KIT LIGAND STIMULATES ACTIVATION OF BOVINE PRIMORDIAL FOLLICLES IN VITRO

Abstract

The regulation of activation of primordial follicles, which reside in the cortex of mammalian ovaries, remains one of the least understood processes of folliculogenesis, especially in humans and domestic animals. When cortical pieces from bovine and baboon ovaries are cultured in serum-free medium containing ITS+ (insulin-transferrin-selenium + bovine serum albumin and linoleic acid) wholesale, spontaneous activation occurs (i.e. primordial follicles initiate growth and develop into primary follicles). In contrast, we recently observed that activation does not occur when bovine cortical pieces are cultured in the absence of insulin (i.e. with TS+), but the follicles remain healthy; this provides a useful model for testing putative stimulators of follicle activation. The objective of this study was to test the hypothesis that kit ligand (Kitlg) stimulates activation of bovine primordial follicles in vitro. Small pieces of cortex were dissected from bovine fetal ovaries (6–8 months gestation; n = 3 fetuses) and cultured for 4 days (4 pieces/treatment/fetus) with 1) ITS+, 2) TS+ or 3) TS+ and Kitlg (10, 50, 100, 500 and 1000 ng/ml). After 4 days of culture, pieces were fixed, embedded in plastic and serially sectioned; every other set of 10 sections was mounted and stained with toluidine blue. One section in each mounted set was analyzed by histological morphometry to determine numbers and sizes of follicles. Only follicles in which the oocyte nucleus was visible were considered; this sampling method ensures that follicles are counted only once. Follicles in each section were classified as primordial, primary, or secondary and as healthy or atretic and the mean number of follicles per histological section was determined for each cortical piece. Pieces fixed on day 0 served as non-cultured controls. After 4 days of culture in TS+ the number (mean ± SEM) of primordial and primary follicles per histological section (8.8 ± 1.4 and 2.7 ± 0.4, respectively) did not differ from day 0 (8.7 ± 0.4 and 2.7 ± 0.6), indicating absence of activation in TS+. As expected, after 4 days cortical pieces cultured in ITS+ had very few primordial follicles (1.3 ± 0.4; P < 0.05 vs. day 0 and TS+) and the number of primary follicles per section had increased to 9.0 ± 1.0, approximately a 3-fold increase relative to day 0 and to TS+ (P < 0.05). When cortical pieces were cultured with TS+ and Kitlg, Kitlg at all doses tested decreased (P < 0.05) the number of primordial follicles and the four highest doses increased (P < 0.05) the number of primary follicles in a dose-dependent fashion, compared to day 0 or to TS+ alone, indicating that Kitlg stimulated activation of bovine primordial follicles. Interestingly, there were more primordial follicles and fewer primary follicles with all doses of Kitlg than with ITS+ (P < 0.05), showing that fewer follicles were activated by Kitlg than by the insulin in ITS+. There were no treatment effects on the mean number of follicles per section or on follicular health; the incidence of atresia in each follicle class was less than 20%. In conclusion, Kitlg stimulates activation of bovine primordial follicles in vitro in the absence of insulin. The fact that fewer follicles were activated by maximally effective doses of Kitlg compared to insulin suggests that the two factors stimulate activation via different mechanisms, but this hypothesis remains to be tested. (poster)