Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis

Abstract

Ovarian granulosa cells are coupled via gap junctions containing connexin43 (Cx43 or α-1 connexin). In the absence of Cx43, granulosa cells stop growing in an early preantral stage. However, the fact that granulosa cells of mature follicles express multiple connexins complicated interpretation of this finding. The present experiments were designed to clarify the role of Cx43 vs. these other connexins in the earliest stages of folliculogenesis. Dye injection experiments revealed that granulosa cells from Cx43 knockout follicles are not coupled, and this was confirmed by ionic current injections. Furthermore, electron microscopy revealed that gap junctions are extremely rare in mutant granulosa cells. In contrast, mutant granulosa cells were able to form gap junctions with wild-type granulosa cells in a dye preloading assay. It was concluded that mutant granulosa cells contain a population of connexons, composed of an unidentified connexin, that do not normally contribute to gap junctions. Therefore, although Cx43 is not the only gap junction protein present in granulosa cells of early preantral follicles, it is the only one that makes a significant contribution to intercellular coupling.

Analysis of Oocyte-Like Cells Differentiated from Porcine Fetal Skin-Derived Stem Cells

“…Gap junctions are intercellular channels formed by end-to-end docking of connexons between adjacent cells, each connexon being a cylindrical hexamer of CXs. Among the *20 members of the CX family in mammals [25], CX37 and CX43 are both essential for ovarian follicle development as revealed by analysis of the respective mouse gene knockouts [26,27]. To further characterize the COC-like structures generated from our differentiation protocol, realtime RT-PCR was performed to assess expression of these 2 CXs.…”

In vivo analysis of undocked connexin43 gap junction hemichannels in ovarian granulosa cells

“…Undocked hemichannels are involved in the release of important signaling molecules, including ATP, NAD + , prostaglandin and glutamate (Stout et al, 2002;Ye et al, 2003;Bruzzone et al, 2001;Cherian et al, 2005;Zhao et al, 2005), suggesting possible roles for gap junction hemichannels in paracrine signaling. Previous studies with Cx43-knockout mice demonstrated an essential role for this connexin in ovarian granulosa cells in supporting folliculogenesis and the development of competent oocytes (Ackert et al, 2001;Gittens et al, 2003;. Although the loss of Cx43 from granulosa cells abolished gap junctional intercellular communication (Gittens et al, 2003;Tong et al, 2006), no evidence has been presented that it is the loss of intercellular channels and not hemichannels, or both, that accounts for the ovarian defects.…”

“…Connexin43 (Cx43, encoded by Gja1) is highly expressed in developing follicles, in which it forms the gap junctions coupling granulosa cells (Gittens et al, 2003;Tong et al, 2006). The indispensable role of this connexin during ovarian folliculogenesis has been clearly demonstrated using knockout mice.…”

Defective Epidermal Barrier in Neonatal Mice Lacking the C-Terminal Region of Connexin43

“…Simon et al (1997) showed, that Cx37 deficient mice lack mature Graafian follicles and develop numerous incomplete corpora lutea. Loss of Cx43 on the other hand resulted in an arrest of follicular growth, with most follicles failing to develop multiple granulosa cell layers characteristic of secondary follicles (Ackert et al, 2001;Gittens et al, 2003). This implies that the presence of Cx37 and functional regulation and/or turnover of Cx43 are necessary for ovulation in late folliculogenesis.…”