Essential role of brc-2 in chromosome integrity of germ cells in C. elegans

brc-2, an ortholog of BRCA2 in Caenorhabditis elegans, is essential in the maintenance of genetic integrity. In C. elegans, cellular location correlates with meiotic progression, and transgene-induced cosuppression is observed in the germ line but not in somatic cells. We used these unique features to dissect the role of brc-2 in the germ line from that in somatic cells. In situ hybridization of wild type animals revealed that brc-2 gene expression was higher in oocytes than in other germline cells, and was barely detectable in mitotic cells. In contrast, germ cells containing multicopies of the brc-2 transgene showed no significant in situ hybridization signal at any oogenesis stage, confirming that brc-2 expression was functionally cosuppressed in the transgenic germ line. RAD-51 foci formation in response to DNA damage was abrogated in brc-2-cosuppressed germ cells, whereas wild-type germ cells showed strong RAD-51 foci formation. These germ cells exhibited massive chromosome fragmentation and decompaction instead of six bivalent chromosomes in diakinesis. Accordingly, lethality was observed after the early stage of germline development. These results suggest that brc-2 plays essential roles in chromosome integrity in early prophase, and therefore is crucial in meiotic progression and embryonic survival.     

Dna2 requirement for normal reproduction of Caenorhabditis elegans is temperature-dependent

To determine the function of Dna2 in a multicellular organism, the Caenorhabditis elegans Dna2 expression was probed and deletion mutant phenotypes were analyzed. Dna2 was localized to the nuclei of C. elegans oocytes and early embryos by immunostaining or green fluorescent protein-tagging. A homozygous dna2 deletion mutant showed a reduced brood size and embryonic lethality, and the phenotypes greatly depended on growth temperature and aggravated in the succeeding generation. The mutant embryos also showed delayed cell divisions, which together with temperature-dependence of the mutant phenotypes supported the well-conserved role of Dna2 in DNA replication.     

Efficacy of Four Nematicides Against the Reproduction and Development of Pinewood Nematode,Bursaphelenchus xylophilus

To understand the efficacy of emamectin benzoate, avermectin, milbemectin, and thiacloprid on the reproduction and development of Bursaphelenchus xylophilus, seven parameters, namely population growth, fecundity, egg hatchability, larval lethality, percent larval development, body size, and sexual ratio, were investigated using sublethal (LC₂₀) doses of these compounds in the laboratory. Emamectin benzoate treatment led to a significant suppression in population size, brood size, and percent larval development with 411, 3.50, and 49.63%, respectively, compared to 20850, 24.33, and 61.43% for the negative control. The embryonic and larval lethality increased obviously from 12.47% and 13.70% to 51.37% and 75.30%, respectively. In addition, the body length was also significantly reduced for both males and females in the emamectin benzoate treatment. Avermectin and milbemectin were also effective in suppressing population growth by increasing larval lethality and reducing larval development, although they did not affect either brood size or embryonic lethality. Body length for both male and female worms was increased by avermectin. Thiacloprid caused no adverse reproductive effects, although it suppressed larval development. Sexual ratio was not affected by any of these four nematicides. Our results indicate that emamectin benzoate, milbemectin, and avermectin are effective against the reproduction of B. xylophilus. We think these three nematicides can be useful for the control of pine wilt disease.     

The RNA-binding proteins PUF-5, PUF-6, and PUF-7 reveal multiple systems for maternal mRNA regulation during C. elegans oogenesis

