Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure

An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.     

Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure

An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.     

Most germ-line mutations in the nevoid basal cell carcinoma syndrome lead to a premature termination of the PATCHED protein, and no genotype-phenotype correlations are evident.

The human homologue of the Drosophila segment polarity gene patched is implicated in the development of nevoid basal cell carcinoma syndrome (NBCCS) and in the genesis of sporadic basal cell carcinomas. In order to examine the phenotypic variability in NBCCS and to highlight functionally important domains of the PTCH protein, we have now screened 71 unrelated NBCCS individuals for mutations in the PTCH exons. We identified 28 mutations that are distributed throughout the entire gene, and most (86%) cause protein truncation. As part of this analysis, we demonstrate that failure of one NBCCS family to show clear linkage to chromosome 9q22.3-31 is most likely due to germinal mosaicism. We have identified three families bearing identical mutations with variable phenotypes, suggesting phenotypic variability in NBCCS is a complex genetic event. No phenotype genotype correlation between the position of truncation mutations and major clinical features was evident. Two missense mutations have been identified, and their location within transmembrane domains supports the notion that PTCH may have a transport function. The preponderance of truncation mutants in the germ line of NBCCS patients suggests that the developmental defects associated with the disorder are most likely due to haploinsufficiency.     

Aurora B expression in post-puberal testicular germ cell tumours

Aurora/Ipl1-related kinases are a conserved family of proteins that are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumours and have been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Previous studies of our group have shown that Aurora B expression is restricted to specific germinal cells. In this study, we have evaluated by immunohistochemical analysis Aurora B expression in post-puberal testicular germ cell tumours (22 seminomas, 2 teratomas, 15 embryonal carcinomas, 5 mixed germinal tumours with a prominent yolk sac tumour component and 1 choriocarcinoma). The Aurora B protein expression was detected in all intratubular germ cell tumours, seminomas and embryonal carcinomas analysed but not in teratomas and yolk sac carcinomas. The immunohistochemical data were further confirmed by Western blot analysis. In addition, the kinase Aurora B was vigorously expressed in GC-1 cells line derived from murine spermatogonia. The block of Aurora B function induced by a pharmacological inhibitor significantly reduced the growth of GC-1 cells suggesting that Aurora B is a potential therapeutic target. J. Cell. Physiol. 221: 435–439, 2009. © 2009 Wiley-Liss, Inc.     

Transgenerational effects from exposure to environmental toxic substances

Exposure of mouse germ cells to radiation and chemicals results in mutation, malformation, cancer and other adverse effects (e.g., functional disorders) in the offspring, though these findings have not been proven in human studies. Environmental toxic substances such as urethane (ethyl carbamate) which had been injected subcutaneously to 50 million people as a co-solvent of analgesics and dioxin (an endocrine disruptor) have been found to be associated with adverse effects in the progeny of mice after parental exposures. There are some reports on congenital malformations in the progeny of fathers who had been exposed to dioxin. However, these substances have not shown mutagenicity in in vitro assay systems such as bacterial systems even with S9, cell transformation assays, etc., in spite of their potent teratogenicity and carcinogenicity in in vivo systems. Urethane was negative in the mouse specific locus test for germ cell mutations, but elicited a significant response at the same loci in the offspring of mice treated during pregnancy. Further, urethane is a mutagen in Drosophila germ cell tests, specifically inducing point mutations. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) does not induce in vivo somatic mutations in mice and rats. It does not induce chromosomal aberrations when the mouse and/or human sperm are treated, but induces mutations at ESTR (expanded simple tandem repeat) loci in mice at low frequencies and also congenital malformations. In this paper, we first present an overview of the results of our studies on transgenerational effects of these toxic substances, compare the results with those obtained after radiation exposure, and then discuss our subsequent studies to reconcile the problems underlying their mutagenicity, teratogenicity and carcinogenicity.     

