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.
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.
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.
A commercially available kit (ApopTag; Intergen, Purchase, NY) was used for the detection of 3′-OH DNA ends in the sections. After washing with distilled water three times for 10 min each, the sections were incubated in the equilibration buffer of the kit for 20 sec at RT. Then, sections were incubated at 37°C for 1 h in a moist chamber with 60 |xl of the working buffer containing terminal deoxynucleotidyl transferase, digoxigenin-11-dUTP The reaction was stopped by incubating the sections in a blocking buffer containing sodium citrate and NaCl at 37°C for 30 min.
The signal obtained after such a background correction was considered to be an antigen-specific signal. For each image, specific antibody staining was merged with nuclear staining (blue) using Soft Imaging System software (Soft Imaging System Corp., Lakewood, CO) and MetaMorph 4.6 software (Universal Imaging Corp., Downington, PA) that caused virtually no pixel shifting during image merger and resulted in shades of red, green, and blue.
The procedure used for immunofluorescence microscopy of rat ovarian sections and porcine oocytes, zygotes, and blastocysts has been described in detail previously. Mouse monoclonal PCNA, polyclonal rabbit antiprohibitin, and polyclonal anti-rat cytochrome P450scc antibodies were used at a dilution of 1:200. The specificity of the antibodies was verified by incubating ovarian sections and oocytes, zygotes, and blastocysts without primary antibodies as well as with NRS.
In all experiments, the zona pellucida was removed from porcine oocytes and embryos by a brief pronase (Protease; Sigma) treatment and then processed for immunofluorescence as described previously. All animal handling procedures in the present study were approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health and the U.S. Department of Agriculture.
Porcine nuclear transfer (NT) was performed as described by Park et al.. Briefly, the cumulus-free oocytes were enucleated in TCM/BSA medium supplemented with 7.5 |xg/ml of cytochalasin B. The first polar body and the surrounding cytoplasm were removed with the aid of a beveled pipette (inner diameter, —25-30 |xm). Enucleated oocytes were kept and micromanipulated in TCM/BSA until NT. A Nikon Diaphot (Nikon Corporation, Tokyo, Japan) equipped with a 40X objective and a pair of Narishige micromanipulators (Narishige International USA, East Meadow, NY) was used for micromanipulations. Donor fibroblasts (fetal fibroblasts were isolated from a 35-day-old pig fetus) were selected according to their size and shape (small cells with smooth membranes).
The OCCs were then washed in modified TCM 199 without FSH or LH and matured for an additional 20 h. Matured oocytes were stripped of cumulus cells and fertilized in 50-|xl drops of modified Tris-buffered medium consisting of 113.1 mM NaCl, 3 mM KCL, 7.5 mM CaCl2, 20 mM Tris, 11 mM glucose, 5 mM sodium pyruvate, 2 mM caffeine, and 0.2% (w/v) BSA.
Subsequently, ovaries were excised, cleared of adhering fat, weighed, and fixed either in 10% neutral buffered formalin for immunohistochemical detection of prohibitin, PCNA, or P450scc or for in situ terminal deoxyn-ucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of ap-optotic cells nuclei (n = 5 rats/group). In addition, granulosa cells from each group (n = 10 rats/group) of animals were harvested by follicle puncture as previously described, washed, and resuspended in 10 mM Hepes buffer (pH 7.4) containing 1 mM EGTA and 2 mM PMSF. Cells were processed for Western blot analyses.