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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.
Pyruvate reduces in vitro the embryotoxic effect of peritoneal fluid from infertile women with endometriosis

Noordin L, San GT, Singh HJ, Othman MS, Hafizah W

Eur J Obstet Gynecol Reprod Biol, 2008 Jan;136(1):67-73.
PMID: 18079036

To ascertain the embryotoxicity of peritoneal fluid from infertile women with endometriosis (PF-E), on mouse embryos in culture and to examine the effect of pyruvate in the culture medium on PF-E induced embryotoxicity.
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.

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