Jia Yuhang, Liu Yufang, Wang Peng, Liu Ziyi, Zhang Runan, Chu Mingxing, Zhao Ayong
College of Animal Science and Technology, College of Veterinary Medicine, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China.
State Key Laboratory of Animal Biotech Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, China.
Animals (Basel). 2024 May 14;14(10):1465. doi: 10.3390/ani14101465.
Neurotrophin receptor B (), also named , belongs to the neurotrophic factor family. Previous studies have shown that is associated with high fertility in mammals. However, the molecular mechanism and regulatory pathway of this neurotrophic factor remain unclear. In this study, overexpression and -siRNA were constructed to detect the effects of on the proliferation and hormone secretion of the ovarian granulosa cells (GCs) of sheep. We successfully isolated follicular phase granulosa cells in vitro from the ovaries of sheep in simultaneous estrus, and the immunofluorescence results confirmed that was expressed in the collected cells. Subsequently, the effect of on the proliferation of sheep granulosa cells was examined via cell transfection experiments. The results showed that the expression of and was significantly increased after overexpression, while the opposite trend was observed after the inhibition of expression ( < 0.05). The EdU and CCK-8 assays showed that the proliferation rate of sheep GCs was significantly increased after overexpression, while the opposite trend was observed after the inhibition of expression ( < 0.05). Moreover, significantly increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein () and hydroxy-δ-5-steroid dehydrogenase (), and cytochrome P450 family 19 subfamily A member 1 (). The ELISA results showed that the secretion levels of E and P significantly increased after overexpression, while the opposite trend was observed after the inhibition of expression ( < 0.05). Previous studies had confirmed that gene belongs to the PI3K-AKT signaling pathway and participates in the signaling of this pathway. This was demonstrated by protein-protein interaction analysis and NTRK2 belongs to the PI3K-AKT pathway. The modification of PI3K and AKT, markers of the PI3K-AKT pathway, via phosphorylation was increased after overexpression in the sheep GCs, while the opposite trend was observed after the inhibition of expression ( < 0.05). Overall, these results suggest that the gene regulates the proliferation of GCs and the secretion of steroid hormones in sheep, and that it influences the phosphorylation level of the PI3K/AKT signaling pathway. These findings provided a theoretical basis and new perspectives for exploring the regulation of gene in the development of ovine follicles.
神经营养因子受体B（），也称为，属于神经营养因子家族。先前的研究表明，与哺乳动物的高繁殖力有关。然而，这种神经营养因子的分子机制和调控途径仍不清楚。在本研究中，构建了过表达载体和 -siRNA，以检测对绵羊卵巢颗粒细胞（GCs）增殖和激素分泌的影响。我们成功地从同期发情绵羊的卵巢中体外分离出卵泡期颗粒细胞，免疫荧光结果证实收集的细胞中表达。随后，通过细胞转染实验检测对绵羊颗粒细胞增殖的影响。结果表明，过表达后和的表达显著增加，而抑制表达后则出现相反的趋势（<0.05）。EdU和CCK-8检测表明，过表达后绵羊GCs的增殖率显著增加，而抑制表达后则出现相反的趋势（<0.05）。此外，显著增加了类固醇生成相关基因的表达，包括类固醇生成急性调节蛋白（）、羟基-δ-5-类固醇脱氢酶（）和细胞色素P450家族19亚家族A成员1（）。ELISA结果表明，过表达后E和P的分泌水平显著增加，而抑制表达后则出现相反的趋势（<0.05）。先前的研究证实基因属于PI3K-AKT信号通路并参与该通路的信号传导。蛋白质-蛋白质相互作用分析证明了这一点，并且NTRK2属于PI3K-AKT通路。在绵羊GCs中过表达后，PI3K-AKT通路的标志物PI3K和AKT通过磷酸化的修饰增加，而抑制表达后则出现相反的趋势（<0.05）。总体而言，这些结果表明基因调节绵羊GCs的增殖和类固醇激素的分泌，并影响PI3K/AKT信号通路的磷酸化水平。这些发现为探索基因在绵羊卵泡发育中的调控提供了理论基础和新的视角。
