Prohibitin immunostaining was found in the cytoplasm of granulosa and theca-interstitial cells, whereas PCNA localized in the nuclei within the preantral follicles (Fig. 2). At this stage of development, prohibitin immunoreactivity was higher in granulosa cells than in theca-interstitial cells. Cells with intense PCNA staining (Fig. 2D, arrow) showed low im-munostaining for prohibitin (Fig. 2A, arrowhead). Some cells showed weak signal intensity for PCNA in the nucleus but exhibited increased prohibitin expression in the cytoplasm. The PCNA immunoreactivity was also evident in oocytes of preantral follicles (Fig. 2, D and G). Interestingly, some cells expressed neither prohibitin nor PCNA.
Similar developmental patterns for prohibitin and PCNA immunoreactivity were observed in granulosa cells from early and large antral follicles. Moreover, a gradient-like spatial expression pattern of prohibitin was evident, with mural granulosa cells being most intensely stained and granulosa cell layers closer to the antral cavity exhibiting lower immunoreactivity (Fig. 2, B, C, E, F, H, and I). As the follicles matured, prohibitin was more abundantly expressed in the theca-interstitial cells (Fig. 2, B and H).
FIG. 3. Immunolocalization of prohibitin and 450scc in preantral, early antral, and large antral follicles from immature rats treated with eCG plus NRS. Using adjacent paraffin sections, prohibitin (A, C, and E) and 450scc (B, D, and F) proteins were localized with corresponding specific antibodies by indirect immunofluorescence method. Negative control (G and H) is also shown. The arrowhead in F denotes the cumulus-oocyte complex. The DNA was counterstained with 4′,6′-diamidino-2-phenylindole (blue). Each photomicrograph is a representative of three serial sections per ovaries per animal (n = 5). a, Antrum; GC, granulosa cell; o, oocyte; TIC, theca-interstitial cell. Bar = 50 ^m (A-F) and 100 ^m (G and H).