Reproductive biologies
Abstract
In eutherians, the placenta plays a critical role in the uptake, storage, and metabolism of lipids. These processes govern the availability of fatty acids to the developing fetus, where inadequate supply has been associated with substandard fetal growth. Whereas lipid droplets are essential for the storage of neutral lipids in the placenta and many other tissues, the processes that regulate placental lipid droplet lipolysis remain largely unknown. To assess the role of triglyceride lipases and their cofactors in determining placental lipid droplet and lipid accumulation, we assessed the role of patatin like phospholipase domain containing 2 (PNPLA2) and comparative gene identification-58 (CGI58) in lipid droplet dynamics in the human and mouse placenta. While both proteins are expressed in the placenta, the absence of CGI58, not PNPLA2, markedly increased placental lipid and lipid droplet accumulation. These changes were reversed upon restoration of CGI58 levels selectively in the CGI58-deficient mouse placenta. Using co-immunoprecipitation, we found that, in addition to PNPLA2, PNPLA9 interacts with CGI58. PNPLA9 was dispensable for lipolysis in the mouse placenta yet contributed to lipolysis in human placental trophoblasts. Our findings establish a crucial role for CGI58 in placental lipid droplet dynamics and, by extension, in nutrient supply to the developing fetus.
Authors
Jennifer Guerrero-Santoro, Mayumi Morizane, Soo-Young Oh, Takuya Mishima, Julie P. Goff, Ibrahim Bildirici, Elena Sadovsky, Yingshi Ouyang, Vladimir A. Tyurin, Yulia Y. Tyurina, Valerian E. Kagan, Yoel Sadovsky
Abstract
Progesterone (P4) is essential for embryo implantation, but the extent to which the pro-gestational effects of P4 depend on the maternal immune compartment is unknown. Here, we investigate whether regulatory T cells (Treg cells) act to mediate luteal phase P4 effects on uterine receptivity in mice. P4 antagonist RU486 administered to mice on days 0.5 and 2.5 post coitum (dpc) to model luteal phase P4 deficiency caused fewer CD4+Foxp3+ Treg cells and impaired Treg functional competence, along with dysfunctional uterine vascular remodeling and perturbed placental development in mid-gestation. These effects were linked with fetal loss and fetal growth restriction, accompanied by a Th1/CD8-skewed T cell profile. Adoptive transfer at implantation of Treg cells - but not T conventional (Tconv) cells – alleviated fetal loss and fetal growth restriction by mitigating adverse effects of reduced P4 signaling on uterine blood vessel remodeling and placental structure, and restoring maternal T cell imbalance. These findings demonstrate an essential role for Treg cells in mediating P4 effects at implantation, and indicate that Treg cells are a sensitive and critical effector mechanism through which P4 drives uterine receptivity to support robust placental development and fetal growth.
Authors
Ella S. Green, Lachlan M. Moldenhauer, Holly M. Groome, David J. Sharkey, Peck Y. Chin, Alison S. Care, Rebecca L. Robker, Shaun R. McColl, Sarah A. Robertson
Abstract
The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a non-adherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells(12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared to spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in presence of estradiol or proinflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.
Authors
Yong Song, Gregory W. Burns, Niraj R. Joshi, Ripla Arora, Ji-Yong Julie Kim, Asgerally T. Fazleabas
Abstract
Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma–/–) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.
Authors
Meaghan J. Griffiths, Sarah A. Marshall, Fiona L. Cousins, Lauren R. Alesi, Jordan Higgins, Saranya Giridharan, Urooza C. Sarma, Ellen Menkhorst, Wei Zhou, Alison S. Care, Jacqueline F. Donoghue, Sarah J. Holdsworth-Carson, Peter A.W. Rogers, Evdokia Dimitriadis, Caroline E. Gargett, Sarah A. Robertson, Amy L. Winship, Karla J. Hutt
Abstract
Multiple morphological abnormalities of the sperm flagella (MMAF) are the most severe form of asthenozoospermia due to impaired axoneme structure in sperm flagella. Dynein arms are necessary components of the sperm flagellar axoneme. In this study, we recruited 3 unrelated consanguineous Pakistani families with multiple MMAF-affected individuals, who had no overt ciliary symptoms. Whole-exome sequencing and Sanger sequencing identified 2 cilia and flagella associated protein 57 (CFAP57) loss-of-function mutations (c.2872C>T, p. R958*; and c.2737C>T, p. R913*) recessively segregating with male infertility. A mouse model mimicking the mutation (c.2872C>T) was generated and recapitulated the typical MMAF phenotype of CFAP57-mutated individuals. Both CFAP57 mutations caused loss of the long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals or from the Cfap57-mutant mouse model while the short transcript was not affected. Subsequent examinations of the spermatozoa from Cfap57-mutant mice revealed that CFAP57 deficiency disrupted the inner dynein arm (IDA) assembly in sperm flagella and that single-headed IDAs were more likely to be affected. Thus, our study identified 2 pathogenic mutations in CFAP57 in MMAF-affected individuals and reported a conserved and pivotal role for the long transcript-encoded CFAP57 in IDAs’ assembly and male fertility.
