Placental cell death patterns exhibit differences throughout gestation in two strains of laboratory mice.

Cell death is an essential physiological process required for the proper development and function of the human placenta. Although the mouse is a commonly used animal model for development studies, little is known about the extent and distribution of cell death in the mouse placenta throughout development and its physiological relevance. In the present study, we report the results of a systematic and quantitative assessment of cell death patterns in the placentae of two strains of laboratory mice commonly used for developmental studies-ICR and C57Bl/6. TUNEL staining revealed that ICR and C57Bl/6 placentae exhibited similar cell death patterns to those reported in human placentae during pregnancy, with comparatively infrequent death observed during early gestation, which increased and became more organized towards term. Interestingly, when comparing strain differences, increased cell death was observed in almost all regions of the inbred C57Bl/6 placentae compared to the outbred ICR strain. Finally, since Bcl-2 ovarian killer (Bok) has been reported to be a key player in human placental cell death, we examined its expression in murine placentae throughout gestation. Bok protein expression was observed in all placental regions and increased towards term in both strains. The results of this study indicate that although strain-specific differences in placental cell death exist, the overall rates and patterns of cell death during murine placentation parallel those previously described in humans. Thus, the murine placenta is a useful model to investigate molecular pathways involved in cell death signaling during human placentation.

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.

Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell-specific Mule knockout (BMKO) mice were generated using the Cre-LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 transactivation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho-ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apoptosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule-deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM-p53 axis to maintain B cell homeostasis under both steady-state and stress conditions.

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.

Decidual PTEN expression is required for trophoblast invasion in the mouse.

Trophoblast invasion likely depends on complex cross talk between the fetal and maternal tissues and may involve the modulation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling activity in maternal decidual cells. In this report, we studied implantation in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice, which lack the PI3K signaling antagonist gene Pten in myometrial and stromal/decidual cells. Primiparous Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice were found to be subfertile because of increased fetal mortality at e11.5. Histopathological analyses revealed a failure of decidual regression in these mice, accompanied by reduced or absent invasion of fetal trophoblast glycogen cells and giant cells, abnormal development of the placental labyrinth, and frequent apparent intrauterine fetal growth restriction. Unexpectedly, the loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN) expression in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells was not accompanied by a detectable increase in AKT phosphorylation or altered expression or activation of PI3K/AKT downstream effectors such as mammalian target of rapamycin or glycogen synthase kinase-3ß. Terminal deoxynucleotidyl transferase-mediated nick end labeling and bromodeoxyuridine incorporation analyses attributed to the lack of decidual regression mainly to decreased apoptosis in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells, rather than to increased proliferation. Remodeling of the maternal vasculature was delayed in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) uteri at e11.5, as evidenced by persistence of vascular smooth muscle and decreased infiltration of uterine natural killer cells. In addition, thickening of the myometrium and disorganization of the muscle fibers were observed before and throughout gestation. Almost all Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice failed to carry a second litter to term, apparently attributable to endometrial hyperplasia and uterine infections. Together, these data demonstrate novel roles of PTEN in the mammalian uterus and its requirement for proper trophoblast invasion and decidual regression.

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.

Embryonic resorption and polycyclic aromatic hydrocarbons: putative immune-mediated mechanisms.

Polycyclic aromatic hydrocarbons (PAHs) are released into the environment as a result of incomplete fossil fuel combustion from industrial furnaces, wood-burning stoves, and automobile exhaust fumes; however, the primary source of human exposure to these compounds is cigarette smoke. Embryonic and fetal loss after treatment with high doses of PAHs have been well documented in animal studies; however, few studies have addressed the reproductive consequences of long-term, low-level exposure to these chemicals. We previously reported that low doses of PAHs administered to ICR mice over a period of 9 weeks prior to conception resulted in early embryonic resorptions, whereby treated dams lost approximately 50% of their litter. During the course of these studies, we observed greater numbers of infiltrating uterine natural killer (uNK) cells into the placenta of PAH-exposed conceptuses. While exposure to high levels of PAHs has been shown to be immunosuppressive, increasing evidence suggests that chronic, low-dose exposure to PAHs may stimulate immune cells. Thus, we hypothesized that low-dose, chronic PAH exposure in our mouse model is mediating embryonic resorption by hyperstimulating maternal immune cells. In this review of the literature, we outline the rationale of our argument and present preliminary data, focussing upon PAH-mediated alterations in uNK cell dynamics and how these changes may be linked to early embryonic resorptions.

