A transcriptomics model of estrogen action in the ovine fetal hypothalamus: evidence for estrogenic effects of ICI 182,780

Estradiol plays a critical role in stimulating the fetal hypothalamus?pituitary?adrenal axis at the end of gestation. Estradiol action is mediated through nuclear and membrane receptors that can be modulated by ICI 182,780, a pure antiestrogen compound. The objective of this study was to evaluate the transcriptomic profile of estradiol and ICI 182,780, testing the hypothesis that ICI 182,780 antagonizes the action of estradiol in the fetal hypothalamus. Chronically catheterized ovine fetuses were infused for 48 h with: vehicle (Control, n = 6), 17β‐estradiol 500 μg/kg/day (Estradiol, n = 4), ICI 182,780 5 μg/kg/day (ICI 5 μg, n = 4) and ICI 182,780 5 mg/kg/day (ICI 5 mg, n = 5). Fetal hypothalami were collected afterward, and gene expression was measured through microarray. Statistical analysis of transcriptomic data was performed with Bioconductor‐R and Cytoscape software. Unexpectedly, 35% and 15.5% of the upregulated differentially expressed genes (DEG) by Estradiol significantly overlapped (P < 0.05) with upregulated DEG by ICI 5 mg and ICI 5 μg, respectively. For the downregulated DEG, these percentages were 29.9% and 15.5%, respectively. There was almost no overlap for DEG following opposite directions between Estradiol and ICI ICI 5 mg or ICI 5 μg. Furthermore, most of the genes in the estrogen signaling pathway after activation of the epidermal growth factor receptor followed the same direction in Estradiol, ICI 5 μg or ICI 5 mg compared to Control. In conclusion, estradiol and ICI 182,780 have estrogenic genomic effects in the developing brain, suggesting the possibility that the major action of estradiol on the fetal hypothalamus involves another receptor system rather than estrogen receptors.Fil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Keller Wood, Maureen. University of Florida; Estados UnidosFil: Wood, Charles E.. University of Florida; Estados Unido

Transcriptomics modeling of the late-gestation fetal pituitary response to transient hypoxia

Background The late-gestation fetal sheep responds to hypoxia with physiological, neuroendocrine, and cellular responses that aid in fetal survival. The response of the fetus to hypoxia represents a coordinated effort to maximize oxygen transfer from the mother and minimize wasteful oxygen consumption by the fetus. While there have been many studies aimed at investigating the coordinated physiological and endocrine responses to hypoxia, and while immunohistochemical or in situ hybridization studies have revealed pathways supporting the endocrine function of the pituitary, there is little known about the coordinated cellular response of the pituitary to the hypoxia. Results Thirty min hypoxia (from 17.0±1.7 to 8.0±0.8 mm Hg, followed by 30 min normoxia) upregulated 595 and downregulated 790 genes in fetal pituitary (123-132 days' gestation; term = 147 days). Network inference of up- and down- regulated genes revealed a high degree of functional relatedness amongst the gene sets. Gene ontology analysis revealed upregulation of cellular metabolic processes (e.g., RNA synthesis, response to estrogens) and downregulation Conclusions The multiple analytical approaches used in this study suggests that the acute response to 30 min of transient hypoxia in the late-gestation fetus results in reduced cellular metabolism and a pattern of gene expression that is consistent with cellular oxygen and ATP starvation. In this early time point, we see a vigorous gene response. But, like the hypothalamus, the transcriptomic response is not consistent with mediation by HIF-1. If HIF-1 is a significant controller of gene expression in the fetal pituitary after hypoxia, it must be at a later time.Fil: Wood, Charles E.. University of Florida; Estados UnidosFil: Chang, Eileen I.. University of Florida; Estados UnidosFil: Richards, Elaine M.. University of Florida; Estados UnidosFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Keller Wood, Maureen. University of Florida; Estados Unido

Single-cell profiling reveals transcriptome dynamics during bovine oocyte growth

Background: Mammalian follicle development is characterized by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterizing gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage. Results: Single-cell RNA-seq libraries were generated from oocytes of known diameters ( 120 μm), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 μm in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100–109 versus 110–119 μm in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase. Conclusions: The current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis

Ketamine reduces inflammation pathways in the hypothalamus and hippocampus following transient hypoxia in the late-gestation fetal sheep

The physiological response to hypoxia in the fetus has been extensively studied with regard to redistribution of fetal combined ventricular output and sparing of oxygen delivery to fetal brain and heart. Previously, we have shown that the fetal brain is capable of mounting changes in gene expression that are consistent with tissue inflammation. The present study was designed to use transcriptomics and systems biology modeling to test the hypothesis that ketamine reduces or prevents the upregulation of inflammation-related pathways in hypothalamus and hippocampus after transient hypoxic hypoxia. Chronically catheterized fetal sheep (122 ± 5 days gestation) were subjected to 30 min hypoxia (relative reduction in PaO2∼50%) caused by infusion of nitrogen into the inspired gas of the pregnant ewe. RNA was isolated from fetal hypothalamus and hippocampus collected 24 h after hypoxia, and was analyzed for gene expression using the Agilent 15.5 k ovine microarray. Ketamine, injected 10 min prior to hypoxia, reduced the cerebral immune response activation to the hypoxia in both brain regions. Genes both upregulated by hypoxia and downregulated by ketamine after hypoxia were significantly associated with gene ontology terms and KEGG pathways that are, themselves, associated with the tissue response to exposure to bacteria. We conclude that the results are consistent with interruption of the cellular response to bacteria by ketamine.Fil: Chang, Eileen I.. University of Florida; Estados UnidosFil: Zarate, Miguel A.. University of Florida; Estados UnidosFil: Arndt, Thomas J.. University of Florida; Estados UnidosFil: Richards, Elaine M.. University of Florida; Estados UnidosFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Keller Wood, Maureen. University of Florida; Estados UnidosFil: Wood, Charles E.. University of Florida; Estados Unido

Maternal blood transcriptome as a sensor of fetal organ maturation at the end of organogenesis in cattle†

Harnessing information from the maternal blood to predict fetal growth is attractive yet scarcely explored in livestock. The objectives were to determine the transcriptomic modifications in maternal blood and fetal liver, gonads, and heart according to fetal weight and to model a molecular signature based on the fetal organs allowing the prediction of fetal weight from the maternal blood transcriptome in cattle. In addition to a contemporaneous maternal blood sample, organ samples were collected from 10 male fetuses at 42 days of gestation for RNA-sequencing. Fetal weight ranged from 1.25 to 1.69 g (mean = 1.44 ± 0.15 g). Clustering data analysis revealed clusters of co-expressed genes positively correlated with fetal weight and enriching ontological terms biologically relevant for the organ. For the heart, the 1346 co-expressed genes were involved in energy generation and protein synthesis. For the gonads, the 1042 co-expressed genes enriched seminiferous tubule development. The 459 co-expressed genes identified in the liver were associated with lipid synthesis and metabolism. Finally, the cluster of 571 co-expressed genes determined in maternal blood enriched oxidative phosphorylation and thermogenesis. Next, data from the fetal organs were used to train a regression model of fetal weight, which was predicted with the maternal blood data. The best prediction was achieved when the model was trained with 35 co-expressed genes overlapping between heart and maternal blood (root-mean-square error = 0.04, R2 = 0.93). In conclusion, linking transcriptomic information from maternal blood with that from the fetal heart unveiled maternal blood as a predictor of fetal development

Ketamine decreases inflammatory and immune pathways after transient hypoxia in late gestation fetal cerebral cortex

Transient hypoxia in pregnancy stimulates a physiological reflex response that redistributes blood flow and defends oxygen delivery to the fetal brain. We designed the present experiment to test the hypotheses that transient hypoxia produces damage of the cerebral cortex and that ketamine, an antagonist of NMDA receptors and a known anti-inflammatory agent, reduces the damage. Late gestation, chronically catheterized fetal sheep were subjected to a 30-min period of ventilatory hypoxia that decreased fetal PaO2 from 17 ± 1 to 10 ± 1 mmHg, or normoxia (PaO2 17 ± 1 mmHg), with or without pretreatment (10 min before hypoxia/normoxia) with ketamine (3 mg/kg, i.v.). One day (24 h) after hypoxia/normoxia, fetal cerebral cortex was removed and mRNA extracted for transcriptomics and systems biology analysis (n = 3-5 per group). Hypoxia stimulated a transcriptomic response consistent with a reduction in cellular metabolism and an increase in inflammation. Ketamine pretreatment reduced both of these responses. The inflammation response modeled with transcriptomic systems biology was validated by immunohistochemistry and showed increased abundance of microglia/macrophages after hypoxia in the cerebral cortical tissue that ketamine significantly reduced. We conclude that transient hypoxia produces inflammation of the fetal cerebral cortex and that ketamine, in a standard clinical dose, reduces the inflammation response.Fil: Chang, Eileen I.. University of Florida; Estados UnidosFil: Zárate, Miguel A.. University of Florida; Estados UnidosFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Provincia de Córdoba. Ministerio de Ciencia y Técnica. Centro de Excelencia en Productos y Procesos de Córdoba; ArgentinaFil: Richards, Elaine M.. University of Florida; Estados UnidosFil: Arndt, Thomas J.. University of Florida; Estados UnidosFil: Keller Wood, Maureen. University of Florida; Estados UnidosFil: Wood, Charles E.. University of Florida; Estados Unido

Modulatory effects of ghrelin on sperm quality alterations induced by a fructose-enriched diet

The objectives of this study were: 1) to evaluate the effects of a fructose enriched diet (FED) on rat3 sperm quality, epididymal function (i.e. oxidative stress and alpha-glucosidase expression) and4 testosterone concentrations; 2) to determine if the administration of ghrelin (Ghrl), reverses the5 effects induced by FED.6 After validating the protocol as an inductor of metabolic syndrome like-symptoms, adult male rats7 were assigned to one of the following treatments for 8 weeks: FED=10% fructose enriched in water8 (v/v); FED+Ghrl=fructose enriched diet plus Ghrl (6 nmol/animal/day, s.c.) from week 6 to 8; or9 C=water without fructose (n=5-10 animals/group).10 FED significantly decreased sperm concentration and motile sperm count/ml vs C (FED:19.0±1.6x106sperm/ml and 834.6±137.0, respectively vs C: 25.8±2.8x10611 and 1300.4±202.4,respectively; p<0.05); ghrelin injection reversed this negative effect (23.5±1.6x10612 sperm/ml and13 1381.7±71.3 respectively). FED resulted in hypogonadism, but Ghrl could not normalize testosterone14 concentrations (C: 1.4±0.1 ng/ml vs FED: 0.8±0.2 ng/ml and FED+Ghrl: 0.6±0.2 ng/ml ;p<0.05).15 Ghrelin did not reverse metabolic abnormalities secondary to FED. FED did not alter epididymal16 expression of antioxidants enzymes (superoxido-dismutase, catalase and glutathione peroxidases ?17 Gpx-). Nevertheless, FED+Ghrl significantly increased the expression of Gpx3 (FED+Ghrl:18 3.47±0.48 vs FED: 0.69±0.28 and C: 1.00±0.14; p<0.05). The expression of neutral alpha19 glucosidase, which is a marker of epididymal function, did not differ between treatments.20 In conclusion, the administration of Ghrl modulated the negative effects of FED on sperm quality,21 possibly by an epididymal increase in Gpx3 expression. However, Ghrl could not neither normalize22 the metabolism of FED animals, nor reverse hypogonadism.Fil: Ramirez, Nicolás David. Universidad Nacional de Córdoba. Facultad de Medicina. Departamento de Fisiología Humana y Física Biomédica. Cátedra de Fisiología Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Luque, Eugenia Mercedes. Universidad Nacional de Córdoba. Facultad de Medicina. Departamento de Fisiología Humana y Física Biomédica. Cátedra de Fisiología Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Jones, Xaviar Michael. Universidad Nacional de Córdoba. Facultad de Medicina. Departamento de Fisiología Humana y Física Biomédica. Cátedra de Fisiología Humana; ArgentinaFil: Torres, Pedro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Moreira Espinoza, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Cantarelli, Verónica Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Ponzio, Marina Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Arja, Ana. Universidad Nacional de Córdoba. Facultad de Medicina. Departamento de Fisiología Humana y Física Biomédica. Cátedra de Fisiología Humana; ArgentinaFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Martini, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentin

Machine-learning methods applied to integrated transcriptomic data from bovine blastocysts and elongating conceptuses to identify genes predictive of embryonic competence

Early pregnancy loss markedly impacts reproductive efficiency in cattle. The objectives were to model a biologically relevant gene signature predicting embryonic competence for survival after integrating transcriptomic data from blastocysts and elongating conceptuses with different developmental capacities and to validate the potential biomarkers with independent embryonic data sets through the application of machine-learning algorithms. First, two data sets from in vivo-produced blastocysts competent or not to sustain a pregnancy were integrated with a data set from long and short day-15 conceptuses. A statistical contrast determined differentially expressed genes (DEG) increasing in expression from a competent blastocyst to a long conceptus and vice versa; these were enriched for KEGG pathways related to glycolysis/gluconeogenesis and RNA processing, respectively. Next, the most discriminative DEG between blastocysts that resulted or did not in pregnancy were selected by linear discriminant analysis. These eight putative biomarker genes were validated by modeling their expression in competent or noncompetent blastocysts through Bayesian logistic regression or neural networks and predicting embryo developmental fate in four external data sets consisting of in vitro-produced blastocysts (i) competent or not, or (ii) exposed or not to detrimental conditions during culture, and elongated conceptuses (iii) of different length, or (iv) developed in the uteri of high- or subfertile heifers. Predictions for each data set were more than 85% accurate, suggesting that these genes play a key role in embryo development and pregnancy establishment. In conclusion, this study integrated transcriptomic data from seven independent experiments to identify a small set of genes capable of predicting embryonic competence for survival

Cholecystokinin, gastrin, cholecystokinin/gastrin receptors, and bitter taste receptor TAS2R14: trophoblast expression and signaling

We investigated expression of cholecystokinin (CCK) in humans and mice, and the bitter taste receptor TAS2R14 in the human placenta. Because CCK and gastrin activate the CCKBR receptor, we also explored placental gastrin expression. Finally, we investigated calcium signaling by CCK and TAS2R14. By RT-PCR, we found CCK/Cck and GAST/Gast mRNA expression in both normal human and mouse placentas, as well as in human trophoblast cell lines (TCL). Although both Cckar and -br mRNA were expressed in the mouse placenta, only CCKBR mRNA was detected in the human placenta and TCL. mRNA expression for TAS2R14 was also observed in the human placenta and TCL. Using immunohistochemistry, CCK protein was localized to the syncytiotrophoblast (ST) and extravillous trophoblast (EVT) in the human term placenta, and to trophoblast glycogen cells in mouse and human placentas. Gastrin and TAS2R14 proteins were also observed in ST and EVT of the human placenta. Both sulfated and nonsulfated CCK elicited a comparable rise in intracellular calcium in TCL, consistent with CCKBR expression. Three TAS2R14 agonists, flufenamic acid, chlorhexidine, and diphenhydramine, also evoked rises in intracellular calcium in TCL. These results establish CCK, gastrin, and their receptor(s) in both human and mouse placentas, and TAS2R14 in the human placenta. Both CCK and TAS2R14 agonists increased intracellular calcium in human TCL. Although the roles of these ligands and receptors, and their potential cross talk in normal and pathological placentas, are currently unknown, this study opens new avenues for placental research.Fil: Taher, Shèdy. University of Florida; Estados UnidosFil: Borja, Yamilette. University of Florida; Estados UnidosFil: Cabanela, Lucía. University of Florida; Estados UnidosFil: Costers, Vincent J.. University of Florida; Estados UnidosFil: Carson Marino, Morgan. University of Florida; Estados UnidosFil: Bailes, Julie C.. University of Florida; Estados UnidosFil: Dhar, Biswadeep. University of Florida; Estados UnidosFil: Beckworth, Mark T.. University of Florida; Estados UnidosFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Post Uiterweer, Emiel D.. University Medical Center Utrecht; Países BajosFil: Conrad, Kirk P.. University of Florida; Estados Unido