Hypoxia activation attenuates progesterone synthesis in goat trophoblast cells via NR1D1 inhibition of StAR expression†.

Trophoblast plays a crucial role in gestation maintenance and embryo implantation, partly due to the synthesis of progesterone. It has been demonstrated that hypoxia regulates invasion, proliferation, and differentiation of trophoblast cells. Additionally, human trophoblasts display rhythmic expression of circadian clock genes. However, it remains unclear if the circadian clock system is present in goat trophoblast cells (GTCs), and its involvement in hypoxia regulation of steroid hormone synthesis remains elusive. In this study, immunofluorescence staining revealed that both BMAL1 and NR1D1 (two circadian clock components) were highly expressed in GTCs. Quantitative real-time PCR analysis showed that several circadian clock genes were rhythmically expressed in forskolin-synchronized GTCs. To mimic hypoxia, GTCs were treated with hypoxia-inducing reagents (CoCl2 or DMOG). Quantitative real-time PCR results demonstrated that hypoxia perturbed the mRNA expression of circadian clock genes and StAR. Notably, the increased expression of NR1D1 and the reduction of StAR expression in hypoxic GTCs were also detected by western blotting. In addition, progesterone secretion exhibited a notable decline in hypoxic GTCs. SR9009, an NR1D1 agonist, significantly decreased StAR expression at both the mRNA and protein levels and markedly inhibited progesterone secretion in GTCs. Moreover, SR8278, an NR1D1 antagonist, partially reversed the inhibitory effect of CoCl2 on mRNA and protein expression levels of StAR and progesterone synthesis in GTCs. Our results demonstrate that hypoxia reduces StAR expression via the activation of NR1D1 signaling in GTCs, thus inhibiting progesterone synthesis. These findings provide new insights into the NR1D1 regulation of progesterone synthesis in GTCs under hypoxic conditions.

Melatonin receptor depletion suppressed hCG-induced testosterone expression in mouse Leydig cells.

Melatonin receptors MT1 and MT2 (genes officially named MTNR1A and MTNR1B, respectively) play crucial roles in melatonin-mediated regulation of circadian rhythms, the immune system, and control of reproduction in seasonally breeding animals. In this study, immunolocalization assay showed that MT1 and MT2 are highly expressed in Leydig cell membrane. To understand the biological function of melatonin receptors in hCG-induced testosterone synthesis, we generated melatonin receptor knockdown cells using specific siRNA and performed testosterone detection after hCG treatment. We found that knockdown of melatonin receptors, especially MTNR1A, led to an obvious decrease (> 60%) of testosterone level. Our further study revealed that knockdown of melatonin receptors repressed expression, at both the mRNA level and the protein level, of key steroidogenic genes, such as p450scc, p450c17 and StAR, which are essential for testosterone synthesis. hCG triggered endoplasmic reticulum (ER) stress to regulate steroidogenic genes' expression and apoptosis. To further investigate the potential roles of melatonin receptors in hCG-induced regulation of ER stress and apoptosis, we examined expression of some crucial ER stress markers, including Grp78, Chop, ATF4, Xbp1, and IRE1. We found that inhibition of melatonin receptors increased hCG-induced expression of Grp78, Chop and ATF4, but not Xbp1 and IRE1, suggesting that hCG may modulate IRE1 signaling pathways in a melatonin receptor-dependent manner. In addition, our further data showed that knockdown of MTNR1A and MTNR1B promoted hCG-induced expression of apoptosis markers, including p53, caspase-3 and Bcl-2. These results suggested that the melatonin receptors MTNR1A and MTNR1B are essential to repress hCG-induced ER stress and cell apoptosis. Our studies demonstrated that the mammalian melatonin receptors MT1 and MT2 are involved in testosterone synthesis via mediating multiple cell pathways.