Quantitative analysis of germline mitosis in adult C. elegans

Certain aspects of the distal gonad of C. elegans are comparable to niche/stem cell systems in other organisms. The distal tip cell (DTC) caps a blind-ended tube; only the distal germ cells maintain proliferation in response to signaling from the DTC via the GLP-1/Notch signaling pathway in the germ line. Fruitful comparison between this system and other stem cell systems is limited by a lack of basic information regarding germ cell division behavior in C. elegans. Here, we explore the spatial pattern of cell division frequency in the adult C. elegans germ line relative to distance from the distal tip. We mapped the positions of actively dividing germline nuclei in over 600 fixed gonad preparations including the wild type and a gain-of-function ligand-responsive GLP-1 receptor mutant with an extended mitotic zone. One particularly surprising observation from these data is that the frequency of cell divisions is lower in distal-most cells-cells that directly contact the distal tip cell body-relative to cells further proximal, a difference that persists in the gain-of-function GLP-1 mutant. These results suggest that cell division frequency in the distal-most cells may be suppressed or otherwise controlled in a complex manner. Further, our data suggest that the presence of an active cell division influences the probability of observing simultaneous cell divisions in the same gonad arm, and that simultaneous divisions tend to cluster spatially. We speculate that this system behaves similarly to niche/stem cell/transit amplifying cell systems in other organisms.     

T-cell responses at the host: tumour interface

The evidence considered here reinforces the conclusion that T-cell responses to tumours involve complex cellular interactions. An attempt to summarize some of these interactions is shown. This emphasizes that not only are the interactions between the effector cell populations complicated, but that the target cell surface is also subject to variation and modification as a result of the immune response. A feature that also emerges from these studies is that most cells apparently responding to or infiltrating a tumour do not necessarily participate in its destruction, and it is in this area that experimental tumour systems have particular value. This also perhaps explains the preoccupation of experimentalists with the identification of 'the' effector cell crucial to tumour rejection. However, there is heterogeneity between systems in terms of the type of rejection response induced, but a logical basis for this heterogeneity is not established. If experimental studies could define the nature of the immune response generated by a tumour in the context of the biological features of the tumour itself, this could lead to the prediction of the immunogenicity and potential for induction of a rejection response for that tumor. Clearly, experimental tumour systems do not provide an exact reflection of the situation with human tumours. However, they may provide systems that illuminate particular aspects of the human response, and give precedents to guide the interpretation of data derived from human systems. This form of assessment is still at an early stage, but developments in the experimental field should provide a framework for the development and exploitation of T-cell responses to tumours.     

The symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton pump

The male germ line stem cell is the only cell type in the adult that can contribute genes to the next generation and is characterized by postnatal proliferation. It has not been determined whether this cell population can be used to deliberately introduce genetic modification into the germ line to generate transgenic animals or whether human somatic cell gene therapy has the potential to accidentally introduce permanent genetic changes into a patient's germ line. Here we report that several techniques can be used to achieve both in vitro and in vivo gene transfer into mouse male germ line stem cells using a retroviral vector. Expression of a retrovirally delivered reporter lacZ transgene in male germ line stem cells and differentiated germ cells persisted in the testis for more than 6 months. At least one in 300 stem cells could be infected. The experiments demonstrate a system to introduce genes directly into the male germ line and also provide a method to address the potential of human somatic cell gene therapy DNA constructs to enter a patient's germ line.     

Glp-3 Is Required for Mitosis and Meiosis in the Caenorhabditis Elegans Germ Line

The germ line is the only tissue in Caenorhabditis elegans in which a stem cell population continues to divide mitotically throughout life; hence the cell cycles of the germ line and the soma are regulated differently. Here we report the genetic and phenotypic characterization of the glp-3 gene. In animals homozygous for each of five recessive loss-of-function alleles, germ cells in both hermaphrodites and males fail to progress through mitosis and meiosis, but somatic cells appear to divide normally. Germ cells in animals grown at 15° appear by DAPI staining to be uniformly arrested at the G2/M transition with <20 germ cells per gonad on average, suggesting a checkpoint-mediated arrest. In contrast, germ cells in mutant animals grown at 25° frequently proliferate slowly during adulthood, eventually forming small germ lines with several hundred germ cells. Nevertheless, cells in these small germ lines never undergo meiosis. Double mutant analysis with mutations in other genes affecting germ cell proliferation supports the idea that glp-3 may encode a gene product that is required for the mitotic and meiotic cell cycles in the C. elegans germ line.     

