家禽颗粒细胞(GCs)的增殖和死亡在卵泡命运和产卵中起决定性作用。卵泡液(FF)含有多种营养物质和遗传物质,以确保卵泡细胞之间的交流。外泌体,作为一种新的细胞间通信,可以携带和运输蛋白质,RNA,和脂质对GC起反应,在各种家畜的FF中发现。家禽中FF的外泌体是否发挥类似作用尚不清楚。在这项研究中,鹅,产蛋量低的家禽,被选中,研究了FF外泌体对GCs增殖和死亡的影响。首先,产卵阶段不仅有大量健康的黄色小卵泡(HSYFs),而且有一些闭锁的黄色小卵泡(ASYFs)。此外,ASYF的GC层变得松散的互连,向内脱离,存活率比HSYFs降低。此外,与HSYFs相比,E2,P4的含量以及铁凋亡相关基因GPX4,FPN1和FTH1的mRNA表达水平显着降低,而COX2,NCOA4,VDAC3mRNA显著增加,在ASYFs的GC层中,线粒体cr的结构消失,外膜破裂。此外,ROS,MDA,ASYFs的GC层中的氧化水平明显高于HSYFs。所有这些都暗示铁死亡可能导致大量GCs死亡和参与卵泡闭锁。其次,FF外泌体分离自HSYFs和ASYFs,分别,并通过TEM鉴定,NTA,和检测外泌体标记蛋白。此外,我们通过追踪CM-Dil发现外泌体被GC吞噬。此外,ASYF-FF外泌体的添加显着提高了MDA含量,Fe2+水平,和GCs中的线粒体膜电位(MMP),从而显著抑制GC的增殖,通过铁凋亡抑制剂铁抑素-1恢复。第三,在FF来源的HSYFs和ASYFs的外泌体之间进行蛋白质组测序。我们获得了1615种差异表达的蛋白质,主要富集在蛋白质转运和铁凋亡途径中。其中,基于差异蛋白质-蛋白质相互作用网络分析,在铁凋亡途径中富集了HMOX1。最后,进一步探讨了HMOX1在调节GCs铁凋亡中的作用。在ASYF-FF的外泌体中观察到高表达的HMOX1比在HSYF-FF中高表达。HMOX1的过表达增加了ATG5、LC3II、和NCOA4的表达和减少FTH1,GPX4,PCBP2,FPN1在铁凋亡途径中的表达,还促进了细胞内Fe2+的积累和MDA的激增,这导致了GCs的铁中毒。HMOX1对铁凋亡的影响可被其抑制剂Znpp阻断。一起来看,在FF中鉴定了重要的蛋白质HMOX1,可以通过外泌体传递给GC,触发铁性凋亡,从而决定卵泡的命运。
The proliferation and death of granulosa cells (GCs) in poultry play a decisive role in follicular fate and egg production. The follicular fluid (FF) contains a variety of nutrients and genetic substances to ensure the communication between follicular cells. Exosomes, as a new intercellular communication, could carry and transport the proteins, RNA, and lipids to react on GCs, which had been found in FF of various domestic animals. Whether exosomes of FF in poultry play a similar role is unclear. In this study,
geese, a poultry with low egg production, were chosen, and the effect of FF exosomes on the proliferation and death of GCs was investigated. Firstly, there were not only a large number of healthy small yellow follicles (HSYFs) but also some atresia small yellow follicles (ASYFs) in the egg-laying stage. Also, the GC layers of ASYFs became loose interconnections, inward detachment, and diminished survival rate than that of HSYFs. Besides, compared to HSYFs, the contents of E2, P4, and the mRNA expression levels of ferroptosis-related genes GPX4, FPN1, and FTH1 were significantly decreased, while COX2, NCOA4, VDAC3 mRNA were significantly increased, and the structure of mitochondrial cristae disappeared and the outer membrane broke in the GC layers of ASYFs. Moreover, the ROS, MDA, and oxidation levels in the GC layers of ASYFs were significantly higher than those of HSYFs. All these hinted that ferroptosis might result in a large number of GCs death and involvement in follicle atresia. Secondly, FF exosomes were isolated from HSYFs and ASYFs, respectively, and identified by TEM, NTA, and detection of exosome marker proteins. Also, we found the exosomes were phagocytic by GCs by tracking CM-Dil. Moreover, the addition of ASYF-FF exosomes significantly elevated the MDA content, Fe2+ levels, and the mitochondrial membrane potential (MMP) in GCs, thus significantly inhibiting the proliferation of GCs, which was restored by the ferroptosis inhibitor ferrostatin-1. Thirdly, the proteomic sequencing was performed between FF-derived exosomes of HSYFs and ASYFs. We obtained 1615 differentially expressed proteins, which were mainly enriched in the protein transport and ferroptosis pathways. Among them, HMOX1 was enriched in the ferroptosis pathway based on differential protein-protein interaction network analysis. Finally, the role of HMOX1 in regulating ferroptosis in GCs was further explored. The highly expressed HMOX1 was observed in the exosomes of ASYF-FF than that in HSYF-FF. Overexpression of HMOX1 increased ATG5, LC3II, and NCOA4 expression and reduced the expression of FTH1, GPX4, PCBP2, FPN1 in the ferroptosis pathway, also promoted intracellular Fe2+ accumulation and MDA surge, which drove ferroptosis in GCs. The effects of HMOX1 on ferroptosis could be blocked by its inhibitor Znpp. Taken together, the important protein HMOX1 was identified in FF, which could be delivered to GCs via exosomes, triggering ferroptosis and thus determining the fate of follicles.