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 (< 60 to > 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.

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.

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.

Transcriptomic profiling of the bovine endosalpinx and endometrium to identify putative embryokines.

The objectives of the present study were to characterize the expression of genes encoding for cell signaling ligands in the bovine endosalpinx and endometrium and analyze spatial changes in gene expression. RNA sequencing was performed for the endosalpinx from the ampulla of the oviduct and endometrium from the upper and middle uterine horn and uterine body at day 2 after ovulation from ipsilateral and contralateral sides relative to the ovulatory ovary. Of the 17,827 unique mRNA transcripts mapped, 2,072 were affected by cranial-caudal position in the reproductive tract and 818 were affected by side (false discovery rate < 0.05). There were 334 genes encoding for cell signaling ligands, with 128 genes having greater than two transcripts per million on average. A total of 81 cell signaling ligand genes were affected by position and 24 were affected by side. A data set of the transcriptome of two to four cell embryos was used to identify cell signaling ligand genes that were highly expressed in the ampulla for which there was high expression of the receptor in the embryo. The most expressed ligand-receptor pairs were PSAP/SORT1, MIF/CXCR4, GPI/AMFR, and KITLG/KIT. These cell signaling ligands, as well as others whose gene is expressed in the endosalpinx and endometrium, may influence early embryonic development. Spatial changes throughout the reproductive tract highlight the distinctive expression profile of the oviduct versus the endometrium, including a set of the identified genes encoding for cell signaling ligands, and highlight the local influence of the ovary. The results also show the continuity of expression for large numbers of genes in the reproductive tract.NEW & NOTEWORTHY Examination of the transcriptome of the endosalpinx and endometrium revealed the degree to which gene expression in the reproductive tract varies spatially. The expression of genes encoding cell signaling molecules that could potentially regulate embryonic development was also identified.

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.

Transfer of bovine embryos into a uterus primed with high progesterone concentrations positively impacts fetal development at 42 days of gestation.

Elevated circulating progesterone (P4) concentrations in the first week after conception have been associated with accelerated post-hatching conceptus elongation. However, the consequences, if any, on the development of the fetus are unknown. The objective of this study was to determine the relationship between early circulating P4 and fetal and placental morphometric characteristics at 42 days of gestation. A previously validated model of asynchronous embryo transfer (ET), known to alter uterine exposure to P4, was used in 107 heifers divided in two replicates (replicate 1: n = 51, replicate 2: n = 56). Heifers were randomly assigned to one of the two following groups: those receiving a Day 7 embryo on Day 7 of the cycle (synchronous; ET_D7, n = 49) and those transferred a Day 7 embryo on Day 9 of the cycle (asynchronous; ET_D9, n = 58). The synchronization protocol was started two days earlier for heifers in the ET_D9 group such that ET was done on the same day for both groups. P4 concentrations were determined from Day 3 after estrus to the day of ET. Pregnant heifers were slaughtered at Day 42 of gestation for fetal and placental morphometric measurements. The effects of the group, replicate, fetal sex, and interactions between these variables on fetal and placental characteristics were determined by ANOVA, while Pearson correlation was employed to assess the linear relationship between P4 concentrations two days before and on the day of ET on the fetal parameters. The uteri of heifers in the ET_D9 group were exposed to higher concentrations (P < 0.0001) of P4 from four days before ET, than heifers in the ET_D7 group. Both group and fetal sex variables impacted on fetal crown-rump length (CRL) (group: P < 0.0001, sex: P = 0.001) and fetal weight (group: P = 0.006, sex: P = 0.003). Fetal sex influenced the amniotic sac area (P = 0.003) and amniotic sac weight (P = 0.004); while the group affected the number of cotyledons (P = 0.0009), and the fetal heart weight (P = 0.018). All these parameters were larger in the ET_D9 group compared with ET_D7, and in males compared with females. There was a positive correlation between P4 concentrations two days before ET and fetal weight and CRL, for each sex or considering all fetuses (R2 ∼0.4, p < 0.05). In conclusion, bovine embryos transferred into a uterus primed with higher P4 concentrations underwent enhanced development reflected in higher weight and size at the beginning of the fetal period.

Placental vascularization in middle and late gestation in the pig.

