早期卵子发生中P小体募集NANOS3的机制

doi:10.16098/j.issn.0529-1356.2025.03.010

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解剖学报 ›› 2025, Vol. 56 ›› Issue (3) : 323-328. DOI: 10.16098/j.issn.0529-1356.2025.03.010

组织学胚胎学发育生物学

早期卵子发生中P小体募集NANOS3的机制

耿鉴薇^1,2 何飞^2,4 马一丹^2,3 周永睿^{1, 2}穆欣艺^1,2*

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Mechanism of P-bodies recruiting NANOS3 during early oogenesis

GENG Jian-wei^1,2 HE Fei^2,4 MA Yi-dan^2,3 ZHOU Yong-rui^1,2 MU Xin-yi^1,2*

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摘要

目的探讨早期卵子发生过程中卵母细胞内NANOS3与P小体（P-bodies）之间的关系以及两者相互作用的机制。方法通过免疫荧光观察新生1日（1dpp）小鼠卵巢卵母细胞中NANOS3和死盒解旋酶6（DDX6）的共定位情况，并利用免疫共沉淀检测两者的相互作用。构建NANOS3和DDX6全长质粒转染HEK293T细胞，通过免疫荧光和免疫共沉淀探讨两者相互作用的机制。NANOS3转染HeLa细胞，通过活细胞成像探究NANOS3是否具有液-液相分离（LLPS）的能力。通过DDX6-免疫沉淀-质谱联用技术（DDX6-IP-MS）鉴定早期卵子发生中P小体募集的蛋白。结果在1dpp小鼠卵母细胞中NANOS3与DDX6共定位并能够相互作用。然而，转染NANOS3和DDX6的HEK293T细胞却无法观察到两者的共定位，但免疫共沉淀仍显示两者存在相互作用。同时，活细胞成像显示，NANOS3在细胞中具有动态的液体样性质，可能通过LLPS促进P小体的形成。最后，DDX6-IP-MS揭示DDX6可能通过结合NANOS3相互作用蛋白Pumilio，将NANOS3募集到P小体中。结论 NANOS3是新生小鼠卵母细胞中P小体的特定组分，可能参与调控早期卵子发生。

Abstract

Objective To explore the relationship between NANOS3 and P-bodies in oocytes and the mechanism of their interaction during early oogenesis. Methods The co-localization of NANOS3 and dead box helicase 6(DDX6) in day post postnatal 1 (1dpp) mouse oocytes was observed by immunofluorescence, and the interaction between NANOS3 and DDX6 was detected by immunoprecipitation. NANOS3 and DDX6 full-length plasmids were constructed to transfect HEK293T cells, and the mechanism of their interaction was investigated by immunofluorescence and immunoprecipitation. NANOS3 transfected HeLa cells to investigate whether NANOS3 had the ability of liquid-liquid phase separation (LLPS) by live-cell imaging. The proteins recruited by P-bodies in early oogenesis were identified by DDX6-immunoprecipitation-mass spectrometry(DDX6-IP-MS). Results NANOS3 and DDX6 colocalized and interacted with each other in 1dpp mouse oocytes. However, the co-localization of NANOS3 and DDX6 was not observed in HEK293T cells that had been transfected, but co-immunoprecipitation still demonstrated an interaction between these two proteins. Besides, live-cell imaging revealed that NANOS3 exhibited dynamic fluid-like properties within cells, which may promote the formation of P-bodies through LLPS. Finally, DDX6-IP-MS revealed that DDX6 might recruit NANOS3 into P-bodies by binding to the NANOS3 interacting protein Pumilio. Conclusion NANOS3 serves as a specific component of P-bodies in neonatal oocytes and may be involved in the regulation of early oogenesis.

关键词

NANOS3

/ P小体 / 早期卵子发生 / 免疫荧光 / 新生小鼠

Key words

NANOS3 / P-body / Early oogenesis / Immunofluorescence / Neonated mouse

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耿鉴薇何飞马一丹周永睿穆欣艺. 早期卵子发生中P小体募集NANOS3的机制[J]. 解剖学报. 2025, 56(3): 323-328 https://doi.org/10.16098/j.issn.0529-1356.2025.03.010

GENG Jian-wei HE Fei MA Yi-dan ZHOU Yong-rui MU Xin-yi. Mechanism of P-bodies recruiting NANOS3 during early oogenesis[J]. Acta Anatomica Sinica. 2025, 56(3): 323-328 https://doi.org/10.16098/j.issn.0529-1356.2025.03.010

中图分类号： R329.1

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