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

Katarzyna Wojtanowicz-Markiewicz,Bartosz Kempisty,Maciej Zabel,Paul Mozdziak,Dorota Bukowska,Sandra Knap,Katarzyna Stefańska,Magdalena Kulus,Ievgenia Kocherova,Michal Jeseta,Hanna Piotrowska-Kempisty,Maurycy Jankowski,Paweł Antosik,Michał Nowicki

doi:10.1155/2020/7120375

Abstract

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.

Highlights

Continuing the cycle of research on gene expression in the porcine luminal epithelium cells in this work, we have tested the expression of genes responsible for the distribution of proteins involved in cellular communication. ese involved gap junction channel connexins: Cx36 (GJD2-gap junction protein delta 2, Cx36 protein), Cx37 (GJA4-gap junctionBioMed Research International protein alpha 4, Cx37 protein), Cx40 (GJD4-gap junction protein delta 4, Cx40 protein), and Cx43 (GJA1-gap junction protein alpha 1, Cx43 protein), as well as simple water channels in the form of aquaporins: AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, AQP 9, AQP 10, and AQP11.Connexins (GJC) are a large family of proteins that form gap junction; a single connexin forms hexagonal structures called connexons
In 1990 studies conducted on the human uterine epithelium, changes in gap junction configuration were observed during the menstrual cycle. e size and distribution of gap junctions in human endometrial epithelial cells varied depending on the phases of the menstrual cycle
We focused on 10 proteins from the AQP fluid transporter family and the 4 best-known connexins so far found in the endometrium. e main aim of the manuscript was to investigate their expression during in vitro culture and relate the results to the data obtained during real-time proliferation assay. e obtained results should provide information about the formation of the protein connections of interest, serving to improve knowledge about the in vitro processes occurring in the endometrial epithelial cells

Summary

Introduction

Continuing the cycle of research on gene expression in the porcine luminal epithelium cells in this work, we have tested the expression of genes responsible for the distribution of proteins involved in cellular communication. ese involved gap junction channel connexins: Cx36 (GJD2-gap junction protein delta 2, Cx36 protein), Cx37 (GJA4-gap junctionBioMed Research International protein alpha 4, Cx37 protein), Cx40 (GJD4-gap junction protein delta 4, Cx40 protein), and Cx43 (GJA1-gap junction protein alpha 1, Cx43 protein), as well as simple water channels in the form of aquaporins: AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, AQP 9, AQP 10, and AQP11.Connexins (GJC) are a large family of proteins that form gap junction; a single connexin forms hexagonal structures called connexons (nexus type connections). E construction of the gap junctions varied, depending on the structure of individual connexons. E protein structure of individual connexons is responsible for the biophysical properties of gap junction connections [1, 3]. E size and distribution of gap junctions in human endometrial epithelial cells varied depending on the phases of the menstrual cycle. In 1990 studies conducted on the human uterine epithelium, changes in gap junction configuration were observed during the menstrual cycle. It results with synchronization of proliferation and differentiation of the endometrial epithelium regulated by the by gap junction mediated cellular adherence. In the early phase of the proliferation, gap junctions were few and of small size, in contrast to the early secretory phase in which the connections were larger and more frequent [4]

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