Maternal Oxycodone Treatment Results in Neurobehavioral Disruptions in Mice Offspring.

Opioid drugs are increasingly being prescribed to pregnant women. Such compounds can also bind and activate opioid receptors in the fetal brain, which could lead to long-term brain and behavioral disruptions. We hypothesized that maternal treatment with oxycodone (OXY), the primary opioid at the center of the current crisis, leads to later neurobehavioral disorders and gene expression changes in the hypothalamus and hippocampus of resulting offspring. Female mice were treated daily with 5 mg OXY/kg or saline solution (control; CTL) for two weeks before breeding and then throughout gestation. Male and female offspring from both groups were tested with a battery of behavioral and metabolic tests to measure cognition, exploratory-like, anxiety-like, voluntary physical activity, and socio-communication behaviors. qPCR analyses were performed for candidate gene expression patterns in the hypothalamus and hippocampus of OXY and CTL derived offspring. Developmental exposure to OXY caused socio-communication changes that persisted from weaning through adulthood. Such offspring also showed cognitive impairments, reduced voluntary physical activity, and weighed more than CTL counterparts. In the hippocampus, prenatal exposure to OXY caused sex-dependent differences in expression of genes encoding opioid receptors and those involved in serotonin signaling. OXY exposure induced changes in neuropeptide hormone expression and the epigenetic modulator, Dnmt3a, in the hypothalamus, which could result in epigenetic changes in this brain region. The findings suggest cause for concern that consumption of OXY by pregnant mothers may result in permanent neurobehavioral changes in their offspring. Further work is needed to determine the potential underpinning epigenetic mechanisms.

Disruption of global hypothalamic microRNA (miR) profiles and associated behavioral changes in California mice (Peromyscus californicus) developmentally exposed to endocrine disrupting chemicals.

Developmental exposure to endocrine disrupting chemicals (EDCs), e.g., bisphenol A (BPA) or genistein (GEN), causes longstanding epigenome effects. MicroRNAs (miRs) regulate which mRNAs will be translated to proteins and thereby serve as the final checkpoint in epigenetic control. Scant amount is known, however, whether EDCs affect neural miRNA (miR) patterns. We aimed to test the hypothesis that developmental exposure of California mice (Peromyscus californicus) to GEN, BPA, or both chemicals influences hypothalamic miR/small RNA profiles and ascertain the extent such biomolecular alterations correlate with behavioral and metabolic changes. California mice were developmentally exposed to GEN (250 mg/kg feed weight, FW), GEN (250 mg/kg FW)+BPA (5 mg/kg FW), low dose (LD) BPA (5 mg/kg FW), or upper dose (UD) BPA (50 mg/kg FW). Adult offspring were tested in a battery of behavioral and metabolic tests; whereupon, mice were euthanized, brains were collected and frozen, small RNAs were isolated from hypothalamic punches, and subsequently sequenced. California mice exposed to one or both EDCs engaged in one or more repetitive behaviors. GEN, LD BPA, and UD BPA altered aspects of ultrasonic and audible vocalizations. Each EDC exposure led to sex-dependent differences in differentially expressed miR/small RNAs with miR7-2, miR146, and miR148a being increased in all female and male EDC exposed groups. Current findings reveal that developmental exposure to GEN and/or BPA affects hypothalamic miR/small RNA expression patterns, and such changes correlate with EDC-induced behavioral and metabolic alterations. miR146 is likely an important mediator and biomarker of EDC exposure in mammals, including humans.

Endocrine disruption of gene expression and microRNA profiles in hippocampus and hypothalamus of California mice: Association of gene expression changes with behavioural outcomes.

