Dorota Bukowska

Analysis of selected transcript levels in porcine spermatozoa, oocytes, zygotes and two-cell stage embryos

It has been suggested that spermatozoa can deliver mRNAs to the oocyte during fertilisation. Using reverse transcription and real-time quantitative polymerase chain reaction analysis (RQ-PCR), we evaluated the presence of clusterin (CLU), protamine 2 (PRM2), calmegin (CLGN), cAMP-response element modulator protein (CREM), methyltransferase 1 (DNMT1), linker histone 1 (H1), protamine 1 (PRM1), TATA box-binding protein associated factor 1 (TAF1) and TATA box-binding protein (TBP) in porcine mature oocytes, zygotes and two-cell stage embryos. Spermatozoa isolated from semen samples of boars contained all transcripts investigated, whereas oocytes contained only CREM, H1, TAF1, and TBP mRNAs. The zygote and two-cell stage embryos contained CLU, CREM, H1, PRM1, PRM2, TAF1 and TBP transcripts. Our observations suggest that porcine spermatozoa may delivery CLU, PRM1 and PRM2 mRNAs to the oocyte, which may contribute to zygotic and early embryonic development.

Cytoplasmic and nuclear maturation of oocytes in mammals – living in the shadow of cells developmental capability

Abstract The pig is a polyestrous animal in which the ovarian cycle lasts about 21 days and results in ovulation of 10-25 oocytes. Ovum reaches 120-150 μm in diameter, with the surrounding corona radiata providing communication with the environment. The zona pellucida is composed of glycoproteins: ZP1, ZP2, ZP3. In the course of oogenesis, RNA and protein accumulation for embryonic development occurs. Maternal mRNA is the template for protein production. Nuclear, cytoplasmic and genomic maturity condition the ability of the ovum to undergo fertilization. There are several differences in protein expression profiles observed between in vitro and in vivo conditions. Oogenesis is the process of differentiating female primary sex cells into gametes. During development gonocytes migrate from the yolk sac into the primary gonads with TGF-1, fibronectin, and laminin regulating this process. Cell cycle is blocked in dictyotene. Primary oocyte maturation is resumed before each ovulation and lasts until the next block in metaphase II. At the moment of penetration of the sperm into the ovum, the metaphase block is broken. The oocytes, surrounded by a single layer of granular cells, form the ovarian follicle. The exchange of signals between the oocyte and the cumulus cells done by gap-junctions, as well as various endo and paracrine signals. The contact between the corona radiata cells provides substances necessary for growth, through the same gap junctions. Studies on follicular cells can be used to amplify the knowledge of gene expression in these cells, in order to open way for potential clinical applications.

Amino acids metabolism and degradation is regulated during porcine oviductal epithelial cells (OECs) primary culture in vitro – a signaling pathways activation approach

Abstract The ovary is part of the reproductive system, possessing very important functions in the reproduction process (ovum and embryo transfer, providing a suitable environment for sperm capacitation, etc.). There are two types of cells in the fallopian tubes: alveolar and secretive cells. These study shows the metabolic processes in pig oviductal epithelial cells associated with the activation of signaling pathways of amino acids metabolism and degradation during long-term in vitro culture. Oviductal epithelial cells from 45 colonies in the anestrous phase of the estrous cycle have been utilized in this study. RNA extract from the OEC primary cultures was pooled after 24h, 7days, 15 days and 30 days from the beginning of culture and the transcriptome investigated by Affymetrix® Porcine Gene 1.1 ST. From the whole transcript that consisted of 2009 different genes, 1537 were upregulated and 995 were downregulated after 7 days of culture, 1471 were upregulated and 1061 were downregulated after 15 days of culture and 1329 were upregulated and 1203 were downregulated after 30 days of culture. The results of these studies provide, for the first time, information on the activation of metabolic pathways of amino acids such as valine, leucine, isoleucine, cysteine, and methionine in the investigated tissue. They also indicate genes that may be OECs-specific genetic markers that are expressed or upregulated during long-term in vitro culture.

The differentiation and transdifferentiation of epithelial cells in vitro – is it a new strategy in regenerative biomedicine?

Abstract In modern medical research, stem cells are one of the main focuses, believed to be able to provide the solution to many currently unsolvable medical cases. However, their extraordinary potential for differentiation creates much obstacles in their potential application in clinical environment, without understanding the whole array of molecular mechanisms that drive the processes associated with their development and maturation. Because of that, there is a large need for studies that concern the most basic levels of those processes. Progenitor stem cells are a favorable target, as they are relatively lineage committed, making the amount of signaling required to reach the final form much lower. Their presence in the adult organism is also an advantage in their potential use, as they can be extracted without the need for storage from the moment of pre-natal development or birth. Epithelial tissues, because of their usual location or function, exhibit extraordinary level of plasticity and proliferative potential. That fact makes them one of the top candidates for use in applications such as tissue engineering, cell based therapies, regenerative and reconstructive medicine. The potential clinical application, however, need to be based on well developed methods, in order to provide an effective treatment without causing major side effects. To achieve that goal, a large amount of research, aiming to analyze the molecular basics of proliferation and differentiation of epithelial stem cells, and stem cells in general, needs to be conducted.

