In vivo repair of DNA damage induced by X-rays in the early stages of mouse fertilization, and the influence of maternal PARP1 ablation.

The early pronucleus stage of the mouse zygote has been characterised in vitro as radiosensitive, due to a high rate of induction of chromosome-type chromosome abnormalities (CA). We have investigated the repair of irradiation induced double strand DNA breaks in vivo by γH2AX foci and first cleavage metaphase analysis. Breaks were induced in sperm and in the early zygote stages comprising sperm chromatin remodelling and early pronucleus expansion. Moreover, the role of PARP1 in the formation and repair of spontaneous and radiation-induced double strand breaks in the zygote was evaluated by comparing observations in C57BL/6J and PARP1 genetically ablated females. The results confirmed in vivo that the rate of chromosome aberration induction by X-rays was approximately 3-fold higher in the zygote than in mouse lymphocytes. This finding was related to a diminished efficiency of double strand break signalling, as shown by a lower rate of γH2AX radiation-induced foci compared to that measured in most other somatic cell types. The spontaneous frequency of CA in PARP1 depleted zygotes was slightly but significantly higher than in wild type zygotes. Also, these zygotes showed some impairment of the radiation-induced DNA Damage Response when exposed closer to the start of S-phase, revealed by a higher number of γH2AX foci and a longer cell cycle delay. The rate of chromosome aberrations, however, was not elevated over that of wild type zygotes, possibly thanks to backup repair pathways and/or selection mechanisms against damaged cells. When comparing with the literature data on irradiation induced CA in mouse zygotes in vitro, the levels of induction were strikingly similar as was the frequency of misrepair of double strand breaks (γH2AX foci). This result can be reassuring for in vitro human gamete and embryo handling, because it shows that culture conditions do not significantly affect double strand DNA break repair.

Parental origin of chromatin in human monopronuclear zygotes revealed by asymmetric histone methylation patterns, differs between IVF and ICSI.

In mouse zygotes, many post-translational histone modifications are asymmetrically present in male and female pronuclei. We investigated whether this principle could be used to determine the genetic composition of monopronuclear human zygotes in conventional IVF and ICSI. First we determined whether male female asymmetry is conserved from mouse to human by staining polypronuclear zygotes with antibodies against a subset of histone N-tail post-translational modifications. To analyze human monopronuclear zygotes, a modification, H3K9me3, was selected that is present in the maternal chromatin. After IVF a total of 45 monopronuclear zygotes were obtained. In 39 (87%) of zygotes a nonuniform staining pattern was observed, proof of a bi-parental origin and assumed to result into a diploid conception. Two zygotes showed no staining for the modification, indicating that the single pronucleus was of paternal origin. Four zygotes contained only maternally derived chromatin. ICSI-derived monopronuclear zygotes (n = 33) could also be divided into three groups based on the staining pattern of their chromatin: (1) of maternal origin (n = 15), (2) of paternal origin (n = 8) or (3) consisting of two chromatin domains as dominating in IVF (n = 10). Our data show that monopronuclear zygotes originating from IVF generally arise through fusion of parental chromatin after sperm penetration. Monopronuclear zygotes derived from ICSI in most cases contain uni-parental chromatin. The fact that chromatin was of paternal origin in 24% of ICSI and in 4% of the IVF zygotes confirms earlier results obtained by FISH on cleavage stages. Our findings are of clinical importance in IVF and ICSI practice.

Maternal effects of the scid mutation on radiation-induced transgenerational instability in mice.

The results of a number of recent studies show that mutation rates in the offspring of irradiated parents are substantially elevated, however, the effect of parental genotype on transgenerational instability remains poorly understood. Here, we have analysed the mutation frequency at an expanded simple tandem repeat (ESTR) locus in the germline and bone marrow of the first-generation male offspring of control and irradiated male mice. The frequency of ESTR mutation was studied in the offspring of two reciprocal matings male symbol scid x female symbol BALB/c and male symbol BALB/c x female symbol scid, which were compared with that in BALB/c mice. In the offspring of the BALB/c x BALB/c and male symbol scid x female symbol BALB/c matings, which were conceived after paternal sperm irradiation, the frequency of ESTR mutation was significantly elevated in both tissues. In contrast, ESTR mutation frequency was only slightly elevated in the offspring of male symbol BALB/c x female symbol scid mating conceived after paternal irradiation. The results of this study suggest that the oocytes of scid females are unable to fully support the repair of double-strand breaks induced in paternal sperm which may in turn result in the elimination of cells/embryos containing high levels of DNA damage, thus partially preventing the manifestation of genomic instability.

Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin.

Rapidly after gamete fusion, the sperm nucleus loses its specific chromatin conformation and the DNA is repopulated with maternally derived nucleosomes. We evaluated the nature of paternally derived nucleosomes and the dynamics of sperm chromatin remodeling in the zygote directly after gamete fusion. We observed histone H4 acetylated at K8 or K12 already prior to full decondensation of the sperm nucleus, suggesting that these marks are transmitted by the spermatozoon. Tracking down the origin of H4K8ac and H4K12ac during spermiogenesis revealed the retention of nucleosomes with these modifications in the chromocenter of elongating spermatids. We show that sperm constitutive heterochromatin is enriched for nucleosomes carrying specific histone modifications which are transmitted to the zygote. Our results suggest an epigenetic mechanism for inheritance of chromosomal architecture. Furthermore, up to pronucleus formation, histone acetylation and phosphorylation build up in a cascade-like fashion in the paternal chromatin. After formation of the pronucleus, a subset of these marks is removed from the heterochromatin, which suggests a reestablishment of the euchromatin-heterochromatin partition.