Neonatal iron status is impaired by maternal obesity and excessive weight gain during pregnancy.

OBJECTIVE: Maternal iron needs increase sixfold during pregnancy, but obesity interferes with iron absorption. We hypothesized that maternal obesity impairs fetal iron status. STUDY DESIGN: Three hundred and sixteen newborns with risk factors for infantile iron deficiency anemia (IDA) were studied to examine obesity during pregnancy and neonatal iron status. Erythrocyte iron was assessed by cord blood hemoglobin (Hb), zinc protoporphyrin/heme (ZnPP/H) and reticulocyte-ZnPP/H, and storage iron by serum ferritin. RESULT: Women with body mass index (BMI) ⩾ 30 kg m(-)(2), as compared with non-obese women, delivered larger offspring with higher reticulocyte-ZnPP/H and lower serum ferritin concentrations (P<0.05 for both). With increasing BMI, the estimated body iron was relatively lower (mg kg(-)(1)) and the ratio of total Hb-bound iron (mg) per total body iron (mg) increased. Maternal diabetes compromised infant iron status, but multivariate analysis demonstrated that obesity was an independent predictor. CONCLUSION: Obesity during pregnancy and excessive weight gain are independent risk factors for iron deficiency in the newborn.

Epigenetic mechanisms may underlie the aetiology of sex differences in mental health risk and resilience.

In this review, we propose that experiential and hormonal influences on biological sex during development may produce differences in the epigenome, and that these differences play an important role in gating risk or resilience to a number of neurological and psychiatric disorders. One intriguing hypothesis is that the framework belying sex differences in the brain creates differences in methylation and demethylation patterns, and these in turn confer risk and resilience to mental health disorders. Here, we discuss these concepts with regard to social behaviour in rodent models and briefly discuss their possible relevance to human disease.

Sex difference in the expression of DNA methyltransferase 3a in the rat amygdala during development.

Sexual differentiation of the neonatal rat brain is regulated by dynamic processes occurring at the level of DNA, resulting in sexually dimorphic gene expression. Steroid hormone receptors act partly in the developing brain by recruiting co-activators, thereby increasing the acetylation of histones and gene expression. Recent data indicate that sexual differentiation of the brain may also result, in part, from differences in promoter methylation patterns of some steroid responsive genes. Methylation of DNA is an epigenetic process that can decrease gene expression without altering the original DNA sequence. DNA cytosine-5-methyltransferases (DNMTs) 1 and 3a are two factors that induce methylation. We investigated whether sex differences in the expression of DNMT1 and DNMT3a were apparent in the amygdala, preoptic area and medial basal hypothalamus at different time points during development. We found that females express significantly more DNMT3a mRNA and protein in the amygdala but not within the preoptic area or the medial basal hypothalamus at postnatal day 1. There were no sex differences in DNMT3a mRNA or protein at postnatal day 10. Furthermore, no sex differences were observed in the expression of DNMT1 at either time point. Because most sex differences in the brain are a result of a higher level of gonadal steroid hormone exposure in males at birth, we examined whether dihydrotestosterone or oestradiol exposure would reduce DNMT3a expression in neonatal female rats. We found that both oestradiol and dihydrotestosterone treatment significantly reduced DNMT3a, but not DNMT1, mRNA expression within the developing amygdala. Our results indicate that sex differences in DNMT3a within the developing amygdala are partly a result of steroid exposure. This suggests that steroid hormone exposures may programme lasting differences in amygdala function by altering the expression of the epigenetic factor, DNMT3a.

Brain sex differences and the organisation of juvenile social play behaviour.

Juvenile social play behaviour is one of the earliest forms of non-mother directed social behaviour in rodents. Juvenile social play behaviour is sexually dimorphic, with males exhibiting higher levels compared to females, making it a useful model to study both social development and sexual differentiation of the brain. As with most sexually dimorphic behaviour, juvenile play behaviour is organised by neonatal steroid hormone exposure. The developmental organisation of juvenile play behaviour also appears to be influenced by the early maternal environment. This review will focus briefly on why and how rats play, some brain regions controlling play behaviour, and how neurotransmitters and the social environment converge within the developing brain to influence sexual differentiation of juvenile play behaviour.

