mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages

During final maturation (between LH surge and ovulation) in vivo, a switch from oestradiol to progesterone dominance within the follicle is well described. The aim was to mimic the in vivo situation during in vitro maturation via the supplementation of different gonadotropins. Groups of 30 cumulus-oocyte complex (abattoir-derived ovaries) were matured in TCM 199 plus different gonadotropins (eCG/hCG; FSH/LH, each in 0.05 or 0.01 IU; only FSH 0.05 IU; without gonadotropins) using a standard protocol without oil overlay. In Experiment 1, denuded oocytes were collected at 0 h (immature) and after 24 h of in vitro maturation (IVM; exhibit first polar body). In Experiment 2, oocytes were collected at different time points [0 (immature), 4, 8, 12, 16, 20, and 24 h] after IVM in eCG/hCG-supplemented medium. They were individually stored at −80°C until analyses. Transcripts of developmental competence (BMP15, GDF9, ZAR1), glucose or steroid metabolism (G6PD, STAR), and progesterone receptors (PGR, PGRMC1/2) were examined in individual oocytes via quantitative RT-PCR (n = 5). For statistical analyses, 1-way ANOVA followed by a Tukey test was used. Relative abundance of BMP15 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group matured for 24 h with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature oocytes. Relative amount of G6PD and PGRMC2 mRNA was significantly lower (P ≤ 0.05) in mature oocytes of the group with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature ones. Relative abundance of GDF9, STAR, and ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group with eCG/hCG, FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins compared with immature oocytes. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in mature oocytes of the group with eCG/hCG than in immature oocytes, FSH/LH 0.01IU, FSH 0.05 IU, and without gonadotropins (Experiment 1). Relative amount of GDF9 transcripts was significantly lower (P ≤ 0.05) in mature oocytes collected after 24 h than in immature ones. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in oocytes collected after 20 and 24 h of IVM than in immature ones. Relative amount of ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h of IVM than in immature oocytes; likewise, they were significantly lower (P ≤ 0.05) in oocytes collected after 12, 16, 20, and 24 h than in oocytes collected after 4 h of IVM. Relative amount of STAR mRNA was significantly lower (P ≤ 0.05) in oocytes collected after 24 h than in immature ones, and significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h than in oocytes collected after 4 h of IVM (Experiment 2). The results suggest a down-regulation of most transcripts during the period of IVM with different gonadotropin supplements with exception of PGR. Furthermore, most transcripts follow a timely regulated mRNA expression pattern during the entire IVM period. We gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369, WR 154/3–1).

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SARS-CoV-2 Infection-Induced Promoter Hypomethylation as an Epigenetic Modulator of Heat Shock Protein A1L (HSPA1L) Gene

Frontiers in Genetics ◽

10.3389/fgene.2021.622271 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jibran Sualeh Muhammad ◽

Narjes Saheb Sharif-Askari ◽

Zheng-Guo Cui ◽

Mawieh Hamad ◽

Rabih Halwani

Keyword(s):

Epithelial Cells ◽

Mrna Expression ◽

Candidate Genes ◽

Epigenetic Regulation ◽

Dna Methyltransferases ◽

Lung Epithelial Cells ◽

Potential Candidate ◽

Blood Samples ◽

Lung Epithelial

Numerous researches have focused on the genetic variations affecting SARS-CoV-2 infection, whereas the epigenetic effects are inadequately described. In this report, for the first time, we have identified potential candidate genes that might be regulated via SARS-CoV-2 induced DNA methylation changes in COVID-19 infection. At first, in silico transcriptomic data of COVID-19 lung autopsies were used to identify the top differentially expressed genes containing CpG Islands in their promoter region. Similar gene regulations were also observed in an in vitro model of SARS-CoV-2 infected lung epithelial cells (NHBE and A549). SARS-CoV-2 infection significantly decreased the levels of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in lung epithelial cells. Out of 14 candidate genes identified, the expression of 12 genes was upregulated suggesting promoter hypomethylation, while only two genes were downregulated suggesting promoter hypermethylation in COVID-19. Among those 12 upregulated genes, only HSPA1L and ULBP2 were found to be upregulated in AZA-treated lung epithelial cells and immune cells, suggesting their epigenetic regulation. To confirm the hypomethylation of these two genes during SARS-CoV-2 infection, their promoter methylation and mRNA expression levels were determined in the genomic DNA/RNA obtained from whole blood samples of asymptomatic, severe COVID-19 patients and equally matched healthy controls. The methylation level of HSPA1L was significantly decreased and the mRNA expression was increased in both asymptomatic and severe COVID-19 blood samples suggesting its epigenetic regulation by SARS-CoV-2 infection. Functionally, HSPA1L is known to facilitate host viral replication and has been proposed as a potential target for antiviral prophylaxis and treatment.

