Fig. 3

Analysis of cellular apoptosis after extended bovine ovary. a Representative confocal images of TUNEL staining in cultured whole bovine ovaries treated with continuous fresh media with or without 500 nM DXR for 24 h and 48 h (Groups 1 and 3). b represent corresponding FITC staining of the images shown in A. All treatment groups were fixed, stained using a TUNEL stain. TUNEL stain indicates cells that are undergoing apoptosis (FITC, green) and Propidium iodide (PI, red) to show the nuclei. Examples of primordial and primary follicles are shown in images A-D, antral follicles (F-H), and stroma (I to L). Control = ovaries cultured in continuous fresh media circulation, DXR = Doxorubicin. H = Hour, scale bars = 100 μm for all images. Brightness was increased to +30 to enhance image visualization. c Representative images of TUNEL staining of cultured whole bovine ovaries perfused with recirculated media at 0, 48, and 96 h (Group 2). A-C represents spectral composite confocal images taken at 100X oil objective and D-I represent confocal images taken at 20X objective. All treatment groups were stained using TUNEL assay. Control = uncultured ovaries. H = Hour, scale bars = 10 μm for the spectral composite confocal images (A-C). and 100 μm for the confocal images (D-I). Brightness was increased to +30 to enhance image visualization. d Summary graph comparing cellular apoptosis after extended culture of bovine ovaries perfused with continuous fresh media with or without 500 nM DXR (A/B), or with recirculated media (C). All treatment groups were fixed, stained using a TUNEL stain and analyzed at 24 h and 48 h time points post-DXR treatment. TUNEL-positivity was quantified in ovarian stroma and granulosa cells irrespective of follicle types. Single circulation fresh media (Group 1) demonstrated the lowest level of cell apoptosis. DXR = Doxorubicin, H = Hour; n = 3 ovaries per treatment group, two-way ANOVA, ** p < 0.01; *** p < 0.001, n.s = non-significant