Developmental variations in environmental influences including endocrine disruptors on pubertal timing and neuroendocrine control: Revision of human observations and mechanistic insight from rodents.

Puberty presents remarkable individual differences in timing reaching over 5 years in humans. We put emphasis on the two edges of the age distribution of pubertal signs in humans and point to an extended distribution towards earliness for initial pubertal stages and towards lateness for final pubertal stages. Such distortion of distribution is a recent phenomenon. This suggests changing environmental influences including the possible role of nutrition, stress and endocrine disruptors. Our ability to assess neuroendocrine effects and mechanisms is very limited in humans. Using the rodent as a model, we examine the impact of environmental factors on the individual variations in pubertal timing and the possible underlying mechanisms. The capacity of environmental factors to shape functioning of the neuroendocrine system is thought to be maximal during fetal and early postnatal life and possibly less important when approaching the time of onset of puberty.

Early developmental actions of endocrine disruptors on the hypothalamus, hippocampus, and cerebral cortex.

Sex steroids and thyroid hormones play a key role in the development of the central nervous system. The critical role of these hormonal systems may explain the sensitivity of the hypothalamus, the cerebral cortex, and the hippocampus to endocrine-disrupting chemicals (EDC). This review examines the evidence for endocrine disruption of glial-neuronal functions in the hypothalamus, hippocampus, and cerebral cortex. Focus was placed on two well-studied EDC, the insecticide dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCB). DDT is involved in neuroendocrine disruption of the reproductive axis, whereas polychlorinated biphenyls (PCB) interact with both the thyroid hormone- and sex steroid-dependent systems and disturb the neuroendocrine control of reproduction and development of hippocampus and cortex. These results highlight the impact of EDC on the developing nervous system and the need for more research in this area.

Multi- and Transgenerational Outcomes of an Exposure to a Mixture of Endocrine-Disrupting Chemicals (EDCs) on Puberty and Maternal Behavior in the Female Rat.

BACKGROUND: The effects of endocrine-disrupting chemicals (EDCs) on fertility and reproductive development represent a rising concern in modern societies. Although the neuroendocrine control of sexual maturation is a major target of EDCs, little is known about the potential role of the hypothalamus in puberty and ovulation disruption transmitted across generations. OBJECTIVES: We hypothesized that developmental exposure to an environmentally relevant dose of EDC mixture could induce multi- and/or transgenerational alterations of sexual maturation and maternal care in female rats through epigenetic reprograming of the hypothalamus. We investigated the transmission of a disrupted reproductive phenotype via the maternal germline or via nongenomic mechanisms involving maternal care. METHODS: Adult female Wistar rats were exposed prior to and during gestation and until the end of lactation to a mixture of the following 13 EDCs: di-n-butyl phthalate (DnBP), di(2-ethylhexyl) phthalate (DEHP), bisphenol A (BPA), vinclozolin, prochloraz, procymidone, linuron, epoxynaxole, dichlorodiphenyldichloroethylene, octyl methoxynimmate, 4-methylbenzylidene camphor (4-MBC), butylparaben, and acetaminophen. Perinatally exposed offspring (F1) were mated with unexposed males to generate germ cell (F2) and transgenerationally exposed (F3 and F4) females. Sexual maturation, maternal behavior, and hypothalamic targets of exposure were studied across generations. RESULTS: Germ cell (F2) and transgenerationally (F3) EDC-exposed females, but not F1, displayed delayed pubertal onset and altered folliculogenesis. We reported a transgenerational alteration of key hypothalamic genes controlling puberty and ovulation (Kiss1, Esr1, and Oxt), and we identified the hypothalamic polycomb group of epigenetic repressors as actors of this mechanism. Furthermore, we found a multigenerational reduction of maternal behavior (F1-F3) induced by a loss in hypothalamic dopaminergic signaling. Using a cross-fostering paradigm, we identified that the reduction in maternal phenotype was normalized in EDC-exposed pups raised by unexposed dams, but no reversal of the pubertal phenotype was achieved. DISCUSSION: Rats developmentally exposed to an EDC mixture exhibited multi- and transgenerational disruption of sexual maturation and maternal care via hypothalamic epigenetic reprogramming. These results raise concerns about the impact of EDC mixtures on future generations. https://doi.org/10.1289/EHP8795.

Right Atrial Myocardial Remodeling in Children With Atrial Septal Defect Involves Inflammation, Growth, Fibrosis, and Apoptosis.

