Role of gap junctions in regulation of progesterone secretion by ovine luteal cells in vitro

To evaluate the role of gap junctions in the regulation of progesterone secretion, two experiments were conducted. In Experiment 1, luteal cells obtained on days 5, 10, and 15 were cultured overnight at densities of 50X10(3), 100X10(3) 300X10(3), and 600 X 10(3) cells/dish in medium containing: (1) no treatment (control), (2) LH, or (3) dbcAMP. In Experiment 2, luteal cells from days 5 and 10 of the estrous cycle were transfected with siRNA, which targeted the connexin (Cx) 43 gene. In Experiment 1, progesterone secretion, Cx43 mRNA expression, and the rates of gap junctional intercellular communication (CJIC), were affected by the day of the estrous cycle, cell density, and treatments (LH or dbcAMP). The changes in progesterone secretion were positively correlated with the changes in Cx43 mRNA expression and the rates of GJIC. Cx43 was detected on the luteal cell borders in every culture, and luteal cells expressed 30-hydroxysteroid dehydrogenase. In Experiment 2, two CA3 gene-targeted sequences decreased CA3 mRNA expression and progesterone production by luteal cells. The changes in CA3 mRNA expression were positively correlated with changes in progesterone concentration in media. Thus, our data demonstrate a relationship between gap junctions and progesterone secretion that was supported by (1) the positive correlations between progesterone secretion and CA3 mRNA expression and GJIC of luteal cells and (2) the inhibition of CA3 mRNA expression by siRNA that resulted in decreased production of progesterone by luteal cells. This suggests that gap junctions may be involved in the regulation of steroidogenesis in the ovine corpus luteum.