Single-cell insights into epithelial morphogenesis in the neonatal mouse uterus.

The uterus is vital for successful reproduction in mammals, and two different types of epithelia (luminal and glandular) are essential for embryo implantation and pregnancy establishment. However, the essential cellular and molecular factors and pathways governing postnatal epithelium maturation, determination, and differentiation in developing uterus are yet to be elucidated. Here, the epithelium of the neonatal mouse uterus was isolated and subjected to single-cell transcriptome (scRNA-seq) analysis. Both the undifferentiated epithelium and determined luminal epithelium were heterogeneous and contained several different cell clusters based on single-cell transcription profiles. Substantial gene expression differences were evident as the epithelium matured and differentiated between postnatal days 1 to 15. Two new glandular epithelium-expressed genes (Gas6 and Cited4) were identified and validated by in situ hybridization. Trajectory analyses provided a framework for understanding epithelium maturation, lineage bifurcation, and differentiation. A candidate set of transcription factors and gene regulatory networks were identified that potentially direct epithelium lineage specification and morphogenesis. This atlas provides a foundation important to discover intrinsic cellular and molecular mechanisms directing uterine epithelium morphogenesis during a critical window of postnatal development.

Single-nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta.

Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.

Single-cell insights into development of the bovine placenta†.

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation.

Metritis and the uterine disease microbiome are associated with long-term changes in the endometrium of dairy cows†.

Cows with metritis (uterine disease) during the first 1 to 2 wk postpartum have lower pregnancy rates when inseminated later postpartum (typically >10 wk). We hypothesized that metritis and the disease-associated uterine microbiome have a long-term effect on endometrial gene expression. Changes in gene expression may inform a mechanism through which disease lowers pregnancy rates. A total of 20 cows were enrolled at 1 to 2 wk postpartum to either metritis (clinical disease; n = 10) or healthy (control; n = 10) groups and randomly assigned to be slaughtered at approximately 80 d and 165 d postpartum (mid-lactation). The microbiome of the reproductive tract was sampled to confirm the presence of pathogens that are typical of metritis. In addition to the original clinical diagnosis, study cows were retrospectively assigned to uterine-disease and control groups based on the composition of their microbiome. There was no effect of early postpartum uterine disease on the uterine microbiome at mid-lactation (time of slaughter). Nonetheless, early postpartum metritis and the disease microbiome were associated with a large number of differentially-expressed genes at mid-lactation primarily in the caruncular compared with the inter-caruncular endometrium. Gene enrichment analysis identified oxidative phosphorylation as the primary pathway increased in caruncular endometrium of diseased cows whereas growth factor signaling pathways were reduced. The current study demonstrated that metritis and a uterine disease microbiome leave a sustained imprint on gene expression in the caruncular endometrium that may explain lower fertility in cows with postpartum uterine disease.

Development of Polarity-Reversed Endometrial Epithelial Organoids.

The uterine epithelium is composed of a single layer of hormone responsive polarized epithelial cells that line the lumen and form tubular glands. Endometrial epithelial organoids (EEO) can be generated from uterine epithelia and recapitulate cell composition and hormone responses in vitro. As such, the development of EEO represents a major advance for facilitating mechanistic studies in vitro. However, a major limitation for the use of EEO cultured in basement membrane extract and other hydrogels is the inner location of apical membrane, thereby hindering direct access to the apical surface of the epithelium to study interactions with the embryo or infectious agents such as viruses and bacteria. Here, a straightforward strategy was developed that successfully reverses the polarity of EEO. The result is an apical-out organoid that preserves a distinct apical-basolateral orientation and remains responsive to ovarian steroid hormones. Our investigations highlight the utility of polarity-reversed EEO to study interactions with E. coli and blastocysts. This method of generating apical-out EEO lays the foundation for developing new in vitro functional assays, particularly regarding epithelial interactions with embryos during pregnancy or other luminal constituents in a pathological or diseased state.

Capture and metabolomic analysis of the human endometrial epithelial organoid secretome.

