76 EFFECT OF CULTURE METHOD ON THE mRNA EXPRESSION BEFORE AND AFTER CRYOPRESERVATION IN BOVINE BLASTOCYSTS

Blastocyst mRNA expression and cryopreservability are thought to be suitable indicators of embryo quality and developmental competence and have been shown to be affected by production methods and culture systems. The aim of the present study was to assess cryosurvival and levels of mRNA expression of selected genes [occludin, desmocollin 2, solute carrier family 2 member 3 (formerly glucose transporter 3), BAX, BCL xL, heat shock protein A1A (formerly heat shock protein 70.1), aquaporin 3, and DNA methyltransferase 1a] of bovine blastocysts derived by 4 different, established culture methods [in vitro production (IVP); multiple-ovulation embryo transfer (MOET); transfer into the heifer oviducts of gametes (GIFT); or in vitro derived cleaved stage embryos (Days 2–7)]. Linear models were used for the comparison of the relative abundances of the blastocyst mRNA transcripts. Separate 1-way ANOVA were used. The production methods were used as factors, except for the comparisons between pre- and post-cryopreservation, where 2-way ANOVA were used. The level of significance was set at P ≤ 0.05. A significant difference in re-expansion rates was found only at 24 h post-thawing, with significantly higher rates in blastocysts produced in vitro compared to embryos of the Days 2–7 group. Levels of mRNA expression were assessed using RT-qPCR. Before cryopreservation of embryos, no significant inter-group differences were seen. However, significantly more desmocollin 2 mRNA expression was detected in embryos of the MOET group compared with blastocysts derived by the other production methods. Post-cryopreservation, blastocysts of 3 embryo production groups (IVP, MOET, Days 2–7) were available for analysis. Compared with levels of mRNA expression before cryopreservation, re-expanded blastocysts after cryopreservation showed a significant up-regulation of heat shock protein A1A transcripts in all groups, and of solute carrier family 2 member 3 transcripts only in the IVP-derived group. The BAX, BCL-xL, occludin, and desmocollin 2 were significantly up-regulated in embryos of the MOET and IVP groups after cryopreservation, as compared with their counterparts before cryopreservation. None of the culture groups showed any pre- v. post-cryopreservation differences in the aquaporin 3 and the DNA methyltransferase 1 mRNA levels. Blastocysts derived by transfer of in vitro derived cleaved stage embryos into the oviduct of synchronised heifers (Days 2–7) did not show any pre- v. post-cryopreservation differences in the mRNA levels of any of the assessed genes. These results merit further investigation. After the process of cryopreservation and thawing, re-expanded embryos of the MOET and IVP groups do increase their mRNA levels to prepare for hatching and further development.

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Insulin regulation of solute carrier family 2 member 1 (glucose transporter 1) expression and glucose uptake in decidualizing human endometrial stromal cells: an in vitro study

Reproductive Biology and Endocrinology ◽

10.1186/s12958-020-00674-0 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Ivika Jakson ◽

Dorina Ujvari ◽

Sebastian Brusell Gidlöf ◽

Angelica Lindén Hirschberg

Keyword(s):

