(342) High Concentrations of Intrafollicular Androstenedione in the Dominant Follicle Upregulates Steroidogenesis, Cell Cycle Arrest, and Fibrosis in Bovine Theca Cells

Abstract Authors: Rachel R. Reith^1,3, Kerri Bochantin-Winders¹, Sarah M. Romereim¹, Scott G. Kurz¹, Renee M. McFee², John S. Davis³, Jennifer R. Wood¹, Andrea S. Cupp¹

1. Department of Animal Science, University of Nebraska-Lincoln, Lincoln, Nebraska, USA


2. School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA


3. Olson Center for Women’s Health, University of Nebraska Medical Center, Omaha, Nebraska,USA

Abstract Text:

Hyperandrogenism, due to excess ovarian androgen synthesis, is observed in women with polycystic ovary syndrome (PCOS) and causes anovulation in women and decreased fertility. A population of beef cows within the University of Nebraska – Lincoln research herd have been identified with naturally occurring elevated intrafollicular concentrations of androstenedione (A4; High A4), anovulation, reduced circulating estrogen and characteristics similar to PCOS patients. Microarray analysis of granulosa cells observed upregulated CYP17A1 expression and predicted cell cycle arrest in granulosa cells of High A4 cows compared to controls. The objective of this study was to identify changes in the thecal cell transcriptome due to high levels of follicular fluid A4 and predict biological functions due to the altered gene expression profile. Based on prior studies, it was hypothesized that high follicular fluid A4 would alter genes associated with inhibition of the cell cycle and proliferation in theca cells, but increase genes associated with steroidogenesis. Cows were classified as Control (n=6) or High A4 (n=6) based on aspirated follicular fluid A4 concentrations following a synchronization protocol. Cows were ovariectomized 36 hours after prostaglandin F2alpha (PG) given during a modified co-synch protocol, and the dominant follicle was aspirated after the ovary was retrieved. Theca cells were isolated from the follicle, RNA was extracted and used on the Affymetrix Bovine GeneChip 1.0 ST Array to measure gene expression. The microarray data was analyzed in R by normalizing microarray probe intensities and identifying outliers before performing differential expression analysis comparing High A4 (n=4) to Control (n=4) cows setting a false discovery rate of 0.05. Differentially expressed genes (DEG; P-adjusted < 0.05) were categorized by function, then used for Ingenuity Pathway Analysis. There were 1117 DEG, 82.3% of which were upregulated. The top categories of upregulated DEG were olfaction, signal transduction, and ion/molecule transport. Upregulated steroidogenesis genes included CYP11A1, CYP17A1, CGA, AKR1C3, and LHCGR. The top categories of downregulated DEG were mitosis/cell cycle transcription regulation, and metabolism. There were 50 significant pathways (Z-score ≥ |2| and P-value < 0.05); 46 were upregulated. Upregulated pathways included collagen and extracellular matrix pathways, olfactory receptors, and G-coupled protein receptors. Downregulated pathways were mitotic prometaphase and cell cycle checkpoints. Taken together these results indicate increased mRNA abundance of genes involved in steroidogenesis in High A4 theca cells which could explain the increased A4 and altered hormonal profiles. The predicted cell cycle arrest in theca cells, and previously in granulosa cells, explain in part the anovulation and reduction in calving rates in High A4 cows. The upregulated collagen genes and pathways appear to result in ovarian fibrosis previously observed in High A4 cows, also a common symptom of PCOS. This fibrosis may also be a contributor to follicular arrest and anovulation. Altogether this analysis demonstrated how high intrafollicular A4 alters the theca cell transcriptome to increase androgen production, induce cell cycle arrest, and cause fibrosis in ovaries, creating a PCOS-like phenotype in these High A4 cows.