Exosomal regulation is intimately involved in key cellular processes, such as migration, proliferation, and adhesion. By participating in the regulation of basic mechanisms, extracellular vesicles are important in intercellular signaling and the functioning of the mammalian reproductive system. The complexity of intercellular interactions in the ovarian follicle is also based on multilevel intercellular signaling, including the mechanisms involving cadherins, integrins, and the extracellular matrix. The processes in the ovary leading to the formation of a fertilization-ready oocyte are extremely complex at the molecular level and depend on the oocyte's ongoing relationship with granulosa cells. An analysis of gene expression from material obtained from a primary in vitro culture of porcine granulosa cells was employed using microarray technology. Genes with the highest expression (LIPG, HSD3B1, CLIP4, LOX, ANKRD1, FMOD, SHAS2, TAGLN, ITGA8, MXRA5, and NEXN) and the lowest expression levels (DAPL1, HSD17B1, SNX31, FST, NEBL, CXCL10, RGS2, MAL2, IHH, and TRIB2) were selected for further analysis. The gene expression results obtained from the microarrays were validated using quantitative RT-qPCR. Exosomes may play important roles regarding intercellular signaling between granulosa cells. Therefore, exosomes may have significant applications in regenerative medicine, targeted therapy, and assisted reproduction technologies.

• Keywords
• cell adhesion, cell migration and proliferation, cellular signaling, extracellular matrix, extracellular vesicles, porcine granulosa cells, transcriptomics,
• MeSH
• Granulosa Cells * metabolism   MeSH
• Oocytes metabolism   MeSH
• Ovarian Follicle * metabolism   MeSH
• Ovary metabolism   MeSH
• Swine MeSH
• Cell Proliferation genetics   MeSH
• Mammals MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Stem cell therapies offer a great promise for regenerative and reconstructive medicine, due to their self-renewal and differentiation capacity. Although embryonic stem cells are pluripotent, their utilization involves embryo destruction and is ethically controversial. Therefore, adult tissues that have emerged as an alternative source of stem cells and perinatal tissues, such as the umbilical cord, appear to be particularly attractive. Wharton's jelly, a gelatinous connective tissue contained in the umbilical cord, is abundant in mesenchymal stem cells (MSCs) that express CD105, CD73, CD90, Oct-4, Sox-2, and Nanog among others, and have the ability to differentiate into osteogenic, adipogenic, chondrogenic, and other lineages. Moreover, Wharton's jelly-derived MSCs (WJ-MSCs) do not express MHC-II and exhibit immunomodulatory properties, which makes them a good alternative for allogeneic and xenogeneic transplantations in cellular therapies. Therefore, umbilical cord, especially Wharton's jelly, is a promising source of mesenchymal stem cells.

• Keywords
• Wharton’s jelly, stem cells, umbilical cord,
• Publication type
• Journal Article MeSH
• Review 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.

• Keywords
• cell division, human, in vitro, ovarian granulosa,
• Publication type
• Journal Article 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
• Cell Differentiation * MeSH
• Antigens, Differentiation biosynthesis   MeSH
• Adult MeSH
• Granulosa Cells metabolism   pathology   MeSH
• Humans MeSH
• Adolescent MeSH
• Osteoblasts metabolism   pathology   MeSH
• Gene Expression Regulation * MeSH
• Oligonucleotide Array Sequence Analysis * MeSH
• Gene Expression Profiling * MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Differentiation MeSH

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.

• Keywords
• cells morphogenesis, granulosa, pig,
• MeSH
• Cell Differentiation genetics   physiology   MeSH
• Granulosa Cells cytology   metabolism   MeSH
• Morphogenesis genetics   physiology   MeSH
• Ovarian Follicle metabolism   MeSH
• Ovary metabolism   MeSH
• Swine MeSH
• Transcriptome genetics   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH