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The effect of sodium selenite on apoptotic gene expression and development of in vitro cultured mouse oocytes in comparison with in vivo obtained oocytes.

ABSTRACT: In vitro maturation (IVM) of oocytes is widely used in assisted reproduction technologies. The present study aimed to improve the in vitro oocyte maturation and its development through enriching the culture media with sodium selenite (SS). Moreover, the effects of SS on the expression of the oocytes apoptosis-related genes were assessed. In this study, male and female NMRI mice were used and after collecting their germinal vesicle (GV) oocytes, they were cultured with SS (experimental group) and without SS (control group). Collected metaphase II oocytes (MII) from the fallopian tube were considered as in vivo group. After in vitro culture, the oocytes were assessed in terms of nuclear maturation. The MII oocytes were inseminated and the development was examined until the blastocyst stage. Also, oocytes were subjected to the molecular analysis for evaluating the expression of BAX, BCL2, P53, and BAD genes using the real-time RT-PCR. The maturation rate was significantly increased in the SS supplemented group compared to the control one. The developmental rate of the embryos was significantly higher for both of the in vivo and SS supplemented groups rather than the control one, however, no significant difference was seen between these rates of the experimental and in vivo groups. Real-time RT-PCR did not show any significant differences in the expression of the apoptosis-related genes for all of the studied groups. The p53 gene was not expressed in any of groups. Sodium selenite improved the oocyte developmental competence but did not change the expression of the apoptosis-related genes in MII oocytes.

SUBMITTER: Malekzadeh Kebria M

PROVIDER: S-EPMC7904118 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The effect of sodium selenite on apoptotic gene expression and development of in vitro cultured mouse oocytes in comparison with in vivo obtained oocytes.

Malekzadeh Kebria Maziar M Salehnia Mojdeh M Zavareh Saeed S Moazzeni Seyyed Saeed SS

Veterinary research forum : an international quarterly journal 20201215 4

In vitro maturation (IVM) of oocytes is widely used in assisted reproduction technologies. The present study aimed to improve the in vitro oocyte maturation and its development through enriching the culture media with sodium selenite (SS). Moreover, the effects of SS on the expression of the oocytes apoptosis-related genes were assessed. In this study, male and female NMRI mice were used and after collecting their germinal vesicle (GV) oocytes, they were cultured with SS (experimen ...[more]

PMID: 33643591

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Project description:Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data.Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly.Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies.

Project description:BackgroundReproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.ResultsAn in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O2) and hypoxia (5% oxygen, 5% O2). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O2 condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile.ConclusionsOur observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O2 had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.

Dataset Information

The effect of sodium selenite on apoptotic gene expression and development of in vitro cultured mouse oocytes in comparison with in vivo obtained oocytes.

Publications

The effect of sodium selenite on apoptotic gene expression and development of <i>in vitro</i> cultured mouse oocytes in comparison with <i>in vivo</i> obtained oocytes.

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