The ovarian granulosa cells (GCs) that form the structure of follicle undergo substantial modification during the various stages of human folliculogenesis. These modifications include morphological changes, accompanied by differential expression of genes, encoding proteins which are mainly involved in cell growth, proliferation and differentiation. Recent data bring a new insight into the aspects of GCs' stem-like specificity and plasticity, enabling their prolonged proliferation and differentiation into other cell types. This manuscript focuses attention on emerging alterations during GC cell cycle - a series of biochemical and biophysical changes within the cell. Human GCs were collected from follicles of women set to undergo intracytoplasmic sperm injection procedure, as a part of remnant follicular fluid. The cells were primarily cultured for 30 days. Throughout this time, we observed the prominent change in cell morphology from epithelial-like to fibroblast-like, suggesting differentiation to other cell types. Additionally, at days 1, 7, 15 and 30, the RNA was isolated for molecular assays. Using Affymetrix® Human Genome U219 Array, we found 2579 human transcripts that were differentially expressed in GCs. From these genes, we extracted 582 Gene Ontology Biological Process (GO BP) Terms and 45 KEGG pathways, among which we investigated transcripts belonging to four GO BPs associated with cell proliferation: "cell cycle phase transition", "G1/S phase transition", G2/M phase transition" and "cell cycle checkpoint". Microarray results were validated by RT-qPCR. Increased expression of all the genes studied indicated that increase in GC proliferation during long-term in vitro culture is orchestrated by the up-regulation of genes related to cell cycle control. Furthermore, observed changes in cell morphology may be regulated by a presented set of genes, leading to the induction of pathways specific for stemness plasticity and transdifferentiation in vitro.

Department of Anatomy Poznan University of Medical Sciences Poznan Poland

Department of Anatomy Poznan University of Medical Sciences Poznan Poland Department of Histology and Embryology Poznan University of Medical Sciences Poznan Poland Department of Obstetrics and Gynecology University Hospital and Masaryk University Brno Czech Republic

Department of Cancer Immunology Chair of Medical Biotechnology Poznan University of Medical Sciences Poznan Poland

Department of Clinical and Experimental Endocrinology of the Medical University of Gdansk Poland

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

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

Department of Pathophysiology Poznan University of Medical Sciences Poznan Poland

Division of Infertility and Reproductive Endocrinology Department of Gynecology Obstetrics and Gynecological Oncology Poznan University of Medical Sciences Poznan Poland