Interplay between paracrine signaling and gap junctional communication in ovarian follicles

Abstract

Intercellular communication is required for ovarian folliculogenesis. This is apparent in mice lacking connexin43 (Cx43, a gap junction protein strongly expressed in granulosa cells), or growth/differentiation factor-9 (GDF9, an oocyte-specific growth factor that stimulates granulosa cell proliferation and differentiation), or in mice expressing a mutant form of Kit ligand (KITL, a paracrine factor that, in the ovary, is secreted by granulosa cells to stimulate oocyte growth). In all of these mutant lines, follicle growth is impaired suggesting a possible interaction between paracrine signaling and gap junctional communication. To assess this possibility, we analyzed gene expression in mutant ovaries. Despite the lack of gap junctional coupling between granulosa cells of Cx43 null mutant ovaries, expression of the genes encoding KITL and its receptor, KIT, is maintained. Furthermore, GDF9 expression is maintained. In GDF9 null mutant ovaries, there is no apparent change in Cx43 expression and, correspondingly, the granulosa cells remain coupled. There is also no increase in granulosa cell apoptosis in ovaries lacking Cx43 or GDF9. Staining for proliferating cell nuclear antigen (PCNA) revealed that the granulosa cells of Cx43 null mutant ovaries have a reduced frequency of DNA synthesis. Using both radiolabeled thymidine incorporation and PCNA staining in vitro, we showed that recombinant GDF9 could restore the proliferation of coupling-deficient granulosa cells to the level of control cells. These results indicate that impaired folliculogenesis in mice lacking Cx43 is due at least in part to reduced responsiveness of granulosa cells to oocyte-derived GDF9, indicating an interaction between these two modes of intercellular communication.

Differential contributions of connexin37 and connexin43 to oogenesis revealed in chimeric reaggregated mouse ovaries

“…Cx43 forms gap junction channels between the granulosa cells of quiescent and growing follicles (Juneja et al, 1999;Valdimarsson et al, 1993;Wiesen and Midgley, 1993) to enable granulosa cell intercommunication (Gittens et al, 2003). Without Cx43, granulosa cell proliferation is hampered because the cells do not respond optimally to mitogenic signals evoked by an oocyte-secreted paracrine factor, growth differentiation factor 9 (GDF9) (Gittens et al, 2005). The fact that female sterility ensues in mice expressing C-terminal truncated Cx43 (Cx43 K258stop ) that gives rise to functional gap junction channels (Maass et al, 2004) leaves open the possibility that Cx43 mediates folliculogenesis via both gap junctional coupling-dependent and -independent mechanisms.…”

“…One-way ANOVA followed by Tukey's test was used to judge the significance of differences in measurements between ovaries. To confirm that the methodology did not allow for wild-type cells to contaminate knockout cell preparations and vice versa, chimeric ovaries lacking Cx43 in somatic cells were constructed and ovarian sections or fixed follicle cultures were treated with a polyclonal antibody raised against the C-terminal tail of Cx43 as described previously (Gittens et al, 2005). Images were captured on either a Zeiss LSM 510 Meta inverted confocal microscope or Zeiss Axioskop 2 microscope equipped with a Retiga 1300 CCD digital camera (Q imaging).…”

“…In part, the function of oocyte-granulosa cell coupling is to support oocyte growth, regulate meiotic maturation and regulate granulosa cell differentiation (Eppig, 1991), whereas the function of granulosa cell coupling is to optimize the proliferative response to paracrine signals emanating from the oocyte (Gittens et al, 2005). Given that Cx37 and Cx43 gap junction channels have different permeability properties (Elfgang et al, 1995), one might predict that different signals pass between cells of different compartments of the developing follicle.…”

Oogenesis defects in a mutant mouse model of oculodentodigital dysplasia

“…In contrast to the full range of follicles, from primordial through to large preovulatory follicles, that are observed in grafted wild-type ovaries, folliculogenesis in mutant ovaries of the C57BL/6 strain did not proceed beyond the primary unilaminar stage (Ackert et al, 2001). Further studies demonstrated that intercellular coupling among granulosa cells was totally abolished in Cx43-null mice, indicating that cell coupling through Cx43 channels is required to sustain granulosa cell proliferation (Gittens et al, 2003;Gittens et al, 2005;Tong et al, 2006). Failure of the Cx43-null follicles to develop multiple layers of granulosa cells was correlated with reduced growth of the oocytes, which were morphologically abnormal, meiotically incompetent and could not be fertilized (Ackert et al, 2001).…”

Studies on Gene Expression and Developmental Competence of Bovine Embryos Produced Under Different Conditions of Heat Stress

“…Immunofluorescence microscopy located Cx37 within gap junction plaques between granulosa cells and the oocyte, and Cx43 between surrounding granulosa cells. Gittens et al [86] reviewed that the intercellular communication is required for ovarian folliculogenesis. Yogo et al [87] reported that the connexin 43 (Cx43)-intermediate gap junctional communication in granulosa cells is crucial for germ line development and postnatal folliculogenesis.…”