Expression of PTX3, HAS2 AND TNFAIP6 genes in relation to real-time proliferation of porcine endometrial luminal epithelial cells in primary cultivation model.

Endometrial cells undergo very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume. Then, if fertilization fails, endometrial cells are liable for apoptosis, preparing new cells that are ready for subsequent processes related to the possibility of embryo implantation and the development of pregnancy. PTX3 and TNFAIP6 are absent or reduced in cultured COCs, resulting in a functional change in COC in vitro. In this work, we want to check how PTX3, HAS2 and TNFAIP6 behave in luminal epithelium primary cell culture. Cells obtained during slaughter from porcine specimens were cultured primarily in vitro for 7 days. Their proliferation patterns were then analysed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes in the expression of the genes of interest were analysed on each of the seven days of the porcine luminal primary cell culture. Our study showed the increased level of PTX3, HAS2 and TN¬FAIP6 expression at the same hours of primary culture. Rt-qPCR showed a higher level of expression of the PTX3 gene in the first 72 h, at the end of the lag phase (in the phase of stasis in which the cells adapt to the new environment and often die). In contrast, TNFAIP6 expression increases about 96 hours when the cells are in the full log phase (logarithmic phase growth) and continue this trend in the plateau phase. We did not observe such drastic changes in the HAS2 expression pattern, which leads us to hypothesize that PTX3 and TNFAIP6 are designed to maintain a constant level of HAS2 in the cell throughout its lifetime. The obtained results could become a point of reference for further in vivo and clinical research.

Balance between epithelial and stromal marker expression and distribution in primary culture model of porcine endometrium during real-time cell proliferation..

The similarity between humans and pigs, when it comes to tissue morphology, makes Sus scrofa not only a good research model, but also a potential source of cells for tissue engineering. Cell samples obtained from the pig donor, could be influenced in vitro, in order to become a source of tissue material for xenotransplantation, reconstructive and regenerative medicine. Significant amounts of data point to especially major similarities in pig and human reproductive systems. Because of that, particular scientific focus is centered on research concerning porcine COCs, theca and granulosa cells in primary cultures. One of the aspects of the reproductive process, that is still largely undiscovered, is the interaction between preimplantation blastocyst and maternal uterine tissues. In this study, we used molecular analysis techniques, such as RT-qPCR and immunocytochemistry, to analyze the expression and distribution of cytokeratin 18 and panCytokeratins 8, 18 and 19 and vimentin in porcine luminal endometrial epithelial cells, coupled with analysis of their behavior in RTCA. The results have confirmed the presence of epithelial, as well as stromal cell markers in the cells, varying in levels at different stages of culture. They have also given insight into the modes of proliferation and differentiation of studied cells in in vitro culture, as well as providing additional proof for the possible mesenchymal transdifferentiation of epithelial cells.

Microfluidic versus molecular assays - different approaches in assessing oocyte developmental competence.

In recent years, molecular techniques have brought about new solutions that focus on the developmental capacity of female oocytes and reproductive performance in the mammalian species. The developmental potency is the ability of oocytes to reach the MII stage following the long stages of folliculo- and oogenesis. The main proteins involved in this process belong to the connexin (Cx) family, which are responsible for the formation of gap junction (GJC) connections between the female gamete and surrounding somatic cells. The Cx are involved in bi-directional transport of small molecules and are therefore responsible for correct oocyte-somatic cell nutrition, proliferation, and differentiation. However, the application of certain molecular techniques often leads to destabilization or destruction of the materials of interest, such as cells or whole tissues. Therefore, the applications of microfluidic methods, which are non-invasive and quantitative, give new opportunities to further this area of biomedical research. Microfluidic research is based on real-time experiments that allow for control and/ or observation of the results during each step. The purpose of this review is to present both positive and negative aspects of molecular-microfluidic methods while describing the role of connexins in oocyte developmental capacity.

The differentiation of mammalian ovarian granulosa cells – living in the shadow of cellular developmental capacity.

The mammalian cumulus-oocyte complex (COCs) promotes oocyte growth and development during long stages of folliculogenesis and oogenesis. Before ovulation, the follicle is formed by a variety of fully differentiated cell populations; cumulus cells (CCs) that tightly surround the female gamete, granulosa cells (GCs) and theca cells (TCs) which build the internal and external mass of the follicular wall. It is well documented that CCs surrounding the oocyte are necessary for resumption of meiosis and full maturation of the gamete. However, the role of the granulosa cells in acquisition of MII stage and/or full fertilization ability is not yet entirely known. In this article, we present an overview of mammalian oocytes and their relationship to the surrounding cumulus and granulosa cells. We also describe the processes of GCs differentiation and developmental capacity. Finally, we describe several markers of mammalian GCs, which could be used for positive identification of isolated cells. The developmental capacity of oocytes and surrounding somatic cells – a “fingerprint” of folliculogenesis and oogenesis.

Association between progesterone and estradiol-17beta treatment and protein expression of pgr and PGRMC1 in porcine luminal epithelial cells: a real-time cell proliferation approach.

The correct functionality (sensitivity and receptivity) of endometrial tissue is regulated by paracrine and endocrine pathways that activate several mediators or metabolic pathways and gene cascades. This study aimed to investigate the influence of E2 and P4 on progesterone receptor (PGR) and progesterone receptor membrane component 1 (PGRMC1) protein expression in porcine luminal epithelial cells and their influence on the proliferation of these cells in real-time. Surface uterine luminal epithelial cells were removed using sterile surgical blades from uterine horns of ten crossbred anestrus gilts. Following treatment with collagenase I, cells were separated and transferred into 48-well E-Plates for use in a realtime cell analyzer (RTCA). The luminal epithelial cells were cultured in vitro (IVC) in standard DMEM cell culture medium and incubated with E2 (10 pg/ml, 40 pg/ml, 500 pg/ml) and P4 (10 ng/ml, 40 ng/ ml, 500 ng/ml). The cell proliferation index was analyzed after 0-240 h, 0-120 h, 120-240 h. After using the RTCA analysis we found increased proliferation of luminal epithelial cells after treatment of low doses of P4 (10 and 40 ng/ml), (P < 0.001). Higher doses of P4 led to decrease of proliferation (P < 0.001). Conversely, higher doses of E2 (500 pg/ml) increased the proliferation index as compared to low doses (10 pg/ml) and control (P < 0.001). Confocal microscopic observations revealed that higher concentrations of E2 upregulate the expression of both PGR and PGRMC1. Additionally, P4 used in lower concentrations stimulated the expression of these receptors, too. Our study presents a new influence of E2 and P4 on the expression of PGR and PGRMC1 and on the real-time proliferation of porcine luminal epithelial cells. The relationship between PGR or PGRMC1 expression and the proliferation of luminal epithelial cells may be influenced (up- or down regulated) by E2 or P4 in a steroid type- and dose-dependent manner.