Inviable Embryos Research Articles

Early-acting inbreeding depression can evolve as an inbreeding avoidance mechanism.

Despite the potential for mechanical, developmental and/or chemical mechanisms to prevent self-fertilization, incidental self-fertilization is inevitable in many predominantly outcrossing species. In such cases, inbreeding can compromise individual fitness. Unquestionably, much of this inbreeding depression is maladaptive. However, we show that when reproductive compensation allows for the replacement of inviable embryos lost early in development, selection can favour deleterious recessive variants that induce 'self-sacrificial' death of inbred embryos. Our theoretical results provide numerous testable predictions which could challenge the assumption that inbreeding depression is always maladaptive. Our work is applicable any species that cannot fully avoid inbreeding, exhibits substantial inbreeding depression, and has the potential to compensate embryos lost early in development. In addition to its general applicability, our theory suggests that self-sacrificial variants might be responsible for the remarkably low realized selfing rates of gymnosperms with high primary selfing rates, as gymnosperms exhibit strong inbreeding depression, have effective reproductive compensation mechanisms, and cannot evolve chemical self-incompatibility.

Conservation of shibire and RpII215 temperature-sensitive lethal mutations between Drosophila and Bactrocera tryoni.

The sterile insect technique can suppress and eliminate population outbreaks of the Australian horticultural pest, Bactrocera tryoni, the Queensland fruit fly. Sterile males mate with wild females that produce inviable embryos, causing population suppression or elimination. Current sterile insect releases are mixed sex, as the efficient removal of unrequired factory-reared females is not yet possible. In this paper, we assessed the known Drosophila melanogaster temperature-sensitive embryonic lethal alleles shibire (G268D, shits1) and RNA polymerase II 215 (R977C, RpII215ts) for potential use in developing B. tryoni genetic sexing strains (GSS) for the conditional removal of females. Complementation tests in D. melanogaster wild-type or temperature-sensitive genetic backgrounds were performed using the GAL4-UAS transgene expression system. A B. tryoni wild-type shibire isoform partially rescued Drosophila temperature lethality at 29°C by improving survivorship to pupation, while expressing B. tryoni shits1 failed to rescue the lethality, supporting a temperature-sensitive phenotype. Expression of the B. tryoni RpII215 wild-type protein rescued the lethality of D. melanogaster RpII215ts flies at 29°C. Overexpressing the B. tryoni RpII215ts allele in the D. melanogaster wild-type background unexpectedly produced a dominant lethal phenotype at 29°C. The B. tryoni shibire and RpII215 wild-type alleles were able to compensate, to varying degrees, for the function of the D. melanogaster temperature-sensitive proteins, supporting functional conservation across species. Shibire and RpII215 hold potential for developing insect strains that can selectively kill using elevated temperatures; however, alleles with milder effects than shits1 will need to be considered.

SKN-1 isoform-c is essential for C. elegans development.

The transcription factor SKN-1 in Caenorhabditis elegans is a critical regulator of various biological processes, impacting development, diet and immune responses, cellular detoxification, and lipid metabolism; thereby playing a pivotal role in regulating the health and lifespan of the organism. The primary isoforms of SKN-1 ( SKN-1 a, SKN-1 b, and SKN-1 c) exhibit distinct functions resembling mammalian Nrf transcription factors. This study investigates the specific role of the SKN-1 c isoform in development by generating mutants with targeted missense mutations in the skn-1 c and skn-1 a isoforms. The skn-1 c Met1Ala mutants, which replaces a start methionine with alanine, renders SKN-1 c non-functional while preserving other isoforms, produced inviable embryos, requiring a balancer chromosome for proper embryonic development. In contrast, skn-1 a Met1Ala mutants, which replaces the start methionine with alanine for this isoform, displayed normal embryonic development and hatching. Moreover, the data suggest that SKN-1 c plays a crucial role in embryonic development, as strains without maternally deposited SKN-1 c lead to embryos that are developmentally arrested. Together, these findings contribute to our understanding of SKN-1 c's specific role in influencing embryogenesis and development in C. elegans.

Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Proteomic Analysis.

The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner.

Reproductive Compensation and Selection among Viable Embryos Drive the Evolution of Polyembryony.

