(318) High Fat Diet is Associated with Significant Gene Expression Changes in Oocytes Lacking the Toll-Like Receptor Signaling Adaptor MyD88

Abstract Authors: Keshari M. Thakali^1,2; Antonia Johnson¹; Suhana Mushtaq¹; Adam Corken^1,2; James D. Sikes¹; Elizabeth C. Wahl¹; Kartik Shankar³; Meghan L. Ruebel⁴

1. Arkansas Children’s Nutrition Center, Little Rock, AR, USA
2. Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA
3. USDA Agricultural Research Service, Responsive Agricultural Food Systems Research Unit, College Station, TX, USA
4. Microbiome and Metabolism Research Unit USDA-ARS, Southeast Area, Arkansas Children’s Nutrition Center, Little Rock, AR, USA

Abstract Text: Maternal obesity is associated with pro-inflammatory signatures in the oocyte, uterus, male blastocysts, and placenta as observed in preclinical models. In both human and rodent models, maternal obesity has been associated with transcriptional changes in ovaries and oocytes involving pro-inflammatory and acute phase response genes, hypothesized to be downstream of the MyD88/Toll-Like Receptor (TLR) signaling pathway. Here we investigated the role of maternal high fat/sucrose diet (HFD) on MyD88/TLR signaling in the mature oocyte. Using Zp3-Cre, we generated oocyte-specific MyD88 knockout mice and fed them a control or HFD (Inotiv TD0.8811, 45% calories from fat) for 6-8 weeks to determine the effect of HFD on oocyte gene expression. Thus, four groups of female mice were generated: control diet or HFD-oocyte MyD88 knockout (MyD88-CKO) and control diet or HFD-wild type (flox). At the end of the diet intervention, mice were super ovulated with PMSG and hCG, and 14-15 hours post hCG, mature (MII) oocytes were collected for RNA sequencing (n=6/group) (Illumina NextSeq 2000). There was no difference in the number of MII oocytes collected between the four groups (avg range 13.9±2.3 to 17.3±1.5 oocytes/mouse, n=10-12/group). In mice fed a control diet, 8 genes were upregulated, and 47 genes were downregulated in MyD88-CKO compared to flox oocytes, and MyD88 was 5-fold downregulated in MyD88-CKO compared to flox. Contrary to our hypothesis, in MyD88-CKO oocytes HFD led to significantly more upregulated genes (1048 vs 25, respectively, ±1.5 fold-change) compared to flox oocytes, and there were no overlapping upregulated genes. In MyD88-CKO compared to flox oocytes, there were fewer down-regulated genes (123 vs 240, respectively, ±1.5 fold-change), and again there were no overlapping genes. Ingenuity pathway analysis revealed significant pathways related to TLR signaling, IL-1 signaling, Death Receptor signaling, TNF signaling, and MAPK signaling, and biological functions related to inflammation and immune response were altered by HFD in MyD88-CKO oocytes. In flox oocytes, significant pathways related to embryonic stem cell pluripotency and gap junction signaling and functions related to cell proliferation, differentiation, and cell survival were altered by HFD. These data suggest that in the oocyte MyD88 may play a key role in regulating TLR effector expression and that in the absence of MyD88, HFD may recruit other TLR effectors to maintain oocyte development, the mechanisms of which remain unclear. These results highlight the importance of maternal dietary pre-conception programming of oocytes with potential implications for programming of offspring health and development. USDA ARS 6026-10700-001-000D