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.

Effects of diazepam on hippocampal blood flow in people at clinical high risk for psychosis.

Elevated hippocampal perfusion has been observed in people at clinical high risk for psychosis (CHR-P). Preclinical evidence suggests that hippocampal hyperactivity is central to the pathophysiology of psychosis, and that peripubertal treatment with diazepam can prevent the development of psychosis-relevant phenotypes. The present experimental medicine study examined whether diazepam can normalize hippocampal perfusion in CHR-P individuals. Using a randomized, double-blind, placebo-controlled, crossover design, 24 CHR-P individuals were assessed with magnetic resonance imaging (MRI) on two occasions, once following a single oral dose of diazepam (5 mg) and once following placebo. Regional cerebral blood flow (rCBF) was measured using 3D pseudo-continuous arterial spin labeling and sampled in native space using participant-specific hippocampus and subfield masks (CA1, subiculum, CA4/dentate gyrus). Twenty-two healthy controls (HC) were scanned using the same MRI acquisition sequence, but without administration of diazepam or placebo. Mixed-design ANCOVAs and linear mixed-effects models were used to examine the effects of group (CHR-P placebo/diazepam vs. HC) and condition (CHR-P diazepam vs. placebo) on rCBF in the hippocampus as a whole and by subfield. Under the placebo condition, CHR-P individuals (mean [±SD] age: 24.1 [±4.8] years, 15 F) showed significantly elevated rCBF compared to HC (mean [±SD] age: 26.5 [±5.1] years, 11 F) in the hippocampus (F(1,41) = 24.7, pFDR < 0.001) and across its subfields (all pFDR < 0.001). Following diazepam, rCBF in the hippocampus (and subfields, all pFDR < 0.001) was significantly reduced (t(69) = -5.1, pFDR < 0.001) and normalized to HC levels (F(1,41) = 0.4, pFDR = 0.204). In conclusion, diazepam normalized hippocampal hyperperfusion in CHR-P individuals, consistent with evidence implicating medial temporal GABAergic dysfunction in increased vulnerability for psychosis.

Protocol for the establishment and characterization of an endometrial-derived epithelial organoid and stromal cell co-culture system.

Postnatal development of the uterus involves the specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. Here, we present a co-culture system to study the effects of stromal-derived factors on epithelial cell growth and differentiation into organoids. First, we describe epithelial cell isolation and organoid growth characterization. Second, we detail a co-culture system that allows the study of stromal-derived paracrine factors on epithelial development. For complete details on the use and execution of this protocol, please refer to Rizo et al.1.

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.

Sperm exposure to accessory gland secretions alters the transcriptomic response of the endometrium in cattle.

In cattle, mating to intact, but not vasectomised, bulls has been shown to modify the endometrial transcriptome, suggesting an important role of sperm in the modulation of the uterine environment in this species. However, it is not clear whether these changes are driven by intrinsic sperm factors, or by factors of accessory gland (AG) origin that bind to sperm at ejaculation. Therefore, the aim of the present study was to determine whether ejaculated sperm, which are suspended in the secretions of the AGs, elicit a different endometrial transcriptomic response than epididymal sperm, which have never been exposed to AG factors. To this end, bovine endometrial explants collected from heifers in oestrus were (co-)incubated for 6 h alone (control), or with epididymal sperm or ejaculated sperm, following which transcriptomic changes in the endometrium were evaluated. Epididymal sperm elicited a more dramatic endometrial response than ejaculated sperm, in terms of the number of differentially expressed genes (DEGs). Indeed, RNA-sequencing data analysis revealed 1912 DEGs in endometrial explants exposed to epididymal sperm compared with control explants, whereas 115 DEGs were detected between endometrial explants exposed to ejaculated sperm in comparison to control explants. The top pathways associated with genes upregulated by epididymal sperm included T cell regulation and TNF, NF-KB and IL17 signalling. Interestingly, ejaculated sperm induced downregulation of genes associated with T cell immunity and Th17 differentiation, and upregulation of genes involved in NF-KB signalling, in comparison to epididymal sperm. These data indicate that factors of AG origin modulate the interaction between sperm and the endometrium in cattle.