The hypothalamus and hippocampus are sensitive to early exposure to endocrine disrupting chemicals (EDCs). Two EDCs that have raised particular concerns are bisphenol A (BPA), a widely prevalent chemical in many common household items, and genistein (GEN), a phyto-oestrogen present in soy and other plants. We hypothesised that early exposure to BPA or GEN may lead to permanent effects on gene expression profiles for both coding RNAs (mRNAs) and microRNAs (miRs), which can affect the translation of mRNAs. Such EDC-induced biomolecular changes may affect behavioural and metabolic patterns. California mice (Peromyscus californicus) male and female offspring were developmentally exposed via the maternal diet to BPA (5 mg kg-1 feed weight low dose [LD] and 50 mg kg-1 feed weight upper dose [UD]), GEN (250 mg kg-1 feed weight) or a phyto-oestrogen-free diet (AIN) control. Behavioural and metabolic tests were performed at 180 days of age. A quantitative polymerase chain reacttion analysis was performed for candidate mRNAs and miRs in the hypothalamus and hippocampus. LD BPA and GEN exposed California mice offspring showed socio-communication impairments. Hypothalamic Avp, Esr1, Kiss1 and Lepr were increased in LD BPA offspring. miR-153 was elevated but miR-181a was reduced in LD BPA offspring. miR-9 and miR-153 were increased in the hippocampi of LD BPA offspring, whereas GEN decreased hippocampal miR-7a and miR-153 expression. Correlation analyses revealed neural expression of miR-153 and miR-181a was associated with socio-communication deficits in LD BPA individuals. The findings reveal a cause for concern such that developmental exposure of BPA or GEN in California mice (and potentially by translation in humans) can lead to long standing neurobehavioural consequences.

Hypothalamic transcriptome of tame and aggressive silver foxes (Vulpes vulpes) identifies gene expression differences shared across brain regions.

The underlying neurological events accompanying dog domestication remain elusive. To reconstruct the domestication process in an experimental setting, silver foxes (Vulpes vulpes) have been deliberately bred for tame vs aggressive behaviors for more than 50 generations at the Institute for Cytology and Genetics in Novosibirsk, Russia. The hypothalamus is an essential part of the hypothalamic-pituitary-adrenal axis and regulates the fight-or-flight response, and thus, we hypothesized that selective breeding for tameness/aggressiveness has shaped the hypothalamic transcriptomic profile. RNA-seq analysis identified 70 differentially expressed genes (DEGs). Seven of these genes, DKKL1, FBLN7, NPL, PRIMPOL, PTGRN, SHCBP1L and SKIV2L, showed the same direction expression differences in the hypothalamus, basal forebrain and prefrontal cortex. The genes differentially expressed across the three tissues are involved in cell division, differentiation, adhesion and carbohydrate processing, suggesting an association of these processes with selective breeding. Additionally, 159 transcripts from the hypothalamus demonstrated differences in the abundance of alternative spliced forms between the tame and aggressive foxes. Weighted gene coexpression network analyses also suggested that gene modules in hypothalamus were significantly associated with tame vs aggressive behavior. Pathways associated with these modules include signal transduction, interleukin signaling, cytokine-cytokine receptor interaction and peptide ligand-binding receptors (eg, G-protein coupled receptor [GPCR] ligand binding). Current studies show the selection for tameness vs aggressiveness in foxes is associated with unique hypothalamic gene profiles partly shared with other brain regions and highlight DEGs involved in biological processes such as development, differentiation and immunological responses. The role of these processes in fox and dog domestication remains to be determined.

Early genistein exposure of California mice and effects on the gut microbiota-brain axis.

Human offspring encounter high amounts of phytoestrogens, such as genistein (GEN), through maternal diet and soy-based formulas. Such chemicals can exert estrogenic activity and thereby disrupt neurobehavioral programming. Besides inducing direct host effects, GEN might cause gut dysbiosis and alter gut metabolites. To determine whether exposure to GEN affects these parameters, California mice (Peromyscus californicus) dams were placed 2 weeks prior to breeding and throughout gestation and lactation on a diet supplemented with GEN (250 mg/kg feed weight) or AIN93G phytoestrogen-free control diet (AIN). At weaning, offspring socio-communicative behaviors, gut microbiota and metabolite profiles were assayed. Exposure of offspring to GEN-induced sex-dependent changes in gut microbiota and metabolites. GEN exposed females were less likely to investigate a novel female mouse when tested in a three-chamber social test. When isolated, GEN males and females exhibited increased latency to elicit their first call, suggestive of reduced motivation to communicate with other individuals. Correlation analyses revealed interactions between GEN-induced microbiome, metabolome and socio-communicative behaviors. Comparison of GEN males with AIN males revealed the fraction of calls above 20 kHz was associated with daidzein, α-tocopherol, Flexispira spp. and Odoribacter spp. Results suggest early GEN exposure disrupts normal socio-communicative behaviors in California mice, which are otherwise evident in these social rodents. Such effects may be due to GEN disruptions on neural programming but might also be attributed to GEN-induced microbiota shifts and resultant changes in gut metabolites. Findings indicate cause for concern that perinatal exposure to GEN may detrimentally affect the offspring microbiome-gut-brain axis.