Suboptimal uterine fluid (UF) composition can lead to pregnancy loss and likely contributes to offspring susceptibility to chronic adult-onset disorders. However, our understanding of the biochemical composition and mechanisms underpinning UF formation and regulation remain elusive, particularly in humans. To address this challenge, we developed a high-throughput method for intraorganoid fluid (IOF) isolation from human endometrial epithelial organoids. The IOF is biochemically distinct to the extraorganoid fluid (EOF) and cell culture medium as evidenced by the exclusive presence of 17 metabolites in IOF. Similarly, 69 metabolites were unique to EOF, showing asymmetrical apical and basolateral secretion by the in vitro endometrial epithelium, in a manner resembling that observed in vivo. Contrasting the quantitative metabolomic profiles of IOF and EOF revealed donor-specific biochemical signatures of organoids. Subsequent RNA sequencing of these organoids from which IOF and EOF were derived established the capacity to readily perform organoid multiomics in tandem, and suggests that transcriptomic regulation underpins the observed secretory asymmetry. In summary, these data provided by modeling uterine luminal and basolateral fluid formation in vitro offer scope to better understand UF composition and regulation with potential impacts on female fertility and offspring well-being.

Wnt7a deficit is associated with dysfunctional angiogenesis in pulmonary arterial hypertension.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is characterised by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. We hypothesised that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH. METHODS: Lung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a -/- mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx). RESULTS: Healthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with the formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced vascular endothelial growth factor (VEGF)-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signalling by facilitating Y1175 tyrosine phosphorylation in vascular endothelial growth factor receptor 2 (VEGFR2) through receptor tyrosine kinase-like orphan receptor 2 (ROR2), a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a -/- mice under either chronic hypoxia or SuHx, global Wnt7a +/- mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodelling. Similar to PAH, Wnt7a +/- PMVECs exhibited an insufficient angiogenic response to VEGF-A that improved with Wnt7a. CONCLUSIONS: Wnt7a promotes VEGF signalling in lung PMVECs and its loss is associated with an insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH.

Basolateral secretions of human endometrial epithelial organoids impact stromal cell decidualization.

Uterine glands and, by inference, their secretions impact uterine receptivity, blastocyst implantation, stromal cell decidualization, and placental development. Changes in gland function across the menstrual cycle are primarily governed by the steroid hormones estrogen (E2) and progesterone (P4) but can also be influenced by extrinsic factors from the stroma. Using a human endometrial epithelial organoid system, transcriptome and proteome analyses identified distinct responses of the organoids to steroid hormones and prostaglandin E2 (PGE2). Notably, P4 and PGE2 modulated the basolateral secretion of organoid proteins, particularly cystatin C (CST3), serpin family A member 3 (SERPINA3), and stanniocalcin 1 (STC1). CST3, but not SERPINA3 or STC1, attenuated the in vitro stromal decidualization response to steroid hormones and PGE2. These findings provide evidence that uterine gland-derived factors impact stromal cell decidualization, which has implications for pregnancy establishment and fertility in women.

Sire modulates developmental kinetics and transcriptome of the bovine embryo.

In brief: The first week of gestation is a period of major pregnancy loss in cattle, this study reveals that the male plays a key role in regulating embryonic development during this time. Abstract: The impact of sire on preimplantation embryonic development in cattle remains poorly understood. This study evaluated differences in embryos produced in vitro from sires with varying capacities to produce blastocysts. Sires classified as high (HP) and low performing (LP) based on their ability to produce embryos were used to better understand how sire regulates embryonic development. By monitoring development, it was determined that the most common arrest stage was the five- to six-cell stage. Embryos (four to six cells) from HP and LP sires were then analyzed for autophagic activity, where embryos for LP sires exhibited increased autophagy than HP-derived embryos. Transcriptome analysis of four-cell embryos found that embryos from LP sires might have issues in sperm mitochondrial clearance, histone retention, and DNA damage, while HP sires had increased expression of genes involved in transcription, chromosome segregation, and cell division. In conclusion, LP sires had an increased proportion of embryos arresting at the five- to six-cell stage, and these embryos had higher rates of cellular stress due to paternal contributions from the spermatozoon.

Gene editing provides a tool to investigate genes involved in reproduction of pigs.

CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.

Sexually dimorphic effects of forkhead box a2 (FOXA2) and uterine glands on decidualization and fetoplacental development.

