Female Lethality Research Articles

Development and utilization of transgenic New World screwworm, Cochliomyia hominivorax

The New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), was the first insect to be effectively controlled using the sterile insect technique (SIT). Recent efforts to improve SIT control of this species have centred on the development of genetically transformed strains using the piggyBac transposon vector system. Eight transgenic strains were produced incorporating an enhanced green fluorescent protein (EGFP) marker gene under polyubiquitin regulation that has the potential for use as a genetic marking system for released males. The transgenic strains were genetically and phenotypically characterized, including for life fitness parameters and mating competitiveness. These characteristics were unique for each strain and thus some strains were deemed suitable for incorporation into SIT eradication programmes. The strain with the best attributes is designated 'CLAY'. Four of the strains, including CLAY, have been successfully cryopreserved so that their original characteristics should be unchanged when further evaluation is required. With the demonstration of efficient germ-line transformation in NWS, allowing production of fit and competitive transformants, it is now possible to consider further transgenic strain development to improve SIT that are currently being tested in other dipteran species. This includes strains that allow genetic marking with fluorescent proteins, genetic sexing by female lethality, male-specific fluorescent sorting and male sterility by testis-specific lethality. The SIT may also be improved upon by new strategies resulting in lethality of offspring of released insects using conditional lethal systems based upon temperature-dependent or dietary tetracycline regulation of lethal gene expression. Both the creation of new NWS transgenic strains and the ecological safety of their release will be enhanced by new vector systems that allow specific genomic targeting of vector constructs and their subsequent immobilization, ensuring transgene and strain stability.

Targeted disruption of the mouse Lipoma Preferred Partner gene

LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp−/− females. Fertility of Lpp−/− males was proven to be normal, however, females from Lpp−/−×Lpp−/− crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp−/− mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp−/− mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.

Acute oral toxicity of colchicine in rats: effects of gender, vehicle matrix and pre‐exposure to lipopolysaccharide

The oral toxicity of a single administration by gavage (10, 20 or 30 mg kg(-1) body weight) of colchicine (COL) was determined in young, mature male and female Sprague-Dawley rats. The effect of COL was evaluated in the presence or absence of additional treatment variables that included vehicle and lipopolysaccharide (LPS) pre-exposure. The vehicle for COL was either Half and Half cream (H & H) or saline, and each group included pretreatment with either saline or a low, minimally toxic dose (83 microg kg(-1) body weight) of LPS. Colchicine toxicity in both male and female age-matched rats was characterized by progressively more severe dose-related clinical signs of toxicity. These included mortality, decreased body weight and feed intake during the first several days after dosing, with recovery thereafter in surviving animals. There were differences in the severity of the toxic response to COL between male and female rats. The most notable sex-related difference was in COL lethality. Female rats were two times more susceptible to the lethal effects of COL than male rats. Saline or H & H delivery vehicles did not result in any apparent qualitative or quantitative differences in COL toxicity. LPS pretreatment significantly potentiated COL lethality in both males and females, although the potentiation in males was greater than in females. LPS pretreatment modestly increased the COL induced anorexic effect in surviving males, but not in surviving female animals. LPS did not appear to modulate either the body weights or clinical signs of COL induced toxicity in surviving males or females.

Essential role of the V-ATPase in male gametophyte development.

Intracellular pH homeostasis is a prerequisite for biological processes and requires the action of proton pumps. The vacuolar H(+)-ATPase (V-ATPase) is involved in regulating pH in endomembrane compartments of all eukaryotic cells. In plants, there is an additional endomembrane proton pump, H(+)-pyrophosphatase (H(+)-PPase). However, the relative roles of the two types of pumps in endomembrane acidification and energization of secondary active transport are unclear. Here, we show that a strong T-DNA insertion allele of VHA-A, the single copy gene encoding the catalytic subunit of the Arabidopsis V-ATPase, causes complete male and partial female gametophytic lethality. Severe changes in the morphology of Golgi stacks and Golgi-derived vesicles in male gametophytes are the first visible symptoms of cell degeneration leading to a failure to develop mature pollen. Similar effects on Golgi morphology were observed in pollen tubes when growth was blocked by Concanamycin A, a specific V-ATPase inhibitor. Taken together, our results suggests that V-ATPase function is essential for Golgi organization and development of the male gametophyte.

