Detection of Apoptosis in Ovarian Cells In Vitro and In Vivo Using the Annexin V-Affinity Assay
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The ability of a cell to undergo apoptosis is crucial during development, tissue homeostasis, and in the pathogenesis and treatment of disease ( 1 ). To study apoptosis, it is important to be able to detect apoptotic cells reliably. Here we describe a method to detect apoptosis in vitro and in vivo on basis of the changes in phospholipid distribution in the plasma membrane that occur during this process. In healthy cells, phosphatidylserine (PS) is maintained predominantly in the inner plasma membrane surface by an aminophospholipid translocase ( 2 ). However, early during apoptosis, PS is translocated from the inner to the outer membrane surface and serves as a trigger for adjacent phagocytes to remove the dying cell ( 3 - 5 ). Exposure of PS can be detected in vitro and in vivo with fluorochrome- or biotin-labeled annexin V, a protein that binds to negatively charged phospholipids in the presence of calcium ions ( 6 , 7 ). In cells that are cultured in suspension, detection of apoptosis on the basis of PS exposure is relatively easy ( 8 ). However, sample handling of adherent cell lines, such as the ovarian cell line PA-1, might interfere with reliable detection of PS exposure. Therefore, we developed a method to detect PS exposure in adherent cell lines by labeling cells in a monolayer with annexin V and harvesting the cells afterwards by mechanical scraping ( 9 ) (Figs. 1 and 2). Fixation of annexin V-labeled cells also allows the study of the relationship between PS exposure and expression of intracellular antigens ( 10 ). We also present a method to detect apoptosis in vivo during follicular maturation in the mouse Fig. 3 ). This method is based on in vivo studies of viable mouse embryos, which indicate that PS exposure is a pancellular phenomenon of apoptosis during mammalian development ( 11 , 12 ).
Fig. 1. Confocal scanning laser microscopy analysis of PA-1 ovary teratocarcinoma cells. Apoptosis was induced by treating the cells for 6 h with 50 μM roscovitine, a cyclin-dependent kinase inhibitor. Cells were labeled with annexin V-Oregon green to detect PS exposure, harvested by mechanical scraping, and labeled with propidium iodide (PI) to detect plasma membrane integrity. A and B show a linear projection of a stack of confocal images of early apoptotic cells after labeling with annexin V. At this stage, the plasma membrane integrity is preserved and, therefore, PI cannot enter the cell. C shows a secondary necrotic PA-1 cell, with clear annexin V staining at the plasma membrane ( C1 ) and with PI staining of the condensed and fragmented chromatin ( C2 ).
Fig. 2. Dotplot of bivariate PI/annexin V flow cytometric analysis of adherent ovary cell line PA-1. Plasma membrane integrity is shown on the X-axis and annexin V immunofluorescence is shown on the Y-axis. Cells were treated with 50 μM roscovitine to induce apoptosis. 6 h after roscovitine treatment, cells were labeled with annexin V-Oregon green, harvested by scraping, and labeled with PI. Four populations of cells can be identified: region R1: vital cells (annexin V negative/PI negative), region R2: apoptotic cells (annexin V positive/PI negative), region R3: dead cells (annexin V positive/ PI positive); and region R4: damaged cells (annexin V negative/PI positive). For technical details, see ref. 9.
Fig. 3. Micrographs of paraffin sections through mice ovaries that were perfused with biotinylated annexin V ( A-F ) or HEPES-buffer only (G and H ). In A, annexin V labeled early apoptotic cells (arrowhead) and late apoptotic-pyknotic (arrow) granulosa cells are shown. During follicle maturation, initially apoptosis is absent (B). At later phases, annexin V labeled apoptotic granulosa cells (C, arrow) were observed in the primary (C) and secondary (D) follicles. Unstained pyknotic cells were also observed (C, arrowhead), presumably these cells were already located in the phagosomes before perfusion with annexin V. Also in the Graafian follicle, apoptotic cells were present in large numbers (E). F shows an enlargement of the boxed area in E. Labeled apoptotic and postapoptotic necrotic cells that have been shed into the antrum are clearly visible (asterisk), as well as unlabeled late postapoptotic necrotic cells. Labeling of ingested (arrowhead) and noningested (arrow) apoptotic cells was absent in ovaries of specimen that were perfused with HEPES-buffer only (G: overview, H: detail of boxed area in G). Scale bars equal 10 μm (A), 25 μm (C, F, and H), 50 μm (B and D) and 100 μm (E and G).
