis. Caspase-3 is an apoptotic enzyme involved in the execution of cell death by apoptosis. Reports have previously demonstrated that the active caspase-3 enzyme is expressed in granulosa cells and oocytes of larger developing follicles, suggesting a role for caspase-3 mediated degeneration in these follicular types. Active caspase-3 has never been detected in cells of small, putatively resting follicles in the normal adult ovary. Using the adult mouse as a model, an initial aim of this study was to determine whether the message and translated pro-protein for caspase-3 is constitutively expressed in cells of small single-layered follicles. A second aim was to confirm that caspase-3 is associated with apoptosis in the ovary by correlating the active form of caspase-3 with the TdT-mediated dUTP nick end-label (TUNEL) assay using confocal microscopy. Results showed that in the smallest single-layered follicles, caspase-3 mRNA was detectable in granulosa cells and oocytes using non-radioactive in situ hybridisation and RT-PCR analysis of laser-dissected follicle isolates. The inactive caspase-3 pro-protein was also localised in oocytes and granulosa cells of small single-layered follicles by immunohistochemistry. In larger multi-layered follicles, granulosa cells expressed both mRNA and pro-protein at all stages of follicle development, while the active form co-localized in cells that were also TUNEL positive. This study confirms that prior to the spontaneous onset of atresia, follicles are poised to rapidly degenerate by the activation of a caspase-3 mediated apoptotic pathway. This study has also shown that in the normal adult ovary the smallest follicles express caspase-3, but as yet there is no evidence for its activation or TUNEL reactivity in constituent cells.
KEY WORDS: apoptosis, follicle, caspase-3, primordial