In metazoans, many mRNAs needed for embryogenesis are produced during oogenesis and must be tightly regulated during the complex events of oocyte development. In C. elegans, translation of the Notch receptor GLP-1 is repressed during oogenesis and is then activated specifically in anterior cells of the early embryo. The KH domain protein GLD-1 represses glp-1 translation during early stages of meiosis, but the factors that repress glp-1 during late oogenesis are not known. Here, we provide evidence that the PUF domain protein PUF-5 and two nearly identical PUF proteins PUF-6 and PUF-7 function during a specific period of oocyte differentiation to repress glp-1 and other maternal mRNAs. Depletion of PUF-5 and PUF-6/7 together caused defects in oocyte formation and early embryonic cell divisions. Loss of PUF-5 and PUF-6/7 also caused inappropriate expression of GLP-1 protein in oocytes, but GLP-1 remained repressed in meiotic germ cells. PUF-5 and PUF-6/7 function was required directly or indirectly for translational repression through elements of the glp-1 3' untranslated region. Oogenesis and embryonic defects could not be rescued by loss of GLP-1 activity, suggesting that PUF-5 and PUF-6/7 regulate other mRNAs in addition to glp-1. PUF-5 and PUF-6/7 depletion, however, did not perturb repression of the maternal factors GLD-1 and POS-1, suggesting that subsets of maternal gene products may be regulated by distinct pathways. Interestingly, PUF-5 protein was detected exclusively during mid to late oogenesis but became undetectable prior to completion of oocyte differentiation. These results reveal a previously unknown maternal mRNA control system that is specific to late stages of oogenesis and suggest new functions for PUF family proteins in post-mitotic differentiation. Multiple sets of RNA-binding complexes function in different domains of the C. elegans germ line to maintain silencing of Notch/glp-1 and other mRNAs.     

A conserved RNA-protein complex component involved in physiological germline apoptosis regulation in C. elegans

Two conserved features of oogenesis are the accumulation of translationally quiescent mRNA, and a high rate of stage-specific apoptosis. Little is understood about the function of this cell death. In C. elegans, apoptosis occurring through a specific ;physiological' pathway normally claims about half of all developing oocytes. The frequency of this germ cell death is dramatically increased by a lack of the RNA helicase CGH-1, orthologs of which are involved in translational control in oocytes and decapping-dependent mRNA degradation in yeast processing (P) bodies. Here, we describe a predicted RNA-binding protein, CAR-1, that associates with CGH-1 and Y-box proteins within a conserved germline RNA-protein (RNP) complex, and in cytoplasmic particles in the gonad and early embryo. The CGH-1/CAR-1 interaction is conserved in Drosophila oocytes. When car-1 expression is depleted by RNA interference (RNAi), physiological apoptosis is increased, brood size is modestly reduced, and early embryonic cytokinesis is abnormal. Surprisingly, if apoptosis is prevented car-1(RNAi) animals are characterized by a progressive oogenesis defect that leads rapidly to gonad failure. Elevated germ cell death similarly compensates for lack of the translational regulator CPB-3 (CPEB), orthologs of which function together with CGH-1 in diverse organisms. We conclude that CAR-1 is of critical importance for oogenesis, that the association between CAR-1 and CGH-1 has been conserved, and that the regulation of physiological germ cell apoptosis is specifically influenced by certain functions of the CGH-1/CAR-1 RNP complex. We propose that this cell death pathway facilitates the formation of functional oocytes, possibly by monitoring specific cytoplasmic events during oogenesis.     

A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis

The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis.     

Cholesterol-producing transgenic Caenorhabditis elegans lives longer due to newly acquired enhanced stress resistance

Because Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterol as an essential nutrient. Supplemented cholesterol undergoes extensive enzymatic modification in C. elegans to form other sterols of unknown function. 7-Dehydrocholesterol reductase (DHCR) catalyzes the reduction of the Delta7 double bond of sterols and is suspected to be defective in C. elegans, in which the major endogenous sterol is 7-dehydrocholesterol (7DHC). We microinjected a human DHCR expression vector into C. elegans, which was then incorporated into chromosome by gamma-radiation. This transgenic C. elegans was named cholegans, i.e., cholesterol-producing C. elegans, because it was able to convert 7DHC into cholesterol. We investigated the effects of changes in sterol composition on longevity and stress resistance by examining brood size, mean life span, UV resistance, and thermotolerance. Cholegans contained 80% more cholesterol than the wild-type control. The brood size of cholegans was reduced by 40% compared to the wild-type control, although the growth rate was not significantly changed. The mean life span of cholegans was increased up to 131% in sterol-deficient medium as compared to wild-type. The biochemical basis for life span extension of cholegans appears to partly result from its acquired resistance against both UV irradiation and thermal stress.     

More is not better: brood size and population growth in a self-fertilizing nematode.

