The amount of the acidic isoform (Fig. 1B, arrowhead) of prohibitin content increased more than twofold in granulosa cells treated with eCG plus NRS compared to those treated with saline (P = 0.0312). Consistent with its effect on the induction of apoptosis in granulosa cells, withdrawal of gonadotropin (treatment with eCG plus anti-eCG) induced a decrease in the acidic isoform of the protein content 48 h after treatment.
Spatial Expression Pattern of Prohibitin During Induction of Follicular Maturation and Atresia
We previously demonstrated in a conditionally immortalized rat granulosa cell line that prohibitin content decreased during cell proliferation but increased in cells undergoing differentiation and at early stages of apoptosis. To determine whether these finding were physiologically relevant in vivo, the cellular distribution pattern of prohibitin expression in the ovarian follicles and the influence of gonadotropic stimulation and withdrawal were examined in ovaries of immature rats treated with eCG plus NRS and with eCG plus anti-eCG. As summarized in Table 1, prohibitin and PCNA (a cofactor of DNA polymerase 8 and cyclin-cdk complexes and used as a marker for cellular proliferation) were immunolocalized in both granulosa and the-ca-interstitial cells in rat ovary 48 h following eCG treatment when follicular development was observed.
FIG. 2. Immunolocalization of prohibitin (red) and PCNA (green) in gonadotropin-stimulated rat ovarian follicles at different stages of development. Ovaries from immature rats treated with eCG plus NRS (48 h) were collected, fixed in 10% formalin, embedded in paraffin, and sectioned for immunohistochemical analyses. Note the intense immunostaining for PCNA in the preantral follicle granulosa cell (arrow) and the decreased staining for prohibitin (arrowhead) as well as the PCNA-positive oocyte nucleus. Preantral follicles (A, D, and G), early antral follicles (B, E, and H), and large antral follicles (C, F, and I) are shown, as is negative control (J, K, and L). 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; N, nucleus; o, oocyte; TIC, theca-interstitial cell. Bar = 50 ^m (A-I) and 100 ^m (J-L).