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Barnetova I.,Institute of Animal Science | Barnetova I.,Center for Cell Therapy and Tissue Repair | Okada K.,Nippon Veterinary and Life Science University
Czech Journal of Animal Science | Year: 2010

Conflicting data still exist regarding the extent of paternal pronuclear DNA demethylation in one cell-stage mammalian embryos. Demethylation of paternal pronuclear DNA was observed in in vivo produced porcine zygotes, whereas in vitro produced embryos do not show any or only weak paternal genome demethylation. In our experiments, we have used and compared two in vitro techniques commonly used for in vitro embryo production (in vitro fertilization and intracytoplasmic sperm injection) and then we evaluated the extent of labelling in both these groups after 5-methylcytosine (5-MeC) or dimethyl H3/K9 labelling. We have found no differences in the methylation pattern between both those techniques used for the production of embryos. Moreover, we did not detect any demethylation of paternal DNA after 5-MeC labelling at all. Contrary to this, labelling with dimethyl H3/K9 antibodies showed differences in labelling intensity between maternal and paternal genomes in 42% of zygotes after in vitro fertilization and in 44% of zygotes after intracytoplasmic sperm injection. Our results indicate that in vitro matured pig oocytes exhibit rather inconsistent methylation patterns. This inconsistency probably resulted from insufficient cytoplasmic maturation of oocytes and to a lesser extent from the in vitro technique for embryo production.

Fulka H.,Institute of Animal Science | Fulka H.,Center for Cell Therapy and Tissue Repair | Hirose M.,RIKEN | Inoue K.,RIKEN | And 7 more authors.
Stem Cells | Year: 2011

ESCs are most commonly derived from embryos originating from oocytes that reached metaphase II. We describe here a novel approach where ESCs with all pluripotency parameters were established from oocytes in which metaphase I was converted, from the cell cycle perspective, directly into metaphase II-like stage without the intervening anaphase to telophase I transition. The resulting embryos initiate development and reach the blastocyst stage from which the ESC lines are then established. Thus, our approach could represent an ethically acceptable method that can exploit oocytes that are typically discarded in in vitro fertilization clinics. Moreover, our results also indicate that the meiotic cell cycle can be converted into mitosis by modulating chromosomal contacts that are typical for meiosis with subsequent licensing of chromatin for DNA replication. © AlphaMed Press.

Vackova I.,Institute of Animal Science | Vackova I.,Center for Cell Therapy and Tissue Repair | Novakova Z.,Institute of Animal Science | Novakova Z.,Center for Cell Therapy and Tissue Repair | And 9 more authors.
Journal of Reproduction and Development | Year: 2011

The present study was designed to extensively characterize cell lines derived from porcine blastocysts by several methodical approaches, including morphological observation, cytogenetic analysis, estimation of alkaline phosphatase activity and detection of specific marker expression at the mRNA/protein level. A comparison was made between the properties of cell lines isolated from in vivo- and in vitro-obtained blastocysts. Our results showed that 57.1% of the in vivo-obtained blastocysts attached to the feeder layer and that 33.3% of them started to grow in a monolayer. The percentage of attached in vitro-produced blastocysts was lower (24.6%), and only 6.9% of them started to grow. Outgrowths from the in vitro-produced blastocysts formed mainly trophectoderm or epithelial-like monolayer, whereas the in vivo-obtained blastocysts formed heterogeneous outgrowths that also contained cells with embryonic stem (ES)-like morphology. Detailed analyses showed that the primary outgrowths with ES-like morphology expressed the pluripotency markers OCT-4 and NANOG and revealed intensive alkaline phosphatase staining, while they did not express markers of differentiation. The majority of passaged cells, including those with ESlike morphology, lacked OCT-4 protein and revealed expression of specific differentiation markers (CYTOKERATIN 18, LAMINS A/C, TRANSFERRIN, α-FETOPROTEIN and GATA-4), although they still expressed NANOG and exhibited weak alkaline phosphatase activity. Moreover, these cells spontaneously differentiated into neural, fibroblast or epithelial-like cells, even in the presence of leukaemia inhibitory factor. Our results show that complex analysis of markers of pluripotency as well as differentiation markers is necessary for proper interpretation of data in porcine embryonic stem cell studies. © 2011 by the Society for Reproduction and Development.

