Tsang, B.,Boone, David L.2009-03-252009-03-2519971997Source: Dissertation Abstracts International, Volume: 59-03, Section: B, page: 0944.9780612261068http://hdl.handle.net/10393/9486http://dx.doi.org/10.20381/ruor-16345Apoptosis is a physiological form of cell death and is the cellular basis of ovarian follicular atresia and luteal regression. Apoptosis has distinct morphological and biochemical features including the degradation of DNA by the action of a Ca$\sp{2+}$/Mg$\sp{2+}$-dependent endonuclease. This characteristic DNA degradation (DNA "ladders") is evident in atretic follicles and regressing luteal tissue of diverse species. Hormones, growth factors and cytokines that induce or suppress follicular atresia or luteal regression also induce or suppress the characteristic apoptotic DNA degradation. In order to elucidate the cellular and molecular mechanisms of ovarian apoptosis, the Ca$\sp{2+}$/Mg$\sp{2+}$-dependent endonuclease responsible for DNA degradation must be identified. The objectives of the present work were to identify the endonuclease responsible for ovarian apoptotic DNA degradation and its possible regulation during follicular atresia and luteal regression. Immature female rats were sequentially treated with diethylstilbestrol (DES; DES group; preantral follicles), DES+eCG (eCG group; antral follicles) or DES+eCG+hCG (hCG group; luteal tissue). There was a developmental pattern of ovarian endonuclease activity such that nuclei from the eCG- and hCG-, but not the DES-group. degraded their DNA in an apoptotic fashion when exposed to Ca$\sp{2+}$ and Mg$\sp{2+}$ in vitro. Nuclear protein extracts contained three nuclease activities: a Mg$\sp{2+}$-dependent 27 kDa protein which was active on single-stranded DNA and did not display a developmental pattern of activity, and a doublet of 32/34 kDa which was Ca$\sp{2+}$/Mg$\sp{2+}$-dependent, active on double- or single-stranded DNA and present in the extracts of eCG and hCG, but not DES, groups. The 32/34 kDa activity was biochemically, immunologically and functionally indistinguishable from deoxyribonuclease I (DNase I). The mRNA encoding DNase I was also present, in a developmental pattern, in the rat ovary. Immunohistochemistry localized DNase I in nuclei of ovarian cells susceptible to apoptosis including luteal cells, granulosa cells from antral but not preantral follicles, and oocytes from preantral but not antral follicles. The possibility that caspase cleavage of actin and/or poly(ADP ribose) polymerase (PARP) regulates the activity of DNase I during ovarian apoptosis was investigated using an anti-eCG antibody in vivo to induce atresia and PGF$\sb{2\alpha}$ to induce luteal regression. Apoptosis during PGF$\sb{2\alpha}$-induced luteal regression, but not anti-eCG antibody-induced atresia, was associated with actin and PARP cleavage. Caspase-3 was immunolocalized in luteal cells and in theca, but not granulosa cells, of healthy follicles, and was also detected in granulosa cells of atretic follicles from anti-eCG antibody treated rats. These results demonstrate that the Ca$\sp{2+}$/Mg$\sp{2+}$-dependent endonuclease responsible for DNA "ladders" in ovarian cells is DNase I. Further, the expression and cellular distribution of DNase I during follicular development correlates with the susceptibility of ovarian cells to undergo apoptosis, which suggests that DNase I is responsible for the DNA "ladders" associated with follicular atresia and luteal regression. Although DNase I activity does not appear to be regulated by the caspase-mediated cleavage of actin or PARP, caspase-3 may be involved in the regulation of DNase I activity during ovarian apoptosis. Studies of the regulation of DNase I expression and activation will provide further insight into the cellular and molecular mechanisms of ovarian follicular atresia and luteal regression.244 p.Biology, Cell.Thesis