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Guo-Min Zhang, Ming-Tian Deng, Zhi-Hai Lei, Yong-Jie Wan, Hai-Tao Nie, Zi-Yu Wang, Yi-Xuan Fan, Feng Wang, and Yan-Li Zhang

During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

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Xiaolei Yao, M A EI-Samahy, Shenhua Xiao, Zhibo Wang, Fanxing Meng, Xiaodan Li, Yongjin Bao, Yanli Zhang, Ziyu Wang, Yixuan Fan, and Feng Wang

Being a novel target of luteinizing hormone (LH), the effect of CREB-binding protein/P300-interacting trans-activator with ED-rich tail member 4 (CITED4) gene on the proliferation, apoptosis, and steroidogenesis of ovarian granulosa cells (GCs) in Hu sheep was investigated. The presence of CITED4, CREB-binding protein (CBP), CCAAT/enhancer-binding protein alpha (C/EBPα) and -beta (C/EBPβ) proteins was demonstrated in GCs and luteal cells. CITED4 protein in GCs was induced by LH, and CITED4 overexpression moderately increased GC responses to LH. In contrast, CITED4 knockdown in GCs decreased prostaglandin (PGE2)-induced LH target gene levels. Moreover, PGE2-stimulated CITED4 mRNA expression was blocked by ERK1/2 inhibition (U0126), suggesting that CITED4 is a downstream target of the ERK1/2 pathway in sheep GCs. In contrast to CITED4 knockdown, CITED4 overexpression promoted GC proliferation, inhibited apoptosis, upregulated cell cycle-related genes, and downregulated apoptosis-related genes. Additionally, CITED4 overexpression induced cell cycle transition from S to G2/M phase. No effect was observed with CITED4 knockdown. CITED4 overexpression increased progesterone (P4) production levels and STAR mRNA expression, whereas CITED4 knockdown decreased P4 production and STAR and 3β-HSD mRNA expression levels. Thus, our results suggest that CITED4 is involved in regulating the expression of LH-induced genes and the ERK1/2 pathway and the proliferation, apoptosis, and steroidogenesis in Hu sheep GCs by modulating the expression of related genes. These findings will help understand the role of CITED4 in follicular development and ovulation of pre-ovulatory follicles.