Jacqui Detmar

Maternal exposure to polycyclic aromatic hydrocarbons diminishes murine ovarian reserve via induction of Harakiri

Maternal smoking during pregnancy is associated with a variety of adverse neonatal outcomes including altered reproductive performance. Herein we provide molecular evidence for a pathway involved in the elimination of the female germline due to prepregnancy and/or lactational exposure to polycyclic aromatic hydrocarbons (PAHs), environmental toxicants found in cigarette smoke. We show that ovaries of offspring born to mice exposed to PAHs contained only a third of the ovarian follicle pool compared with offspring of unexposed female mice. Activation of the cell death pathway in immature follicles of exposed females was mediated by the aryl hydrocarbon receptor (Ahr), as ovarian reserve was fully rescued by maternal cotreatment with the Ahr antagonist, resveratrol, or by inactivation of the Ahr gene. Furthermore, in response to PAHs, Ahr-mediated activation of the harakiri, BCL2 interacting protein (contains only BH3 domain), was necessary for execution of cell death. This pathway appeared to be conserved between mouse and human, as xenotransplanted human ovarian cortex exposed to PAHs responded by activation of the identical cell death cascade. Our data indicate that maternal exposure to PAHs prior to pregnancy and/or during lactation compromises ovarian reserve of female offspring, raising the concern about the transgenerational impact of maternal smoking on ovarian function in the human.

Fetal growth restriction triggered by polycyclic aromatic hydrocarbons is associated with altered placental vasculature and AhR-dependent changes in cell death

Maternal cigarette smoking is considered an important risk factor associated with fetal intrauterine growth restriction (IUGR). Polycyclic aromatic hydrocarbons (PAHs) are well-known constituents of cigarette smoke, and the effects of acute exposure to these chemicals at different gestational stages have been well established in a variety of laboratory animals. In addition, many PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a cellular xenobiotic sensor responsible for activating the metabolic machinery. In this study, we have applied a chronic, low-dose regimen of PAH exposure to C57Bl/6 female mice before conception. This treatment caused IUGR in day 15.5 post coitum (d15.5) fetuses and yielded abnormalities in the placental vasculature, resulting in significantly reduced arterial surface area and volume of the fetal arterial vasculature of the placenta. However, examination of the small vasculature within the placental labyrinth of PAH-exposed dams revealed extensive branching and enlargement of these vessels, indicating a possible compensatory mechanism. These alterations in vascularization were accompanied by reduced placental cell death rates, increased expression levels of antiapoptotic Xiap, and decreased expression of proapoptotic Bax, cleaved poly(ADP-ribose) polymerase-1, and active caspase-3. AhR-deficient fetuses were rescued from PAH-induced growth restriction and exhibited no changes in the labyrinthine cell death rate. The results of this investigation suggest that chronic exposure to PAHs is a contributing factor to the development of IUGR in human smokers and that the AhR pathway is involved.

Embryonic loss due to exposure to polycyclic aromatic hydrocarbons is mediated by Bax

The high miscarriage rates observed in women smokers raises the possibility that chemicals in cigarette smoke could be detrimental to embryo development. Previous studies have established that polycyclic aromatic hydrocarbons (PAHs), transactivate the arylhydrocarbon receptor (AhR), leading to cell death. Herein we show that PAH exposure results in murine embryo cell death, acting as a potential mechanism underlying cigarette-smoking-induced pregnancy loss. Cell death was preceded by increases in Bax levels, activation of caspase-3 and decreased litter size. Chronic exposure of females to PAHs prior to conception impaired development, resulting in a higher number of resorptions. This embryonic loss could not be prevented by the disruption of Hrk, but was diminished in embryos lacking Bax. We conclude that exposure of early embryos to PAHs reduces the allocation of cells to the embryonic and placental lineages by inducing apoptosis in a Bax-dependent manner, thus compromising the developmental potential of exposed embryos.

Vessel tortuousity and reduced vascularization in the fetoplacental arterial tree after maternal exposure to polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and the main toxicants found in cigarettes. Women are often exposed to PAHs before pregnancy, typically via prepregnancy smoking. To determine how prepregnancy exposure affects the fetoplacental vasculature of the placenta, we exposed female mice to PAHs before conception, perfused the fetoplacental arterial trees with X-ray contrast agent, and imaged the vasculature ex vivo by microcomputed tomography (micro-CT) at embryonic day 15.5. Automated vascular segmentation and flow calculations revealed that in control trees, <40 chorionic plate vessels (diameter>180 μm) gave rise to ∼1,300 intraplacental arteries (50-180 μm), predicting an arterial vascular resistance of 0.37±0.04 mmHg·s·μl(-1). PAH exposure increased vessel curvature of chorionic plate vessels and significantly increased the tortuousity ratio of the tree. Intraplacental arteries were reduced by 17%, primarily due to a 27% decrease in the number of arteriole-sized (50-100 μm) vessels. There were no changes in the number of chorionic vessels, the depth or span of the tree, the diameter scaling coefficient, or the segment length-to-diameter ratio. PAH exposure resulted in a tree with a similar size and dichotomous branching structure, but one that was comparatively sparse so that arterial vascular resistance was increased by 30%. Assuming the same pressure gradient, blood flow would be 19% lower. Low flow may contribute to the 23% reduction observed in fetal weight. New insights into the specific effects of PAH exposure on a developing arterial tree were achieved using micro-CT imaging and automated vascular segmentation analysis.

