The ovarian follicle is the basic functional unit of the ovary, comprising theca cells and granulosa cells (GCs). Two different types of GCs, mural GCs and cumulus cells (CCs), serve different functions during folliculogenesis. Mural GCs produce oestrogen during the follicular phase and progesterone after ovulation, while CCs surround the oocyte tightly and form the cumulus oophurus and corona radiata inner cell layer. CCs are also engaged in bi-directional metabolite exchange with the oocyte, as they form gap-junctions, which are crucial for both the oocyte's proper maturation and GC proliferation. However, the function of both GCs and CCs is dependent on proper follicular angiogenesis. Aside from participating in complex molecular interplay with the oocyte, the ovarian follicular cells exhibit stem-like properties, characteristic of mesenchymal stem cells (MSCs). Both GCs and CCs remain under the influence of various miRNAs, and some of them may contribute to polycystic ovary syndrome (PCOS) or premature ovarian insufficiency (POI) occurrence. Considering increasing female fertility problems worldwide, it is of interest to develop new strategies enhancing assisted reproductive techniques. Therefore, it is important to carefully consider GCs as ovarian stem cells in terms of the cellular features and molecular pathways involved in their development and interactions as well as outline their possible application in translational medicine.

• Keywords
• cumulus cells, follicular angiogenesis, granulosa cells, miRNA, stem cells, translational medicine,
• MeSH
• Neovascularization, Physiologic * MeSH
• Stem Cells metabolism   MeSH
• Cumulus Cells metabolism   MeSH
• Humans MeSH
• Primary Ovarian Insufficiency metabolism   MeSH
• Polycystic Ovary Syndrome metabolism   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

• Keywords
• cortical granule, microarray, molecular markers, oocyte maturation, pig,
• MeSH
• Cell Differentiation * MeSH
• Cytoplasmic Granules metabolism   MeSH
• In Vitro Oocyte Maturation Techniques methods   MeSH
• Cells, Cultured MeSH
• Oocytes cytology   metabolism   MeSH
• Swine MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

• Keywords
• Microarray, Mitochondrial activity, Oocyte maturation, Pig,
• MeSH
• Eosine Yellowish-(YS) chemistry   MeSH
• Hematoxylin chemistry   MeSH
• Hormones genetics   metabolism   MeSH
• In Vitro Oocyte Maturation Techniques * MeSH
• Cells, Cultured MeSH
• Lipids analysis   MeSH
• Oocytes growth & development   metabolism   MeSH
• Oxazines chemistry   MeSH
• Swine MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Brilliant Cresyl Blue MeSH Browser
• Eosine Yellowish-(YS) MeSH
• Hematoxylin MeSH
• Hormones MeSH
• Lipids MeSH
• Oxazines MeSH

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.

• Keywords
• SIRT1, differentiation, granulosa cells, mesenchymal stem cells, resveratrol,
• MeSH
• Models, Biological MeSH
• Cell Differentiation * drug effects   MeSH
• Granulosa Cells cytology   drug effects   metabolism   MeSH
• Humans MeSH
• Mesenchymal Stem Cells cytology   MeSH
• Resveratrol chemistry   pharmacology   MeSH
• Signal Transduction drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Resveratrol 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.

• Keywords
• Granulosa cells, Microarray, Ovarian follicle, Pig, Primary culture,
• MeSH
• Cell Cycle genetics   MeSH
• Granulosa Cells cytology   MeSH
• Cells, Cultured MeSH
• Ovarian Follicle cytology   MeSH
• Swine MeSH
• Gene Expression Profiling MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

In the ovarian follicle, maturation of the oocyte increases in the presence of somatic cells called cumulus cells (CCs). These cells form a direct barrier between the oocyte and external environment. Thanks to bidirectional communication, they have a direct impact on the oocyte, its quality and development potential. Understanding the genetic profile of CCs appears to be important in elucidating the physiology of oocytes. Long-term in vitro culture of CCs collected from patients undergoing controlled ovarian stimulation during in vitro fertilization procedure was conducted. Using microarray expression analysis, transcript levels were assessed on day 1, 7, 15, and 30 of culture. Apoptosis and aging of CCs strictly influence oocyte quality and subsequently the outcome of assisted reproductive technologies (ART). Thus, particular attention was paid to the analysis of genes involved in programmed cell death, aging, and apoptosis. Due to the detailed level of expression analysis of each of the 133 analyzed genes, three groups were selected: first with significantly decreased expression during the culture; second with the statistically lowest increase in expression; and third with the highest significant increase in expression. COL3A1, SFRP4, CTGF, HTR2B, VCAM1, TNFRSF11B genes, belonging to the third group, were identified as potential carriers of information on oocyte quality.

• Keywords
• cumulus cells, gene expression, human, programmed cell death,
• MeSH
• Principal Component Analysis MeSH
• Biomarkers metabolism   MeSH
• Cell Death genetics   MeSH
• Cell Culture Techniques methods   MeSH
• Time Factors MeSH
• Adult MeSH
• Gene Ontology MeSH
• Gene Regulatory Networks MeSH
• Cumulus Cells cytology   metabolism   MeSH
• Humans MeSH
• Gene Expression Regulation MeSH
• Reproducibility of Results MeSH
• Gene Expression Profiling * MeSH
• Cellular Senescence genetics   MeSH
• Cell Shape genetics   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers 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

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