Journal Article

INTRK2/I Promotes Sheep Granulosa Cells Proliferation and Reproductive Hormone Secretion and Activates the PI3K/AKT Pathway

Jia, Yuhang, Wang, Peng, Zhang, Runan, Liu, Ziyi, Zhao, Ayong, Chu, Mingxing, Liu, Yufang

2024

Overview

Neurotrophin receptor B (NTRK2) is a member of the neurotrophic tyrosine receptor kinase (NTRK) family, and recent studies have shown that the NTRK2 gene plays an important role in mammalian reproduction. The expression of the NTRK2 gene is often closely related to the production and quality of gametes in mammals. In the present study, we found that overexpression of the NTRK2 gene upregulated the expression of genes associated with cell proliferation and genes associated with steroid synthesis in sheep granulosa cells, and then verified this result by examining cell proliferation levels and hormone levels. We further demonstrated that NTRK2 activates the PI3K-AKT signaling pathway, which is well known to regulate cell proliferation. These findings suggest that NTRK2 plays a role in regulating the proliferation and hormone secretion of sheep GCs through the PI3K-AKT pathway. These studies provide valuable insights into the regulatory role of the NTRK2 gene in follicular development in sheep. Neurotrophin receptor B (NTRK2), also named TRKB, belongs to the neurotrophic factor family. Previous studies have shown that NTRK2 is associated with high fertility in mammals. However, the molecular mechanism and regulatory pathway of this neurotrophic factor remain unclear. In this study, NTRK2 overexpression and NTRK2-siRNA were constructed to detect the effects of NTRK2 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 NTRK2 was expressed in the collected cells. Subsequently, the effect of NTRK2 on the proliferation of sheep granulosa cells was examined via cell transfection experiments. The results showed that the expression of CDK4 and CyclinD2 was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). The EdU and CCK-8 assays showed that the proliferation rate of sheep GCs was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Moreover, NTRK2 significantly increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase (HSD3B1), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The ELISA results showed that the secretion levels of E[sub.2] and P[sub.4] significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Previous studies had confirmed that NTRK2 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 NTRK2 overexpression in the sheep GCs, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Overall, these results suggest that the NTRK2 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 NTRK2 gene in the development of ovine follicles.

• Publisher: MDPI AG
• Subject: Gonadotropin