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Gap Junctional Communication and Connexin43 Expression in Relation to Apoptotic Cell Death and Survival of Granulosa Cells

Dmitri V. Krysko¹, Sylvie Mussche¹, Luc Leybaert and Katharina D'Herde

Department of Human Anatomy, Embryology, Histology and Medical Physics (DVK,SM,KD), and Department of Physiology and Pathophysiology (LL), Ghent University, Ghent, Belgium

Correspondence to: Dmitri V. Krysko, Dept. of Human Anatomy, Embryology, Histology and Medical Physics, Faculty of Medicine, Ghent University, Godshuizenlaan 4, B-9000 Ghent, Belgium. E-mail: dmitri.krysko{at}UGent.be

Ovarian follicular atresia in all vertebrates is mediated via apoptosis that is initiated in the granulosa cell layer. Here we investigated the relation between connexin expression, cell coupling, and apoptosis in avian granulosa cells. Results from qualitative and quantitative immunocytochemical analysis and Western blotting of connexin43 (Cx43) and electron microscopic observations of gap junctions were compared with functional data on gap junctional coupling obtained by fluorescence recovery after photobleaching in four experimental groups: a control group of freshly isolated granulosa cells, 24-hr serum-free cultures as the apoptosis-inducing condition, and two other groups in which apoptosis was inhibited by either hormone substitution or exposure to elevated extracellular calcium. Our work shows that apoptosis induction in granulosa cells is accompanied by an increased level of cell coupling and that decreasing cell coupling with the gap junction blocker -glycyrrhetinic acid dose-dependently inhibits apoptosis. The level of Cx43 expression was inversely related to the apoptotic index, suggesting that Cx43 expression plays a role in granulosa cell survival. Our study supports the hypothesis that gap junctional coupling plays a role in propagating a cell death message and suggests a role for Cx43 expression per se in granulosa cell survival. (J Histochem Cytochem 52:1199–1207, 2004)

Key Words: granulosa cells • apoptosis • gap junctions • connexin43 • FRAP

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