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.

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.

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.

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.

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.

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.

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.

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.

D1 dopamine receptor agonist (SKF-38393) induction of Fos immunoreactivity in progestin receptor-containing areas of female rat brain.

Injection of dopamine or dopamine receptor subtype agonists facilitates the expression of lordosis in estrogen-primed female rats. The D1 receptor specific agonist, SKF-38393, facilitates lordosis in estradiol-primed female rats via a process that requires progestin receptors. Based on these data, neuronal response to the D1 receptor agonist SKF-38393 was assessed by expression of the immediate early gene protein, Fos. In the first experiment we examined the modulation of Fos expression by D1 agonists in progestin receptor-containing areas of estradiol-primed female rat brain. In the second experiment we examined if there are progestin if there are progestin receptor-containing cells that respond to stimulation of D1 receptors with increased Fos expression. Ten to 14 days following ovariectomy and stereotaxic surgery, animals were injected with 5 micrograms estradiol benzoate. Forty eight h later they were injected intracerebroventricularly with 100 ng of SKF-38393 or saline. One h following injection animals were perfused, and brain sections immunostained for Fos protein. Results from the first experiment suggest that SKF-38393 increased the total number of Fos immunoreactive cells in the mid-ventromedial hypothalamic nucleus/ventrolateral portion (VMHVL), the caudal VMHVL, the paraventricular hypothalamic nucleus and the caudate putamen. In the medial preoptic area, the rostral VMHVL and the arcuate hypothalamic nucleus, there was a significant increase in the number of darkly stained Fos-immunoreactive cells following the SKF-38393 treatment. In the second study, SKF-38393 increased the number of progestin receptor-containing cells which contained Fos immunoreactivity in the caudal VMHVL. The results suggest potential sites of action for the facilitation of sexual behavior by centrally administered D1 agonists.

Progesterone-independent activation of rat brain progestin receptors by reproductive stimuli.

Activation of steroid hormone receptors by steroid hormones alters both the physiology and behavior of animals. Steroid hormone receptors (e.g., progestin receptors) can also be activated in the absence of steroid hormones by pharmacological treatment with neurotransmitters or neuropeptides. However, it is not known if progesterone-independent activation of brain progestin receptors occurs under natural, physiological, conditions. We report that increases in reproductive behavior and brain immediate early gene expression in female rats induced by mating stimuli can be blocked by prior treatment with progesterone antagonists in the absence of circulating progesterone. This suggests that progestin receptors are activated in a progesterone-independent manner by a physiologically relevant stimulus in female rats, thus implicating a novel pathway by which mating stimuli and other environmental influences could activate steroid receptors to influence neuronal response and behavior.

Progesterone treatment increases Fos-immunoreactivity within some progestin receptor-containing neurons in localized regions of female rat forebrain.

In female rats, the sequential release of estradiol and progesterone from the ovaries is required for the expression of sexual behavior during the estrous cycle. Many of the neuronal effects of estradiol and progesterone involve estrogen and progestin receptors. Treatment with a behaviorally-effective dose of estradiol increases Fos expression, suggestive of neuronal response, and subsequent treatment with a behaviorally-effective dose of progesterone further increases Fos expression within a few hours in female rat brain. In order to determine if neurons that respond to progesterone with increase Fos expression also contain progestin receptors, we used a double-label immunofluorescent technique to label both progestin receptors and Fos protein following progesterone or vehicle treatment of estradiol-primed female rats. As shown previously, progesterone treatment increased Fos expression in progestin receptor-containing regions, such as the ventromedial nucleus of the hypothalamus and the medial preoptic area. In addition, progesterone treatment induced a statistically-significant increase in Fos-immunoreactivity within progestin receptor-containing cells in the medial preoptic area and the ventromedial nucleus of the hypothalamus, but not in the arcuate nucleus. Therefore, many but not all of the neurons that respond to progesterone with increased Fos expression also contain progestin receptor-immunoreactivity. The progesterone-induced Fos expression within progestin receptor-containing neurons may or may not be associated with the effects of progesterone on sexual or other reproductive behaviors, as it remains to be tested. However, the Fos expression provides a useful marker to aid in identification of neurons that respond to a behaviorally-relevant dose of progesterone.

Reproductively-relevant stimuli induce Fos-immunoreactivity within progestin receptor-containing neurons in localized regions of female rat forebrain.

An experiment was conducted to determine if neurons that respond to stimuli associated with mating in female brain also contain progestin receptors. We found that a portion of the neurons that respond to stimuli associated with mating also contains progestin receptors. While the appropriate hormonal conditions are important for sexual receptivity, somatosensory information provided by the male also influences sexual behavior. One important stimulus provided by the male during copulation is vaginal-cervical stimulation (VCS). VCS has been shown to elicit many different behavioral and endocrine changes in female rats, such as increases in lordosis, pseudopregnancy, and termination of sexual receptivity. VCS also increases the expression of the immediate early gene product, Fos, in areas associated with reproduction. A portion of the neurons responding to VCS with increased Fos-immunoreactivity (Fos-IR) in female rat forebrain also contains estrogen receptors, illustrating that hormonal and mating-stimuli converge in a population of cells. As progesterone also plays an important role in female sex behavior, it is important to determine if some of the neurons also integrate information concerning serum progesterone levels and social interactions. Thus, we used a dual immunofluorescent technique to label both Fos-IR and progestin receptor-immunoreactivity (PR-IR) in the brains of estradiol-primed, ovariectomized female rats following VCS manually applied by the experimenter. Many of the neurons that respond to VCS with increased Fos-IR within the medial preoptic area, the arcuate nucleus, and the progestin receptor-rich areas of the rostral and caudal ventromedial nucleus of the hypothalamus also contain PR-IR.

Progesterone enhances an estradiol-induced increase in Fos immunoreactivity in localized regions of female rat forebrain.

In female rats, the onset of reproductive behavior depends on the sequential presence of estradiol followed by progesterone. Although treatment with high doses of estradiol has been shown to increase immunostaining for the Fos protein, an immediate early gene product that is expressed upon cellular activation, another report conflicts with this finding. However, the previous reports agree that subsequent treatment with progesterone has no apparent effect on Fos expression. In order to resolve this discrepancy and investigate possible effects of progesterone, we used Fos immunocytochemistry combined with computer-aided image analysis. In experiment one, we found that treatment with 5 micrograms of estradiol increased Fos immunoreactivity (Fos-IR) within a section of the medial preoptic area and the dorsal medial hypothalamus. Subsequent treatment with 500 micrograms of progesterone 1 hr before perfusion increased the intensity of the immunostaining within the medial preoptic area and the dorsal medial hypothalamus, although it had no significant effect on Fos-IR cell number. In experiment 2, a lower concentration of Fos antiserum was used in order to diminish the immunostaining sensitivity to a level in which no increase of Fos-IR cell number was observed after treatment with estradiol. Under these immunocytochemical conditions, subsequent treatment with progesterone increased the number of Fos-IR cells in the medial preoptic area, the dorsal medial hypothalamus and the steroid receptor-rich area lateral to the ventromedial hypothalamus. Thus, treatment with behaviorally effective doses of both estradiol and progesterone induces Fos expression in localized regions of female rat brain.