Conditional knockout of leptin receptor in the female reproductive tract reduces fertility due to parturition defects in mice.

Leptin is required for fertility, including initiation of estrous cycles. It is therefore challenging to assess the role of leptin signaling during pregnancy. Although neuron-specific transgene approaches suggest that leptin signaling in the central nervous system is most important, experiments with pharmacologic inhibition of leptin in the uterus or global replacement of leptin during pregnancy suggest leptin signaling in the reproductive tract may be required. Here, conditional leptin receptor knockout (Lepr cKO) with a progesterone receptor-driven Cre recombinase was used to examine the importance of leptin signaling in pregnancy. Lepr cKO mice have almost no leptin receptor in uterus or cervix, and slightly reduced leptin receptor levels in corpus luteum. Estrous cycles and progesterone concentrations were not affected by Lepr cKO. Numbers of viable embryos did not differ between primiparous control and Lepr cKO dams on Days 6.5 and 17.5 of pregnancy, despite a slight reduction in the ratio of embryos to corpora lutea, showing that uterine leptin receptor signaling is not required for embryo implantation. Placentas of Lepr cKO dams had normal weight and structure. However, over four parities, Lepr cKO mice produced 22% fewer live pups than controls, and took more time from pairing to delivery by their fourth parity. Abnormal birth outcomes of either dystocia or dead pups occurred in 33% of Lepr cKO deliveries but zero control deliveries, and the average time to deliver each pup after crouching was significantly increased. Thus, leptin receptor signaling in the reproductive tract is required for normal labor and delivery.

Early onset preeclampsia in a model for human placental trophoblast.

We describe a model for early onset preeclampsia (EOPE) that uses induced pluripotent stem cells (iPSCs) generated from umbilical cords of EOPE and control (CTL) pregnancies. These iPSCs were then converted to placental trophoblast (TB) representative of early pregnancy. Marker gene analysis indicated that both sets of cells differentiated at comparable rates. The cells were tested for parameters disturbed in EOPE, including invasive potential. Under 5% O2, CTL TB and EOPE TB lines did not differ, but, under hyperoxia (20% O2), invasiveness of EOPE TB was reduced. RNA sequencing analysis disclosed no consistent differences in expression of individual genes between EOPE TB and CTL TB under 20% O2, but, a weighted correlation network analysis revealed two gene modules (CTL4 and CTL9) that, in CTL TB, were significantly linked to extent of TB invasion. CTL9, which was positively correlated with 20% O2 (P = 0.02) and negatively correlated with invasion (P = 0.03), was enriched for gene ontology terms relating to cell adhesion and migration, angiogenesis, preeclampsia, and stress. Two EOPE TB modules, EOPE1 and EOPE2, also correlated positively and negatively, respectively, with 20% O2 conditions, but only weakly with invasion; they largely contained the same sets of genes present in modules CTL4 and CTL9. Our experiments suggest that, in EOPE, the initial step precipitating disease is a reduced capacity of placental TB to invade caused by a dysregulation of O2 response mechanisms and that EOPE is a syndrome, in which unbalanced expression of various combinations of genes affecting TB invasion provoke disease onset.

Skeletal muscle specific mitochondrial dysfunction and altered energy metabolism in a murine model (oim/oim) of severe osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder with patients exhibiting bone fragility and muscle weakness. The synergistic biochemical and biomechanical relationship between bone and muscle is a critical potential therapeutic target, such that muscle weakness should not be ignored. Previous studies demonstrated mitochondrial dysfunction in the skeletal muscle of oim/oim mice, which model a severe human type III OI. Here, we further characterize this mitochondrial dysfunction and evaluate several parameters of whole body and skeletal muscle metabolism. We demonstrate reduced mitochondrial respiration in female gastrocnemius muscle, but not in liver or heart mitochondria, suggesting that mitochondrial dysfunction is not global in the oim/oim mouse. Myosin heavy chain fiber type distributions were altered in the oim/oim soleus muscle with a decrease (-33 to 50%) in type I myofibers and an increase (+31%) in type IIa myofibers relative to their wildtype (WT) littermates. Additionally, altered body composition and increased energy expenditure were observed oim/oim mice relative to WT littermates. These results suggest that skeletal muscle mitochondrial dysfunction is linked to whole body metabolic alterations and to skeletal muscle weakness in the oim/oim mouse.