Map positions of third chromosomal female sterile and lethal mutations ofDrosophila melanogaster

Chromosomal mutations induced by ethyl methanesulfonate (EMS) treatment can cause female sterility or maternal-effect lethality in Drosophila. EMS is particularly useful to researchers because it creates mutations independent of position effects. However, because researchers have little control over the chromosomal site of mutation, post-mutagenic genetic mapping is required to determine the cytological location of the mutation. To make a valuable set of mutants more useful to the research community, we have mapped the uncharacterized part of the female-sterile - maternal-effect lethal Tubingen collection. We mapped 49 female-sterile - maternal-effect lethal alleles and 72 lethal alleles to individual deficiency intervals on the third chromosome. In addition, we analyzed the phenotype of ovaries resulting from female sterile mutations. The observed phenotypes range from tumorous ovaries and early blocks in oogenesis, to later blocks, slow growth, blocks in stage 10, to apparently full development of the ovary. The mapping and phenotypic characterization of these 121 mutations provide the necessary information for the researcher to consider a specific mutant as a candidate for their gene of interest.

A New Hybrid Rescue Allele in Drosophila melanogaster

Crosses of Drosophila melanogaster females to males of its sibling species Drosophila simulans, Drosophila mauritiana and Drosophila sechellia produce no sons and daughters that are viable only at low temperatures. We describe here a novel rescue allele Df(1)EP307-1-2 isolated on the basis of its suppression of high temperature hybrid female lethality. Df(1)EP307-1-2 also rescues hybrid males to the pharate adult stage, the same stage at which it is lethal to D. melanogaster pure species males. Molecular analysis indicates that Df(1)EP307-1-2 is associated with a deletion of about 61 kb in the 9D region of the X chromosome. The structure of Df(1)EP307-1-2 suggests that it was formed by a process similar to P-element induced male recombination.

Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway.

Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1(-/-) mice, which lacked the pathway's ubiquitin ligase E3alpha, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2(-/-) mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2(-/-) genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2(-/-) females died as embryos, whereas UBR2(-/-) males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2(-/-) testes was normal and spermatogonia were intact as well, but UBR2(-/-) spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2(-/-) spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.

Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse

The DNA damage-dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, homo- and heterodimerize and are both involved in the base excision repair (BER) pathway. Here, we report that mice carrying a targeted disruption of the PARP-2 gene are sensitive to ionizing radiation. Following alkylating agent treatment, parp-2(-/-)-derived mouse embryonic fibroblasts exhibit increased post-replicative genomic instability, G(2)/M accumulation and chromosome mis-segregation accompanying kinetochore defects. Moreover, parp-1(-/-)parp-2(-/-) double mutant mice are not viable and die at the onset of gastrulation, demonstrating that the expression of both PARP-1 and PARP-2 and/or DNA-dependent poly(ADP-ribosyl) ation is essential during early embryogenesis. Interestingly, specific female embryonic lethality is observed in parp-1(+/-)parp-2(-/-) mutants at E9.5. Meta phase analyses of E8.5 embryonic fibroblasts highlight a specific instability of the X chromosome in those females, but not in males. Together, these results support the notion that PARP-1 and PARP-2 possess both overlapping and non-redundant functions in the maintenance of genomic stability.

GENETIC DISSECTION OF HYBRID INCOMPATIBILITIES BETWEEN DROSOPHILA SIMULANS AND D. MAURITIANA. III. HETEROGENEOUS ACCUMULATION OF HYBRID INCOMPATIBILITIES, DEGREE OF DOMINANCE, AND IMPLICATIONS FOR HALDANE'S RULE

The genetic basis of Haldane's rule was investigated through estimating the accumulation of hybrid incompatibilities between Drosophila simulans and D. mauritiana by means of introgression. The accumulation of hybrid male sterility (HMS) is at least 10 times greater than that of hybrid female sterility (HFS) or hybrid lethality (HL). The degree of dominance for HMS and HL in a pure D. simulans background is estimated as 0.23–0.29 and 0.33–0.39, respectively; that for HL in an F1 background is unlikely to be very small. Evidence obtained here was used to test the Turelli-Orr model of Haldane's rule. Composite causes, especially, faster-male evolution and recessive hybrid incompatibilities, underlie Haldane's rule in heterogametic male taxa such as Drosophila (XY male and XX female). However, if faster-male evolution is driven by sexual selection, it contradicts Haldane's rule for sterility in heterogametic-female taxa such as Lepidoptera (ZW female and ZZ male). The hypothesis of a faster-heterogametic-sex evolution seems to fit the current data best. This hypothesis states that gametogenesis in the heterogametic sex, instead of in males per se, evolves much faster than in the homogametic sex, in part because of sex-ratio selection. This hypothesis not only explains Haldane's rule in a simple way, but also suggests that genomic conflicts play a major role in evolution and speciation.

Association between loci with deleterious alleles and distorted sex ratios in an inbred line of tilapia (Oreochromis aureus).

Three microsatellite markers (UNH159, UNH231, and UNH216) were examined for association with both deleterious genes and sex-ratio distortions in a full-sib family of 222 progeny from the fourth generation of a meiogynogenetic tilapia line (Oreochromis aureus). The three markers were mapped previously to different linkage groups and were shown to be associated with genes with deleterious alleles in this line. A restricted maximum likelihood model was used for analysis of major effects and their interactions on sex ratio and viability. This model was based on selective mortality of genders, ignoring effects of possible sex-determining genes. The results showed that deleterious genes linked to UNH216 and UNH231 exert higher lethality in females than in males (P < .0005 and P < .05, respectively). UNH159 was not associated directly with sex ratio distortion, but acts strongly as a modifier of sex ratio in combination with UNH216 and UNH231. Each of the three loci was found to have a significant effect on viability (P < .05) in the maximum likelihood analysis. The deleterious single-locus effects act strongly against females, while most of the epistatic interactions exert higher lethality in males. This contradiction results in a close to 1:1 sex ratio at maturity. The genetic mechanism and significance of such a balance between genders are still unknown. A detailed analysis of sex-specific lethality may be applied by screening in appropriate series of matings and fine mapping with additional markers. Our data suggest that UNH216 and UNH231 are linked to sex ratio distortion genes and that UNH159 may be linked to a modifier of these genes.

Prospects for using genetic transformation for improved SIT and new biocontrol methods.

The genetic manipulation of non-drosophilid insect species is possible by the creation of recombinant DNA constructs that can be integrated into host genomes by several transposon-based vector systems. This technology will allow the development and testing of a variety of systems that can improve existing biological control methods, and the development of new highly efficient methods. For programs such as sterile insect technique (SIT), transgenic strains may include fluorescent protein marker genes for detection of released insects, and conditional gene expression systems that will result in male sterility and female lethality for genetic sexing. Conditional expression systems include the yeast GAL4 system and the bacterial Tet-off and Tet-on systems that can, respectively, negatively or positively regulate expression of genes for lethality or sterility depending on a dietary source of tetracycline. Importantly, strains for male sterility must also incorporate an effective system for genetic sexing, since typically, surviving females would remain fertile. Models for the use of these expression systems and associated genetic material come from studies in Drosophila and, while many of these systems should be transferable to other insects, continued research will be necessary in insects of interest to clone genes, optimize germ-line transformation, and perform vector stability studies and risk assessment for their release as transgenic strains.