Introduction: Myocardial remodeling due to large atrial septum defect (ASD) is macroscopically characterized by dilation of the right-sided cardiac cavities secondary to volume overload, the cellular mechanisms of which are not yet understood. We postulated that inflammation, fibrosis, and cell death are actors of right atrial remodeling secondary to ASD. Patients and Methods: In 12 children with large ASD (median age: 63 months), expression of genes coding for proteins involved in the response to cell stress and -protection, inflammation, growth and angiogenesis, fibrosis, and apoptosis was assessed by RT-PCR in right atrial myocardial biopsies taken during cardiac surgery. The presence of cytokines in myocardial cells was confirmed by immunohistochemistry and effective apoptosis by TUNEL assay. Results: In all patients investigated, a cellular response to early mechanical stress with the initiation of early protective mechanisms, of inflammation (and its control), -growth, and -angiogenesis, of fibrosis and apoptosis was present. The apoptotic index assessed by TUNEL assay averaged 0.3%. Conclusions: In children with large ASD, macroscopic right atrial remodeling relates to cellular mechanisms involving the expression of numerous genes that either still act to protect cells and tissues but that also harm as they initiate and/or sustain inflammation, fibrosis, and cell death by apoptosis. This may contribute to long term morbidity in patients with ASD.

Persistent vs Transient Alteration of Folliculogenesis and Estrous Cycle After Neonatal vs Adult Exposure to Bisphenol A.

Exposure to bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical (EDC), is known to produce variable effects on female puberty and ovulation. This variability of effects is possibly due to differences in dose and period of exposure. Little is known about the effects of adult exposure to environmentally relevant doses of this EDC and the differences in effect after neonatal exposure. This study sought to compare the effects of neonatal vs adult exposure to a very low dose or a high dose of BPA for 2 weeks on ovulation and folliculogenesis and to explore the hypothalamic mechanisms involved in such disruption by BPA. One-day-old and 90-day-old female rats received daily subcutaneous injections of corn oil (vehicle) or BPA (25 ng/kg/d or 5 mg/kg/d) for 15 days. Neonatal exposure to both BPA doses significantly disrupted the estrous cycle and induced a decrease in primordial follicles. Effects on estrous cyclicity and folliculogenesis persisted into adulthood, consistent with a disruption of organizational mechanisms. During adult exposure, both doses caused a reversible decrease in antral follicles and corpora lutea. A reversible disruption of the estrous cycle associated with a delay and a decrease in the amplitude of the LH surge was also observed. Alterations of the hypothalamic expression of the clock gene Per1 and the reproductive peptide phoenixin indicated a disruption of the hypothalamic control of the preovulatory LH surge by BPA.

Oxytocin facilitates female sexual maturation through a glia-to-neuron signaling pathway.

It has been earlier proposed that oxytocin could play a facilitatory role in the preovulatory LH surge in both rats and humans. We here provide evidence that oxytocin also facilitates sexual maturation in female rats. The administration of an oxytocin antagonist for 6 d to immature female rats decreased GnRH pulse frequency ex vivo and delayed the age at vaginal opening and first estrus. The in vitro reduction in GnRH pulse frequency required chronic blockade of oxytocin receptors, because it was not acutely observed after a single injection of the antagonist. Hypothalamic explants exposed to the antagonist in vitro showed a reduced GnRH pulse frequency and failed to respond to oxytocin with GnRH release. Prostaglandin E(2) (PGE(2)) mimicked the stimulatory effect of oxytocin on GnRH pulse frequency, and inhibition of PG synthesis blocked the effect of oxytocin, suggesting that oxytocin accelerates pulsatile GnRH release via PGE(2). The source of PGE(2) appears to be astrocytes, because oxytocin stimulates PGE(2) release from cultured hypothalamic astrocytes. Moreover, astrocytes express oxytocin receptors, whereas GnRH neurons do not. These results suggest that oxytocin facilitates female sexual development and that this effect is mediated by a mechanism involving glial production of PGE(2).

Mechanisms of interaction of endocrine-disrupting chemicals with glutamate-evoked secretion of gonadotropin-releasing hormone.

