Environmental Exposure of the Mouse Germ Line: DNA Adducts in Spermatozoa and Formation of De Novo Mutations during Spermatogenesis

Background

Spermatozoal DNA damage is associated with poor sperm quality, disturbed embryonic development and early embryonic loss, and some genetic diseases originate from paternal de novo mutations. We previously reported poor repair of bulky DNA-lesions in rodent testicular cells.

Methodology/Principal Findings

We studied the fate of DNA lesions in the male germ line. B[a]PDE-N²-dG adducts were determined by liquid chromatography-tandem mass spectrometry, and de novo mutations were measured in the cII-transgene, in Big Blue®mice exposed to benzo[a]pyrene (B[a]P; 3×50 mg/kg bw, i.p.). Spermatozoa were harvested at various time-points following exposure, to study the consequences of exposure during the different stages of spermatogenesis. B[a]PDE-N²-dG adducts induced by exposure of spermatocytes or later stages of spermatogenesis persisted at high levels in the resulting spermatozoa. Spermatozoa originating from exposed spermatogonia did not contain DNA adducts; however de novo mutations had been induced (p = 0.029), specifically GC-TA transversions, characteristic of B[a]P mutagenesis. Moreover, a specific spectrum of spontaneous mutations was consistently observed in spermatozoa.

Conclusions/Significance

A temporal pattern of genotoxic consequences following exposure was identified, with an initial increase in DNA adduct levels in spermatozoa, believed to influence fertility, followed by induction of germ line de novo mutations with possible consequences for the offspring.     

小鼠原生殖细胞的研究进展

在动物的整个生命周期中,生殖细胞提供了各代间的连续性。对于多数动物来说,其生殖细胞不是在生殖腺中产生的,其前身原生殖细胞（ＰｒｉｍｏｒｄｉａｌＧｅｒｍＣｅｌ简称ＰＧＣ）是迁移到正在发育着的生殖腺中的。近年来,由于ＰＧＣ体外培养技术的不断完善,ＰＧＣ的...     

Interactions between Germ Cells and Extracellular Matrix Glycoproteins during Migration and Gonad Assembly in the Mouse Embryo

Cells are known to bind to individual extracellular matrix glycoproteins in a complex and poorly understood way. Overall strength of adhesion is thought to be mediated by a combinatorial mechanism, involving adhesion of a cell to a variety of binding sites on the target glycoproteins. During migration in embryos, cells must alter their overall adhesiveness to the substrate to allow locomotion. The mechanism by which this is accomplished is not well understood. During early development, the cells destined to form the gametes, the primordial germ cells (PGCs), migrate from the developing hind gut to the site where the gonad will form. We have used whole-mount immunocytochemistry to study the changing distribution of three extracellular matrix glycoproteins, collagen IV, fibronectin, and laminin, during PGC migration and correlated this with quantitative assays of adhesiveness of PGCs to each of these. We show that PGCs change their strength of adhesion to each glycoprotein differentially during these stages. Furthermore, we show that PGCs interact with a discrete tract of laminin at the end of migration. Closer analysis of the adhesion of PGCs to laminin revealed that PGCs adhere particularly strongly to the E3 domain of laminin, and blocking experiments in vitro suggest that they adhere to this domain using a cell surface proteoglycan.     

Allele-Specific Non-CpG Methylation of the Nf1 Gene during Early Mouse Development

Recent reports of cytosine methylation occurring at CpA and CpT dinucleotides in murine ES cells as well as in Drosophila have renewed interest in methylation at sites other than CpGs. Our examination of the murine neurofibromatosis type 1 gene by sodium bisulfite genomic sequencing has revealed non-CpG methylation primarily in the oocyte and the maternally derived allele of the 2-cell embryo, with markedly lower levels found in sperm. Non-CpG methylation was not found in later stages of embryo development or in adult tissue. Our results suggest that maternal-specific de novo non-CpG methylation has occurred sometime between ovulation and formation of the 2-cell embryo, while during the same period the paternally derived allele has undergone site-specific active demethylation. Our data demonstrate both stage and parent-of-origin specific changes in methylation patterns within the neurofibromatosis type 1 coding region-involving cytosines located at both CpG and non-CpG dinucleotides.     

Prdm1在哺乳动物胚胎和生殖细胞发育中的作用

Prdm1（PR domain zinc finger protein 1）,又称为Blimpl（B—lymphocyte-induced maturation protein-1）,是一个具有锌指结构的转录因子,通过调控多个基因的表达影响哺乳动物多种类型细胞的发育分化。从1991年发现至今,有关Prdm1的研究进展迅速,Prdm1在促进B细胞向浆细胞终末分化过程中的作用已经得到共识。但是,在小鼠及其他哺乳动物的胚胎发育过程中,尤其是关于Prdm1在生殖细胞发育分化中的作用机理研究则起步相对较晚。近期发现,在哺乳动物胚胎发育过程中,Prdm1在原始生殖细胞的形成、干细胞全能性的维持以及其他组织器官的形成中都发挥了重要的作用。     

Expression of Calcium-Binding Proteins in Layer 1 Reelin-Immunoreactive Cells during Rat and Mouse Neocortical Development

