Figure 4.

Regulation of transcriptional activities of the Xlr5c-like promoter. Preovulatory granulosa cells were isolated from gonadotropin-primed immature rats (48 h-post-PMSG). A) The cells were transfected with empty luciferase reporter vector (LUC), 1879-bp (−1841/+37), 1379-bp (−1841/+37), and 579-bp (−541/+37) Xlr5c-like luciferase reporter constructs, treated with FSK+PMA and further cultured for 8 h. B) The cells were transiently transfected with wild type, mutant-A, mutant-B, and mutant-AB Xlr5c-like luciferase reporter constructs, and stimulated with FSK+PMA for 8 h. C) ChIP detection of PGR binding to the rat Xlr5c-like promoter region in periovulatory granulosa cells. Chromatin samples prepared from 0 h or 12 h after hCG stimulation were immunoprecipated with antibodies for SP1, SP3, PGR. The immunoprecipitated chromatins were analyzed by PCR using the primers (arrows) designed to amplify fragments spanning SP1, SP3 and PGR binding motifs in the Xlr5c-like promoter. The experiment was repeated at least 3 times with different granulosa cell samples. D) Preovulatory granulosa cells transfected with the wild type 1379-bp (−1841/+37) Xlr5c-like luciferase reporter construct were treated without or with RU486 (PGR antagonist; 1 μM) and then stimulated with FSK+PMA for 12 h. Firefly luciferase activity was normalized to Renilla luciferase activity in each sample. Experiments for A, B, and D were repeated 4 times, each with different granulosa cell sample (mean ± SEM). Bars with no common superscripts in each panel are significantly different (p < 0.05).