Talpur Hira Sajjad, Worku Tesfaye, Rehman Zia Ur, Dad Rahim, Bhattarai Dinesh, Bano Iqra, Liang Aixin, He Changjiu, Yang Liguo
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China.
Department of Veterinary Physiology & Biochemistry, Sindh Agriculture University, Tandojam, Sindh, Pakistan.
Reprod Biol. 2017 Dec;17(4):380-388. doi: 10.1016/j.repbio.2017.10.005. Epub 2017 Oct 31.
Melatonin receptor 1 (MT1) performs a critical role in the regulation of the animal reproductive system, particularly in follicular growth, and has a considerable effect on reproductive performance. However, the role that MT1 plays in regulating hormones associated with reproduction remains unclear. This study was designed to examine the physiological role of constitutive MT1 silencing and follicle stimulating hormone (FSH) treatment in reproduction, making use of mouse granulosa cells (mGCs) as a model. To understand the constitutive role of MT1 in ovarian physiology, the RNAi-Ready pSIREN-RETROQ-ZsGreen Vector mediated recombinant pshRNA was used to silence MT1 gene expression. Furthermore, we observed that the expression of MT1 was successfully inhibited both at the protein and mRNA levels (P<0.001). We demonstrated that RNAi-B-mediated MT1 down-regulation significantly promoted apoptosis (P<0.001), inhibited proliferation, and regulated the cell cycle at the S-phase; conversely, FSH treatment partially aided the apoptotic effect and improved proliferation but showed a significant effect at the S-phase of the cell cycle. Transitory knockdown of MT1 proved essential in the function of mGCs, as it significantly decreased cyclic adenosine monophospahte (cAMP) level and increased cell apoptosis. Following knockdown of MT1, the expression of Bax was significantly up-regulated (P<0.001), but Bcl-2 was slightly down-regulated, both at the transcriptional and at translational levels. Moreover, the silencing of MT1 and its constitutive effect on FSH significantly promoted an increase in estradiol (P<0.001) and slightly decreased the concentration of progesterone. Together, our data indicates that MT1 suppression leads to interference in the normal physiological function of the ovary by enhancing follicular apoptosis, inhibiting proliferation, and influencing hormonal signaling, whereas constitutive FSH treatment counteracted the negative down-regulatory effects of MT1 on mGCs.
褪黑素受体1（MT1）在动物生殖系统的调节中发挥着关键作用，尤其是在卵泡生长方面，并且对生殖性能有显著影响。然而，MT1在调节与生殖相关激素方面所起的作用仍不清楚。本研究旨在利用小鼠颗粒细胞（mGCs）作为模型，研究组成型MT1沉默和促卵泡激素（FSH）处理在生殖中的生理作用。为了解MT1在卵巢生理中的组成型作用，使用RNAi-Ready pSIREN-RETROQ-ZsGreen载体介导的重组pshRNA来沉默MT1基因表达。此外，我们观察到MT1的表达在蛋白质和mRNA水平均成功受到抑制（P<0.001）。我们证明，RNAi-B介导的MT1下调显著促进细胞凋亡（P<0.001），抑制细胞增殖，并在S期调节细胞周期；相反，FSH处理部分辅助了凋亡作用并改善了增殖，但在细胞周期的S期显示出显著作用。MT1的瞬时敲低被证明对mGCs的功能至关重要，因为它显著降低了环磷酸腺苷（cAMP）水平并增加了细胞凋亡。MT1敲低后，Bax的表达在转录和翻译水平均显著上调（P<0.001），但Bcl-2略有下调。此外，MT1的沉默及其对FSH的组成型作用显著促进了雌二醇的增加（P<0.001），并略微降低了孕酮浓度。总之，我们的数据表明，MT1抑制通过增强卵泡凋亡、抑制增殖和影响激素信号传导导致对卵巢正常生理功能的干扰，而组成型FSH处理抵消了MT1对mGCs的负下调作用。