Morphology and gene expression in mouse placentas lacking leptin receptors.

In the pregnant mouse, the hormone leptin is primarily produced by adipose tissue and does not significantly cross the placenta into fetal circulation. Nonetheless, leptin treatment during gestation affects offspring phenotypes. Leptin treatment also affects placental trophoblast cells in vitro, by altering proliferation, invasion and nutrient transport. The goal of the present study was to determine whether the absence of placental leptin receptors alters placental development and gene expression. Leprdb-3j+ mice possessing only one functional copy of the leptin receptor were mated to obtain wildtype, Leprdb-3j+ and Leprdb-3j/db-3j conceptuses, which were then transferred to wildtype recipient dams. Placentas were collected at gestational d18.5 to examine placental morphology and gene expression. Placentas lacking functional leptin receptor had reduced weights, but were otherwise morphologically indistinguishable from control placentas. Relative mRNA levels, however, were altered in Leprdb-3j/db-3j placentas, particularly transcripts related to amino acid and lipid metabolism and transport. Consistent with a previous in vitro study, leptin was found to promote expression of stathmin, a positive regulator of trophoblast invasion, and of serotonin receptors, potential mediators of offspring neurological development. Overall placental leptin receptor was found not to play a significant role in morphological development of the placenta, but to regulate placental gene expression, including in metabolic pathways that affect fetal growth.

Use of a human embryonic stem cell model to discover GABRP, WFDC2, VTCN1 and ACTC1 as markers of early first trimester human trophoblast.

Human placental development during early pregnancy is poorly understood. Many conceptuses are lost at this stage. It is thought that preeclampsia, intrauterine growth restriction and other placental syndromes that manifest later in pregnancy may originate early in placentation. Thus, there is a need for models of early human placental development. Treating human embryonic stem cells (hESCs) with BMP4 (bone morphogenic protein 4) plus A83-01 (ACTIVIN/NODAL signaling inhibitor) and PD173074 (fibroblast growth factor 2 or FGF2 signaling inhibitor) (BAP conditions) induces differentiation to the trophoblast lineage (hESCBAP), but it is not clear which stage of trophoblast differentiation these cells resemble. Here, comparison of the hESCBAP transcriptome to those of trophoblasts from human blastocysts, trophoblast stem cells and placentas collected in the first-third trimester of pregnancy by principal component analysis suggests that hESC after 8 days BAP treatment most resemble first trimester syncytiotrophoblasts. To further test this hypothesis, transcripts were identified that are expressed in hESCBAP but not in cultures of trophoblasts isolated from term placentas. Proteins encoded by four genes, GABRP (gamma-aminobutyric acid type A receptor subunit Pi), WFDC2 (WAP four-disulfide core domain 2), VTCN1 (V-set domain containing T-cell activation inhibitor 1) and ACTC1 (actin alpha cardiac muscle 1), immunolocalized to placentas at 4-9 weeks gestation, and their expression declined with gestational age (R2 = 0.61-0.83). None are present at term. Expression was largely localized to syncytiotrophoblast of both hESCBAP cells and placental material from early pregnancy. WFDC2, VTCN1 and ACTC1 have not previously been described in placenta. These results support the hypothesis that hESCBAP represent human trophoblast analogous to that of early first trimester and are a tool for discovery of factors important to this stage of placentation.

