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

ABSTRACT: In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory "aged" oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.

SUBMITTER: Fujimine-Sato A

PROVIDER: S-EPMC8395835 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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

Fujimine-Sato Ayako A Kuno Takashi T Higashi Keiko K Sugawara Atsushi A Hiraga Hiroaki H Takahashi Aiko A Tanaka Keiko K Yokoyama Emi E Shiga Naomi N Watanabe Zen Z Yaegashi Nobuo N Tachibana Masahito M

International journal of molecular sciences 20210816 16

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume ...[more]

PMID: 34445470

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Project description:Study questionDoes the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF?Summary answerEmbryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen.What is known alreadyART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations.Study design, size, durationZygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested.Participants/materials, setting, methodsMitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM).Main results and the role of chanceEmbryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development.Large scale dataN/A.Limitations, reasons for cautionFindings in mice embryos might be different from human embryos.Wider implications of the findingsThis study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming.Study funding/competing interest(s)This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.

Project description:Somatic cell nuclear transfer and transcription factor overexpression can induce reprogramming of somatic cells, whereby one cell fate is changed into another cell fate of choice. Yet the efficiency of this process for generating functional cells is low, limiting their therapeutic applications. The persistence of transcriptional memory from the cell's prior identity is thought to be a major hindrance to effective reprogramming and differentiation to functional cell types. To explore the effects of transcriptional memory on cell fate specification of reprogrammed cells, we analyzed epidermal tissue development in nuclear transfer embryos derived from endoderm nuclei. Our findings reveal variations in the success rate of differentiation to functional cells across cell types in cloned embryos. While some cell types, such as goblet cells, differentiated normally, a specific subset of cells resisted cell fate reprogramming, adopting a new endoderm-like state and disrupting normal body patterning. Furthermore, inefficient transcriptional reprogramming correlated with reduced basal stem cell populations, faulty differentiation of basal stem cell-derived fates, and increased cell death in the epidermis of cloned embryos. Crucially, we identified that the memory of active transcriptional states linked to key endoderm transcription factors plays a significant role in these issues. Mimicking active state transcriptional memory of these genes through the forced expression of Sox17b and Foxa4 in the epidermis of fertilized embryos produced the same defects. On the contrary, reducing transcriptional memory by interfering with the expression of Sox17b led to the rescue of observed epidermal defects. In summary, our study suggests that transcriptional memory tends to persist predominantly in specific reprogrammed cell types, hindering their differentiation into functional cells and embryonic structures. This underscores the critical need to assess and reduce transcriptional memory from the previous somatic identity during reprogramming, to efficiently generate functional cell types for therapeutic applications.

Dataset Information

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

Publications

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

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