Genes involved in hormone metabolism and cellular response in human ovarian granulosa cells.

Endocrinal interactions are one of the most crucial regulatory mechanisms that maintain the state of homeostasis in humans. Processes such as oogenesis, folliculogenesis, menstruation and pregnancy remain under hormonal control. A key role in folliculogenesis is played by granulosa cells. Moreover, granulosa cells take part in corpus luteum formation after ovulation. Because of that, it is important to understand the ways in which the granulosa cells, associated with those processes, respond to hormonal stimulus. In the present study, a transcriptomic analysis of human granulosa cells (GCs) was carried out with the use of expression microarrays. The results were validated by RT-qPCR. The total RNA was isolated after 1st, 7th, 15th and 30th days of long-term primary cultures. The main focus of this work was placed on the genes belonging to "Response to estradiol", "Response to follicle-stimulating-hormone", "Cellular response to hormone stimulus", "Cellular hormone metabolic process" and "Hormone biosynthetic process" gene ontology groups. These groups of genes have been associated with GC hormone metabolism and cellular response to hormones. Eighty genes belonging to these groups were identified. Those that were members of more than one of the analyzed gene ontology groups, or exhibited unique expression patterns, were selected for further analysis. All of the selected genes were described, with their expression patterns detailed. In this manuscript, two gene expression patterns have been described. The first one showed large downregulation of genes in the later stages of culture, with the second one presenting upregulation of expression after day 1 of IVC. The present research was focused on six genes found to be the most important for steroidogenesis: STAR, POR, CYP11A1, ADM, GCLC, IL1B, as well as three genes of higher expression at the later stages of long-term in vitro culture: NR2F2, BMP4, COL1A1. The main goal of the presented study was to select genes involved in response to hormonal stimulus and hormone metabolism in GC long-term in vitro culture.

Human Wharton's jelly mesenchymal stem cells: properties, isolation and clinical applications.

Human Wharton's jelly mesenchymal stem cells (WJ-MSCs) exhibit CD29, CD79 and CD105 markers, characteristic for mesenchymal cell lines. Under the influence of the appropriate factors, WJ-MSCs can be dedifferentiated to osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes, glial cells and dopaminergic neurons. Wharton's jelly (WJ) is one of the potential sources of mesenchymal stem cells (MSCs) - obtaining these cells does not raise moral or ethical objections, because the umbilical cord (UC) is a regular waste material. The expression of the OCT-4 and Nanog proteins, which are characteristic for WJ-MSCs may indicate that these cells have retained some embryonic character. The collected data suggests that WJMSCs show increased division and telomerase activity compared to bone marrow MSCs (BM-MSCs). The published results showed no human leucocyte antigen (HLA) class II expression, with the possibility of HLA class I modification by WJ-MSCs, allowing for the transplantation of these cells both within the same and other species - which allows the use of human cells in animal models. The results of selected studies indicate that WJ-MSCs can be an essential element of regenerative medicine of the 21st century.

Transcriptomic profile of cell cycle progression genes in human ovarian granulosa cells.

The ovarian granulosa cells (GCs) that form the structure of follicle undergo substantial modification during the various stages of human folliculogenesis. These modifications include morphological changes, accompanied by differential expression of genes, encoding proteins which are mainly involved in cell growth, proliferation and differentiation. Recent data bring a new insight into the aspects of GCs' stem-like specificity and plasticity, enabling their prolonged proliferation and differentiation into other cell types. This manuscript focuses attention on emerging alterations during GC cell cycle - a series of biochemical and biophysical changes within the cell. Human GCs were collected from follicles of women set to undergo intracytoplasmic sperm injection procedure, as a part of remnant follicular fluid. The cells were primarily cultured for 30 days. Throughout this time, we observed the prominent change in cell morphology from epithelial-like to fibroblast-like, suggesting differentiation to other cell types. Additionally, at days 1, 7, 15 and 30, the RNA was isolated for molecular assays. Using Affymetrix® Human Genome U219 Array, we found 2579 human transcripts that were differentially expressed in GCs. From these genes, we extracted 582 Gene Ontology Biological Process (GO BP) Terms and 45 KEGG pathways, among which we investigated transcripts belonging to four GO BPs associated with cell proliferation: "cell cycle phase transition", "G1/S phase transition", G2/M phase transition" and "cell cycle checkpoint". Microarray results were validated by RT-qPCR. Increased expression of all the genes studied indicated that increase in GC proliferation during long-term in vitro culture is orchestrated by the up-regulation of genes related to cell cycle control. Furthermore, observed changes in cell morphology may be regulated by a presented set of genes, leading to the induction of pathways specific for stemness plasticity and transdifferentiation in vitro.

Balance between epithelial and stromal marker expression and distribution in primary culture model of porcine endometrium during real-time cell proliferation..

The similarity between humans and pigs, when it comes to tissue morphology, makes Sus scrofa not only a good research model, but also a potential source of cells for tissue engineering. Cell samples obtained from the pig donor, could be influenced in vitro, in order to become a source of tissue material for xenotransplantation, reconstructive and regenerative medicine. Significant amounts of data point to especially major similarities in pig and human reproductive systems. Because of that, particular scientific focus is centered on research concerning porcine COCs, theca and granulosa cells in primary cultures. One of the aspects of the reproductive process, that is still largely undiscovered, is the interaction between preimplantation blastocyst and maternal uterine tissues. In this study, we used molecular analysis techniques, such as RT-qPCR and immunocytochemistry, to analyze the expression and distribution of cytokeratin 18 and panCytokeratins 8, 18 and 19 and vimentin in porcine luminal endometrial epithelial cells, coupled with analysis of their behavior in RTCA. The results have confirmed the presence of epithelial, as well as stromal cell markers in the cells, varying in levels at different stages of culture. They have also given insight into the modes of proliferation and differentiation of studied cells in in vitro culture, as well as providing additional proof for the possible mesenchymal transdifferentiation of epithelial cells.

Enzyme linked receptor protein signaling pathway is one of the ontology groups that are highly up-regulated in porcine oocytes before in vitro maturation.

Before being able to fully participate in the processes associated with its function as a female gamete, the oocyte needs to undergo a range of changes to achieve its mature form. These morphological, biochemical and metabolomic processes are induced by the somatic tissues surrounding the oocyte, through the expression of specific transcription and growth factors. The maturation of the oocyte is highly important for the proceedings that lead to successful fertilization, early embryonic development and implantation. Domestic pigs were used as models for our study, with the cumulus-oocyte complexes obtained from the ovaries that were recovered at slaughter. After shedding of the cumulus, oocytes were assessed with BCB test, with the viable ones chosen to undergo in vitro maturation. With the use of expression microarrays, we analyzed gene expression before and after IVM and detected major changes in both genes that were proven to be associated with oocyte maturation before (FOS, VEGFA, CHRDL1, TGFBR3, FST, INSR, ID1, TXNIP, SMAD4, MAP3K1, EIF2AK3 and KIT) and genes not previously linked with reproduction associated processes (MYO1E, PHIP, KLF10 and SHOC2). All the genes were briefly described, with consideration of possible involvement of the newly discovered elements of the transcriptome in the process of oocyte maturation.