Fecundity is impaired in a mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by mutations in genes encoding type I collagen or proteins that process it. Women with OI have a small, but significant increase in risk of serious pregnancy complications including uterine rupture. Here, the OI mouse, Col1a2oim/oim , was used to examine the effects of collagen mutation on establishment and maintenance of pregnancy. Picrosirius birefringence was faint in Col1a2oim/oim uteri, indicating diminished collagen in the myometrium and endometrium. There was some evidence of increased uterine gland number (p = .055) and size (p = .12) in (p = .055) virgin uteri, though the they were not significantly different than controls. There were no differences in the number of corpora lutea, or the time from pairing to delivery of pups between Col1a2oim/oim and control dams, suggesting that ovulation and conception occur normally. However, when examined at Gestation Day 6.5 (postimplantation), gestation Day 10.5 (midpregnancy), and Postnatal Days 1-2, Col1a2oim/oim dams had significantly fewer viable pups than controls overall. In pairwise comparisons, the loss was only significant in the postnatal group, suggesting the gradual loss of pups over time. Overall, the Col1a2oim/oim mouse data suggest that OI impairs uterine function in pregnancy in a way that affects a small but significant number of fetuses.

Transcription Factor PLAGL1 Is Associated with Angiogenic Gene Expression in the Placenta.

During pregnancy, the placenta is important for transporting nutrients and waste between the maternal and fetal blood supply, secreting hormones, and serving as a protective barrier. To better understand placental development, we must understand how placental gene expression is regulated. We used RNA-seq data and ChIP-seq data for the enhancer associated mark, H3k27ac, to study gene regulation in the mouse placenta at embryonic day (e) 9.5, when the placenta is developing a complex network of blood vessels. We identified several upregulated transcription factors with enriched binding sites in e9.5-specific enhancers. The most enriched transcription factor, PLAGL1 had a predicted motif in 233 regions that were significantly associated with vasculature development and response to insulin stimulus genes. We then performed several experiments using mouse placenta and a human trophoblast cell line to understand the role of PLAGL1 in placental development. In the mouse placenta, Plagl1 is expressed in endothelial cells of the labyrinth layer and is differentially expressed in placentas from mice with gestational diabetes compared to placentas from control mice in a sex-specific manner. In human trophoblast cells, siRNA knockdown significantly decreased expression of genes associated with placental vasculature development terms. In a tube assay, decreased PLAGL1 expression led to reduced cord formation. These results suggest that Plagl1 regulates overlapping gene networks in placental trophoblast and endothelial cells, and may play a critical role in placental development in normal and complicated pregnancies.

Developmental origins of ovarian disorder: impact of maternal lean gestational diabetes on the offspring ovarian proteome in mice†.

Gestational diabetes mellitus (GDM) is an obstetric disorder affecting approximately 10% of pregnancies. The four high-fat, high-sucrose (HFHS) mouse model emulates GDM in lean women. Dams are fed a HFHS diet 1 week prior to mating and throughout gestation resulting in inadequate insulin response to glucose in mid-late pregnancy. The offspring of HFHS dams have increased adiposity, thus, we hypothesized that maternal metabolic alterations during lean GDM would compromise ovarian function in offspring both basally and in response to a control or HFHS diet in adulthood. Briefly, DLPL were lean dams and control diet pups; DLPH were lean dams and HFHS pups; DHPL were HFHS dams and control diet pups; and DHPH were HFHS dams and HFHS pups. A HFHS challenge in the absence of maternal GDM (DLPL vs. DLPH) increased 3 and decreased 30 ovarian proteins. Maternal GDM in the absence of a dietary stress (DLPL vs. DHPL) increased abundance of 4 proteins and decreased abundance of 85 proteins in the offspring ovary. Finally, 87 proteins increased, and 4 proteins decreased in offspring ovaries due to dietary challenge and exposure to maternal GDM in utero (DLPL vs. DHPH). Canopy FGF signaling regulator 2, deleted in azoospermia-associated protein 1, septin 7, and serine/arginine-rich splicing factor 2 were altered across multiple offspring groups. Together, these findings suggest a possible impact on fertility and oocyte quality in relation to GDM exposure in utero as well as in response to a western diet in later life.

Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta.

