Neuropeptide y regulates proliferation and apoptosis in granulosa cells in a follicular stage-dependent manner

Abstract

Background: The complex regulatory mechanism involved in ovarian follicular development is not completely understood. Neuronal neuropeptide Y (NPY) is involved in the regulation of feeding behavior, energy homeostasis, and reproduction behavior, while its function in ovarian follicular development is not clear. The objective of this study was to investigate if and how NPY regulates follicle development in the ovary. Methods: All experiments were performed using Sprague Dawley rats. To understand NPY expression pattern at different stages of follicular development, NPY content was assessed using immunohistochemistry in individual follicles. NPY and its receptors expression pattern were evaluated in granulosa cells isolated from preantral (PA), early antral (EA) and late antral follicles (LAF). The influence of NPY on granulosa cell proliferation and apoptosis were further assessed in vitro, using Ki67- and TUNEL-positivity assays. To investigate whether NPY induced-proliferation in EA granulosa cells is mediated through the activation of NPY receptor Y5 (NPY5R) and Mitogen-activated protein kinase (MEK) signal pathway, EA granulosa cells were treated with NPY5R antagonist (CGP71683) and MEK inhibitors (PD98059 and U0126), and Ki67-positive cells were assessed. Results: NPY protein expression was follicular stage-dependent and cell type-specific. NPY signal intensity in EA was higher than those in PA and LAF. Antral granulosa cells showed the highest signal intensity compared to mural granulosa cells, cumulus cells and theca cells. Granulosa cells NPY protein content and mRNA abundance were higher in EA than in LAF. NPY receptor contents in granulosa cells were follicular stage-dependent. While NPY reduced apoptosis of EA granulosa cells, it increased the proliferation through NPY5R and MEK pathway. In contrast, in LAF granulosa cells, NPY reduced proliferation and increased the number of apoptotic cells, with no significant effects on PA granulosa cells. Conclusion: This study is the first to evaluate the intraovarian role of NPY in granulosa cells at various stage of follicular development. These results indicate that NPY regulates granulosa cells proliferation and apoptosis in a follicular stage-dependent and autocrine manner. NPY may play a role in pathogenesis of ovarian follicular disorders.