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The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

The oogenesis and folliculogenesis are closely linked and occur simultaneously in the growing ovarian follicles. Biochemical and morphological changes in oocytes (OC) and surrounding granulosa cells (GCs) are highly complex and depend on many factors, including intercellular communication. GCs are cells with many functions, often crucial for the proper viability of the oocyte and subsequent positive fertilization. The purpose of this study was to analyze gene expression in porcine GCs, to define differentially expressed genes belongs to the "cell growth", "aging", "positive regulation of cell death", "apoptotic process", "regulation of cell death", "cell death" and "negative regulation of cell death" ontology groups during the short - term primary in vitro culture. Microarrays were employed to study the transcriptome contained in the total RNA of the cultured GCs. Ovaries were obtained after slaughter, from 40 gilts of swine aged 170 days. The cells were obtained through puncture of the ovaries, collection of follicular fluid, removal of the cumulus - oocyte complexes and centrifugation. The cells were then cultured in vitro. The RNA material was obtained before the culture was established (0h) and then after 48h, 96h and 144h of its course. From 182 differently expressed genes belonging to the these ontology groups, we have selected POSTN, FN1, FMOD, ITGB3, DCN, SERPINB2, SFRP2, IGFBP5, EMP1, and CCL2 which were upregulated, as well as DAPL1, ESR1, IHH, TGFBR3, PPARD, PDK4, TXNIP, IFIT3, CSRNP3, and TNFSF10 genes whose expression was downregulated during the time of in vitro culture of the GCs. The significance of the differential gene expression is to provide new information on the molecular aspects of in vitro granulosa cell culture., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte-granulosa cells-are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h-before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: "Cell morphogenesis", "cell differentiation" and "cell development" were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1 , CXCL10 , NEBL , IHH , TGFBR3 , SCUBE1 , DAB1 , ITM2A , MCOLN3 , IGF1 which are most downregulated and PDPN , CAV1 , TMOD1 , TAGLN , IGFBP5 , ITGB3 , LAMB1 , FN1 , ITGA2 , POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.