Glucose Uptake ◽

Stromal Cells ◽

Glucose Transporter ◽

Mrna Levels ◽

Solute Carrier Family ◽

Endometrial Stromal Cells ◽

Glucose Transporter 1 ◽

Protein Levels ◽

Solute Carrier

Abstract Background Solute carrier family 2 member 1 (SLC2A1; previously known as glucose transporter 1), is the most abundant glucose transporter in human endometrium and is up-regulated during decidualization, whereas high insulin may have a negative impact on this process. The present study aimed to investigate the effect of insulin on the expression of SLC2A1 and glucose uptake in decidualizing human endometrial stromal cells. Methods We induced in vitro decidualization of endometrial stromal cells obtained from regularly menstruating healthy non-obese women. The cells were treated with increasing concentrations of insulin, and the involvement of the transcription factor forkhead box O1 (FOXO1) was evaluated using a FOXO1 inhibitor. SLC2A1 mRNA levels were measured by Real-Time PCR and protein levels were evaluated by immunocytochemistry. Glucose uptake was estimated by an assay quantifying the cellular uptake of radioactive glucose. One-way ANOVA, Dunnett’s multiple comparisons test and paired t-test were used to determine the statistical significance of the results. Results We found that insulin dose-dependently decreased SLC2A1 mRNA levels and decreased protein levels of SLC2A1 in decidualizing human endometrial stromal cells. Transcriptional inactivation of FOXO1 seems to explain at least partly the down-regulation of SLC2A1 by insulin. Glucose uptake increased upon decidualization, whereas insulin treatment resulted in a slight inhibition of the glucose uptake, although not significant for all insulin concentrations. Conclusions These results indicate an impairment of decidualization by high concentrations of insulin. Future studies will determine the clinical significance of our results for endometrial function and decidualization in women with insulin resistance and hyperinsulinemia.

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Evaluation of Reference Genes and Age Estimation of Forensically Useful Aldrichina grahami (Diptera: Calliphoridae) During Intrapuparial Period

Journal of Medical Entomology ◽

10.1093/jme/tjaa144 ◽

2020 ◽

Author(s):

Zhuoying Liu ◽

Han Han ◽

Wei Chen ◽

Shiwen Wang ◽

Fanming Meng ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Ribosomal Protein ◽

Developmental Stage ◽

Mrna Expression ◽

Ribosomal Rna ◽

Reference Genes ◽

28S Rrna ◽

Mrna Levels ◽

Gene Markers

Abstract The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (β-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

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Cloning of the gene encoding peptide-binding protein 74 shows that it is a new member of the heat shock protein 70 family.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3598 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3598-3610 ◽

Cited By ~ 104

Author(s):

S Z Domanico ◽

D C DeNagel ◽

J N Dahlseid ◽

J M Green ◽

S K Pierce

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Antigen Processing ◽

Peptide Binding ◽

Sequence Information ◽

Mrna Levels ◽

Mature Protein ◽

Hsp70 Mrna ◽

Hsp70 Family

We have previously described peptide-binding proteins of 72 and 74 kDa (PBP72/74), which have been implicated as playing a role in antigen processing and are serologically related to the 70-kDa heat shock protein (hsp70) family. Here we report the cloning and sequencing of the cDNA encoding PBP74 in mice and in humans, accomplished by using amino acid sequence information obtained from the purified protein. We show that PBP74 is highly homologous to members of the hsp70 family but, significantly, is not identical to any known member of this family. Inspection of the cDNA nucleotide sequence indicates that it encodes a 46-residue N-terminal peptide which is not present in the mature protein. Transcription and translation in vitro of the PBP74 cDNA verified that it encodes a form of PBP74 which is larger than the mature protein. The presequence does not conform to known motifs for organelle-targeting sequences, and at present, its function is not known. By confocal microscopy, PBP74 was localized to cytoplasmic vesicles but not to the nucleus, mitochondria, or plasma membrane by using antibodies specific for the N-terminal 16 residues of PBP74. By RNA filter hybridization analysis, PBP74 mRNAs are detected in all cell types tested. Exposure of cells to heat shock does not result in an increase in the mRNA levels of PBP74, unlike the dramatic increase observed for the stress-inducible hsp70 mRNA. Thus, PBP74 appears to be a constitutive, new member of the hsp70 family.

