Three-dimensional culture in a bioengineered matrix and somatic cell complementation to improve growth and survival of bovine preantral follicles

Abstract

STRUCTURED ABSTRACTPurposeNovel assisted reproductive technologies that capitalize on the preantral follicle population and utilize stem cell biology are greatly warranted. In this study, we explored the use of poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture, and examined the propensity of bovine embryonic stem cells (bESCs) to differentiate towards gonadal somatic cells. Together, these technological advances can be complemented to develop a sustainable and dynamic follicle culture system similar to the ovarian microenvironment.MethodsBovine preantral follicles at the primary and early secondary stages were cultured in two-dimensional (2D) control or encapsulated in PEG hydrogels (3D) to assess growth and then co-cultured in PEG hydrogels with bovine ovarian cells (BOCs) to determine cell and follicle viability. In a separate experiment, we tested protocols to modulate WNT, FGF and BMP4 signaling to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad-like fate and measured gene and protein expression of key lineage markers.ResultsPrimary follicles (40-60 µm), but not early secondary follicles (61-70 µm), grew over the 10-day culture period in PEG hydrogels compared to 2D control. However, follicles co-encapsulated with BOCs in hydrogels did not maintain viability and BOCs lost stromal cell signature over the culture period. When bESCs were induced towards gonadal somatic cells under WNT signaling activation, bFGF and BMP4 were dispensable to upregulate intermediate mesoderm (LHX1) and early coelomic epithelium/bipotential gonad markers (OSR1,GATA4,WT1). Higher BMP4 concentrations upregulated the lateral plate mesoderm markerFOXF1. Expression ofPAX3did not change over the course of the culture, indicating that the paraxial mesoderm lineage was not induced.ConclusionsCulture of preantral follicles at the primary stage in PEG hydrogels resulted in follicle growth compared to 2D system, although the benefit of the 3D system was inconsistent. BOCs did not maintain their identity in the PEG hydrogels; collectively, these results demonstrate that the PEG hydrogels can be a potential culture system for early stage follicles after refinements are implemented. These refinements could include the addition of ESC-derived ovarian somatic cells using protocols such as the one described here.CAPSULE SUMMARYWe demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.