Reproduction Abstracts

Introduction: Primordial follicles are relatively quiescent structures that form the basis of the ovarian reserve. Maintenance of the quiescent state, and conversely, release from this state towards irreversible growth, involves mechanisms that are currently unresolved. Two transcription factors have been implicated alongside this process in granulosa cells (GCs). Specifically, Smad3 was recently identified in the nuclei of GCs in small single-layered follicles whereas Foxl2 is a fundamental regulator of GC viability and phenotype. Since Smad3 interacts with Foxl2 in other models, we aimed to determine whether these two transcription factors co-operate in GCs to directly regulate the cell cycle regulator CyclinD2.

Methods: To study the protein expression pattern of Smad3, Foxl2 and CyclinD2 throughout the different follicle stages, immature mouse ovaries (containing high proportions of small follicles) were immuno-fluorescently labelled and imaged by high-resolution confocal microscopy. Follicles were staged and >3700 GC nuclei were analysed using Image J. A proximity ligation assay (PLA) was used to visualise a direct interaction between the two proteins. In parallel, samples of whole ovaries were used for ChIP-PCR and qPCR to determine if Foxl2 and Smad3 directly bound to the gene promoter of CyclinD2 in vivo.

Results and discussion: Smad3, Foxl2 and CyclinD2 proteins co-localised in GC nuclei and followed a similar pattern of expression in small follicles. Specifically, staining intensity increased in transitional and primary stages, followed by a reduction in expression as follicles formed multiple layers. In addition, CyclinD2 promoter fragments were detectable in Foxl2 and Smad3-bound chromatin complexes in neonatal ovary samples. Together these findings suggest that both transcription factors are regulating CyclinD2 in a co-operative manner; however, a direct interaction was not evident by PLA. The relationships presented here further implicate the TGFβ pathway during follicle activation, where recruitment of cofactors may be important for signalling in this context.