Although TUNEL alone cannot distinguish between apoptosis and necrosis, we and others have previously demonstrated that apoptosis is the form of cell death prevalent during follicular atresia and luteal regression. For this reason, TUNEL-positive cells in ovarian sections were considered apoptotic rather than necrotic in the present studies. Caspase-3 and apoptosis were localized in ovarian follicles of rats treated with anti-eCG antiserum, or in luteal tissue from rats treated with PGF2a (Fig. 5). As previously observed, the CL exhibited intense staining for caspase-3 (Fig. 5A), and there was no apparent difference in the extent of caspase-3 immunostaining in CL that were positive or negative for apoptosis identified by TUNEL (Fig. 5B).
Conversely, follicles that contained TUNEL-positive apoptotic granulosa cells (Fig. 5, D and F) displayed immunostaining for caspase-3 in both the granulosa and theca layers (Fig. 5, С and E), while TUNEL-negative follicles exhibited no caspase-3 in granulosa cells (Fig. 5C). TUNEL and caspase-3 localization were done in adjacent sections, so it is not possible to assess the colocalization of caspase-3 and apoptosis in individual cells. However, TUNEL-positive follicles contained scattered apoptotic granulosa cells (Fig. 5F), and these follicles displayed scattered caspase-3 staining, in a similar pattern, in the adjacent section (Fig. 5E). buy asthma inhalers
FIG. 4. Localization of caspase-3 in the rat ovary. Ovaries removed from rats 48 h after eCG injection (ovaries enriched with antral follicles), or 7 days after eCG + hCG injection (ovaries enriched with CL) were processed for histology as described in Materials and Methods. Cleared and rehydrated sections were incubated with normal rabbit serum (control; A and D) or rabbit anti-CPP32 antibody (5 p,g/ml; В, С, E, and F), washed, and incubated with biotinylated goat anti-rabbit IgG. The sections were again washed, incubated with avidin-biotin-HRP, and developed with dimethylaminoazoben-zene (DAB)/CuS04. Positive immunostaining (black) was observed in the theca cells (t) and oocytes (arrowhead) of follicles (В, C, and E) and in luteal cells (cl) of the CL (E and F). Immunostaining for caspase-3 was weak or absent from granulosa cells (gc; В and C) and interstitial tissue (i; В and E). In positively stained cells, caspase-3 was localized in the cytoplasm (c), whereas the nucleus (n) contained little or no staining for this enzyme (F). A, B, D, and E) X100. С and F) X640. One representative of five experiments is shown.
FIG. 5. Localization of caspase-3 and apoptosis in follicles and luteal tissue. Caspase-3 (left panels) and apoptosis (right panels) were localized in histological sections, with the anti-CPP32 antibody and TUNEL, respectively, as described in the Materials and Methods. A) There was no difference in caspase-3 staining between two neighboring CL (a) and (b), which both displayed intense caspase-3 immunoreactivity, although in an adjacent section (B), one CL was TUNEL-positive (b) while the other was TUNEL-negative (a). C) TUNEL-negative follicles (c) displayed typical caspase-3 staining in theca (t) but not granulosa (gc) cells. However, a TUNEL-positive follicle (d) contained scattered caspase-3-positive granulosa cells. D) In an adjacent section, scattered TUNEL-positive granulosa cells were evident in the follicle containing caspase-3-positive (d), but not the caspase-3-negative (c) granulosa cells. E) In a TUNEL-positive follicle, positive caspase-3 staining was evident in some granulosa cells (arrowhead) while others did not display caspase-3 immunoreactivity (arrow). F) In an adjacent section, some granulosa cells were TUNEL-positive (arrowhead) while others were not (arrow). A-D) X100. E and F) X640. One representative of five experiments is shown.