Piotr Celichowski, Karol Jopek, Marta Szyszka, Paulina Milecka, Ludwik K. Malendowicz, Marianna Tyczewska and Marcin Ruciński
Nampt (Nicotinamidephosphoribosyltransferase - also known as visfatin/PBEF) is the enzyme that regulates the NAD+ level, therefore influencing many metabolic pathways within the cells. As circulating cytokine, extracellular Nampt (eNampt) exerts pro-inflammatory, pro-chemotactic, pro-angiogenic and insulin-like effects; however the mechanism of eNampt action is still unclear.Earlier studies have shown that eNampt exerts a stimulating effect on the proliferation of many cancer cell lines. However, the effect of this cytokine on cell proliferation in primary culture is little known. Therefore, the aim of the study was to analyse the influence of eNampt on the proliferation of rat adrenocortical cells in primary culture and to investigate similar influence of eNampt on the line H295R of human adrenal corticocarcinoma cells. Proliferation of the examined cells was assessed using the RTCA (Real Time Cell Analyzer) method. The obtained results indicate that eNampt stimulates the proliferation of H295R cells, but does not change the proliferation of cultured rat adrenocortical cells. In primary culture of rat adrenocortical cells, Fk866 (specific Nampt inhibitor) does not modify the rate of proliferation of tested cells. In H295R cells the addition of Fk866 alone inhibits proliferative activity and stimulates apoptosis. Fk866 also inhibits the stimulating effect of eNampt on H295R cell proliferation.
Katarzyna Stefańska, Ievgenia Kocherova, Sandra Knap, Magdalena Kulus, Piotr Celichowski and Michal Jeseta
The oviduct is a part of female reproductive tract that is essential for successful fertilization and early embryo development. It is lined with epithelium consisting of two types of cells: ciliated and secretory. The primary function of ciliated oviductal epithelial cells (OECs) is to support the transport of gametes and embryos through the ovary, whereas secretory OECs produce components of the oviductal fluid. Undoubtedly, the oviductal epithelium plays a major part in the early aspects of pregnancy development, from providing an optimal environment for gametes and embryos to supporting fertilization. Therefore, our aim was to gain a better insight into the genetic changes underlying function of these cells. We have harvested OECs from crossbred gilts (n=45), at the age of about nine months and which displayed two regular estrous cycles, and established long-term primary culture of porcine OECs. Microarray analysis was utilized to determine differentially expressed genes during day 1, 7, 15 and 30 of cultivation, with our results revealing54 differentially expressed genes belonging to three ontology groups: „maintenance of location”, „maintenance of protein location” and „maintenance of protein location in cell”. Since the biochemistry and morphology of epithelial cells may change during long term cultivation, we conclude that our results are a reflection of these changes and help to shed a light on porcine OECs properties in in vitro environment.
Running title: Maintenance of cellular protein location in porcine epithelial oviductal cells
Sylwia Borys-Wójcik, Ievgenia Kocherova, Piotr Celichowski, Małgorzata Popis, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Michał Nowicki and Bartosz Kempisty
A wide variety of mechanisms controlling oligomerization are observed. The dynamic nature of protein oligomerization is important for bioactivity control. The oocyte must undergo a series of changes to become a mature form before it can fully participate in the processes associated with its function as a female gamete. The growth of oocytes in the follicular environment is accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). It has been shown that oocytes tested before and after in vitro maturation (IVM) differ significantly in the transcriptomic and proteomic profiles. The aim of this study was to determine new proteomic markers for the oligomerization of porcine oocyte proteins that are associated with cell maturation competence. The Affymetrix microarray assay was performed to examine the gene expression profile associated with protein oligomerization in oocytes before and after IVM. In total, 12258 different transcriptomes were analyzed, of which 419 genes with lower expression in oocytes after IVM. We found 9 genes: GJA1, VCP, JUP, MIF, MAP3K1, INSR, ANGPTL4, EIF2AK3, DECR1, which were significantly down-regulated in oocytes after IVM (in vitro group) compared to oocytes analyzed before IVM (in vivo group). The higher expression of genes involved in the oligomerization of the protein before IVM indicates that they can be recognized as important markers of biological activation of proteins necessary for the further growth and development of pig embryos.
Katarzyna Stefańska, Małgorzata Józkowiak, Paweł Antosik, Dorota Bukowska, Piotr Celichowski, Małgorzata Bruska, Michał Nowicki, Bartosz Kempisty, Jana Zakova, Marie Machatkova and Michal Jeseta
Correct maturation of the oocyte is crucial for further fertilization and embryogenesis. It comprises of both nuclear and cytoplasmic maturation, during which the proteins, nutrients and mRNAs are assembled. Cumulus cells are connected with the oocyte via gap-junctions, which enable bi-directional transfer of molecules, forming cumulus-oocyte complex (COC). The expression pattern in CCs is thought to resemble the genes expressed in the oocyte. The CCs surrounding the gamete of high developmental competence have an increased expression of apoptotic markers. Therefore, our aim in this study was to determine whether any apoptosis-related genes are upregulated in porcine oocytes before or after IVM. We isolated COCs from 45 pubertal crossbred gilts, performed brilliant cresyl blue (BCB) staining and analyzed the gene expression pattern in oocytes before and after IVM with the use of microarray analysis. The results include 419 differentially expressed transcripts, 25 of which belong to „regulation of apoptosis” and „regulation of cell death” GO BP terms. This set of genes includes BCLAF1, EIF2AK3, KLF10, MIF, MAP3K1, NOTCH2, TXNIP and APP, all of which have been upregulated in immature porcine oocytes. Our results suggest that they play part in porcine oocyte maturation and could be used as potential markers of female gamete’s developmental competence. This knowledge could serve as a basis to improve ART in pigs.
Agata Chamier-Gliszczyńska, Sandra Kałużna, Katarzyna Stefańska, Piotr Celichowski, Paweł Antosik, Dorota Bukowska, Małgorzata Bruska, Jana Zakova, Marie Machatkova, Michal Jeseta and Michał Nowicki
The formation of mammalian oocytes begins in the ovary during fetal development. The proper development of oocytes requires close communication with surrounding somatic cells, the substances they emit allow proper maturation of oocytes. Somatic cumulus (CC) cells and oocytes form cumulus-oocyte (COC) complexes.
In this study, the Affymetrix microarray analysis was used to investigate changes in gene expression occurring in oocytes before and after in vitro maturation (IVM). The aim of the study was to examine oocyte genes involved in two ontological groups, “regulation of cell migration” and “regulation of cell proliferation” discovered by the microarray method.
We found a reduced expression of all 28 genes tested in the ontological groups: ID2, VEGFA, BTG2, CCND2, EDNRA, TGFBR3, GJA, LAMA2, RTN4, CDK6, IHH, MAGED1, INSR, CD9, PTGES, TXNIP, ITGB1, SMAD4, MAP3K1, NOTCH2 , IGFBP7, KLF10, KIT, TPM1, PLD1, BTG3, CD47 and MITF. We chose the most regulated genes down the IVM culture, and pointed out those belonging to two ontological groups.
Increased expression of the described genes before IVM maturation may indicate the important role of these genes in the process of ovum maturation. After the maturation process, the proteins produced by them did not play such an important role. In summary, the study provides us with many genes that can serve as molecular markers of oocyte processes associated with in vitro maturation. This knowledge can be used for detailed studies on the regulation of oocyte maturation processes.
Running title: Genes regulating cellular migration and proliferation in porcine oocytes
Katarzyna Stefańska, Sandra Knap, Magdalena Kulus, Ievgenia Kocherova, Piotr Celichowski, Michal Jeseta, Marie Machatkova, Dorota Bukowska and Paweł Antosik
Oxygen metabolism is crucial in establishing successful pregnancy, since excessive amount of reactive oxygen species (ROS) may exert deleterious effects on the developing embryo. There are several defense mechanisms against oxidative stress in the female reproductive tract, including production of antioxidant enzymes by oviductal epithelial cells (OECs). Undoubtedly, OECs play major part in female fertility and may also serve as an in vitro model of the oviduct. Therefore, the aim of this study was to investigate the expression of genes involved in oxygen metabolism. We have isolated OECs from oviducts of crossbred gilts (n=45) and maintained their in vitro culture for 30 days, collecting their RNA at days 1, 7, 15 and 30. The gene expression was determined with the use of Affymetrix® Porcine Gene 1.1 ST Array Strip. Our results revealed 166 differentially expressed genes belonging to four ontology groups: „cellular response to oxidative stress”, “cellular response to oxygen-containing compound”, “cellular response to oxygen levels” and “cellular response to reactive oxygen species”, most of which are also involved in other major processes in the organism. However, our findings provide a valuable insight into porcine reproductive biology and may be utilized in optimization of assisted reproduction techniques.
Running title: Genes involved in oxygen metabolism in oviductal epithelial cells
Joanna Budna, Piotr Celichowski, Sandra Knap, Maurycy Jankowski, Magdalena Magas, Mariusz J. Nawrocki, Piotr Ramlau, Andrzej Nowicki, Magdalena Rojewska, Błażej Chermuła, Michal Jeseta, Paweł Antosik, Dorota Bukowska, Małgorzata Bruska, Maciej Zabel, Michał Nowicki and Bartosz Kempisty
The process of reproduction requires several factors, leading to successful fertilization of an oocyte by a single spermatozoon. One of them is the complete maturity of an oocyte, which is acquired during long stages of folliculogenesis and oogenesis. Additionally, the oviduct, composed of oviductal epithelial cells (OECs), has a prominent influence on this event through sperm modification and supporting oocyte’s movement towards uterus. OECs were isolated from porcine oviducts. Cells were kept in primary in vitro culture for 30 days. After 24h and on days 7, 15 and 30 cells were harvested, and RNA was isolated. Transcript changes were analyzed using microarrays. Fatty acids biosynthetic process and fatty acids transport ontology groups were selected for analysis and described. Results of this study indicated that majority of genes in both ontology groups were up-regulated on day 7, 15 and 30 of primary in vitro culture. We analyzed genes involved in fatty acids biosynthetic process, including: GGT1, PTGES, INSIG1, SCD, ACSL3, FADS2, FADS1, ACSS2, ALOX5AP, ACADL, SYK, ACACA, HSD17B8, FADS3, OXSM, and transport, including: ABCC2, ACSL4, FABP3, PLA2G3, PPARA, SYK, PPARD, ACACA and P2RX7. Elevated levels of fatty acids in bovine and human oviducts are known to reduce proliferation capacity of OECs and promote inflammatory responses in their microenvironment. Most of measured genes could not be connected to reproductive events. However, the alterations in cellular proliferation, differentiation and genes expression during in vitro long-term culture were significant. Thus, we can treat them as putative markers of changes in OECs physiology.
Mariusz J. Nawrocki, Joanna Budna, Piotr Celichowski, Ronza Khozmi, Artur Bryja, Wiesława Kranc, Sylwia Borys, Sylwia Ciesiółka, Sandra Knap, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty
The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the compound process responsible for proper oocytes transport and successful fertilization. However, the main mechanisms which regulated this process are still not entirely known. Moreover, the OECs metabolism, which may be identified as the “cellular activity” marker, is poorly recognized. In this study we investigated the fructose and mannose metabolic pathway in porcine OECs primary long-term cultured in vitro.
In our study, we employ a primary long term in vitro culture (IVC) and microarray approach (the Affymetrix microarray were used for analysis of transcriptomic profile of OECs) for expression levels analysis.
We found that from the whole analyzed transcriptome, 1537 genes were upregulated and 995 were down regulated after 7 days of culture, 1471 genes were upregulated and 1061 were downregulated after 15 days of culture and 1329 genes were upregulated and 1203 were downregulated after 30 days of culture. Moreover, the differential expression of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 at 24 hours, 7 day, 15 day and 30 day, was also observed.
We suggested that fructose and mannose metabolism may be important molecular bio-marker of porcine OECs capability in in vitro model. The metabolic profile is significantly accompanied by cells proliferation in vitro. The transcriptomic profile of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 expression may be identified as “fingerprint” of fructose and mannose metabolism in OECs as well as involved in cellular in vitro developmental capacity in pigs.
Wiesława Kranc, Piotr Celichowski, Joanna Budna, Ronza Khozmi, Artur Bryja, Sylwia Ciesiółka, Marta Rybska, Sylwia Borys, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty
The mammalian oocytes maturation is the compound process that involves morphological and molecular changes. These modifications include storage of macromolecules, which are crucial for proteins biosynthesis during periimplantation stages of embryo development. This study was aimed to investigate the genes expression profile encoding macromolecules important for regulation of proper porcine oocytes maturation.
The porcine oocytes were collected from large ovarian follicles and analyzed both before and after in vitro maturation (IVM). Additionally, to check the developmental competence status, brilliant crezyl blue test (BCB) was performed. The obtained cDNA was used for biotin labeling and fragmentation by AffymetrixGeneChip® WT Terminal Labeling and Hybridization (Affymetrix). The preliminary analysis of the scanned chips was performed using AffymetrixGeneAtlasTM Operating Software. The created CEL files were imported into downstream data analysis software.
In results, we found expression of 419 different genes, 379 genes were down-regulated and 40 genes were up-regulated in relation to the oocyte transcriptome before in vitro procedure. We observed up-regulation of all genes involved in “positive regulation of macromolecule metabolic process” before IVM as compared to transcriptional profile analyzed after IVM.
In conclusion, we suggested that genes encoding proteins involved in macromolecule metabolism are important for achieving of porcine oocytes maturational stage. Moreover, the “activity of macromolecules metabolism” is much more increased in immature oocytes.
Artur Bryja, Marta Dyszkiewicz-Konwińska, Maurycy Jankowski, Piotr Celichowski, Katarzyna Stefańska, Agata Chamier-Gliszczyńska, Małgorzata Popis, Katarzyna Mehr, Dorota Bukowska, Paweł Antosik, Małgorzata Bruska, Maciej Zabel, Michał Nowicki and Bartosz Kempisty
The oral mucosa is a compound tissue composed of several cells types, including fibroblasts and keratinocytes, that are characterized by different morphology, as well as biochemical and metabolomic properties. The oral mucosal cells are the most important factors mediated between transport and drugs delivery. The changes in cellular ion homeostasis may significantly affect the bioavailability of administrated drugs and their transport across the mucous membrane. Therefore we investigated the expression profile of genes involved in ion transport and homeostasis in porcine buccal pouch mucosal cells.
The oral mucosa was separated surgically and isolated enzymatically. The cells were examined during long-term in vitro culture (IVC). The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation and next, the gene expression profile was measured using Affymetrix microarray assays.
In the results, we can extract genes belonging to four ontology groups: “ion homeostasis”, “ion transport”, “metal ion transport”, and “inorganic ion homeostasis”. For TGFB1 and CCL2, we observed up-regulation after 7 days of IVC, down-regulation after 15 days of IVC and upregulation again after 30 days of IVC. The ATP13A3, ATP1B1, CCL8, LYN, STEAP1, PDPN, PTGS2, and SLC5A3genes showed high activity after day 7 of IVC, and in the days 15 and 30 of IVC showed low activity.
We showed an expression profile of genes associated with the effects of ion influence on the porcine normal oral mucosal cell development in IVC. These studies may be the starting point for further research into oral diseases and will allow for the comparison of the gene expression profile of normal and disease altered cells.