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.

Expression Changes in Fatty acid Metabolic Processrelated Genes in Porcine Oocytes During in Vitro Maturation

Abstract Mammalian oocytes undergo compound processes of nuclear and cytoplasmic maturation that allow them to reach MII stage. Only fully mature, oocyte can be successfully fertilized by a single spermatozoon. Fatty acids, apart from their role in cellular metabolism, inflammation and tissue development, have positive and detrimental effects on oocyte maturation, fertilization, blastocyst cleavage rate and embryo development in mammals. Using microarrays, we have analyzed the expression changes in fatty acids- -related genes during in vitro maturation of porcine oocytes. The oocytes were recovered from ovaries of 45 pubertal crossbred Landrace gilts and subsequently subjected to BCB test. For further analyses, only granulosa cell-free BCB+ oocytes were used and divided into two groups. The first one, described as “before IVM”, was directly exposed to molecular assays, the second one, described as “after IVM”, was first in vitro matured and then subjected to a second BCB test. Oocytes, if classified as BCB+, were then passed to corresponding molecular analyses. We found significant down-regulation of genes involved in fatty acid metabolic process, such as: ACSL6, EPHX2, FADS2, PTGES, TPI1, TBXAS1, NDUFAB1, MIF, ACADSB and DECR1 in porcine oocytes analyzed after IVM, in comparison to those analyzed before IVM. In conclusion, apart from poor data available concerning analyzed genes in relation to reproductive events, significant changes in their expression point to their potential role as an oocyte developmental competence markers in pigs. Introducing molecular diagnostics of oocytes could be the prospective tool for selection of best gametes, leading to improved outcomes of in vitro fertilization.

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.

Splenic Leiomyoma in Dog

Abstract Leiomyoma is a benign tumour, originating from smooth muscles cells. This tumor commonly involves the uterus, vagina, stomach, intestine, urinary bladder and other organs. Only a few cases of splenic leiomyoma in dogs have been reported in the available literature. Much more frequently malignant leiomyosarcoma was found. The aim of this study was to compile rare clinical case of splenic leiomyoma in dog, which developed with no clinical signs and no abnormalities in blood findings. A 14-year-old, spayed bitch was examined with ultrasonography, where lesions on the spleen were identified. Based on the clinical findings (blood test in norm, no metastases in X-ray examination) surgical removal of spleen was recommended. Two fragments of tumors were prepare for histopathological examination. The lesion was described as smooth muscle benign tumor, therefore a diagnosis of leiomyoma was made. About a year after splenectomy no signs of metastases were present in a ultrasound and X-ray examinations. This report indicates the necessity of taking the occurrence of benign lesions in the spleen into account. Splenectomy based on the presence of tumor lesion should be associated with histopathological examination to identify the nature of change. This clinical case, despite a marked morphological lesion shown during intraoperative examination, was benign with successful prognosis.

Characteristic of factors influencing the proper course of folliculogenesis in mammals

Abstract Folliculogenesis is the process of ovarian follicle formation,, taking presence during foetal period. During the follicular development, oogoniums undergo meiosis and oocytes are formed. In the ovaries of new born sows, primary and secondary follicles are present and, 90 days after birth, tertiary follicles appear. During development in the ovarian follicles growth of granulosa cells and differentiation of the thecal cells can be observed. A cavity filled with follicular fluid appears. Granulosa cells are divided into: mural cells and corona radiata, which together with the oocyte form the cumulus oophorus. Corona radiata cells, mural layers and oolemma contact each other by a network of gap junctions. Secreted from the pituitary gland, FSH and LH gonadotropin hormones act on receptors located in granular and follicular cells. In the postnatal life tertiary follicles and Graafian follicles are formed. When the follicle reaches a diameter of 1 mm, further growth depends on the secretion of gonadotropins. Mature ovarian follicles produce: progestins, androgens and oestrogens. The growth, differentiation and steroidogenic activity of ovarian follicles, in addition to FSH and LH, is also affected by prolactin, oxytocin, steroid and protein hormones, numerous proteins from the cytokine and interleukin family, metabolic hormones like insulin, glucocorticoids, leptin, thyroid hormones and growth hormones. Despite numerous studies, many processes related to folliculogenesis have not been discovered Learning the mechanisms regulating reproductive processes would allow to easily distinguish pathological processes and discover more and more genes and mechanisms of their expression in cells that build ovarian follicles.