The aims of this study were to determine the changes in the capillary area density in relation to fetal development, to determine immunoexpression of angiogenic factors and to compare their mRNA expression throughout pig gestation. Samples were collected from the maternal-chorioallantoic interface at days 40, 77, 85 and 114 of pregnancy for immunohistochemistry analysis and the measurement of mRNA expression of VEGFA, ANGPT1, ANGPT2, FGF2 and its receptors KDR, TEK, FGFR1, FGFR2respectively. Morphometric measurement of blood vessels was performed. We found a significant increase in capillary area density throughout gestation (P< 0.05). On the maternal side, at day 77, we observed a significant increase in the number of vessels from small vascular areas (P < 0.05) and the vascular area was significantly higher on day 85 (P < 0.05). On the fetal side, the number of vessels and the vascular area increased between days 40 and 77 (P < 0.05) and between days 77 and 114 (P < 0.05), respectively. Immunohistochemical findings revealed intense VEGFA staining and a trend for increased expression towards the end of gestation (P < 0.05). We also demonstrated a high VEGFA, FGF2, FGFR1, ANGPT1 and ANGPT2mRNA expression at day 77 (P < 0.05). In conclusion, our findings suggest that an active angiogenesis would be present even until late-middle gestation at day 77 of pregnancy with the predominance of angiogenic stimulation by VEGFA/KDR, FGF2/FGFR1 and a balance between ANGPT1 and ANGPT2/TEK. Lay summary: Critical moments occur at different stages of placental formation in pigs, where the expression of angiogenic factors, that is, molecules that stimulate the formation of blood vessels must be adequate to promote their development. This exchange is necessary to cover the increasing nutritional demands of fetuses in continuous development. Determining the changes in the area of capillary density in relation to fetal development and the expression of angiogenic factors throughout pregnancy in pigs could contribute to understanding the causes of fetal loss. Placental samples were obtained at gestational days 40, 77, 85 and 114 (n = 7, 10, 7 and 5, respectively). We found that the capillary area density increases accompanying fetal growth with advancing gestation and an increase in capillary area density in late-middle gestation, around day 77, is due to the expansion in the number of small blood vessels on the maternal side. The present findings suggest that an intense angiogenesis would be present even until late-middle gestation at day 77 of pregnancy, with the predominance of angiogenic stimulation by specific molecules that promote this process.

Effect of age on the vascular proteome in middle cerebral arteries and mesenteric resistance arteries in mice.

Aging is associated with hypertension and brain blood flow dysregulation, which are major risk factors for cardiovascular and neurodegenerative diseases. Structural remodeling, endothelial dysfunction, or hypercontractility of resistance vessels may cause increased total peripheral resistance and hypertension. Recent studies showed that G protein- and RhoA/Rho-kinase pathways are involved in increased mean arterial pressure (MAP) and arterial tone in middle-aged mice. We aimed to characterize the age-dependent changes in the vascular proteome in normal laboratory mice using mass spectrometry and bioinformatics analyses on middle cerebral arteries and mesenteric resistance arteries from young (3 months) vs. middle-aged (14 months) mice. In total, 31 proteins were significantly affected by age whereas 172 proteins were differentially expressed by vessel type. Hierarchical clustering revealed that 207 proteins were significantly changed or clustered by age. Vitamin B6 pathway, Biosynthesis of antibiotics, Regulation of actin cytoskeleton and Endocytosis were the top enriched KEGG pathways by age. Several proteins in the RhoA/Rho-kinase pathway changed in a manner consistent with hypertension and dysregulation of cerebral perfusion. Although aging had a less profound effect than vessel type on the resistance artery proteome, regulation of actin cytoskeleton, including the RhoA/Rho-kinase pathway, is an important target for age-dependent hypertension.

Maternal intake of alpha-lipoic acid prevents development of symptoms associated with a fructose-rich diet in the male offspring in Wistar rats.

