Perinatal Exposure to Methoxychlor Affects Reproductive Function and Sexual Behavior in Mice.

Numerous chemicals derived from human activity are now disseminated in the environment where their exert estrogenic endocrine disrupting effects, and therefore represent major health concerns. The present study explored whether Methoxychlor (MXC), an insecticide with xenoestrogens activities, given during the perinatal period (from gestational day 11 to postnatal day 8) and at an environmentally dose [20 µg/kg (body weight)/day], would affect reproductive physiology and sexual behavior of the offspring in mice. While MXC exposure did not induce any differences in the weight gain of animals from birth to 4 months of age, a clear difference (although in opposite direction according to the sexes) was observed on the anogenital distance between intact and exposed animals. A similar effect was also observed on preputial separation and vaginal opening, which reflects, respectively, in males and females, puberty occurrence. The advanced puberty observed in females was associated with an enhanced expression of kisspeptin cells in the anteroventral periventricular region of the medial preoptic area. Exposure to MXC did not induce in adult females changes in the estrous cycle or in the weight of the female reproductive tract. By contrast, males showed reduced weight of the epididymis and seminiferous vesicles associated with reduced testosterone levels and seminiferous tubule diameter. We also showed that both males and females showed deficits in mate preference tests. As a whole, our results show that MXC impacts reproductive outcomes.

Sex chromosome complement influences vulnerability to cocaine in mice.

Women acquire cocaine habits faster and are more motivated to obtain drug than men. In general, female rodents acquire intravenous cocaine self-administration (SA) faster and show greater locomotor responses to cocaine than males. Sex differences are attributed to differences in circulating estradiol. We used the four core genotype (FCG) mouse to ask whether sex chromosome complement influences vulnerability to cocaine's reinforcing and/or locomotor-activating effects. The FCG cross produces ovary-bearing mice with XX or XY genotypes (XXF, XYF) and testes-bearing mice with XX or XY genotypes (XXM, XYM). A greater percentage of gonadal females acquired cocaine SA via infusions into jugular catheters as compared with XYM mice, but XXM mice were not significantly different than any other group. Discrimination of the active versus inactive nose poke holes and cocaine intake were in general greater in gonadal females than in gonadal males. Progressive ratio tests for motivation revealed an interaction between sex chromosomes and gonads: XYM mice were more motivated to self-administer cocaine taking more infusions than mice in any other group. Locomotor responses to cocaine exposure revealed effects of sex chromosomes. After acute exposure, activity was greater in XX than in XY mice and the reverse was true for behavioral sensitization. Mice with XY genotypes displayed more activity than XX mice when given cocaine after a 10-day drug-free period. Our data demonstrate that sex chromosome complement alone and/or interacting with gonadal status can modify cocaine's reinforcing and locomotor-activating effects. These data should inform current studies of sex differences in drug use.

Kisspeptin system in ovariectomized mice: Estradiol and progesterone regulation.

The kisspeptin system is clustered in two main groups of cell bodies (the periventricular region, RP3V and the arcuate nucleus, ARC) that send fibers mainly to the GnRH neurons and in a few other locations, including the paraventricular nucleus, PVN. In physiological conditions, gonadal hormones modulate the kisspeptin system with expression changes according to different phases of the estrous cycle: the highest being in estrus phase in RP3V and PVN (positive feedback), and in ARC during the diestrus phase (negative feedback). In this work we wanted to study these hormonal fluctuations during the estrous cycle, investigating the role played by progesterone (P) or estradiol (E2), alone or together, on the kisspeptin system. Gonadectomized CD1 female mice were treated with P, E2 or both (E2 + P), following a timing of administration that emulates the different phases of estrous cycle, for two cycles of 4 days. As expected, the two cell groups were differentially affected by E2; the RP3V group was positively influenced by E2 (alone or with the P), whereas in the ARC the administration of E2 did not affect the system. However P (alone) induced a rise in the kisspeptin immunoreactivity. All the treatments significantly affected the kisspeptin innervation of the PVN, with regional differences, suggesting that these fibers arrive from both RP3V and ARC nuclei.

Revisiting the neural role of estrogen receptor beta in male sexual behavior by conditional mutagenesis.

Estradiol derived from neural aromatization of gonadal testosterone plays a key role in the perinatal organization of the neural circuitry underlying male sexual behavior. The aim of this study was to investigate the contribution of neural estrogen receptor (ER) ß in estradiol-induced effects without interfering with its peripheral functions. For this purpose, male mice lacking ERß in the nervous system were generated. Analyses of males in two consecutive tests with a time interval of two weeks showed an effect of experience, but not of genotype, on the latencies to the first mount, intromission, pelvic thrusting and ejaculation. Similarly, there was an effect of experience, but not of genotype, on the number of thrusts and mating length. Neural ERß deletion had no effect on the ability of males to adopt a lordosis posture in response to male mounts, after castration and priming with estradiol and progesterone. Indeed, only low percentages of both genotypes exhibited a low lordosis quotient. It also did not affect their olfactory preference. Quantification of tyrosine hydroxylase- and kisspeptin-immunoreactive neurons in the preoptic area showed unaffected sexual dimorphism of both populations in mutants. By contrast, the number of androgen receptor- and ERα-immunoreactive cells was significantly increased in the bed nucleus of stria terminalis of mutant males. These data show that neural ERß does not play a crucial role in the organization and activation of the neural circuitry underlying male sexual behavior. These discrepancies with the phenotype of global ERß knockout models are discussed.

Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta.

Ovarian oestradiol is essential for pubertal maturation and adult physiology of the female reproductive axis. It acts at central and peripheral sites through two main oestrogen receptors (ER) α and ß. Here we investigate the role of ERß on central effects of oestradiol, by generating a mouse line specifically lacking the ERß gene in neuronal and glial cells. Central ERß deletion delays the age at vaginal opening and first oestrous and reduces uterine weight without affecting body growth. Analysis of factors necessary for pubertal progression shows reduced levels of Kiss1 transcripts at postnatal (P) day 25 in the preoptic area, but not in the mediobasal hypothalamus (MBH) of mutant females. In agreement with these data, the number of kisspeptin-immunoreactive neurons was decreased by 57-72% in the three subdivisions of the rostral periventricular area of the third ventricle (RP3V), whereas the density of kisspeptin-immunoreactive fibres was unchanged in the arcuate nucleus of mutant mice. These alterations do not involve changes in ERα mRNAs in the preoptic area and protein levels in the RP3V. The number and distribution of GnRH-immunoreactive cells were unaffected, but gonadotropin-releasing hormone (GnRH) transcript levels were higher in the P25 preoptic area of mutants. At adulthood, mutant females have normal oestrous cyclicity, kisspeptin system and exhibit unaltered sexual behaviour. They display, however, reduced ovary weight and increased anxiety-related behaviour during the follicular phase. This argues for the specific involvement of central ERß in the regulation of pubertal onset in female reproduction, possibly through prepubertal induction of kisspeptin expression in the RP3V.

Neuroendocrine and behavioral effects of maternal exposure to oral bisphenol A in female mice.

Bisphenol A (BPA) is a widespread estrogenic compound. We investigated the effects of maternal exposure to BPA at reference doses on sexual behavior and neuroendocrine functions of female offspring in C57BL/6J mice. The dams were orally exposed to vehicle alone or vehicle-containing BPA at doses equivalent to the no observed adverse effect level (5 mg/kg body weight per day) and tolerable daily intake (TDI, 0.05 mg/kg body weight per day) level from gestational day 15 until weaning. Developmental exposure to BPA increased the lordosis quotient in naive females exposed to BPA at the TDI dose only. BPA exposure had no effect on olfactory preference, ability to express masculine behaviors or number of calbindin-positive cells, a sexually dimorphic population of the preoptic area. BPA at both doses selectively increased kisspeptin cell number in the preoptic periventricular nucleus of the rostral periventricular area of the third ventricle in adult females. It did not affect the number of GNRH-positive cells or percentage of kisspeptin appositions on GNRH neurons in the preoptic area. These changes were associated with higher levels of estradiol (E2) at the TDI dose while levels of LH, estrus cyclicity, ovarian and uterine weights, and fertility remained unaffected. Delay in the time of vaginal opening was observed during the postnatal period at TDI dose, without any alteration in body growth. This shows that developmental exposure to BPA at reference doses did not masculinize and defeminize the neural circuitry underlying sexual behavior in female mice. The TDI dose specifically exacerbated responses normally induced by ovarian E2, through estrogen receptor α, during the postnatal/prepubertal period.

Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol a in male mice.

There are human reproduction concerns associated with extensive use of bisphenol A (BPA)-containing plastic and, in particular, the leaching of BPA into food and beverages. In this context, it remains unclear whether and how exposure to BPA interferes with the developmental organization and adult activation of male sexual behavior by testosterone. We evaluated the developmental and adult exposure to oral BPA at doses equivalent to the no-observed-adverse-effect-level (5 mg/kg body weight per day) and tolerable daily intake (TDI) (50 µg/kg body weight per day) on mouse sexual behavior and the potential mechanisms underlying BPA effects. Adult exposure to BPA reduced sexual motivation and performance at TDI dose only. Exposed males took longer to initiate mating and reach ejaculation despite normal olfactory chemoinvestigation. This deficiency was not restored by sexual experience and was associated with unchanged circulating levels of testosterone. By contrast, developmental exposure to BPA at TDI or no-observed-adverse-effect-level dose did not reduce sexual behavior or alter the neuroanatomical organization of the preoptic area. Disrupting the neural androgen receptor resulted in behavioral and neuroanatomical effects similar to those induced by adult exposure to TDI dose. Moreover, adult exposure of mutant males to BPA at TDI dose did not trigger additional alteration of sexual behavior, suggesting that BPA and neural androgen receptor mutation share a common mechanism of action. This shows, for the first time, that the neural circuitry underlying male sexual behavior is vulnerable to chronic adult exposure to low dose of BPA and suggests that BPA could act in vivo as an antiandrogenic compound.

