Olga Mudrak

Chromosome architecture in the decondensing human sperm nucleus

Whereas recent studies demonstrated a well-defined nuclear architecture in human sperm nuclei, little is known about the mode of DNA compaction above the elementary structural unit of nucleoprotamine toroids. Here, using fluorescence in-situ hybridization (FISH) with arm-specific DNA probes of chromosomes 1, 2 and 5, we visualized arm domains and established hierarchical levels of sperm chromatin structures. The compact chromosome territories, which in sperm have a preferred intranuclear localization, have an extended conformation represented by a 2000 nm chromatin fiber. This fiber is composed of a 1000 nm chromatin thread bent at 180° near centromere. Two threads of 1000 nm, representing p-arm and q-arm chromatin, run in antiparallel fashion and join at the telomeres. Each 1000 nm thread, in turn, resolves into two rows of chromatin globules 500 nm in diameter interconnected with thinner chromatin strands. We propose a unified comprehensive model of chromosomal and nuclear architecture in human sperm that, as we suggest, is important for successful fertilization and early development.

Characterisation of a human sperm cell subpopulation marked by the presence of the TSH2B histone

During the process of mammalian spermiogenesis, a significant reorganisation of the chromatin structure occurs involving the sequential substitution of somatic histones with protamines. In the human sperm nucleus, approximately 15% of the basic nuclear protein complement is maintained as histones. Human testis/sperm-specific histone H2B (hTSH2B) is a variant of the histone H2B expressed exclusively in spermatogenic germline cells and present in some mature sperm cells. Thus, this protein marks a subpopulation of sperm cells in the ejaculate. Using indirect immunofluorescence, we examined the influence of hTSH2B on zona pellucida binding and sperm head decondensation in amphibian egg cell-free extract. As suggested by previous studies, we found that hTSH2B can be localised in only approximately 30% of sperm cells within a given ejaculate. We established that the presence of hTSH2B does not influence sperm zona pellucida binding capacity. Finally, we found that decondensation occurred more rapidly and to a greater extent in those cells containing hTSH2B. We propose that the presence or absence of hTSH2B within spermatozoa influences pronuclei formation and the activation of paternal genes following fertilisation and during early embryonic development.

Positioning of Chromosomes in Human Spermatozoa Is Determined by Ordered Centromere Arrangement

The intranuclear positioning of chromosomes (CHRs) is a well-documented fact; however, mechanisms directing such ordering remain unclear. Unlike somatic cells, human spermatozoa contain distinct spatial markers and have asymmetric nuclei which make them a unique model for localizing CHR territories and matching peri-centromere domains. In this study, we established statistically preferential longitudinal and lateral positioning for eight CHRs. Both parameters demonstrated a correlation with the CHR gene densities but not with their sizes. Intranuclear non-random positioning of the CHRs was found to be driven by a specific linear order of centromeres physically interconnected in continuous arrays. In diploid spermatozoa, linear order of peri-centromeres was identical in two genome sets and essentially matched the arrangement established for haploid cells. We propose that the non-random longitudinal order of CHRs in human spermatozoa is generated during meiotic stages of spermatogenesis. The specific arrangement of sperm CHRs may serve as an epigenetic basis for differential transcription/replication and direct spatial CHR organization during early embryogenesis.

Reorganisation of human sperm nuclear architecture during formation of pronuclei in a model system

By fertilisation, two terminally differentiated cells, namely the egg and spermatozoon, are combined to create a totipotent zygote. During this process, the inactive sperm nucleus is transformed into a functional male pronucleus. Recent studies demonstrate that human sperm chromatin has an elaborate multilevel organisation, but almost nothing is known about how sperm chromosomes are transformed during fertilisation. Because of ethical reasons and technical complications, experimentation with human embryos is generally unworkable and adequate model systems are necessary to study the formation of male pronuclei. Here, we analyse remodelling of human sperm chromatin and chromosome architecture in Xenopus egg extracts using immunofluorescent localisation of protamines and centromere protein A, as well as fluorescence in situ hybridisation localisation of major alpha-satellite DNA and whole chromosome territory (CT). We demonstrate noticeable relocalisation of centromeres and remodelling of CT during the decondensation-recondensation cycle, mimicking cellular events that occur in the paternal genome in vivo during fertilisation.

