Uterine selection of human embryos at implantation

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca2+ signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca2+ fluxes whereas low-quality embryos caused a heightened and prolonged Ca2+ response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation

Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages. © 2013 Rogers et al

Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.</p

Regulation of endometrial regeneration; mechanisms contributing to repair and restoration of tissue integrity following menses

The human endometrium is a dynamic, multi-cellular tissue that lines the inside of the uterine cavity. During a woman’s reproductive lifespan the endometrium is subjected to cyclical episodes of proliferation, angiogenesis, differentiation/decidualisation, shedding (menstruation), repair and regeneration in response to fluctuating levels of oestrogen and progesterone secreted by the ovaries. The endometrium displays unparalleled, tightly regulated, tissue remodelling resulting in a healed, scar-free tissue following menses or parturition. Mechanisms responsible for initiation of menses have been well documented: following progesterone withdrawal there is an increase in inflammatory mediators, focal hypoxia and induction and activation of matrix-degrading enzymes. In contrast, the molecular and cellular changes responsible for rapid, regulated, tissue repair at a time when oestrogen and progesterone are low are poorly understood. Histological studies using human menstrual phase endometrium have revealed that tissue destruction and shedding occur in close proximity to re-epithelialisation/repair. It has been proposed that re-epithelialisation involves proliferation of glandular epithelial cells in the remaining basal compartment; there is also evidence for a contribution from the underlying stroma. A role for androgens in the regulation of apoptosis of endometrial stromal cells has been proposed but the impact of androgens on tissue repair has not been investigated. Studies using human xenografts and primates have been used to model some aspects of the impact of progesterone withdrawal but simultaneous shedding (menses) and repair have not been modelled in mice; the species of choice for translational biomedical research. In the course of the studies described in this thesis, the following aims have been addressed: 1. To establish a model of menses in the mouse which mimics menses in women, namely; simultaneous breakdown and repair, overt menstruation, immune cell influx, tissue necrosis and re-epithelialisation. 2. To use this model to determine if the stromal cell compartment contributes to endometrial repair. 3. To examine the impact of androgens on the regulation of menses (shedding) and repair. An informative mouse model of endometrial breakdown that was characterised by overt menses, as well as rapid repair, was developed. Immunohistological evidence for extensive tissue remodelling including active angiogenesis, transient hypoxia, epithelial cell-specific proliferation and re-epithelialisation were obtained by examining uterine tissues recovered during an “early window of breakdown and repair” (4 to 24 hours after progesterone withdrawal). Novel data included identification of stromal cells that expressed epithelial cell markers, close to the luminal surface following endometrial shedding, suggesting a role for mesenchymal to epithelial transition (MET) in re-epithelialisation of the endometrium. In support of this idea, array and qRTPCR analyses revealed dynamic changes in expression of mRNAs encoded by genes known to be involved in MET during the window of breakdown and repair. Roles for hypoxia and tissue-resident macrophages in breakdown and tissue remodelling were identified. Treatment of mice with dihydrotestosterone to mimic concentrations of androgens circulated in women at the time of menses had an impact on the timing and duration of endometrial breakdown. Array analysis revealed altered expression of genes implicated in MET and angiogenesis/inflammation highlighting a potential, previously unrecognised role for androgens in regulation of tissue turnover during menstruation. In summary, using a newly refined mouse model new insights were obtained, implicating androgens and stromal MET in restoration of endometrial tissue homeostasis during menstruation. These findings may inform development of new treatments for disorders associated with aberrant repair such as heavy menstrual bleeding and endometriosis

The role of DNA insertions in phenotypic differentiation between humans and other primates

What makes us human is one of the most interesting and enduring questions in evolutionary biology. To assist in answering this question, we have identified insertions in the human genome which cannot be found in five comparison primate species: Chimpanzee, gorilla, orangutan, gibbon, and macaque. A total of 21,269 nonpolymorphic human-specific insertions were identified, of which only 372 were found in exons. Any function conferred by the remaining 20,897 is likely to be regulatory. Many of these insertions are likely to have been fitness neutral; however, a small number has been identified in genes showing signs of positive selection. Insertions found within positively selected genes show associations to neural phenotypes, which were also enriched in the whole data set. Other phenotypes that are found to be enriched in the data set include dental and sensory perception-related phenotypes, features which are known to differ between humans and other apes. The analysis provides several likely candidates, either genes or regulatory regions, which may be involved in the processes that differentiate humans from other apes

A fish-specific transposable element shapes the repertoire of p53 target genes in zebrafish.

Transposable elements, as major components of most eukaryotic organisms' genomes, define their structural organization and plasticity. They supply host genomes with functional elements, for example, binding sites of the pleiotropic master transcription factor p53 were identified in LINE1, Alu and LTR repeats in the human genome. Similarly, in this report we reveal the role of zebrafish (Danio rerio) EnSpmN6_DR non-autonomous DNA transposon in shaping the repertoire of the p53 target genes. The multiple copies of EnSpmN6_DR and their embedded p53 responsive elements drive in several instances p53-dependent transcriptional modulation of the adjacent gene, whose human orthologs were frequently previously annotated as p53 targets. These transposons define predominantly a set of target genes whose human orthologs contribute to neuronal morphogenesis, axonogenesis, synaptic transmission and the regulation of programmed cell death. Consistent with these biological functions the orthologs of the EnSpmN6_DR-colonized loci are enriched for genes expressed in the amygdala, the hippocampus and the brain cortex. Our data pinpoint a remarkable example of convergent evolution: the exaptation of lineage-specific transposons to shape p53-regulated neuronal morphogenesis-related pathways in both a hominid and a teleost fish

Beta-keratins of turtle shell are glycine-proline-tyrosine rich proteins similar to those of crocodilians and birds

This study presents, for the first time, sequences of five beta-keratin cDNAs from turtle epidermis obtained by means of 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses. The deduced amino acid sequences correspond to distinct glycine-proline-serine-tyrosine rich proteins containing 122-174 amino acids. In situ hybridization shows that beta-keratin mRNAs are expressed in cells of the differentiating beta-layers of the shell scutes. Southern blotting analysis reveals that turtle beta-keratins belong to a well-conserved multigene family. This result was confirmed by the amplification and sequencing of 13 genomic fragments corresponding to beta-keratin genes. Like snake, crocodile and avian beta-keratin genes, turtle beta-keratins contain an intron that interrupts the 5'-untranslated region. The length of the intron is variable, ranging from 0.35 to 1.00 kb. One of the sequences obtained from genomic amplifications corresponds to one of the five sequences obtained from cDNA cloning; thus, sequences of a total of 17 turtle beta-keratins were determined in the present study. The predicted molecular weight of the 17 different deduced proteins range from 11.9 to 17.0 kDa with a predicted isoelectric point of 6.8-8.4; therefore, they are neutral to basic proteins. A central region rich in proline and with beta-strand conformation shows high conservation with other reptilian and avian beta-keratins, and it is likely involved in their polymerization. Glycine repeat regions, often containing tyrosine, are localized toward the C-terminus. Phylogenetic analysis shows that turtle beta-keratins are more similar to crocodilian and avian beta-keratins than to those of lizards and snakes