Ecosystems adapt differently to global warming through microclimatic factors. Mires are sensitive wetland habitats that strongly rely on local soil properties, making them a good model to understand how local climatic parameters counteract the effects of climate change. We quantified the temperature buffering effect in waterlogged mire soils as compared with adjacent dry soils. We buried dataloggers at 5 cm depth in waterlogged and dry points in eight mires of the Cantabrian Mountains (Spain, southwestern Europe) and recorded soil temperatures for ca. 5 years. We also compared our local measures with air temperatures predicted by the CHELSA model. Waterlogged soils had less diurnal thermal amplitude (-2.3 degrees C), less annual thermal amplitude (-5.1 degrees C), cooler summer maxima (-4.3 degrees C) and warmer winter minima (+0.8 degrees C). CHELSA air temperatures only correlated significantly (p < .05) with winter minimum soil temperatures (Pearson's r > .83), and CHELSA predictions were less accurate (higher root-mean-square error [RMSE]) for waterlogged soils, except for the summer maxima. We conclude that mire soils show a thermal buffer effect that insulates them from the surrounding landscape. This effect is stronger at the warm end of the climatic spectrum, that is, during summer and at lower elevations. These results highlight the potential refugial character of mires under global warming, and the need to integrate microclimate measurements into climate change models.

Introduction: The use of flavors in electronic cigarettes appeals to adults and never-smoking youth. Consumption has rapidly increased over the last decade, and in the U.S. market alone, there are over 8000 unique flavors. The U.S. Food and Drug Administration (FDA) has begun to regulate e-liquids, but many have not been tested, and their impact, both at the cellular level, and on human health remains unclear. Methods: We tested e-liquids on the human cell line HEK293T and measured toxicity, mitochondria! membrane potential (Delta Psi(m)), reactive oxygen species production (ROS), and cellular membrane potential (V-m) using high-throughput screening (HTS) approaches. Our HTS efforts included single-dose and 16-point dose-response curves, which allowed testing of AO commercially available e-liquids in parallel to provide a rapid assessment of cellular effects as a proof of concept for a fast, preliminary toxicity method. We also investigated the chemical composition of the flavors via gas chromatography-mass spectrometry. Results: We found that e-liquids caused a decrease in Delta Psi(m) and V-m and an increase in ROS production and toxicity in a dose-dependent fashion. In addition, the presence of five specific chemical components: vanillin, benzyl alcohol, acetoin, cinnamaldehyde, and methyl-cyclopentenolone, but not nicotine, were linked with the changes observed in the cellular traits studied. Conclusion: Our data suggest that Delta Psi(m), ROS, V-m, and toxicity may be indicative of the extent of cell death upon e-liquid exposure. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.

Endometrial cell co-culture (ECC) with single embryo may reflect endometrium responses in vivo. Bovine Day-6 in vitro-produced morulae were cultured until Day-8 in modified synthetic oviductal fluid (mSOF), or on the epithelial side of ECC. Expression of epithelial- and stromal-cell transcripts was analyzed by RT-PCR in ECC with one male (ME) or female embryo (FE). Concentrations of ARTEMIN (ARTN) and total protein were determined in epithelial cell-conditioned medium. ECCs yielded embryos with more cells in the inner cell mass than embryos cultured in mSOF. Embryos altered transcript expression only in epithelial cells, not in stromal ones. Thus, ME induced larger reductions than FE and controls (i.e., no embryos cultured) in hexose transporter solute carrier family 2 member 1 (SLC2A1) and member 5 (SLC2A5), connective tissue growth factor (CTGF), artemin (ARTN), and interferon alpha and beta receptors subunit IFNAR1 and IFNAR2. FE reduced SLC2A1 and SLC2A5, and increased ARTN expression with respect to controls. ME tended to reduce total protein concentration (P < 0.082) in ECC-conditioned medium, while ARTN protein and gene expressions strongly correlated (R > 0.90; P < 0.05) in the group of ME or FE, but not in controls (without embryo). Isolated male and female embryos may differentially release signaling factors that induce sexually dimorphic responses in endometrial cells.

The interleukin-1 (IL1) system likely mediates mammalian embryo maternal communication. In cattle, we have reported that the uterine fluid of heifers carrying early embryos shows downregulated IL1 beta (IL1B), which could lead to reduced NFkB expression and dampening of maternal innate immune responses. In this work, we assessed the expression of IL 1 beta (IL1B) and its receptor, interleukin 1 receptor type I (IL1R1) in the bovine endometrium and embryos by RT-PCR, immunohistochemistry and Western blot at the time of blastocyst development. Day 8 endometrium, both collected from animals after transfer of day 5 embryos (ET) and sham transferred (ST), showed IL1B and IL1R1 mRNA transcription and protein co-localization. Similarly, day 8 blastocyst, from ET animals and entirely produced in vitro, showed IL1R1 mRNA transcription and IL1B and IL1R1 protein co-localization. IL1B mRNA was detected in the analyzed blastocysts, but at very low levels that precluded its quantification. IL1B and IL1R1 immunostaining was observed in luminal epithelial cells, glandular epithelium and stromal cells. The presence of embryos increased endometrial IL1B protein locally, while no differences regarding IL1R1 protein and IL1B and IL1R1 mRNA were detected. These results suggest that the early preimplantation bovine embryo in the maternal tract might interact with the maternal immune system through the IL1 system. Such a mechanism may allow the embryo to elicit local endometrial responses at early stages, which are required for the development of a receptive endometrium. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

