Connexin43 in Rat Oocytes: Developmental Modulation of Its Phosphorylation1

Abstract

It is well established that the 43-kDa connexin (Cx43) is predominantly expressed by ovarian somatic cells, whereas the identity of the connexins contributed by the oocyte to form gap junctions with its neighboring cells is not fully elucidated. Our study aimed to examine oocytes for the expression and regulation of Cx43 throughout oogenesis. Growing and fully grown rat oocytes that were meiotically incompetent and competent, respectively, were examined. Fully grown oocytes were analyzed either before or after reinitiation of meiosis as well as at the second meiotic metaphase. Immunofluorescent analysis of zona pellucida-free oocytes using conventional and confocal microscopy demonstrated a characteristic pattern of punctuated staining of Cx43 on the oolema. Immunogold electron microscopy localized Cx43 to the oocyte surface and the microvillar processes. Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed similar amounts of Cx43 gene and protein in oocytes of different developmental stages. However, a relative increase in the phosphorylated forms of the protein was observed in fully grown oocytes that had completed their maturation. Our findings demonstrate that rat oocytes express a developmentally regulated Cx43. They further suggest that homotypic gap junctions that consist of Cx43 may be present between rat oocytes and their adjacent cumulus cells.

Effect of serum and cumulus cell expansion on marker gene transcripts in bovine cumulus-oocyte complexes during maturation in vitro

“…Cx43 mRNA and protein has previously been detected in bovine cumulus cells (12,14), but the protein was not detected in bovine oocytes in two studies (44,45), while Cx43 mRNA has been detected in bovine oocytes during IVM (13). Recently, Cx43 mRNA and protein have been detected and localized to gap junctions in rat oocytes (46). In the current experiments, Cx43 mRNA relative abundance was affected by maturation medium.…”

“…The lack of effect of time on Cx43 mRNA abundance was not unanticipated because, although Cx43 gap junctions decrease between the bovine cumulus cell and oocyte by 6 hours of maturation, there are still numerous functional gap junctions in cumulus cells at 24 hours of maturation (12). Despite unchanged abundance of Cx43 mRNA and protein in rat oocytes during maturation, the phosphorylation status of the Cx43 protein changed (46), which may have affected gap junction function. Overall, these factors may limit the usefulness of Cx43 mRNA as a marker gene.…”

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

“…The same is true in the rat, but in that species Cx43 was also demonstrated in oocytes (Granot et al, 2002). The ovaries of mice lacking Cx43 contain follicles that arrest development in preantral stages with oocytes that do not undergo meiotic maturation in vitro (Ackert et al, 2001).…”

“…Thus, three important conclusions can be drawn from these experiments: (1) folliculogenesis defects in Cx43 knockout ovaries are not due to deficiencies inherent to oocytes at the onset of folliculogenesis, but to deficiencies in the granulosa cells with which they associate postnatally; (2) Cx43 expression by growing oocytes is dispensable for oocyte and follicle development; and (3) Cx43 absence from granulosa cells alone is sufficient to compromise both follicle development and, at least indirectly, oocyte development. As the oocyte was shown to regulate the progression of follicle development using this same technique (Eppig et al, 2002) and as Cx43 has been identified in rat oocytes (Granot et al, 2002), we expected to see folliculogenesis disruptions in 43 -/-oo/43 +/+ gc ovaries. Instead, the results demonstrated that granulosa cells can influence both oocyte and follicle development.…”

Changes in Expression and Localization of Connexin 43 mRNA and Protein in Porcine Ovary Granulosa Cells during Follicular Atresia

“…Gap junctions are essential for maintaining interactions between cells within a tissue and their homeostasis, and regulate growth/proliferation and death [24,25]. In ovarian follicles, gap junctions are considered to play i m p o r t a n t r o l e s i n f o l l i c u l a r g r o w t h a n d development and in the prevention of follicular atresia in developing preantral secondary follicles [13][14][15][16][17][18][19][20][21][22][23]. Although the role of gap junctions in mammalian follicles has been investigated, there is insufficient information on whether or not Cx43, which is a dominant gap junction protein in porcine follicles [16,17] and has multiple phosphorylated/ activated and non phosphorylated/native forms [30], is involved in granulosa cell apoptosis during atresia in porcine ovaries.…”