The hypothesis was that maternal intake of the antioxidant alpha-lipoid acid (ALA), during the developmental period of the hypothalamic orexigenic neurons, causes a permanent beneficial effect in offspring metabolism. Pregnant Wistar rats were fed with standard diet (food) + ALA (0.4% wt/wt) from day 14 of gestation to day 20 of lactation (n = 4) or food (n = 4). At 3 months of age, male offspring born from ALA-fed rats or controls (CT) were randomly assigned to be fed with food + 10% fructose solution in drinking water (F) or food + tap water (C), resulting in four groups: ALAF, ALAC, CTF, and CTC (n = 5/group). Food intake and body weight (BW) were measured twice a week for 31 days. Metabolites' levels in blood, mRNA expressions of Npy, Agrp (hypothalamus), Fasn, Srebf1, Ppard, and Pparg (liver), and the antioxidant capacity of the liver were determined. Results significance was set at p < 0.05. Average BW gain, daily BW gain, and intraabdominal fat tissue at necropsy were higher in CTF group followed by CTC, ALAF, and ALAC groups. There were no differences between groups in Kcal intake per day. mRNA expressions of hypothalamic and hepatic genes and plasmatic levels of glucose and triglycerides were higher in CTF group followed by ALAF, CTC, and ALAC groups. Fructose intake affected the oxidative capacity of the liver, but this effect was not observed in the ALAF group. In conclusion, maternal ALA intake protected the adult offspring to develop metabolic symptoms associated with high fructose in the drinking water.

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 rat sperm quality, epididymal function (i.e. oxidative stress and alpha-glucosidase expression) and testosterone concentrations; 2) to determine if the administration of ghrelin (Ghrl), reverses the effects induced by FED. After validating the protocol as an inductor of metabolic syndrome like-symptoms, adult male rats were assigned to one of the following treatments for 8 weeks: FED = 10% fructose enriched in water (v/v); FED + Ghrl = fructose enriched diet plus Ghrl (6 nmol/animal/day, s.c.) from week 6-8; or C = water without fructose (n = 5-10 animals/group). FED significantly decreased sperm concentration and motile sperm count/ml vs C (FED: 19.0 ± 1.6 × 106sperm/ml and 834.6 ± 137.0, respectively vs C: 25.8 ± 2.8 × 106 and 1300.4 ± 202.4, respectively; p < 0.05); ghrelin injection reversed this negative effect (23.5 ± 1.6 × 106sperm/ml and 1381.7 ± 71.3 respectively). FED resulted in hypogonadism, but Ghrl could not normalize testosterone 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). Ghrelin did not reverse metabolic abnormalities secondary to FED. FED did not alter epididymal expression of antioxidants enzymes (superoxido-dismutase, catalase and glutathione peroxidases -Gpx-). Nevertheless, FED + Ghrl significantly increased the expression of Gpx3 (FED + Ghrl: 3.47 ± 0.48 vs FED: 0.69 ± 0.28 and C: 1.00 ± 0.14; p < 0.05). The expression of neutral alpha-glucosidase, which is a marker of epididymal function, did not differ between treatments. In conclusion, the administration of Ghrl modulated the negative effects of FED on sperm quality, possibly by an epididymal increase in Gpx3 expression. However, Ghrl could not neither normalize the metabolism of FED animals, nor reverse hypogonadism.

Dickkopf-related protein 1 is a progestomedin acting on the bovine embryo during the morula-to-blastocyst transition to program trophoblast elongation.

Progesterone regulates the endometrium to support pregnancy establishment and maintenance. In the ruminant, one action of progesterone early in pregnancy is to alter embryonic development and hasten the process of trophoblast elongation around day 14-15 of pregnancy, which is required for maternal recognition of pregnancy. Here we demonstrate that the WNT antagonist DKK1, whose expression is increased by progesterone treatment, can act on the bovine embryo during day 5 to 7.5 of development (the morula to blastocyst stage) to promote embryonic elongation on day 15 of pregnancy. Embryos were produced in vitro and exposed to 0 or 100 ng/ml recombinant human DKK1 from day 5 to 7.5 of culture. Blastocysts were transferred into synchronized recipient cows on day 7.5 (n = 23 for control and 17 for DKK1). On day 15, cows were slaughtered and embryos recovered by flushing the uterus. Embryo recovery was n = 11 for controls (48% recovery) and n = 11 for DKK1 (65% recovery). Except for two DKK1 embryos, all embryos were filamentous. Treatment with DKK1 increased (P = 0.007) the length of filamentous embryos from 43.9 mm to 117.4 mm and the intrauterine content of the maternal recognition of pregnancy signal IFNT (P = 0.01) from 4.9 µg to 16.6 µg. Determination of differentially expressed genes (DEG), using the R environment, revealed 473 DEG at p < 0.05 but none at FDR < 0.05, suggesting that DKK1 did not strongly modify the embryo transcriptome at the time it was measured. However, samples clustered apart in a multidimensional scaling analyisis. Weighted gene co-expression analysis of the transcriptome of filamentous embryos revealed a subset of genes that were related to embryo length, with identification of a significant module of genes in the DKK1 group only. Thus, several of the differences between DKK1 and control groups in gene expression were due to differences in embryo length. In conclusion, DKK1 can act on the morula-to-blastocyst stage embryo to modify subsequent trophoblast elongation. Higher pregnancy rates associated with transfer of DKK1-treated embryos may be due in part to enhancements of trophoblast growth and antiluteolytic signaling through IFNT secretion. Given that progesterone can regulate both timing of trophoblast elongation and DKK1 expression, DKK1 may be a mediator of progesterone effects on embryonic development.