Regulation of progestin receptor expression in the developing rat brain by a dopamine d1 receptor antagonist.

Steroid receptors within the developing brain influence a variety of cellular processes that endure into adulthood, altering both behaviour and physiology. Therefore, it is important to understand how steroid receptor expression is regulated during early brain development. Most studies indicate that oestradiol, by acting upon oestrogen receptors, increases the expression of progestin receptors in the developing brain. We have recently observed an additional mechanism by which dopamine can increase the expression of progestin receptors in developing female rat brain. That is, we found that a dopamine D1 receptor agonist can further increase progestin receptor expression by activating oestrogen receptors in a ligand-independent manner within restricted areas of female brain; however, it is unclear whether dopamine D1 receptors are involved in the normally occurring expression of progestin receptors in developing male and female brain. To investigate this, we examined whether a dopamine D1 receptor antagonist can disrupt the normal developmental expression of progestin receptors in both male and female rat brain. We report that treatment with a dopamine D1 receptor antagonist reduces progestin receptor expression within some, but not all, regions of the developing rat brain in a sex-specific manner. Some of the current findings also suggest that dopamine might be acting to prevent sex differences in progestin receptor expression in some areas while contributing to a sex difference in other areas.

Effective use of microarrays in neuroendocrine research.

The development of microarray technology makes it possible to simultaneously assay the expression level of hundreds to tens of thousands of mRNA transcripts in one experiment. Genome-wide transcriptional analysis has increasing importance for many areas of neuroendocrinology research. The expense and technical complexity of microarray experiments can make it difficult to navigate the terrain of rival platforms and technologies. In this review, we provide a practical view and comparison of various microarray technologies. Affymetrix arrays, high-density cDNA arrays, membrane arrays and experimental design and data analysis are all discussed by researchers currently using these techniques to study gene regulation in neuroendocrine tissues.

Altered gene expression in mice selected for high maternal aggression.

We previously applied selective breeding on outbred mice to increase maternal aggression (maternal defense). In this study, we compared gene expression within a continuous region of the central nervous system (CNS) involved in maternal aggression (hypothalamus and preoptic regions) between lactating selected (S) and nonselected control (C) mice (n= 6 per group). Using microarrays representing over 40,000 genes or expressed sequence tags, two statistical algorithms were used to identify significant differences in gene expression: robust multiarray and the probe logarithmic intensity error method. Approximately 200 genes were identified as significant using an intersection from both techniques. A subset of genes was examined for confirmation by real-time polymerase chain reaction (PCR). Significant decreases were found in S mice for neurotensin and neuropeptide Y receptor Y2 (both confirmed by PCR). Significant increases were found in S mice for neuronal nitric oxide synthase (confirmed by PCR), the K+ channel subunit, Kcna1 (confirmed by PCR), corticotrophin releasing factor binding protein (just above significance using PCR; P= 0.051) and GABA A receptor subunit 1A (not confirmed by PCR, but similar direction). S mice also exhibited significantly higher levels of the neurotransmitter receptor, adenosine A1 receptor and the transcription factors, c-Fos, and Egr-1. Interestingly, for 24 genes related to metabolism, all were significantly elevated in S mice, suggesting altered metabolism in these mice. Together, this study provides a list of candidate genes (some previously implicated in maternal aggression and some novel) that may play an important role in the production of this behavior.

Sex differences in Fos protein expression in the neonatal rat brain.