The normal form of the nematode Caenorhabditis elegans is a self-fertilizing hermaphrodite, which produces from the same germ-line tissue first a limited number of sperm and then a larger number of oocytes. Self-progeny brood sizes are determined by the number of sperm, and most of the oocytes remain unfertilized. Therefore it might seem selectively advantageous to increase the number of sperm, and hence the size of the brood. A mutation that leads to a 50% increase in sperm production allows a comparison of population growth rates between the wild type (mean brood 327 progeny) and the mutant (mean brood 499 progeny). Wild-type populations grow faster, as measured by food consumption, indicating that increased brood size is not advantageous. The mutant appears to be at a disadvantage because the additional spermatogenesis leads to a delay in the onset of oogenesis, and hence to an increase in the minimum generation time. In support of the notion of an optimal brood size, it was found that different natural isolates of this species have self-fertilities similar to that of the standard laboratory strain, in the range 250-350 progeny per worm.     

Genetic control of sex determination in the germ line of Caenorhabditis elegans

The nematode Caenorhabditis elegans normally exists as one of two sexes: self-fertilizing hermaphrodite or male. Development as hermaphrodite or male requires the differentiation of each tissue in a sex-specific way. In this review, I discuss the genetic control of sex determination in a single tissue of C. elegans: the germ line. Sex determination in the germ line depends on the action of two types of genes:--those that act globally in all tissues to direct male or female development and those that act only in the germ line to specify either spermatogenesis or oogenesis. First, I consider a tissue-specific sex-determining gene, fog-1, which promotes spermatogenesis in the germ line. Second, I consider the regulation of the hermaphrodite pattern of germ-line gametogenesis where first sperm and then oocytes are produced.     

Deficiency of Bloom's syndrome protein causes hypersensitivity of C. elegans to ionizing radiation but not to UV radiation, and induces p53-dependent physiological apoptosis

Caenorhabditis elegans him-6 mutants, which show a high incidence of males and partial embryonic lethality, are defective in the orthologue of human Bloom's syndrome protein (BLM). When strain him-6(e1104) containing a missense him-6 mutation was irradiated with gamma-rays during germ cell development or embryogenesis, embryonic lethality was higher than in the wild type, suggesting a critical function of the wild type gene in mitotic and pachytene stage germ cells as well as in early embryos. Even in the absence of gamma-irradiation, apoptosis was elevated in the germ cells of the him-6 strain and this increase was dependent on a functional p53 homologue (CEP-1), suggesting that spontaneous DNA damage accumulates due to him-6 deficiency. However, induction of germline apoptosis by ionizing radiation was not significantly affected by the deficiency, indicating that HIM-6 has no role in the induction of apoptosis by exogenous DNA damage. We conclude that the C. elegans BLM orthologue is involved in DNA repair in promeiotic cells undergoing homologous recombination, as well as in actively dividing germline and somatic cells.     

Maternal-Effect Lethal Mutations on Linkage Group II of Caenorhabditis Elegans

We have analyzed a set of linkage group (LG) II maternal-effect lethal mutations in Caenorhabditis elegans isolated by a new screening procedure. Screens of 12,455 F1 progeny from mutagenized adults resulted in the recovery of 54 maternal-effect lethal mutations identifying 29 genes. Of the 54 mutations, 39 are strict maternal-effect mutations defining 17 genes. These 17 genes fall into two classes distinguished by frequency of mutation to strict maternal-effect lethality. The smaller class, comprised of four genes, mutated to strict maternal-effect lethality at a frequency close to 5 X 10(-4), a rate typical of essential genes in C. elegans. Two of these genes are expressed during oogenesis and required exclusively for embryogenesis (pure maternal genes), one appears to be required specifically for meiosis, and the fourth has a more complex pattern of expression. The other 13 genes were represented by only one or two strict maternal alleles each. Two of these are identical genes previously identified by nonmaternal embryonic lethal mutations. We interpret our results to mean that although many C. elegans genes can mutate to strict maternal-effect lethality, most genes mutate to that phenotype rarely. Pure maternal genes, however, are among a smaller class of genes that mutate to maternal-effect lethality at typical rates. If our interpretation is correct, we are near saturation for pure maternal genes in the region of LG II balanced by mnC1. We conclude that the number of pure maternal genes in C. elegans is small, being probably not much higher than 12.     