In previous studies, we detected a dichlorodiphenyltrichloroethane (DDT) derivative in the serum of children with sexual precocity after migration from developing countries. Recently, we reported that DDT stimulated pulsatile gonadotropin-releasing hormone (GnRH) secretion and sexual maturation in the female rat. The aim of this study was to delineate the mechanisms of interaction of endocrine-disrupting chemicals including DDT with GnRH secretion evoked by glutamate in vitro. Using hypothalamic explants obtained from 15-day-old female rats, estradiol (E2) and DDT caused a concentration-related increase in glutamate-evoked GnRH release while p,p'-dichlorodiphenyldichloroethene and methoxychlor had no effect. The effective DDT concentrations in vitro were consistent with the serum concentrations measured in vivo 5 days after exposure of immature rats to 10 mg/kg/day of o,p'-DDT. Bisphenol A induced some stimulatory effect, whereas no change was observed with 4-nonylphenol. The o,p'-DDT effects in vitro were prevented partially by a selective antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptors. A complete prevention of o,p'-DDT effects was caused by an estrogen receptor (ER) antagonist as well as an aryl hydrocarbon receptor (AHR) antagonist and inhibitors of protein kinases A and C and mitogen-activated kinases. While an intermittent incubation with E2 caused no change in amplification of the glutamate-evoked GnRH release for 4 h, continuous incubation with E2 or o,p'-DDT caused an increase of this amplification after 3.5 h of incubation. In summary, DDT amplifies the glutamate-evoked GnRH secretion in vitro through rapid and slow effects involving ER, AHR, and AMPA receptor mediation.

Neuroendocrine disruption without direct endocrine mode of action: Polychloro-biphenyls (PCBs) and bisphenol A (BPA) as case studies.

Endocrine disruption is commonly thought to be restricted to a direct endocrine mode of action i.e. the perturbation of the activation of a given type of hormonal receptor by its natural ligand. Consistent with the WHO definition of an endocrine disrupter, a key issue is the "altered function(s) of the endocrine system". Such altered functions can result from different chemical interactions, beyond agonistic or antagonistic effect at a given receptor. Based on neuroendocrine disruption by polychlorinated biphenyls and bisphenol A, this paper proposes different mechanistic paradigms that can result in adverse health effects. They are a consequence of altered endocrine function(s) secondary to chemical interaction with different steps in the physiological regulatory processes, thus accounting for a possibly indirect endocrine mode of action.

Delayed Neuroendocrine Sexual Maturation in Female Rats After a Very Low Dose of Bisphenol A Through Altered GABAergic Neurotransmission and Opposing Effects of a High Dose.

Rat sexual maturation is preceded by a reduction of the interpulse interval (IPI) of GnRH neurosecretion. This work aims at studying disruption of that neuroendocrine event in females after early exposure to a very low dose of bisphenol A (BPA), a ubiquitous endocrine disrupting chemical. Female rats were exposed to vehicle or BPA 25 ng/kg·d, 25 µg/kg·d, or 5 mg/kg·d from postnatal day (PND)1 to PND5 or PND15. Exposure to 25 ng/kg·d of BPA for 5 or 15 days was followed by a delay in developmental reduction of GnRH IPI studied ex vivo on PND20. After 15 days of exposure to that low dose of BPA, vaginal opening tended to be delayed. In contrast, exposure to BPA 5 mg/kg·d for 15 days resulted in a premature reduction in GnRH IPI and a trend toward early vaginal opening. RNA sequencing analysis on PND20 indicated that exposure to BPA resulted in opposing dose effects on the mRNA expression of hypothalamic genes involved in gamma aminobutyric acid A (GABAA) neurotransmission. The study of GnRH secretion in vitro in the presence of GABAA receptor agonist/antagonist confirmed an increased or a reduced GABAergic tone after in vivo exposure to the very low or the high dose of BPA, respectively. Overall, we show for the first time that neonatal exposure to BPA leads to opposing dose-dependent effects on the neuroendocrine control of puberty in the female rat. A very low and environmentally relevant dose of BPA delays neuroendocrine maturation related to puberty through increased inhibitory GABAergic neurotransmission.

In vitro paradigms for the study of GnRH neuron function and estrogen effects.