Cajal-Retzius cells in layer 1 of the developing cerebral cortex and their product of secretion, reelin, an extracellular matrix protein, play a crucial role in establishing the correct lamination pattern in this tissue. As many studies into reelin signaling routes and pathological alterations are conducted in murine models, we used double-labeling and confocal microscopy to compare the distribution of the cell-specific markers, calretinin and calbindin, in reelin-immunoreactive cells during postnatal rat and mouse neocortical development. In the rat, neither calretinin nor calbindin colocalized with reelin in Cajal-Retzius cells at P0-P2. From P5 to P14, the colocalization of reelin and calretinin was commonly found in presumptive rat subpial piriform cells. These cells progressively lacked calretinin expression and persisted into adulthood as part of the pool of layer 1 reelin-positive interneurons. Conversely, in the mouse, reelin-immunoreactive Cajal-Retzius cells colocalized with calretinin and/or calbindin. Subpial piriform cells containing reelin and calretinin were identified at P5-P7, but lacked calretinin expression at P14. In adult mice, as in the rat, reelin-immunoreactive cells did not colocalize with calcium-binding proteins. Our results reveal a complex neurochemical profile of layer 1 cells in the rat neocortex, which makes using a single calcium-binding protein as a marker of rodent reelin-immunoreactive cells difficult.     

p53蛋白在周期调节蛋白A1变异引起的雄性小鼠生殖细胞凋亡中的作用

为研究p5 3蛋白在周期调节蛋白A1(cyclinA1)变异引起的雄性小鼠生殖细胞凋亡中的作用 ,以p5 3基因敲除的小鼠和周期调节蛋白A1基因敲除的小鼠杂交 ,获取同胎生单基因变异和双基因同时变异的雄性后代共 4组 12只 .比较它们的性腺和生殖细胞发育 ,并用TUNEL染色法观察和比较生殖细胞的凋亡情况 .在睾丸最大横切面上观察到 :周期调节蛋白A1变异组凋亡细胞最多 (348± 10 4个 ) ,明显高于p5 3 周期调节蛋白A1双基因变异组 (12 1± 38个 ) ,t=3 2 5 79,P =0 0 4 72 .p5 3变异组凋亡细胞最少 (45± 2 4个 ) ,配对t检验显示有非常显著性差异 ,t=8 4 0 13,P =0 0 0 35 .这一研究结果提示 ,p5 3基因可能在雄性生殖细胞的发育中起监视作用 ,并在周期调节蛋白A1变异引起发育异常时启动p5 3途径造成异常细胞的凋亡 .     

小鼠胚胎干细胞向生殖细胞分化的研究

小鼠胚胎干细胞(ESC)在体外可以分化为多种细胞类型,其中包括各阶段的生殖细胞,甚至精细胞和成熟卵母细胞。ESC向生殖细胞分化的效率受到包括生长因子、激素和体细胞等多种因素的影响,在体外形成的是雌性配子还是雄性配子与ESC是XX型还是XY型没有必然联系。简要综述了小鼠生殖细胞在体内外的分化发育、性别决定和增殖等,并总结和展望了ESC向生殖细胞分化研究面临的问题和应用前景。     

小鼠生殖细胞体外分化与发育机制

小鼠作为发育机制的模式动物,其生殖细胞分化与发育的研究一直是发育生物学研究的重点之一。主要综述了小鼠原始生殖细胞的起源、迁移与增殖的机制,以及原始生殖细胞向生殖细胞的分化,卵母细胞与精子的发生与发育机理,讨论了胚胎干细胞向生殖细胞体外诱导分化以及生殖细胞体外培养的应用前景。     

Mitochondrial DNA Heteroplasmy and Purifying Selection in the Mammalian Female Germ Line

Inherited mutations in the mitochondrial (mt)DNA are a major cause of human disease, with approximately 1 in 5000 people affected by one of the hundreds of identified pathogenic mtDNA point mutations or deletions. Due to the severe, and often untreatable, symptoms of many mitochondrial diseases, identifying how these mutations are inherited from one generation to the next has been an area of intense research in recent years. Despite large advances in our understanding of this complex process, many questions remain unanswered, with one of the most hotly debated being whether or not purifying selection acts against pathogenic mutations during germline development.     

Dullard/Ctdnep1 Modulates WNT Signalling Activity for the Formation of Primordial Germ Cells in the Mouse Embryo

Dullard/Ctdnep1 is a member of the serine/threonine phosphatase family of the C-terminal domain of eukaryotic RNA polymerase II. Embryos lacking Dullard activity fail to form primordial germ cells (PGCs). In the mouse, the formation of PGCs is influenced by BMP4 and WNT3 activity. Although Dullard is reputed to negatively regulate BMP receptor function, in this study we found mutations in Dullard had no detectable effect on BMP4 and p-Smad activity. Furthermore Dullard mutations did not influence the dosage-dependent inductive effect of Bmp4 in PGC formation. However, Dullard may function as a positive regulator of WNT signalling. Combined loss of one copy each of Dullard and Wnt3 had a synergistic effect on the reduction of PGC numbers in the compound heterozygous embryo. In addition, loss of Dullard function was accompanied by down-regulation of WNT/β-catenin signalling activity and a reduction in the level of Dishevelled 2 (Dvl2). Therefore, Dullard may play a role in the fine-tuning of WNT signalling activity by modulating the expression of ligands/antagonists and the availability of Dvl2 protein during specification of the germ cell lineage.