Effect of β-Hydroxybutyrate on Gene Expression in the Hypothalamus and Pituitary of Sheep

Negative energy balance (NEB) and metabolic dysfunctions have been shown to impair reproduction; however, the mechanisms and molecular pathways that couple energy status to reproductive function are poorly defined. Previous studies have indicated an association between elevated beta-hydroxybutyrate (BHB) and reproduction efficiency. To identify molecular pathways that couple metabolic dysfunctions to reproduction, 10 wethers were randomly assigned to be centrally injected into the lateral ventricle through intracerebroventricular cannulas with 1 mL of β-hydroxybutyric acid sodium salt solution (BHB; 12,800 µmol/L) or saline solution (CON; 0.9% NaCl). Two hours post injection, wethers were humanely euthanized and hypothalamus and pituitary were harvested for transcriptome characterization using RNA-sequencing. Ribonucleic acid was extracted from hypothalamus and pituitary and deeply sequenced (hypothalamus: 468,912,732 reads; pituitary: 515,106,092 reads) using an Illumina Hi-Seq platform and aligned to the Bos taurus and Ovis aries genomes using BLAST. Of the total raw reads, 87% (hypothalamus) and 90.5% (pituitary) mapped to the reference Ovis aries genome. Within these two sets of reads, approximately 56% in hypothalamus and 69% in pituitary mapped to either known or putative genes. Fragments per kilobase of transcripts per million normalized counts were averaged and ranked to identify the transcript expression level. Gene Ontology analysis of these two gene sets (using DAVID Bioinformatics Resources) identified biological process functions related to genes shared between tissues, as well as functional categories with tissue-specific enrichment. Injection of BHB altered (P < 0.05) expression of 11 genes in the pituitary and 44 genes in the hypothalamus. Three genes (zinc finger and BTB domain containing 16, (ZBTB16) FK506 binding protein 5 (FKBP5), and eukaryotic elongation factor 2 kinase (EEF2K)) were influenced (P < 0.05) by BHB-injection in both tissues. Of these 3 genes, FKBP5 is of particular interest as a mediator of fertility during NEB, due to its role in modulating stress at the hypothalamus-pituitary-adrenal axis. Functional enrichment analyses revealed that BHB injection altered expression of genes in pathways related to stimulus perception, inflammation and cell cycle control. The set of genes altered by BHB creates a foundation from which to identify the signaling pathways that suppress fertility during NEB or periods of metabolic dysfunctions.