Changes in insulin-like growth factor-II/mannose-6-phosphate receptor during growth and atresia of ovine ovarian follicles

To assess a potential role of insulin-like growth factor (IGF)-II/mannose-6-phosphate (M6P) receptor in the ewe ovary, [125I]IGF-II binding was assessed on whole ovarian membranes and on ovarian sections. Competition studies with IGF-I, IGF-II, and insulin were performed to estimate the specificity of [125I]IGF-II binding. On the ovarian sections, labeling was quantified after microautoradiography by image analysis. In addition, immunohistochemistry experiments using a specific polyclonal antibody raised against the IGF-II/M6P receptor were performed on ovarian sections. Specific and high-affinity binding sites for IGF-II were present on whole ovarian membranes and on ovarian sections. On membranes, the Kd value was close to 0.2 nM. IGF-I was approximately 1000-fold less potent than IGF-II in displacing binding of [125I]IGF-II from whole membranes as well as from granulosa and thecal cells on ovarian sections. Insulin did not displace [125I]IGF-II binding. Cross-linking experiments followed by SDS-PAGE under reducing conditions revealed that [125I]IGF-II binding predominantly involved a 260-kDa protein, the size of which is compatible with that of the IGF-II/M6P receptor. Furthermore, both autoradiography and immunohistochemistry experiments demonstrated that IGF-II/M6P receptor was present at high levels in granulosa of atretic follicles and in theca of healthy follicles. Conversely, very low levels were found in granulosa of healthy follicles and in theca of atretic follicles. The involvement of the IGF-II/M6P receptor in ovarian tissue remodeling and in mediating IGF-II action during the processes of folliculogenesis and atresia remains to be determined