Atypical Forms of Incontinentia Pigmenti in Male Individuals Result from Mutations of a Cytosine Tract in Exon 10 of NEMO ( IKK-γ)

Familial incontinentia pigmenti (IP [MIM 308310]), or Bloch-Sulzberger syndrome, is an X-linked dominant and male-lethal disorder. We recently demonstrated that mutations in NEMO (IKK-gamma), which encodes a critical component of the NF-kappaB signaling pathway, were responsible for IP. Virtually all mutations eliminate the production of NEMO, causing the typical skewing of X inactivation in female individuals and lethality in male individuals, possibly through enhanced sensitivity to apoptosis. Most mutations also give rise to classic signs of IP, but, in this report, we describe two mutations in families with atypical phenotypes. Remarkably, each family included a male individual with unusual signs, including postnatal survival and either immune dysfunction or hematopoietic disturbance. We found two duplication mutations in these families, at a cytosine tract in exon 10 of NEMO, both of which remove the zinc (Zn) finger at the C-terminus of the protein. Two deletion mutations were also identified in the same tract in additional families. However, only the duplication mutations allowed male individuals to survive, and affected female individuals with duplication mutations demonstrated random or slight skewing of X inactivation. Similarly, NF-kappaB activation was diminished in the presence of duplication mutations and was completely absent in cells with deletion mutations. These results strongly indicate that male individuals can also suffer from IP caused by NEMO mutations, and we therefore urge a reevaluation of the diagnostic criteria.

The Drosophila melanogaster hybrid male rescue gene causes inviability in male and female species hybrids.

The Drosophila melanogaster mutation Hmr rescues inviable hybrid sons from the cross of D. melanogaster females to males of its sibling species D. mauritiana, D. simulans, and D. sechellia. We have extended previous observations that hybrid daughters from this cross are poorly viable at high temperatures and have shown that this female lethality is suppressed by Hmr and the rescue mutations In(1)AB and D. simulans Lhr. Deficiencies defined here as Hmr(-) also suppressed lethality, demonstrating that reducing Hmr(+) activity can rescue otherwise inviable hybrids. An Hmr(+) duplication had the opposite effect of reducing the viability of female and sibling X-male hybrid progeny. Similar dose-dependent viability effects of Hmr were observed in the reciprocal cross of D. simulans females to D. melanogaster males. Finally, Lhr and Hmr(+) were shown to have mutually antagonistic effects on hybrid viability. These data suggest a model where the interaction of sibling species Lhr(+) and D. melanogaster Hmr(+) causes lethality in both sexes of species hybrids and in both directions of crossing. Our results further suggest that a twofold difference in Hmr(+) dosage accounts in part for the differential viability of male and female hybrid progeny, but also that additional, unidentified genes must be invoked to account for the invariant lethality of hybrid sons of D. melanogaster mothers. Implications of our findings for understanding Haldane's rule-the observation that hybrid breakdown is often specific to the heterogametic sex-are also discussed.

Flex, an X-linked female-lethal mutation in Drosophila melanogaster controls the expression of Sex-lethal.

The Sex-lethal (Sxl) gene is required in Drosophila females for sexual differentiation of the soma, for gem cell differentiation and dosage compensation. We have isolated three new alleles of female-lethal-on-X (flex), an X-linked female-lethal mutation and have characterized its function in sex determination. SXL protein is missing in flex/flex embryos, however transcription from both Sxl(Pe), the early Sxl promoter and Sxl(Pm), the late maintenance promoter, is normal in flex homozygotes. In flex/flex embryos, Sxl mRNA is spliced in the male mode. Analysis of flex germline clones shows that it also functions in oogenesis, but in contrast to Sxl mutants that show an early arrest tumorous phenotype, flex mutant egg chambers develop to stage 10. In flex ovarian clones, Sxl RNA is also spliced in the male form. Hence, flex is a sex-specific regulator of Sxl functioning in both the soma and the germline. Genetic interaction studies show that flex does not enhance female lethality of Sxl loss-of-function alleles but it rescues the male-specific lethality of both of the gain-of-function Sxl mutations, Sxl(M1 )and Sxl(M4.) In contrast to mutations in splicing regulators of Sxl, the female lethality of flex is not rescued by either Sxl(M1 )or Sxl(M4). Based on these observations, we propose that flex regulates Sxl at a post-splicing stage and regulates either its translation or the stability of the SXL protein.