Opposing effects of S-equol supplementation on metabolic and behavioral parameters in mice fed a high-fat diet.

Phytoestrogens, such as daidzein and genistein, may be used to treat various hormone-dependent disorders. Daidzein can be metabolized by intestinal microbes to S-equol. However, not all individuals possess bacteria producing this metabolite, resulting in categorization of equol vs nonequol producers. Past human and rodent studies have suggested that supplementation of this compound might yield beneficial metabolic and behavioral effects. We hypothesized that administration of S-equol to diet-induced obese male and female mice would mitigate potential diet-induced metabolic and comorbid neurobehavioral disorders. To test this possibility, we placed 5-week-old C57 mice on a high-fat diet (HFD) to mimic the diet currently consumed by many Western adults. Animals were randomly assigned to S-equol supplementation (10 mg/kg body weight) or vehicle control group. After 4 weeks on HFD with or without S-equol supplementation, metabolic and behavioral phenotyping was performed. Although the initial hypothesis proposed that S-equol treatment would improve metabolic and neurobehavioral outcomes, this supplementation instead exacerbated aspects of HFD-induced metabolic disease, as indicated by suppressed physical activity in treated individuals, reduced energy expenditure in treated males, and serum chemistry changes (hyperglycemia in treated individuals; hyperinsulinemia and hypoleptinemia in treated males). Conversely, S-equol individuals exhibited less anxiety-like and depressive-like behaviors, as evidenced by increased exploratory time in the elevated plus maze by treated males and increased time spent mobile in the tail suspension test for treated individuals. In summary, S-equol may be beneficial in mitigating depression and anxiety disorders in individuals, but for indeterminate reasons, supplementation may worsen facets of metabolic disorders in obese individuals.

Effects of different dietary n-6/n-3 polyunsaturated fatty acid ratios on boar reproduction.

BACKGROUND: N-3 and N-6 polyunsaturated fatty acids are widely used in reproduction, yet few studies have addressed the effects of dietary n-6/n-3 ratios on boar reproduction. The present study aimed to determine the effects of different dietary n-6/n-3 ratios on the reproductive performance of breeding boars. Thirty-two boars with body weights of 15.0 ± 1.4 kg were divided into four treatments (C, T1, T2, T3) and fed diets with different n-6/n-3 fatty acid ratios (29.06:1, 20.07:1, 1:1, 1:17.96, respectively) for 174 days. RESULTS: The highest testis index was observed for treatment T2. Sperm density and total sperm number per ejaculate in the T2 treatment were significantly higher than those in all other treatments, whereas the sperm deformity rate was the lowest. Interestingly, the fatty acid compositions and ratios of sperm were consistent with dietary treatments. Acid phosphatase and fructose concentration of seminal plasma, and the total superoxide dismutase and glutathione peroxidase of sperm in T2 were higher than those in other treatments. The concentration of testosterone and prostaglandin E2 increased in boars fed on diets supplemented with fatty acids as compared with boars subjected to the C group treatment, reaching a peak at n-6/n-3 fatty acid ratios of 1:1. Furthermore, higher expression of Δ(6)-fatty acid desaturase and peroxisome proliferator activated receptor-α in spermatozoa of the T2 treatment were observed, indicating more vigorous metabolism and intensive hormonal regulation. CONCLUSIONS: Our data suggest that the ideal n-6/n-3 ratio in the diet of breeding boars is 1:1, and proper balancing of n-6/n-3 fatty acids plays an important role in male reproduction.

Glycine supplementation in vitro enhances porcine preimplantation embryo cell number and decreases apoptosis but does not lead to live births.

