Granulosa cells (GCs) have many functions and are fundamental for both folliculogenesis and oogenesis, releasing hormones and communicating directly with the oocyte. Long-term in vitro cultures of GCs show significant stem-like characteristics. In the current study, RNA of human ovarian granulosa cells was collected at 1, 7, 15 and 30 days of long-term in vitro culture. Understanding the process of differentiation of GCs towards different cell lineages, as well as the molecular pathways underlying these mechanisms, is fundamental to revealing other possible stemness markers of this type of cell. Identifying new markers of GC plasticity may help to understand the aetiology and recurrence of a wide variety of diseases and health conditions and reveal possible clinical applications of the ovarian tissue cells, affecting not only the reproductive ability but also sex hormone production. Granulosa cells were the subject of this study, as they are readily available as remnant material leftover after in vitro fertilisation procedures and exhibit significant stem-like characteristics in culture. The change in gene expression was investigated through a range of molecular and bioinformatic analyses. Expression microarrays were used, allowing the identification of groups of genes typical of specific cellular pathways. This candidate gene study focused on ontological groups associated with muscle cell morphogenesis, structure, development and differentiation, namely, "muscle cell development", "muscle cell differentiation", "muscle contraction", "muscle organ development", "muscle organ morphogenesis", "muscle structure development", "muscle system process" and "muscle tissue development". The results showed that the 10 most upregulated genes were keratin 19, oxytocin receptor, connective tissue growth factor, nexilin, myosin light chain kinase, cysteine and glycine-rich protein 3, caveolin 1, actin, activating transcription factor 3 and tropomyosin, while the 10 most downregulated consisted of epiregulin, prostaglandin-endoperoxide synthase 2, transforming growth factor, interleukin, collagen, 5-hydroxytryptmine, interleukin 4, phosphodiesterase, wingless-type MMTV integration site family and SRY-box 9. Moreover, ultrastructural observations showing heterogeneity of granulosa cell population are presented in the study. At least two morphologically different subpopulations were identified: large, light coloured and small, darker cells. The expression of genes belonging to the mentioned ontological groups suggest the potential ability of GCs to differentiate and proliferate toward muscle lineage, showing possible application in muscle regeneration and the treatment of different diseases.

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

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

Department of Obstetrics and Gynecology University Hospital and Masaryk University 20 Jihlavská St 62500 Brno Czech Republic

Department of Toxicology Poznan University of Medical Sciences 30 Dojazd St 60 631 Poznań Poland

Department of Veterinary Surgery Institute of Veterinary Medicine Nicolaus Copernicus University in Torun 1 Lwowska St 87 100 Toruń 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

Physiology Graduate Program North Carolina State University Raleigh NC 27695 USA

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

The School of Medicine Medical Sciences and Nutrition University of Aberdeen Aberdeen AB25 2ZD UK

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