Female embryonic lethality in Msh2-Trp53 nullizygous mice is strain dependent.

Female embryonic lethality in Msh2-Trp53 nullizygous mice is strain dependent.

Modelling in Escherichia coli of mutations in mitoribosomal protein S12: novel mutant phenotypes of rpsL.

The rpsL gene of Escherichia coli encodes the highly conserved rps12 protein of the ribosomal accuracy centre. We have used the E. coli gene to model the phenotypic effects of specific substitutions found in the mitochondrial gene for rps12. Variants created by in vitro mutagenesis were tested in two different plasmid vector systems, in both streptomycin-sensitive and streptomycin-resistant hosts. A substitution with respect to eubacterial rps12 (K87-->Q), found in all metazoan and fungal mitochondrial orthologues thus far studied, is associated with low-level resistance to streptomycin and a modest (15%) drop in translational elongation rate, but without significant effects on translational accuracy. An amino-acid replacement at a highly conserved leucine residue (L56-->H), associated with the phenotype of sensitivity to mechanical vibration and hemizygous female lethality in Drosophila, creates a functionally inactive but structurally stable protein that is not assembled into ribosomes. The presence in the cell of the mutant, but not wild-type, rpsL greatly downregulates the level of a prominent polypeptide of approximately 50 kDa. These results indicate novel structure-function relationships in rps12 genes affecting translational function, ribosome assembly and drug sensitivity, and indicate a novel regulatory pathway that may influence ribosome biogenesis.

Strain-dependent recovery of open-field behavior and striatal dopamine deficiency in the mouse MPTP model of Parkinson's disease.

The neurotoxin MPTP can damage dopamine systems in the brains of rodents, cats, or monkeys, and is therefore widely used to model degenerative processes that underlie human Parkinson's disease. Here, we investigated the relationships between behavioral and neurochemical effects of systemic MPTP treatment in C57Bl/6 and Balb/c mice. Initially, different doses of MPTP were used to determine which of them might be useful to establish severe striatal dopamine depletions. These data showed that four injections of 20mg/kg at two hour intervals, were more efficient than 10 or 15mg/kg per injection. However, this dose was not usable due to its severe lethality in females. In contrast, 4x 15mg/kg had a low risk of lethality and led to substantial dopamine depletions, which were more severe in the neostriatum than the ventral striatum, and more severe in C57 than in Balb mice. In the first open field test, which was performed two hours after the last injection, this treatment led to severe behavioral inactivation in all parameters taken (distance and speed of locomotion, peripheral activity, frequency and duration of rearing). This effect was seen in both strains and gender. Thereafter, recovery differed between strains, since Balb mice, which had sustained the smaller lesions, had completely recovered on the subsequent day, whereas similar recovery took longer in C57 mice. On the fourth day, all groups appeared largely normal; however, the measure of rearing behavior still showed a deficit in C57 mice. This deficit on day 4 was correlated with neostriatal dopamine depletion; that is, the larger the lesion, the less the number and duration of rearings. Interestingly, these relationships were also observed with respect to ventral striatal dopamine damage, which was correlated with the rearing deficit not only on day 4, but also on day 1. These data will be discussed with respect to mechanisms of toxicity, functional recovery, and the function of striatal dopamine systems.