Protection of internal (TTAGGG)n repeats in Chinese hamster cells by telomeric protein TRF1

Chinese hamster cells have large interstitial (TTAGGG) bands (ITs) which are unstable and should be protected by an unknown mechanism. Here, we expressed in Chinese hamster V79 cells green fluorescent protein (GFP)-tagged human TRF1, and found that a major fraction of GFP-TRF1 bound to ITs is diffusionally mobile. This fraction strongly decreases after treatment of cells with wortmannin, a protein kinase inhibitor, and this drug also increases the frequency of chromosome aberrations. Ionizing radiation does not induce detectable translocation of GFP-TRF1 to the sites of random double-strand breaks visualized using antibodies against histone γ-H2AX. TRF1 is known to be eliminated from telomeres by overexpression of tankyrase 1 which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding tankyrase 1 and found that the frequency of chromosome rearrangements is increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 is involved in sequence-specific protection of internal nontelomeric (TTAGGG)n repeats.

Kinetics of human male pronuclear development in a heterologous ICSI model

PurposeTo evaluate human sperm nuclear chromatin decondensation in a heterologous ICSI system using hamster ova injected with human sperm.Materials and methodsFrozen hamster oocytes were injected with Triton X-100 treated sperm and fixed at different time points post ICSI. Oocytes injected with non-treated sperm served as controls. Male pronuclear decondensation was evaluated after staining with DAPI.ResultsSperm cells with partially destroyed membranes and depletion of the acrosome decondense more rapidly and to a greater extent than membrane/acrosome intact cells. Marked variability in pronuclear size was observed for any time point post ICSI, which most probably reflects the heterogeneity in the mature human sperm population.ConclusionRemodeling of male gamete nuclei in this heterologous ICSI mimics events that occur during natural fertilization in humans and therefore this approach may be used for studies of human sperm chromosomes transformations.

Chromosome Positioning in Spermatozoa

Similar to interphase chromosomes, mammalian supercompact and genetically inactive sperm chromatin is arranged in non-overlapping chromosome territories. Recent works demonstrate that chromosome territories in sperm have defined and nonrandom intranuclear positioning. It is hypothesized that preferred localization of chromosomes in sperm together with well-organized chromosome architecture provide epigenetic signature to genome, which might be important at fertilization and early development. Here, we describe relevant experimental data with primary attention to the studies related to human spermatozoa. Possible implications of sperm chromosome positioning for modern reproductive technologies are discussed.

Organization of Chromosomes During Spermatogenesis and in Mature Sperm

One of the prominent features of male germ cell differentiation is a profound sequential, structural, and spatial reorganization of the chromosomes, which manifests in diverse events, such as chromatin remodeling, DNA recombination and repair, dynamic movements of chromosomes, and chromosome domains (centromeres and telomeres) within the nucleus. Divergent states of male germ cell chromosomes during proliferation, meiosis, and final differentiation to sperm are accompanied and supported by unprecedented in-scale changes in the chromatin protein composition.

Preferable location of chromosomes 1, 29, and X in bovine spermatozoa

Chromosome positioning in sperm nucleus may have a functional significance by influencing the sequence of post-fertilization events. In this study we present data on preferential locations of chromosomes 1, 29 and X in Bos taurus spermatozoa. Here we demonstrate that the position of X chromosome in the sperm nucleus is more restricted as compared to the position of chromosome 1, which is about of the same size. Our data support the concept of the functional significance of genome architecture in male germline cells.

Erratum to: Kinetics of human male pronuclear development in a heterologous ICSI model

Erratum to: J Assist Reprod Genet DOI 10.1007/s10815-010-9402-y The original version of this article unfortunately contained a mistake. The article should include this information as the Acknowledgement part: This work was supported by grant from The Jones Foundation and, in part, by a National Institutes of Health grant (HD-042748) to A.Z.