A. Schrevens

Histochemical Demonstration of Apoptotic Cells in the Chicken Embryo Using Annexin V

This study describes the use of biotinylated annexin V for the histochemical detection of apoptotic cells in cultured chicken embryos during gastrulation. This method is based on the Ca2+-dependent binding of annexin V to phosphatidylserine, a negatively charged phospholipid, located at the inner leaflet of the cell membrane in living cells. However, in the early stages of apoptosis, phosphatidylserine is translocated to the outer layer of the cell membrane and can then be recognized by annexin V. Applying this method in cultured chicken embryos during gastrulation, we obtained labelling of apoptotic cells in the three germ layers. In the epiblast and mesoblast, labelling was predominantly present in the region lateral to the primitive streak. At the level of the germinal crescent, labelled cells were also found in the epiblast. Labelled cells in the deep layer, which is a heterogeneous tissue layer composed of endophyll, sickle endoblast and definitive endobl ast, were rather scarce. The distribution of cells, as observed in this study after labelling with annexin V in light microscopy and confocal laser scanning microscopy, is consistent with distributions reported by other authors using other approaches and with our previous observations made with the TUNEL technique and by electron microscopy after fixation in a tannic acid-based fixative. The main advantages of this method over other more sophisticated methods is its easiness and rapidity of execution and the fact that both early and late stages of apoptosis are detected.

Immunohistochemical localization of transforming growth factor-β1 and β2 during folliculogenesis in the quail ovary

SummaryImmunohistochemical methods were used to show the presence and distribution of transforming growth factor-β1 and β2 during folliculogenesis in quail ovarian tissues. The results indicated that both transforming growth factor-β subtypes are present. Immunolabelling for transforming growth factor-β1 demonstrated that prelampbrush oocytes are immunoreactive in the Balbiani complex, and developing and pre-ovulatory oocytes in the zona radiata. Immunolabelling was also associated with granulosa cells. The number of stained granulosa cells decreased during folliculogenesis. In the pre-ovulatory follicles, immunolabelling was found predominantly in the theca interna. Immunolabelling for transforming growth factor-β2 was associated with the zona radiata of developing and preovulatory follicles, and with stromal interstitial cells. Moderate immunoreactivity was found in the Balbiani complex of prelampbrush oocytes. Weak immunolabelling was localized in the granulosa cells of prelampbrush follicles, and in a few cells of the theca interna of pre-ovulatory follicles. The immunolocalization of transforming growth factor-β1 and-β2 in the quail ovary supports their autocrine and/or paracrine role in avian ovarian processes.

Localization of apoptotic cells by direct immunogold detection of digoxigenin-labeled genomic DNA in semithin sections.

In this study, which correlates apoptosis with avian ovarian physiology, we modified an in situ DNA nick end-labeling method using immunogold reagents to detect apoptotic cells in semithin sections of quail ovaries embedded in glycol methacrylate resin. Special attention was paid to the prevention of background staining. The results are comparable with those of the ApopTag peroxidase kit in paraffin-embedded ovaries.