Sex differences in the brain and behaviour are mostly a result of transient increases in testosterone during the perinatal period. Testosterone influences brain development primarily through aromatization to oestradiol and subsequent binding to oestrogen receptors. Although some studies report that steroid hormones regulate the expression of the inducible transcription factor, Fos, in developing brain, it is not known if there is a sex difference in Fos expression. Changes in Fos protein can be used as an indicator of neuronal/genomic activity. Thus, it provides a useful tool to identify brain regions responding directly or indirectly to steroid hormones. In a first experiment, we examined Fos protein expression in the developing male and female rat brain using western immunoblotting. Dissections were taken from male and female rat pups on the day of birth (postnatal day 0; PN 0), PN1, PN5, PN11 or PN20. Although there was no difference on PN 0, males expressed significantly greater levels of Fos protein on PN1, PN5 and PN20. In a second experiment, we localized the sex difference in Fos protein expression using immunocytochemistry. We found that males expressed significantly higher levels of Fos within a variety of brain regions. These data indicate a sex difference in Fos protein expression during brain development, suggesting a potential role for Fos in differentiating male from female rat brain.

Sex differences in the developing brain: crossroads in the phosphorylation of cAMP response element binding protein.

Although it is widely known that steroid hormones differentiate the brain, little is known about the signal transduction pathways that are influenced by steroid hormones during development. This review focuses on divergence in the phosphorylation of cAMP response element binding protein (CREB) in the developing male and female rat brain. At birth, males have an increased phosphorylation of CREB compared to females. As CREB mediates changes in cellular morphology, function and survival rates, its activation may underlie an important event in steroid-mediated sexual differentiation of the brain. The importance of CREB is further supported by a sex difference in the expression of the nuclear receptor coactivator, CREB-binding protein, a critical factor involved in the genomic actions of CREB. This suggests that the developing male brain may be in a hyper-responsive state to factors that lead to increased phosphorylation of CREB, resulting in divergent responses in males versus females. An example of this divergence is the response to GABA. In the male rat brain, GABA action leads to increased phosphorylation of CREB; whereas GABA action in the female brain leads to decreased phosphorylation of CREB. The potential consequences of this divergence in the regulation of CREB are discussed in relation to adult sexually dimorphic brain morphology, physiology and behaviour.

Excitatory actions of GABA in developing brain are mediated by l-type Ca2+ channels and dependent on age, sex, and brain region.

Although GABA is the major inhibitory neurotransmitter in adult brain, it exerts depolarizing actions in developing neurons that include activation of voltage-gated calcium channels. The depolarizing actions of GABA serve an obvious trophic function, but the specific physiological significance of excitatory versus inhibitory GABA action has been largely ignored. We previously demonstrated that estradiol enhances the magnitude and duration of calcium influx through L-type voltage-gated calcium channels following GABA(A) receptor activation in neonatal hypothalamic neurons. This has led us to propose that GABA action represents a major divergence point in steroid-mediated sexual differentiation of rat brain. Presently, we examined sex differences in phosphorylation of the calcium-regulated transcription factor, cyclic AMP response element binding protein, following activation of the GABA(A) receptor with muscimol, in vivo. Muscimol given 30 min before killing significantly increased the number of neurons exhibiting phosphorylated cyclic AMP response element binding protein in newborn male hypothalamus and CA1 hippocampus but decreased phosphorylated cyclic AMP response element binding protein in most brain regions in females. Muscimol-induced increases in phosphorylated cyclic AMP response element binding protein in hypothalamus and hippocampus of newborn males were attenuated by pretreatment with the L-type voltage-gated calcium channel blocker, nimodipine, suggesting that calcium influx is involved in phosphorylation of cyclic AMP response element binding protein in neonate brain. Muscimol treatment had no effect on hypothalamic or hippocampal phosphorylated cyclic AMP response element binding protein levels in juvenile males and females. These results are consistent with a divergence in male and female rat brain in the calcium-mediated cellular response to muscimol that is restricted to the early neonatal period, a time critical for estradiol-mediated sexual differentiation.

Expression of cAMP response element binding protein-binding protein in the song control system and hypothalamus of adult European starlings (Sturnus vulgaris).