Evidence for shared molecular pathways of dysregulated decidualization in preeclampsia and endometrial disorders revealed by microarray data integration.

Microarray data of chorionic villous samples (CVSs) obtained from women of ∼11.5 gestational weeks who developed preeclampsia with severe features (sPE; PE-CVS) revealed a molecular signature of impaired endometrial maturation (decidualization) before and during early pregnancy. Because endometrial disorders are also associated with aberrant decidualization, we asked whether they share molecular features with sPE. We employed microarray data integration to compare the molecular pathologies of PE-CVS and endometrial disorders, as well as decidua obtained postpartum from women with sPE. Eight public databases were reanalyzed with R software to determine differentially expressed genes (DEGs) in pathologic tissues relative to normal controls. DEGs were then compared to explore overlap. Shared DEGs were examined for enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Principal component and network analyses were subsequently applied to selected DEGs. There was significant overlap of DEGs changing in the same direction for PE-CVS and endometrial disorders, suggesting common molecular pathways. Shared DEGs were enriched for cytokine-cytokine receptor interaction. Genes in this pathway revealed expression patterns forming 2 distinct clusters, one for normal and the other pathologic endometrium. The most affected hub genes were related to decidualization and NK cell function. Few DEGs were shared by PE-CVS, and PE decidua obtained postpartum. sPE may be part of a biologic continuum of "endometrial spectrum disorders."-Rabaglino, M. B., Conrad, K. P. Evidence for shared molecular pathways of dysregulated decidualization in preeclampsia and endometrial disorders revealed by microarray data integration.

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), 17ß-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.

Immunohistochemical determination of Ang-1, Ang-2 and Tie-2 in placentas of sows at 30, 60 and 114 days of gestation and validation through a bioinformatic approach.

Angiopoietins (Ang-1, Ang-2) participate in vascular development and placental growth, both bind to Tie-2. This study aimed to determine the localization of angiopoietins in placental development of sows by immunohistochemistry and to validate the gene expression during gestation through a bioinformatic approach. Samples were collected from fifteen maternal-fetal interface from approximately 30 (n = 5), 60 (n = 5) and 114 (n = 5) days of gestation for immunohistochemistry. A bioinformatic approach was performed by re-analysis of public datasets to determine the increase or decrease of genes involved in angiogenesis during pregnancy. There was no significant statistical difference of Ang-1 during gestation, although there was a tendency to increase from mid- to term-gestation (P = 0.7680). A notable decrease of Ang-2 was observed from early- to term-pregnancy (P ≤ 0.05), consistent with the gene expression determined through bioinformatics. Furthermore, there were greater abundances of Tie-2 at both early and at term periods, but lesser abundances at mid-gestation (P ≤ 0.05). The bioinformatics approach indicated that genes related to biological processes such as angiogenesis (i.e., development and morphogenesis of blood vessels) were expressed to a greater extent in early gestation as compared with later in gestation. The Ang-1 gene expression related to cell maturation, response to hypoxia and apoptosis, however, increased as gestation period advanced. In conclusion, angiopoietins may have an important role in the vascular development thus ensuring adequate placental growth in sows. The presence of angiopoietins in the trophoblast suggests a specific role for these pro-angiogenic factors in the tissue formation at the maternal-fetal interface.

