Gene by environment interaction mouse model reveals a functional role for 5-hydroxymethylcytosine in neurodevelopmental disorders

Mouse knockouts of Cntnap2 exhibit altered neurodevelopmental behavior, deficits in striatal GABAergic signaling and a genome-wide disruption of an environmentally sensitive DNA methylation modification (5-hydroxymethylcytosine, 5hmC) in the orthologs of a significant number of genes implicated in human neurodevelopmental disorders. We tested adult Cntnap2 heterozygous mice (Cntnap2+/-, lacking behavioral or neuropathological abnormalities) subjected to a prenatal stress and found that prenatally stressed Cntnap2+/- female mice showed repetitive behaviors and altered sociability, similar to the homozygote phenotype. Genomic profiling revealed disruptions in hippocampal and striatal 5hmC levels that were correlated to altered transcript levels of genes linked to these phenotypes (e.g., Reln, Dst, Trio, and Epha5). Chromatin-immunoprecipitation coupled with high-throughput sequencing and hippocampal nuclear lysate pull-down data indicated that 5hmC abundance alters the binding of the transcription factor CLOCK in the promoters of these genes (e.g., Palld, Gigyf1, and Fry), providing a mechanistic role for 5hmC in gene regulation. Together, these data support gene by environment hypotheses for the origins of mental illness and provide a means to identify the elusive factors contributing to complex human diseases.

Comparative Phosphoproteomic Profiling in Prefrontal Cortex of Prenatal Stressed Offspring Rats

Background: Many investigations indicate that prenatal stress caused depressive-like disturbances in offspring rats. The underlying pathogenic mechanisms have not yet been fully unravelled. The PFC has been shown to play a role in susceptibility to stress during fetal, thus we focus our attention on differential protein phosphorylation in this region of PS-S(Susceptibility to PS) offspring rats.Method: SPT was used to screen for susceptibility to PS. The validity of prenatally stressed model was verified by other common depression-like behaviors. We used MS-based TMT quantitative proteomics in combination with phosphopeptide enrichment method to compare phosphoproteomic profiling in prefrontal cortex of of PS-S and CON offspring rats. Results: Totally, 3418 phosphoproteins, 8404 phosphopeptides and 12175 phosphosites were identified in this analysis. According to the screening criteria, 902 phosphopeptides increased and 609 decreased in the PFC of PS-S group compared to the control rats. GO enrichment analysis indicated that the main enriched terms in CC category were ‘synapse part’, ‘myelin sheath’, ‘synapse’, ‘neuron part’ and ‘axon’. The phosphorproteins enriched in MF and BP category were mainly related to cytoskeleton and projection morphogenesis associated proteins. KEGG pathway enrichment analyses identified 30 significant KEGG pathways, the top five pathways included salivary secretion, Endocrine and other factor-regulated calcium reabsorption, Pancreatic secretion and Insulin secretion. Motifs such as......_S_P...RR, ......_S_PE...., ......_S_PV...., ......_S_P.H...and ..S..._S_PT....were the top five motifs enriched in phosphorylated sites.Conclusion: PS may induce depressive-like behaviors in offspring rats through regulating the phosphorylation of protein mainly related to synapse, myelin sheath, neuron and cytoskeleton. And the phosphorylation of related proteins may act as key pathogenic hits. Data are available via ProteomeXchange with identifier PXD026563.

Association of Melatonin Administration in Pregnant Ewes with Growth, Redox Status and Immunity of Their Offspring

In this study, the effects of melatonin treatment on growth, redox status and immunity in prenatally stressed newborn lambs were evaluated. Thirty-seven newborn lambs were allocated into two groups (melatonin-MEL and control-CON), based on whether their mothers were treated with melatonin implants or not, respectively. All pregnant ewes were exposed to heat stress. The body weight of lambs was recorded at birth (L0), and then on days 15 (L15) and 40 (L40). Redox biomarkers [total antioxidant capacity (TAC), glutathione (GSH), thiobarbituric acid reactive substances (TBARS)] were assayed in blood samples collected from lambs on days L0, L1, L2, L5, L10 and L40. Chemical analysis and antioxidant capacity were evaluated in colostrum and milk samples collected at the same time points with blood samples. Cytokines (IL-1β, IL-6, IL-10, IFN-γ) and immunoglobulin (IgG) were assayed in blood and colostrum samples collected from ewes on days L0 and L1, and in lambs’ blood on days L0, L1 and L2. The results revealed that body weight gain of newborn lambs did not differ between the two groups (p > 0.05). Better redox status was found in MEL lambs until L2, as well as higher antioxidant capacity in the colostrum of MEL ewes compared to CON ones on day L0 (p < 0.05). In MEL ewes’ colostrum, higher protein content was measured on day L0 and higher fat content on L1 compared to CON group (p < 0.05). The highest level of IL-6 was found in MEL ewes on L1, with a concomitant increase of IL-10 level in MEL lambs in comparison to CON lambs on L2. Moreover, CON colostrum resulted in a higher level of IL-10 within time, coupled with an increased level of IgG found in lambs’ plasma on L2 (p = 0.04). This study indicated that melatonin could be administered as antioxidant and immune-modulatory regime in prenatally stressed offspring in order to cope with the crucial first days of their life. This effect of melatonin was also amplified by crosstalk between IL-6, IL-10 and IgG production, resulting in an improved quality of produced milk.

