Abstract
Granulosa cells (GCs) are a population of somatic cells whose role after ovulation is progesterone production. GCs were collected from patients undergoing controlled ovarian stimulation during an in vitro fertilization procedure, and they were maintained for 1, 7, 15, and 30 days of in vitro primary culture before collection for further gene expression analysis. A study of genes involved in the biological processes of interest was carried out using expression microarrays. To validate the obtained results, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) was performed. The direction of changes in the expression of the selected genes was confirmed in most of the examples. Six ontological groups (“cell cycle arrest”, “cell cycle process”, “mitotic spindle organization”, “mitotic spindle assembly checkpoint”, “mitotic spindle assembly”, and “mitotic spindle checkpoint”) were analyzed in this study. The results of the microarrays obtained by us allowed us to identify two groups of genes whose expressions were the most upregulated (FAM64A, ANLN, TOP2A, CTGF, CEP55, BIRC5, PRC1, DLGAP5, GAS6, and NDRG1) and the most downregulated (EREG, PID1, INHA, RHOU, CXCL8, SEPT6, EPGN, RDX, WNT5A, and EZH2) during the culture. The cellular ultrastructure showed the presence of structures characteristic of mitotic cell division: a centrosome surrounded by a pericentric matrix, a microtubule system, and a mitotic spindle connected to chromosomes. The main goal of the study was to identify the genes involved in mitotic division and to identify the cellular ultrastructure of GCs in a long-term in vitro culture. All of the genes in these groups were subjected to downstream analysis, and their function and relation to the ovarian environment are discussed. The obtained results suggest that long-term in vitro cultivation of GCs may lead to their differentiation toward another cell type, including cells with cancer-like characteristics.
Highlights
In the last phase of preovulatory follicle growth, mature oocytes are accompanied by somatic cells known as granulosa cells (GCs), with facilitation of the final steps of estrogen synthesis being their main function
Affymetrix microarrays were used to determine the changes in expression of the GC transcriptome between the 1st, 7th, 15th, and 30th day of in vitro culture
In many of the studies conducted on GCs, it has been suggested that the secretory properties of GCs in primary culture can be used in a coculture with embryos [32]
Summary
In the last phase of preovulatory follicle growth, mature oocytes are accompanied by somatic cells known as granulosa cells (GCs), with facilitation of the final steps of estrogen synthesis being their main function. GCs are an important factor in the comprehension of the folliculogenesis process [2,3]. Understanding their transcriptome may be an important step in expanding knowledge about their possible application to medicine of the 21st century. This includes the significant stem-like potential of in vitro cultured GCs (described by Kossowska-Tomaszczuk et al.), which could be employed in the fields of regenerative and reconstructive medicine [4,5,6,7]. An analysis of GC gene expression in long-term in vitro cultures could help to define new therapeutic goals in the rapidly expanding field of infertility treatment [8]
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