Ovarian Granulosa Cells (GCs) are known to proliferate in the developing follicle and undergo several biochemical processes during folliculogenesis. They represent a multipotent cell population that has been differentiated to neuronal cells, chondrocytes, and osteoblasts in vitro. However, progression and maturation of GCs are accompanied by a reduction in their stemness. In the developing follicle, GCs communicate with the oocyte bidirectionally via gap junctions. Together with neighboring theca cells, they play a crucial role in steroidogenesis, particularly the production of estradiol, as well as progesterone following luteinization. Many signaling pathways are known to be important throughout the follicle development, leading either towards luteinization and release of the oocyte, or follicular atresia and apoptosis. These signaling pathways include cAMP, PI3K, SMAD, Hedgehog (HH), Hippo and Notch, which act together in a complex manner to control the maturation of GCs through regulation of key genes, from the primordial follicle to the luteal phase. Small molecules such as resveratrol, a phytoalexin found in grapes, peanuts and other dietary constituents, may be able to activate/inhibit these signaling pathways and thereby control physiological properties of GCs. This article reviews the current knowledge about granulosa stem cells, the signaling pathways driving their development and maturation, as well as biological activities of resveratrol and its properties as a pro-differentiation agent.
- MeSH
- biologické modely MeSH
- buněčná diferenciace * účinky léků MeSH
- folikulární buňky cytologie účinky léků metabolismus MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie MeSH
- resveratrol chemie farmakologie MeSH
- signální transdukce účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The involvement of the mTOR system/enzyme sirtuin 1 (SIRT1) intracellular signaling system in the control of ovarian functions and its role in mediating hormonal action on the ovary has been proposed, but this hypothesis should be supported by a demonstrated influence of hormones on mTOR/SIRT1. Therefore, the aim of our in vitro experiments was to examine the effect of the known hormonal regulators of ovarian functions, such as follicle-stimulating hormone (FSH), oxytocin (OT) and insulin-like growth factor I (IGF-I), on mTOR/SIRT1. The accumulation of SIRT1 in porcine ovarian granulosa cells cultured with and without these hormones (at doses of 1, 10 or 100 ng.ml-1) was evaluated using immunocytochemistry. It was observed that the addition of FSH (at 10 ng.ml-1 but not at 1 or 100 ng/ml) and OT (at all tested doses) increased the expression of SIRT1 in ovarian cells. In addition, 100 ng.ml-1, but not at 1 or 10 ng.ml-1, of IGF-I decreased SIRT1 accumulation. Our observations are the first demonstration that hormones can directly regulate the ovarian mTOR/SIRT1 system and that this system could mediate the action of hormonal regulators on the ovary.
- MeSH
- apoptóza účinky léků MeSH
- folikulární buňky cytologie účinky léků metabolismus MeSH
- folikuly stimulující hormon farmakologie MeSH
- insulinu podobný růstový faktor I farmakologie MeSH
- kultivované buňky MeSH
- ovarium cytologie účinky léků metabolismus MeSH
- oxytocin farmakologie MeSH
- prasata MeSH
- proliferace buněk účinky léků MeSH
- sirtuin 1 biosyntéza genetika metabolismus MeSH
- uterotonika farmakologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The aim of our study was to examine the direct influence of plant polyphenol resveratrol and oil-related environmental contaminant benzene on ovarian hormone release, as well as the ability of resveratrol to prevent the effect of benzene. Porcine ovarian granulosa cells were cultured with and without resveratrol (0, 1,10 or 100 ug/ml) alone or in combination with 0.1% benzene. The release of progesterone, oxytocin and prostaglandin F was measured by enzyme immunoassay (EIA). Benzene promoted the release of progesterone, oxytocin and prostaglandin F. Resveratrol, when given alone, stimulated both progesterone and prostaglandin F, but not the oxytocin output. Moreover, resveratrol prevented and even inverted the stimulatory action of benzene on all analysed hormones. These observations demonstrate the direct influence of both benzene and resveratrol on porcine ovarian hormone release, as well as the ability of resveratrol to prevent the benzene action on the ovary.
- MeSH
- benzen farmakologie MeSH
- folikulární buňky cytologie účinky léků metabolismus MeSH
- kultivované buňky MeSH
- oxytocin metabolismus MeSH
- prasata MeSH
- progesteron metabolismus MeSH
- prostaglandiny F metabolismus MeSH
- resveratrol farmakologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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 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.
- 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
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.
- MeSH
- buněčný cyklus * MeSH
- folikulární buňky cytologie MeSH
- lidé MeSH
- ovariální folikul cytologie MeSH
- transkriptom * MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články 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.
