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Fulltext Effect of DNase treatment on RNA extraction from preimplantation murine embryos

Norhazlin J, Nor-Ashikin MN, Hoh BP, Sheikh Abdul Kadir SH, Norita S, Mohd-Fazirul M, et al.

Genet. Mol. Res., 2015;14(3):10172-84.
PMID: 26345954 DOI: 10.4238/2015.August.28.1

The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase-treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.

Matched MeSH terms: Blastocyst/metabolism*
Fulltext Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos

Dasiman R, Rahman NS, Othman S, Mustafa MF, Yusoff NJ, Jusoff WH, et al.

Med Sci Monit Basic Res, 2013 Oct 04;19:258-66.
PMID: 24092420 DOI: 10.12659/MSMBR.884019

BACKGROUND: This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM).
MATERIAL/METHODS: Fifty female mice, aged 4-6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3.
RESULTS: The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos.
CONCLUSIONS: Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.

Matched MeSH terms: Blastocyst/metabolism*
Fulltext Epigenetic modification does not determine the time of POU5F1 transcription activation in cloned bovine embryos

Jafari S, Hosseini SM, Hajian M, Forouzanfar M, Jafarpour F, Abedi P, et al.

J Assist Reprod Genet, 2011 Nov;28(11):1119-27.
PMID: 22020531 DOI: 10.1007/s10815-011-9638-1

To investigate the effect of epigenetic modification on pattern, time and capacity of transcription activation of POU5F1, the key marker of pluripotency, in cloned bovine embryos.

Matched MeSH terms: Blastocyst/metabolism
Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent

Jafari S, Hosseini MS, Hajian M, Forouzanfar M, Jafarpour F, Abedi P, et al.

Mol. Reprod. Dev., 2011 Aug;78(8):576-84.
PMID: 21721066 DOI: 10.1002/mrd.21344

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.

Matched MeSH terms: Blastocyst/metabolism
Effects of thyroxine on expression of proteins related to thyroid hormone functions (TR-α, TR-β, RXR and ERK1/2) in uterus during peri-implantation period

Sayem ASM, Giribabu N, Muniandy S, Salleh N

Biomed Pharmacother, 2017 Dec;96:1016-1021.
PMID: 29221723 DOI: 10.1016/j.biopha.2017.11.128

INTRODUCTION: Thyroid hormone is known to play important role during embryo implantation, however mechanisms underlying its actions in uterus during peri-implantation period has not been fully identified. In this study, we hypothesized that thyroid hormone could affect expression of proteins related to its function, where these could explain mechanisms for its action in uterus during this period.
METHODS: Female rats, once rendered hypothyroid via oral administration of methimazole (0.03% in drinking water) for twenty-one days were mated with fertile euthyroid male rats at 1:1 ratio. Pregnancy was confirmed by the presence of vaginal plug and this was designated as day-1. Thyroxine (20, 40 and 80 μg/kg/day) was then subcutaneously administered to pregnant, hypothyroid female rats for three days. A day after last injection (day four pregnancy), female rats were sacrificed and expression of thyroid hormone receptors (TR-α and β), retinoid X receptor (RXR) and extracellular signal-regulated kinase (ERK1/2) in uterus were quantified by Western blotting while their distribution in endometrium was visualized by immunofluorescence.
RESULTS: Expression of TRα-1, TRβ-1 and ERK1/2 proteins in uterus increased with increasing doses of thyroxine however no changes in RXR expression was observed. These proteins were found in the stroma with their distribution levels were relatively higher following thyroxine treatment.
CONCLUSIONS: Increased expression of TRα-1, TRβ-1 and ERK1/2 at day 4 pregnancy in thyroxine-treated hypothyroid pregnant rats indicate the importance of thyroxine in up-regulating expression of these proteins that could help mediate the uterine changes prior to embryo implantation.

Matched MeSH terms: Blastocyst/metabolism