Prenatal maternal glucocorticoid exposure modifies sperm miRNA profiles across multiple generations in the guinea-pig.

Maternal stress and glucocorticoid exposure during pregnancy have multigenerational effects on neuroendocrine function and behaviours in offspring. Importantly, effects are transmitted through the paternal lineage. Altered phenotypes are associated with profound differences in transcription and DNA methylation in the brain. In the present study, we hypothesized that maternal prenatal synthetic glucocorticoid (sGC) exposure in the F0 pregnancy will result in differences in miRNA levels in testes germ cells and sperm across multiple generations, and that these changes will associate with modified microRNA (miRNA) profiles and gene expression in the prefrontal cortex (PFC) of subsequent generations. Pregnant guinea-pigs (F0) were treated with multiple courses of the sGC betamethasone (Beta) (1 mg kg-1; gestational days 40, 41, 50, 51, 60 and 61) in late gestation. miRNA levels were assessed in testes germ cells and in F2 PFC using the GeneChip miRNA 4.0 Array and candidate miRNA measured in epididymal sperm by quantitative real-time PCR. Maternal Beta exposure did not alter miRNA levels in germ cells derived from the testes of adult male offspring. However, there were significant differences in the levels of four candidate miRNAs in the sperm of F1 and F2 adult males. There were no changes in miRNA levels in the PFC of juvenile F2 female offspring. The present study has identified that maternal Beta exposure leads to altered miRNA levels in sperm that are apparent for at least two generations. The fact that differences were confined to epididymal sperm suggests that the intergenerational effects of Beta may target the epididymis. KEY POINTS: Paternal glucocorticoid exposure prior to conception leads to profound epigenetic changes in the brain and somatic tissues in offspring, and microRNAs (miRNAs) in sperm may mediate these changes. We show that there were significant differences in the miRNA profile of epididymal sperm in two generations following prenatal glucocorticoid exposure that were not observed in germ cells derived from the testes. The epididymis is a probable target for intergenerational programming. The effects of prenatal glucocorticoid treatment may span multiple generations.

Prenatal Glucocorticoid Exposure Results in Changes in Gene Transcription and DNA Methylation in the Female Juvenile Guinea Pig Hippocampus Across Three Generations.

Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery, to mature the fetal lung and decrease neonatal morbidity. sGC also profoundly affect the fetal brain. The hippocampus expresses high levels of glucocorticoid (GR) and mineralocorticoid receptor (MR), and its development is affected by elevated fetal glucocorticoid levels. Antenatal sGC results in neuroendocrine and behavioral changes that persist in three generations of female guinea pig offspring of the paternal lineage. We hypothesized that antenatal sGC results in transgenerational changes in gene expression that correlate with changes in DNA methylation. We used RNASeq and capture probe bisulfite sequencing to investigate the transcriptomic and epigenomic effects of antenatal sGC exposure in the hippocampus of three generations of juvenile female offspring from the paternal lineage. Antenatal sGC exposure (F0 pregnancy) resulted in generation-specific changes in hippocampal gene transcription and DNA methylation. Significant changes in individual CpG methylation occurred in RNApol II binding regions of small non-coding RNA (snRNA) genes, which implicates alternative splicing as a mechanism involved in transgenerational transmission of the effects of antenatal sGC. This study provides novel perspectives on the mechanisms involved in transgenerational transmission and highlights the importance of human studies to determine the longer-term effects of antenatal sGC on hippocampal-related function.

Antenatal Glucocorticoid Exposure Results in Sex-Specific and Transgenerational Changes in Prefrontal Cortex Gene Transcription that Relate to Behavioural Outcomes.

Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery to reduce respiratory distress syndrome in the newborn. The prefrontal cortex (PFC) is important in regulating stress responses and related behaviours and expresses high levels of glucocorticoid receptors (GR). Further, antenatal exposure to sGC results in a hyperactive phenotype in first generation (F1) juvenile male and female offspring, as well as F2 and F3 juvenile females from the paternal lineage. We hypothesized that multiple courses of antenatal sGC modify gene expression in the PFC, that these effects are sex-specific and maintained across multiple generations, and that the gene sets affected relate to modified locomotor activity. We performed RNA sequencing on PFC of F1 juvenile males and females, as well as F2 and F3 juvenile females from the paternal lineage and used regression modelling to relate gene expression and behavior. Antenatal sGC resulted in sex-specific and generation-specific changes in gene expression. Further, the expression of 4 genes (C9orf116, Calb1, Glra3, and Gpr52) explained 20-29% of the observed variability in locomotor activity. Antenatal exposure to sGC profoundly influences the developing PFC; effects are evident across multiple generations and may drive altered behavioural phenotypes.

Gestational age-dependent gene expression profiling of ATP-binding cassette transporters in the healthy human placenta.

