NET1 是调控小鼠卵母细胞纺锤体组装和肌动蛋白动态的关键调节因子。

NET1 is a critical regulator of spindle assembly and actin dynamics in mouse oocytes.

机构信息

State Key Laboratory of Animal Biotech Breeding, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China.

出版信息

Reprod Biol Endocrinol. 2024 Jan 2;22(1):5. doi: 10.1186/s12958-023-01177-4.

DOI:10.1186/s12958-023-01177-4

PMID:38169395

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10759572/

Abstract

BACKGROUND

Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation.

METHODS

NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation.

RESULTS

Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects.

CONCLUSIONS

Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.

摘要

背景

神经上皮转化基因 1（NET1）是 RhoA 亚家族鸟嘌呤核苷酸交换因子，调控广泛的生物学过程。然而，其在减数分裂卵母细胞中的作用尚不清楚。我们在此证明 NET1-HACE1-RAC1 通路介导了卵母细胞成熟过程中减数分裂的缺陷。

方法

通过特异性小干扰 RNA 降低小鼠卵母细胞中的 NET1 水平。通过免疫染色和共聚焦显微镜分析观察纺锤体组装、染色体排列、肌动蛋白帽和染色体铺片。我们还应用了质谱和 Western blot 分析进行这项研究。

结果

我们的结果表明，NET1 在 GV 期定位于核内，在 GVBD 后，NET1 定位于细胞质，主要分布在染色体周围，与减数分裂进程一致。NET1 位于细胞质中，并在 MI 和 MII 期大量聚集在纺锤体上。缺乏 Net1 的小鼠卵母细胞表现出异常的第一极体排出和不对称分裂缺陷。我们还确定 Net1 耗竭导致小鼠卵母细胞中 RAC1 蛋白表达减少，而 NET1 通过 HACE1 保护 RAC1 免受降解，这对肌动蛋白动力学和减数分裂纺锤体形成至关重要。重要的是，外源性 RAC1 在 Net1 耗尽的卵母细胞中的表达显著挽救了这些缺陷。