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.

• Klíčová slova
• Granulosa cells, Microarray, Ovarian follicle, Pig, Primary culture,
• MeSH
• buněčný cyklus genetika   MeSH
• folikulární buňky cytologie   MeSH
• kultivované buňky MeSH
• ovariální folikul cytologie   MeSH
• prasata MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• cell division, human, in vitro, ovarian granulosa,
• Publikační typ
• časopisecké články MeSH

Under physiological conditions, human ovarian granulosa cells (GCs), are responsible for a number of processes associated with folliculogenesis and oogenesis. The primary functions of GCs in the individual phases of follicle growth are: Hormone production in response to follicle stimulating hormone (FSH), induction of ovarian follicle atresia through specific molecular markers and production of nexus cellular connections for communication with the oocyte. In recent years, interest in obtaining stem cells from particular tissues, including the ovary, has increased. Special attention has been paid to the novel properties of GCs during long‑term in vitro culture. It has been demonstrated that the usually recycled material in the form of follicular fluid can be a source of cells with stem‑like properties. The study group consisted of patients enrolled in the in vitro fertilization procedure. Total RNA was isolated from GCs at 4 time points (after 1, 7, 15 and 30 days of culture) and was used for microarray expression analysis (Affymetrix® Human HgU 219 Array). The expression of 22,480 transcripts was examined. The selection of significantly altered genes was based on a P‑value <0.05 and expression higher than two‑fold. The leucine rich repeat containing 17, collagen type I α1 chain, bone morphogenetic protein 4, twist family bHLH transcription factor 1, insulin like growth factor binding protein 5, GLI family zinc finger 2 and collagen triple helix repeat containing genes exhibited the highest changes in expression. Reverse‑transcription‑quantitative PCR was performed to validate the results obtained in the analysis of expression microarrays. The direction of expression changes was validated in the majority of cases. The presented results indicated that GCs have the potential of cells that can differentiate towards osteoblasts in long‑term in vitro culture conditions. Increased expression of genes associated with the osteogenesis process suggests a potential for uninduced change of GC properties towards the osteoblast phenotype. The present study, therefore, suggests that GCs may become an excellent starting material in obtaining stable osteoblast cultures. GCs differentiated towards osteoblasts may be used in regenerative and reconstructive medicine in the future.

• MeSH
• buněčná diferenciace * MeSH
• diferenciační antigeny biosyntéza   MeSH
• dospělí MeSH
• folikulární buňky metabolismus   patologie   MeSH
• lidé MeSH
• mladiství MeSH
• osteoblasty metabolismus   patologie   MeSH
• regulace genové exprese * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů * MeSH
• stanovení celkové genové exprese * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• diferenciační antigeny MeSH

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.

• Klíčová slova
• Differentiation, Granulosa cells, Microarrays, Proliferation, Stem cells,
• MeSH
• adhezní spoje metabolismus   MeSH
• buněčná adheze genetika   MeSH
• buněčná diferenciace genetika   MeSH
• dospělí MeSH
• folikulární buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• ovarium cytologie   MeSH
• proliferace buněk genetika   MeSH
• upregulace * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH