Cytoplasmic volume of recipient oocytes affects the nucleus reprogramming and the developmental competence of HMC buffalo (<i>Bubalus bubalis</i>) embryos

Abstract

The present study was undertaken to examine the effects of cytoplasmic volume on nucleus reprogramming and developmental competence of buffalo handmade cloning (HMC) embryos. We found that both HMC embryos derived from ~150% cytoplasm or ~225% cytoplasm resulted in a higher blastocyst rate and total cell number of blastocyst in comparison with those from ~75% cytoplasm (25.4 ± 2.0, 27.9 ± 1.6% vs. 17.9 ± 3.1%; 150 ± 10, 169 ± 12 vs. 85 ± 6, P<0.05). Meanwhile, the proportions of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) were also increased in the embryos derived from ~150 or ~225% enucleated cytoplasm compared to those from ~75% cytoplasm. Moreover, HMC embryos derived from ~225% cytoplasm showed a decrease of global DNA methylation from the 2-cell to the 4-cell stage in comparison with those of ~75% cytoplasm (P<0.05). Furthermore, the expression of embryonic genome activation (EGA) relative genes (eIF1A and U2AF) in HMC embryos derived from ~225% cytoplasm at the 8-cell stages was also found to be enhanced compared with that of the ~75% cytoplasm. Two of seven recipients were confirmed to be pregnant following transfer of blastocysts derived from ~225% cytoplasm, and one healthy cloned calf was delivered at the end of the gestation period, whereas no recipients were pregnant after the transfer of blastocysts derived from ~75% cytoplasm. These results indicate that the cytoplasmic volume of recipient oocytes affects donor nucleus reprogramming, and then further accounted for the developmental ability of the reconstructed embryos.

Exploration of the Cytoplasmic Function of Abnormally Fertilized Embryos via Novel Pronuclear-Stage Cytoplasmic Transfer

“…This observation indicated the hypothesis that an affected reprogramming process or improper maternal-zygotic transition might be the consequence of an inappropriate cytoplast activation due to the lack of sperm contribution. Despite the cytoplasmic volume being important for the nucleus reprogramming and developmental competence of embryos [50], proper progress of the post-fertilization cytoplasmic event may also be crucial to support subsequent embryo development. However, further study is needed to clarify the mechanism responsible for the difference in cytoplasmic function among abnormally fertilized embryos and, thus, the biological marker indicating cytoplasmic function.…”

Effect of Additional Cytoplasm of Cloned Embryo on In Vitro Developmental Competence and Reprogramming Efficiency in Mice

“…The cloned embryo efficiency is related to gene expression, histone methylation, and acetylation. Moreover, cytoplasmic volume and donor cells positively affect embryonic development and nuclear reprogramming (Beyhan et al, 2007;Liu et al, 2018). In a previous study, the reconstructed cloned embryo, using CICT, showed that gene expression levels of DNMT 1 and DNMT 3a were significantly lower than in the traditional cloning method, indicating that CICT can improve epigenetic reprogramming.…”