Evidence for alternative pathways of granulosa cell heath in healthy and slightly atretic bovine antral follicles

Granulosa cell death is an early feature of atresia; however, there are man y apparent contradictions in the literature concerning the mode of granulos a cell death. We have therefore examined this process in bovine healthy and atretic antral follicles, using a variety of established techniques. Light and electron microscopic observations indicated the presence of pyknotic o r shrunken nuclei in both the membrana granulosa and the antrum. In the mem brana granulosa, these nuclei were frequently crescent shaped and uniformly electron dense and were approximately the same size as healthy nuclei, all of which are typical of early apoptosis. However, these nuclei were within the membranes of a healthy granulosa cell, suggesting that phagocytosis by a neighboring granulosa cell is an unusually early event in the apoptotic pathway of granulosa cells. In the membrana granulosa, pyknotic nuclei stai ned intensely with hematoxylin but weakly with the DNA-intercalating stain propidium iodide. A percentage of these pyknotic nuclei stained by TUNEL (t erminal deoxy-UTP nick end-labeling). However, in the antrum, the pyknotic nuclei and larger globules of DNA stained intensely with both hematoxylin a nd propidium iodide, but were not TUNEL positive. The comet assay of cell d eath produced a streak tail of randomly nicked DNA, rather than the plume o f low mol wt apoptotic DNA. Globules collected from fresh follicular fluid stained intensely with propidium iodide and were shown by PAGE to contain D NA, the majority of which was high mol wt. In conclusion, granulosa cells w ithin the membrana granulosa die by apoptosis, with phagocytosis by a neigh boring cell preceding any potential budding of the nucleus or cell itself. Granulosa cells near the antrum are sloughed off into the antrum, and their death has features more consistent with that of other cell types that unde rgo death as a result of terminal differentiation.