Glands of the uterus are essential for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands of the uterus and a critical regulator of glandular epithelium (GE) differentiation, development, and function. Mice with a conditional deletion of FOXA2 in the adult uterus, created using the lactotransferrin iCre (Ltf-iCre) model, have a morphologically normal uterus with glands, but lack FOXA2-dependent GE-expressed genes, such as leukemia inhibitory factor (LIF). Adult FOXA2 conditional knockout (cKO; LtfiCre/+Foxa2f/f ) mice are infertile due to defective embryo implantation arising from a lack of LIF, a critical implantation factor of uterine gland origin. However, intraperitoneal injections of LIF can initiate embryo implantation in the uterus of adult FOXA2 cKO mice with pregnancies maintained to term. Here, we tested the hypothesis that FOXA2-regulated genes in the uterine glands impact development of the decidua, placenta, and fetus. On gestational day 8.5, the antimesometrial and mesometrial decidua transcriptome was noticeably altered in LIF-replaced FOXA2 cKO mice. Viable fetuses were reduced in FOXA2 cKO mice on gestational days 12.5 and 17.5. Sex-dependent differences in fetal weight, placenta histoarchitecture, and the placenta and metrial gland transcriptome were observed between control and FOXA2 cKO mice. The transcriptome of the placenta with a female fetus was considerably more altered than the placenta with a male fetus in FOXA2 cKO dams. These studies reveal previously unrecognized sexually dimorphic effects of FOXA2 and uterine glands on fetoplacental development with potential impacts on offspring health into adulthood.

Prss29 Cre recombinase mice are useful to study adult uterine gland function.

All mammalian uteri contain glands in their endometrium that develop only or primarily after birth. In mice, those endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful for the study of uterine gland function during pregnancy. Utilizing CRISPR-Cas9 genome editing, Cre recombinase was inserted into the endogenous serine protease 29 precursor (Prss29) gene. Both Prss29 mRNA and Cre recombinase activity was specific to the GE of the mouse uterus following implantation, but was absent from other areas of the female reproductive tract. Next, Prss29-Cre mice were crossed with floxed forkhead box A2 (Foxa2) mice to conditionally delete Foxa2 specifically in the endometrial glands. Foxa2 was absent in the glands of the post-implantation uterus, and Foxa2 deleted mice exhibited complete infertility after their first pregnancy. These results establish that Prss29-Cre mice are a valuable resource to elucidate and explore the functions of glands in the adult uterus.

Inserting Cre recombinase into the Prolactin 8a2 gene for decidua-specific recombination in mice.

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here, we describe a new decidua-specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG/+ or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

Notch effector recombination signal binding protein for immunoglobulin kappa J signaling is required for the initiation of endometrial stromal cell decidualization†.

The Notch signaling pathway is required for reproductive success. This pathway activates its transcriptional effector, recombination signal binding protein for immunoglobulin kappa J (Rbpj), to induce transcription of its target genes. This signaling pathway is required for successful decidualization, implantation, and uterine repair following parturition. To identify the compartmental specific roles of the Notch signaling pathway in the establishment of pregnancy, we generated epithelial and decidual stromal cell specific knockouts of Rbpj utilizing lactoferrin iCre and Prl8A2 iCre, respectively. Both conditional knockout mouse models were fertile. The Rbpj epithelial knockout mice displayed 27% resorption sites at E15.5, but this did not significantly impact the number of live born pups compared with controls. In addition, the Rbpj epithelial knockout mice displayed increased estrogen signaling in their stromal compartment. Given that both mouse models exhibited fertility comparable to control animals, the epithelial and stromal specific nature of the iCre recombinases utilized, and previously published Rbpj total uterine knockout mouse models, we conclude that Notch effector Rbpj signaling is required at the initiation of pregnancy to support decidualization in stromal cells, but that Rbpj is not required in the epithelial compartment nor is it required for post-implantation pregnancy success.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Uterine glands impact embryo survival and stromal cell decidualization in mice.

Uterine glands are essential for the establishment of pregnancy and have critical roles in endometrial receptivity to blastocyst implantation, stromal cell decidualization, and placentation. Uterine gland dysfunction is considered a major contributing factor to pregnancy loss, however our understanding of how glands impact embryo survival and stromal cell decidualization is incomplete. Forkhead box A2 (FOXA2) is expressed only in the glandular epithelium and regulates its development and function. Mice with a conditional deletion of FOXA2 in the uterus are infertile due to defective embryo implantation arising from a lack of leukemia inhibitory factor (LIF), a critical factor of uterine gland origin. Here, a glandless FOXA2-deficient mouse model, coupled with LIF repletion to rescue the implantation defect, was used to investigate the roles of uterine glands in embryo survival and decidualization. Studies found that embryo survival and decidualization were compromised in glandless FOXA2-deficient mice on gestational day 6.5, resulting in abrupt pregnancy loss by day 7.5. These findings strongly support the hypothesis that uterine glands secrete factors other than LIF that impact embryo survival and stromal cell decidualization for pregnancy success.