In songbirds, the initiation of song behaviour and the neural substrate of this system are highly influenced by gonadal steroids. Receptors for gonadal steroid hormones, such as androgens and oestrogens, have been localized within select nuclei of the song system. An important step in steroid receptor action is the recruitment of nuclear receptor coactivators. The coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), has been implicated in both androgen and oestrogen receptor transactivation. Although the role of CBP in transcriptional mechanisms has been widely studied, little is known about CBP expression in the brain. The association between the distribution of CBP and oestrogen receptors in the hippocampus has been related to long-term memory. However, the distribution of brain CBP has not been related to the expression of gonadal steroid receptors in a system as relevant to reproductive behaviour as the avian song system. Western immunoblotting of European starling (Sturnus vulgaris) brain tissue reveals a band at 265 kDa. Immunohistochemical localization of CBP in starling brain indicates wide, but heterogeneous expression. CBP-immunoreactive (CBP-ir) cells define the boundaries of song control nuclei. In HVc (sometimes called the High Vocal Center) and the robust nucleus of the archistriatum (RA), there is a higher density of CBP-ir cells within the boundaries of these nuclei than in adjacent neostriatum or archistriatum, for HVc and RA, respectively. We also report that the distribution of CBP-ir cells varies among different nuclei within the song control system. CBP-ir cells within area X (also a part of the song system) and HVc are densely packed into clusters, whereas cells can be easily discriminated in RA. CBP is also highly expressed in hypothalamic areas, indicating that areas rich in steroid receptors also contain CBP. These data suggest that CBP is important for modulating transcriptional activities in the song system and other sites in the songbird brain that express gonadal steroid receptors.

Ligand-independent activation of progestin receptors: relevance for female sexual behaviour.

Traditionally, steroid receptors were believed to be activated only by ligand binding; however, recent studies indicate that steroid receptors can also be activated by mechanisms that do not require ligand, referred to as ligand-independent activation. Specifically, progestin receptors can be activated in vitro and in vivo after treatment with neurotransmitters, such as dopamine, in the absence of progesterone. Furthermore, mating-related stimuli can also lead to ligand-independent activation of progestin receptors in female rat brain. This finding indicates that environmental stimuli can influence steroid receptor-dependent processes in the absence of circulating hormone. This review focuses on studies indicating that progestin receptors can be activated in the absence of progesterone to influence neuronal response and sexual behaviour in rodents.

Oestradiol increases phosphorylation of a dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32) in female rat brain.

Recent studies suggest that oestrogen and progestin receptors may be activated by the neurotransmitter dopamine, as well as by their respective ligands. Because intracerebroventricular infusion of D(1), but not D(2), dopaminergic receptor agonists increases oestrous behaviour in oestradiol-primed rats, we wanted to determine if treatment with oestradiol alters the activity of D(1) receptor-associated processes in steroid receptor-containing areas in female rat brain. One D(1) receptor-associated phosphoprotein that may be influenced by oestradiol is a dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000 (DARPP-32). Because DARPP-32 is phosphorylated in response to dopamine acting via a cAMP-dependent protein kinase, it provides a useful marker to examine where in the brain a particular stimulus might be altering the activity of D(1) receptor-containing neurones. To determine if oestradiol alters the phosphorylation of DARPP-32, we stained immunocytochemically brain sections of female rats treated with behaviourally relevant doses of oestradiol or oil vehicle with an antibody that detects only the threonine 34-phosphorylated form of DARPP-32. Behaviourally effective doses of oestradiol increase the phosphorylation of DARPP-32 within the medial preoptic nucleus, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus and the ventromedial nucleus of the hypothalamus, 48 h after treatment. These data suggest that oestradiol increases the activity of D(1) dopamine receptor-associated processes in oestrogen receptor-containing areas of female rat forebrain.

Excitatory versus inhibitory GABA as a divergence point in steroid-mediated sexual differentiation of the brain.