The ATP-binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal-fetal interface. We and others have demonstrated a gestational age-dependent expression pattern of two ABC transporters, P-glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational-age dependent manner in normal human pregnancy. Using the TaqMan® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.

The DNA methylation landscape of enhancers in the guinea pig hippocampus.

AIM: To determine the state of methylation of DNA molecules in the guinea pig hippocampus that are associated with either poised or active enhancers. METHODS: We used sequential chromatin immunoprecipitation-bisulfite-sequencing with an antibody to H3K4me1 to map the state of methylation of DNA that is found within enhancers. Actively transcribing transcription start sites were mapped by chromatin immunoprecipitation-sequencing with an antibody to RNApolII-PS5. Total DNA methylation was mapped using reduced representation bisulfite sequencing. RESULTS: DNA that overlaps with H3K4me1 binding regions in the genome is heavily methylated. However, DNA molecules that are found in H3K4me1 chromatin are hypomethylated, while DNA found in enhancers that are associated with active transcription is further demethylated. Differential methylation in enhancers is spotted in single CGs, bimodal and corresponds to transcription factor binding sites. CONCLUSION: Our study delineates the DNA methylation status of H3K4 me1-bound regions in the hippocampus in active and inactive genes.

Chorioamnionitis Induces a Specific Signature of Placental ABC Transporters Associated with an Increase of miR-331-5p in the Human Preterm Placenta.

BACKGROUND/AIMS: The ATP-binding cassette (ABC) transporters mediate drug biodisposition and immunological responses in the placental barrier. In vitro infective challenges alter expression of specific placental ABC transporters. We hypothesized that chorioamnionitis induces a distinct pattern of ABC transporter expression. METHODS: Gene expression of 50 ABC transporters was assessed using TaqMan® Human ABC Transporter Array, in preterm human placentas without (PTD; n=6) or with histological chorioamnionitis (PTDC; n=6). Validation was performed using qPCR, immunohistochemistry and Western blot. MicroRNAs known to regulate P-glycoprotein (P-gp) were examined by qPCR. RESULTS: Up-regulation of ABCB9, ABCC2 and ABCF2 mRNA was detected in chorioamnionitis (p<0.05), whereas placental ABCB1 (P-gp; p=0.051) and ABCG2 (breast cancer resistance protein-BCRP) mRNA levels (p=0.055) approached near significant up-regulation. In most cases, the magnitude of the effect significantly correlated to the severity of inflammation. Upon validation, increased placental ABCB1 and ABCG2 mRNA levels (p<0.05) were observed. At the level of immunohistochemistry, while BCRP was increased (p<0.05), P-gp staining intensity was significantly decreased (p<0.05) in PTDC. miR-331-5p, involved in P-gp suppression, was upregulated in PTDC (p<0.01) and correlated to the grade of chorioamnionitis (p<0.01). CONCLUSIONS: Alterations in the expression of ABC transporters will likely lead to modified transport of clinically relevant compounds at the inflamed placenta. A better understanding of the potential role of these transporters in the events surrounding PTD may also enable new strategies to be developed for prevention and treatment of PTD.

Prenatal Glucocorticoid Exposure Modifies Endocrine Function and Behaviour for 3 Generations Following Maternal and Paternal Transmission.

Fetal exposure to high levels of glucocorticoids programs long-term changes in the physiologic stress response and behaviours. However, it is not known whether effects manifest in subsequent generations of offspring following maternal (MT) or paternal (PT) transmission. We treated pregnant guinea pigs with three courses of saline or synthetic glucocorticoid (sGC) at a clinically relevant dose. Altered cortisol response to stress and behaviours transmitted to juvenile female and male F2 and F3 offspring from both parental lines. Behavioural effects of sGC in F1-F3 PT females associated with altered expression of genes in the prefrontal cortex and hypothalamic paraventricular nucleus (PVN). Exposure to sGC programmed large transgenerational changes in PVN gene expression, including type II diabetes, thermoregulation, and collagen formation gene networks. We demonstrate transgenerational programming to F3 following antenatal sGC. Transmission is sex- and generation-dependent, occurring through both parental lines. Paternal transmission to F3 females strongly implicates epigenetic mechanisms of transmission.

Programming of stress pathways: A transgenerational perspective.

