Perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters sex-dependent expression of hepatic CYP2C11

The cytochrome P450 (CYP) isoform CYP2C11 is specifically expressed in the liver of adult male rats, and 5alpha-reductase is specifically expressed in the liver of the adult female rats. The sexually dimorphic expressions of these hepatic enzymes are regulated by the sex-dependent profiles of the circulating growth hormone (GH). However, it is not well known whether hormonal imprinting or activation factors in the neonatal brain influence the sexually dimorphic expression patterns of hepatic enzymes. We therefore examined the effect of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sex-dependent expressions of hepatic enzymes. Pregnant rats were treated with TCDD at a dose of 0, 200, or 800 ng/kg on gestation day 15, exposing the pups to the chemical. Although the expression of CYP2C11 protein in the livers of male pups on postnatal day (PND) 49 was significantly higher than that of the controls, but the 5alpha-reductase activities in the livers of female pups were not altered by exposure to TCDD. Focusing on perinatal periods, testosterone and estrogen levels significantly increased in the brain of male pups on PND 2. The results suggest that the alteration of testosterone and estrogen levels affect hormonal imprinting in the neonatal brain of male pups, and thus induces a change in the level of male-specific hepatic CYP2C11. We conclude that perinatal exposure to TCDD at low doses may change the sexual differentiation of the neonatal brain in male rats.     

Sexual dimorphism of rat liver nuclear proteins: regulatory role of growth hormone

Many genes are expressed in mammalian liver in a sexually dimorphic manner. DNA microarray analysis has shown that growth hormone (GH) and its sex-dependent pattern of pituitary secretion play a major role in establishing the sexually dimorphic patterns of liver gene expression. However, GH may exert effects on protein post-translational modification and nuclear localization that are not reflected at the mRNA level. To investigate these potential effects of GH, we used two-dimensional gel electrophoresis followed by LC-MS/MS to: 1) identify rat liver nuclear proteins whose abundance or state of post-translational modification displays sex-dependent differences; and 2) determine the role of the plasma GH profile in establishing these differences. Nuclear extracts prepared from livers of individual male (n=9) and female (n=5) adult rats, and from males given GH by continuous infusion for 7 days to feminize liver gene expression (n=5 rats), were resolved by two-dimensional electrophoresis. Image analysis of SYPRO Ruby-stained gels revealed 165 sexually dimorphic protein spots that differ in normalized volume between male and female groups by >1.5-fold at p<0.05. Sixty of these proteins exhibited female-like changes in spot abundance following continuous GH treatment. Comparison of male and GH-treated male groups revealed 130 proteins that displayed >1.5-fold differences in abundance, with 60 of these GH-responsive spots being sexually dimorphic. Thus, GH plays an important role in establishing the sex-dependent differences in liver nuclear protein content. Twenty-eight of the sexually dimorphic and/or GH-regulated protein spots were identified by LC-MS/MS. Proteins identified include regucalcin, nuclear factor 45, and heterogeneous nuclear ribonucleoproteins A3, D-like, and K, in addition to proteins such as GST, normally associated with cytosolic extracts but also reported to be localized in the nucleus.     

Hormonally mediated epigenetic changes to steroid receptors in the developing brain: Implications for sexual differentiation

The establishment of sex-specific neural morphology, which underlies sex-specific behaviors, occurs during a perinatal sensitive window in which brief exposure to gonadal steroid hormones produces permanent masculinization of the brain. In the rodent, estradiol derived from testicular androgens is a principal organizational hormone. The mechanism by which transient estradiol exposure induces permanent differences in neuronal anatomy has been widely investigated, but remains elusive. Epigenetic changes, such as DNA methylation, allow environmental influences to alter long-term gene expression patterns and therefore may be a potential mediator of estradiol-induced organization of the neonatal brain. Here we review data that demonstrate sex and estradiol-induced differences in DNA methylation on the estrogen receptor α (ERα), estrogen receptor β (ERβ), and progesterone receptor (PR) promoters in sexually dimorphic brain regions across development. Contrary to the overarching view of DNA methylation as a permanent modification directly tied to gene expression, these data demonstrate that methylation patterns on steroid hormone receptors change across the life span and do not necessarily predict expression. Although further exploration into the mechanism and significance of estradiol-induced alterations in DNA methylation patterns in the neonatal brain is necessary, these results provide preliminary evidence that epigenetic alterations can occur in response to early hormone exposure and may mediate estradiol-induced organization of sex differences in the neonatal brain.     

