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To determine whether endogenous KITL was responsible for the spontaneous activation of primordial follicles, rabbit ovarian cortical explants were treated with KITL-neutralizing antibody. There was no change in the developmental distribution of follicles in explants treated with neutralizing antibody compared with untreated cultured explants (Fig. 6). Therefore, neutralizing antibody did not prevent spontaneous follicle activation. (more…)

Following 8 days of culture in vitro, the developmental distribution of follicles in cortical explants changed significantly (P < 0.05). In untreated cultured explants, the primordial follicle population decreased to comprise only 25.2% 6 2.7% of all of the follicles present, and the percentages of early primary, primary, and preantral follicles increased significantly (P < 0.05) to 58.1% 6 1.4%, 11.6% 6 1.4%, and 5.2% 6 0.8%, respectively (Fig. 6). These data indicate that a large proportion of rabbit primordial follicles spontaneously activate and develop in vitro. (more…)

Effects of Culture and Recombinant Mouse KITL Treatment on the Survival of Follicles in Rabbit Ovarian Cortical Explants In preculture rabbit ovarian cortical explants, follicles were generally healthy and showed no signs of atresia. Following 8 days in vitro, approximately 50% of follicles exhibited signs of degeneration (Fig. 5). However, the proportion of total follicles that were healthy in the rabbit ovarian cortical explants did not differ significantly between the cultured treatment groups (Fig. 5). These results indicate that follicle survival was not enhanced by recombinant mouse KITL (50 ng/ml and 150 ng/ml), nor was follicular atresia promoted when the KITL/KIT interaction was inhibited by KITL-neutralizing antibody. (more…)

Many oocytes of antral follicles appeared to express reduced levels of KIT protein compared with those of smaller growing follicles (Fig. 4b). Intensely stained granules also were detected at the oolemma and in the cytoplasm of oocytes from primordial through antral follicles (Fig. 4, b-d). KIT protein was weakly detected in the theca cells of some preantral and antral follicles (not shown). All other somatic cells within the ovary were free of KIT immunoreactivity. Specific staining was not detected when antiserum was substituted with preimmune serum (Fig. 4a). (more…)

KIT Protein Expression in Juvenile Rabbit Ovaries At 2 wks of age, KIT protein was detected at very low levels in the outer cortex of the ovary, whereas higher concentrations of the protein were localized toward the inner region of the ovary (not shown). Very weak KIT immunostaining was observed within naked oocytes located closest to the ovarian epithelium. Closer to the medulla the intensity of KIT immunoreactivity was variable, with KIT protein expression detected in some naked oocytes. (more…)

While some of the germ cell clusters in the outer cortex were entirely devoid of staining, others contained a mixture of Kit-positive and Kit-negative oocytes (Fig. 3b). Kit mRNA was not detected in any somatic cells within 2-wk-old rabbit ovarian tissue (Fig. 3, a and b). (more…)

The cuboidal granulosa cells of most activated and growing follicles expressed high levels of KITL protein (Fig. 2, c and d). No difference in granulosa cell staining intensity was detected within or between preantral follicles. In contrast, the granulosa cells of large antral follicles appeared to express varying levels of KITL (not shown). In most large antral follicles, granulosa cell KITL expression was very low, whereas in very few follicles at this stage low to moderate KITL protein expression was evident in both mural and cumulus granulosa cells (not shown). (more…)

KITL Protein Expression in Juvenile Rabbit Ovaries Similar to the results obtained by in situ hybridization, in the 2-wk-old rabbit ovary intense KITL immunostaining was observed in the outer cortex, with considerably less staining evident toward the medulla (not shown). KITL protein was strongly localized to the somatic cells associated with nests of naked oocytes, whereas the oocytes within the nests exhibited very diffuse staining. The somatic cells of the stroma located between the clusters of germ cells did not stain for KITL protein. KITL immunoreactivity was weakly detected in the granulosa cells and oocytes of the newly formed follicles located toward the medulla of the 2-wk-old ovary. (more…)

The pattern of staining in ovarian tissue from 4- to 12-wk-old rabbits remained the same among age groups, with comparatively low levels of expression in primordial follicles, high expression levels in growing preantral follicles, and variable expression levels in antral follicles (Fig. 1, c-e). Kitl mRNA occasionally was observed in the flattened granulosa cells surrounding some primordial follicles (Fig. 1, c and d). In early primary follicles the cuboidal granulosa cells nearly always expressed Kitl (Fig. 1d), whereas the flattened granulosa cells within the same follicle often remained free of staining (Fig. 1d). Growing follicles from the primary stage onward expressed high levels of Kitl in almost every granulosa cell (Fig. 1e). (more…)

Moreover, a series of three lysine residues (amino acids 213, 214, and 215) adjacent to the transmembrane domain of KITL are present in the rabbit, mouse, rat, and bovine amino acid sequence. These charged residues may serve to anchor the mature protein in the membrane (see Supplemental Fig. 1). (more…)