摘要
氯喹(chloroquine,CQ)和雷帕霉素(rapamycin,RAPA)是实验室常用的自噬抑制剂和增效剂.小鼠、斑马鱼等模式生物中的研究发现改变自噬水平会对精子和卵子发生产生影响.以曼氏无针乌贼为对象,通过建立精巢和卵巢的体外自噬抑制(CQ)和自噬增效(RAPA)组织培养模型,系统比较不同浓度CQ和RAPA处理后精巢、卵巢中自噬(LC3、BECN1)、凋亡(p53、Cas3)、无脊椎动物缝隙连接蛋白基因(Inx4)、雄性标记(Sox3、Sox30)和雌性标记(Nanos-1、FTZ-F1)基因表达的影响.结果表明CQ处理引起曼氏无针乌贼的精巢和卵巢的自噬水平显著抑制,自噬标记基因LC3 显著升高,BECN1 显著下降;Inx4 的变化趋势与自噬标记基因基本一致,表明Inx4 与自噬之间存在密切关系;凋亡标记基因p53 和Cas3 显著升高,表明自噬抑制引起凋亡通路被激活;雄性标记基因Sox4 和Sox30 的表达变化相对滞后于自噬和凋亡相关基因,表明自噬抑制对配子发生也产生了显著影响.自噬增效(RAPA)同样引起了自噬标记基因(LC3、BECN1)、无脊椎动物缝隙连接蛋白基因(Inx4)、凋亡基因(p53、Cas3)、雄性(Sox3、Sox30)和雌性标记基因(Nanos-1、FTZ-F1)均发生了显著变化,表明自噬对曼氏无针乌贼精子发生和卵子发生有重要作用.研究结果首次在海洋无脊椎动物中揭示了Inx4 与自噬之间存在密切关系,且二者对配子发生有直接影响,为进一步研究自噬和Inx4 对包括头足类在内的海洋无脊椎动物配子发生的调控作用提供了重要的基础数据.
Abstract
Chloroquine(CQ)and rapamycin(RAPA)are commonly used autophagy inhibitors and poten-t iators in the laboratory.Studies in model organisms such as mice and zebrafish have found that altering the level of autophagy can have an effect on spermatogenesis and oogenesis.In this study,we used Sepiella japonica to systematically compare the effects of autophagy(LC3,BECN1),apoptosis(p53,Cas3),invertebrate gap function protein gene(Inx4),male marker(Sox3,Sox30)and female marker(Nanos-1,FTZ-F1)gene expression after treatment with different concentrations of CQ and RAPA in testis and ovaries by establishing in vitro au-tophagy-inhibiting(CQ)and autophagy-potentiating(RAPA)tissue-culture models in testis and ovary.The re-sults showed that CQ treatment caused a significant inhibition of autophagy level in testis and ovary of S.japonica,a significant increase of autophagy marker gene LC3 and a significant decrease of BECN1;the trend of change of Inx4 was basically consistent with that of autophagy marker genes,suggesting that there was a close relationship between Inx4 and autophagy;apoptosis marker genes p53 and Cas3 were significantly elevat-ed,indicating that the The expression changes of male marker genes Sox4 and Sox30 lagged behind those of autophagy and apoptosis-related genes,indicating that autophagy inhibition also had a significant effect on ga-metogenesis.Autophagy potentiation(RAPA)likewise caused significant changes in autophagy marker genes(LC3,BECN1),invertebrate gap function protein gene(Inx4),apoptosis genes(p53,Cas3),male marker genes(Sox3,Sox30)and female marker genes(Nanos-1,FTZ-F1),suggesting that autophagy exerted a significant ef-fect on S.japonica spermatogenesis and oogenesis.The results of this study reveal for the first time in marine invertebrates the close relationship between Inx4 and autophagy,and the direct influence of both on gametoge-nesis,which provides important basic data for further research on the regulation of autophagy and Inx4 on ga-metogenesis in marine invertebrates including cephalopods.
万方数据