Mercury‐associated DNA hypomethylation in polar bear brains via the LUminometric Methylation Assay: a sensitive method to study epigenetics in wildlife

In this paper we describe a novel approach that may shed light on the genomic DNA methylation of organisms with non‐resolved genomes. The LUminometric Methylation Assay (LUMA) is permissive for genomic DNA methylation studies of any genome as it relies on the use of methyl‐sensitive and ‐insensitive restriction enzymes followed by polymerase extension via Pyrosequencing technology. Here, LUMA was used to characterize genomic DNA methylation in the lower brain stem region from 47 polar bears subsistence hunted in central East Greenland between 1999 and 2001. In these samples, average genomic DNA methylation was 57.9% ± 6.69 (SD; range was 42.0 to 72.4%). When genomic DNA methylation was related to brain mercury (Hg) exposure levels, an inverse association was seen between these two variables for the entire study population (P for trend = 0.17). After dichotomizing animals by gender and controlling for age, a negative trend was seen amongst male animals (P for trend = 0.07) but no associations were found in female bears. Such sexually dimorphic responses have been found in other toxicological studies. Our results show that genomic DNA methylation can be quantitatively studied in a highly reproducible manner in tissue samples from a wild organism with a non‐resolved genome. As such, LUMA holds great promise as a novel method to explore consequential questions across the ecological sciences that may require an epigenetic understanding.     

Sex differences in epigenetic mechanisms may underlie risk and resilience for mental health disorders

Alterations in the epigenetic programming of sex differences in the brain may underlie sexually dimorphic neurodevelopmental disorders. Sex differences have been found in DNA methyltransferases 3a, DNA methylation patterns, MeCP2, and nuclear corepressor within the developing brain. Natural variations in these epigenetic mechanisms have profound consequences on gene expression and brain function. Exogenous or endogenous perturbations during development may impact these epigenetic processes and alter the trajectory of the developing brain and confer sexually dimorphic risk and resilience for developing a neurological or mental health disorder.     

Gene expression and DNA methylation changes in the hypothalamus and hippocampus of adult rats developmentally exposed to bisphenol A or ethinyl estradiol: a CLARITY-BPA consortium study

Bisphenol A (BPA), an endocrine disrupting chemical (EDC), is a ubiquitous pollutant. As part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), we sought to determine whether exposure of Sprague-Dawley rats to 2,500 μg/kg/day BPA (BPA) or 0.5 μg/kg/day ethinyl estradiol (EE) from gestational day 6 through postnatal day 21 induces behavior-relevant gene expression and DNA methylation changes in hippocampus and hypothalamus at adulthood. RNA and DNA were isolated from both regions. Expression of ten genes (Dnmt1, Dnmt3a, Dnmt3b, Esr1, Esr2, Avp, Ar, Oxt, Otr, and Bdnf) presumably altered by early-life BPA/EE exposure was examined. Three genes (Bdnf, Dnmt3b, and Esr1) were studied for DNA methylation changes in their putative 5? promoter regions. Molecular changes in hippocampus were correlated to prior Barnes maze performance, including sniffing correct holes, distance traveled, and velocity. Exposure to BPA and/or EE disrupted patterns of sexually dimorphic gene expression/promoter DNA methylation observed in hippocampus and hypothalamus of controls. In the hippocampus of female offspring, BPA exposure resulted in hypermethylation of the putative 5? promoter region of Bdnf, while EE exposure induced hypomethylation. Bdnf methylation was weakly associated with Bdnf expression in hippocampi of female rats. Hippocampal Bdnf expression in females showed a weak negative association with sniffing correct hole in Barnes maze. Hippocampal expression of Avp, Esr2, Oxt, and Otr was strongly associated with velocity of control rats in Barnes maze. Findings suggest BPA exposure induced non-EE-like gene expression and epigenetic changes in adult rat hippocampi, a region involved in spatial navigation.     

Effects of testosterone on brain mRNA levels of steroid 5alpha-reductase isozymes in early postnatal life of rat

The enzyme 5alpha-reductase (5alpha-R) (EC 1.3.99.5) exists as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2), and both are present in the brain. 5alpha-R1 has been proposed as a constitutive enzyme that essentially plays a catabolic and neuron protective role whereas 5alpha-R2 has been associated with sexually dimorphic functions of the male. In this work, we studied the effects of testosterone (T), the masculinizing hormone of the central nervous system (CNS), on mRNA levels of both 5alpha-R isoforms in the prefrontal cortex of male and female rats during the postnatal sexual differentiation of the CNS in the rat, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). We found an increase in 5alpha-R2 mRNA levels in both male and female rats after T treatment, while 5alpha-R1 mRNA levels were decreased in the same experimental conditions. Our results clearly indicated that T regulates the expression of both 5alpha-R1 and 5alpha-R2 genes in an opposite manner and independently of the sex. This could point to a crucial role of T in the sexual dimorphism for both 5alpha-R isozymes in the neonatal brain. These results open up a new research line that could improve understanding of the role of 5alpha-R isozymes in the physiology of the CNS.     