Preferential localization of SSEA-4 in interfaces between blastomeres of mouse preimplantaion embryos

The monoclonal antibody 6E2 raised against the embryonal carcinoma cell line NCR-G3 had been shown to also react with human germ cells. Thin-layer chromatography (TLC) immunostaining revealed that 6E2 specifically reacts with sialosylglobopentaosylceramide (sialylGb5), which carries an epitope of stage-specific embryonic antigen-4 (SSEA-4), known as an important cell surface marker of embryogenesis. The immunostaining of mouse preimplantation embryos without fixation showed that the binding of 6E2 caused the clustering and consequent accumulation of sialylGb5 at the interface between blastomeres. These results suggest that SSEA-4 actively moves on the cell surface and readily accumulates between blastomeres after binding of 6E2.     

Maternal effects of general and regional specificity on embryos of Drosophila melanogaster caused by dunce and rutabaga mutant combinations

Summary The developmental patterns of embryos produced by female germ line cells homozygous for null-enzyme mutations of dunce and for dunce in combination with each of three different rutabaga mutations are compared with the normal pattern. At least three discrete developmental defects at progressive stages following fertilization can be identified and correlated with the loss of adenylate cyclase activity caused by rutabaga mutations, suggesting that the defects are caused by elevated cyclic AMP levels in female germ line cells. The earliest defect occurs soon after fertilization and affects DNA replication and mitosis, prevents nuclear migration, and leads to large polyploid nuclei. A later defect prevents cleavage nuclei from migrating into, or dividing in, the posterior region of the egg. The last affects the developmental behavior or fate of blastoderm cells. Some of these defects mimic those produced by previously described maternal-effect mutations.     

Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases

Direct genomic manipulation at a specific locus is still not feasible in most vertebrate model organisms. In vertebrate cell lines, genomic lesions at a specific site have been introduced using zinc-finger nucleases (ZFNs). Here we adapt this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdr (also known as kdra). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early-stage embryos are easily accessible.     

Establishment of three human breast epithelial cell lines derived from carriers of the 999del5 <Emphasis Type="Italic">BRCA2</Emphasis> icelandic founder mutation

Summary Germ line mutations in BRCA1 and BRCA2 account for a large proportion of inherited breast and ovarian cancer. Both genes are involved in DNA repair by homologous recombination and are thought to play a vital role in maintaining genomic stability. A major drawback for long-term functional studies of BRCA in general and BRCA2 in particular has been a lack of representative human breast epithelial cell lines. In the present study, we have established three cell lines from two patients harboring the 999del5 germ line founder mutation in the BRCA2 gene. Primary cultures were established from cellular outgrowth of explanted tissue and subsequently transfected with a retroviral construct containing the HPV-16 E6 and E7 oncogenes. Paired cancer-derived and normal-derived cell lines were established from one patient referred to as BRCA2-999del5-2T and BRCA2-999del5-2N, respectively. In addition, one cell line was derived from cancer-associated normal tissue from another patient referred to as BRCA2-999del5-1N. All three cell lines showed characteristics of breast epithelial cells as evidenced by expression of breast epithelial specific cytokeratins. Cytogenetic analysis showed marked chromosomal instability with tetraploidy and frequent telomeric association. In conclusion, we have established three breast cpithelial cell lines from two patients carrying the BRCA2 Icelandic 999del5 founder mutation. These cell lines from the basis for further studies on carcinogenesis and malignant progression of breast cancer on a defined genetic background. Agla J. Rubner Fridriksdottir and Thorarinn Gudjonsson contributed equally to this study.     

Germ line specific factors in chemical mutagenesis

Chemical mutagenesis test results have not revealed evidence of germ line specific mutagens. However, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend upon the particular cell stage exposed. Many factors inherent in the germ line can be speculated to influence chemical transport to, and interaction with, target cell populations to result in mutagenic outcomes. The level of uncertainty regarding the general operation of such factors, in combination with the limited availability of chemical test data designed to address comparative somatic and germ cell mutagenesis, leaves open the question of whether there are mutagens specifically affecting germ cells. This argues for a conservative approach to interpreting germ cell risk from somatic cell mutation analysis.     

