Levels of Prohibitin Protein in Granulosa Cells During Follicular Development and Atresia
We previously demonstrated that the administration of eCG to immature female rats followed by an antibody against the gonadotropin induces follicular atresia and granulosa cell apoptosis. To determine whether prohi-bitin protein content in granulosa cells in vivo is modulated by gonadotropin, cells were isolated from rats treated with saline, eCG plus NRS, or eCG plus anti-eCG and analyzed by Western blot analysis.
The results revealed the presence of immunoreactive proteins corresponding to prohibitin protein content in extracts of rat ovarian granulosa cells (Fig. 1A). Prohibitin content was significantly higher in granulosa cells isolated from the group treated with eCG plus NRS compared to the group treated with saline (P = 0.0375) and with eCG plus anti-eCG (P = 0.0425). Den-sitometric analyses of prohibitin levels on one-dimensional gels revealed an increase greater than twofold in prohibitin expression in granulosa cells isolated from ovaries of rats treated with eCG plus NRS (Fig. 1A).
Samples of the respective protein extracts were also utilized in two-dimensional Western blot analyses to determine whether changes in prohibitin isoforms occur during gonadotropin stimulation. Two polypeptide species from the 30-kDa protein (Fig. 1B) were delineated.
TABLE 1. Descriptive evaluation of prohibitin immunoreactivity in individual rat ovarian compartments during gonadotropin stimulation (G) and gonadotropin withdrawal (GW) in the rat*
* The intensity of the immunostaining was assessed on scale in which the highest and lowest intensities were indicated by 4+ and —, respectively. GC, Granulosa cells; TIC, theca interstitial cells.
FIG. 1. Western blot analysis of protein level for prohibitin in granulosa cells. Fifty micrograms of protein from granulosa cells treated with saline, eCG plus NRS, or eCG plus anti-eCG were applied to each lane and analyzed for protein level for prohibitin by Western blot analysis. Samples were further focused for 16 000 volt-hours with a mixture of pH 3-10 and pH 5-7 ampholyte, and the second-dimensional Western blot procedure detected prohibitin-immunoreactive spots. Representative blots were scanned using NIH Image software. The bar graphs represent the mean ± SEM of results from three replicate experiments after normalization of data against cyclophilin A protein. One-dimensional Western blot analysis: *P = 0.0375 (compared to saline), *P = 0.0425 (compared to eCG plus anti-eCG); two-dimensional Western blot analysis: *P = 0.0312 (compared to saline). Arrowheads indicate acidic isoform.