ESA-SRB-AOTA 2019

Granulosa cell transcriptome of neonatal mice provides new clues for primordial follicle activation (#123)

Emmalee A Ford 1 2 3 , Emma L Beckett 3 4 , Shaun D Roman 2 3 4 5 , Eileen M McLaughlin 2 6 7 , Jessie M Sutherland 1 2 3
  1. School of Biomedical Science and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia
  2. Priority Research Centre for Reproductive Science, Newcastle, NSW, Australia
  3. Hunter Medical Research Institue, New Lambton, NSW, Australia
  4. School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia
  5. Priority Research Centre for Drug Development, University of Newcastle, Callaghan, NSW, Australia
  6. School of Biological Science, University of Auckland, Auckland, New Zealand
  7. School of Science and Technology, University of Canberra, Bruce, ACT, Australia

The dormant population of primordial follicles is determined at birth and serves as the reservoir for future fertility. Of equal importance to fertility is the rate that primordial follicles activate and enter folliculogenesis, yet our understanding of the biochemical processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signalling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, ovaries from C57BL6 mice were enzymatically dissociated at two time points spanning the initial wave of primordial follicle activation. Post-natal day 1 (PND1) ovaries yielded primordial granulosa cells, and post-natal day 4 (PND4) ovaries yielded a mixed population of both primordial and primary granulosa cells. RNA was isolated from these two time points and sequenced using the Illumina NextSeq 500 System. Differential expression was defined as a log2 fold change of 1 in either direction, and a significance level of p≤0.05. Differential expression analysis revealed a total of 132 differentially expressed genes between PND1 and PND4 granulosa cells. Biological network modelling was performed using Ingenuity Pathway Analysis and confirmed the upregulation of regulatory genes present within pathways, established (Figla, MapK) and mostly unexplored (Wnt, Eif4e), during the primordial-to-primary transition. Validation of transcriptomic findings was carried out via quantitative RT-PCR of candidates among the top 8 differentially expressed genes. We provide preliminary evidence of a novel role for Fzrb, a precursor to the Wnt signalling pathway, as contributing to promoting primordial follicle activation. The transcript showed a 3.5-fold increase in PND4 granulosa cells relative to PND1 (p=0.023), with the same trend in qPCR (p<0.001). Advances in primordial follicle activation research may contribute to the development of novel fertility preservation strategies for women at risk of premature reproductive decline.