Dietary manipulation implicates lipid signaling in the regulation of germ cell maintenance in C. elegans

Reproduction in C. elegans relies on continuously proliferating germ cells which, during germline development, exit mitosis, undergo meiosis and differentiate into gametes. Supplementing the diet of C. elegans with dihommogamma-linolenic acid (20:3n-6, DGLA), a long chain omega-6 polyunsaturated fatty acid, results in sterile worms that lack germ cells. The effect is remarkably specific for DGLA, as eicosapentaenoic acid (20:5n-3, EPA) and other long-chain polyunsaturated fatty acids with similar physical properties have little or no effect on fertility. Germ cells undergoing mitosis during larval stages are especially sensitive to DGLA, but exposure to DGLA during adulthood also reduces germ cells and brood size, in part by inducing inappropriate apoptosis of meiotic germ cells. Mutant strains with defects in fatty acid desaturation and elongation display altered susceptibility to DGLA, indicating that the sterility effect of the dietary lipid depends on the amount of DGLA present in membranes as well as on the capacity to convert DGLA to other fatty acids. We propose that DGLA produces a signal that interacts with one or more pathways regulating germ cell survival. Our DGLA findings are the first report of a role for a specific fatty acid affecting the development and maintenance of germ cells in C. elegans.     

银鲫种系细胞标记分子Vasa: cDNA克隆及其抗体制备

种系细胞始自胚胎发育早期,是动物生殖及生殖工程的基础。为研究鱼类的种系细胞提供标记分子,我们克隆并鉴定了银鲫的vasacDNA即Cagvasa。CagvasacDNA全长2771碱基(nt),编码的蛋白为银鲫Vasa即CagVasa,全长701个氨基酸(aa)。CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列,在C端Vasa所特有的8个功能域俱全。银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%,89%,61%-66%和50%的同源性。卵巢切片的RNA原位杂交揭示,Cagvasa限于种系细胞,且表达水平呈现出低-高-低的动态变化:即两头低(卵原细胞跟Ⅳ期成熟卵子),中间高(Ⅱ-Ⅲ期卵子)。为分析鱼类种系细胞提供手段,我们用310aa的N端序列产生细菌的重组蛋白来免疫大白兔,获得了抗Vasa的多克隆抗体αVasa。Western免疫印迹表明,αVasa特异性地识别一个鱼类性腺的蛋白,该蛋白的分子量为75kD,仅见于银鲫的性腺和卵子。卵巢切片的组织免疫荧光共聚焦显微分析表明,抗体αVasa只对种系细胞染色:卵原细胞着色最深,卵母细胞和早期的卵子都浓染,成熟卵则浅染。类似情况亦见之于精子发生早期阶段的雄性种系细胞。卵巢和精巢的体细胞则不着色。因此,Cagvasa编码的当是Vasa同源蛋白,为银鲫种系细胞的第一个标记分子。我们的研究表明,抗体αVasa染色灵敏度高,特异性好,当是鉴别银鲫及其它鲤科鱼类的种系细胞的有效手段     

A DNA repair gene of Caenorhabditis elegans: a homolog of human XPF

The xeroderma pigmentosum complementation group F (XPF) protein is a structure-specific endonuclease in a complex with ERCC1 and is essential for nucleotide excision repair (NER). We report a single cDNA of Caenorhabditis elegans (C. elegans) encoding highly similar protein to human XPF and other XPF members. We propose to name the corresponding C. elegans gene xpf. Messenger RNA for C. elegans xpf is 5'-tagged with a SL2 splice leader, suggesting an operon-like expression for xpf. Using RNAi, we showed that loss of C. elegans xpf function caused hypersensitivity to ultra-violet (UV) irradiation, as observed in enhanced germ cell apoptosis and increased embryonic lethality. This study suggests that C. elegans xpf is conserved in evolution and plays a role in the repair of UV-damaged DNA in C. elegans.     

Conditional Tek Promoter-Driven Deletion of Arginyltransferase in the Germ Line Causes Defects in Gametogenesis and Early Embryonic Lethality in Mice

Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis—capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene—a problem that has not previously been reported for this commercial mouse strain—a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.     

