Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions

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Abstract
Numerous gap junctions exist between cumulus cells and between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap Junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.