During oocyte maturation, granulosa cells (GCs) respond to fluctuating hormone levels in the ovary. The study aims to reveal metabolism and activity patterns of isolated and cultured GCs, reflecting in vivo processes. A downregulation of GARNL3 and ARRDC4 across all time points (48 h, 96 h, and 144 h) suggests reduced cell signaling and response to external stimuli, which may be related to the isolation and in vitro culturing of GCs from the complex ovarian microenvironment. The consistent elevation of LOX underscores its role in extracellular matrix (ECM) cross-linking, crucial for oocyte quality, whereas FN1 and ITGB3 highlight cellular adhesion and ECM interaction during adaptation to in vitro conditions. The study further demonstrates that ANKRD1 and SLC1A1 are upregulated over time in vitro, indicating cellular differentiation and metabolic alterations. Furthermore, proteoglycan and MAPK signaling pathways are identified as key players in cell-to-cell and cell-to-ECM interactions. GSEA revealed heightened activity in vasculature development, the TGF-β signaling pathway, cell development, and lipid response. The findings suggest that while GCs in vitro mimic in vivo processes related to ECM remodeling and oocyte development, they also exhibit a tendency towards aging. The research emphasizes that isolated GCs in vitro exhibit time-dependent activity shifts related to cellular differentiation, ECM remodeling, and lipid metabolism, which also have implications for the understanding of reproductive physiology and pathologies.

Center for Gynecology Obstetrics and Infertility Treatment Pastelova Pastelowa 8 60 198 Poznan Poland

Collegium Medicum University of Zielona Gora 65 046 Zielona Gora Poland

Department of Anatomy and Histology Collegium Medicum Institute of Health Sciences 65 046 Zielona Gora Poland

Department of Anatomy Poznan University of Medical Sciences 60 781 Poznan Poland

Department of Basic and Preclinical Sciences Institute of Veterinary Medicine Nicolaus Copernicus University in Torun 87 100 Torun Poland

Department of Diagnostics and Clinical Sciences Institute of Veterinary Medicine Nicolaus Copernicus University in Torun 87 100 Torun Poland

Department of Histology and Embryology Poznan University of Medical Sciences 60 812 Poznan Poland

Department of Medical Biochemistry Faculty of Medicine Wroclaw Medical University 50 368 Wroclaw Poland

Department of Obstetrics and Gynecology University Hospital and Masaryk University 602 00 Brno Czech Republic

Department of Toxicology Poznan University of Medical Sciences 60 812 Poznan Poland

Department of Veterinary Surgery Institute of Veterinary Medicine Nicolaus Copernicus University in Torun 87 100 Torun Poland

Division of Anatomy Department of Human Morphology and Embryology Faculty of Medicine Wroclaw Medical University 50 368 Wroclaw Poland

Division of Philosophy of Medicine and Bioethics Department of Social Sciences and the Humanities Poznan University of Medical Sciences 60 812 Poznan Poland

Physiology Graduate Faculty North Carolina State University Raleigh NC 27695 USA

Prestage Department of Poultry Science North Carolina State University Raleigh NC 27695 USA

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