Spermatogenesis in Drosophila. A genetic approach to cellular and subcellular differentiation.

Y chromosomal fertility genes are essential for spermatogenesis, but those genes which code for major structural components of the spermatozoon and those controlling sperm morphogenesis must be located on a different chromosome. In the past, it had been questioned whether it would be possible to achieve a meaningful classification of male sterile mutations by light microscopy. I now show, however, that comparison of 244 autosomal male sterile mutants of Drosophila hydei with 400 similar mutants in D. melanogaster not only allows such a classification on the basis of the apparent targets, but also permits a genetic dissection of sperm morphogenesis. Differentiation of male germ cells is best characterized as spermeoteleosis, since male sterile mutations have the effect of aborting spermatogenesis rather than changing the cellular fate of the germ cells. In contrast to earlier proposals concerning sequential determinative events during this process, male sterile mutations can block spermatogenesis at nearly every stage, and not, as previously postulated, exclusively at the transitions between gonial, meiotic, and postmeiotic stages. Male sterile mutations can modify the topology of the organelles of a spermatid, and they can also affect the different components (i.e., nucleus, axoneme, nebenkern) of a germ cell to quite different degrees, leading to characteristic pleiotropic phenotypes. Some male sterile mutations can decouple the development of the different components of a germ cell, i.e., they may lead to a heterochrony of the development of the different subcellular structures, or they may permit the differentiation of some components of a germ cell even in the complete absence of an organelle. Thus, it is possible to describe spermatogenesis as the concerted, but not interdependent, execution of separate developmental programs for the particular components of male germ cells.     

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.     

性腺母细胞瘤的分子遗传机制研究进展

性腺母细胞瘤(Gonadoblastoma, GB)是一种由性索和生殖细胞演化而来的罕见原位性腺肿瘤,与性腺遗传物质异常有密切联系。80%的GB患者表现为46,XY女性表型,其余为45,XY 和46,XX性别发育异常患者等。35%的GB会进一步演化为无性细胞瘤和精原细胞瘤等恶性肿瘤。由于表型与遗传的异质性,GB的分子遗传机制还未完全揭示。越来越多的研究显示GB的发生与性别分化和决定调控基因(如SRY、WT1、SOX9、Foxl2和TSPY等)之间存在密切关联,且表现出遗传与表观遗传调控相互作用。本文综述了GB的临床表现、病理特征、诊断与治疗措施,总结了性腺遗传异常导致GB的分子遗传与表观遗传调控机制,分析并归纳参与GB形成相关基因的共同表达调控网络,指出了当前研究中的障碍与不足,为进一步研究GB致病分子机制提供新思路。     

Elevated Mutagenicity in Meiosis and Its Mechanism

Diploid germ cells produce haploid gametes through meiosis, a unique type of cell division. Independent reassortment of parental chromosomes and their recombination leads to ample genetic variability among the gametes. Importantly, new mutations also occur during meiosis, at frequencies much higher than during the mitotic cell cycles. These meiotic mutations are associated with genetic recombination and depend on double‐strand breaks (DSBs) that initiate crossing over. Indeed, sequence variation among related strains is greater around recombination hotspots than elsewhere in the genome, presumably resulting from recombination‐associated mutations. Significantly, enhanced mutagenicity in meiosis may lead to faster divergence during evolution, as germ‐line mutations are the ones that are transmitted to the progeny and thus have an evolutionary impact. The molecular basis for mutagenicity in meiosis may be related to the repair of meiotic DSBs by polymerases, or to the exposure of single‐strand DNA to mutagenic agents during its repair.     

