Poster Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Cumulus-oocyte interactions are required for oocyte NAD+ homeostasis   (#454)

Bettina P Mihalas 1 , Sonia Bustamante 1 , Prakruti Sirigeri 1 , Ananya Vuyyuru 1 , Hemna Govindaraju 1 , Frost R Emily 1 , Irene E Sucquart 1 , Fabrizzio Horta 1 , William L Ledger 1 2 , Michael Bertoldo 1 , Lindsay E Wu 1 , Robert B Gilchrist 1 2
  1. UNSW Sydney, Kensington, NSW, Australia
  2. Fertility & Research Center, The Royal Hospital for Women, Randwick, New South Wales, Australia

The metabolic exchange between oocytes and their somatic supporting cells is essential for the oocyte to acquire developmental competence. Nicotinamide adenine dinucleotide (NAD+) is a critical metabolite for oocyte quality and declines with age. In this study, we examined the role of cumulus cells in regulating NAD+ metabolism in mouse and human oocytes. We first disrupted communication between oocytes and cumulus cells from young mice by mechanical removal of cumulus cells (DO) and chemical inhibition of gap junctions (200 µM CBX) in cumulus-oocyte complexes (COCs).  Quantitative mass spectrometry revealed significant decreases in NAD+ in oocytes (CBX: -16.01±4.25 fmol, DO: -15.86±2.53 fmol), while the NAD+ precursor, nicotinamide mononucleotide (NMN), increased (CBX: +15.76±3.11 fmol, DO: +15.69±3.44 fmol). No other measured NAD+ precursors were altered. Isotype tracing of in vitro supplemented deuterium-labelled-NMN (d4-major-NMN) also revealed a significant decrease in NAD+ synthesis from d4-major-NMN upon COC communication disruption (CBX: -34.74±9.82 fmol, DO: -22.31±1.82 fmol). To examine the role of cumulus-derived NAD+ metabolism in mediating age-related oxidative stress (OS) we examined the metabolic response to ageing and OS. Quantitative metabolomics from young (6 weeks) and old (12 months) mice, known to have elevated levels of OS, revealed a decrease in NAD+ in the cumulus cells of older mice (-52.00±13.44 fmol). Similarly, there was a decrease in NAD+ in cumulus cells when COCs were challenged with 100 µM H202 (-102.9±46.18 fmol). These data suggest that cumulus cell-derived NAD+ contributes to neutralizing oocyte OS. Supporting this, NMN supplementation during the in vitro maturation of oocytes from older mice reduced OS (H2DCFDA: -12.65±4.87 AU), restored mitochondrial membrane potential (JC-1: +0.18±0.08) and glutathione levels (monochlorobimane: +11.28±3.43 AU). Reciprocal experiments in human oocytes, discarded from fertility treatment, are underway. Taken together, these data suggest that communication­ with cumulus cells regulates NAD+ metabolism in oocytes supporting key aspects of oocyte quality.