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Brief report: Expression of TNF-α in chronic periapical lesions correlates with expression of bacterial chaperonin-60

Vojnosanitetski pregled ◽

10.2298/vsp201106052s ◽

2021 ◽

pp. 52-52

Author(s):

Jelena Stanisic-Zindovic ◽

Branko Mihailovic ◽

Filip Djordjevic ◽

Marija Milovanovic ◽

Nebojsa Arsenijevic ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Mrna Expression ◽

Mrna Levels ◽

Periapical Lesions ◽

Quantitative Expression ◽

Tnf Α ◽

Chaperonin 60 ◽

Anti Inflammatory ◽

Anti Inflammatory Cytokine

Background/Aim: The aim of this study is to determine the quantitative expression of the bacterial heat shock protein, Chaperonin-60 (Cpn60) and pro-inflammatory and anti-inflammatory cytokine in periapical tissue, obtained from individuals with chronic periapical lesions and to determine the correlation between the expression of the bacterial heat shock protein and the expression of these cytokines. Methods. The study was performed on 18 periapical lesions and 6 control samples of healthy periapical tissue, taken at the Clinic of Dental Medicine, Faculty of Medical 4 Sciences University of Pristina, Kosovska Mitrovica. The levels of mRNA expression of pro- and anti- inflammatory cytokines and bacterial heat shock protein were determined by real time quantitative RT-PCR. Results. Analysis revealed significantly higher mRNA levels of TNF-? and Cpn60 in the tissue of periapical lesions compared with normal periapical tissue (P <0.05). Contrary to these results, the mRNA expression of anti-inflammatory IL-10 was significantly higher in the samples of normal periapical tissue compared with the mRNA levels of this cytokine in the tissue of periapical lesions (P <0.001). Expression of Cpn60 is in strong correlation with TNF-? expression in periapical lesions. Conclusion. Cpn60 released from bacteria in periapical tissue could be a strong stimulator of inflammatory response and one of the important players in the pathogenesis of periapical lesions.

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Expression of Heat Shock Protein 72 in Peritoneal Leukocytes is Induced by Peritoneal Dialysis

Peritoneal Dialysis International ◽

10.1177/089686080702700314 ◽

2007 ◽

Vol 27 (3) ◽

pp. 288-295 ◽

Cited By ~ 6

Author(s):

Lukasz Marzec ◽

Tomasz Liberek ◽

Michal Chmielewski ◽

Ewa Bryl ◽

Jacek M. Witkowski ◽

...

Keyword(s):

Peritoneal Dialysis ◽

Heat Shock ◽

Heat Shock Protein ◽

Peripheral Blood ◽

Peritoneal Macrophages ◽

Mrna Levels ◽

Blood Monocytes ◽

Heat Shock Protein 72 ◽

Peritoneal Leukocytes

Background One of the main limitations of peritoneal dialysis (PD) is deterioration of functional and morphological characteristics of the peritoneum. This complication appears to be related to the low biocompatibility profile of PD fluids. Recently, induction of the heat shock protein (HSP) stress response was demonstrated in cultured human mesothelial cells exposed to PD fluid in vitro. We investigated whether expression of heat shock protein 72 (HSP-72) in peritoneal macrophages is induced upon exposure to PD fluid during continuous ambulatory PD. Methods Peritoneal leukocytes were isolated from 4-hour dwell dialysate; peripheral blood mononuclear cells (PBMC) and peripheral blood monocytes isolated from the same patients were used as a control. In separate experiments, PBMC from healthy individuals were exposed in vitro to different PD fluids or to culture media. Expression of HSP-72 was assessed by Western immunoblotting, flow cytometry, and reverse-transcription polymerase chain reaction analysis. Results Macrophages and leukocytes isolated from dialysis effluent expressed significantly increased HSP-72 and mRNA levels compared to blood monocytes and PBMC of the same patients. In vitro exposure of PBMC to fresh PD fluids resulted in significantly higher expression of HSP-72 compared to those incubated in culture medium. PBMC exposed in vitro to standard lactate-buffered dialysis fluids also expressed significantly more HSP-72 compared to cells exposed to bicarbonate/lactate-buffered fluids. Conclusion Our results indicate that exposure to PD fluids during dialysis triggers a shock response in peritoneal cells, which is manifested by significantly increased HSP-72 expression at both protein and mRNA levels. Analysis of this protein expression in peritoneal macrophages could be a new, convenient, and relevant way to assess the biocompatibility of PD fluids ex vivo.

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