Effects of chemical and physical agents on recombination events in cells of the germ line of male and female Drosophila melanogaster

Genotoxic agents can induce mutations as well as recombination in the genetic material. The fruit fly Drosophila melanogaster was one of the first assay systems to test physical and chemical agents for recombinogenic effects. Such effects can be observed in cells of the germ line as well as in somatic cells. At present information is available on 54 agents, among them 48 chemicals that have been tested in cells of the germ line of males and/or females. Effects on meiotic recombination in female germ cells cannot simply be classified as positive or negative since for a number of agents, depending on the chromosome region studied, recombination frequencies may be increased, unaffected or decreased. The male germ line of D. melanogaster represents a unique situation because meiotic recombination does not occur. Among 25 agents tested in male germ cells 24 did induce male recombination, among them alkylating, intercalating and cross-linking agents, direct-acting ones as well as compounds needing metabolic activation. With several compounds the frequency of induced recombination is highest in the heterochromatic regions near the centromeres. In brood pattern analyses, e.g., after exposure of adult males to ionizing radiation, the first appearance of crossover progeny is indicative of the sampling of exposed spermatocytes. In premeiotic cells of the male and the female germ line mitotic recombination can occur. Upon clonal expansion of the recombinant cells, clusters of identical crossovers can be observed.     

Chemical mutagenesis and fine-structure functional analysis of the mouse genome

Heritable mutations constitute important raw materials for mammalian developmental genetics and general genome studies. Mutations induced by high-efficiency chemical mutagenesis of germ cells in mice can be used in genetic and molecular studies to complement physical-mapping strategies and to examine the nature and extent of the functional complexities hidden within the mammalian genome.     

Models and assumptions underlying genetic risk assessment

Various methods employed for estimating the genetic risks of radiation are reviewed. With the doubling-dose method, genetic damage is expressed as an increase in cases of known genetic disease. The actual doubling dose is based on figures obtained with the mouse. There have been no recent data on induced mutation frequencies. Recent results suggest that the prevalence figure for multifactorial disease may be at least one order of magnitude higher than before. Various assumptions underlying the doubling-dose concept are discussed in the light of recent findings on: (1) spontaneous mutations resulting from insertion elements, and (2) the comparability between spontaneous and induced mutations. The so-called direct method makes use of figures for induction of dominant mutations affecting the skeleton and the lens of the eye in the mouse, and of translocation induction in monkeys. Induction rates are converted to overall rates of induced dominant effects in man by applying certain assumptions. The proportionality between dose and effect is the basis for all genetic risk assessments. The possible significance of data on human lymphocytes indicating a threshold below 4 rad and the induction of repair enzymes by low radiation doses is discussed. The parallelogram approach is based on the principle that estimates can be obtained on the amount of genetic damage that cannot always be assessed directly. Thus mutations in mouse germ cells can be predicted by using mutation frequencies in cultured mammalian cells and O6-ethylguanine adducts. Measurement of haemoglobin mutations in human and mouse erythrocytes, and of HPRT-deficient mutations in lymphocytes of man and mouse should make more precise estimates of mutation frequencies in human germ cells possible. The development of a database on mutations in somatic cells of the mouse, their induction frequencies and molecular nature are considered an important priority. Used in combination with mouse germ-cell mutation frequencies, they should enable more precise risk estimates on the basis of mutations in somatic cells of man.     

Cell-autonomous and inductive signals can determine the sex of the germ line of drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.     

Comparison of types of chemically induced genetic changes in mammals

The present paper reviews the currently available in vivo systems for detection of chemically induced mutations and chromosome aberrations and summarizes the data of the relevant tests for mammalian germ-cell mutations (specific-locus test and heritable translocation test). The value of in vivo screening tests (somatic mutations and sperm abnormalities) for predicting specific-locus mutations is illustrated by comparing doubling doses. The results from the mammalian germ-cell mutation tests (specific-locus test and heritable translocation test) constitute the base-line for an assessment of predictability. Radiation and chemically induced specific-locus mutations differ in a number of respects, suggesting a need for caution in making risk estimates for chemical mutagen exposures in terms of radiation-equivalent doses. In vivo nondisjunction tests are discussed. Finally, unsolved problems and difficulties in generalizing qualitative and quantitative correlations between test systems are outlined. It is concluded that even qualitative predictions from data on somatic cells to germ cells are at best insecure because germ-cell specificity cannot be foretold, not to mention the fact that quantitative extrapolations from the results of in vivo screening tests, in general, are fraught with even more uncertainties. There is an acute need for collection of more data from studies involving germ cells.     