Sex difference in volume of the ventromedial nucleus of the hypothalamus in the rat

The volume of the ventromedial nucleus of the hypothalamus (VMN) of normal male rats was significantly greater than that of normal female rats. Castration of day 1 neonatal males significantly reduced the volume of the VMN to a level comparable with that of normal females. However, the VMN volume was no longer influenced by castration on day 7. Injection of 1.25 mg testosterone propionate to 5- to 15-day-old females did not have any significant effect on the volume of the VMN. These results indicate that the volume of the VMN is sexually dimorphic and is modified by internal secretion of neonatal testes.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone‐filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 502–510, 2003     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

Galectin-1 Enhances Astrocytic BDNF Production and Improves Functional Outcome in Rats Following Ischemia

Galectin-1, an endogenous mammalian lectin, has been implicated in a variety of CNS disorders. However, its role in cerebral ischemia is still elusive. In the present study, we investigated the effect of recombinant galectin-1 on production of astrocytic brain-derived neurotrophic factor (BDNF) and functional recovery following ischemia. Endogenous galectin-1 was found to be markedly upregulated, paralleled with increased astrocytic BDNF production under ischemic conditions both in vitro and in vivo. Administration of galectin-1significantly enhanced the expression and secretion of astrocytic BDNF in dose dependent manner. Moreover, rats subjected to photochemical cerebral ischemia showed reduced neuronal apoptosis in ischemic boundary zone and improved functional recovery after brain infusion of galectin-1 (1 μg/days, 7 days). These results suggest that induction of BDNF in astrocytes by galectin-1 may be a promising intervention to attenuate brain damage after stroke.     

Systemic Evaluation of Hypoxic-Ischemic Brain Injury in Neonatal Rats

The objective of the study was to evaluate the systematically rat model of neonatal hypoxic-ischemic brain damage. The right carotid arteries of 7-day-old healthy Wistar rats were ligated, and then, the rats were subjected to an environment with 8 % of oxygen. Four weeks after the birth, neurobehavioral test, water maze test, and motor-evoked potential and neuropathologic examinations were performed. The footprint analysis showed significantly larger and instable paces in the hypoxic-ischemic group (P < 0.05); the time that rats crossed the balance beam in the hypoxic-ischemic group was longer than the control group (P < 0.05). The water maze test showed that the escape latency of hypoxic-ischemic group was significantly longer than that of control group (P < 0.05). The hindlimb quadriceps compound muscle-evoked potential CMEP of rats in hypoxic-ischemic group showed that the wave amplitude was lower than that of control group (P < 0.05). HE staining showed visible periventricular leukomalacia in hypoxic-ischemic groups; disrupted nuclear membrane was detected in the IH group with transelectronmicroscopy; Immunohistochemistry: compared with control group, MBP-positive neurocytes decreased, glial fibrillary acidic protein positive neurocytes increased in the periventricular zone (P < 0.05). Carotid artery ligation combining the hypoxic chamber created a reliable and stable rat model of neonatal hypoxic-ischemic brain damage and can be used for experimental research related to management of cerebral palsy.     

Sex differences in brain developing in the presence or absence of gonads

Brain sexual differentiation results from the interaction of genetic and hormonal influences. This study used a unique agonadal mouse model to determine relative contributions of genetic and gonadal hormone influences in the differentiation of selected brain regions. SF-1 knockout (SF-1 KO) mice are born without gonads and adrenal glands and are not exposed to endogenous sex steroids during fetal/neonatal development. Consequently, male and female SF-1 KO mice are born with female external genitalia and if left on their own, die shortly after birth due to adrenal insufficiency. In this study, SF-1 KO mice were rescued by neonatal adrenal transplantation to examine their brain morphology in adult life. To determine potential brain loci that might mediate functional sex differences, we examined the area and distribution of immunoreactive calbindin and neuronal nitric oxide synthase in the preoptic area (POA) and ventromedial nucleus of the hypothalamus, two areas previously reported to be sexually dimorphic in the mammalian brain. A sex difference in the positioning of cells containing immunoreactive calbindin in a group within the POA was clearly gonad dependent based on the elimination of the sex difference in SF-1 KO mice. Several other differences in the area of ventromedial hypothalamus and in POA were maintained in male and female SF-1 KO mice, suggesting gonad-independent genetic influences on sexually dimorphic brain development.     

