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Effect of ovarian disaggregation on adult murine follicle yield and viability
Follicles are isolated from ovaries for numerous reasons, including in vitro maturation, but adult murine yields are <2 follicles/mg. This study aimed to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n=7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV 590U/mL), or animal origin free collagenase IV (AOF 590U/mL) or AOF 1180U/mL. Isolated follicles were stained with nuclear DAPI, mitochondrial CMXRos, or alpha-tubulin-FITC antibody. Follicle diameters and staining were measured using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles; 17±10 follicles/mg ovarian tissue. All disaggregation methods released more secondary follicles (86±20 per ovary, p<0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles whereas AOF disaggregation caused more damage (p<0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (p<0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. We improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function and used these to optimise ovarian disaggregation.
RD16398 Accepted 08 April 2017
© CSIRO 2017