The diameter profiles of follicles and corpora lutea are illustrated in Figure 1, and the method for densitometric quantitative analysis is illustrated in Figure 2. The specificity of the immunohistochemical staining was confirmed by adsorbing primary antiserum with excess of follistatin and by omitting the secondary antibody treatment during the staining procedure (Fig. 3). A positive reaction (brown staining) detected after primary antiserum treatment of follicles and corpora lutea was undetectable after the antiserum was adsorbed with follistatin or the secondary antibody was omitted from the staining procedure (Fig. 3). Data and statistical results are summarized separately for follicles (see Figs. 4, 6, and 7) and corpora lutea (see Figs. 5 and 8). buy cheap antibiotics
As evidenced by diameter profiles (Fig. 1), all dominant follicles of wave 1 were in the growing phase on D3W1, the early-static phase on D6W1, the late-static phase on D1W2, and the regressing phase on D>17. Subordinate follicles of wave 1 were in the early-static phase on D3W1 and the regressing phase on D6W1. All preselection follicles of wave 2 on D1W2, and the preovulatory follicles on D>17, were in the growing phase. Subordinate follicles of the ovulatory wave were regressing on D>17.
FIG. 1. Diameter profiles (mean ± SEM) of the corpus luteum and dominant, largest subordinate, and second-largest subordinate follicles of wave 1 (anovulatory) and wave 2 (ovulatory); the dotted lines indicate the mean days of ovariectomy on Day 3 of wave 1 (D3W1), Day 6 of wave 1 (D6W1), Day 1 of wave 2 (D1W2), and during proestrus a 17 days postovulation (Da17). The numbers in parentheses are the number of corpora lutea and follicles of each type collected for each period. Breaks in the profiles indicate that data were included only from those heifers that were ovariectomized at the end of that segment.
FIG. 2. The technique for densitometric measurement of the follistatin reaction in the granulosa layer of ovarian follicles. Positive reaction was localized in the granulosa layer (a); the theca interna was used as a nonreactive reference area (b). Mean gray-scale values were measured in a box (values below the box on each figure; black = 0, white = 255) encompassing 34 X 167 picture elements (0.06-mm2 area) from the granulosa layer (a, c) and theca interna (b, d) of sections stained with the primary antiserum (a, b) and those stained with follistatin-adsorbed primary antiserum (c, d). Total absorptive index was calculated by using the values of reactive area versus nonreactive area (a vs. b), and nonspecific absorptive index (calculated from c vs. d) was subtracted to obtain the specific absorptive index for the granulosa layer.
FIG. 3. Micrographs showing the specificity of follistatin immunohistochemical staining (brown color) of a growing dominant follicle (a-c) and a mid-diestrous corpus luteum (d-f). When primary antiserum was used, intense staining was detected in the granulosa layer of the follicle (a) and in the luteal cells of the corpus luteum (d). The labeling was not detected when the same tissues were stained with follistatin-adsorbed antiserum (b, e) or when the secondary antibody was omitted from the staining procedure (c, f). Bar = 50 mm.