Most in vitro culture conditions are less-than-optimal for embryo development. Here, we used a transcriptional-profiling database to identify culture-induced differences in gene expression in porcine blastocysts compared to in vivo-produced counterparts. Genes involved in glycine transport (SLC6A9), glycine metabolism (GLDC, GCSH, DLD, and AMT), and serine metabolism (PSAT1, PSPH, and PHGDH) were differentially expressed. Addition of 10 mM glycine to the culture medium (currently containing 0.1 mM) reduced the abundance of SLC6A9 transcript and increased total cell number, primarily in the trophectoderm lineage (P = 0.003); this was likely by decreasing the percentage of apoptotic nuclei. As serine and glycine can be reversibly metabolized by serine hydroxymethyltransferase 2 (SHMT2), we assessed the abundance of SHMT2 transcript as well as its functional role by inhibiting it with aminomethylphosphonic acid (AMPA), a glycine analog, during in vitro culture. Both AMPA supplementation and elevated glycine decreased the mRNA abundance of SHMT2 and tumor protein p53 (TP53), which is activated in response to cellular stress, compared to controls (P ≤ 0.02). On the other hand, mitochondrial activity of blastocysts, mtDNA copy number, and abundance of mitochondria-related transcripts did not differ between control and 10 mM glycine culture conditions. Despite improvements to these metrics of blastocyst quality, transfer of embryos cultured in 10 mM glycine did not result in pregnancy whereas the transfer of in vitro-produced embryos cultured in control medium yielded live births. Mol. Reprod. Dev. 83: 246-258, 2016. © 2016 The Authors.

Low phytoestrogen levels in feed increase fetal serum estradiol resulting in the "fetal estrogenization syndrome" and obesity in CD-1 mice.

BACKGROUND: Although estrogenic chemicals can disrupt development of the reproductive system, there is debate about whether phytoestrogens in soy are beneficial, benign, or harmful. OBJECTIVES: We compared reproductive and metabolic characteristics in male and female mice reared and maintained on non-soy low-phytoestrogen feed or soy-based high-phytoestrogen feed. METHODS: The low-phytoestrogen diet was non-soy PMI 5K96 (verified casein diet), and the high-phytoestrogen diet consisted of soy-based PMI 5008 during pregnancy and lactation and soy-based PMI 5001 maintenance feed after weaning. RESULTS: In fetuses whose mothers consumed the low-phytoestrogen PMI 5K96 feed, we found a paradoxical significant elevation in endogenous serum estradiol, which was associated postnatally with adverse reproductive outcomes referred to as the "fetal estrogenization syndrome (FES)". In females, this syndrome included early puberty and increased uterine responsiveness to estrogen, and in males, it included reduced testis, epididymis, and seminal vesicle size, but an enlarged prostate. The low-phytoestrogen-fed males and females were lighter at birth, but, between weaning and adulthood, they became obese and developed abnormally high serum leptin levels; these males, but not females, showed impaired glucose regulation. CONCLUSIONS: Removing phytoestrogens from mouse feed produces an obese phenotype consistent with metabolic syndrome, and the associated reproductive system abnormalities are consistent with FES due to elevated endogenous fetal estradiol. Laboratory rodents may have become adapted to high-phytoestrogen intake over many generations of being fed soy-based commercial feed; removing all phytoestrogens from feed leads to alterations that could disrupt many types of biomedical research.

Effects of diets enriched in omega-3 and omega-6 polyunsaturated fatty acids on offspring sex-ratio and maternal behavior in mice.

There have been many trials describing the effects of polyunsaturated fatty acids (PUFA) on fecundity, neonatal development, and maternal behavior in humans, but few controlled studies in rodents. We examined the effects of a maternal diet high in omega 3 (N-3) or omega 6 (N-6) PUFA on NIH Swiss mice. Female mice were ad libitum fed one of three complete and balanced diets (N-3, enriched in menhaden oil; N-6, enriched in corn oil; C, control diet, Purina 5015) from age 4 wk until the end of the study. Mice were bred at approximately 19 wk and 27 wk of age, providing a total of 838 pups from 129 litters in two experiments. After weaning their pups from parity 1, behavior of dams was assessed on elevated-plus and open-field mazes. Although the fraction of male pups from the N-3 and C groups was not different from 0.5, dams on the N-6 diet birthed more daughters than sons (213 vs. 133; P < 0.001). Although maternal stress has been reported to favor birth of daughters, the behavior of N-6 dams was not different from controls. By contrast, the N-3 dams displayed greater anxiety, spending less time in the open arms and more time in the closed arms of the elevated maze and traveling less distance and exhibiting less exploratory behavior in the open field (P < 0.05). N-3 dams tended to produce smaller litters than C dams, and N-3-suckled pups gained less weight (P < 0.05). In conclusion, the N-3 diet had negative effects on murine fecundity and maternal behavior, whereas the N-6 diet favored birth of daughters.