Epigenetic Characteristics of Paternal Chromatin in Interspecies Zygotes

Abstract

Abstract. 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. Key words: Epigenetics, Interspecific, Mouse, Pig, Zygote (J. Reprod. Dev. 56: [601][602][603][604][605][606] 2010) he transformation of the sperm head into a male pronucleus represents a radical process during which highly compacted and condensed chromatin associated with protamines gradually decondenses, protamines are replaced with histones and certain epigenetic marks are removed [1]. Concomitantly, a new nuclear envelope is formed around the decondensing chromatin, and replication of DNA begins later on. This radical transformation converts the terminally differentiated state of the sperm into a paternal genome of a totipotent zygote. These processes are much less radical in the maternal genome. Thus, fertilization represents a major remodeling event that is accompanied by epigenetic changes of both parental genomes. Among the most prominent events studied so far is the global demethylation of paternal DNA [2][3][4][5]. It has been shown first in the mouse that levels of methylation differ between both pronuclei. Whilst the paternal pronucleus showed a negative signal after labeling against 5-methylcytosine (5-MeC), the maternal pronucleus remained heavily labeled [6,7]. These processes were studied in some other species, and it has been suggested that this paternal genome demethylation step is a common phenomenon for all mammals [8]. However, some additional experiments have challenged this view. Thus, in the sheep and init...

H3S10 Phosphorylation Marks Constitutive Heterochromatin During Interphase in Early Mouse Embryos Until the 4-Cell Stage

“…It is only at the 2-cell stage that the pericentromeric heterochromatin of different chromosomes clusters together, forming the characteristic heterochromatin domains called chromocenters [31,32]. Two very well established markers of pericentromeric heterochromatin are the epigenetic modification H3K9me3 and the protein HP1β, which have been widely used to study chromatin arrangement and nuclear organization in preimplantation embryos [33][34][35][36][37][38]. However, there is a clear parental asymmetry in early preimplantation embryos until the 4-cell stage, and H3K9me3 (as well as HP1β) constitutes the major marker for maternal pericentric heterochromatin only.…”

Active DNA demethylation in mammalian preimplantation embryos: new insights and new perspectives

“…Based on these results we can speculate that the factors determining the loss of PPN 5-meC signals may exist in the sperm itself. But based on the results obtained from interspecies zygote-like cells (Beaujean et al, 2004a, b;Barnetova et al, 2010), the mouse ooplasm was also able to partially reduce the 5-meC signal intensity of PPN from sheep sperm, which failed to lose 5-meC signals in the sheep ooplasm (Beaujean et al, 2004a, b). In addition, 5-hmC signals could also be detected in the nucleus of mouse somatic cell nuclear transfer (SCNT) zygote-like cells (Wossidlo et al, 2011).…”

“…Normal fertilization Signal lost Mayer et al (2000); IVF Signal lost Santos et al (2002); Zona pellucida laser microdissection-facilitated IVF Signal lost Peters et al (2009) ICSI Signal lost Beaujean et al (2004a, b); ROSI/elongating spermatid injection Signals partially lost 6 h after injection, but resumed 10 h after injection Ohta et al (2009) Zygote-like cells Parthenogenesis Signal not lost Barton et al (2001) Androgenesis Barnetova et al (2010) All data above are derived from indirect immunofluorescence labeling of pronuclear 5-meCs. The data for 5-hmCs still await replenishing.…”