A search for additional X-linked genes affecting sex determination inDrosophila melanogaster

The geneSex-lethal (Sxl) plays a pivotal role inDrosophila sexual development. Once activated in response to the X: A ratio signal in XX embryos,Sxl participates in appropriate implementation of all known aspects of sexual differentiation. We have attempted to identify new X-linked genes involved in sex determination, especially those involved in the regulation ofSxl. Since misregulation ofSxl, or that of the genes that regulate it, leads to female-specific lethality, or synergistic female-lethal gene interactions, or both, we used these criteria to screen about 10,000 EMS-treated chromosomes for (i) recessive female-specific lethality or (ii) enhanced female lethality intransheterozygous combination withSxl. Four potentially useful mutations—Sxldlf, fl-35, fl-46, 1–43—were recovered and a few of their properties were characterized. Approximate map positions of these mutations were determined by meiotic mapping. To understand their probable position(s) in the hierarchy of genes regulating sex determination, we studied dose-dependent interactions between them and mutations in genes known to affect sex determination by generating double and triple heterozygotes. These studies suggest that (i)Sxldlf is not defective in the ‘early’ regulation or functions ofSxl, and (ii)fl-35, fl-46 and1–43 are unlikely to be a part of the X: A ratio signal, i.e. they are not needed for the transcriptional activation ofSxl. On the other hand, they could be affecting post-transcriptional processing ofSxl transcripts.

Female embryonic lethality in mice nullizygous for both Msh2 and p53.

The mutator hypothesis of tumorigenesis suggests that loss of chromosomal stability or maintenance functions results in elevated mutation rates, leading to the accumulation of the numerous mutations required for multistep carcinogenesis. The human DNA mismatch repair (MMR) genes are highly conserved homologues of the Escherichia coli MutHLS system, which contribute to genomic stability by surveillance and repair of replication misincorporation errors and exogenous DNA damage. Mutations in one of these MMR genes, hMSH2, account for about half of all cases of genetically linked hereditary non-polyposis colorectal cancer. Loss of function of p53 has also been proposed to increase cellular hypermutability, thereby accelerating carcinogenesis, although a clear role for p53 in genomic instability remains controversial. p53 is mutated frequently in a wide range of human cancers, including colonic tumours. Both Msh2- and p53-targeted knockout mice are viable and susceptible to cancer. Here we demonstrate that combined Msh2 and p53 ablation (Msh2-/-p53-/-) results in developmental arrest of all female embryos at 9.5 days. In contrast, male Msh2-/-p53-/- mice are viable, but succumb to tumours significantly earlier (t1-2 is 73 days) than either Msh2-/- or p53-/- littermates. Furthermore, the frequency of microsatellite instability (MSI) in tumours from Msh2-/-p53-/- mice is not significantly different than in Msh2-/- mice. Synergism in tumorigenesis and independent segregation of the MSI phenotype suggest that Msh2 and p53 are not genetically epistatic.

A murine model of Menkes disease reveals a physiological function of metallothionein

Human Menkes disease and the murine Mottled phenotype are X-linked diseases that result from copper deficiency due to mutations in a copper-effluxing ATPase, designated ATP7A. Male mice with the Mottled-Brindled allele (Mo-brJ) accumulate copper in the intestine, fail to export copper to peripheral organs and die a few weeks after birth. Much of the intestinal copper is bound by metallothionein (MT). To determine the function of MT in the presence of Atp7a deficiency, we crossed Mo-brJ females with males that bear a targeted disruption of the Mt1 and Mt2 genes (Mt-/-). On an Mt -/- background, most Mo-brJ males as well as heterozygous Mo-brJ females die before embryonic day 11. The lethality in Mo-brJ females can be explained by preferential inactivation of the paternal X chromosome in extraembryonic tissues and resultant copper toxicity in the absence of MT. In support of this hypothesis, cell lines derived from Mt -/-, Mo-brJ embryos are very sensitive to copper toxicity.