- MeSH
- buněčná diferenciace genetika fyziologie MeSH
- folikulární buňky cytologie metabolismus MeSH
- morfogeneze genetika fyziologie MeSH
- ovariální folikul metabolismus MeSH
- ovarium metabolismus MeSH
- prasata MeSH
- transkriptom genetika MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Proper course of folliculogenesis and oogenesis have an enormous impact on female fertility. Both processes take place in the ovary and involve not only the maturing germ cell, but also few types of somatic cells that assist the ovarian processes and mediate the dialog with the oocyte. These cells, granulosa and theca, are heavily involved in essential reproductive processes, such as ovulation, fertilization, and embryo implantation. In this study, we have used the expressive microarray approach to analyze the transcriptome of porcine granulosa cells, during short-term in vitro culture. We have further selected differentially expressed gene ontologies, involved in cell proliferation, migration, adhesion, and tissue development, namely, "cell-cell adhesion," "cell motility," "cell proliferation," "tissue development," and "tissue migration" to screen them for the possibility of discovery of new markers of those processes. A total of 303 genes, expression of which varied significantly in different culture periods and belonged to the analyzed ontology groups, were detected, of which 15 that varied the most (between 0 and 48 h of culture) were selected for validation. As the validation confirmed the transcriptomic patterns, 10 genes of biggest changes in expression (CAV1, IGFBP5, ITGB3, FN1, ITGA2, LAMB1, POSTN, FAM83D, KIF14, and CDK1) were analyzed, described, and referred to the context of the study, with the most promising new markers and further proof for the viability of the currently recognized ones detailed. Overall, the study provided valuable insight into the molecular functioning of in vitro granulosa cell cultures.
- MeSH
- biologické markery metabolismus MeSH
- buněčná adheze genetika MeSH
- folikulární buňky cytologie metabolismus fyziologie MeSH
- kultivované buňky MeSH
- pohyb buněk genetika MeSH
- prasata MeSH
- proliferace buněk genetika MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The physiological processes that drive the development of ovarian follicle, as well as the process of oogenesis, are quite well known. Granulosa cells are major players in this occurrence, being the somatic element of the female gamete development. They participate directly in the processes of oogenesis, building the cumulus-oocyte complex surrounding the ovum. In addition to that, they have a further impact on the reproductive processes, being a place of steroid sex hormone synthesis and secretion. It is known that the follicle development creates a major need for angiogenesis and blood vessel development in the ovary. In this study, we use novel molecular approaches to analyze markers of these processes in porcine granulosa cultured primarily in vitro. The cells were recovered from mature sus scrofa specimen after slaughter. They were then subjected to enzymatic digestion and culture primarily for a short term. The RNA was extracted from cultures in specific time periods (0h, 24h, 48h, 96h, and 144h) and analyzed using expression microarrays. The genes that exhibited fold change bigger than |2|, and adjusted p-value lower than 0.05, were considered differentially expressed. From these, we have chosen the members of "angiogenesis," "blood vessel development," "blood vessel morphogenesis," "cardiovascular system development," and "vasculature development" for further selection. CCL2, FGFR2, SFRP2, PDPN, DCN, CAV1, CHI3L1, ITGB3, FN1, and LOX which are upregulated, as well as CXCL10, NEBL, IHH, TGFBR3, SCUBE1, IGF1, EDNRA, RHOB, PPARD, and SLITRK5 genes whose expression is downregulated through the time of culture, were chosen as the potential markers, as their expression varied the most during the time of culture. The fold changes were further validated with RT-qPCR. The genes were described, with special attention to their possible function in GCs during culture. The results broaden the general knowledge about GC's in vitro molecular processes and might serve as a point of reference for further in vivo and clinical studies.
- MeSH
- cévy růst a vývoj metabolismus MeSH
- folikulární buňky cytologie metabolismus MeSH
- fyziologická neovaskularizace genetika MeSH
- lidé MeSH
- morfogeneze genetika MeSH
- oocyty růst a vývoj MeSH
- oogeneze genetika MeSH
- ovariální folikul růst a vývoj MeSH
- ovarium růst a vývoj metabolismus MeSH
- prasata MeSH
- primární buněčná kultura MeSH
- proteosyntéza genetika MeSH
- vývojová regulace genové exprese genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Granulosa cells (GCs) have many functions in the endocrine system. Most notably, they produce progesterone following ovulation. However, it has recently been proven that GCs can change their properties when subjected to long‑term culture. In the present study, GCs were collected from hyper‑stimulated ovarian follicles during in vitro fertilization procedures. They were grown in vitro, in a long‑term manner. RNA was collected following 1, 7, 15 and 30 days of culture. Expression microarrays were used for analysis, which allowed to identify groups of genes characteristic for particular cellular processes. In addition, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was performed to validate the obtained results. Two ontological groups characteristic for processes associated with the development and morphogenesis of the heart were identified during the analyses: 'Heart development' and 'heart morphogenesis'. The results of the microarrays revealed that the highest change in expression was demonstrated by the lysyl Oxidase, oxytocin receptor, nexilin F‑actin binding protein, and cysteine‑rich protein 3 genes. The lowest change was exhibited by odd‑skipped related transcription factor 1, plakophilin 2, transcription growth factor‑β receptor 1, and kinesin family member 3A. The direction of changes was confirmed by RT‑qPCR results. In the present study, it was suggested that GCs may have the potential to differentiate towards other cell types under long‑term in vitro culture conditions. Thus, genes belonging to the presented ontological groups can be considered as novel markers of proliferation and differentiation of GCs towards the heart muscle cells.
- MeSH
- buněčná diferenciace genetika MeSH
- buněčné kultury * MeSH
- buněčný rodokmen genetika MeSH
- folikulární buňky cytologie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- lysyloxidasa genetika MeSH
- morfogeneze genetika MeSH
- ovariální folikul cytologie metabolismus MeSH
- ovulace genetika MeSH
- progesteron genetika MeSH
- receptory oxytocinu genetika MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
