Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
Biol Reprod. 2023 Nov 15;109(5):720-735. doi: 10.1093/biolre/ioad094.
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.
滋养层在妊娠维持和胚胎着床中起着至关重要的作用，部分原因是其合成孕激素。已经证明，缺氧调节滋养层细胞的侵袭、增殖和分化。此外，人类滋养层细胞表现出昼夜节律基因的节律性表达。然而，尚不清楚昼夜节律系统是否存在于山羊滋养层细胞（GTC）中，以及其是否参与缺氧调节甾体激素合成。在这项研究中，免疫荧光染色显示 BMAL1 和 NR1D1（两个昼夜节律钟组件）在 GTC 中高度表达。实时定量 PCR 分析显示，几个昼夜节律钟基因在福司可林同步的 GTC 中呈节律性表达。为了模拟缺氧，用缺氧诱导剂（CoCl2 或 DMOG）处理 GTC。实时定量 PCR 结果表明，缺氧扰乱了昼夜节律钟基因和 StAR 的 mRNA 表达。值得注意的是，在缺氧的 GTC 中也检测到 NR1D1 的表达增加和 StAR 表达的减少。此外，缺氧的 GTC 中孕激素分泌明显下降。NR1D1 激动剂 SR9009 显著降低了 GTC 中 StAR 的 mRNA 和蛋白表达水平，并显著抑制了孕激素的分泌。此外，NR1D1 拮抗剂 SR8278 部分逆转了 CoCl2 对 GTC 中 StAR 和孕激素合成的 mRNA 和蛋白表达水平的抑制作用。我们的结果表明，缺氧通过激活 GTC 中的 NR1D1 信号降低 StAR 表达，从而抑制孕激素合成。这些发现为 NR1D1 在缺氧条件下调节 GTC 中孕激素合成提供了新的见解。
