Intercellular Communication via Connexin43 Gap Junctions Is Required for Ovarian Folliculogenesis in the Mouse

Abstract

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.

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.…”

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

“…In addition to dysmorphogenesis of the heart leading to early neonatal death (31,38), offspring lacking Cx43 exhibit pathophysiological features in a variety of organs. These include slowed cardiac conduction and increased susceptibility to arrhythmias (8,14,22), precataractogenic lesions in the lenses (12), delayed ossification and osteoblast dysfunction (21), impaired hematopoiesis (27), reduced germ cell numbers in the fetal gonads (17), and disrupted gametogenesis in both sexes (1,32). It is the latter consequence of the loss of Cx43 that is the focus of the present report.…”

“…As follicles grow and reach maturity, the expanding granulosa cell population continues to express Cx43 (19,35). Given the presence of this connexin from the onset of folliculogenesis, it was not surprising that developing follicles arrest in preantral stages when Cx43 is absent (1). However, interpretation of this finding was complicated by the fact that granulosa cells lacking Cx43 retain the ability to form gap junctions when paired with wild-type granulosa cells in a dye preloading assay (1).…”

“…Given the presence of this connexin from the onset of folliculogenesis, it was not surprising that developing follicles arrest in preantral stages when Cx43 is absent (1). However, interpretation of this finding was complicated by the fact that granulosa cells lacking Cx43 retain the ability to form gap junctions when paired with wild-type granulosa cells in a dye preloading assay (1). Although dye transfer was limited to first-order cells, this suggested the presence of another connexin(s).…”

Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization

“…In order to justify or negate this notion, the present study was carried out to examine the developmental competence of OPU-derived COCs by studying the expression profile of a panel of genes, related to oocyte proper, cumulus-oocyte interaction, fertilization, embryonic development and apoptosis, both at germinal vesicle (GV) stage as well as after in vitro maturation (IVM). The panel of genes selected for expression analysis included genes associated with: follicular development and ovulation like, CONNEXIN37, CONNEXIN43, BMP15, GDF9, bFGF (Gospodarowicz et al, 1985;Goodenough et al, 1996;Fair et al, 1997;Simon et al, 1997;Elvin et al, 1999;Otsuka et al, 2000;Ackert et al, 2001;Nilsson et al, 2001;Granot and Dekel, 2002;Gittens et al, 2003;Moore and Shimasaki, 2005); Fertilization like, ZP2 and ZP3 (Chen et al, 2011;Shah et al, 2015); Embryonic-genome activation like, ZAR1, HSF1 (Christians et al, 2000;Wu et al, 2003); and Embryonic survival like, BAX, BID, BCL-XL, MCL1 (Chang and Yang, 2000;Jin et al, 2007;Goovartaes et al, 2011). The developmental competence was further elucidated by comparing embryonic developmental potential of Group 1 and Group 2 oocytes, following in vitro fertilization (IVF) and culture of the presumed zygotes.…”