The human ovarian granulosa cells (GCs) surround the oocyte and form the proper architecture of the ovarian follicle. The ability of GCs to proliferate and differentiate in the conditions of in vitro culture has been proven. However, there is still a large field for extensive investigation of molecular basics, as well as marker genes, responsible for these processes. This study aimed to find the new marker genes, encoding proteins that regulate human GCs in vitro capability for proliferation and differentiation during long-term primary culture. The human follicular GCs were collected from hyper-stimulated ovarian follicles during IVF procedures and transferred to a long-term in vitro culture. The culture lasted for 30 days, with RNA samples isolated at days 1, 7, 15, 30. Transcriptomic analysis was then performed with the use of Affymetrix microarray. Obtained results were then subjected to bioinformatical evaluation and sorting. After subjecting the datasets to KEGG analysis, three differentially expressed ontology groups "cell differentiation" (GO:0030154), "cell proliferation" (GO:0008283) and "cell-cell junction organization" (GO:0045216) were chosen for further investigation. All three of those ontology groups are involved in human GCs' in vitro lifespan, proliferation potential, and survival capability. Changes in expression of genes of interest belonging to the chosen GOs were validated with the use of RT-qPCR. In this manuscript, we suggest that VCL, PARVA, FZD2, NCS1, and COL5A1 may be recognized as new markers of GC in vitro differentiation, while KAT2B may be a new marker of their proliferation. Additionally, SKI, GLI2, FERMT2, and CDH2 could also be involved in GC in vitro proliferation and differentiation processes. We demonstrated that, in long-term in vitro culture, GCs exhibit markers that suggest their ability to differentiate into different cells types. Therefore, the higher expression profile of these genes may also be associated with the induction of cellular differentiation processes that take place beyond the long-term primary in vitro culture.

Department of Anatomy Poznan University of Medical Sciences 6 Święcickiego St 60 781 Poznań Poland

Department of Anatomy Poznan University of Medical Sciences 6 Święcickiego St 60 781 Poznań Poland Department of Biomaterials and Experimental Dentistry Poznan University of Medical Sciences 70 Bukowska St 60 812 Poznań Poland

Department of Anatomy Poznan University of Medical Sciences 6 Święcickiego St 60 781 Poznań Poland Department of Histology and Embryology Poznan University of Medical Sciences 6 Święcickiego St 60 781 Poznań Poland Department of Obstetrics and Gynecology University Hospital and Masaryk University Jihlavská 20 625 00 Brno Czech Republic

Department of Clinical and Experimental Endocrinology Medical University of Gdańsk 7 Dębinki St 80 211 Gdańsk Poland

Department of Histology and Embryology Poznan University of Medical Sciences 6 Święcickiego St 60 781 Poznań Poland

Department of Obstetrics and Gynecology University Hospital and Masaryk University Jihlavská 20 625 00 Brno Czech Republic

Department of Pathophysiology Poznań University of Medical Sciences 8 Rokietnicka St 60 806 Poznan Poland

Division of Histology and Embryology Department of Human Morphology and Embryology Wroclaw Medical University Wroclaw Poland Division of Anatomy and Histology University of Zielona Góra Zielona Góra Poland

Division of Infertility and Reproductive Endocrinology Department of Gynecology Obstetrics and Gynecological Oncology Poznan University of Medical Sciences 33 Polna St 60 535 Poznań Poland

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