Secretory Profile Analysis of Human Granulosa Cell Line Following Gonadotropin Stimulation

Granulosa cell (GC) differentiation, stimulated by FSH and LH, drives oocyte maturation and follicle development. FSH promotes GC proliferation, and LH triggers ovulation. In clinical practice, hCG is used to mimic LH. Despite various controlled ovarian stimulation (COS) protocols employing exogenous gonadotropins and GnRH analogs to prevent premature ovulation, their effectiveness and safety remain debated. To identify markers predicting a positive treatment response, the secretome of gonadotropin-stimulated GC using the human granulosa-like tumor cell line (KGN) via proteomics was analyzed. Additionally, a novel 2D-FFT quantitative method was employed to assess cytoskeleton fiber aggregation and polymerization, which are critical processes for GC differentiation. Furthermore, the activation of key kinases, focal adhesion kinase (FAK), and Rho-associated coiled-coil-containing protein kinase 1 (ROCK-1), which are implicated in cytoskeleton dynamics and hormone signaling, was evaluated. The proteomic analysis revealed significant modulation of proteins involved in extracellular matrix organization, steroidogenesis, and cytoskeleton remodeling. Notably, the combined FSH/hCG treatment led to a dynamic upregulation of the semaphorin pathway, specifically semaphorin 7A. Finally, a significant reorganization of the cytoskeleton network and signaling was detected. These findings enhance our understanding of folliculogenesis and suggest potential novel molecular markers for predicting patient responses to gonadotropin stimulation.