Clonal analysis of the tissue specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female-sterile mutation Fs(1)K1237

Using the newly isolated, germ line-dependent dominant female-sterile mutation Fs(1)K1237, we have characterized the germ line or somatic line dependence of 25 X-linked recessive female-sterile mutations. Since Fs(1)K1237/+ females fail to lay eggs, only germ line cells which lose Fs(1)K1237 as a result of X-ray-induced mitotic recombination are capable of producing eggs. Such recombination events will render genes on the homologous chromosome homozygous. If this chromosome carries a recessive female-sterile mutation, the fertility will be restored only if the altered function is not required in the germ line. Using this test, we have classified 25 recessive female-sterile mutations: 12 affect germ line function, 12 affect somatic line function, and one gave an ambiguous result for which an explanation is proposed. For a few of the somatic line-dependent mutants, we found that some eggs derived from germ line clones showed the same phenotype as eggs laid by females homozygous for the recessive female-sterile mutation. These results are discussed in terms of a coincident production of clones in the follicle cells.     

No extension of lifespan by ablation of germ line in Drosophila

Increased reproduction is frequently associated with a reduction in longevity in a variety of organisms. Traditional explanations of this 'cost of reproduction' suggest that trade-offs between reproduction and longevity should be obligate. However, it is possible to uncouple the two traits in model organisms. Recently, it has been suggested that reproduction and longevity are linked by molecular signals produced by specific reproductive tissues. For example, in Caenorhabditis elegans, lifespan is extended in worms that lack a proliferating germ line, but which possess somatic gonad tissue, suggesting that these tissues are the sources of signals that mediate lifespan. In this study, we tested for evidence of such gonadal signals in Drosophila melanogaster. We ablated the germ line using two maternal effect mutations: germ cell-less and tudor. Both mutations result in flies that lack a proliferating germ line but that possess a somatic gonad. In contrast to the findings from C. elegans, we found that germ line ablated females had reduced longevity relative to controls and that the removal of the germ line led to an over-proliferation of the somatic stem cells in the germarium. Our results contrast with the widely held view that it is downstream reproductive processes such as the production and/or laying of eggs that are costly to females. In males, germ line ablation caused either no difference, or a slight extension, in longevity relative to controls. Our results indicate that early acting, upstream reproductive enabling processes are likely to be important in determining reproductive costs. In addition, we suggest that the specific roles and putative patterns of molecular signalling in the germ line and somatic tissues are not conserved between flies and worms.     

Ageing and mammalian mitochondrial genetics

Mitochondrial DNA (mtDNA) is essential for the ability of mammalian cells to generate a functional oxidative phosphorylation system. Mutations in mtDNA occur in human disease and also during ageing. Here, we address three questions concerning the occurrence and accumulation of mtDNA mutations during the lifespan of the mammalian cell. What sort of mutations accumulate with age in humans and other mammals? How is the female germ line spared from the accumulation of such mutations as occurs in many somatic tissues, so that neonates normally start life with a ‘clean sheet'? Is the occurrence of mtDNA mutations associated with the functional decline of cells and tissues during ageing? We argue that mtDNA mutations in somatic cells do not just reflect a passive imprint of ageing, but they are causally associated with the loss of bioenergetic function during the ageing process.     

Mutations in Nop60B,the Drosophila homolog of human dyskeratosis congenita 1, affect the maintenance of the germ-line stem cell lineage during spermatogenesis

Spermatogenesis in Drosophila is maintained by germ-line stem cells. These cells undergo self-renewing divisions and also generate daughter gonial cells, whose function is to amplify the germ cell pool. Gonial cells subsequently differentiate into spermatocytes that undergo meiosis and generate haploid gametes. To elucidate the circuitry that controls progression through spermatogenic stem cell lineages, we are identifying mutations that lead to either excess germ cells or germ cell loss. From a collection of male sterile mutants, we identified P-element-induced hypomorphic alleles of nop60B, a gene encoding a pseudouridine synthase. Although null mutations are lethal, our P element-induced alleles generate viable, but sterile flies, exhibiting severe testicular atrophy. Sterility is reversed by P-element excision, and the atrophy is rescued by a Nop60B transgene, confirming identity of the gene. Using cell-type-specific markers, we find that testicular atrophy is due to severe loss of germ cells, including stem cells, but much milder effects on the somatic cells, which are themselves maintained by a stem cell lineage. We show that Nop60B activity is required intrinsically for the maintenance of germ-line stem cells. The relationship of these phenotypes to the human syndrome Dyskeratosis congenita, caused by mutations in a Nop60B homolog, is discussed.