282 Nutritional Monitoring of Prenatally Stressed and Translocated Brahman Heifers

One drought mitigation strategy is transporting livestock to non-drought locations. Our objective was to evaluate the effects of prenatal stress and translocation on growing Bos indicus heifers. Twelve heifers born in spring 2019 at Overton, TX (1245 mm annual precipitation) were transported ~700 km to Sonora, TX (610 mm annual precipitation) in April 2020. Six heifers (283±10 kg) were born to dams subjected to transportation stress during mid-gestation (PNS) and 6 (279±17 kg) were born to non-stressed dams (CON). Heifers grazed a series of 24-ha native range pastures (aboveground forage biomass; 1508±390 kg/ha) and were sampled (non-shrunk BW/BCS score, feces) at 2-wk intervals from May through September. Fecal samples were collected from the ground or the rectum of each animal and stored at -20o C until processed for near infrared spectroscopy (NIRS) and prediction of diet crude protein (CP) and digestible organic matter (DOM). Inputs to a grazing animal nutrition model for prediction of BW included diet CP and DOM, age, and weather. Differences between groups for BW and nutritional parameters were determined by analysis of variance or paired t-test. Both groups gained BW (22±4 kg) throughout the study, there were no differences (P &gt; 0.1) due to treatment. Diet CP and DOM were affected by date (P &lt; 0.01) as diet quality declined from spring to fall. Percent diet CP was greater (P &lt; 0.05) in PNS than CON, especially during July and August (6.94±0.10 vs 6.23±0.17, respectively). Corresponding values for diet DOM were (59.53±0.55 vs 59.14±0.43, respectively; P = 0.09). Observed vs model-predicted weight was different (P &lt; 0.05) for both groups when using CP-based outputs, but not when using metabolizable protein-based outputs (P &gt; 0.1). In summary, PNS heifers selected a diet of greater CP than CON, and tended to select a diet greater in DOM.

The biochemical estimation of the nitric oxide system in prenatally stressed rats

Introduction. Pregnancy development following unfavorable conditions could facilitate disorders of nitric oxide (NO) production during offspring’s postnatal life and «program» offspring’s cardiovascular diseases. Investigation of particular features and mechanisms of nitric oxide synthesis and action disorders following prenatal stress will promote expansion of considerations about pathogenesis of different cardiovascular diseases and propose new approaches to their prevention and management.The aim of the investigation is to assess the nature of nitric oxide synthesis and action in mature rats whose mothers were exposed to chronic «unpredictable» stress during pregnancy. Materials and methods. Pregnant rats were subdivided into the «control» and «stress» groups (in 20 animals). The rats from the «stress» group were exposed to multiple different stressors at various intervals, such as 1-day famine; 20-min. immobilization in the water at room temperature; 1-day contact with cats’ excrements. In the blood serum of 3-mo offspring (n=96, including «control» males – 24, «control» females – 26, «stress» males – 22, «stress» females – 24) concentration of the stable products of NO degradation – nitrates/nitrites (NO3–/NO2–), endothelial (eNOS) and inducible (iNOS) isoforms of the NO-synthase, inhibitor of NO-synthase asymmetric dimethylargininne (ADMA), cyclic guanosine monophosphate (cGMP), lipid peroxidation products – diene conjugates (DC) and malonic dialdehyde (MDA) and C-reactive protein (hsCRP) was detected. Results. The decrease of eNOS and cGMP concentration (by 12.9 and 31.9 %, respectively), increase of iNOS, hsCRP and ADMA concentration (by 49.9, 20.3 и 63.1 %, respectively) without statistically significant fluctuation in the NO3–/NO2– level and accumulation of DC and MDA by 21.1 % and 1.5 times in a prenatally stressed male rats’ blood serum were found (as compared with «control» male rats). In a blood serum of female rats, whose mothers were exposed to chronic «unpredictable» stress during pregnancy, a tendency to eNOS concentration decreasing, and increase of iNOS by 30.6 %, hsCRP by 23.9 % and MDA by 2.3 times without statistically significant changes in cGMP, ADMA, NO3–/NO2–, and DC concentration were detected (as compared with «control» female rats). Conclusion. Identified changes of the nitric oxide system synthesis and action in the prenatally stressed male rats could argue the high risk of their cardiovascular system lesion.

Correction of Free Radical Blood Imbalance and Behaviour in Prenatally Stressed Rats with α-Tocopherol

The research involved female mongrel white rats (n = 16) and their offspring (sexually mature males, n = 75). Pregnant females were divided into 4 groups: control (intact animals), stress (from the 16th to the 19th day of pregnancy females were subjected to 3-hour immobilization in plastic cases), stress+α-tocopherol (against the background of immobilization, females received α-tocopherol at a dose of 1 mg per 100 g of weight starting from the 2nd day of pregnancy), and α-tocopherol (females received α-tocopherol in the same dose and regimen as the group above). The control and stress groups received vegetable oil instead of α-tocopherol. In 3-month-old female offspring, we studied behaviour and determined the levels of products of free radical modification of proteins and lipids, total antioxidant activity as well as superoxide dismutase and ceruloplasmin activity in the blood plasma. Prenatal stress increased the exploratory and motor activity in offspring and modified the level of free radical homeostasis in the serum of male rats: there was an increase in the level of products of oxidative modification of proteins, thiobarbituric acid reactive substances concentration, end products of lipid peroxidation (Schiff bases), total antioxidant activity and ceruloplasmin activity, as well as a decrease in superoxide dismutase activity. Alpha-tocopherol taken by females during pregnancy brought all the oxidation parameters modified by immobilization to the values of the control group; the levels of primary products of oxidative modification of proteins and lipid peroxidation decreased two-fold, compared with the stress group. The activity of superoxide dismutase and ceruloplasmin returned to the control values, but the total antioxidant activity remained at the level of the stress group. Thus, there is reason to assume that α-tocopherol has a significant corrective effect on the intensity of free radical processes in prenatally stressed white male rats.