RESUMO
To search for the origin of nutrition in the amnion, we focused attention on both endocytotic and autophagic pathways. Using ultrastructural and biochemical methods, we examined 20 human amnions at term gestation. The uptake of horseradish peroxidase (HRP) was used for the detection of endocytosis. Transfection of the LC3-GFP plasmid and staining with monodansylcadaverine (MDC) and LysoTracker red (LTR) were used to demonstrate the formation of autophagic vacuoles. In addition, two autophagic genes, beclin 1 and Atg5, were assayed by RT-PCR. Within the amniotic epithelial (AE) cells, autophagic vacuoles contained organelles and cytoplasmic components and were enclosed by a double membrane. They contained autophagosomes with transfected LC3-GFP that stained positive for MDC and autolysosomes that stained positive for LTR. Endocytosis was an extremely active process in the cellular uptake of fluid and fluid contents and led to formation of vesicles and endosomes, which were found to be positive by HRP test. Many uniform vesicles were collected in the multivesicular bodies (MVBs). Finally, both endosomes and autophagosomes were fused and degraded by lysosomes. The data also demonstrated that large autophagosomes engulfed some endosomes or MVBs. Transcription of beclin 1 and Atg5 occurred in the amnion at term gestation. Taken together, these results show that AE cells have active endocytotic and autophagic capacities and that lysosomes are involved in the intracellular degradation of endosomes and autophagosomes. Sometimes the autophagic and endocytotic pathways converge. This study suggests that of endocytosis and autophagy activities in AE cells can be induced by nutrient limitation and are probably also evoked in response to some hormones in the amniotic fluid. Activation of both endocytotic and autophagic pathways plays different roles in the ability of the cell to acquire nutrients needed for its survival.
