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The expression and localization of GATA-4 and GATA-6 messenger RNAs and proteins were assessed in porcine ovaries at different stages of the estrous cycle. RT-PCR and Western blot analyses revealed that GATA-4 and GATA-6 transcripts and proteins were strongly expressed in granulosa cells isolated from antral follicles, intact antral follicles, corpora hemorrhagica (CH) and midluteal phase corpora lutea (CL). Immunoblot analyses showed two predominant proteins with molecular masses of approximately 53 and 55 kDa for GATA-4 and one 55 kDa protein for GATA-6. Immunohistochemical studies revealed GATA-4 and GATA-6 nuclear staining in granulosa cells of healthy primordial, primary, and different-sized antral follicles. The percentage of immunopositive thecal cell nuclei increased with follicular development. In CH and CL, luteal cells displayed nuclear immunoreactivity for both transcription factors. Regressing CL showed a decrease in GATA immunopositive cells. Immunoreactivity for GATA-4 and GATA-6 was present in most blood vessels. In electrophoretic mobility shift assays, nuclear protein extracts isolated from granulosa cells and CL exhibited both GATA-4 and GATA-6 binding to a GATA consensus oligonucleotide with GATA-4 being the predominant binding protein. GATA-4 and GATA-6 DNA binding was elevated in granulosa cell nuclear extracts from preovulatory (8-10 mm) follicles. Cotransfection of primary cultures of luteinizing granulosa cells with GATA-4 or GATA-6 expression vectors increased the activity of the porcine StAR gene promoter significantly, but did not significantly activate the inhibin alpha gene promoter. The detection of GATA-4 and GATA-6 mRNAs and proteins in porcine ovaries and the identification of a least one possible target gene may help to establish roles for these GATA factors in follicular development and luteal function.
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