The comet assay in male reproductive toxicology

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.     

Genetic toxicology of 1,2-dibromo-3-chloropropane (DBCP)

1,2-Dibromo-3-chloropropane (DBCP) is a nematocide, which has been used extensively as a soil fumigant in agriculture. Since sterility was found among male workers involved in the manufacture of DBCP, great concern has been focused on the genetic hazards of DBCP. DBCP gave positive results in many tests such as microbial, in vitro cytogenetics, and Drosophila studies. In mammalian test systems, DBCP caused chromosomal aberrations in the bone marrow cells and dominant-lethal mutations in germ cells in rats. In mice, there were no signs of DBCP-induced heritable mutation in germ cells, although point mutations were detected in somatic cells. The occurrence of Y-chromosomal non-disjunction was indicated in DBCP-exposed male workers by an increased number of sperm containing 2 Y-chromosomes.     

The frequency of dominant cataract and recessive specific-locus mutations in mice derived from 80 or 160 mg ethylnitrosourea per kg body weight treated spermatogonia

A systematic comparison of the frequency of dominant cataract and recessive specific-locus mutations in mice has been extended to include results for 80 and 160 mg ethylnitrosourea per kg body weight spermatogonial treatment. The frequency of confirmed dominant cataract mutations in the historical control, 80 and 160 mg/kg ethylnitrosourea treatment groups was 1/22594, 8/5090 and 14/6435, respectively. The frequency of recessive specific-locus mutations in the same dose groups was, respectively, 19/227805, 20/13274 and 35/8658. These present results confirm previous results, which indicate that ethylnitrosourea is effective in inducing both recessive specific-locus and dominant cataract mutations although the per locus mutation rate to recessive alleles was observed to be approximately 6 times greater than the per locus mutation rate to dominant alleles. The exclusion of certain classes of lens opacity variant phenotypes, previously demonstrated not to be due to a dominant mutation, from the group of suspected dominant cataract mutations subjected to a genetic confirmation test has greatly improved the efficiency of the test. A total of 23 dominant cataract mutations were confirmed from a group of 67 phenotypic variants. Of the 23 confirmed dominant cataract mutations, 8 were shown to have reduced transmission to the following generation of offspring expressing the mutant phenotype. These results are also consistent with previous results for ethylnitrosourea or radiation treatment in which it was shown that approximately one-third of the recovered mutations have reduced penetrance. One group of dominant cataract mutations, with phenotypic effects on the polar, sub-capsular or corneal regions, is overly represented in the group of recovered mutations with a reduced transmission of offspring expressing the mutant phenotype. Two hypotheses are suggested for this observation, both dependent on the fact that the regions affected indicate that the mutations are expressed later in the development of the eye. Either all carrier individuals have not expressed the phenotype at the time of examination and classification, or later acting mutations are more subject to environmental interactions resulting in more variable expression. Finally, it is argued that a dominant cataract mutation test represents a most practicable protocol to screen for induced dominant mutations in germ cells of the mouse. The imposition of the criterion that suspected variants be subjected to a genetic confirmation test has at least two advantages beside the fact that results represent unambiguous mutational events.(ABSTRACT TRUNCATED AT 400 WORDS)     

A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype.

The genetic analysis of a gene at a late developmental stage can be impeded if the gene is required at an earlier developmental stage. The construction of mosaic animals, particularly in Drosophila, has been a means to overcome this obstacle. However, the phenotypic analysis of mitotic clones is often complicated because standard methods for generating mitotic clones render mosaic tissues that are a composite of both mutant and phenotypically normal cells. We describe here a genetic method (called EGUF/hid) that uses both the GAL4/UAS and FLP/FRT systems to overcome this limitation for the Drosophila eye by producing genetically mosaic flies that are otherwise heterozygous but in which the eye is composed exclusively of cells homozygous for one of the five major chromosome arms. These eyes are nearly wild type in size, morphology, and physiology. Applications of this genetic method include phenotypic analysis of existing mutations and F(1) genetic screens to identify as yet unknown genes involved in the biology of the fly eye. We illustrate the utility of the method by applying it to lethal mutations in the synaptic transmission genes synaptotagmin and syntaxin.     