Identifying female phenotypes that promote behavioral isolation in a sexually dimorphic species of fish Etheostoma zonale

In sexually dimorphic species characterized by exaggerated male ornamentation, behavioral isolation is often attributed to female preferences for conspecific male signals. Yet, in a number of sexually dimorphic species, male mate choice also results in behavioral isolation. In many of these cases, the female traits that mediate species boundaries are unclear. Females in sexually dimorphic species typically lack many of the elaborate traits that are present in males and that are often used for taxonomic classification of species. In a diverse and largely sexually dimorphic group of fishes called darters (Percidae: Etheostoma), male mate choice contributes to behavioral isolation between a number of species; however, studies addressing which female traits males prefer are lacking. In this study, we identified the dominant female pattern for two sympatric species, Etheostoma zonale and Etheostoma barrenense, using pattern energy analysis, and we used discriminate function analysis to identify which aspects of female patterning can reliably classify species. We then tested the role of female features in male mate choice for E. zonale, by measuring male preference for computer animations displaying the identified (species-specific) conspecific features. We found that the region above the lateral line is important in mediating male mate preferences, with males spending a significantly greater proportion of time with animations exhibiting conspecific female patterning in this region than with animations exhibiting heterospecific female patterning. Our results suggest that the aspects of female phenotypes that are the target of male mate choice are different from the conspicuous male phenotypes that traditionally characterize species.     

Neonatal estradiol exposure to female rats changes GABAA receptor expression and function,and spatial learning during adulthood

Exposure of female rats to estradiol during the perinatal period has profound effects on GABAergic neurotransmission that are crucial to establish sexually dimorphic brain characteristics. We previously showed that neonatal β-estradiol 3-benzoate (EB) treatment decreases brain concentrations of the neurosteroid allopregnanolone, a potent positive modulator of extrasynaptic GABA_A receptors (GABA_AR). We thus evaluated whether neonatal EB treatment affects GABA_AR expression and function in the hippocampus of adult female rats. Neonatal EB administration increased the expression of extrasynaptic α4/δ subunit-containing GABA_ARs and the modulatory action of THIP on tonic currents mediated by these receptors. The same treatment decreased the expression of synaptic α1/α4/γ2 subunit-containing receptors, as well as phasic currents. These effects of neonatal EB treatment are not related to ambient allopregnanolone concentrations per se, given that vehicle-treated rats in diestrus, which have opposite neurosteroid levels than EB-treated rats, show similar changes in GABA_ARs. Rather, these changes may represent a compensatory mechanism to counteract the long-term reduction in allopregnanolone concentrations, induced by neonatal EB. Given that both α4/δ receptors and allopregnanolone are involved in memory consolidation, we evaluated whether neonatal EB treatment alters performance in the Morris water maze test during adulthood. Neonatal EB treatment decreased the latency and the cumulative search error to reach the platform, as well as thigmotaxis, suggesting improved learning, and also enhanced memory performance during the probe trial. These enduring changes in GABA_AR plasticity may be relevant for the regulation of neuronal excitability in the hippocampus and for the etiology of psychiatric disorders that originate in development and show sex differences.     

Long-term effects of combined neonatal and adolescent stress on brain-derived neurotrophic factor and dopamine receptor expression in the rat forebrain

Altered brain-derived neurotrophic factor (BDNF) signalling and dopaminergic neurotransmission have been shown in the forebrain in schizophrenia. The ‘two hit’ hypothesis proposes that two major disruptions during development are involved in the pathophysiology of this illness. We therefore used a ‘two hit’ rat model of combined neonatal and young-adult stress to assess effects on BDNF signalling and dopamine receptor expression. Wistar rats were exposed to neonatal maternal separation (MS) stress and/or adolescent/young-adult corticosterone (CORT) treatment. At adulthood the medial prefrontal cortex (mPFC), caudate putamen (CPu) and nucleus accumbens (NAc) were analysed by qPCR and Western blot. The ‘two hit’ combination of MS and CORT treatment caused significant increases in BDNF mRNA and protein levels in the mPFC of male, but not female rats. BDNF mRNA expression was unchanged in the CPu but was significantly reduced by CORT in the NAc. DR3 and DR2 mRNA were significantly up-regulated in the mPFC of two-hit rats and a positive correlation was found between BDNF and DR3 expression in male, but not female rats. DR2 and DR3 expression were significantly increased following CORT treatment in the NAc and a significant negative correlation between BDNF and DR3 and DR2 mRNA levels was found. Our data demonstrate male-specific two-hit effects of developmental stress on BDNF and DR3 expression in the mPFC. Furthermore, following chronic adolescent CORT treatment, the relationship between BDNF and dopamine receptor expression was significantly altered in the NAc. These results elucidate the long-term effects of ‘two hit’ developmental stress on behaviour.