Preclinical Models of Attention Deficit Hyperactivity Disorder: Neurobiology, Drug Discovery, and Beyond.

OBJECTIVE: We offer an overview of ADHD research using mouse models of nicotine exposure. METHOD: Nicotine exposure of C57BL/6 or Swiss Webster mice occurred during prenatal period only or during the prenatal and the pre-weaning periods. Behavioral, neuroanatomical and neurotransmitter assays were used to investigate neurobiological mechanisms of ADHD and discover candidate ADHD medications. RESULTS: Our studies show that norbinaltorphimine, a selective kappa opioid receptor antagonist is a candidate novel non-stimulant ADHD treatment and that a combination of methylphenidate and naltrexone has abuse deterrent potential with therapeutic benefits for ADHD. Other studies showed transgenerational transmission of ADHD-associated behavioral traits and demonstrated that interactions between untreated ADHD and repeated mild traumatic brain injury produced behavioral traits not associated with either condition alone. CONCLUSION: Preclinical models contribute to novel insights into ADHD neurobiology and are valuable tools for drug discovery and translation to benefit humans with ADHD.

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.

Impact of Sire on Embryo Development and Pregnancy.

The use of in vitro embryo production (IVP) has increased globally, particularly in the United States. Although maternal factors influencing embryo development have been extensively studied, the influence of the sire is not well understood. Sperm plays a crucial role in embryo development providing DNA, triggering oocyte maturation, and aiding in mitosis. Current sire fertility measurements do not consistently align with embryo production outcomes. Low-fertility sires may perform well in IVP systems but produce fewer pregnancies. Testing sires in vitro could identify characteristics affecting embryo development and pregnancy loss risk in IVP and embryo transfer programs.

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.

The development of new biomarkers of spermatozoa quality in cattle.

There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high (n = 4) or low (n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater (p < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development.

Extensive rewiring of the gene regulatory interactions between in vitro-produced conceptuses and endometrium during attachment.

Pregnancy loss is a significant problem when embryos produced in vitro are transferred to a synchronized uterus. Currently, mechanisms that underlie losses of in vitro-produced embryos during implantation are largely unknown. We investigated this problem using cattle as a model of conceptus attachment by analyzing transcriptome data of paired extraembryonic membrane and endometrial samples collected on gestation days 18 and 25, which spans the attachment window in cattle. We identified that the transfer of an in vitro-produced embryo caused a significant alteration in transcript abundance of hundreds of genes in extraembryonic and endometrial tissues on gestation days 18 and 25, when compared to pregnancies initiated by artificial insemination. Many of the genes with altered transcript abundance are associated with biological processes that are relevant to the establishment of pregnancy. An integrative analysis of transcriptome data from the conceptus and endometrium identified hundreds of putative ligand-receptor pairs. There was a limited variation of ligand-receptor pairs in pregnancies initiated by in vitro-produced embryos on gestation day 18, and no alteration was observed on gestation day 25. In parallel, we identified that in vitro production of embryos caused an extensive alteration in the coexpression of genes expressed in the extraembryonic membranes and the corresponding endometrium on both gestation days. Both the transcriptional dysregulation that exists in the conceptus or endometrium independently and the rewiring of gene transcription between the conceptus and endometrium are a potential component of the mechanisms that contribute to pregnancy losses caused by in vitro production of embryos.

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.

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.

Estrogen receptor alpha regulates uterine epithelial lineage specification and homeostasis.

Postnatal development of the uterus involves specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. This study utilized mouse genetic models of Esr1 deletion, endometrial epithelial organoids (EEO), and organoid-stromal co-cultures to decipher the role of Esr1 in uterine epithelial development. Organoids derived from wild-type (WT) mice developed a normal single layer of columnar epithelium. In contrast, EEO from Esr1 null mice developed a multilayered stratified squamous type of epithelium with basal cells. Co-culturing Esr1 null epithelium with WT uterine stromal fibroblasts inhibited basal cell development. Of note, estrogen treatment of EEO-stromal co-cultures and Esr1 conditional knockout mice increased basal epithelial cell markers. Collectively, these findings suggest that Esr1 regulates uterine epithelium lineage plasticity and homeostasis and loss of ESR1 promotes altered luminal-to-basal differentiation driven by ESR1-mediated paracrine factors from the stroma.

Tryptophan Stabilization of a Biochemical Carbocation Evaluated by Analysis of π Complexes of 3-Ethylindole with the t-Butyl Cation.

Understanding how the highly unstable carbocation intermediates in terpenoid biosynthesis are stabilized and protected during their transient existence in enzyme active sites is an intriguing challenge which has to be addressed computationally. Our efforts have focused on evaluating the stabilization afforded via carbocation-π complexation between a biochemical carbocation and an aromatic amino acid residue. This has involved making measurements on an X-ray structure of an enzyme active site that shows a π donor proximate to a putative carbocation site and using these to build models which are analyzed computationally to provide an estimated stabilization energy (SE). Previously, we reported estimated SEs for several such carbocation-π complexes involving phenylalanine. Herein, we report the first such estimate involving tryptophan as the π donor. Because there was almost no published information about indole as a π-complexation donor, we first located computationally equilibrium π and σ complexes of 3-ethylindole with the t-butyl cation as relevant background information. Then, measurements on the X-ray structure of the enzyme CotB2 complexed with geranylgeranyl thiodiphosphate (GGSPP), specifically on the geometric relationship of the putative carbocation at C15 of GGSPP to W186, were used to build a model that afforded a computed SE of -15.3 kcal/mol.

Editorial: Height and Stimulant Treatment in Attention-Deficit/Hyperactivity Disorder Youth: Mind the Gap.

In this issue of the Journal, the important story of height and weight issues related to stimulant medication in attention-deficit/hyperactivity disorder (ADHD) is yet again addressed, in the largest study to date.1 Controversy related to potential growth decrements in youth with ADHD receiving stimulants has been around for decades. In fact, in a quick PubMed search of the literature, we identified more than 100 studies, 15 reviews, and meta-analyses in this area. In a general score card, about three-fourths of studies seem to support some degree of decrement, but clinical significance has yet to be determined. What is not being debated anymore is the short- and longer-term effectiveness of stimulants for ADHD with reductions in mood disorders, suicide, substance use/disorders, criminality, motor vehicle accidents, injuries, concussions, and academic failure documented in the Swedish and other register studies.2 Many of these improvements are linked with a decrease in major morbidity and even mortality. Hence, we need to put the issue of potential stimulant-related growth velocity changes in the context of the very real adverse outcomes associated with non-treatment of ADHD.

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.

Online speech assessment of the psychotic spectrum: Exploring the relationship between overlapping acoustic markers of schizotypy, depression and anxiety.

BACKGROUND: Remote assessment of acoustic alterations in speech holds promise to increase scalability and validity in research across the psychosis spectrum. A feasible first step in establishing a procedure for online assessments is to assess acoustic alterations in psychometric schizotypy. However, to date, the complex relationship between alterations in speech related to schizotypy and those related to comorbid conditions such as symptoms of depression and anxiety has not been investigated. This study tested whether (1) depression, generalized anxiety and high psychometric schizotypy have similar voice characteristics, (2) which acoustic markers of online collected speech are the strongest predictors of psychometric schizotypy, (3) whether including generalized anxiety and depression symptoms in the model can improve the prediction of schizotypy. METHODS: We collected cross-sectional, online-recorded speech data from 441 participants, assessing demographics, symptoms of depression, generalized anxiety and psychometric schizotypy. RESULTS: Speech samples collected online could predict psychometric schizotypy, depression, and anxiety symptoms with weak to moderate predictive power, and with moderate and good predictive power when basic demographic variables were added to the models. Most influential features of these models largely overlapped. The predictive power of speech marker-based models of schizotypy significantly improved after including symptom scores of depression and generalized anxiety in the models (from R2 = 0.296 to R2 = 0. 436). CONCLUSIONS: Acoustic features of online collected speech are predictive of psychometric schizotypy as well as generalized anxiety and depression symptoms. The acoustic characteristics of schizotypy, depression and anxiety symptoms significantly overlap. Speech models that are designed to predict schizotypy or symptoms of the schizophrenia spectrum might therefore benefit from controlling for symptoms of depression and anxiety.