Authors
Ao Ma, Jianteng Zhou, Haider Ali, Tanveer Abbas, Imtiaz Ali, Zubair Muhammad, Sobia Dil, Jing Chen, Xiongheng Huang, Hui Ma, Daren Zhao, Beibei Zhang, Yuanwei Zhang, Wasim Shah, Basit Shah, Ghulam Murtaza, Furhan Iqbal, Muzammil Ahmad Khan, Asad Khan, Qing Li, Bo Xu, Limin Wu, Huan Zhang, Qinghua Shi
Abstract
Preeclampsia is a serious pregnancy disorder that lacks effective treatments other than delivery. Improper sensing of oxygen changes during placentation by prolyl hydroxylases (PHD), specifically PHD2, causes placental Hypoxia-Inducible Factor-1 (HIF1) buildup and abnormal downstream signaling in early-onset preeclampsia; yet therapeutic targeting of HIF1 has never been attempted. Here we generated a conditional (placenta-specific) knockout of Phd2 in mice (Phd2-/- cKO) to reproduce HIF1 excess and to assess anti-HIF therapy. Conditional deletion of Phd2 in the junctional zone (JZ) during pregnancy increased placental HIF1 content, resulting in abnormal placentation, impaired remodeling of the uterine spiral arteries, and fetal growth restriction. Pregnant dams developed new-onset hypertension at mid-gestation (E9.5) in addition to proteinuria and renal and cardiac pathology, hallmarks of severe preeclampsia in humans. Daily injection of acriflavine, a small-molecule inhibitor of HIF1, to pregnant Phd2-/- cKO mice from E7.5 (prior to hypertension) or E10.5 (after hypertension has been established) to E14.5 corrected placental dysmorphologies and improved fetal growth. Moreover, it reduced maternal blood pressure and reverted renal and myocardial pathology. Thus, therapeutic targeting of the HIF pathway may improve placental development and function, as well as maternal and fetal health, in preeclampsia.
Authors
Julien Sallais, Chanho Park, Sruthi Alahari, Tyler Porter, Ruizhe Liu, Merve Kurt, Abby Farrell, Martin Post, Isabella Caniggia
Abstract
Uterine fibroids (leiomyomas) affect Black women disproportionately in terms of prevalence, incidence, and severity of symptoms. The causes of this racial disparity are essentially unknown. We hypothesized that myometria of Black women are more susceptible to developing fibroids and examined the transcriptomic and DNA methylation profiles of myometria and fibroids from Black and White women for comparison. Myometrial samples cluster by race in both their transcriptome and DNA methylation profiles, whereas fibroid samples only cluster by race in the latter. More differentially expressed genes (DEGs) were detected in the Black and White myometrial sample comparison than in the fibroid comparison. Leiomyoma gene set expression analysis identified four clusters of DEGs, including a cluster of 24 genes with higher expression in myometrial samples from Black women. One of the DEGs in this group, VWF, was significantly hypomethylated at two CpG probes that are near a putative enhancer site in myometrial samples from Black women and in all fibroids and that correlate with VWF expression levels. These results suggest that the molecular basis for the disparity in fibroid disease between Black and White women could be found in the myometria before fibroid development and not in the fibroids themselves.