AbstractSimple polyembryony, where one gametophyte produces multiple embryos with different sires but the same maternal haplotype, is common among vascular plants. We develop an infinite-sites, forward population genetics model showing that together polyembryony's two benefits-"reproductive compensation" achieved by providing a backup for inviable embryos and the opportunity to favor the fitter of surviving embryos-can favor its evolution. Our model tests how these factors can favor the evolution of polyembryony and how these underlying benefits of polyembryony shape the genetic load under a range of biological parameters. While these two benefits are difficult to disentangle in nature, we construct variant models of polyembryony that either only include or only exclude the opportunity for reproductive compensation. We find that reproductive compensation strongly favors the evolution of polyembryony and that polyembryony is favored much more weekly in its absence, suggesting that the benefit of a backup embryo is the major force favoring polyembryony. Remarkably, we find nearly identical results in cases in which mutations impact either embryonic or postembryonic fitness (no pleiotropy) and in cases in which mutations have identical effects on embryonic and postembryonic fitness (extreme pleiotropy). Finally, we find that the consequences of polyembryony depend on its function-polyembryony results in a decrease in mean embryonic fitness when acting as a mechanism of embryo compensation and ultimately increases mean embryonic fitness when we exclude this potential benefit.

Cell biology: Centromere instability and replication stress underlie Xenopus hybrid incompatibility

Crosses between closely related species of frogs result in inviable embryos that die following catastrophic cell divisions. A new study identifies defects in centromere maintenance and DNA replication stress as key forces driving this incompatibility.

Transcriptome analysis of egg viability in rainbow trout, Oncorhynchus mykiss

BackgroundMaternal transcripts are accumulated in the oocyte during oogenesis to provide for protein synthesis from oocyte maturation through early embryonic development, when nuclear transcription is silenced. The maternal mRNAs have short poly(A) tails after undergoing post-transcriptional processing necessary for stabilizing them for storage. The transcripts undergo cytoplasmic polyadenylation when they are to be translated. Transcriptome analyses comparing total mRNA and elongated poly(A) mRNA content among eggs of different quality can provide insight into molecular mechanisms affecting egg developmental competence in rainbow trout. The present study used RNA-seq to compare transcriptomes of unfertilized eggs of rainbow trout females yielding different eyeing rates, following rRNA removal and poly(A) retention for construction of the libraries.ResultsThe percentage of embryos to reach the 32-cell stage at 24 h post fertilization was significantly correlated to family eyeing rate, indicating that inviable embryos were developmentally compromised before zygotic genome activation. RNA sequencing identified 2 differentially expressed transcripts (DETs) from total mRNA sequencing comparing females with low-quality (< 5% eyeing), medium-quality (30–50% eyeing), and high-quality (> 80% eyeing) eggs. In contrast, RNA sequencing from poly(A) captured transcripts identified 945 DETs between low- and high-quality eggs, 1012 between low- and medium-quality eggs, and only 2 between medium- and high-quality eggs. The transcripts of mitochondrial genes were enriched with polyadenylated transcript sequencing and they were significantly reduced in low-quality eggs. Similarly, mitochondrial DNA was reduced in low-quality eggs compared with medium- and high-quality eggs. The functional gene analysis classified the 945 DETs between low- and high-quality eggs into 31 functional modules, many of which were related to ribosomal and mitochondrial functions. Other modules involved transcription, translation, cell division, apoptosis, and immune responses.ConclusionsOur results indicate that differences in egg quality may be derived from differences in maternal nuclear transcript activation and cytoplasmic polyadenylation before ovulation, as opposed to accumulation and storage of maternal nuclear transcripts during oogenesis. Transcriptome comparisons suggest low-quality eggs suffered from impaired oxidative phosphorylation and translation. The DETs identified in this study provide insight into developmental competence in rainbow trout eggs.

Analysis of Meiotic Sister Chromatid Cohesion in Caenorhabditis elegans.