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

BioMed Research International ◽

10.1155/2020/7120375 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Katarzyna Wojtanowicz-Markiewicz ◽

Magdalena Kulus ◽

Sandra Knap ◽

Ievgenia Kocherova ◽

Maurycy Jankowski ◽

...

Keyword(s):

Cell Culture ◽

Epithelial Cells ◽

Primary Cell Culture ◽

Water Movement ◽

Inorganic Ions ◽

Primary Cell ◽

Lag Phase ◽

Luminal Epithelial Cells

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.

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Study Effects of Drug Treatment and Physiological Physical Stimulation on Surfactant Protein Expression of Lung Epithelial Cells Using a Biomimetic Microfluidic Cell Culture Device

Micromachines ◽

10.3390/mi10060400 ◽

2019 ◽

Vol 10 (6) ◽

pp. 400 ◽

Cited By ~ 1

Author(s):

Ting-Ru Lin ◽

Sih-Ling Yeh ◽

Chien-Chung Peng ◽

Wei-Hao Liao ◽

Yi-Chung Tung

Keyword(s):

Cell Culture ◽

Epithelial Cells ◽

Drug Treatment ◽

Surfactant Protein ◽

Lung Epithelial Cells ◽

Single Chip ◽

Physical Stimulation ◽

Lung Epithelial

This paper reports a biomimetic microfluidic device capable of reconstituting physiological physical microenvironments in lungs during fetal development for cell culture. The device integrates controllability of both hydrostatic pressure and cyclic substrate deformation within a single chip to better mimic the in vivo microenvironments. For demonstration, the effects of drug treatment and physical stimulations on surfactant protein C (SPC) expression of lung epithelial cells (A549) are studied using the device. The experimental results confirm the device’s capability of mimicking in vivo microenvironments with multiple physical stimulations for cell culture applications. Furthermore, the results indicate the critical roles of physical stimulations in regulating cellular behaviors. With the demonstrated functionalities and performance, the device is expected to provide a powerful tool for further lung development studies that can be translated to clinical observation in a more straightforward manner. Consequently, the device is promising for construction of more in vitro physiological microenvironments integrating multiple physical stimulations to better study organ development and its functions.

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Effect of ganglioside GT1b treatment during porcine in vitro maturation on embryonic development and mRNA expression pattern in cumulus cell

Reproduction Abstracts ◽

10.1530/repabs.1.p093 ◽

2014 ◽

Author(s):

Seon-Ung Hwang ◽

Sang-Hwan Hyun

Keyword(s):

Embryonic Development ◽

Mrna Expression ◽

Expression Pattern ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Mrna Expression Pattern ◽

Ganglioside Gt1b

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111. LIF REGULATION OF EPITHELIAL CELL FUCOSYLTRANSFERASE EXPRESSION IN MOUSE ENDOMETRIUM DURING EARLY PREGNANCY

Reproduction Fertility and Development ◽

10.1071/srb09abs111 ◽

2009 ◽

Vol 21 (9) ◽

pp. 30

Author(s):

