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  • Author: Dorota Bukowska x
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Ontology groups representing angiogenesis and blood vessels development are highly up-regulated during porcine oviductal epithelial cells long-term real-time proliferation – a primary cell culture approach

Abstract

The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the group of compound processes responsible for proper oocyte transport and successful fertilization. The cellular interactions between cumulus-oocyte complexes (COCs) and oviductal epithelial cells (OECs) are crucial for this unique mechanism. In the present study we have analyzed angiogenesis and blood vessel development processes at transcript levels. By employing microarrays, four ontological groups associated with these mechanisms have been described. Differentially expressed genes belonging to the “angiogenesis”, “blood circulation”, “blood vessel development” and “blood vessel morphogenesis” GO BP terms were investigated as a potential markers for the creation of new blood vessels in cells under in vitro primary culture conditions.

Open access
Fatty Acids Related Genes Expression Undergo Substantial Changes in Porcine Oviductal Epithelial Cells During Long-Term Primary Culture

Abstract

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.

Open access
Does Porcine Oocytes Maturation in Vitro is Regulated by Genes Involved in Transforming Growth Factor Beta Receptor Signaling Pathway?

Summary

The oocyte growth and development in follicular environment are substantially accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). During these processes, the mammalian gametes reach full maturational stage and may be further successfully fertilized by single spermatozoon. These unique mechanisms are regulated by expression of clusters of genes and their biochemical signaling pathways.

In this article we described differential expression pattern of transforming growth factor beta (TGFB) gene superfamily in porcine oocytes before and after in vitro maturation (IVM).

We performed Affymetrix® microarray assays to investigate the TGFB-related genes expression profile in porcine immature oocytes and gametes cultured for 44h in vitro.

In results we found 419 different genes, 379 genes with lower expression, and 40 genes characterized by increased RNA profile. Moreover, significant up-regulation of 6 genes belonging to TGFB signaling pathway such as: TGFBR3, SMAD4, FOS, KLF10, ID1, MAP3K1 in immature porcine oocytes (before IVM), was also observed.

It may be suggested that genes involved in TGFB-related signaling pathway are substantially regulated before IVM. Furthermore, these genes may play a significant role during early stages of nuclear and/or cytoplasmic porcine oocytes maturation. The investigated transcripts may be also recommended as the markers of oocytes maturational capability in pigs.

Open access
Ion homeostasis and transport are regulated by genes differentially expressed in porcine buccal pouch mucosal cells during long-term culture in vitro – a microarray approach

Abstract

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.

Open access
Cation homeostasis and transport related gene markers are differentially expressed in porcine buccal pouch mucosal cells during long-term cells primary culture in vitro

Abstract

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.

Open access
Positive Regulation of Macromolecule Metabolic Process Belongs to the Main Mechanisms Crucial for Porcine Oocytes Maturation

Summary

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.

Open access
Genes involved in angiogenesis and circulatory system development are differentially expressed in porcine epithelial oviductal cells during long-term primary in vitro culture – a transcriptomic study

Abstract

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.

Open access
Epithelium morphogenesis and oviduct development are regulated by significant increase of expression of genes after long-term in vitro primary culture – a microarray assays

Abstract

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.

Open access
Selected aspects of endometritis – pyometra complex in dogs – current troubles and treatment perspectives

Abstract

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.

Open access
Does migrative and proliferative capability of epithelial cells reflect cellular developmental competence?

Abstract

Mammalian epithelial and epithelial-like cells are significantly involved in various processes associated with tissue development, differentiation and oncogenesis. Because of that, high number of research is focused on identifying cells that express stem-like or progenitor characteristics. Identifying such cells and recognizing their specific markers, would open new clinical opportunities in transplantology and oncology. There are several epithelia characterized by their ability to rapidly proliferate and/or differentiate. Due to their function or location they are subject to cyclic changes involving processes of apoptosis and regeneration. Literature presenting well-structured studies of these types of epithelia was analyzed in order to compare various results and establish if epithelial cells’ migrative and proliferative ability indicates their stemness potential. Endometrial, ovarian, oviductal and oral mucosal epithelia were analyzed with most of the publications delivering relatively unified results. The ability to rapidly proliferate/differentiate usually indicated the presence of some kind of stem/stem-like/progenitor cells. Most of the papers focused on pinpointing the exact location of these kind of cells, or analyzing specific markers that would be used for their future identification. There have also been substantial proportion of research that focused on discovering growth factors or intercellular signals that induced proliferation/differentiation in analyzed epithelia. Most of the research provided valuable insights into the modes of function and characteristics of the analyzed tissue, outlining the importance of such study for the possible clinical application of in vitro derived cell cultures.

Open access