Oral Presentation ESA-SRB 2023 in conjunction with ENSA

Assessing the consequences of heightened pro-AMH processing on female reproduction in mouse. (#64)

Shreya Maskey 1 2 , William A Stocker 2 , Adam Hagg 1 , Amy L Winship 3 , Craig A Harrison 2 , Kelly L Walton 1 2
  1. School of Biomedical Sciences, The University of Queesland, St. Lucia, Queesland, Australia
  2. Department of Physiology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
  3. Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

Within the mammalian ovary, anti-Müllerian hormone (AMH) is produced by granulosa cells of growing follicles and primarily acts to limit the activation of dormant primordial follicles. AMH is initially synthesised as a pro-AMH protein that undergoes proteolytic maturation to release the mature bioactive dimer, which binds and signals via AMH receptors. However, pro-AMH cleavage is largely inefficient and as a result, the pro-AMH form is much more abundant in circulation compared to the mature form. This study hypothesised that enhancing pro-AMH cleavage efficiency would increase AMH bioactivity both in vitro and in vivo. Firstly, we used site-directed mutagenesis to enhance the furin cleavage site in pro-AMH (443RTGR445 to 443RKKR445) and verified that these modifications improved the yield of bioactive mature AMH (in AMH responsive HEK293T cells). Next, we aimed to determine whether improving pro-AMH processing in vivo translated to enhanced AMH-mediated actions in the mouse ovary. Here, we used CRISPR/Cas9 to generate an AMHRKKR/RKKR mouse on a C57BL/6J background. Analyses of 12-week-old AMHRKKR/RKKR mice revealed that the ovaries were significantly lower in mass (-25%, p<0.05) relative to ovaries from littermate AMHWT/WT controls. Similarly, the ovaries from 24-week-old AMHRKKR/RKKR mice tended to be lower in mass compared to the ovaries from control AMHWT/WT females. Histological analyses hope to reveal the cause of the shrunken ovaries in the AMHRKKR/RKKR mice. Despite the overt differences in ovarian masses, oestrus cycling, female fertility, and serum AMH and inhibin B levels were unaltered across genotypes. Furthermore, no overt differences in the testes were observed in adult male AMHRKKR/RKKR mice compared with AMHWT/WT controls. Our findings to date support that the optimisation of pro-AMH processing alters the size of reproductive organs in female mice, and ongoing investigations hope to unveil the mechanisms driving this phenotype.

  1. Visser JA, Schipper I, Laven JS, Themmen AP. Anti-Müllerian hormone: an ovarian reserve marker in primary ovarian insufficiency. Nat Rev Endocrinol. 2012;8(6):331-41.
  2. di Clemente N, Jamin SP, Lugovskoy A, Carmillo P, Ehrenfels C, Picard JY, et al. Processing of anti-mullerian hormone regulates receptor activation by a mechanism distinct from TGF-beta. Mol Endocrinol. 2010;24(11):2193-206.
  3. Pankhurst MW, Chong YH, McLennan IS. Relative levels of the proprotein and cleavage-activated form of circulating human anti-Müllerian hormone are sexually dimorphic and variable during the life cycle. Physiol Rep. 2016;4(9).