Sp1 regulates steroidogenic genes and LHCGR expression in primary human luteinized granulosa cells.

Luteinizing hormone and human chorionic gonadotropin (hCG) bind to the luteinizing hormone/chorionic gonadotropin receptor (LHCGR). LHCGR is required to maintain corpus luteum function but the mechanisms involved in the regulation of LHCGR in human luteal cells remain incompletely understood. This study aimed to characterize the expression of LHCGR mRNA in primary human luteinized granulosa cells (hLGCs) obtained from patients undergoing in vitro fertilization and to correlate LHCGR expression with the response of hLGCs to hCG by assessing the expression of genes known to be markers of hCG actions. The results show that LHCGR expression is low in freshly isolated cells but recovers rapidly in culture and that hCG maintains LHCGR expression, suggesting a positive feedback loop. The activity of a LHCGR-LUC reporter increased in cells treated with hCG but not with follicle-stimulating hormone. Treatment with hCG also stimulated the expression of genes involved in steroidogenesis in a time-dependent manner. LHCGR promoter expression was found to be regulated by SP1, which we show is highly expressed in hLGCs. Moreover, SP1 inhibition prevented the stimulation of steroidogenic genes and the increase in LHCGR-LUC reporter activity by hCG. Finally, we provide evidence that a complex formed by SP1 and GATA4 may play a role in the maintenance of LHCGR expression. This report reveals the mechanisms involved in the regulation of the LHCGR and provides experimental data demonstrating that the proximal region of the LHCGR promoter is sufficient to drive the expression of this gene in primary hLGCs.

Progesterone production is affected by unfolded protein response (UPR) signaling during the luteal phase in mice.

AIMS: We examined whether the three unfolded protein response (UPR) signaling pathways, which are activated in response to endoplasmic reticulum (ER)-stress, are involved in progesterone production in the luteal cells of the corpus luteum (CL) during the mouse estrous cycle. MAIN METHODS: The luteal phase of C57BL/6 female mice (8 weeks old) was divided into two stages: the functional stage (16, 24, and 48 h) and the regression stage (72 and 96 h). Western blotting and reverse transcription (RT)-PCR were performed to analyze UPR protein/gene expression levels in each stage. We investigated whether ER stress affects the progesterone production by using Tm (0.5 µg/g BW) or TUDCA (0.5 µg/g BW) through intra-peritoneal injection. KEY FINDINGS: Our results indicate that expressions of Grp78/Bip, p-eIF2α/ATF4, p50ATF6, and p-IRE1/sXBP1 induced by UPR activation were predominantly maintained in functional and early regression stages of the CL. Furthermore, the expression of p-JNK, CHOP, and cleaved caspase3 as ER-stress mediated apoptotic factors increased during the regression stage. Cleaved caspase3 levels increased in the late-regression stage after p-JNK and CHOP expression in the early-regression stage. Additionally, although progesterone secretion and levels of steroidogenic enzymes decreased following intra-peritoneal injection of Tunicamycin, an ER stress inducer, the expression of Grp78/Bip, p50ATF6, and CHOP dramatically increased. SIGNIFICANCE: These results suggest that the UPR signaling pathways activated in response to ER stress may play important roles in the regulation of the CL function. Furthermore, our findings enhance the understanding of the basic mechanisms affecting the CL life span.