Oral Presentation ESA-SRB Conference 2015

NAD+ availability is critical for meiotic maturation in mouse oocytes  (#96)

Laurin Lau 1 , Angelique Riepsamen 1 , Wei Guo Loh 1 , Jonathan Ho 1 2 , Dave Listijono 1 , Lindsay Wu 2 , Hayden Homer 1
  1. School of Women's & Children's Health, University of New South Wales, Sydney, NSW, Australia
  2. School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia

Sirtuins are NAD+-dependent deacetylases with a growing repertoire of functions, which are critically dependent upon NAD+ availability in somatic cells. Here we study the effects of altering NAD+ levels in oocytes using FK866, a highly specific inhibitor of the NAD+-generating enzyme, NAMPT, as well as a genetic approach involving transgenic mice overexpressing another NAD+-generating enzyme, NMNAT1 (NMNAT-Tg mice). We first investigated in vitro meiotic maturation, which stretches from germinal vesicle breakdown (GVBD) to polar body extrusion (PBE). GVBD was markedly inhibited in wild-type (WT) oocytes cultured in 30µM FK866 (P≤0.001). Significantly, GVBD rates for FK866-treated NMNAT-Tg oocytes (67%) were comparable to untreated controls and almost three-fold higher than FK866-treated WT littermates (23%; P=0.0034) reinforcing the importance of NAD+ homeostasis in oocytes and supporting that FK866-induced effects reflected NAD+ de-regulation. The impact on PBE was even more stark as PBE rates at 10h, 12h and 14h were drastically reduced by FK866 (P<0.0001), attaining maximal rates by 20h post-GVBD of only 16% versus 92% in controls. Although there was a clear trend towards improved PBE in FK866-treated NMNAT-Tg oocytes (two-fold higher), this was less marked than improvements observed for GVBD, suggesting that late stages of maturation are especially vulnerable to NAD+ de-regulation. Sirtuins are critical regulators of mitochondrial function and of glutathione production for antioxidant defences in somatic cells. We therefore examined mitochondrial activity, glutathione and levels of reactive oxygen species (ROS) during FK866 treatment using MitoTracker, MCB (monochlorobimane) and 2’,7’-dichlorofluorescin diacetate (DCFDA), respectively. FK866-treated oocytes exhibited significantly reduced MitoTracker (P=0.008) and MCB (P<0.0001) fluorescence suggesting that reduced NAD+ availability led to reduced mitochondrial activity and less reducing power. ROS levels were also reduced (P<0.0001), likely secondary to lower mitochondrial activity. Collectively, these data show that reduced NAD+ availability severely impairs meiotic maturation producing defects consistent with deregulated sirtuin activity.