Guangxi Medical University, Nanning, 530021, China.
Department of the Reproductive Medicine Research Center, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, China.
J Assist Reprod Genet. 2023 Mar;40(3):491-508. doi: 10.1007/s10815-023-02765-4. Epub 2023 Mar 4.
To explore the underlying mechanism of primordial follicle loss in the early period following ovarian tissue transplantation (OTT).
BNIP3 was selected through bioinformatic protocols, as the hub gene related to autophagy during OTT. BNIP3 and autophagy in mice ovarian grafts and in hypoxia-mimicking KGN cells were detected using immunohistochemistry, transmission electron microscopy (TEM), western blotting, qPCR, and fluorescence staining. The regulatory role played by BNIP3 overexpression and the silencing of KGN cells in autophagy via the mTOR/ULK1 pathway was investigated.
Ultrastructure examination showed that autophagic vacuoles increased after mice ovarian auto-transplantation. The BNIP3 and autophagy-related proteins (Beclin-1, LC3B, and SQSTM1/p62) in mice ovarian granulosa cells of primordial follicle from ovarian grafts were altered compared with the control. Administration of an autophagy inhibitor in mice decreased the depletion of primordial follicles. In vitro experiments indicated that BNIP3 and autophagy activity were upregulated in KGN cells treated with cobalt chloride (CoCl). The overexpression of BNIP3 activated autophagy, whereas the silencing of BNIP3 suppressed it and reversed the autophagy induced by CoCl in KGN cells. Western blotting analysis showed the inhibition of mTOR and activation of ULK1 in KGN cells treated with CoCl and in the overexpression of BNIP3, and the opposite results following BNIP3 silencing. The activation of mTOR reversed the autophagy induced by BNIP3 overexpression.
BNIP3-induced autophagy is crucial in primordial follicle loss during OTT procedure, and BNIP3 is a potential therapeutic target for primordial follicle loss after OTT.
探讨卵巢组织移植（OTT）早期原始卵泡丢失的潜在机制。
通过生物信息学方案选择 BNIP3 作为与 OTT 期间自噬相关的关键基因。采用免疫组织化学、透射电子显微镜（TEM）、western blot、qPCR 和荧光染色检测 BNIP3 和小鼠卵巢移植物以及缺氧模拟 KGN 细胞中的自噬。研究 BNIP3 过表达和 KGN 细胞通过 mTOR/ULK1 通路对自噬的沉默对 KGN 细胞自噬的调节作用。
超微结构检查显示，小鼠卵巢自体移植后自噬小体增加。与对照组相比，卵巢移植物中原始卵泡的颗粒细胞 BNIP3 和自噬相关蛋白（Beclin-1、LC3B 和 SQSTM1/p62）发生改变。在小鼠中给予自噬抑制剂可减少原始卵泡的耗竭。体外实验表明，钴氯化物（CoCl）处理的 KGN 细胞中 BNIP3 和自噬活性上调。BNIP3 的过表达激活自噬，而 BNIP3 的沉默抑制自噬，并逆转 CoCl 诱导的 KGN 细胞自噬。Western blot 分析显示，CoCl 处理和 BNIP3 过表达的 KGN 细胞中 mTOR 抑制和 ULK1 激活，以及 BNIP3 沉默后的相反结果。mTOR 的激活逆转了 BNIP3 过表达诱导的自噬。
BNIP3 诱导的自噬在 OTT 过程中原始卵泡丢失中起关键作用，BNIP3 是 OTT 后原始卵泡丢失的潜在治疗靶点。
