Deciphering early folliculogenesis: Jun Amino Terminal Kinase (JNK) plays a key role in preantral follicle growth

Abstract

Objective: The factors regulating the preantral follicle growth are largely unknown. Our preliminary data indicated that extracellular matrix (ECM) and integrin signaling pathways might play a role in preantral follicle growth (1). JNK is one of the key signaling molecules involved in integrin signaling. In this study, we sought to determine whether ECM and integrins regulate preantral ovarian follicle growth by signaling via JNK.Design: Controlled laboratory study involving rodent ovarian tissue, granulosa cells, and isolated preantral follicles.Materials and Methods: Ovarian sections from 8 cycling rats (4 months old) as well as spontaneously immortalized rat granulosa cells (SIGC) were stained with anti-phospho-c-Jun antibodies to determine the activation of c-Jun by JNK. Synchronized SIGC were treated with a JNK inhibitor, SP600125 (JNKi), and a FACS analysis was performed to determine progression through mitosis. To determine the role of JNK on preantral follicle development, preantral follicles were isolated by an enzymatic and mechanical dissection method from 3-week-old mice. They were then cultured in DMEM-F12 supplemented with 10% FBS, FSH, and growth factors for 9 days with or without 100uM JNKi. Follicle measurements were performed using an image analyzer every other day.Results: Phospho-c-jun was specifically expressed in mitotic granulosa cells of growing follicles (Fig. 1), and mitotic SIGC as shown by immunohistochemistry and immunofluorescence, respectively. JNKi arrested granulosa cell proliferation at G2/M phase in a dose-dependent manner as shown by FACS analysis (Fig. 2). Likewise, JNKi treatment resulted in the arrest and reversal of preantral follicle growth in vitro (Day 0: 125.2±6.5μm vs. day 9: 114.56±5.8μm, p < 0.001). Whereas in controls, follicles continued to grow (144.88±17.5μm vs. 164.34±19.1μm, p = 0.001). View Large Image Figure ViewerDownload (PPT)Conclusion: These data strongly suggest a key role for JNK in preantral ovarian follicle growth in a rodent model. Considered together with previous data (1), ECM and integrins may regulate preantral follicle growth via JNK. Furthermore, pharmacological manipulation of preantral follicle growth via JNK inhibitors or stimulators may bode well for future therapeutic applications and in vitro growth.1. Oktay et al. Biol Reprod 2000(63):457.Supported by: NIH HDO43339-01 (to K.O). Objective: The factors regulating the preantral follicle growth are largely unknown. Our preliminary data indicated that extracellular matrix (ECM) and integrin signaling pathways might play a role in preantral follicle growth (1). JNK is one of the key signaling molecules involved in integrin signaling. In this study, we sought to determine whether ECM and integrins regulate preantral ovarian follicle growth by signaling via JNK. Design: Controlled laboratory study involving rodent ovarian tissue, granulosa cells, and isolated preantral follicles. Materials and Methods: Ovarian sections from 8 cycling rats (4 months old) as well as spontaneously immortalized rat granulosa cells (SIGC) were stained with anti-phospho-c-Jun antibodies to determine the activation of c-Jun by JNK. Synchronized SIGC were treated with a JNK inhibitor, SP600125 (JNKi), and a FACS analysis was performed to determine progression through mitosis. To determine the role of JNK on preantral follicle development, preantral follicles were isolated by an enzymatic and mechanical dissection method from 3-week-old mice. They were then cultured in DMEM-F12 supplemented with 10% FBS, FSH, and growth factors for 9 days with or without 100uM JNKi. Follicle measurements were performed using an image analyzer every other day. Results: Phospho-c-jun was specifically expressed in mitotic granulosa cells of growing follicles (Fig. 1), and mitotic SIGC as shown by immunohistochemistry and immunofluorescence, respectively. JNKi arrested granulosa cell proliferation at G2/M phase in a dose-dependent manner as shown by FACS analysis (Fig. 2). Likewise, JNKi treatment resulted in the arrest and reversal of preantral follicle growth in vitro (Day 0: 125.2±6.5μm vs. day 9: 114.56±5.8μm, p < 0.001). Whereas in controls, follicles continued to grow (144.88±17.5μm vs. 164.34±19.1μm, p = 0.001). Conclusion: These data strongly suggest a key role for JNK in preantral ovarian follicle growth in a rodent model. Considered together with previous data (1), ECM and integrins may regulate preantral follicle growth via JNK. Furthermore, pharmacological manipulation of preantral follicle growth via JNK inhibitors or stimulators may bode well for future therapeutic applications and in vitro growth. 1. Oktay et al. Biol Reprod 2000(63):457. Supported by: NIH HDO43339-01 (to K.O).