Elucidating somatic interstitial cell and ovarian follicle crosstalk: insights from a cross species model of murine follicle development and primary human ovarian interstitial cells

Ovarian follicles activate and develop in a dynamic environment consisting of a complex and mostly uncharacterized interstitial cell (IC) population. Murine studies have shown that ICs co-cultured alongside follicles improves follicle growth. Using a unique cross species model, we test the hypothesis that this growth advantage is directed by paracellular crosstalk between ICs and follicles. Ovarian tissue fragments donated under an IRB approved protocol from consented individuals across the pubertal transition (age range: 8.87 to 12.14 years) undergoing ovarian tissue cryopreservation were digested to obtain primary human ovarian ICs. ICs were seeded onto tissue culture treated plates and treated with mitomycin C to pause proliferation at confluency. Secondary follicles isolated from CD1 mice were encapsulated in 0.5% alginate and cultured in two groups: co-culture alongside ICs or in conditioned media derived from ICs. For each condition, there were three additional sub-groups: follicles alone (control), pre-pubertal derived ICs (pre), and post-pubertal derived ICs (post). Follicles were monitored over 8 days by brightfield microscopy and assayed for diameter and survival. Media was collected for proteomics analysis. P < 0.05 determined by two-way ANOVA using Tukey’s correction for multiple comparisons was considered statistically significant. Results are reported as mean ± standard error. ICs were heterogenous and contained theca cells, endothelial cells, smooth muscle cells, and granulosa cells. Follicles in all conditions grew from day 0 to day 8, and survival across all conditions was at least 90 ± 10%. By day 4, follicles co-cultured with ICs were significantly (pre, 245.7 ± 8.7 μm) or trended towards significantly (post, 238.5 ± 8.0 μm) larger than control (205.4 ± 8.3 μm). This continued through day 6 (control, 236.8 ± 8.6 μm; pre, 272.8 ± 7.9 μm; post, 267.0 ± 8.4μm) and day 8 (control, 241.5 ± 9.1 μm; pre, 281.6 ± 8.1 μm; post, 274.9 ± 7.9 μm). Follicles grown in conditioned media exhibited no significant difference between groups at days 2 (control, 170.3 ± 4.0 μm; pre, 175.5 ± 3.6 μm; post, 185.4 ± 6.0 μm), 4 (control, 208.6 ± 6.0 μm; pre, 214.0 ± 5.0 μm; post, 219.6 ± 7.9 μm), 6 (control, 234.6 ± 6.9 μm; pre, 245.2 ± 5.9 μm; post, 242.7 ± 6.1 μm) or 8 (control, 247.6 ± 8.5 μm; pre, 261.7 ± 6.9 μm; post, 255.6 ± 6.5 μm). No difference was detected between control in the co-culture condition and any of the sub-groups in the conditioned media group at days 0, 2, 4, 6 or 8. Proteomic analysis is ongoing. Improved growth in the presence of ICs only occurred in the context of co-culture, supporting the hypothesis that signaling directed by crosstalk between ICs and follicles rather than IC intrinsic signaling promote follicle growth in this in vitro system.