PSVII-21 Effect of urea on gene expression and viability in bovine granulosa cells cultured in vitro

Abstract Feeding dairy cows with imbalanced, high protein diets can increase levels of urea in serum and ovarian follicular fluid. However, whether and precisely how this compound influences the reproductive physiology of the animals has not yet been determined. The main objective of this study was to evaluate the effect of urea on the viability and function of cultured bovine granulosa cells (bGCs). The bGCs, collected by follicular aspiration, were disrupted until single-cell suspensions were achieved and then cultured in vitro with different urea concentrations (0, 2.5, 5, 8 mM). After 24 hours in culture, bGC growth and viability were evaluated, and frozen for gene expression analysis. Freshly isolated cells (without culture) were collected as a reference. The BioMark™ qPCR platform (Fluidigm Inc.) for gene expression analysis was used to compare the expression levels of 42 genes in non-cultured, and cultured/treated bGCs. The genes evaluated were from the following functional groups: GC-specific, steroid hormone biosynthesis, urea cycle, solute channels/transporters, immediate/early stress response, and ‘housekeeping’. Microscopic evaluation of cultured bGCs showed that urea had a substantial effect on cell morphology. Cell viability was not different between 0 mM (control) and 2.5 mM urea-treated cells (P>0.1), or between 5 mM and 8 mM urea-treated cells (P>0.1). Surprisingly, the 5 mM and 8 mM urea-treated cells showed higher viability than the cells in the control and 2.5 mM groups. Only one gene showed significant differences between control and urea-treated cells (MAPK14; P=0.033). However, 10 genes (AMH, AQP7, CYP19A1, FSHR, GJA1, INHBA, PTGS2, PTX3, TAF11, VNN2) were differentially expressed (P