Barnetova I.,Institute of Animal Science | Barnetova I.,Center for Cell Therapy and Tissue Repair | Vackova I.,Institute of Animal Science | Vackova I.,Center for Cell Therapy and Tissue Repair | Firla P.,Institute of Animal Science
Czech Journal of Animal Science | Year: 2012

The process of active paternal chromatin demethylation after fertilization in the pig is not fully understood and very inconsistent data have been published by different research groups. We have applied the interspecies intracytoplasmic sperm injection (iICSI) to evaluate remodeling capabilities of porcine oocytes in more details. We injected mouse frozen-thawed sperm heads into porcine in vitro matured or ovulated oocytes, respectively. Embryos produced by intracytoplasmic sperm injection (ICSI) of boar spermatozoa into porcine ovulated oocytes (intraspecies) served as controls. Zygotes with 2-pronuclei were labeled with antibodies against certain epigenetic modifications (5-methylcytosine, 5-MeC; heterochromatin protein 1, HP1; trimethylation of H3/K4, H3/K4-me3; and dimethylation of H3/K9, H3/K9-me2). The labeling patterns were not different between zygotes produced from in vitro matured and ovulated oocytes. Both pronuclei were symmetrically labeled with 5-MeC, HP1, and H3/K4-me3 antibodies. Asymmetrical labeling was observed only with H3/K9-me2 antibody. The labeling of interspecies zygotes was similar to that of intraspecies zygotes. Moreover, the DNA demethylation was observed neither in control zygotes (intraspecies). The only difference observed between zygotes produced from in vitro matured and ovulated oocytes was in their ability to be activated. Intraspecies zygotes produced from ovulated oocytes were able to form the paternal pronucleus without additional activation; the zygotes produced from in vitro matured oocytes formed the paternal pronucleus only after additional activation with electric pulses. Our results show that the remodeling abilities of in vitro matured and ovulated oocytes are essentially similar. Moreover, it seems that reasons of inconsistent data reporting the active demethylation in the pig are more complicated and they are not associated exclusively with the oocyte quality.

Barnetova I.,Institute of Animal Science | Barnetova I.,Center for Cell Therapy and Tissue Repair | Fulka H.,Institute of Animal Science | Fulka H.,Center for Cell Therapy and Tissue Repair | And 2 more authors.
Journal of Reproduction and Development | Year: 2010

Both the sperm and oocyte are terminally differentiated cells, but within a very short post-fertilization period, their genomes are converted into a totipotent zygote. The process of this transformation has been studied in a number of mammals as well as in the pig, for which very inconsistent results have been published. To clarify these inconsistencies, we have used the interspecies intracytoplasmic sperm injection technique for embryo production and subsequent paternal genome remodeling evaluation. First, we injected boar sperm heads into ovulated and in vitro matured mouse oocytes. The boar spermatozoa consistently decondense in ovulated oocytes and form fully developed pronuclei with demethylated DNA (5-methylcytosine; 5-MeC). Additional labeling against other histone modifications (H3/K9 dimethylation, H3/K4 trimethylation) and HP1 (Heterochromatin Protein 1) revealed similarity to those changes that are typical for natural mouse zygotes. On the other hand, no decondensation and formation of male pronuclei were observed, in spite of obvious oocyte activation, in in vitro matured oocytes. For this reason, we have evaluated the reprogramming parameters of in vitro matured mouse oocytes in more detail. In mouse zygotes (intraspecific), both pronuclei were consistently formed, but no sperm head chromatin demethylation was detected after 5-MeC labeling. Our observations suggest that porcine sperm heads are capable of undergoing active demethylation in in vivo matured mouse oocytes. On the other hand, in vitro matured oocytes possess much lower sperm remodeling capabilities. © 2010 by the Society for Reproduction and Development.

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