Emerging role for dysregulated decidualization in the genesis of preeclampsia.

In normal human placentation, uterine invasion by trophoblast cells and subsequent spiral artery remodeling depend on cooperation among fetal trophoblasts and maternal decidual, myometrial, immune and vascular cells in the uterine wall. Therefore, aberrant function of anyone or several of these cell-types could theoretically impair placentation leading to the development of preeclampsia. Because trophoblast invasion and spiral artery remodeling occur during the first half of pregnancy, the molecular pathology of fetal placental and maternal decidual tissues following delivery may not be informative about the genesis of impaired placentation, which transpired months earlier. Therefore, in this review, we focus on the emerging prospective evidence supporting the concept that deficient or defective endometrial maturation in the late secretory phase and during early pregnancy, i.e., pre-decidualization and decidualization, respectively, may contribute to the genesis of preeclampsia. The first prospectively-acquired data directly supporting this concept were unexpectedly revealed in transcriptomic analyses of chorionic villous samples (CVS) obtained during the first trimester of women who developed preeclampsia 5 months later. Additional supportive evidence arose from investigations of Natural Killer cells in first trimester decidua from elective terminations of women with high resistance uterine artery indices, a surrogate for deficient trophoblast invasion. Last, circulating insulin growth factor binding protein-1, which is secreted by decidual stromal cells was decreased during early pregnancy in women who developed preeclampsia. We conclude this review by making recommendations for further prospectively-designed studies to corroborate the concept of endometrial antecedents of preeclampsia. These studies could also enable identification of women at increased risk for developing preeclampsia, unveil the molecular mechanisms of deficient or defective (pre)decidualization, and lead to preventative strategies designed to improve (pre)decidualization, thereby reducing risk for preeclampsia development.

Maternal Triclosan consumption alters the appetite regulatory network on Wistar rat offspring and predispose to metabolic syndrome in the adulthood.

The objectives of this study were to evaluate the effects of maternal oral exposure to the antibacterial Triclosan (TCS) during gestation and lactation on the metabolic status of the adult offspring and on the expression of main genes controlling the appetite regulatory network. Pregnant rats were fed ad-libitum with ground food + TCS (1 mg/kg) from day 14 of gestation to day 20 of lactation (n=3) or ground food (n=3). After litter reduction, 12 males and 12 females born from the TCS exposed rats (TCS, n=24) or not (Control, n=24) were used to evaluate monthly body weight, food intake, plasma levels of cholesterol, glucose and triglycerides, and the hypothalamic mRNA expression of agouti-related protein (Agrp), neuropeptide Y (Npy) and propiomelanocortin (Pomc). Body weight for rats in the TCS group was 12.5% heavier for males at 4 months (p<0.001) and 19% heavier for females at 8 months (p=0.01). Food intake was significantly higher for rats in the TCS group at 5 months of age (p<0.01). Cholesterol and glucose levels were significantly higher for rats in the TCS group at 8 months (p<0.05). mRNA expression of Npy and Agrp were significantly increased in hypothalami of rats in the TCS group at 2 months for males or 8 months for females (p<0.05). In conclusion, low doses of oral TCS consumption by the pregnant and lactating dam increase the hypothalamic expression of the orexigenic neuropeptides Npy and Agrp in the offspring and alter their metabolic status during adulthood, resembling development of the metabolic syndrome.

Genomic Effect of Triclosan on the Fetal Hypothalamus: Evidence for Altered Neuropeptide Regulation.

