Development of an efficient and effective protocol for the isolation and culture of bovine primordial follicles

BACKGROUND-AIM The ability to grow undifferentiated oocytes in vitro from primordial follicles (PMF) would increase the supply of fully grown oocytes, to be destined to downstream applications in the livestock industry and fertility preservation programs. To date, the production of living offspring using in vitro development of oocytes from the PMF reserve has only been achieved in mice, providing the proof of principle of the potential value of follicle culture as a source of fully grown oocytes. However, culture systems to produce mature oocytes from PMF are still experimental. One of the main limiting factor in PMFs in vitro culture’s efficiency is follicle death occurring shortly after isolation from surrounding tissue. Therefore, we hypothesize that counteracting the cell-death signaling network(s) triggered upon isolation from the surrounding ovarian cortex should improve the outcome of PMF in vitro culture. To pursue this objective, we started by developing a reliable and efficient protocol for the isolation of viable PMF from the bovine ovarian cortex. METHODS Fragments of ovarian cortex of about 2 cm2 and 1 mm thick removed from slaughter-derived heifers (14-22 months) and adult cows (48-60 months) were chopped up into small fragments with a blade, dispersed in manipulation medium and homogenized. The resultant homogenate was then passed through a serial sieves system up to the lower limit of 30 μm. In the first set of experiments, homogenization speed and time were optimized to reduce follicle damage and foam formation. The second set of experiments was conducted to evaluate the viability after 16 hrs of culture by live/death staining and TUNEL and Caspases 3/7 assay. RESULTS The obtained results indicate that the number of follicles isolated from 2 cm2 and 1 mm thick ovarian cortex is inversely related to the age (mean of 100 vs. 40 from heifers and adult cows, respectively). Moreover, PMFs revealed a 30% decline in viability after culture compared to the freshly isolated ones. Finally, live/death staining showed two different patterns, suggestive of different modes of PMF cell death. CONCLUSIONS Our protocol can allow isolation of a high number of PMF per animal, higher than previously reported, and poses the basis for a morphological and functional characterization of isolated PMFs.