The objective of this work was to determine whether metabolic fingerprinting of spent bovine embryo culture media using Fourier transform infrared spectroscopy (FTIR) correlates with embryonic sex. Embryos were produced in vitro from oocytes collected from cows slaughtered in an abattoir. Day-6 embryos were individually cultured in synthetic oviduct fluid for 24 h, prior to the time (Day-7) intended for embryo transfer or cryopreservation. Culture medium was analyzed by FTIR. Embryos were sexed by a PCR procedure based on amelogenin gene amplification or transferred to a recipient and sex observed at birth. Media samples from embryos diagnosed as male (n = 47) or female (n = 70) were individually collected and evaluated using FTIR. The spectra obtained were analyzed according to metabolomic profile of embryo culture media and embryonic sex. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (female), sensitivity (male) and area under the ROC curve (AUC). Performance of sex prediction (%) was high within early blastocysts + blastocysts (74.4 ± 10.2, accuracy; 0.749 ± 0.099, AUC) and excellent for expanded blastocysts (86.0 ± 12.6, accuracy; 0.898 ± 0.094, AUC). A combination of metabolomic and bioinformatic analysis provides a non-invasive mean of embryonic sex analysis.

Tumor necrosis factor (TNF) alpha likely mediates embryomaternal communication in mammals. In bovine, we have previously found that the uterine fluid of heifers that carried early embryos shows downregulation in the TNF and nuclear factor κB system. In this work, we assessed the expression of TNF and its receptor TNFR2 in the bovine endometrium and embryos during blastocyst development. Moreover, to explore the endometrial immune response to early embryos, we analyzed the number of CD45 leukocytes in the bovine endometrium. Day 8 endometrium and blastocyst recovered from animals after transfer of Day 5 embryos showed TNF and TNFR2 mRNA transcription and protein colocalization. The presence of embryos increased endometrial TNF and TNFR2 protein, whereas endometrial leukocytes decreased. Blastocysts exposed to the uterine tract had undetectable levels of TNF and lower levels of TNFR2 mRNA. These results suggest that the endometrium might lower the TNF concentration in the blastocyst by (1) regulating TNF secretion into the uterine fluid and (2) inducing decreased TNF and TNFR2 mRNA transcription in the embryo. Thus, TNF and TNFR2 might participate in early embryomaternal communication.

Asymmetry in the cow affects ovarian function and pregnancy. In this work we studied ovarian and uterine asymmetry. Synchronised animals, in which in vitro-produced embryos (n=30-60) had been transferred on Day 5 to the uterine horn ipsilateral to the corpus luteum (CL), were flushed on Day 8. Ovulatory follicle diameter, oestrus response and total protein flushed did not differ between sides. However, a corpus luteum in the right ovary led to plasma progesterone concentrations that were higher than when it was present in the left ovary. Fewer embryos were recovered from the left than the right horn. Among 60 uterine proteins identified by difference gel electrophoresis, relative abundance of nine (acyl-CoA dehydrogenase, very long chain; twinfilin, actin-binding protein, homologue 1; enolase 1; pyruvate kinase isozymes M1/M2 (rabbit); complement factor B Bb fragment ; albumin; fibrinogen gamma-B chain; and ezrin differed (P<0.05) between horns. Glucose concentration was higher, and fructose concentration lower, in the left horn. In a subsequent field trial, pregnancy rates after embryo transfer did not differ between horns (51.0±3.6, right vs 53.2±4.7, left). However, Day 7 blood progesterone concentrations differed (P=0.018) between pregnant and open animals in the left (15.9±1.7 vs 8.3±1.2) but not in the right horn (12.4±1.3 vs 12.4±1.2). Progesterone effects were independent of CL quality (P=0.55). Bilateral genital tract asymmetry in the cow affects progesterone, proteins and hexoses without altering pregnancy rates.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (FranceAUC=0.751±0.039, SpainAUC=0.718±0.024), while cumulative data decreased the AUC (AUC=0.604 ± 0.029). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: FranceAUC=0.690±0.044; SpainAUC<0.55; cumulativeAUC=0.747±0.032). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET.

Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes.