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
Marta Rybska, Sandra Knap, Katarzyna Stefańska, Maurycy Jankowski, Agata Chamier-Gliszczyńska, Małgorzata Popis, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Bartosz Kempisty and Jędrzej M. Jaśkowski
The superfamily of transforming growth factors β (TGF-β) consists of cytokines that are crucial in regulating the organism’s biological functions and includes three isoforms of TGF-β protein, Anti-Müllerian Hormone (AMH), inhibin A and B, activins, 20 bone morphogenetic proteins (BMP1-20) and 9 growth factors (GDF1-9). Their signal transduction pathway involves three types of membrane receptors that exhibit a serine/threonine kinase activity, as well as the Smad proteins. After ligand binding, the Smad proteins are phosphorylated and translocated to the nucleus, where they interact with transcription factors and affect gene expression. TGF-β family members are involved in cell growth and differentiation, as well as chemo-taxis and apoptosis, and play an important role during an inflammation. Defects in TGF-β proteins or in their signalling pathway underlie many severe diseases, such as systemic lupus, systemic scleroderma, bronchial asthma, atherosclerosis, hyperthyroidism or cancer. These factors are also crucial in mammal reproductive functions, as they are involved in folliculogenesis, steroidogenesis, ovulation, maternal-embryo interaction, embryo development and uterine decidualization. Their defects result in issues with fertility. This review focuses on the relevance of TGF-β family members in a mammal reproduction with an emphasis on three TGF-β isoforms, inhibins A and B, GDF-9 and their signal transduction pathway.
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.
Agata Chamier-Gliszczyńska, Maciej Brązert, Patrycja Sujka-Kordowska, Małgorzata Popis, Katarzyna Ożegowska, Katarzyna Stefańska, Ievgeniia Kocherova, Piotr Celichowski, Magdalena Kulus, Dorota Bukowska, Leszek Pawelczyk, Małgorzata Bruska, Paweł Antosik, Michał Nowicki and Bartosz Kempisty
An oviduct is an essential organ for gamete transport, oocyte maturation, fertilization, spermatozoon capacitation and early embryo development. The epithelium plays an important role in oviduct functioning. The products of secretory cells provide an optimal environment and influence gamete activities and embryonic development. The oviduct physiology changes during the female cycle, thus, the ratio of the secreted molecules in the oviduct fluid differs between phases. In this study, a differential gene expression in porcine oviduct epithelial cells was examined during the long-term primary in vitro culture. The microarray expression analysis revealed 2552 genes, 1537 of which were upregulated and 995 were downregulated after 7 days of culture, with subsequent changes in expression during 30 day-long culture. The obtained genes were classified into 8 GO BP terms, connected with angiogenesis and circulatory system development, extracted by DAVID software. Among all genes, 10 most up-regulated and 10 most down-regulated genes were selected for further investigation. Interactions between genes were indicated by STRING software and REACTOME FIViz application to the Cytoscape 3.6.0 software. Most of the genes belonged to more than one ontology group. Although studied genes are mostly responsible for angiogenesis and circulatory system development, they can also be found to be expressed in processes connected with fertilization and early embryo development. The latter function is focused on more, considering the fact that these genes were expressed in epithelial cells of the fallopian tube which is largely responsible for reproductive processes.
Artur Bryja, Marta Dyszkiewicz-Konwińska, Maurycy Jankowski, Piotr Celichowski, Katarzyna Stefańska, Agata Chamier-Gliszczyńska, Blanka Borowiec, Katarzyna Mehr, Dorota Bukowska, Paweł Antosik, Małgorzata Bruska, Maciej Zabel, Michał Nowicki and Bartosz Kempisty
The mucous membrane is composed of two layers. The layer of stratified squamous epithelium and the underlying layer of the connective tissue. The epithelium is composed of keratinocytes that are in different stages of differentiation, depending on their localization. In our research, after isolation of primary in vitro cultured buccal pouch mucosal cells, we observed keratinocytes in various stages of differentiation and fibroblasts. These cells, depending on the ionic dynamics, may be subject to different morphological and biochemical transformations. Understanding the expression profile of the normal oral mucosal tissue is important for further research into the effects of biomaterials on the mucosal cells, their growth, proliferation, and differentiation.
The porcine buccal pouch mucosal cells were used in this study. The oral mucosa was separated surgically and isolated enzymatically. The cells were in vitro cultured for 30 days, and after each step of in vitro culture (7 days, 15 days, 30 days), samples were collected for isolation of total RNA. The gene expression profile was measured using Affymetrix microarray assays.
In results, we observed genes belonging to two ontology groups: cation homeostasis and cation transport. These genes were up-regulated after 7 days of in vitro culture as compared to down-regulation after 15 and 30 days of in vitro culture. These results suggested that dynamic growth, proliferation and cell adhesion are more intense in the first 7 days of in vitro culture. We also observed, for the first time, the expression of ATP13A3 in porcine oral mucosal cells.
Katarzyna Stefańska, Agata Chamier-Gliszczyńska, Maurycy Jankowski, Piotr Celichowski, Magdalena Kulus, Magdalena Rojewska, Paweł Antosik, Dorota Bukowska, Małgorzata Bruska, Michał Nowicki, Bartosz Kempisty, Michal Jeseta and Jana Zakova
The correct oviductal development and morphogenesis of its epithelium are crucial factors influencing female fertility. Oviduct is involved in maintaining an optimal environment for gametes and preimplantation embryo development; secretory oviductal epithelial cells (OECs) synthesize components of oviductal fluid. Oviductal epithelium also participates in sperm binding and its hyperactivation. For better understanding of the genetic bases that underlay porcine oviductal development, OECs were isolated from porcine oviducts and established long-term primary culture. A microarray approach was utilized to determine the differentially expressed genes during specific time periods. Cells were harvested on day 7, 15 and 30 of in vitro primary culture and their RNA was isolated. Gene expression was analyzed and statistical analysis was performed. 48 differentially expressed genes belonging to “tube morphogenesis”, “tube development”, “morphogenesis of an epithelium”, “morphogenesis of branching structure” and “morphogenesis of branching epithelium” GO BP terms were selected, of which 10 most upregulated include BMP4, ARG1, SLIT2, FGFR1, DAB2, TNC, EPAS1, HHEX, ITGB3 and LOX. The results help to shed light on the porcine oviductal development and its epithelial morphogenesis, and show that after long-term culture the OECs still proliferate and maintain their tube forming properties.
Edyta Porowska, Magdalena Kulus, Maurycy Jankowski, Ievgenia Kocherova, Michal Jeseta, Agata Chamier-Gliszczyńska, Katarzyna Stefańska, Blanka Borowiec, Dorota Bukowska, Klaus P. Brüssow, Bartosz Kempisty and Paweł Antosik
Pyometra is the most common gynecological disease in female dogs. It usually occurs in middle age female dogs, usually about two months after the completion of heat. This disease is the accumulation of purulent fluid inside the uterus. Etiology of pyometra is not fully understood. It is assumed, that pyometra is a result of hormonal disorders in the endometrium combined with bacterial superinfection. The diagnosis is based on the interview, clinical examination, additional laboratory tests and ultrasound or x-ray of the abdomen. There are two treatments: ovariohysterectomy and conservative treatment with pharmacological agents for example prostaglandin, aglepriston, antibiotics with a broad spectrum of action. Currently conducted molecular studies have a large influence on the development of the present knowledge on the pathogenesis and course of pyometra, whose conclusions may be used to change the current therapeutic protocols.