190 JNK SIGNALLING IN FOLLICLE ACTIVATION

Abstract

Primordial follicles are maintained in a quiescent state until they receive a signal to activate and join the growing pool. It is essential that the rate of follicle activation is well coordinated as it determines the reproductive lifespan of the female. This finely tuned process is under the control of a variety of positive and negative factors, and there is evidence that the JNK pathway is involved. The aim of this study was to assess the effect of a JNK inhibitor (SP600125) on follicle activation in vitro. Ovaries from 6 prepubertal sheep were dissected into 1-mm³ fragments and cultured in the presence or absence of SP600125 (0, 5, or 25 µM; SP0, SP5, and SP25, respectively). After 0, 2, 5, or 9 days, fragments were fixed, sectioned, and analysed by histological morphometry to determine the number and type of follicles in addition to TUNEL analysis for apoptosis. In total, 21 584 follicles were assessed. Follicles were classified as either primordial, intermediate, primary, secondary, or antral. Culture media were also assayed for steroid content. After multinomial regression analysis, there were no differences in the rate of follicle activation between groups on Day 2 of culture. However, after 5 days of culture there were significantly more primordial follicles in SP25 (69 ± 9.15%; P < 0.01) and SP5 (52 ± 8.12%; P < 0.05) groups when compared with SP0 (34 ± 3.98%). At the end of culture (Day 9), these differences continued to be observed (SP25: 63 ± 8.47%; SP5: 51 ± 6.62%; SP0: 34 ± 5.61%; P < 0.05). On Day 9 there were more intermediate follicles following treatment with SP0 (52 ± 3.27%) compared with SP5 and SP25 (42 ± 4.72% and 31 ± 7.41% respectively; P < 0.05). There was also a tendency for more primary follicles on Day 9 for the control (11 ± 4.29%) when compared with the other treatments (4 to 7%; P = 0.06). The proportion of apoptotic primordial follicles was greatest in the SP25 group when compared with SP5 and SP0 at all timepoints (P < 0.05). After 2 days of culture, progesterone concentration was highest in the SP25 group (4.1 ± 0.45 ng mL^–1) compared with the SP5 and SP0 groups (2.9 ± 0.39 ng mL^–1 and 1.6 ± 0.79 ng mL^–1, respectively; P < 0.05). However, this dose effect was lost by the end of culture. There was no effect of the inhibitor on oestradiol synthesis. By using SP600125, we have observed a dose-dependent block on follicle activation. These results suggest that the JNK pathway is a novel regulator of follicle activation and development. However, further confirmation is required to determine its specific action. The mechanisms by which SP25 promotes progesterone synthesis and increases apoptosis are unknown and also require clarification.