Apoptosis was evident as an increase in the extent of DNA ladders in ovarian follicles treated with anti-eCG antiserum, or in luteal tissue from rats treated with PGF2a (Fig. 1). Actin cleavage to produce the 41-kDa product from the intact 42-kDa form was evident in protein extracts from apoptotic luteal tissue (Fig. 2B) but not follicular granulosa cells (Fig. 2A). In addition, after longer exposure of the film to the membrane, a cleavage product of approximately 30 kDa could be observed in the extracts from regressing luteal cells but not atretic follicular cells (Fig. 2C). Similarly, cleavage of PARP from the intact 113-kDa protein to produce an 85-kDa fragment was not evident in anti-eCG-treated rat ovaries (Fig. ЗА), but the 85-kDa fragment was observed in protein extracts from rats treated with PGF2a (Fig. 3B).
Caspase-3 was localized in follicles and luteal tissue of ovaries from the eCG and hCG groups (Fig. 4). Positive immunostaining was observed in luteal cells of the CL and in the theca and oocytes of follicles (Fig. 4, В, С, E, and F). Immunostaining for caspase-3 was weak or absent in granulosa cells and interstitial tissue. In the intensely staining theca and luteal cells, caspase-3 was localized in the cytoplasm whereas the nucleus contained little or no staining for this enzyme (Fig. 4, С and F). Buy Asthma Inhalers Online
FIG. 1. Low-moiecular-weight DNA degradation during induced follicular atresia and luteal regression. Follicular atresia (A) following injection of anti-eCG antibody (control: rats injected with normal serum). Luteal regression (B) following injection of Lutalyse (control: rats injected with saline) as described in the Materials and Methods. DNA was isolated, end-labeled with [32P]dCTP, and resolved by electrophoresis. Representative results of three experiments.
FIG. 2. Actin cleavage during induced follicular atresia and luteal regression. Follicular atresia (A) following injection of anti-eCG antibody (lane 2) or normal serum as control (lane 1). Luteal regression (B) following injection of Lutalyse (lane 4) or saline as control (lane 3) as described in the Materials and Methods. Total cell protein was extracted, and 40 |xg was resolved by 9% SDS-PAGE, electrotransferred to nitrocellulose, im-munoblotted with anti-actin antibody, and visualized by ECL, as described in the Materials and Methods. Actin degradation is indicated by the production of a 41-kDa fragment from the intact 42-kDa actin (B), and also by the production of a 30-kDa fragment visualized after longer exposure of the ECL-treated membrane to x-ray film (C). Representative results of three experiments.
FIG. 3. Western analysis of PARP cleavage during induced follicular atresia and luteal regression. Protein extracts (40 jxg) were resolved by electrophoresis, electrotransferred to nitrocellulose membranes, and immunoblotted with anti-PARP antibody, as described. PARP cleavage is indicated by the production of the 85-kDa fragment from the intact 113-kDa protein. Numbers on the right indicate molecular weight standards (X10~3). Representative result of 3 experiments.