During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstntm1Sjl/+) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstntm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2oim), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2oim/+ offspring from natural mating of Mstntm1Sjl/+ dams to Col1a2oim/+sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2oim/+ dams to Col1a2oim/+ sires. Finally, increased bone biomechanical strength of Col1a2oim/+ offspring that had been transferred into Mstntm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta.

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure.

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

Effects of acute exposure to a high-fat, high-sucrose diet on gestational glucose tolerance and subsequent maternal health in mice.

Gestational diabetes mellitus (GDM) is a common obstetric complication. Half of women who have GDM will go on to develop type 2 diabetes. Understanding the mechanisms by which this occurs requires an animal model of GDM without ongoing diabetes at conception. C57Bl/6J mice react acutely to a high-fat, high-sucrose (HFHS) challenge. Here, we hypothesized that a periconceptional HFHS challenge will induce glucose intolerance during gestation. C57Bl/6J female mice were placed on an HFHS either 1 or 3 weeks prior to mating and throughout pregnancy. Intraperitoneal glucose tolerance tests, insulin measurements, and histological analysis of pancreatic islets were used to assess the impact of acute HFHS. C57Bl/6J females fed HFHS beginning 1 week prior to pregnancy became severely glucose intolerant, with reduced insulin response to glucose, and decreased pancreatic islet expansion during pregnancy compared to control mice. These GDM characteristics did not occur when the HFHS diet was started 3 weeks prior to mating, suggesting the importance of acute metabolic stress. Additionally, HFHS feeding resulted in only mild insulin resistance in nonpregnant females. When the diet was discontinued at parturition, symptoms resolved within 3 weeks. However, mice that experienced glucose intolerance in pregnancy became glucose intolerant more readily in response to a HFHS challenge later in life than congenic females that experienced a normal pregnancy, or that were fed the same diet outside of pregnancy. Thus, acute HFHS challenge in C57Bl/6 mice results in a novel, nonobese, animal model that recapitulates the long-term risk of developing type 2 diabetes following GDM.

Placental development in a mouse model of spinal muscular atrophy.

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder, leading to fatal loss of motor neurons. It is caused by loss of function of the SMN gene, which is expressed throughout the body, and there is increasing evidence of dysfunction in non-neuronal tissues. Birthweight is one of most powerful prognostic factors for infants born with SMA, and intrauterine growth restriction is common. In the SMNΔ7 mouse model of SMA, pups with the disease lived 25% longer when their mothers were fed a higher fat, "breeder" diet. The placenta is responsible for transport of nutrients from mother to fetus, and is a major determinant of fetal growth. Thus, the present study tested the hypothesis that placental development is impaired in SMNΔ7 conceptuses. Detailed morphological characterization revealed no defects in SMNΔ7 placental development, and expression of key transcription factors regulating mouse placental development was unaffected. The intrauterine growth restriction observed in SMA infants likely does not result from impaired placental development.

The evolution of the placenta.

The very apt definition of a placenta is coined by Mossman, namely apposition or fusion of the fetal membranes to the uterine mucosa for physiological exchange. As such, it is a specialized organ whose purpose is to provide continuing support to the developing young. By this definition, placentas have evolved within every vertebrate class other than birds. They have evolved on multiple occasions, often within quite narrow taxonomic groups. As the placenta and the maternal system associate more intimately, such that the conceptus relies extensively on maternal support, the relationship leads to increased conflict that drives adaptive changes on both sides. The story of vertebrate placentation, therefore, is one of convergent evolution at both the macromolecular and molecular levels. In this short review, we first describe the emergence of placental-like structures in nonmammalian vertebrates and then transition to mammals themselves. We close the review by discussing the mechanisms that might have favored diversity and hence evolution of the morphology and physiology of the placentas of eutherian mammals.

Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4.

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7(+) within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7(+) and in which the majority of cells are transiently CDX2(+). Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.

Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

Fluorescence-activated nuclear sorting (FANS) of nuclei from in vitro-generated syncytiotrophoblast.

