Mammalian sperm nuclear organization: resiliencies and vulnerabilities
Persisting nucleosomes in sperm
Several reports have shown that histones could be found in mature sperm in variable quantities depending on the species. For example, it was estimated that around 1% of the sperm DNA is still associated with histones in mice, hamster, stallion, and bull spermatozoa [82–84] while it may go up to 10–15% in human sperm [85]. More recent immunoprecipitation studies (yet to be published) narrowed this difference down and reported that in human sperm persisting histones constitute about 5 to 7% of the DNA sequences. This is still substantially more than the other mammals studied to date. The reason for the higher percentage of histones in humans sperm is not yet understood. Some authors suggest an inefficient spermatogenetic program in human as a possible reason. Others postulate that human sperm needs to maintain more paternal chromosomal regions to be readily accessible for the onset of the developmental programme post-fertilization. Initially considered as remnants of an incomplete histone replacement process during spermatogenesis these persisting histones are now considered critical for the early transcriptional reactivation of the paternal genome [86, 87]. This notion is backed by the observation that the persisting histones in sperm can be found in the zygote [88].
The presence of paternal persisting histones after protamine-histone exchange, fertilization followed by decondensation [89, 90] may reveal an important functional role for these proteins in the early embryo development [91, 92].
In mice and human spermatozoa, immunocytochemical approaches reveal localized histones at the periphery of the nucleus as well as in the post-acrosomal and basal domains of the sperm head [93–95]. The basal localization of the histone signal resembles that of the nuclear annulus [75]. This structure is seen as a component of the sperm nuclear matrix, acting as an anchor for the sperm DNA via the sperm MARs (localized among toroids) and the histone-rich telomeres [94, 96]. In mouse sperm we recently reported that nuclear domains rich in matrix proteins are also histone-rich regions of lower compaction [95, 97].
The fact that specific locations in the mature sperm nucleus house these histones supports the notion of an ordered process for the maintenance. This is backed by genome-wide analyses including chromatin immunoprecipitation (ChIP) studies, DNA microarrays and high-throughput sequencing revealing organized specific regions in mouse and human sperm. Nucleosomes were found enriched in 2 types of genomic regions. One region concerns large areas of DNA up to 100 kb in length that punctuate the protamine-associated chromatin (see Fig. 1). Ward [98] suggested that these segments of DNA are organized in a less condensed state with a solenoid-like conformation resembling the somatic chromatin. Additionally, high histone levels were found in interspersed short DNA sequences (around 1 kb-long) that correspond to the small DNA linkers bridging toroids together [95, 97, 98]. Interestingly, the latter regions were also reported to be associated with the sperm nuclear matrix [95, 97, 98]. ChIP studies utilizing histone antibodies to recover the histone-associated DNA sequences in mature sperm revealed histones to be significantly enriched at the promoters of: genes coding for microRNAs, genes involved in early embryonic development (e.g. transcription factors, HOX genes, signaling protein, etc.…), genes subjected to genomic imprinting, and genes involved in spermatogenesis [99]. However, conflicting data from different groups showed that persisting histones were associated with intergenic sequences outside of the gene regulatory regions [95, 100]. To date, there is no consensus as to what specific DNA sequences in sperm are associated with nucleosomes. This is essentially due to the way DNA sequences were recovered from mature sperm during the ChIP assays. Due to the extreme difficulty in recovering mature sperm DNA associated with the intrinsic properties of the compacted nucleus, strong reducing conditions are required to obtain the halo sperm phenotype followed by microccocal nuclease (Mnase) digestion to retrieve the long loops of DNA exiting the sperm nucleus. The soluble DNA fraction is then separated from the insoluble portion that also contains the nuclear debris. This operation leads to the recovery of sperm DNA sequences associated with histones which mainly corresponds to the solenoid regions embedded within the toroids of protamines (see above). Following this type of sperm DNA retrieval protocol it is not possible to recover the sperm DNA associated with the nuclear matrix as it is discarded during the preparation. However, with more aggressive sperm DNA retrieval conditions such as sonication, it is possible to obtain some of the histone-associated DNA sequences attached to the sperm nuclear matrix. The use of different extraction techniques may thus explain the present discrepancy in the literature of sperm DNA histone-associated sequences in the species that have been investigated.
Another important question is why peripheral regions of the mature sperm nucleus maintain association with histones? Due to their lower state of compaction and their peripheral localization, these nuclear domains will be the first ones to be susceptible to DNA damage from external stressors. So what is the evolutionary reason for the maintainance of such regions of fragility? One reason could be that these peripheral regions are the first ones targeted by the oocyte-driven centripetal decondensing processes of the sperm nucleus after fertilization. Accordingly, these regions may have to be retained in a pre-decondensed state either to facilitate the decondensation process or/and to remain transcriptionally active very early in the zygote developmental program. Regardless of the reasons, the paternal DNA present in these peripheral less tightly packed regions of the sperm nucleus will be more likely to incur damage. This observation has been verified experimentally in transgenic mouse models where it was observed that the regions sensitive to high post-testicular oxidative damage match the distribution of nucleosomes [95, 98].
A more detailed analysis of histone variants in these loci, where histones are maintained, showed a precise distribution of these epigenetic marks. For example, the testis-specific H2B variant (TH2B) was found enriched in the promoters of genes involved in sperm cell maturation, function, capacitation and fertilization, but never in the promoters of genes controlling embryonic development [89]. As another example, the H2A.Z variant was mainly found in pericentromeric heterochromatin domains [101]. Conversely, the promoters of developmental transcription factors genes were found enriched in H3K4me2 marks (transcriptionally permissive marks consisting in dimethylation of histone H3 on lysine 4) while H3K9me3 marks (transcriptionally repressive marks consisting in trimethylation of histone H3 on lysine 9) were not found localized near genes, but in pericentromeric genomic regions [99]. In conclusion, histone content and PTM of these paternal-borne nucleosomes represent distinct epigenetic characteristics that are unique to the sperm nucleus. Notably, protamines (Prm1 and Prm2) also carry multiple PTM that have led some authors to propose that like the somatic cell “histone code”, there might also be a sperm “protamine code”, unique to the sperm nucleus [102]. Thus, PTM of sperm persisting histones as well as protamines may constitute a complex epigenetic signature driving embryo development and potentially transgenerational inheritance, as persisting paternal histones may be transmitted to the next generation.