Chromosome-wide nucleosome replacement and H3.3 incorporation during mammalian meiotic sex chromosome inactivation.

In mammalian males, the first meiotic prophase is characterized by formation of a separate chromatin domain called the sex body. In this domain, the X and Y chromosomes are partially synapsed and transcriptionally silenced, a process termed meiotic sex-chromosome inactivation (MSCI). Likewise, unsynapsed autosomal chromatin present during pachytene is also silenced (meiotic silencing of unsynapsed chromatin, MSUC). Although it is known that MSCI and MSUC are both dependent on histone H2A.X phosphorylation mediated by the kinase ATR, and cause repressive H3 Lys9 dimethylation, the mechanisms underlying silencing are largely unidentified. Here, we demonstrate an extensive replacement of nucleosomes within unsynapsed chromatin, depending on and initiated shortly after induction of MSCI and MSUC. Nucleosomal eviction results in the exclusive incorporation of the H3.3 variant, which to date has primarily been associated with transcriptional activity. Nucleosomal exchange causes loss and subsequent selective reacquisition of specific histone modifications. This process therefore provides a means for epigenetic reprogramming of sex chromatin presumably required for gene silencing in the male mammalian germ line.

DNA double-strand break repair in parental chromatin of mouse zygotes, the first cell cycle as an origin of de novo mutation.

In the human, the contribution of the sexes to the genetic load is dissimilar. Especially for point mutations, expanded simple tandem repeats and structural chromosome mutations, the contribution of the male germline is dominant. Far less is known about the male germ cell stage(s) that are most vulnerable to mutation contraction. For the understanding of de novo mutation induction in the germline, mechanistic insight of DNA repair in the zygote is mandatory. At the onset of embryonic development, the parental chromatin sets occupy one pronucleus (PN) each and DNA repair can be regarded as a maternal trait, depending on proteins and mRNAs provided by the oocyte. Repair of DNA double-strand breaks (DSBs) is executed by non-homologous end joining (NHEJ) and homologous recombination (HR). Differentiated somatic cells often resolve DSBs by NHEJ, whereas embryonic stem cells preferably use HR. We show NHEJ and HR to be both functional during the zygotic cell cycle. NHEJ is already active during replacement of sperm protamines by nucleosomes. The kinetics of G1 repair is influenced by DNA-PK(cs) hypomorphic activity. Both HR and NHEJ are operative in S-phase, HR being more active in the male PN. DNA-PK(cs) deficiency upregulates the HR activity. Both after sperm remodeling and at first mitosis, spontaneous levels of gammaH2AX foci (marker for DSBs) are high. All immunoflurescent indices of DNA damage and DNA repair point at greater spontaneous damage and induced repair activity in paternal chromatin in the zygote.

gammaH2AX signalling during sperm chromatin remodelling in the mouse zygote.

In the mouse, the paternal post-meiotic chromatin is assumed to be devoid of DNA repair after nuclear elongation and protamine-induced compaction. Hence, DNA lesions induced thereafter will have to be restored upon gamete fusion in the zygote. Misrepair of such lesions often results in chromosome type aberrations at the first cleavage division, suggesting that the repair event takes place prior to S-phase. During this stage of the zygotic cell cycle, the paternal chromatin transits from a protamine- to a nucleosome-based state. We addressed the question whether the canonical signalling pathway to DNA double strand breaks (DSBs), the phosphorylated form of histone H2AX (gammaH2AX) is active during chromatin restructuring of the male genetic complement in the zygote. Here, we describe the detailed characterization of gammaH2AX signalling in the early stages of zygotic development up to the appearance of the pronuclei. We have found the gammaH2AX signalling pathway to be already active during sperm chromatin remodelling after gamete fusion in a dose dependent manner, reflecting the amount of DSBs present in the sperm nucleus after in vivo male irradiation. Using DNA damaging compounds to induce lesions in the early zygote, differences in DSB sensitivity and gammaH2AX processing between paternal and maternal chromatin were found, suggesting differences in DNA repair capacity between the parental chromatin sets.

Proliferation of meniscal fibrochondrocytes cultured on a new polyurethane scaffold is stimulated by TGF-ß.

The aim of this study was to investigate if newly developed polyurethane (PU) scaffolds are suitable as scaffold for cell-seeded meniscus tissue engineered constructs. Scaffolds were seeded with goat meniscal fibrochondrocytes and cultured to assess changes in biological and mechanical properties. Furthermore, the effect of TGF-ß on these properties was investigated. PU scaffolds were made from poly d/l lactide and caprolactone as soft segments and 1,4-butanediisocyanate for the urethane hard segments. The porosity of the scaffolds was 95%. Isolated goat meniscal fibrochondrocytes were seeded on the scaffolds and cultured with or without the addition of 10 ng/mL TGF-ß in standard culture medium. After 2, 4, and 6 weeks of culture, scaffolds were analyzed for cell proliferation, matrix synthesis, and mechanical properties. Scanning electron microscopy and histology showed that the scaffolds had an interconnected isotropic pore structure. Without the addition of TGF-ß, cells did not proliferate during the culture period and isolated meniscus fibrochondrocytes were more frequently located in the peripheral parts of the scaffold. Fibrochondrocytes supplemented with TGF-ß were distributed throughout the construct. Clustered cells were surrounded by matrix which stained slightly positive for glycosaminoglycans (GAGs). Also, collagen production was increased significantly after 4 and 6 weeks of culture compared to cultures without TGF-ß and also more GAG staining was found after 4 and 6 weeks in the sections of the TGF-ß stimulated cultures. Despite the increase in matrix production, the compressive stiffness of the constructs was not increased during the culture period. Meniscal fibrochondrocytes were able to adhere to the PU scaffold. However, the scaffold itself does not stimulate proliferation and matrix production. The addition of TGF-ß resulted in a strong induction of both proliferation and extracellular matrix production.

Motile human normozoospermic and oligozoospermic semen samples show a difference in double-strand DNA break incidence.

BACKGROUND: Among ICSI children de novo structural chromosome aberrations of male descent are increased. Misrepair of double-strand DNA breaks (DSBs) is a prerequisite for such aberrations to occur. To date, no absolute assessment of the number of DSBs in human sperm nuclei after gamete fusion has been described. METHODS: Using man-mouse heterologous ICSI and gammaH2AX immunofluorescent staining, capable of detecting a single DSB, the number of lesions in ICSI selected sperm from normozoospermic men (n = 2) and oligozoospermic patients (n = 3) was quantified. A comparison with a subfertile male mouse model (n = 5) has been made. In addition, the fate of morphologically normal ejaculated immotile sperm after ICSI was examined. RESULTS: A significant increase in the fraction of sperm cells bearing DSBs was found in oligozoospermic semen compared with that from normozoospermic men (P < 0.01). The majority of morphologically normal immotile human sperm showed excess gammaH2AX staining and nuclear disintegration. However, some had a non-deviant DSB pattern. CONCLUSIONS: The increased fraction of DSB-positive sperm in both human and mouse oligozoospermic semen is adding to the surmise that semen from oligozoospermic patients has a reduced chromatin quality, causally related to reduced preimplantation embryo development. The use of ejaculated immotile sperm for in vitro reproduction is debatable due to sperm DNA degradation.