Kisspeptin: a new neuronal target of primer pheromones in the control of reproductive function in mammals.

Pheromones are known to trigger either short-term behavioral responses, usually referred to as "releaser effects", or more long-term physiological changes, known as "primer effects", which especially affect reproductive function at the level of the gonadotrope axis. The precise mechanisms through which pheromones interact with the gonadotrope axis in the hypothalamus is not fully known. We propose that the neuropeptide Kisspeptin, could be a specific target of primer pheromones, allowing these pheromones to modulate the gonadotrope axis and GnRH activity. This emerging hypothesis is discussed in the context of puberty acceleration in female mice and the male effect in female ungulates (sheep or goat). These examples have been chosen to illustrate the diversity of the reproductive contexts in mammals and potential mechanisms affected by primer effects at the level of the gonadotrope axis.

Synergic effects of estradiol and progesterone on regulation of the hypothalamic neuronal nitric oxide synthase expression in ovariectomized mice.

Nitric oxide (NO) is a gaseous neurotransmitter that plays an important role in the regulation of sexual behavior in rodents. NO is produced, within the central nervous system, by the enzyme neural NO synthase (nNOS) whose expression is influenced by gonadal hormones. In previous studies, we demonstrated that part of the nitrergic hypothalamic and limbic system is influenced, in physiological conditions, by the hormonal fluctuations during the estrous cycle, but we were unable to distinguish among the role played by progesterone (P) or estradiol (E(2)) in inducing these changes. In the present study, we investigated the effects of E(2) and P (alone or together) on the nitrergic system of gonadectomized female mice, following a timing of administration that emulates the different phases of estrous cycle. In parallel, we tested the influence of the two hormones on sexual behavior, confirming that P works in synergistic fashion with E(2) to facilitate female receptivity. The quantitative analysis of nNOS-ir system demonstrated a statistically significant variation in the number of positive cells only in those part of the limbic-hypothalamic nitrergic system that are affected in cycling females, i.e. the bed nucleus of the stria terminalis, the arcuate nucleus and the medial preoptic area, with the highest number of positive neurons observed in E(2)+P group. The variable effects of E(2) and P may depend on the different distribution of their receptors within the analyzed nuclei, but the relationships among variations of estrogen and progesterone levels and in vivo modulation of nNOS expression remain unknown and needed further investigations.

Estrous cycle influences the expression of neuronal nitric oxide synthase in the hypothalamus and limbic system of female mice.

BACKGROUND: Nitric oxide plays an important role in the regulation of male and female sexual behavior in rodents, and the expression of the nitric oxide synthase (NOS) is influenced by testosterone in the male rat, and by estrogens in the female. We have here quantitatively investigated the distribution of nNOS immunoreactive (ir) neurons in the limbic hypothalamic region of intact female mice sacrificed during different phases of estrous cycle. RESULTS: Changes were observed in the medial preoptic area (MPA) (significantly higher number in estrus) and in the arcuate nucleus (Arc) (significantly higher number in proestrus). In the ventrolateral part of the ventromedial nucleus (VMHvl) and in the bed nucleus of the stria terminalis (BST) no significant changes have been observed. In addition, by comparing males and females, we observed a stable sex dimorphism (males have a higher number of nNOS-ir cells in comparison to almost all the different phases of the estrous cycle) in the VMHvl and in the BST (when considering only the less intensely stained elements). In the MPA and in the Arc sex differences were detected only comparing some phases of the cycle. CONCLUSION: These data demonstrate that, in mice, the expression of nNOS in some hypothalamic regions involved in the control of reproduction and characterized by a large number of estrogen receptors is under the control of gonadal hormones and may vary according to the rapid variations of hormonal levels that take place during the estrous cycle.

Rapid changes on nitrinergic system in female mouse hippocampus during the ovarian cycle.

Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei including the hippocampus, where some neurons express estrogen receptors. Nitric oxide plays a wide range of functions in the nervous system generally by acting as a neurotransmitter-like molecule. It has been demonstrated that long-term treatments with estradiol in ovariectomized females and with testosterone in castrated males induce neuronal nitric oxide synthase (nNOS) expression in rat hypothalamus, whereas changes in nNOS immunoreactivity or in associated NADPH-diaphorase activity were observed both in hypothalamus and in amygdala during different phases of estrous cycle. Estradiol could induce nNOS expression in several brain regions in rodents. Therefore, to clarify if the hippocampal NO producing system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity on two-month-old intact female mice. Immunoreactive cells were observed in all hippocampal subregions: the higher number was detected in the pyramidal layer of CA1 region and in polymorph layer of dentate gyrus. The number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant in CA1, CA2 and CA3 regions. These results suggest that the nitrinergic system is a target for estrogen action in the hippocampus, and that this action may take place in physiological conditions according to the short-term variations of gonadal hormones during the estrous cycle.