Mammalian spermatogenesis investigated by genetic engineering.

Genes involved in mammal spermatogenesis can now be identified through mutants created by genetic engineering. Information has been obtained on male meiosis, but also on the factors regulating the proliferation, maintenance and differentiation of male germ cells. Its has also increased our knowledge of the germ cell phenotype emerging from an altered germ cell genotype. This review is focused on data from genes expressed in male germ cells and on the question of how germ cells and Sertoli cells cope with the molecular lesions induced. The conservation of a wild-type phenotype of male germ cells in mutant mice is discussed, and how the mouse genetic background can lead to different germ cell phenotypes for a given gene mutation.     

Review of the molecular characteristics of gene mutations of the germline and somatic cells of the human

Molecular analyses of the limited number of de novo germinal mutations identified in humans indicate that an array of alterations in gene structure can be generated. Similar conclusions are derived from the large data set obtained from molecular analyses of alleles that segregate in the human population and cause genetic diseases. The molecular alterations include nucleotide substitutions as well as insertions, deletions and other rearrangements of the DNA. The lesions may be located in the coding or the noncoding regions of genes or may involve the flanking sequences. The insertions and deletions involve fragments ranging from single nucleotides to many kilobases, and involve both unique sequences and repetitive elements. The nature of the lesions observed to date as either de novo mutations or segregating variants suggests there are locus-specific characteristics of the alterations in DNA structure that are recovered as genetic diseases. Differences in mutation spectra among genetic loci appear to reflect both the structure of the target sequences and the relationship between gene structure and gene function. No induced germinal mutations have been identified, thus no data are available that reveal the relationships between mutagenic exposures and the molecular fingerprints of the lesion induced in the human germ cell and transmitted to the subsequent generations. In contrast, the prospects for analyzing the roles of genetic target, exposure history and individual responsiveness to exposure in creating particular molecular lesions in somatic cells are excellent, both for alterations of single nucleotides and for major alterations of gene structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Avoiding bad genes: oxidatively damaged DNA in germ line and mate choice

August Weismann proposed that genetic changes in somatic cells cannot pass to germ cells and hence to next generations. Nevertheless, evidence is accumulating that some environmental effects can promote heritable changes in the DNA of germ cells, which implies that some somatic influence on germ line is possible. This influence is mostly detrimental and related to the presence of oxidative stress, which induces mutations and epigenetic changes. This effect should be stronger in males due to the particular characteristics of sperm. Here, we propose the hypothesis that females are able to avoid males with oxidatively damaged DNA in the germ line by using oxidative-dependent (pre- and post-mating) signals. This new hypothesis may shed light on unsolved questions in evolutionary biology, such as the benefits of polyandry, the lek paradox, or the role of sexual selection on the evolution of aging.     

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.     

Elimination of deleterious mutations in plastid genomes by gene conversion

Asexual reproduction is believed to be detrimental, mainly because of the accumulation of deleterious mutations over time, a hypothesis known as Muller's ratchet. In seed plants, most asexually reproducing genetic systems are polyploid, with apomictic species (plants forming seeds without fertilization) as well as plastids and mitochondria providing prominent examples. Whether or not polyploidy helps asexual genetic systems to escape Muller's ratchet is unknown. Gene conversion, particularly when slightly biased, represents a potential mechanism that could allow asexual genetic systems to reduce their mutation load in a genome copy number-dependent manner. However, direct experimental evidence for the operation of gene conversion between genome molecules to correct mutations is largely lacking. Here we describe an experimental system based on transgenic tobacco chloroplasts that allows us to analyze gene conversion events in higher plant plastid genomes. We provide evidence for gene conversion acting as a highly efficient mechanism by which the polyploid plastid genetic system can correct deleterious mutations and make one good genome out of two bad ones. Our finding that gene conversion can be biased may provide a molecular link between asexual reproduction, high genome copy numbers and low mutation rates.