Large, multinucleated cells, like syncytiotrophoblasts (STB), are not readily analyzed by standard methods used for single cells, such as single-cell RNA-sequencing and fluorescence-activated cellular sorting (FACS). Here we have demonstrated that fluorescence-activated nuclear sorting (FANS) is suitable to analyze nuclei from STB. Human pluripotent stem cells (PSCs) can be differentiated into a mixed trophoblast populations comprising approximately 20 % STB by treatment with BMP4 (Bone Morphogenetic Protein-4), plus A83-01 and PD173074, inhibitors of activin and FGF2 signaling, respectively (the BAP model) in about a week. Here we demonstrate that FANS can be used to separate two types of STB nuclei from the nine different clusters of trophoblast nuclei previously identified in the BAP model by single nucleus RNA sequencing (snRNAseq). Rather than using cell surface markers, as in FACS, transcription factors in various combinations were employed to target specific nuclear types. Nuclei were isolated at d 8 of BAP differentiation of H1 human embryonic stem cells and fixed in 4 % paraformaldehyde. After permeabilization in 0.1 % triton X-100, nuclei were incubated for 3 and 1 h at 4 °C with primary and secondary antibodies respectively and nuclear samples were then subjected to FANS. By using markers identified by snRNA and immunohistochemistry, nuclei were first sorted into a Topoisomerase-1, or TOP1, bright population and then into the two STB subpopulations by using antibodies to JUNB (Jun B Proto-Oncogene) and TFCP2L1 (Transcription Factor CP2 Like 1). The protocol established here is simple, straightforward, and efficient and can be used on a relatively large scale to sort individual subtypes of nuclei from mixed populations of trophoblasts for further analysis.

RISING STARS: Approaches to modeling placental function in preeclampsia in vitro and in vivo.

Modeling preeclampsia remains difficult due to the nature of the disease and the unique characteristics of the human placenta. Members of the Hominidae superfamily have a villous hemochorial placenta that is different in structure from those of other therian mammals, including the mouse hemochorial placenta, making this common animal model less ideal for studying this disease. Human placental tissues delivered from pregnancies complicated by preeclampsia are excellent for assessing the damage the disease causes but cannot answer how or when the disease begins. Symptoms of preeclampsia manifest halfway through pregnancy or later, making it currently impossible to identify preeclampsia in human tissues obtained from an early stage of pregnancy. Many animal and cell culture models recapitulate various aspects of preeclampsia, though none can on its own completely capture the complexity of human preeclampsia. It is particularly difficult to uncover the cause of the disease using models in which the disease is induced in the lab. However, the many ways by which preeclampsia-like features can be induced in a variety of laboratory animals are consistent with the idea that preeclampsia is a two-stage disease, in which a variety of initial insults may lead to placental ischemia, and ultimately systemic symptoms. The recent development of stem cell-based models, organoids, and various coculture systems have brought in vitro systems with human cells ever closer to recapitulating in vivo events that lead to placental ischemia.

Effect of Genetically Reduced Maternal Myostatin on Late Gestation Maternal, Fetal, and Placental Metabolomes in Mice.

Myostatin (gene symbol: Mstn) is an autocrine and paracrine inhibitor of muscle growth. Pregnant mice with genetically reduced levels of myostatin give birth to offspring with greater adult muscle mass and bone biomechanical strength. However, maternal myostatin is not detectable in fetal circulations. Fetal growth is dependent on the maternal environment, and the provisioning of nutrients and growth factors by the placenta. Thus, this study examined the effect of reduced maternal myostatin on maternal and fetal serum metabolomes, as well as the placental metabolome. Fetal and maternal serum metabolomes were highly distinct, which is consistent with the role of the placenta in creating a specific fetal nutrient environment. There was no effect from myostatin on maternal glucose tolerance or fasting insulin. In comparisons between pregnant control and Mstn+/- mice, there were more significantly different metabolite concentrations in fetal serum, at 50, than in the mother's serum at 33, confirming the effect of maternal myostatin reduction on the fetal metabolic milieu. Polyamines, lysophospholipids, fatty acid oxidation, and vitamin C, in fetal serum, were all affected by maternal myostatin reduction.