The isolation of sufficient numbers of mouse oocytes to develop new IVM and vitrification methods is expensive and time-consuming. Here we developed a fast, inexpensive method for collecting and characterising follicles to be used as a source of immature oocytes.

Ovaries from 6-10 week old mice (n=8) were quartered and allocated to collagenase IV (2mg/mL) for 0, 15, 30, 60 min (n=4) or to collagenase II (0, 0.5, 1, 2 mg/mL) for 30 or 60 min (n=4). After exposure, tissues were mechanically disaggregated for 5 min to release follicles. These were then photographed before staining with Mitotracker Red CMXRos to localise mitochondria, or α-tubulin-FITC antibody or DAPI to localise nuclei. The photographs of freshly isolated or stained follicles were analysed using Image J. Freshly isolated follicles were classified by diameter using an objective, automated method with reference to published standards [1].

Disaggregation with 2mg/ml collagenase II for 30 and 60 min yielded high numbers of follicles per unit weight of ovarian tissue; 34±8 and 31±8 respectively compared to 19±12 or 11±4 from control, mechanically disaggregated ovarian tissue. Yields after 30min were 12±8 primary, 10±6 secondary and 0.5±0.6 antral follicles (n=4). CMXRos and α-tubulin immunolocalisation was predominantly to the outer cytoplasmic sub-membrane regions of follicular granulosa cells and oocytes, whereas DAPI staining was localised to the nuclei. Preliminary quantification of staining indicates that there was no significant difference between 30 and 60 minute enzyme exposures with respect to mitochondria or α-tubulin.

The low numbers of primordial follicles and primary follicles retrieved were striking, and further optimisation of methods are required to isolate these follicle cohorts and to increase follicular yield. The disaggregation method did not affect mitochondrial, cytoskeletal or nuclear morphology, and the yield of secondary and antral follicles was sufficient to supply oocytes for IVM and vitrification experiments.