In sexually reproducing organisms, the formation of healthy gametes (sperm and eggs) requires the proper establishment and release of meiotic sister chromatid cohesion (SCC). SCC tethers replicated sisters from their formation in premeiotic S phase until the stepwise removal of cohesion in anaphase of meiosis I and II allows the separation of homologs and then sisters. Defects in the establishment or release of meiotic cohesion cause chromosome segregation errors that lead to the formation of aneuploid gametes and inviable embryos. The nematode Caenorhabditis elegans is an attractive model for studies of meiotic sister chromatid cohesion due to its genetic tractability and the excellent cytological properties of the hermaphrodite gonad. Moreover, mutants defective in the establishment or maintenance of meiotic SCC nevertheless produce abundant gametes, allowing analysis of the pattern of chromosome segregation. Here I describe two approaches for analysis of meiotic cohesion in C. elegans. The first approach relies on cytology to detect and quantify defects in SCC. The second approach relies on PCR and restriction digests to identify embryos that inherited an incorrect complement of chromosomes due to aberrant meiotic chromosome segregation. Both approaches are sensitive enough to identify rare errors and precise enough to reveal distinctive phenotypes resulting from mutations that perturb meiotic SCC in different ways. The robust, quantitative nature of these assays should strengthen phenotypic comparisons of different meiotic mutants and enhance the reproducibility of data generated by different investigators.

A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans.

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging.

Mitotic fidelity requires transgenerational action of a testis-restricted HP1.

Sperm-packaged DNA must undergo extensive reorganization to ensure its timely participation in embryonic mitosis. Whereas maternal control over this remodeling is well described, paternal contributions are virtually unknown. In this study, we show that Drosophila melanogaster males lacking Heterochromatin Protein 1E (HP1E) sire inviable embryos that undergo catastrophic mitosis. In these embryos, the paternal genome fails to condense and resolve into sister chromatids in synchrony with the maternal genome. This delay leads to a failure of paternal chromosomes, particularly the heterochromatin-rich sex chromosomes, to separate on the first mitotic spindle. Remarkably, HP1E is not inherited on mature sperm chromatin. Instead, HP1E primes paternal chromosomes during spermatogenesis to ensure faithful segregation post-fertilization. This transgenerational effect suggests that maternal control is necessary but not sufficient for transforming sperm DNA into a mitotically competent pronucleus. Instead, paternal action during spermiogenesis exerts post-fertilization control to ensure faithful chromosome segregation in the embryo.

Joint Molecule Resolution Requires the Redundant Activities of MUS-81 and XPF-1 during Caenorhabditis elegans Meiosis

The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in Caenorhabditis elegans, SPO-11-generated double stranded breaks are resolved to generate a single crossover per bivalent and the remaining recombination intermediates are resolved as noncrossovers. We discovered that early recombination intermediates are limited by the C. elegans BLM ortholog, HIM-6, and in the absence of HIM-6 by the structure specific endonuclease MUS-81. In the absence of both MUS-81 and HIM-6, recombination intermediates persist, leading to chromosome breakage at diakinesis and inviable embryos. MUS-81 has an additional role in resolving late recombination intermediates in C. elegans. mus-81 mutants exhibited reduced crossover recombination frequencies suggesting that MUS-81 is required to generate a subset of meiotic crossovers. Similarly, the Mus81-related endonuclease XPF-1 is also required for a subset of meiotic crossovers. Although C. elegans gen-1 mutants have no detectable meiotic defect either alone or in combination with him-6, mus-81 or xpf-1 mutations, mus-81;xpf-1 double mutants are synthetic lethal. While mus-81;xpf-1 double mutants are proficient for the processing of early recombination intermediates, they exhibit defects in the post-pachytene chromosome reorganization and the asymmetric disassembly of the synaptonemal complex, presumably triggered by crossovers or crossover precursors. Consistent with a defect in resolving late recombination intermediates, mus-81; xpf-1 diakinetic bivalents are aberrant with fine DNA bridges visible between two distinct DAPI staining bodies. We were able to suppress the aberrant bivalent phenotype by microinjection of activated human GEN1 protein, which can cleave Holliday junctions, suggesting that the DNA bridges in mus-81; xpf-1 diakinetic oocytes are unresolved Holliday junctions. We propose that the MUS-81 and XPF-1 endonucleases act redundantly to process late recombination intermediates to form crossovers during C. elegans meiosis.

A Hypomorphic Mutation Reveals a Stringent Requirement for the ATM Checkpoint Protein in Telomere Protection During Early Cell Division in Drosophila

Using Drosophila as a model system, we identified a stringent requirement for the conserved function of Ataxia Telangiectasia Mutated (ATM) in telomere protection during early embryonic development. Animals homozygous for a hypomorphic mutation in atm develop normally with minimal telomere dysfunction. However, mutant females produce inviable embryos that succumb to mitotic failure caused by covalent fusions of telomeric DNA. Interestingly, although the atm mutation encodes a premature stop codon, it must not have eliminated the production of the mutant protein, and the mutant protein retains kinase activity upon DNA damage. Moreover, although the embryonic phenotype of this mutation resembles that of hypomorphic mutations in the MRN complex, the function of MRN appears normal in the atm embryos. In contrast, there is a prominent reduction of the level of HipHop, an essential member of the Drosophila capping complex. How ATM functions in telomere protection remains poorly understood. The amenability of Drosophila embryos to molecular and biochemical investigations ensures that this newly identified mutation will facilitate future studies of ATM in telomere maintenance.

