Expression of Selected Connexin and Aquaporin Genes and Real-Time Proliferation of Porcine Endometrial Luminal Epithelial Cells in Primary Culture Model.

Luminal epithelial cells are the first embryonic-maternal contact site undergoing very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume, with the help of aquaporins that provide a transcellular path of rapid water movement during the secretion and absorption of fluids, as well as connexins enabling the flow of inorganic ions and small molecules. In this work, we have examined how AQPs and Cx's behave in luminal epithelium primary cell culture. Cells obtained from porcine specimen during slaughter were primarily in vitro cultured for 7 days. Their proliferation patterns were then analyzed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes of gene of interest expression were analyzed on each of the seven days of the porcine luminal primary cell culture. Our study showed that the significant changes were noted in the case of Cx43, whose level of protein expression and distribution increases after 120 hours of culture, when the cells enter the lag phase, and maintains an upward trend until the end of the culture. We noted an increase in AQP4, AQP7, AQP8, and AQP11 levels throughout the entire culture period, while the largest differences in expression were found in AQP3, AQP4, and AQP10. The obtained results could become a point of reference for further in vivo and clinical research. Experiments conducted with these proteins showed that they influence the endometrial fluid content during the oestrous cycle and participate in the process of angiogenesis, which intensifies during endometrial development.

Expression pattern of new genes regulating female sex differentiation and in vitro maturational status of oocytes in pigs.

The processes underlying maturation of mammalian oocytes are considered crucial for the oocytes ability to undergo monospermic fertilization. The same factors of influence are suggested to impact the development of sex associated characteristics, allowing sex differentiation to progress during embryonic growth. The primary aim of the study was to analyze the gene ontology groups involved in regulation of porcine oocytes' response to endogenous stimuli. The results obtained would indicate potential genes influencing sex differentiation. Additionally, they could help to determine new genetic markers, expression profile of which is substantially regulated during porcine oocytes' in vitro maturation. To achieve that, porcine oocytes were collected for analysis before and after in vitro maturation. Pigs were used as they are a readily available model that presents significant similarity to humans in terms of physiology and anatomy. Microarray analysis of oocytes, before and after in vitro maturation was performed and later validated by RT-qPCR. We have particularly detected and analyzed genes belonging to gene ontology groups associated with hormonal stimulation during maturation of the oocytes, that exhibited significant change in expression (fold change ≥ |2|; p < 0.05) namely "Female sex differentiation" (CCND2, MMP14, VEGFA, FST, INHBA, NR5A1), "Response to endogenous stimulus" (INSR, ESR1, CCND2, TXNIP, TACR3, MMP14, FOS, AR, EGR2, IGFBP7, TGFBR3, BTG2, PLD1, PHIP, UBE2B) and "Response to estrogen stimulus" (INSR, ESR1, CCND2, IHH, TXNIP, TACR3, MMP14). Some of them were characteristic for just one of the described ontologies, while some belonged into multiple ontological terms. The genes were analyzed, with their relation to the processes of interest explained. Overall, the study provides us with a range of genes that might serve as molecular markers of in vitro maturation associated processes of the oocytes. This knowledge might serve as a reference for further studies and, after further validation, as a potentially useful knowledge in assessment of the oocytes during assisted reproduction processes.

"Positive Regulation of RNA Metabolic Process" Ontology Group Highly Regulated in Porcine Oocytes Matured In Vitro: A Microarray Approach.

The cumulus-oocyte complexes (COCs) growth and development during folliculogenesis and oogenesis are accompanied by changes involving synthesis and accumulation of large amount of RNA and proteins. In this study, the transcriptomic profile of genes involved in "oocytes RNA synthesis" in relation to in vitro maturation in pigs was investigated for the first time. The RNA was isolated from oocytes before and after in vitro maturation (IVM). Interactions between differentially expressed genes/proteins belonging to "positive regulation of RNA metabolic process" ontology group were investigated by STRING10 software. Using microarray assays, we found expression of 12258 porcine transcripts. Genes with fold change higher than |2| and with corrected p value lower than 0.05 were considered as differentially expressed. The ontology group "positive regulation of RNA metabolic process" involved differential expression of AR, INHBA, WWTR1, FOS, MEF2C, VEGFA, IKZF2, IHH, RORA, MAP3K1, NFAT5, SMARCA1, EGR1, EGR2, MITF, SMAD4, APP, and NR5A1 transcripts. Since all of the presented genes were downregulated after IVM, we suggested that they might be significantly involved in regulation of RNA synthesis before reaching oocyte MII stage. Higher expression of "RNA metabolic process" related genes before IVM indicated that they might be recognized as important markers and specific "transcriptomic fingerprint" of RNA template accumulation and storage for further porcine embryos growth and development.