Oocyte specific transcription regulators, nobox and figla, are identified as key contributors to the decline in fecundity associated with ovarian aging

Abstract

Advanced maternal age (AMA; ≥35 years) is associated with a decline in fecundity that is largely attributed to loss of oocyte number and quality. The aim of this study was to explore at a molecular level the relationship between aging, ovarian environment and oocyte quality. Longitudinal research study. Young outbred CD1 female mice (3-4 months old; Young) and naturally aged outbred CD1 female mice (10-12 months old; Aged) were super-ovulated and oocytes collected (n = 10 group) for quantitative immunofluorescence of endoplasmic reticulum (ER) stress indicators, pIRE1 and ATF6. Total RNA was isolated from unstimulated ovaries (n = 6 per group), sequence libraries were prepared using the TruSEQ Total RNA library kit (Illumina) and sequenced on the Illumina NovaSEQ 6000. Differentially expressed genes (DEGs) were generated using edgeR, with an FDR cutoff of 5% (q value <0.01) and Student’s t-test significance at p<0.001, followed by Ingenuity Pathway Analysis (Qiagen). Oocyte numbers significantly declined with natural aging (mean: Aged = 4.6, Young = 12.9; p<0.0001). Total RNA sequencing revealed 281 significant DEGs in Aged versus Young ovaries (120 increased and 161 decreased; p<0.0001). Unsupervised hierarchical clustering of the 281 DEGs cleanly separated the ovaries according to female age. Enriched pathway analysis revealed signaling pathways including Citrulline-Nitric Oxide Cycle, VEGF Family Ligand Receptor Interactions, and HIF1α signaling. Nitric oxide is a common signaling molecule in these pathways, and has been shown to maintain diplotene arrest in pre-ovulatory oocytes. Aged ovaries displayed a significant decrease in nitric oxide gene expression (p<0.0001) that could lead to premature meiotic resumption. Interestingly, a significant increase in the proportion of immature oocytes was also observed with natural aging (mean: Aged = 79.1%, Young = 24.0%; p<0.0001). Oocyte specific upstream transcription factors, NOBOX and FIGLA, were identified as significantly inhibited in Aged ovaries (p<0.0001). Vital oocyte genes targeted by these regulators include Gdf9 (folliculogenesis), H1foo (germinal vesicle maturation), Rfpl4 (oogenesis) and Zp3 (zona pellucida glycoprotein), all significantly decreased in Aged ovaries (P<0.0001). Additionally, FIGLA plays a role promoting ubiquitin-mediated proteolysis via genes Fbxw21 and Nlrp4f, both significantly decreased in Aged ovaries (p<0.0001). Inhibition of protein degradation results in cellular stress identified by increased intensity of staining and nuclear localization of ER stress indicators, ATF6 (p<0.01) and pIRE1 (p<0.05), in Aged oocytes. This study revealed at a molecular level the widespread, damaging impacts of natural aging on ovarian function, including significant transcriptomic and protein expression changes that directly contribute to the decline in oocyte quality and overall fecundity. Ongoing studies are focused on manipulating identified genomic targets with the potential to slow down ovarian aging.