Current and Future Estimates of Marginal Emission Factors for Indian Power Generation.

Emission factors from Indian electricity remain poorly characterized, despite known spatial and temporal variability. Limited publicly available emissions and generation data at sufficient detail make it difficult to understand the consequences of emissions to climate change and air pollution, potentially missing cost-effective policy designs for the world's third largest power grid. We use reduced-form and full-form power dispatch models to quantify current (2017-2018) and future (2030-2031) marginal CO2, SO2, NOX, and PM2.5 emission factors from Indian power generation. These marginal emissions represent emissions changes due to small changes in demand. For 2017-2018, spatial variability in marginal CO2 emission factors range 3 orders of magnitude across India's states. There is limited seasonal and intraday variability with coal generation likely to meet changes in demand more than half the time in more than half of the states. Assuming the Government of India approximate 2030 targets, the median marginal CO2 emission factor across states decreases by approximately a factor of 2, but emission factors still span 3 orders of magnitude across states. Under 2030-2031 assumptions there is greater seasonal and intraday variability by up to factors of two and four, respectively. Estimates provide emission factors to evaluate interventions such as electric vehicles, increased air conditioning, and energy efficiency.

Frontal Cortical Monoamine Release, Attention, and Working Memory in a Perinatal Nicotine Exposure Mouse Model Following Kappa Opioid Receptor Antagonism.

Perinatal nicotine exposure (PNE) produces frontal cortical hypo-dopaminergic state and attention and working memory deficits consistent with neurodevelopmental disorders such as attention deficit hyperactivity disorder (ADHD). Methylphenidate alleviates ADHD symptoms by increasing extracellular dopamine and noradrenaline. Kappa opioid receptor (KOR) antagonism may be another mechanism to achieve the same results because KOR activation inhibits frontal cortical dopamine release. We administered the selective KOR antagonist norbinaltorphimine (norBNI) (20 mg/kg; intraperitoneal) or methylphenidate (0.75 mg/kg; intraperitoneal) to PNE mouse model and examined frontal cortical monoamine release, attention, and working memory. Both compounds increased dopamine and noradrenaline release but neither influenced serotonin release. Both compounds improved object-based attention and working memory in the PNE group, with norBNI's effects evident at 2.5 h and 5.5 h but absent at 24 h. Methylphenidate's effects were evident at 0.5 h but not at 2.5 h. norBNI's effects temporally coincided with frontal cortical c-Jun N-terminal kinase phosphorylation. norBNI did not alter tissue dopamine content in the nucleus accumbens, offering preliminary support for lack of reinforcement.

Self-renewing endometrial epithelial organoids of the human uterus.

The human endometrium is essential in providing the site for implantation and maintaining the growth and survival of the conceptus. An unreceptive endometrium and disrupted maternal-conceptus interactions can cause infertility due to pregnancy loss or later pregnancy complications. Despite this, the role of uterine glands in first trimester human pregnancy is little understood. An established organoid protocol was used to generate and comprehensively analyze 3-dimensional endometrial epithelial organoid (EEO) cultures from human endometrial biopsies. The derived EEO expand long-term, are genetically stable, and can be cryopreserved. Using endometrium from 2 different donors, EEO were derived and then treated with estrogen (E2) for 2 d or E2 and medroxyprogesterone acetate (MPA) for 6 d. EEO cells were positive for the gland marker, FOXA2, and exhibited appropriate hormonal regulation of steroid hormone receptor expression. Real-time qPCR and bulk RNA-sequencing analysis revealed effects of hormone treatment on gene expression that recapitulated changes in proliferative and secretory phase endometrium. Single-cell RNA sequencing analysis revealed that several different epithelial cell types are present in the EEO whose proportion and gene expression changed with hormone treatment. The EEO model serves as an important platform for studying the physiology and pathology of the human endometrium.