Joseph R. Kurian

Sex Differences in Epigenetic Regulation of the Estrogen Receptor-α Promoter within the Developing Preoptic Area

Sex differences in the brain are largely organized by a testicular hormone surge that occurs in males shortly after birth. Although this hormone surge is transient, sex differences in brain and behavior are lasting. Here we describe a sex difference in DNA methylation of the estrogen receptor-alpha (ERalpha) promoter region within the developing rat preoptic area, with males exhibiting more DNA methylation within the ERalpha promoter than females. More importantly, we report that simulating maternal grooming, a form of maternal interaction that is sexually dimorphic with males experiencing more than females during the neonatal period, effectively masculinizes female ERalpha promoter methylation and gene expression. This suggests natural variations in maternal care that are directed differentially at males vs. females can influence sex differences in the brain by creating sexually dimorphic DNA methylation patterns. We also find that the early estradiol exposure may contribute to sex differences in DNA methylation patterns. This suggests that early social interaction and estradiol exposure may converge at the genome to organize lasting sex differences in the brain via epigenetic differentiation.

Mecp2 Organizes Juvenile Social Behavior in a Sex-Specific Manner

Methyl-CpG-binding protein 2 (MeCP2) binds methylated DNA and recruits corepressor proteins to modify chromatin and alter gene transcription. Mutations of the MECP2 gene can cause Rett syndrome, whereas subtle reductions of MeCP2 expression may be associated with male-dominated social and neurodevelopmental disorders. We report that transiently decreased amygdala Mecp2 expression during a sensitive period of brain sexual differentiation disrupts the organization of sex differences in juvenile social play behavior. Interestingly, neonatal treatment with Mecp2 small interfering RNA within the developing amygdala reduced juvenile social play behavior in males but not females. Reduced Mecp2 expression did not change juvenile sociability or anxiety-like behavior, suggesting that this disruption is associated with subtle behavioral modification. This suggests that Mecp2 may have an overlooked role in the organization of sexually dimorphic behaviors and that male juvenile behavior is particularly sensitive to Mecp2 disruption during this period of development.

Sex Difference in Mecp2 Expression During a Critical Period of Rat Brain Development

Pervasive developmental disorder is a classification covering five related conditions including the neurodevelopmental disorder Rett syndrome (RTT) and autism. Of these five conditions, only RTT has a known genetic cause, with mutations in Methyl-CpG-binding protein 2 (MeCP2), a global repressor of gene expression, responsible for the majority of RTT cases. However, recent evidence indicates that reduced MeCP2 expression or activity is also found in autism and other disorders with overlapping phenotypes. Considering the sex difference in autism diagnosis, with males diagnosed four times more often than females, we questioned if a sex difference existed in the expression of MeCP2, in particular within the amygdala, a region that develops atypically in autism. We found that male rats express significantly less mecp2 mRNA and protein than females within the amygdala, as well as the ventromedial hypothalamus (VMH), but not within the preoptic area (POA) on post-natal day 1 (PN1). At PN10 these differences were gone; however, on this day males had more mecp2 mRNA than females within the POA. The transient sex difference of mecp2 expression during the steroid-sensitive period of brain development suggests that mecp2 may participate in normal sexual differentiation of the rat brain. Considering the strong link between MeCP2 and neurodevelopmental disorders, the lower levels of mecp2 expression in males may also underlie a biological risk for mecp2-related neural disorders.

Recent Discoveries on the Control of Gonadotrophin-Releasing Hormone Neurones in Nonhuman Primates

Since Ernst Knobil proposed the concept of the gonadotrophin‐releasing hormone (GnRH) pulse‐generator in the monkey hypothalamus three decades ago, we have made significant progress in this research area with cellular and molecular approaches. First, an increase in pulsatile GnRH release triggers the onset of puberty. However, the question of what triggers the pubertal increase in GnRH is still unclear. GnRH neurones are already mature before puberty but GnRH release is suppressed by a tonic GABA inhibition. Our recent work indicates that blocking endogenous GABA inhibition with the GABAA receptor blocker, bicuculline, dramatically increases kisspeptin release, which plays an important role in the pubertal increase in GnRH release. Thus, an interplay between the GABA, kisspeptin, and GnRH neuronal systems appears to trigger puberty. Second, cultured GnRH neurones derived from the olfactory placode of monkey embryos exhibit synchronised intracellular calcium, [Ca2+]i, oscillations and release GnRH in pulses at approximately 60‐min intervals after 14 days in vitro (div). During the first 14 div, GnRH neurones undergo maturational changes from no [Ca2+]i oscillations and little GnRH release to the fully functional state. Recent work also shows GnRH mRNA expression increases during in vitro maturation. This mRNA increase coincides with significant demethylation of a CpG island in the GnRH 5′‐promoter region. This suggests that epigenetic differentiation occurs during GnRH neuronal maturation. Third, oestradiol causes rapid, direct, excitatory action in GnRH neurones and this action of oestradiol appears to be mediated through a membrane receptor, such as G‐protein coupled receptor 30.

Unusual Dehydroxylation of Antimicrobial Amidoxime Prodrugs by Cytochrome b5 and NADH Cytochrome b5 Reductase

Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble cytochrome b5 and NAD cytochrome b5 reductase, expressed in Escherichia coli. Kinetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to cytochrome b5 and b5 reductase and by chemical inhibitors of b5 reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain cytochrome b5/b5 reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed cytochrome b5/b5 reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. Km values were similar to those for microsomes, and Vmax values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with cytochrome b5 or b5 reductase alone or with cytochrome P450 reductase alone or with cytochrome b5. These results indicate that cytochrome b5 and b5 reductase play a direct role in metabolic activation of DB289 to furamidine.

Tonic Control of Kisspeptin Release in Prepubertal Monkeys: Implications to the Mechanism of Puberty Onset

Previously we have shown that a reduction in γ-amino butyric acid (GABA) inhibition is critical for the mechanism initiating puberty onset because chronic infusion of the GABA(A) receptor antagonist, bicuculline, significantly increased GnRH release and accelerated the timing of menarche and first ovulation in female rhesus monkeys. Because previous studies in our laboratory indicate that in prepubertal female monkeys, kisspeptin release in the medial basal hypothalamus is low, whereas kisspeptin-10 can stimulate GnRH release, we hypothesized that a low level of kisspeptin release prior to puberty onset is due to tonic GABA inhibition. To test this hypothesis we examined the effects of bicuculline infusion on kisspeptin release using a microdialysis method. We found that bicuculline at 1 μM dramatically stimulates kisspeptin release in the medial basal hypothalamus of prepubertal monkeys but had little effect on kisspeptin release in midpubertal monkeys. We further examined whether bicuculline-induced GnRH release is blocked by the presence of the kisspeptin antagonist, peptide 234. We found that inhibition of kisspeptin signaling blocked the bicuculline-induced stimulation of GnRH release, suggesting that kisspeptin neurons may relay inhibitory GABA signals to GnRH neurons. This implies that a reduction in tonic GABA inhibition of GnRH release is, at least in part, mediated through kisspeptin neurons.

Body Weight Impact on Puberty: Effects of High-Calorie Diet on Puberty Onset in Female Rhesus Monkeys

Secular trends toward a declining age at puberty onset with correlated changes in body weight have been reported in economically advanced countries. This has been attributed to excess calorie intake along with reduced physical activity in children. However, because the timing of puberty in humans is also influenced by other factors, such as genetic traits, living conditions, geographical location, and environmental chemicals, it is difficult to distinguish the effect of diet and body size from other factors in a human population. Here we report that feeding juvenile female rhesus monkeys born and raised at the Wisconsin National Primate Research Center with a high-calorie diet results in acceleration of body growth and precocious menarche. The monkeys fed a high-calorie diet also had an elevated body mass index. The most significant treatment effects on circulating hormones were increased leptin and IGF-I levels throughout the experiment. The findings of this study suggest the importance of close monitoring of juvenile feeding behaviors as an important intervention to reduce the prevalence of precocious development and metabolic diseases in adulthood.

Neonatal MeCP2 is important for the organization of sex differences in vasopressin expression

Several neurodevelopmental disorders are marked by atypical Methyl-CpG-binding protein 2 (MeCP2) expression or function; however, the role of MeCP2 is complex and not entirely clear. Interestingly, there are sex differences in some of these disorders, and it appears that MeCP2 has sex-specific roles during development. Specifically, recent data indicate that a transient reduction in MeCP2 within developing amygdala reduces juvenile social play behavior in males to female-typical levels. These data suggest that MeCP2 within the amygdala is involved in programming lasting sex differences in social behavior. In the present study, we infused MeCP2 or control siRNA into the amygdala of male and female rats during the first three days of postnatal life in order to assess the impact of a transient reduction in MeCP2 on arginine vasopressin (AVP), a neural marker that is expressed differentially between males and females and is linked to a number of social behaviors. The expression of AVP, as well as several other genes, was measured in two-week old and adult animals. Two-week old males expressed more AVP and galanin mRNA in the amygdala than females, and a transient reduction in MeCP2 eliminated this sex difference by reducing the expression of both gene products in males. A transient reduction in MeCP2 also decreased androgen receptor (AR) mRNA in two-week old males. In adulthood, control males had more AVP-immunoreactive (AVP-ir) cells than females in the centromedial amygdala (CMA), bed nucleus of the stria terminalis (BST) and in the fibers that project from these cells to the lateral septum (LS). A transient reduction in MeCP2 eliminated this sex difference. Interestingly, there were no lasting differences in galanin or AR levels in adulthood. Reducing MeCP2 levels during development did not alter estrogen receptorα, neurofilament or Foxg1. We conclude that a transient reduction in MeCP2 expression in the developing male amygdala has a transient impact on galanin and AR expression but a lasting impact on AVP expression, highlighting the importance of MeCP2 in organizing sex differences in the amygdala.

Adverse effects of EMLA (lidocaine/prilocaine) cream and efficacy for the placement of jugular catheters in hospitalized cats.

EMLA is a lidocaine/prilocaine cream used for topical analgesia in human pediatric patients. The purpose of this study was to establish the safety of EMLA in clinically ill cats, to measure systemic absorption and to determine whether EMLA reduced the need for sedation for the placement of jugular catheters. Thirty-one cats were randomized to either a placebo or EMLA cream group. Cream was applied to a 10 cm2 area over the jugular vein, with 1 h of occlusive dressing. Neither anesthetic was systemically absorbed in any cat, and no adverse clinical signs were observed. Struggling during catheter placement was less in the EMLA-treated cats compared to placebo, but did not reach significance (P=0.06). Jugular catheters were successfully placed in 60% of EMLA-treated cats and 38% of placebo cats; this difference was not statistically significant and may not justify the added steps of EMLA cream administration for this purpose. However, EMLA does appear to be safe in clinically ill cats, and may be useful for other applications such as for skin mass removal or repeated venepuncture.

Epigenetic Control of Gonadotropin Releasing Hormone Neurons

Epigenetic modifications to the genome, including DNA methylation and histone modifications, occur in response to external stimuli. Reproductive function is highly sensitive to environmental conditions including season, diet, hormonal changes, and exposure to chemical contaminants. GnRH neurons, which play a key role in reproduction, are particularly sensitive to various environmental stimuli. We recently reported that the rhesus monkey GnRH gene exhibits distinct epigenetic changes during embryonic development. More recently, we further found that a similar epigenetic phenomenon occurs across puberty. In this article we highlight recent findings, including those of afferent inputs, to describe the epigenetic control of GnRH circuit development as a link between the environment and reproductive function.