Whereas adult sex differences in brain morphology and behavior result from developmental exposure to steroid hormones, the mechanism by which steroids differentiate the brain is unknown. Studies to date have described subtle sex differences in levels of proteins and neurotransmitters during brain development, but these have lacked explanatory power for the profound sex differences induced by steroids. We report here a major divergence in the response to injection of the gamma-aminobutyric acid type A (GABA(A)) agonist, muscimol, in newborn male and female rats. In females, muscimol treatment primarily decreased the phosphorylation of cAMP response element binding protein (CREB) within the hypothalamus and the CA1 region of the hippocampus. In contrast, muscimol increased the phosphorylation of CREB in males within these same brain regions. Within the arcuate nucleus, muscimol treatment increased the phosphorylation of CREB in both females and males. Thus, the response to GABA can be excitatory or inhibitory on signal-transduction pathways that alter CREB phosphorylation depending on the sex and the region in developing brain. This divergence in response to GABA allows for a previously unknown form of steroid-mediated neuronal plasticity and may be an initial step in establishing sexually dimorphic signal-transduction pathways in developing brain.

CNS region-specific oxytocin receptor expression: importance in regulation of anxiety and sex behavior.

The oxytocin receptor (OTR) is differentially expressed in the CNS. Because there are multiple mechanisms by which the OTR can be transcriptionally induced, we hypothesized that differences in OTR expression may be explained by activation of distinct signal transduction pathways and may be critical for the control of anxiety and sex behaviors. To determine the regulation and functional significance of this expression, we infused female rats with modifiers of protein kinases before assaying for behavior and oxytocin receptor binding. In the ventromedial nucleus of the hypothalamus (VMH), estrogen-dependent induction of oxytocin receptors required protein kinase C activation, and oxytocin infused here promoted female sex behavior but had no effect on anxiety. In contrast, dopamine controlled tonic oxytocin receptor expression in the central nucleus of the amygdala (cAmyg) through activation of protein kinase A, and oxytocin infused here was anxiolytic but had no effect on female sex behavior. Therefore, we have identified brain region-specific regulation of the OTR in the VMH and cAmyg. Distinct signal transduction pathways regulating receptor expression and binding in each brain region may mediate in part the ability of oxytocin to exert these differential behavioral effects.

Sex difference in the phosphorylation of cAMP response element binding protein (CREB) in neonatal rat brain.

On the day of birth, a surge in testicular testosterone release in male rats is critical for sexual differentiation of the brain. Steroid hormones function by binding to intracellular steroid receptors and altering gene expression; however, little is known about the signal transduction pathways altered as a consequence of steroid hormone action in developing brain. We investigated whether the increase in testosterone at birth alters the phosphorylation of CREB, a major signal transduction protein. Adjacent brain sections from male and female pups were immunocytochemically stained for serine(133) phosphorylated CREB (pCREB) or total CREB on the day of birth. Males had more pCREB-immunoreactive positive cells than females in the medial preoptic area, ventromedial nucleus of the hypothalamus, the arcuate nucleus, and the CA1 region of the hippocampus, but not in two thalamic nuclei. There was no sex difference in total CREB immunoreactive cell number. To determine if the sex difference in pCREB persisted 24 h after birth and whether the difference was due to testosterone, newborn female pups were injected with 100 microg of testosterone propionate, and male and control female pups were injected with vehicle. Twenty-four hours later, adjacent brain sections were immunocytochemically stained for either pCREB or CREB. We found that males and testosterone-treated females had more pCREB in the ventromedial nucleus of the hypothalamus contrasted to control females. There were no group differences in pCREB or CREB in any other area examined. These results indicate that some of the effects of testosterone in developing brain occur via pathways associated with the phosphorylation of CREB.

Antisense oligodeoxynucleotides as a tool in developmental neuroendocrinology.

