P–175 Possible role of NAD+ metabolism in oocyte aging: insights from gene expression profiles in reproductive aged oocytes
Authors: Emidio G. D , Konstantinidou F , Artini P G , Gatta V , Tatone C 
Published: August 2021

Abstract

Study question
Does reproductive aging alter the expression of genes involved in NAD+ metabolism in the mammalian oocyte?

Summary answer
We found that aging alters the expression of thirty genes encoding for NAD+-producing and NAD+-consuming enzymes pathway in mouse MII oocytes.

What is known already
NAD, a multifunctional metabolite in living cells, is known to convert between its oxidized NAD+ and reduced NADH forms during nutrients breakdown; the intracellular NAD+/NADH redox state reflects cell ability in generating ATP energy. NAD+ is utilized by proteins that control gene expression, DNA repair, apoptosis, mitochondrial biogenesis (i.e. sirtuins). Raising NAD+ by inducing its biosynthesis leads to sirtuins activation, so directly linking the cellular redox state with signalling events. The NAD+ pool is set by a critical balance between NAD+ biosynthetic and NAD+ consuming pathways. NAD(P)H levels declined in aged oocytes and NAD+ precursors seem to counteract ovarian aging.

Study design, size, duration
Pools of 25 oocytes at MII stage were obtained from young (4–8 weeks) and reproductively aged (48–52 weeks) CD–1 mice and processed for the analysis of 41 genes participating in NAD+ biosynthetic and NAD+ consuming pathways NAD+ pathways. Each experiment was performed three times and data were subjected to bioinformatic analysis to unravel potential age-related effects on NAD metabolism.

Participants/materials, setting, methods
Mice were superovulated by intraperitoneal injection of PMSG followed by hCG 48h apart. MII oocytes were isolated by 0.3 mg/ml hyaluronidase. RNA was obtained from each sample by Arcturus PicoPure Kit, and reverse transcribed. Each cDNA was analysed in triplicate by employing a NAD Metabolism H41 Predesigned panel for use with SYBR® Green, containing 41 genes of the NAD pathway, 2 housekeeping genes and 6 control probes. Raw data were analysed by DataAssist software.

Main results and the role of chance
The comparison between aged and young oocytes were focused on genes showing an absolute fold change (FC) &lt;0.7 or &gt; 1.4, a present call in at least the 50% of experiments and a p-Value &lt;0.05 (ANOVA). Excluding transcripts showing a concordant value &lt; 80%, n.30 differentially expressed genes (DEGs) were found: n.26 transcripts down-expressed and n.4 genes up-regulated. Data obtained by Ingenuity Pathways Analysis (IPA) software (Ingenuity Systems) provide evidence that NAD+ biosynthesis in aged oocytes is severely compromised.

Limitations, reasons for caution
Our results on animal model must be taken with caution. Validation of NAD+ precursor or activators of NAD+ biosynthesis in vivo administration is required.
Wider implications of the findings: Present results demonstrate that aging affect oocyte genes involved in the regulation of NAD+ availability and supports the hypothesis that modulation of NAD+ metabolism may be an “anti-aging” strategy. Overall, these data laid the foundation for a central role of NAD+ metabolism in the maintenance of oocyte competenc

Trial registration number
Not applicable