Oral Presentation ESA-SRB-NZSE-CaSR 2026 in conjunction with ENSA

Maternal NAD reserves in oocytes shape pre-implantation embryo development (143719)

Derek Y Wong 1 , Bettina P Mihalas 1 , Lake-Ee Quek 2 , Russell Pickford 3 , Denise Lin 1 , Ananya Vuyyuru 1 , Fabrizzio Horta 1 , Lindsay E Wu 4 , Robert B Gilchrist 1
  1. Discipline of Women’s Health, School of Clinical Medicine, University of New South Wales , Sydney , NSW, Australia
  2. Sydney Mass Spectrometry , University of Sydney, Sydney , NSW, Australia
  3. Bioanalytical Mass Spectrometry Facility, University of New South Wales , Sydney , NSW, Australia
  4. School of Biomedical Sciences, University of New South Wales , Sydney , NSW, Australia

 

Background

The early embryo is largely dependent on accumulated macromolecular stores from the oocyte to sustain development. It has been shown that nicotinamide adenine dinucleotide (NAD) levels decline drastically in embryos. Given the importance of NAD in many cellular and metabolic processes, we hypothesised that oocyte NAD reserves could play a role in supporting embryo development.

 

Methods

Murine oocytes underwent in vitro maturation (IVM) and fertilisation (IVF). We generated >4000 oocyte and embryo samples across experiments at key developmental stages (GV oocytes to 4-cell embryos) for targeted NAD metabolome profiling using liquid chromatography–mass spectrometry (LC/MS/MS).

 

Results

LC/MS/MS showed that immature oocytes contained 4 times more NAD than other developmental stages; however, >80% of NAD was rapidly depleted after maturation and fertilisation (p=0.0027). The depletion was not due to redox or phosphorylation, as NADH and NADP did not increase. NAD can be broken down to nicotinamide by enzymatic activities. Consistent with this, nicotinamide levels increased by 2-fold from GV to 2-cell (p=0.0041). To determine the NAD-consuming processes, we inhibited candidate NAD-consuming enzymes (PARPs, SIRTs, and CD38), none of which rescued the NAD decline in 2-cell embryos, suggesting that other uncharacterised mechanisms deplete NAD. We further investigated whether oocyte NAD reserves influence NAD availability in embryos. Inhibition of NAD synthesis during IVM significantly reduced NAD content in MII oocytes, zygotes, 2-cell and 4-cell embryos (p<0.0001). Isotope-tracing experiments using deuterium-4 nicotinamide during IVM revealed that ~50% of blastocyst NAD was derived from oocytes. Additionally, inhibition of NAD synthesis during IVM, but not during fertilisation, significantly reduced blastocyst formation rates by 30% (p<0.0001), while NMN supplementation in reproductively aged oocytes during IVM trended towards 10% higher blastocyst rates (p=0.138).

 

Conclusion

Our results suggest that oocyte NAD reserves constitute an important component of an oocyte’s developmental competence to reach later embryo development stages.