Kinetics of human male pronuclear development in a heterologous ICSI model.

PURPOSE: To evaluate human sperm nuclear chromatin decondensation in a heterologous ICSI system using hamster ova injected with human sperm. MATERIALS AND METHODS: Frozen 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. RESULTS: Sperm 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. CONCLUSION: Remodeling 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.

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.

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.

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.

Telomeres in mammalian male germline cells.

Telomeres are terminal chromosomal domains that protect chromosome ends from degradation and fusion and promote complete replication of DNA. Telomeres are involved in the regulation of cellular replicative lifespan and tumorigenesis. These important functions of the telomeres have evoked high interest: numerous studies have resulted in a detailed description of telomere composition and structure in somatic cells. Much less is known about telomeres in germline cells. Emerging novel features and unique behavior of telomeres in the process of gamete differentiation suggest that they may have additional germline-specific function(s). This review describes recent studies revealing changes in the telomere organization in the course of differentiation from the germline stem cells to mature sperm in mammals. Similarities and differences between somatic and spermatogenic cells in telomere nuclear localization, protein composition, DNA length, telomerase activity, and chromatin structure are discussed. The exceptional features of the germline telomeres may be important for regulation of telomerase activity during spermatogenesis, homologous chromosome pairing during recombination, as well as for male pronucleus development and ordered chromosome withdrawal post-fertilization.

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.

Protamine withdrawal from human sperm nuclei following heterologous ICSI into hamster oocytes.

During late stages of spermatogenesis in mammals, most histones bound to DNA are replaced by protamines (PRM), which results in formation of supercondensed and genetically inert sperm chromatin. At fertilization, mature spermatozoon penetrates oocyte and chromatin is remodeled "back" from nucleoprotamine to nucleohistone state. While being crucial for activation of male genome and ultimately for initiation of embryonic development, this process is poorly studied, especially in humans. Data on model animals concerning PRM to histones exchange post fertilization are few and contradictory. As direct experimentation with human embryos is impossible due to ethical, legal and technical reasons, we evaluate the timing and mode of PRM removal in a heterologous ICSI system using hamster ova injected with human sperm. Localization of human PRM 1 and 2 in hybrid zygotes was established using immunofluorescence. We observed a marked zygote to zygote variability in male pronuclei size for any time point post ICSI and demonstrated that PRM removal correlates with the developing pronuclei area rather than time after injection. Overall, the disappearance of protamines from sperm is rather rapid and most likely completed within 1 hr. We propose that the critical characteristic influencing PRM removal after heterologous fertilization is the intrinsic heterogeneity of the human sperm population. The same yet unexplored variance may be one of the reasons for canceled, delayed or aberrant early embryonic development during natural or artificial fertilization in humans.

Sperm chromatin released by nucleases.

In human spermatozoa, 15-20% of histones are retained in the nucleus to coexist with protamines. Hypothetically, nucleohistone regions of sperm chromatin mark DNA sequences for distinctive processing during fertilization and early embryogenesis. The structural organization and molecular composition of nucleohistones in human spermatozoa is poorly studied. Here, we isolate and characterize fractions of sperm chromatin that are solubilized by endogenous and micrococcal nucleases. Chromatin isolated by either nuclease have a nucleosomal organization with the periodicity of approximately 195 bp (endogenous nuclease digest) and approximately 189 bp (micrococcal nuclease digest), which is similar to that of somatic cells. A distinct feature of sperm nucleohistone is its specific compact supra-nucleosomal organization that was demonstrated by two-dimensional electrophoresis and by atomic force microscopy. The latter technique showed compacted fiber arrays composed of globular particles with the prevailing diameter of approximately 16 nm. A rough estimation indicates that histones may cover continuous stretches of >50 kbp of sperm DNA. This initial characterization of sperm chromatin solubilized by nucleases is important for our understanding of the bipartite structural organization of the paternal genome.

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 degrees 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.

Characterization of nucleosomes consisting of the human testis/sperm-specific histone H2B variant (hTSH2B).

We have reported earlier the occurrence of a specific histone H2B variant in human testis and sperm. Here we have structurally characterized this protein, its association with the rest of the histone octamer, and its effects on the nucleosome structure. We show that a reconstituted octamer consisting of hTSH2B and a stoichiometric complement of histones H2A, H3, and H4 exhibits a lower stability compared to the reconstituted native counterpart consisting of H2B. In contrast, the hTSH2B containing octamers are able to form nucleosome core particles which are structurally and dynamically indistinguishable from those reconstituted with octamers consisting of only native histones. Furthermore, the presence of hTSH2B in the nucleosome does not affect its ability to bind to linker histones.

Non-random positioning of chromosomes in human sperm nuclei.

In human spermatozoa, the arrangement of chromosomes is non-random. Characteristic features are association of centromeres in the interior chromocenter and peripheral location of telomeres. In this paper, we have investigated the highest level of order in DNA packing in sperm--absolute and relative intranuclear chromosome positioning. Asymmetrical nuclear shape, existence of a defined spatial marker, and the haploid complement of chromosomes facilitated an experimental approach using in situ hybridization. Our results showed the tendency for non-random intranuclear location of individual chromosome territories. Moreover, centromeres demonstrated specific intranuclear position, and were located within a limited area of nuclear volume. Additionally, the relative positions of centromeres were non-random; some were found in close proximity, while other pairs showed significantly greater intercentromere distances. Therefore, a unique and specific adherence may exist between chromosomes in sperm. The observed chromosome order is discussed in relation to sperm nuclei decondensation, and reactivation during fertilization.

Nature of telomere dimers and chromosome looping in human spermatozoa.

Specific and well-organized chromosome architecture in human sperm cells is supported by the prominent interactions between centromeres and between telomeres. The telomere-telomere interactions result in telomere dimers that are positioned at the nuclear periphery. It is unknown whether composition of sperm telomere dimers is random or specific. We now report that telomere dimers result from specific interactions between the two ends of each chromosome. FISH using pairs of subtelomeric DNA probes that correspond to the small and long arms of seven human chromosomes demonstrates that subtelomeres of one chromosome are brought together. Statistical analysis confirmed that telomere associations could not result from the random proximity of DNA sequences. Therefore, chromosomes in human sperm nuclei adopt a looped conformation. This higher-order chromosome structure is most likely required for chromosome withdrawal/decondensation during the early fertilization events leading to zygote formation.

Novel human testis-specific histone H2B encoded by the interrupted gene on the X chromosome.

Testis-specific histones are synthesized and accumulated at specific stages of mammalian spermatogenesis. Their proposed functions range from facilitation of the replacement of somatic histones by protamines to epigenetic control of gene transcription. Several testis histone variants were characterized in mouse and rat; however, few are known in humans. Here we report the identification and characterization of a novel human histone 2B gene (TH2B-175) located at Xq22.2, which encodes a highly divergent H2B variant. The TH2B-175 gene contains two introns and is transcribed exclusively in testis, where the spliced polyadenylated mRNA was detected. Genomic PCR, Southern blot analysis, and BLAST-based searches indicate that TH2B-175 evolved in the primate lineage or has been lost in rodents. In transfected Chinese hamster cells, GFP-tagged TH2B-175 targeted to large fluorescent bodies that partially colocalize with the interstitial telomeric blocks. Therefore, TH2B-175 may have telomere-associated functions and participate in the telomere-binding complex in the human sperm [1].

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization.

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.