Antisense oligodeoxynucleotides have been highly successful agents at modulating gene expression in the adult brain and widely exploited in the field of neuroendocrinology. We have also used this technique in the developing brain to explore the role of select proteins during sensitive periods of development, particularly those influenced by steroid hormones. Presented here are the technical details of using antisense oligodeoxynucleotides in the neonatal brain, as well as a review of some of our successes and failures. Our goal is to illustrate the relative ease of use of this technique in neonates and demonstrate the power such an approach offers so that other investigators will also begin to take advantage of this useful tool.

Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior.

Steroid hormone action during brain development exerts profound effects on reproductive physiology and behavior that last into adulthood. A variety of in vitro studies indicate that steroid receptors require nuclear receptor coactivators for efficient transcriptional activity. To determine the functional significance of the nuclear receptor coactivator SRC-1 in developing brain, we investigated the consequence of reducing SRC-1 protein during sexual differentiation of the brain. We report that reducing SRC-1 protein interferes with the defeminizing actions of estrogen in neonatal rat brain. Our data indicate that SRC-1 protein expression is critically involved in the hormone-dependent development of normal male reproductive behavior and brain morphology.

Progesterone, but not progesterone-independent activation of progestin receptors by a mating stimulus, rapidly decreases progestin receptor immunoreactivity in female rat brain.

Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal-cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity is decreased in the forebrain of estradiol-primed ovariectomized (OVX) rats within 1 h after a subcutaneous injection of progesterone, a time by which rapid down-regulation of progestin receptors does not seem to have occurred. In estradiol-primed OVX rats, VCS also decreased progestin receptor immunoreactivity within 1 h in the medial preoptic area, but not in any other area examined. To determine if the decrease in immunoreactivity by VCS was due to adrenal secretions or by ligand-independent activation of progestin receptors, we repeated the experiment in estradiol-primed OVX/adrenalectomized rats. Prior removal of the adrenal glands blocked the rapid decrease in progestin receptor immunoreactivity, even though data from other experiments suggest that progestin receptors are activated by VCS at this time. These studies suggest the possibility that progestin receptors may be affected differentially by progesterone-dependent or by progesterone-independent pathways. This raises the possibility that activation of progestin receptors by these two distinct pathways may lead to different neuronal consequences.

Mating-related stimulation induces phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein-32 in progestin receptor-containing areas in the female rat brain.

Vaginal-cervical stimulation induces a number of physiological and behavioral events, including the facilitation of mating behavior. Although the facilitation of one component of mating behavior, lordosis, by vaginal-cervical stimulation does not require the presence of progesterone, it appears to be mediated by neural progestin receptors. Abundant evidence suggests that dopamine may play a role in the neural circuitry activated by vaginal-cervical stimulation, including the mating-induced release of dopamine in progestin receptor-containing areas of the brain, changes in the activational state of progestin receptors because of dopamine D1 receptor stimulation, facilitation of lordosis by D1 receptor stimulation in estradiol-primed rats via progesterone-independent events, and D1 agonist-induced neuronal responses in progestin receptor-containing areas and cells. We tested the hypothesis that vaginal-cervical stimulation induces phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32; Mr = 32,000), a protein phosphorylated predominantly in response to the stimulation of D1 receptors. At 9 d after ovariectomy, female rats were injected subcutaneously with a behaviorally effective dose of estradiol benzoate. At 48 hr later they received vaginal-cervical or control (perineal) stimulation, and they were perfused 1 hr later. Vaginal-cervical stimulation increased the number of cells expressing pDARPP-32 immunoreactivity by 92% in the medial preoptic nucleus, 134% in the caudal ventromedial hypothalamic nucleus, 123% in the posterodorsal medial amygdala, and 103% in the bed nucleus of the stria terminalis. These results suggest that some of the neuronal effects of vaginal-cervical stimulation, and perhaps other social or environmental stimuli, are mediated by phosphorylation of DARPP-32, perhaps via stimulation of D1 receptors, within progestin receptor-containing areas.