Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

BACKGROUND: The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. METHODS: nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. RESULTS: In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. CONCLUSIONS: Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

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.

Cellular and molecular features of EDC exposure: consequences for the GnRH network.

The onset of puberty and the female ovulatory cycle are important developmental milestones of the reproductive system. These processes are controlled by a tightly organized network of neurotransmitters and neuropeptides, as well as genetic, epigenetic and hormonal factors, which ultimately drive the pulsatile secretion of gonadotropin-releasing hormone. They also strongly depend on organizational processes that take place during fetal and early postnatal life. Therefore, exposure to environmental pollutants such as endocrine-disrupting chemicals (EDCs) during critical periods of development can result in altered brain development, delayed or advanced puberty and long-term reproductive consequences, such as impaired fertility. The gonads and peripheral organs are targets of EDCs, and research from the past few years suggests that the organization of the neuroendocrine control of reproduction is also sensitive to environmental cues and disruption. Among other mechanisms, EDCs interfere with the action of steroidal and non-steroidal receptors, and alter enzymatic, metabolic and epigenetic pathways during development. In this Review, we discuss the cellular and molecular consequences of perinatal exposure (mostly in rodents) to representative EDCs with a focus on the neuroendocrine control of reproduction, pubertal timing and the female ovulatory cycle.

Endocrine disrupters and possible contribution to pubertal changes.

The onset of puberty strongly depends on organizational processes taking place during the fetal and early postnatal life. Therefore, exposure to environmental pollutants such as Endocrine disrupting chemicals (EDCs) during critical periods of development can result in delayed/advanced puberty and long-term reproductive consequences. Human evidence of altered pubertal timing after exposure to endocrine disrupting chemicals is equivocal. However, the age distribution of pubertal signs points to a skewed distribution towards earliness for initial pubertal stages and towards lateness for final pubertal stages. Such distortion of distribution is a recent phenomenon and suggests environmental influences including the possible role of nutrition, stress and endocrine disruptors. Rodent and ovine studies indicate a role of fetal and neonatal exposure to EDCs, along the concept of early origin of health and disease. Such effects involve neuroendocrine mechanisms at the level of the hypothalamus where homeostasis of reproduction is programmed and regulated but also peripheral effects at the level of the gonads or the mammary gland.

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.

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.

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.

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 mechanisms controlling female puberty: new approaches, new concepts.

Sexual development and mature reproductive function are controlled by a handful of neurones that, located in the basal forebrain, produce the decapeptide luteinizing hormone releasing hormone (LHRH). LHRH is released into the portal system that connects the hypothalamus to the pituitary gland and act on the latter to stimulate the synthesis and release of gonadotrophin hormones. The pubertal activation of LHRH release requires coordinated changes in excitatory and inhibitory inputs to LHRH-secreting neurones. These inputs are provided by both transsynaptic and glia-to-neurone communication pathways. Using cellular and molecular approaches, in combination with transgenic animal models and high-throughput procedures for gene discovery, we are gaining new insight into the basic mechanisms underlying this dual control of LHRH secretion and, hence, the initiation of mammalian puberty. Our results suggest that the initiation of puberty requires reciprocal neurone-glia communication involving excitatory amino acids and growth factors, and the coordinated actions of a group of transcriptional regulators that appear to represent a higher level of control governing the pubertal process.

Minireview: the neuroendocrine regulation of puberty: is the time ripe for a systems biology approach?

The initiation of mammalian puberty requires an increase in pulsatile release of GnRH from the hypothalamus. This increase is brought about by coordinated changes in transsynaptic and glial-neuronal communication. As the neuronal and glial excitatory inputs to the GnRH neuronal network increase, the transsynaptic inhibitory tone decreases, leading to the pubertal activation of GnRH secretion. The excitatory neuronal systems most prevalently involved in this process use glutamate and the peptide kisspeptin for neurotransmission/neuromodulation, whereas the most important inhibitory inputs are provided by gamma-aminobutyric acid (GABA)ergic and opiatergic neurons. Glial cells, on the other hand, facilitate GnRH secretion via growth factor-dependent cell-cell signaling. Coordination of this regulatory neuronal-glial network may require a hierarchical arrangement. One level of coordination appears to be provided by a host of unrelated genes encoding proteins required for cell-cell communication. A second, but overlapping, level might be provided by a second tier of genes engaged in specific cell functions required for productive cell-cell interaction. A third and higher level of control involves the transcriptional regulation of these subordinate genes by a handful of upper echelon genes that, operating within the different neuronal and glial subsets required for the initiation of the pubertal process, sustain the functional integration of the network. The existence of functionally connected genes controlling the pubertal process is consistent with the concept that puberty is under genetic control and that the genetic underpinnings of both normal and deranged puberty are polygenic rather than specified by a single gene. The availability of improved high-throughput techniques and computational methods for global analysis of mRNAs and proteins will allow us to not only initiate the systematic identification of the different components of this neuroendocrine network but also to define their functional interactions.