M. J. Jasper ◽

A. Care ◽

J. D. Aplin ◽

S. A. Robertson

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Mrna Expression ◽

Seminal Vesicle ◽

Diphtheria Toxin ◽

Embryo Implantation ◽

Mouse Uterus ◽

Uterine Epithelial Cells

Fucosyltransferase (FUT) enzymes are key regulators of glycosylated structures mediating embryo attachment to uterine epithelial cells at implantation. The identity of local regulatory signals is unknown. We have previously shown that macrophage co-culture significantly increases epithelial cell FUT2 and FUT4 mRNA expression in vitro, and the effect of co-culture is replicated with macrophage conditioned media. We aimed to define the identity of macrophage-secreted agents active in regulating FUT expression in mouse uterine epithelial cells, and to investigate the importance of macrophages for FUT expression in vivo. FUT1, FUT2, and FUT4 mRNAs were measured by qRT-PCR and data was normalised to β-actin mRNA in mouse uterine epithelial cells after culture with cytokines known to be secreted by macrophages. mRNA was also quantified in luminal epithelium laser-microdissected from mouse uterus on day 4 after mating with intact males or seminal vesicle deficient (SVX) males, to induce normal or depleted uterine macrophage populations respectively. Lectin staining on day 4 pc was quantified using ImageJ software in an alternate model of transient, systemic macrophage ablation following diphtheria toxin administration to CD11b-DTR transgenic mice. Epithelial FUT2 mRNA expression was specifically enhanced in vitro by addition of rLIF (2 ng/ml) (mean relative expression ± SEM, control 100 ± 5.6; rLIF 162.1 ± 11.5). Depletion of macrophages by mating with seminal vesicle deficient males reduced epithelial FUT2 mRNA expression on day 4 pc (intact 100 ± 9.1; SVX 73.5 ± 8.6). Depletion of macrophages in the CD11b-DTR mouse model caused a 30% reduction in the expression of the resulting glycoprotein epitope (α1,2 fucose) as observed by intensity of endometrial epithelial UEA-1 staining (control 100 ± 10; CD11b-DTR 72 ± 9) 24 hr post diphtheria toxin administration. In conclusion, these data demonstrate that endometrial epithelial FUT2 mRNA synthesis in preparation for embryo implantation is mediated via LIF and potentially other factors secreted from macrophages recruited during the inflammatory response to insemination. Uterine macrophage abundance and phenotype may thus be a determinant of receptivity to implantation.

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Increased mRNA expression of selected pro-inflammatory factors in inflamed bovine endometrium in vivo as well as in endometrial epithelial cells exposed to Bacillus pumilus in vitro

Reproduction Fertility and Development ◽

10.1071/rd14219 ◽

2016 ◽

Vol 28 (7) ◽

pp. 982 ◽

Cited By ~ 12

Author(s):

Martina A. Gärtner ◽

Sarah Peter ◽

Markus Jung ◽

Marc Drillich ◽

Ralf Einspanier ◽

...

Keyword(s):

Epithelial Cells ◽

Mrna Expression ◽

Bacillus Pumilus ◽

Cultured Cells ◽

Inflammatory Factors ◽

Endometrial Cells ◽

Subclinical Endometritis ◽

Endometrial Epithelial Cells

Endometrial epithelium plays a crucial role in the first immune response to invading bacteria by producing cytokines and chemokines. The aim of this study was to investigate the first inflammatory response of the endometrium in vivo and in vitro. Gene expression of several pro-inflammatory factors and Toll-like receptors (TLR2, -4, -6) was determined in endometrial cytobrush samples obtained from healthy cows and cows with clinical or subclinical endometritis. Endometrial epithelial cells were co-cultured with an isolated autochthonous uterine bacterial strain Bacillus pumilus. Total RNA was extracted from in vivo and in vitro samples and subjected to real-time reverse transcription polymerase chain reaction. CXC ligands (CXCL) 1/2 and CXC chemokine receptor (CXCR) 2 mRNA expression was higher in cows with subclinical endometritis and CXCL3 mRNA expression was higher in cows with clinical endometritis compared with healthy cows. B. pumilus induced cell death of epithelial cells within 24 h of co-culturing. The presence of B. pumilus resulted in significantly higher mRNA expression of interleukin 1α (IL1A), IL6, IL8, CXCL1–3 and prostaglandin–endoperoxide synthase 2 in co-cultured cells compared with untreated controls. The maximum increase was mainly detected after 2 h. These results support the hypothesis that bacterial infection of endometrial cells might induce prompt synthesis of pro-inflammatory cytokines resulting in a local inflammatory reaction.

