(159) Progesterone Regulates Lipid Metabolism in Bovine Uterine Epithelium

Progesterone Regulates Lipid Metabolism in Bovine Uterine Epithelium

A. Sharma¹, D. Hernández-Saavedra^2,3, and M. Dean^1,3

1. Department of Animal Sciences, University of Illinois Urban-Champaign, USA

2. Department of Health Kinesiology, University of Illinois Urban-Champaign, USA

3. Division of Nutritional Sciences, University of Illinois Urban-Champaign, USA

 

Abstract Text:

Uterine secretions, called histotroph, contain a mixture of amino acids, carbohydrates, lipids, proteins, and growth factors that are secreted into uterine lumen to support the growth of conceptus during early pregnancy in cows. It is well known that the early pregnant uterus is highly regulated by elevated concentrations of progesterone (P4). Therefore, it is essential to understand how P4 stimulates the synthesis and secretion of each component of histotroph. We previously showed that P4 increased NAPDH production in bovine uterine epithelium. One role of NADPH is to support lipid synthesis. However, little is known about how lipid metabolism is regulated in the uterine epithelium. Thus, we hypothesized that lipid metabolism in the bovine uterine epithelium is regulated by P4. To test this hypothesis, we reanalyzed a previous RNAseq dataset and found that P4 increased expression of genes related to substrates for lipid production, lipogenesis, and lipid trafficking. Next, we treated immortalized bovine uterine epithelial (BUTE) cells with vehicle (control) or P4 (10 µM) for 48 hours. After 48 hours vehicle control (n=5) and P4 treated BUTE cells pellets (n=5), and their corresponding media were collected and subjected to untargeted LC-MS profiling to analyze the global lipid content. Simultaneously, BioTracker 488 Green lipid droplet dye was used to stain the in vitro cultured BUTE cells to observe lipid droplets. Additionally, immunohistochemistry of PPARG (peroxisome proliferator-activated receptor gamma) the key transcription factor that regulates lipid metabolism was performed in the cow uterine epithelial tissue collected on Day 1 or 11 of the estrus cycle. LC-MS profiling identified a total of 1,607 lipids based on ion mobility, m/z ratio and MS/MS spectral matching. The obtained peak height values proportional to lipid amount across control and P4 treated BUTE cells and media samples were statistically analyzed for differentially altered lipids using MetaboAnalyst 6.0 software. In P4 treated BUTE cells, a total of 35 lipids were altered significantly (p< 0.05) of which 10 were high and 17 (p< 0.05, log2FC (≤-1.5 or ≥1.5) were low compared to control BUTE cells. In P4 treated BUTE cell culture media, a total of 61 lipids were significantly altered (p< 0.05) out of which 20 lipids were significantly higher and 4 were significantly lower (p< 0.05, log2FC (≤-1.5 or ≥1.5) than in control BUTE cell media. Lipid ontology (LION) enrichment analysis was performed on lipidomics data to identify the lipid-associated terms. P4 treated BUTE cells were significantly enriched (FDR q value< 0.05) with lipids associated with saturated fatty acids, lipid mediated signaling, lipid storage, and lipid droplet formation. Whereas the P4 conditioned media showed significant enrichment of alkyl diacylglycerols relative to controls (FDR q value< 0.05). Further, staining of in vitro cultured BUTE cells showed increased lipid droplets in P4 treated BUTE cells. In vivo, the immunostaining of PPARG in the uterine epithelium was more intense on day 11 than day 1 of the estrus cycle. Together these results indicate that P4 might be involved in modulating lipid metabolism in uterine epithelium leading to more triglyceride synthesis or storage plausibly via upregulation of PPARG transcription factor. Currently we are investigating the regulatory pathways that might be involved in P4 mediated regulation of lipid metabolism and subsequent lipid secretion, which could be essential for maintaining growth of conceptus. This work was funded by NIH grant R21HD112772 to MD.