The elaboration of in vitro paradigms has enabled direct study of GnRH secretion and the regulation of this process. Common findings using different models are the pulsatile nature and calcium-dependency of GnRH secretion, the excitatory effect of glutamate, and the inhibitory or excitatory effect of GABA. Among the different paradigms, the fetal olfactory placode cultures exhibit the unique property of migration in vitro and may retain the capacity to undergo maturational changes in vitro. The short-term incubation of hypothalamic explants obtained at different ages enables one to study developmental changes as well. Estrogens may have important roles in the regulation of GnRH function and can act indirectly via the neighboring neuronal/glial apparatus and directly on GnRH neurons at the cell body and terminal levels. A direct effect is supported by the recent localization of ERalpha and ERbeta transcripts in GnRH neurons using most paradigms. Discrepant effects of estrogens on GnRH neurons were observed since GnRH biosynthesis is inhibited while GnRH secretion can be either stimulated, unaffected, or reduced. It is likely that the regulatory role of sex steroids including estradiol is very complex since it could involve direct and indirect effects on GnRH neurons through genomic and/or non-genomic mechanisms.

Alteration of rat fetal cerebral cortex development after prenatal exposure to polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are environmental contaminants that persist in environment and human tissues. Perinatal exposure to these endocrine disruptors causes cognitive deficits and learning disabilities in children. These effects may involve their ability to interfere with thyroid hormone (TH) action. We tested the hypothesis that developmental exposure to PCBs can concomitantly alter TH levels and TH-regulated events during cerebral cortex development: progenitor proliferation, cell cycle exit and neuron migration. Pregnant rats exposed to the commercial PCB mixture Aroclor 1254 ended gestation with reduced total and free serum thyroxine levels. Exposure to Aroclor 1254 increased cell cycle exit of the neuronal progenitors and delayed radial neuronal migration in the fetal cortex. Progenitor cell proliferation, cell death and differentiation rate were not altered by prenatal exposure to PCBs. Given that PCBs remain ubiquitous, though diminishing, contaminants in human systems, it is important that we further understand their deleterious effects in the brain.

Pubertal timing after neonatal diethylstilbestrol exposure in female rats: neuroendocrine vs peripheral effects and additive role of prenatal food restriction.

We studied the effects of neonatal exposure to diethylstilbestrol (DES) on pubertal timing in female rats. We examined associated neuroendocrine changes and effects of prenatal food restriction. Age at vaginal opening was advanced after exposure to 10 µg/kg/d of DES and delayed after 1 µg/kg/d (subcutaneous injections). Using this lower dose, pulsatile GnRH secretion was slower at 25 days of age. Both doses reduced KiSS1 mRNA levels at 15 days of age. Using functional Kisspeptin promoter assay, 1 or 10 µM DES reduced or increased KISS1 transcription, respectively. Leptin stimulatory effect on GnRH secretion in vitro (15 days of age) was reduced after prenatal food restriction and neonatal DES exposure (higher dose), both effects being cumulative. Thus, alterations in pubertal timing by DES neonatally are not unequivocally toward precocity, the level of exposure being critical. We provide evidence of neuroendocrine disruption and interaction with prenatal food availability.

Neuroendocrine disruption of pubertal timing and interactions between homeostasis of reproduction and energy balance.

The involvement of environmental factors such as endocrine disrupting chemicals (EDCs) in the timing of onset of puberty is suggested by recent changes in age at onset of puberty and pattern of distribution that are variable among countries, as well as new forms of sexual precocity after migration. However, the evidence of association between early or late pubertal timing and exposure to EDCs is weak in humans, possibly due to heterogeneity of effects likely involving mixtures and incapacity to assess fetal or neonatal exposure retrospectively. The neuroendocrine system which is crucial for physiological onset of puberty is targeted by EDCs. These compounds also act directly in the gonads and peripheral sex-steroid sensitive tissues. Feedbacks add to the complexity of regulation so that changes in pubertal timing caused by EDCs can involve both central and peripheral mechanisms. In experimental conditions, several neuroendocrine endpoints are affected by EDCs though only few studies including from our laboratory aimed at EDC involvement in the pathophysiology of early sexual maturation. Recent observations support the concept that EDC cause disturbed energy balance and account for the obesity epidemic. Several aspects are linking this system and the reproductive axis: coexisting neuroendocrine and peripheral effects, dependency on fetal/neonatal programming and the many factors cross-linking the two systems, for instance leptin, adiponectin, Agouti Related Peptide (AgRP). This opens perspectives for future research and, hopefully, measures preventing the disturbances of homeostasis caused by EDCs.

Early maturation of gonadotropin-releasing hormone secretion and sexual precocity after exposure of infant female rats to estradiol or dichlorodiphenyltrichloroethane.