Authors
Emmanuel N. Paul, Joshua A. Grey, Tyler J. Carpenter, Zachary B. Madaj, Kin H. Lau, Scott A. Givan, Gregory W. Burns, Ronald L. Chandler, Ganesa R. Wegienka, Hui Shen, Jose M. Teixeira
Abstract
Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. One of every 4 preterm neonates is born to a mother with intra-amniotic inflammation driven by invading bacteria. However, the molecular mechanisms underlying this hostile immune response remain unclear. Here, we used a translationally relevant model of preterm birth in Nlrp3-deficient and -sufficient pregnant mice to identify what we believe is a previously unknown dual role for the NLRP3 pathway in the fetal and maternal signaling required for the premature onset of the labor cascade leading to fetal injury and neonatal death. Specifically, the NLRP3 sensor molecule and/or inflammasome is essential for triggering intra-amniotic and decidual inflammation, fetal membrane activation, uterine contractility, and cervical dilation. NLRP3 also regulates the functional status of neutrophils and macrophages in the uterus and decidua, without altering their influx, as well as maternal systemic inflammation. Finally, both embryo transfer experimentation and heterozygous mating systems provided mechanistic evidence showing that NLRP3 signaling in both the fetus and the mother is required for the premature activation of the labor cascade. These data provide insights into the mechanisms of fetal-maternal dialog in the syndrome of preterm labor and indicate that targeting the NLRP3 pathway could prevent adverse perinatal outcomes.
Authors
Kenichiro Motomura, Roberto Romero, Jose Galaz, Li Tao, Valeria Garcia-Flores, Yi Xu, Bogdan Done, Marcia Arenas-Hernandez, Derek Miller, Pedro Gutierrez-Contreras, Marcelo Farias-Jofre, Siddhesh Aras, Lawrence I. Grossman, Adi L. Tarca, Nardhy Gomez-Lopez
Abstract
Accurate estimate of fetal maturity could provide individualized guidance for delivery of complicated pregnancies. However, current methods are invasive, have low accuracy, and are limited to fetal lung maturation. To identify diagnostic gestational biomarkers, we performed transcriptomic profiling of lung and brain, as well as cell-free RNA from amniotic fluid of preterm and term rhesus macaque fetuses. These data identify new and prior associated gestational age differences in distinct lung and neuronal cell populations when compared to existing single-cell and bulk RNA-Seq data. Comparative analyses found hundreds of genes coincidently induced in lung and amniotic fluid, and dozens in brain and amniotic fluid. This data enabled creation of computational models that accurately predicted lung compliance from amniotic fluid and lung transcriptome of preterm fetuses treated with antenatal corticosteroids. Importantly, antenatal steroids induced off target gene expression changes in the brain, impinging upon synaptic transmission, neuronal and glial maturation, which could have long term consequences on brain development. Cell-free RNA in amniotic fluid may provide a substrate of global fetal maturation markers for personalized management of at-risk pregnancies.
Authors
Augusto F. Schmidt, Daniel Schnell, Kenneth P. Eaton, Kashish Chetal, Paranthaman S. Kannan, Lisa A. Miller, Claire A. Chougnet, Daniel T. Swarr, Alan H. Jobe, Nathan Salomonis, Beena D. Kamath-Rayne
Abstract
Although published studies have demonstrated that interferon epsilon (IFNε) has a crucial role in regulating protective immunity in the mouse female reproductive tract (mFRT), expression and regulation of IFNε in the human female reproductive tract (hFRT) have not been characterised. To characterise human IFNε, we obtained hFRT samples from a well- characterized cohort of women, enabling us to comprehensively assess ex vivo IFNε expression in the hFRT at various stages of the menstrual cycle. We found that among the various types of IFNs, IFNε is uniquely selectively and constitutively expressed in the hFRT epithelium. It has distinct expression patterns in the surface and glandular epithelia of the upper hFRT compared with basal layers of the stratified squamous epithelia of the lower hFRT. There is cyclical variation of IFNε expression in the endometrial epithelium of the upper hFRT and not in the distal FRT, consistent with selective endometrial expression of the progesterone receptor and regulation of the IFNE promoter by progesterone. Since we show IFNε stimulates important protective IFN-regulated genes (IRGs) in FRT epithelium, this characterisation is a key element in understanding the mechanisms of hormonal control of mucosal immunity.
Authors
Nollaig M. Bourke, Sharon L. Achilles, Stephanie U-Shane Huang, Helen E. Cumming, San S. Lim, Irene Papageorgiou, Linden J. Gearing, Ross Chapman, Suruchi Thakore, Niamh E. Mangan, Sam Mesiano, Paul J. Hertzog
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