Reproduction Abstracts

Introduction: The molecular mechanisms involved in regulating growth of small, gonadotrophin-independent follicles are poorly understood. We have previously shown that the canonical TGFβ signalling intermediate, Smad3 is highly expressed in granulosa cells (GCs) of single-layered follicles. Furthermore, a reduction in the overall expression of Smad3 is associated with the onset of multi-layering and increased granulosa cell proliferation, suggesting modulation of TGFβ signalling is an important molecular event as follicles initiate growth. Strap has been identified in other tissues as an intracellular TGFβ receptor antagonist; therefore, we aimed to determine the expression and role of this protein in the context of early follicle development.

Methods: Gene and protein expression of Strap and Smad3 were analysed by qPCR and immunofluorescence/confocal microscopy in mouse ovaries at 4, 8 and 16 days of age (d4–16). Using an established culture model, neonatal (d4) mouse ovary fragments were used to examine the effects of Strap mRNA knock down using siRNA, and Strap protein inhibition by immuno-neutralisation. Effects of these treatments were compared with control groups after 7 days by measurements of oocyte size and immuno-fluorescent labelling of anti-Mullerian hormone (Amh) as a marker of GC development.

Results and discussion: Strap and Smad3 mRNA revealed similar patterns of expression levels, being highest in d4 ovaries containing mainly primordial follicles, and significantly reduced in d16 ovaries containing many multi-layered preantral follicles. Despite the reduction in transcript expression, both proteins were weakly detectable in GCs of multi-layered follicles, where they co-localised. Interestingly, mean oocyte diameters were significantly increased in ovary fragments cultured with either Strap siRNA (28.2 μm vs 24.2 μm; non-targeting control) or Strap antibodies (31.4 μm vs 28.5 μm; IgG control) after 7 days. In the latter experiment, Amh staining was increased in the anti-Strap group relative to control, supporting an overall inhibitory role for Strap on early follicle development.