Dynamic shapes of the zygote and two-cell mouse and human

ABSTRACT Mouse zygote morphokinetics were measured during interphase, the mitotic period, cytokinesis, and two-cell stage. Sequences of rounder–distorted–rounder shapes were revealed, as were changing patterns of cross section area. A calcium chelator and an actin-disrupting agent inhibited the area changes that occurred between pronuclear envelope breakdown and cytokinesis. During cell division, two vortices developed in each nascent cell and they rotated in opposite directions at each end of the cell, a pattern that sometimes persisted for up to 10 h. Exchange with the environment may have been promoted by these shape and area cycles and persisting circulation in the cytoplasm may have a similar function between a cell's interior and periphery. Some of these movements were sporadically also seen in human zygotes with abnormal numbers of pronuclei and the two-cell stages that developed from these compromised human zygotes.

Parental Conflicting Role Mediates Regulation of The Chromatin Structure in The Mouse Zygote

Parental pronuclei (PN) are asymmetrical in several points but the underlying mechanism for this is still unclear. Recently, a theory has been become broadly accepted that sperm are more than mere vehicles to carry the paternal haploid genome into oocytes. Here, in order to reveal the formation mechanisms for parental asymmetrically relaxed chromatin structure in zygotes, we investigated histone mobility in parthenogenetic-, androgenic-, ROSI-, ELSI-, tICSI-, and ICSI-zygotes with several numbers of PNs with the use of zygotic fluorescence recovery after photobleaching, a method previous established by our group. The results showed that sperm played a role to cause chromatin compaction in both parental PNs. Interestingly, during spermiogenesis, male germ cells acquired this ability and its resistance. On the other hand, oocytes harbored chromatin relaxation ability. Furthermore, the chromatin relaxation factor was competed for between PNs. Thus, these results indicated that the parental asymmetrically relaxed chromatin structure was established as a result of a competition between the PNs for the chromatin relaxation factor that opposed the chromatin compaction effect by sperm. Together, it was suggested that parental germ cells cooperated for their just arisen newborn zygotes by playing a distinct role in the regulation of chromatin structure.

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote

Mechanisms that ensure repair of double-stranded DNA breaks play a key role in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found in the form of concatemer consisting of multiple co-integrated transgene copies. Here we investigated contribution of various DSB repair pathways to the concatemer formation. We injected a pool of linear DNA molecules carrying unique barcodes at both ends into mouse zygotes and obtained 10 transgenic embryos with transgene copy number ranging from 1 to 300 copies. Sequencing of the barcodes allowed us to assign relative positions to the copies in concatemers and to detect recombination events that happened during integration. Cumulative analysis of approximately 1000 integrated copies revealed that more than 80% of copies underwent recombination when their linear ends were processed by SDSA or DSBR. We also observed evidence of double Holliday junction (dHJ) formation and crossing-over during the formation of concatemers. Additionally, sequencing of indels between copies showed that at least 10% of the DNA molecules introduced into the zygote are ligated by non-homologous end joining (NHEJ). Our barcoding approach documents high activity of homologous recombination after exogenous DNA injection in mouse zygote.

Dimethylated histone H3 lysine 9 is dispensable for the interaction between developmental pluripotency-associated protein 3 (Dppa3) and ten-eleven translocation 3 (Tet3) in somatic cells

Both developmental pluripotency-associated protein 3 (Dppa3/Stella/PGC7) and dioxygenase ten-eleven translocation 3 (Tet3) are maternal factors that regulate DNA methylation reprogramming during early embryogenesis. In the mouse zygote, dimethylated histone H3 lysine 9 (H3K9me2) attracts Dppa3 to prevent Tet3-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here, we addressed the interplay between Dppa3 and Tet3 or H3K9me2 in somatic cells. In mouse NIH3T3 cells, the exogenously expressed Dppa3 preferentially accumulated in the cytoplasm and had no effect on Tet3-mediated 5hmC generation. In HeLa cells, the expressed Dppa3 was predominantly localised in the nucleus and could partially suppress Tet3-induced 5hmC accumulation, but this suppressive function was not correlated with H3K9me2. Co-immunoprecipitation assays further revealed an interaction of Dppa3 with Tet3 but not with H3K9me2 in HeLa cells. In cloned zygotes from somatic cells, Dppa3 distribution and 5hmC accumulation in nuclei were not affected by H3K9me2 levels. Taken together, these results suggest that H3K9me2 is not functionally associated with Dppa3 and Tet3 in somatic cells or somatic cell cloned embryos.

PLK1 regulates spindle formation kinetics and APC/C activation in mouse zygote

SummaryPolo-like kinase 1 (PLK1) is involved in essential events of cell cycle including mitosis in which it participates in centrosomal microtubule nucleation, spindle bipolarity establishment and cytokinesis. Although PLK1 function has been studied in cycling cancer cells, only limited data are known about its role in the first mitosis of mammalian zygotes. During the 1-cell stage of mouse embryo development, the acentriolar spindle is formed and the shift from acentriolar to centrosomal spindle formation progresses gradually throughout the preimplantation stage, thus providing a unique possibility to study acentriolar spindle formation. We have shown previously that PLK1 activity is not essential for entry into first mitosis, but is required for correct spindle formation and anaphase onset in 1-cell mouse embryos. In the present study, we extend this knowledge by employing quantitative confocal live cell imaging to determine spindle formation kinetics in the absence of PLK1 activity and answer the question whether metaphase arrest at PLK1-inhibited embryos is associated with low anaphase-promoting complex/cyclosome (APC/C) activity and consequently high securin level. We have shown that inhibition of PLK1 activity induces a delay in onset of acentriolar spindle formation during first mitosis. Although these PLK1-inhibited 1-cell embryos were finally able to form a bipolar spindle, not all chromosomes were aligned at the metaphase equator. PLK1-inhibited embryos were arrested in metaphase without any sign of APC/C activation with high securin levels. Our results document that PLK1 controls the onset of spindle assembly and spindle formation, and is essential for APC/C activation before anaphase onset in mouse zygotes.

Aurora kinase A is essential for correct chromosome segregation in mouse zygote

SummaryAurora-A kinase (AURKA), a member of the serine/threonine protein kinase family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little information is known about AURKA in the early cleavage mouse embryo with respect to acentrosomal spindle assembly. In vitro experiments in which AURKA was inactivated with specific inhibitor MLN8237 during the early stages of embryogenesis documented gradual arrest in the cleavage ability of the mouse embryo. In the AURKA-inhibited 1-cell embryos, spindle formation and anaphase onset were delayed and chromosome segregation was defective. AURKA inhibition increased apoptosis during early embryonic development. In conclusion these data suggest that AURKA is essential for the correct chromosome segregation in the first mitosis as a prerequisite for normal later development after first cleavage.