Matrix-free three-dimensional culture of bovine secondary follicles to antral stage: Impact of media formulation and epidermal growth factor (EGF).

Disrupted/disordered ovarian steroidogenesis is associated with several fertility disorders such as polycystic ovary syndrome in humans and cystic ovarian disease in cattle. Methods to interrogate theca cell processes as part of follicular development are necessary to further research into treatments for these types of disorders. Multilayer follicles of dairy-breed cows were placed into culture in a novel matrix-free 3D system using round bottom low-attachment plates. Follicles were first cultured in the presence of two types of media previously used for in vitro follicle maturation (basal α-MEM and basal T-199). After the optimal media was identified, impact of supplementation of epidermal growth factor (EGF) on growth and survival of bovine secondary follicles to antral stage was evaluated. No differences were observed in growth and survival of follicles cultured in basal α-MEM media or basal T-199 media, although T-199 media's high phenol red content made assessment of follicles difficult. Further studies were then performed with α-MEM media. Three cohorts of follicles were observed based on time to antrum formation: ≤ 5 days (fast), 6-19 days (slow), or survived but did not form an antrum by 21 days (no). Supplementation of EGF to the basal α-MEM media dramatically improved follicle survival rates in culture (defined as follicles that either formed an antrum or did not form an antrum but did not die during 21 day culture period) from 29% to 95.7% (Chi-square p < 0.0001). However, in follicles that survived to form an antrum there were no differences in proportion of fast, slow and no antrum follicles after addition of EGF (Chi-square p > 0.7). Fast antrum follicles treated with EGF plateaued in size earlier in culture compared to controls (p = 0.013). Slow and no antrum follicles were larger in diameter during EGF culture than controls (p's < 0.0001). Many follicles cultured in this matrix-free system that formed an antrum approached 1.5-2 mm in size, an improvement from previous single follicle culture methods used for bovine pre-antral follicles in vitro. In addition, follicles displayed functional steroidogenesis in vitro producing measureable levels of estradiol and androstenedione. This matrix-free 3D culture system provides an excellent in vitro model to explore processes associated with folliculogenesis in cattle.