RNA synthesised during oogenesis and early embryogenesis in an insect egg (Euscelis plebejus)

Summary RNA labelled during oogenesis or early embryogenesis was isolated from eggs of the leaf hopperEuscelis plebejus. The polyadenylated RNA fraction deposited during early oogenesis accounted for approximately 2.7% of the total RNA content of the newly laid egg. This fraction differed significantly in molecular weight (15–32 S) from poly(A)-containing RNA synthesised between early cleavage and early germ anlage stages (4–20S). Locally injected³H-uridine spread through the egg within approximately 3 h. A considerable fraction (25–35%) of label injected as³H-uridine during early cleavage was recovered in DNA at subsequent stages (10–20 h later); labelled RNA was not found prior to the cellular blastoderm stage. When the yolk-endoplasm was separated from the blastoderm cells, only the latter contained demonstrable amounts of RNA synthesised by the embryo. Of the precursor incorporated into embryonic RNA, approximately 10% was found in the polyadenylated fraction at the early blastoderm stage, but only 3% at the early germ anlage stage. No differences in size distribution of polyadenylated RNA were evident between anterior and posterior halves of the early germ anlage stage.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans

The Caenorhabditis elegans gene mag-1 can substitute functionally for its homolog mago nashi in Drosophila and is predicted to encode a protein that exhibits 80% identity and 88% similarity to Mago nashi (P. A. Newmark et al., 1997, Development 120, 3197-3207). We have used RNA-mediated interference (RNAi) to analyze the phenotypic consequences of impairing mag-1 function in C. elegans. We show here that mag-1(RNAi) causes masculinization of the germ line (Mog phenotype) in RNA-injected hermaphrodites, suggesting that mag-1 is involved in hermaphrodite germ-line sex determination. Epistasis analysis shows that ectopic sperm production caused by mag-1(RNAi) is prevented by loss-of-function (lf) mutations in fog-2, gld-1, fem-1, fem-2, fem-3, and fog-1, all of which cause germ-line feminization in XX hermaphrodites, but not by a her-1(lf) mutation which causes germ-line feminization only in XO males. These results suggest that mag-1 interacts with the fog, fem, and gld genes and acts independently of her-1. We propose that mag-1 normally allows oogenesis by inhibiting function of one or more of these masculinizing genes, which act during the fourth larval stage to promote transient sperm production in the hermaphrodite germ line. When the Mog phenotype is suppressed by a fog-2(lf) mutation, mag-1(RNAi) also causes lethality in the progeny embryos of RNA-injected, mated hermaphrodites, suggesting an essential role for mag-1 during embryogenesis. The defective embryos arrest during morphogenesis with an apparent elongation defect. The distribution pattern of a JAM-1::GFP reporter, which is localized to boundaries of hypodermal cells, shows that hypodermis is disorganized in these embryos. The temporal expression pattern of the mag-1 gene prior to and during morphogenesis appears to be consistent with an essential role of mag-1 in embryonic hypodermal organization and elongation.     

Germ cell sex and cell cycle

Germ cells are the only cells in the body capable of transferring an individual's genetic and epigenetic information to the next generation. However, the developmental processes that provide the foundation for male and female germ line development and later gamete production are complex and poorly understood. In mice the primordial germ cells enter the bipotential gonad at E10.5 and, in response to the testicular or ovarian micro-environment, commit to spermatogenesis or oogenesis. This paper reviews progress in understanding the molecular processes underlying the early stages of male and female germ line development.     

Caenorhabditis elegans dna-2 is involved in DNA repair and is essential for germ-line development

Caenorhabditis elegans germ cell proliferation and development were severely damaged in second generation dna-2 homozygotes. Even in the first generation, a much higher incidence of aberrant chromosomes in oocytes and resultantly higher embryonic lethality were found vs. wild type, when DNA breaks were induced by gamma-rays or camptothecin. The deficiency of dna-2 in combination with RNA interference on mre-11 gene expression synergistically aggravated germ-line development, especially oocyte formation. These results suggest that C. elegans Dna-2 is involved in a DNA repair pathway paralleling homologous recombination or non-homologous end joining with mre-11 participation.