Expansion of the fetoplacental vasculature in late gestation is strain dependent in mice

How the fetoplacental arterial tree grows and expands during late gestational development is largely unknown. In this study, we quantified changes in arterial branching in the fetal exchange region of the mouse placenta during late gestation, when capillarization increases rapidly. We studied two commonly used mouse strains, CD1 and C57Bl/6 (B6), at embryonic days (E)13.5, 15.5, and 17.5. B6 mice differ from CD1 mice by exhibiting a blunted fetal weight gain in late gestation. We found that B6 capillarization and interhemal membrane thinning were reduced and placental hypoxia-inducible factor-1α and VEGF-A expression were higher than CD1 near term. Automated vascular segmentation of microcomputed tomography data sets revealed that the number of arterial vessels ≥50 μm remained constant during late gestation in both strains, despite large increases in downstream capillary volume quantified by stereology (+65% in B6 mice and +200% in CD1 mice). Arterial diameters expanded in both strains from E13.5 to E15.5; however, diameters continued to expand to E17.5 in B6 mice only. The diameter scaling coefficient at branch sites was near optimal (−3.0) and remained constant in CD1 mice, whereas it decreased, becoming abnormal, in B6 mice at term (−3.5 ± 0.2). Based on arterial tree geometry, resistance remained constant throughout late gestation (∼0.45 mmHg·s·μl−1) in CD1 mice, whereas it decreased by 50% in late gestation in B6 mice. Quantification of the fetoplacental vasculature revealed significant strain-dependent differences in arterial and capillary expansion in late gestation. In both strains, enlargement of the fetoplacental arterial tree occurred primarily by increased arterial diameters with no change in segment numbers in late gestation.

The E3 ubiquitin ligase Mule acts through the ATM–p53 axis to maintain B lymphocyte homeostasis

Genetic manipulation reveals that Mule is vital for B cell development, proliferation, and homeostasis as a result of its ability to regulate p53 and ATM.

Developmental consequences of alternative Bcl-x splicing during preimplantation embryo development

Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments. We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro. Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts. Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl‐x transcript. Cell death activation in human blastocysts was accompanied by changes in Bcl‐x splicing, favoring production of Bcl‐xS, an activator of cell death. Expression of Bcl‐xS was detected in a subset of human blastocysts that show particular clustering in dying and/or dead cells. Altering the Bcl‐xL/Bcl‐xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl‐xS, with concomitant downregulation of Bcl‐xL, compromised developmental potential and committed a subset of cells to undergoing cell death. This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content. In addition, altered Bcl‐x splicing in favor of Bcl‐xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development. Thus, we conclude that inappropriate culture conditions affect Bcl‐x isoform expression, contributing to compromised preimplantation embryo development.

Enhanced endocytotic and transcytotic activity in the rat endometrium prior to embryo implantation

Background Understanding the mechanisms by which fluid absorption and secretion occur in the endometrium is clinically important since conditions that deregulate this process reduce fertility. It has been suggested that luminal epithelial cells induce a crucial step in the process of embryo implantation called uterine closure via endocytotic fluid uptake. Uterine lumen closure is a key step in the process of embryo implantation and is absent in some infertile strains of mice. Methods To investigate the process of uterine closure a ferritin-based tracer, used as a marker of endocytosis, was injected into the uterine lumen on day 5 of pregnancy when closure occurs. Results Unexpectedly, luminal epithelial uptake of tracer was minimal on day 5 of pregnancy discrediting endocytosis as the induction method of uterine closure. In contrast, ferritin was found deep in the stromal portion of the endometrium in pre-pregnant animals. Conclusions We have shown for the first time that uterine closure is not induced by luminal epithelial cell driven endocytosis. Another novel finding of this study was the passage of the tracer ferritin up to 15 cells deep into the endometrium suggesting an as yet unstudied mechanism by which information can be transported from the uterine lumen to the underlying stroma.