Introduction Gap junctions are clusters of intercellular channels that aggregate at sites of close cell-cell apposition, connecting the cytoplasm of adjacent cells and permitting the passage of inorganic ions, second messengers, and small metabolites (less than ~1000 daltons) between cells (Bruzzone et al., 1996). The primary unit of the gap-junction channel is the connexon, a hexameric structure of oligomerized connexin (Cx) subunits that forms a hemichannel in the plasma membrane. The end-to-end docking of two compatible hemichannels from neighbouring cells forms the intercellular channel (White and Paul, 1999). The connexins are members of a closely related family of integral membrane proteins with at least 19 members in the mouse genome (Willecke et al., 2002). Most cell types express multiple connexins, but whether different connexin isoforms can hetero-oligomerize into functional channels is dependent upon the compatibility of the connexins involved. This situation arises in vivo in granulosa cells of adult mice and rats where Cx43 and Cx45 are co-localized to the same gap-junctional plaques (Kidder and Mhawi, 2002; Okuma et al., 1996). It also occurs in cardiac myocytes that express Cx40 and Cx43, where the two connexins co-localize in gap-junction plaques (Gros et al., 1994). In 1995, Elfgang and others published an important series of experiments that investigated which connexins could form functional heterotypic channels in vitro (Elfgang et al., 1995). Gap-junctional-communication- deficient HeLa cells were transfected with seven murine connexin expression vectors, and these engineered cells were then cultured in pairs and assessed for dye coupling. Of specific interest to our study, Cx43 demonstrated the ability to form functional heterotypic channels with Cx37 as well as functional Cx43 homotypic channels. This in vitro model is particularly useful when determining the ability of different connexins to form functional gap-junction channels with one another, because although two connexins may co-localize in the same plaque, they could be forming distinct channels, due to their inability to form heterotypic channels. Gap-junctional intercellular communication (GJIC) is a selective process mediated by the connexin isoform(s) that comprise each hemichannel within a gap junction. This communication selectivity and the implied physiological significance of such a system is demonstrated by disease states caused by specific connexin mutations and the phenotypes exhibited by connexin-knockout mice, suggesting that vital intercellular signalling is mediated by specific connexin isoforms (White and Paul, 1999). The mammalian cumulus-oocyte complex (COC) represents a clear example of a multicellular unit that exhibits expression of multiple connexins and is reliant upon GJIC for proper development (Ackert et al., 2001; Juneja et al., 1999; Kidder and Mhawi, 2002; Simon et al., 1997). The COC is housed in the mature ovarian follicle. It consists of a developing oocyte 2699 Studies of mice with targeted disruptions of specific connexin genes have revealed that at least two connexins, connexin37 (Cx37) and connexin43 (Cx43), play essential roles in ovarian follicle development. To explore the respective roles of these two connexins in gap-junctional communication between the developing murine oocyte and its surrounding cumulus granulosa cells, we used confocal immunofluorescence microscopy and oocyte preloading functional assays. Immunofluorescence microscopy located Cx37 within gap-junction plaques between granulosa cells and the oocyte, and Cx43 between surrounding granulosa cells. Preloading assays combining denuded oocytes and cultured granulosa cells expressing or lacking Cx37 or Cx43 revealed that Cx37 must be present in both cell types for the establishment of heterocellular gap-junctional coupling. Furthermore, immunofluorescence microscopy of cultured granulosa cells after incubation with denuded oocytes showed that the oocyte induces the formation of gap junctions containing Cx37 at the surface of granulosa cells. Continuous Cx37 expression in granulosa cells was confirmed using RT-PCR. Together, these results indicate that the growing murine oocyte is functionally coupled with granulosa cells by homotypic gap junctions composed of Cx37, and that the formation and/or stabilization of Cx37 junctions is selectively induced at the oocyte-granulosa interface by cell contact. Key words: Gap junctions, Cx37, Cx43, Ovarian follicle, Oocyte, Cumulus granulosa cell, Connexin 43 Summary Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface Gregory I. Veitch 1,2 , Joanne E. I. Gittens 1,2,5 , Qing Shao 4 , Dale W. Laird 1,4, * and Gerald M. Kidder 1,2,3,5, * ,‡ Departments of 1 Physiology and Pharmacology, 2 Obstetrics and Gynaecology, 3 Paediatrics, and 4 Anatomy and Cell Biology, The University of Western Ontario, London, Ontario N6A 5C1, Canada 5 Child Health Research Institute, 800 Commissioners Road East, London, Ontario N6C 2V5, Canada *These authors contributed equally to this work ‡ Author for correspondence (e-mail: gerald.kidder@fmd.uwo.ca) Accepted 26 January 2004 Journal of Cell Science 117, 2699-2707 Published by The Company of Biologists 2004 doi:10.1242/jcs.01124 Research Article