Embryonic fate after somatic cell nuclear transfer in non-enucleated goldfish oocytes is determined by first cleavages and DNA methylation patterns

Alexandra Depincé,Charlène Rouillon,Pierre-Yves Le Bail,Catherine Labbé

doi:10.1038/s41598-021-83033-2

Abstract

Reducing the variability in nuclear transfer outcome requires a better understanding of its cellular and epigenetic determinants, in order to ensure safer fish regeneration from cryobanked somatic material. In this work, clones from goldfish were obtained using cryopreserved fin cells as donor and non-enucleated oocytes as recipients. We showed that the high variability of clones survival was not correlated to spawn quality. Clones were then characterized for their first cleavages pattern in relation to their developmental fate up to hatching. The first cell cycle duration was increased in clones with abnormal first cleavage, and symmetric first two cleavages increased clone probability to reach later on 24 h- and hatching-stages. At 24 h-stage, 24% of the clones were diploids and from donor genetic origin only. However, ploidy and genetic origin did not determine clones morphological quality. DNA methylation reprogramming in the promoter region of pou2, nanog, and notail marker genes was highly variable, but clones with the nicest morphologies displayed the best DNA methylation reprogramming. To conclude, non-enucleated oocytes did allow authentic clones production. The first two cell cycles were a critical determinant of the clone ability to reach hatching-stage, and DNA methylation reprogramming significantly influenced clones morphological quality.

Highlights

One great difficulty with nuclear transfer in fish resides in oocytes large size and opacity, owing to the chorion and yolk reserves, which impedes the removal of maternal DNA
The objective of this work was to understand to what extend early cellular defects and reprogramming failures will affect embryo development after SCNT in fish, as understanding the cause of abnormalities in clones is expected to facilitate the choice of relevant strategies to improve nuclear transfer outcome
The present study explored the characteristics of goldfish embryos obtained by somatic cell nuclear transfer

Summary

Introduction

One great difficulty with nuclear transfer in fish resides in oocytes large size and opacity, owing to the chorion and yolk reserves, which impedes the removal of maternal DNA. The occurrence of such outcome in our system has never been quantified Another major issue of SCNT is the reprogramming of the donor differentiated cells so that an embryonic profile of gene expression can be established in the clones. Epigenetic actors necessary for proper embryonic pattern to take place, including DNA methylation, are proposed to be dynamically reprogrammed during early embryo development in m ammals[23] and in fish[24]. Despite this supposedly favorable 10 mitosis lapse, fish clones suffer the same drop in survival after EGA13,25 as mammalian ones. Epigenetic reprogramming of the clones was estimated from DNA methylation level in the promoter region of three marker genes, pou[2], nanog and notail, chosen according to their differentially methylated pattern between embryos and adult tissues

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