2.5. Follicle density, survival and activation

Initial follicle densities and subsequent activation and survival were quantified through immunofluorescent staining for FOXO3a, an oocyte activation marker which exports from the nucleus to the cytoplasm when activated (Supplemental Fig. 1). Hydrogels containing follicles were fixed for 1 h in 4% paraformaldehyde at room temperature then washed in PBS (3×, 15 min each). Samples were transferred to a 25% w/v solution of Optimal Cutting Temperature (OCT) medium in water for 24 h at 4 °C, followed by 24 h each in 50% OCT solution then 100% OCT at 4 °C. Samples were cryosectioned on a Microm HM 525 cryostat at −21 °C to make 200 μm thick sections. Samples were blocked for 12 h at 4 °C in 5% normal goat serum in PBS. Primary antibodies for FOXO3a (Cell Signaling Technology, 2497S) were diluted 1:250 in blocking solution and incubated for 12 h at 4 °C. Samples were washed in blocking solution (3×, 1 h each) then incubated with Hoechst 33342 nuclear stain (Thermo Fisher) and secondary antibodies (Thermo Fisher, AlexaFluor 647 A-21244) diluted 1:500 in blocking solution for 12 h at 4 °C. Samples were washed in PBS (3×, 1 h each) then mounted in concave slides with Prolong Diamond (Thermo Fisher). Tile scan z-stack images were captured on a Leica SP8 laser scanning confocal microscope to obtain full images of the entire cryosectioned sample.

Follicle activation was quantified from hydrogels cultured for two days to allow sufficient time for activation and to obtain accurate counts of healthy follicles which survived the dissociation and encapsulation process. Dormant and activated oocytes were manually counted from stained cryosections, in which activated oocytes exhibited distinct cytoplasmic staining of FOXO3a (Supplemental Fig. 1), and dormant oocytes were identified by the presence of a comparatively large cell (>10 μm) surrounded by at least two squamous cells with flattened nuclei visualized with Hoechst. Follicle activation after 2 days of culture was calculated by dividing the number of oocytes with cytoplasmic FOXO3a staining by the total sum of oocytes counted within each cryosection.

Encapsulated follicle densities at the start of culture and follicle survival after 18 days of culture were based on follicle densities within cryosections. Oocytes were identified based on size and visual appearance or FOXO3a staining. Denuded oocytes which appeared healthy (clear and round) were included in density calculations for their potential to be rescued and enveloped in cell aggregates. The volume of the cryosection was calculated from the cross-sectional area, measured in ImageJ, and the height of the z-stack image. This volume was then used to calculate the density of follicles (oocytes/μL) as presented in Supplemental Fig. 1, and an average density across multiple cryosections was extrapolated to the entire 5 μL gel for determining the approximate number of encapsulated follicles at the start of culture. Follicle densities after 18 days of culture were similarly determined from multiple cryosections, and this density was used to determine the approximate number of remaining oocytes in the entire hydrogel. Follicle survival was then calculated by dividing this approximate number of oocytes by the calculated average number of starting follicles in the hydrogel determined from samples after 2 days of culture.