The Molecular Mechnism Of Autophagy Regulated By Follicle Stimulating Hormone In Porcine Granulosa Cells

In this experiment, FSH in autophagy regulation and its molecular pathway were explored in mammalian follicle cells. During the culture of porcine follicles in vitro, FSH treatment accentuated autophagy. The porcine primary granulosa cells were used to pursue the underlying mechanisms. We found that FSH up-regulated the mRNA and protein expressions of beclin-1, the key regulator of autophagy. Pretreatment with LY294002 (specific inhibitor of P13K/AKT) and SP600125 (specific inhibitor of SAPK/JNK) suppressed FSH-induced beclin-1 expression. Moreover, the JNK substrate c-Jun, a well-defined transcriptional factor of beclin-1, was phosphorylated by FSH, which was translocated into the nucleus and bound to the beclin-1 promoter region. However, these effects were prevented by pretreatment with the above two inhibitors, and c-Jun knockdown with siRNA also blocked the increase of beclin-1 protein by FSH. It indicated that beclin-1 was up-regulated through P13K/JNK/c-Jun pathway. Meanwhile, the contributions of FSH-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Nevertheless, the inhibition of autophagy with chloroquine or SP600125 obviated the steroid production induced by FSH although the treatment did not affect the expressions of StAR and P450scc. In conclusion, FSH can promote progesterone production through up-regulated autophagy in porcine granulosa cells, in addition to that mediated by steroidogenesis enzymes.The transcription factor nuclear factor-KB (NF-?B) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-kB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-?B (I?B) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-?B in porcine granulosa cells. Moreover, inhibition of NF-?B by FSH or another specific inhibitor of NF-?B, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-?B by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-?B increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development.