Transcription profiling by array of five isolated size matched populations of preantral follicles from normal human ovaries to evaluate the expression of TGF-beta superfamily growth factors, their receptors and downstream SMAD signalling molecules during early human folliculogenesis

In mammals, members of the transforming growth factor-beta (TGF-beta) superfamily are known to have key roles in the regulation of follicular growth and development. The aim of the study was to evaluate the expression of TGF-beta superfamily growth factors, their receptors and downstream SMAD signalling molecules during early human folliculogenesis. Human preantral follicles were enzymatically isolated from surplus ovarian tissue obtained from women having ovarian cortical tissue frozen for fertility preservation. A total of 348 human preantral follicles, ranging from 40 to 200 um in diameter, were isolated from ovarian tissue obtained from 15 women, aged 24–34 years. Isolated preantral follicles were grouped according to diameter in five size-matched populations spanning the primordial, primary and secondary stage follicles, and analyzed by whole-genome microarray analysis. Selected proteins/genes were analysed by immunocytochemistry and quantitative RT-PCR. TGF-beta superfamily genes with overall highest mRNA expressions levels included growth differentiation factors 9 (GDF9), bone morphogenic protein-15 (BMP15), BMP6, BMP-receptor-2 (BMPR2), anti-Müllerian hormone receptor 2 (AMHR2), TGFbetaR3, inhibin-alpha (INHA), and intracellular SMAD3 and SMAD4. Moreover, genes which were differentially expressed from the primordial to the late secondary stage follicles included GDF9 (p<0.01), BMP15 (p<0.001), AMH (p<0.0001), INHBB (p<0.05), TGFbetaR3 (p<0.05) and SMAD4 (p<0.05). Collectively, these data indicate that the active TGF-beta superfamily pathways in early human folliculogenesis consist of primarily GDF9 combined with synergistic effects of BMP15 through the BMPR2 and intracellular activation of SMAD3 and SMAD4, and that AMH and Inhibin B are engaged in intrafollicular events from the onset of follicular growth.