Mre11-Rad50-Nbs complex is required to cap telomeres during Drosophila embryogenesis

Using Drosophila as a model system, we identified here a stringent requirement for Mre11-Rad50-Nbs (MRN) function in telomere protection during early embryonic development. Animals homozygous for hypomorphic mutations in either mre11 or nbs develop normally with minimal telomere dysfunction. However, they produce inviable embryos that succumb to failure of mitosis caused by covalent fusion of telomeric DNA. Interestingly, the molecular defect is not the absence of MRN interaction or of Mre11 nuclease activities, but the depletion of the maternal pool of Nbs protein in these embryos. Because of Nbs depletion, Mre11 and Rad50 (MR) are excluded from chromatin. This maternal effect lethality in Drosophila is similar to that seen in mice carrying hypomorphic mrn mutations found in human patients, suggesting a common defect in telomere maintenance because of the loss of MRN integrity.

Deleted in cancer 1 (DICE1) is an essential protein controlling the topology of the inner mitochondrial membrane in C. elegans

DICE1 (deleted in cancer 1), first identified in human lung carcinoma cell lines, is a candidate tumor suppressor, but the details of its activity remain largely unknown. We have found that RNA interference of its C. elegans homolog (DIC-1) produced inviable embryos with increased apoptosis, cavities in cells and abnormal morphogenesis. In the dic-1(RNAi) germ line, ced-3-dependent apoptosis increased, and cell cavities appeared at the late-pachytene/oogenic stage, leading to defective oogenesis. Immunofluorescence microscopy of DIC-1 revealed its ubiquitous expression in the form of cytoplasmic foci, and cryoelectron microscopy narrowed down the location of the foci to the inner membrane of mitochondria. After dic-1 RNAi, mitochondria had an irregular morphology and contained numerous internal vesicles. Homozygous embryos from a heterozygous dic-1 mother arrested at the L3 larval stage, in agreement with the essential role of DIC-1 in mitochondria. In summary, C. elegans DIC-1 plays a crucial role in the formation of normal morphology of the mitochondrial cristae/inner membrane. Our results suggest that human DICE1 may have several functions in multiple intracellular locations.

Identification of genes controlling malpighian tubule and other epithelial morphogenesis in Drosophila melanogaster.

The Drosophila Malpighian tubule is a model system for studying genetic mechanisms that control epithelial morphogenesis. From a screen of 1800 second chromosome lethal lines, by observing uric acid deposits in unfixed inviable embryos, we identified five previously described genes (barr, fas, flb, raw, and thr) and one novel gene, walrus (wal), that affect Malpighian tubule morphogenesis. Phenotypic analysis of these mutant embryos allows us to place these genes, along with other previously described genes, into a genetic pathway that controls Malpighian tubule development. Specifically, wal affects evagination of the Malpighian tubule buds, fas and thr affect bud extension, and barr, flb, raw, and thr affect tubule elongation. In addition, these genes were found to have different effects on development of other epithelial structures, such as foregut and hindgut morphogenesis. Finally, from the same screen, we identified a second novel gene, drumstick, that affects only foregut and hindgut morphogenesis.

FEMALE FECUNDITY IN A HYBRID ZONE BETWEEN TWO CHROMOSOME RACES OF THE SCELOPORUS GRAMMICUS COMPLEX (SAURIA, PHRYNOSOMATIDAE).