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.

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.

Hypoxic switch in mitochondrial myeloid cell leukemia factor-1/Mtd apoptotic rheostat contributes to human trophoblast cell death in preeclampsia.

Preeclampsia, a disorder of pregnancy, is characterized by increased trophoblast cell death and altered trophoblast-mediated remodeling of myometrial spiral arteries resulting in reduced uteroplacental perfusion. Mitochondria-associated Bcl-2 family members are important regulators of programed cell death. The mechanism whereby hypoxia alters the mitochondrial apoptotic rheostat is essential to our understanding of placental disease. Herein, myeloid cell leukemia factor-1 (Mcl-1) isoform expression was examined in physiological/pathological models of placental hypoxia. Preeclamptic placentae were characterized by caspase-dependent cleavage of death-suppressing Mcl-1L and switch toward cell death-inducing Mcl-1S. In vitro, Mcl-1L cleavage was induced by hypoxia-reoxygenation in villous explants, whereas Mcl-1L overexpression under hypoxia-reoxygenation rescued trophoblast cells from undergoing apoptosis. Cleavage was mediated by caspase-3/-7 because pharmacological caspase inhibition prevented this process. Altitude-induced chronic hypoxia was characterized by expression of Mcl-1L; resulting in a reduction of apoptotic markers (cleaved caspase-3/-8 and p85 poly-ADP-ribose polymerase). Moreover, in both physiological (explants and high altitude) and pathological (preeclampsia) placental hypoxia, decreased trophoblast syncytin expression was observed. Hence, although both pathological and physiological placental hypoxia are associated with slowed trophoblast differentiation, trophoblast apoptosis is only up-regulated in preeclampsia, because of a hypoxia-reoxygenation-induced switch in generation of proapoptotic Mcl-1 isoforms.

Pinopodes are present in Lif null and Hoxa10 null mice.

OBJECTIVE: To assess pinopode formation in Lif null and Hoxa10 null mice with infertility secondary to failed implantation. DESIGN: Controlled animal experiment. SETTING: Animal research and laboratory facility. ANIMAL(S): Lif null, Hoxa10 null, and ICR mice and Sprague-Dawley rats. INTERVENTION(S): Endometrial tissue was collected during the peri-implantation period and after ovariectomy. MAIN OUTCOME MEASURE(S): Endometrial epithelial tissue was examined under scanning-electron microscopy and assigned a score depending on the number of pinopodes present. RESULT(S): Pinopode scores in ICR, Lif null, and Hoxa10 null mice were comparable throughout the peri-implantation period, rising on day 3.5 of pregnancy and remaining elevated through to day 7.5, suggesting that pinopodes are not a good indicator of receptivity in mice. In contrast, pinopode scores in rats clearly demarcated the window of receptivity, appearing on day 4 of pregnancy and declining sharply on day 6. Pinopode scores were low in E(2)-treated ovariectomized mice, but unexpectedly, pinopode scores in vehicle-injected ovariectomized ICR mice were markedly elevated. CONCLUSION(S): Lif null and Hoxa10 null mice, in which implantation is impaired, have a similar number of pinopodes to fertile ICR mice. Pinopodes do not define a window of implantation in mice.

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.

Molecular mechanisms of trophoblast survival: from implantation to birth.

Fetal development depends upon a coordinated series of events in both the embryo and in the supporting placenta. The initial event in placentation is appropriate lineage allocation of stem cells followed by the formation of a spheroidal trophoblastic shell surrounding the embryo, facilitating implantation into the uterine stroma and exclusion of oxygenated maternal blood. In mammals, cellular proliferation, differentiation, and death accompany early placental development. Programmed cell death is a critical driving force behind organ sculpturing and eliminating abnormal, misplaced, nonfunctional, or harmful cells in the embryo proper, although very little is known about its physiological function during placental development. This review summarizes current knowledge of the cell death patterns and molecular pathways governing the survival of cells within the blastocyst, with a focus on the trophoblast lineage prior to and after implantation. Particular emphasis is given to human placental development in the context of normal and pathological conditions. As molecular pathways in humans are poorly elucidated, we have also included an overview of pertinent genetic animal models displaying defects in trophoblast survival.