Triclosan (TCS), an antibacterial compound commonly added to personal care products, could be an endocrine disruptor at low doses. Although TCS has been shown to alter fetal physiology, its effects in the developing fetal brain are unknown. We hypothesize that exposure to TCS during fetal life could affect fetal hypothalamic gene expression. The objective of this study was to use transcriptomics and systems analysis to identify significantly altered biological processes in the late gestation ovine fetal hypothalamus after direct or indirect exposure to low doses of TCS. For direct TCS exposure, chronically catheterized late gestation fetal sheep were infused with vehicle (n = 4) or TCS (250 µg/d; n = 4) iv. For indirect TCS exposure, TCS (100 µg/kg · d; n = 3) or vehicle (n = 3) was infused into the maternal circulation. Fetal hypothalami were collected after 2 days of infusion, and gene expression was measured through microarray. Hierarchical clustering of all samples according to gene expression profiles showed that samples from the TCS-treated animals clustered apart from the controls. Gene set enrichment analysis revealed that fetal hypothalamic genes stimulated by maternal and fetal TCS infusion were significantly enriching for cell cycle, reproductive process, and feeding behavior, whereas the inhibited genes were significantly enriching for chromatin modification and metabolism of steroids, lipoproteins, fatty acids, and glucose (P < .05). In conclusion, short-term infusion of TCS induces vigorous changes in the fetal hypothalamic transcriptomics, which are mainly related to food intake pathways and metabolism. If these changes persist to postnatal life, they could result in adverse consequences in adulthood.

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 of protein phosphorylation, protein metabolism, and mitosis. Genes found to be at the center of the network of upregulated genes included genes important for purine binding and signaling. At the center of the downregulated network were genes involved in mRNA processing, DNA repair, sumoylation, and vesicular trafficking. Transcription factor analysis revealed that both up- and down-regulated gene sets are enriched for control by several transcription factors (e.g., SP1, MAZ, LEF1, NRF1, ELK1, NFAT, E12, PAX4) but not for HIF-1, which is known to be an important controller of genomic responses to hypoxia. 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.

Mechanisms for the adverse effects of late gestational increases in maternal cortisol on the heart revealed by transcriptomic analyses of the fetal septum.

We have previously shown in sheep that 10 days of modest chronic increase in maternal cortisol resulting from maternal infusion of cortisol (1 mg/kg/day) caused fetal heart enlargement and Purkinje cell apoptosis. In subsequent studies we extended the cortisol infusion to term, finding a dramatic incidence of stillbirth in the pregnancies with chronically increased cortisol. To investigate effects of maternal cortisol on the heart, we performed transcriptomic analyses on the septa using ovine microarrays and Webgestalt and Cytoscape programs for pathway inference. Analyses of the transcriptomic effects of maternal cortisol infusion for 10 days (130 day cortisol vs 130 day control), or ∼25 days (140 day cortisol vs 140 day control) and of normal maturation (140 day control vs 130 day control) were performed. Gene ontology terms related to immune function and cytokine actions were significantly overrepresented as genes altered by both cortisol and maturation in the septa. After 10 days of cortisol, growth factor and muscle cell apoptosis pathways were significantly overrepresented, consistent with our previous histologic findings. In the term fetuses (∼25 days of cortisol) nutrient pathways were significantly overrepresented, consistent with altered metabolism and reduced mitochondria. Analysis of mitochondrial number by mitochondrial DNA expression confirmed a significant decrease in mitochondria. The metabolic pathways modeled as altered by cortisol treatment to term were different from those modeled during maturation of the heart to term, and thus changes in gene expression in these metabolic pathways may be indicative of the fetal heart pathophysiologies seen in pregnancies complicated by stillbirth, including gestational diabetes, Cushing's disease and chronic stress.

Transcriptomics of the fetal hypothalamic response to brachiocephalic occlusion and estradiol treatment.

Estradiol (E2) is a well-known modulator of fetal neuroendocrine activity and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic artery occlusion (BCO) or sham occlusion. Half of the fetuses received subcutaneous pellets that increased plasma E2 concentrations within the physiological range. Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Analysis of the data by ANOVA revealed that E2 differentially regulated (DR) 561 genes, and BCO DR 894 genes compared with control and E2+BCO DR 1,153 genes compared with BCO alone (all P < 0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis but did not significantly involve genes known to directly respond to the estrogen receptor. Brachiocephalic occlusion upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain and downregulated neuropeptide synthesis. E2 upregulated immune- and apoptosis-related pathways after BCO and reduced kinase and epigenetic pathway responses to the BCO. Responses to BCO are different from responses to hypoxic hypoxia suggesting that mechanisms of responses to these two forms of brain hypoxia are distinct. We conclude that cerebral ischemia caused by BCO might stimulate lymphocyte infiltration into the brain and that this response appears to be modified by estradiol.