(327) Spatial Regulation of the Follicle Stimulating Hormone Receptor coordinates FSH Signaling in Human Granulosa Cells 

Mahsa Rasekhi^1, Susmita Barman¹, John S. Davis^1,2 

1 – Olson Center for Women’s Health, Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE, USA

2 – Department of Veterans Affairs Nebraska-Western Iowa Healthcare System, Omaha, NE, USA  

Abstract Text:

In vitro fertilization (IVF) success rates for women over 35 ages have remained unchanged despite significant technological advancement. This observation underscores the need for innovative therapeutic strategies targeting follicle-stimulating hormone (FSH) and its G-protein-coupled receptor (GPCR) to enhance IVF outcomes. A deeper understanding of FSH/FSHR-mediated signaling mechanisms is essential for advancing assisted reproductive technologies. Over the past two decades, our understanding of G-protein-coupled receptor signaling has evolved significantly. It is now recognized that GPCR signaling is not limited to the plasma membrane but also occurs within specific intracellular compartments, hence highlighting its critical role in spatial organization. FSHR exerts its effects mainly through the Gas/cAMP/PKA signaling cascade, but it can also engage multiple pathways through diverse mechanism/s. One such mechanism is the internalization and subsequent trafficking to endosomal compartments, which in turn stimulate alternate signaling networks. To investigate the trafficking fate following internalization in FSHR signaling in human granulosa cells, we hypothesized that Dyngo-4a, an inhibitor of dynamin-dependent endocytosis, and Pitstop-2, a clathrin-mediated endocytosis inhibitor, modulate FSH-induced FSHR signal transduction in human granulosa cells (hGCs), altering downstream pathway activation through their effects on endocytosis of the FSHR. In this study, we utilized primary human granulosa cells alongside a human granulosa cell line, hGRC1cells, that stably overexpresses FSHR to study receptor signaling and trafficking dynamics. Using high-resolution confocal microscopy, we observed that upon FSH stimulation, the FSHR undergoes rapid (< 5 minutes) internalization into very early endosomes (VEEs). As internalization progresses, the FSHR colocalizes to early endosomes marked by Early Endosome Antigen 1 (EEA1) within 5 to 10 minutes, indicating receptor trafficking through the endosomal pathway. These VEEs are enriched with Adaptor Protein Phosphotyrosine Interacting with Pleckstrin Homology Domain and Leucine Zipper 1 (APPL1), a key regulator of receptor recycling. Analysis of our images revealed a significant decrease in FSHR colocalization with APPL1-positive endosomes after 10 minutes, coinciding with its accumulation in EEA1-positive compartments. After 15 minutes, the FSHR relocates to different compartments, which will be examined in our further research to elucidate the trafficking fate of FSHR after stimulation.

To assess the impact of FSH-mediated FSHR internalization, hGCs were pre-treated with either Dyngo-4a or Pitstop2 (30 µM) for 30 to 60 minutes and then stimulated with FSH (1-100 ng/ml) for up to 24 hours. Forskolin and 8-bromo-cAMP were employed as FSHR-independent controls. The effects of Dyngo-4a and Pitstop-2 were assessed by measurements of cAMP and progesterone, as well as by western blotting and immunocytochemistry. Pre-treatment with either Dyngo-4a or Pitstop-2 for 60 minutes reduced FSH-stimulated cAMP accumulation (P < 0.05) and the phosphorylation of CREB, ERK, and Akt (P < 0.05). Furthermore, FSH-induced progesterone synthesis was significantly reduced (P< 0.05) in hGCs pretreated with Dyngo-4a. These results suggest that FSH mediated endocytosis of FSHR is essential for efficient cAMP production and granulosa cell function. This research illustrates the dynamic nature of FSHR trafficking, whereby FSH mediated FSHR endocytosis is crucial for effective cAMP generation and subsequent signaling cascades in granulosa cells. This novel study sheds light on the intricate mechanisms governing the spatial regulation of FSHR signaling and its influence on granulosa cell function, presenting promising opportunities to enhance IVF success rates for older women.