Individuals of the F5 and FM2 cytotypes of the Sceloporus grammicus complex form a narrow zone of parapatric hybridization near Tulancingo, Hidalgo, Mexico. Reproductive parameters were examined among chromosomally parental and hybrid females to assess the degree to which reduced clutch size is correlated with the level of chromosomal heterozygosity. Although clutch size in the two parental groups was highly correlated with female body size, this was not the case for females with intermediate karyotypes. These females displayed increased levels of infertility manifested as smaller clutches and as inviable embryos. F1 females produced the smallest average clutches and suffered the most precipitous fecundity loss (up to 75%). The number of heterozygous marker chromosomes and heterozygosity at chromosome 2 had significant effects on the number of viable embryos. Analysis of embryo karyotypes revealed the production of triploid offspring and an excess number of embryos heterozygous at chromosome 1. Differences in viability, among females heterozygous for the same number of chromosomes, suggest that genetic background of the female and/or sire may be an important factor in determining reproductive success.

THE GENETIC STRUCTURE OF A HYBRID ZONE BETWEEN TWO CHROMOSOME RACES OF THE SCELOPORUS GRAMMICUS COMPLEX (SAURIA, PHRYNOSOMATIDAE) IN CENTRAL MEXICO.

The F5 (2n = 34) and FM2 (2n = 44-46) chromosome races of the Sceloporus grammicus complex form a parapatric hybrid zone in the Mexican state of Hidalgo, characterized by steep concordant clines among three diagnostic chromosome markers across a straight-line distance of about 2 km. Here, we show that this zone is actually structured into local patches in which hybridization extends over an extremely irregular front. The distribution of hybrid-index (HI) scores across the transect reveals some hybridization at almost all localities mapped in a central 7 km × 3 km area. Pooling the central samples produces both a strong heterozygote deficit for all diagnostic markers and strong linkage disequilibria between all pairwise combinations of these (unlinked) markers. Moreover, a highly significant association exists between the habitat on which each individual was caught and its karyotype (F5 chromosomes are more likely to be found on oak). Analysis of genotype frequencies over a range of spatial scales shows that there is no significant heterozygote deficit or habitat association within local areas of less than about 200 m; however, there is significant linkage disequilibrium over the smallest scales (R = D (pquv)1/2 = 0.29, support limits, 0.18-0.36) over 100 m. These patterns suggest that lizards mate and choose habitats randomly within local patches. This conclusion is supported by mark-recapture estimates of dispersal (≈ 80 m in a generation) and by inference of matings from embryo and maternal karyotypes. Closer examination of the two-dimensional pattern reveals a convoluted cline for all three markers, with a width of 830 m (support limits 770 m-930 m). This cline width, combined with the strength of local linkage disequilibrium, implies a dispersal rate of σ = 160 m in a generation and an effective selection pressure of 30% on each chromosome marker. The proportion of inviable embryos is greater in females from the center of the hybrid zone; this is caused by effects associated with both karyotype and location. The hybrid zone is likely to be maintained by selection against chromosomal heterozygotes, by other kinds of selection against hybrids, and by selection adapting the chromosome races to different habitats. The structure of the contact may be caused by both random drift and by selection in relation to habitat.

Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos

The mago nashi (mago) locus is a newly identified strict maternal effect, grandchildless-like, gene in Drosophila melanogaster. In homozygous mutant mago females reared at 17 degrees C, mago+ function is reduced, the inviable embryos lack abdominal segments and 84-98% of the embryos die. In contrast, at 25 degrees C, some mago alleles produce a novel gene product capable of inducing the formation of symmetrical double abdomen embryos. Reciprocal temperature-shift experiments indicate that the temperature-sensitive period is during oogenetic stages 7-14. Furthermore, embryos collected from mago1 homozygous females contain no apparent functional posterior determinants in the posterior pole. In viable F1 progeny from mago mutant females, regardless of genotype and temperature, polar granules are reduced or absent and germ cells fail to form (the grandchildless-like phenotype). Thus, we propose that the mago+ product is a component of the posterior determinative system, required during oogenesis, both for germ cell determination and delineation of the longitudinal axis of the embryo.

Tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster.

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.

Filicidal ring-Y chromosomes in D. melanogaster.

Ring-Y chromosomes are recovered infrequently from crosses of ring-Y-bearing males to females of certain strains (OSTER 1964). Experiments described here have unveiled a diverse class of genes that exert a maternal effect on the behavior during cleavage of these "filicidal" ring chromosomes. Cytological observations of inviable embryos have revealed that the ring-Y chromosome causes gross disorganization of the cleavage nuclei. This inviability may be equivalent to the "dominant lethality" attributed to unstable ring-X chromosomes (HINTON 1955; PASZTOR 1971). Mapping studies indicate that no single region of the normal Y is solely responsible for the unusual behavior of ring-Y chromosomes.