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In vivo HPV 16 E5 mRNA: Expression pattern in patients with squamous intra-epithelial lesions of the cervix

Journal of Clinical Virology ◽

10.1016/j.jcv.2011.06.007 ◽

2011 ◽

Vol 52 (2) ◽

pp. 79-83 ◽

Cited By ~ 7

Author(s):

Laura Lorenzon ◽

Francesca Mazzetta ◽

Aldo Venuti ◽

Antonio Frega ◽

Maria Rosaria Torrisi ◽

...

Keyword(s):

Mrna Expression ◽

Expression Pattern ◽

Hpv 16 ◽

Mrna Expression Pattern ◽

Epithelial Lesions

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Interdependence between Chromogranin-A, Alternatively Activated Macrophages, Tight Junction Proteins and the Epithelial Functions. A Human and In-Vivo/In-Vitro Descriptive Study

International Journal of Molecular Sciences ◽

10.3390/ijms21217976 ◽

2020 ◽

Vol 21 (21) ◽

pp. 7976

Author(s):

Nour Eissa ◽

Hayam Hussein ◽

Diane M. Tshikudi ◽

Geoffrey N. Hendy ◽

Charles N. Bernstein ◽

...

Keyword(s):

Epithelial Cells ◽

Mrna Expression ◽

Chromogranin A ◽

Intestinal Inflammation ◽

Collagen Deposition ◽

Activated Macrophages ◽

Alternatively Activated Macrophages ◽

Alternatively Activated

Background: Ulcerative colitis (UC) is characterized by altered chromogranin-A (CHGA), alternatively activated macrophages (M2) and intestinal epithelial cells (IECs). We previously demonstrated that CHGA is implicated in colitis progression by regulating the macrophages. Here, we investigated the interplay between CHGA, M2, tight junctions (TJ) and IECs in an inflammatory environment. Methods: Correlations between CHGA mRNA expression of and TJ proteins mRNA expressions of (Occludin [OCLN], zonula occludens-1 [ZO1], Claudin-1 [CLDN1]), epithelial associated cytokines (interleukin [IL]-8, IL-18), and collagen (COL1A2) were determined in human colonic mucosal biopsies isolated from active UC and healthy patients. Acute UC-like colitis (5% dextran sulphate sodium [DSS], five days) was induced in Chga-C57BL/6-deficient (Chga−/−) and wild type (Chga+/+) mice. Col1a2 TJ proteins, Il-18 mRNA expression and collagen deposition were determined in whole colonic sections. Naïve Chga−/− and Chga+/+ peritoneal macrophages were isolated and exposed six hours to IL-4/IL-13 (20 ng/mL) to promote M2 and generate M2-conditioned supernatant. Caco-2 epithelial cells were cultured in the presence of Chga−/− and Chga+/+ non- or M2-conditioned supernatant for 24 h then exposed to 5% DSS for 24 h, and their functional properties were assessed. Results: In humans, CHGA mRNA correlated positively with COL1A2, IL-8 and IL-18, and negatively with TJ proteins mRNA markers. In the experimental model, the deletion of Chga reduced IL-18 mRNA and its release, COL1A2 mRNA and colonic collagen deposition, and maintained colonic TJ proteins. Chga−/− M2-conditioned supernatant protected caco-2 cells from DSS and oxidative stress injuries by improving caco-2 cells functions (proliferation, viability, wound healing) and by decreasing the release of IL-8 and IL-18 and by maintaining the levels of TJ proteins, and when compared with Chga+/+ M2-conditioned supernatant. Conclusions: CHGA contributes to the development of intestinal inflammation through the regulation of M2 and epithelial cells. Targeting CHGA may lead to novel biomarkers and therapeutic strategies in UC.

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