The embryo and fetus are highly responsive to the gestational environment. Glucocorticoids (GC) represent an important class of developmental cues and are crucial for normal brain development. Levels of GC in the fetal circulation are tightly regulated. They are maintained at low levels during pregnancy, and increase rapidly at the end of gestation. This surge in GC is critical for maturation of the organs, specifically the lungs, brain and kidney. There are extensive changes in brain epigenetic profiles that accompany the GC surge, suggesting that GC may drive regulation of gene transcription through altered epigenetic pathways. The epigenetic profiles produced by the GC surge can be prematurely induced as a result of maternal or fetal stress, as well as through exposure to synthetic glucocorticoids (sGC). This is highly clinically relevant as 10% of pregnant women are at risk for preterm labour and receive treatment with sGC to promote lung development in the fetus. Fetal overexposure to GC (including sGC) has been shown to cause lasting changes in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis leading to altered stress responses, and mood and anxiety disorders in humans and animals. In animal models, GC exposure is associated with transcriptomic and epigenomic changes that influence behaviour, HPA function and growth. Importantly, programming by GC results in sex-specific effects that can be inherited over multiple generations via paternal and maternal transmission.

Cocaine exposure prior to pregnancy alters the psychomotor response to cocaine and transcriptional regulation of the dopamine D1 receptor in adult male offspring.

There is evidence that maternal experience prior to pregnancy can play an important role in behavioral, physiological, and genetic programming of offspring. Likewise, exposure to cocaine in utero can result in marked changes in central nervous system function of offspring. In this study, we examined whether exposure of rat dams to cocaine prior to pregnancy subsequently alters indices of behavior, physiology, and gene expression in offspring. Multiple outcome measures were examined in adult male offspring: (1) behavioral expression of cocaine-induced psychomotor activation; (2) levels of corticosterone in response to immobilization stress; and (3) expression of multiple genes, including dopamine receptor D1 (DRD1) and D2 (DRD2), glucocorticoid receptor (GR), and corticotropin-releasing factor (CRF), in functionally relevant brain regions. Adult Sprague-Dawley females were exposed to cocaine (15-30 mg/kg, i.p.) or saline for 10 days, and were then mated to drug naïve males of the same strain. Separate groups of adult male offspring were tested for their acute psychomotor response to cocaine (0, 15, 30 mg/kg, i.p.), corticosterone responsivity to 20 min of immobilization stress, and expression of multiple genes using quantitative PCR. Offspring of dams exposed to cocaine prior to conception exhibited increased psychomotor sensitivity to cocaine, and upregulated gene expression of DRD1 in the medial prefrontal cortex (mPFC). Neither stress-induced corticosterone levels nor gene expression of GR or CRF genes were altered. These data suggest that cocaine exposure before pregnancy can serve to enhance psychomotor sensitivity to cocaine in offspring, possibly via alterations in dopamine function that include upregulation of the DRD1.

Electrochemical detection of interaction between Thioflavin T and acetylcholinesterase.

The electrochemical oxidation of the benzothiazole dye Thioflavin T (ThT) was found to be modulated by its interaction with electric eel acetylcholinesterase (AChE). Modifications of AChE by trace amounts of small molecule inhibitors such as carbachol and paraoxon were detectable electrochemically using minimal reagents and with greater sensitivity than attainable through conventional fluorescence approaches. This property appears to be unique to ThT, since its closely related neutral derivative BTA-1 only interacts with AChE, but is not significantly affected by the presence of small molecule inhibitors.

Electrochemical oxidation of benzothiazole dyes for monitoring amyloid formation related to the Alzheimer's disease.

Alzheimer's disease (AD) is associated with the formation and deposition of amyloid fibrils. A better understanding of the oligomeric intermediates on the pathway to fibrilization is highly desired, but efficient methods for their detection are lacking. We have studied the interfacial properties of amyloid peptides (Abeta-40 and Abeta-42) and the course of their aggregation in vitro in the presence of the benzothiazole dyes Thioflavin T (4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline) chloride, ThT) and BTA-1 ([2-(4'-(methylamino)phenyl) benzothiazole]) using electrochemical techniques. The intercalative properties of these dyes between the beta-sheets of amyloids have been well-documented using fluorescence-based systems, but their electrochemistry is reported here for the first time. ThT is positively charged and water-soluble, whereas BTA-1 is neutral and hydrophobic. Immediate and significantly different electrochemical characteristics of these dyes were observed in the presence of amyloid peptides. A decrease of the BTA-1 oxidation signal was observed upon incubation with Abeta-40. Incubation of BTA-1 with Abeta-42 results in an increased rate of exponential decay, which was in agreement with the known rapid aggregation properties of Abeta-42. The aggregation of amyloid peptides with ThT resulted in an unexpected increase in signal after 24 h of incubation, consistent for both peptides. The results of the electrochemical trials were confirmed using simultaneous fluorescence analysis of the same incubated amyloid samples. The very early changes in the interfacial behavior of the amyloid peptides after the first few minutes of incubation were attributed to the fast oligomerization of the peptides with the disruption of the intercalative properties of the benzothiazole dyes between the beta-sheets. The subsequent changes in the electrochemical signals can be related to the onset of intercalation between the fibrils. Our results demonstrate the utility of electrochemical oxidation signals of the benzothiazole dyes as a new and simple tool for the investigation of amyloid formation related to the AD.