Investigation on the impact of sphingolipid disrupting agents on follicle-stimulating hormone receptor function

With over 121 million unintended pregnancies occurring each year worldwide, the study of sexual reproduction hormones is necessary to provide better contraceptive measures. A hormone responsible for sexual reproduction in males and females is follicle-stimulating hormone (FSH). When FSH binds to its receptor (FSHR), signaling cascades are activated, resulting in spermatogenesis in males and oogenesis in females. FSHR resides in microdomains of the cell membrane called lipid rafts, characterized by their high concentrations of cholesterol and sphingolipids. Lipid rafts have been shown to play a role in controlling and regulating the signaling of proteins that reside in them, such as FSHR. Recent research suggests that sphingosine-1-phosphate mediates FSH-induced cell viability but not steroidogenesis. Due to this, we hypothesized that disruption of the sphingolipids would lead to alteration in FSHR signaling, connecting the role of sphingosine-1-phosphate to lipid raft structure. HEK293 cells stably expressing FSHR were treated with myriocin (Myr) or Fumonisin B1 (FB1) to disrupt sphingolipid synthesis. Cells were put through a 30-minute time course of FSH treatment and FSHR signaling was evaluated through western blots monitoring the activation of the p44/42 MAPK pathway. Compared to the control, it was seen that FB1 decreased activation of the p44/42 MAPK pathway due to a reduction in band intensity, while Myr increased activation due to an increase in band intensity. Cyclic adenosine monophosphate (cAMP) levels were observed through the use of a luciferase assay, where it was seen that both FB1 and Myr led to a decrease in cAMP levels compared to the control. Collectively, these results demonstrate that FSHR signaling is dependent on sphingolipids, most likely through their role in lipid raft structure and function. Results from this research provide a better understanding of sexual reproductive pathways, hopefully assisting in the creation of more effective and readily available contraceptives.