An increase in the frequency of pulsatile gonadotropin-releasing hormone (GnRH) secretion in vitro and a reduction in LH response to GnRH in vivo characterize hypothalamic-pituitary maturation before puberty in the female rat. In girls migrating for international adoption, sexual precocity is frequent and could implicate former exposure to the insecticide dichlorodiphenyltrichloroethane (DDT), since a long-lasting DDT derivative has been detected in the serum of such children. We aimed at studying the effects of early transient exposure to estradiol (E(2)) or DDT in vitro and in vivo in the infantile female rat. Using a static incubation system of hypothalamic explants from 15-day-old female rats, a concentration- and time-dependent reduction in GnRH interpulse interval (IPI) was seen during incubation with E(2) and DDT isomers. These effects were prevented by antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)/kainate receptors and estrogen receptor. Also, o,p'-DDT effects were prevented by an antagonist of the aryl hydrocarbon orphan dioxin receptor (AHR). After subcutaneous injections of E(2) or o,p'-DDT between Postnatal Days (PNDs) 6 and 10, a decreased GnRH IPI was observed on PND 15 as an ex vivo effect. After DDT administration, serum LH levels in response to GnRH were not different from controls on PND 15, whereas they tended to be lower on PND 22. Subsequently, early vaginal opening (VO) and first estrus were observed together with a premature age-related decrease in LH response to GnRH. After prolonged exposure to E(2) between PNDs 6 and 40, VO occurred at an earlier age, but first estrus was delayed. We conclude that a transient exposure to E(2) or o,p'-DDT in early postnatal life is followed by early maturation of pulsatile GnRH secretion and, subsequently, early developmental reduction of LH response to GnRH that are possible mechanisms of the subsequent sexual precocity. The early maturation of pulsatile GnRH secretion could involve effects mediated through estrogen receptor and/or AHR as well as AMPA/kainate subtype of glutamate receptors.

Factors accounting for perinatal occurrence of pulsatile gonadotropin-releasing hormone secretion in vitro in rats.

Our aim was to study the inhibitory and facilitatory factors possibly accounting for the undetectable activity of the GnRH pulse generator in late fetal life in vitro and its awakening in early postnatal life. Gamma aminobutyric acid (GABA(A)) receptor antagonism using SR 95 531 did not cause any secretory pulse in fetal explants, whereas a significant stimulation of GnRH pulse frequency was obtained at 5 and 15 days. GnRH secretory response to repeated N-methyl-D-aspartate (NMDA) stimulation showed progressive disappearance, indicating that the inhibitory autofeedback was operating. GnRH release caused by glutamine was respectively 9% and 20% of that evoked by glutamate in fetal and 5-day-old rats whereas both amino acids were equally active at 15 days. Explants obtained after cesarean section performed at onset of labor did not show any secretory pulse, while pulses could be observed with explants obtained 2 h after vaginal delivery. Incubation of fetal explants with oxytocin (10(-8) M) or prostaglandin E2 (PGE2) (10(-6) M) resulted in occurrence of GnRH secretory pulses. A facilitatory effect of the oxytocin was shown to persist on Days 1, 5, and 15 and inhibitory effects of an oxytocin receptor antagonist provided some evidence of endogenous oxytocin involvement. We conclude that, in the fetal rat hypothalamus, GnRH inhibitory autofeedback and GABAergic inputs do not account for the absence of pulsatile GnRH secretion in vitro. A low rate of glutamate biosynthesis from glutamine is a possibly limiting factor. Oxytocin and PGE2 can play a facilitatory role in the postpartal occurrence of pulsatile GnRH secretion.

Early onset of puberty: tracking genetic and environmental factors.

Under physiological conditions, factors affecting the genetic control of hypothalamic functions are predominant in determining the individual variations in timing of pubertal onset. In pathological conditions, however, these variations can involve different genetic susceptibility and the interaction of environmental factors. The high incidence of precocious puberty in foreign children migrating to Belgium and the detection in their plasma of a long-lasting 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) residue suggest the potential role of environmental endocrine disrupting chemicals in the early onset of puberty. This hypothesis was confirmed by experimental data showing that temporary exposure of immature female rats to DDT in vivo results in early onset of puberty. We compared the gene expression profile of hypothalamic hamartoma associated or not with precocious puberty in order to identify gene networks responsible for both hamartoma-dependent sexual precocity and the onset of normal human puberty. In conclusion, pathological variations in the timing of puberty may provide unique information about the interactions of either environmental conditions or genetic susceptibility with the hypothalamic mechanism controlling the onset of sexual maturation, as shown by examples of precocious puberty following exposure to endocrine disrupters or due to hypothalamic hamartoma.