Neuronal activity of aromatase enzyme in non-copulating male rats

There are apparently normal male rats that fail to initiate copulation; these animals are called non-copulating (NC) males. Several research groups have demonstrated that conversion of testosterone to oestradiol (aromatisation) in specific brain areas known to be involved in the control of masculine sexual behaviour is fundamental in the control of masculine sexual behaviour. The aim of the present study was to test the hypothesis that the concentration of aromatase activity (AA) in the brain is lower in NC males than in copulating males (C). We quantified AA in several brain nuclei and also evaluated whether NC rats have altered concentrations of testosterone in their plasma. We found that AA was reduced in the medial preoptic nuclei (MPN) of NC male rats vs C males. In addition, NC and C male rats had similar plasma levels of testosterone. These data suggest that reduced levels of AA in the MPN could be a crucial factor associated with lack of male coital behaviour in rats.     

Partial demasculinization of several brain regions in adult male (XY) rats with a dysfunctional androgen receptor gene

The adult rat posterodorsal medial amygdala (MePD) is sexually dimorphic in regional volume and neuronal soma size, both of which are larger in males than in females. This sexual dimorphism is entirely dependent on adult circulating levels of testicular androgens, and both androgen and estrogen treatment can masculinize MePD structure. We examined male rats that are rendered androgen-insensitive by the testicular feminization mutation (tfm) of the androgen receptor (AR) gene to determine how a dysfunctional AR affects this and other brain sexual dimorphisms. In adult wild-type rats, the MePD in males had a greater regional volume, rostrocaudal extent, and soma size than in females. In genetic males, defective ARs affected some but not all of these indices: MePD volume and soma size in tfm males were intermediate between those of wild-type males and females, but the rostrocaudal extent of the MePD was unaffected by the mutation, being as great in tfm males as in wild-type males. Regional volume and soma size in the suprachiasmatic nucleus was reduced in tfm males compared with wild-type males, suggesting that AR normally affects this region in male rats. Interestingly, whereas volume of the sexually dimorphic nucleus of the preoptic area was unaffected by the tfm allele, soma size in this region was reduced in tfm males compared with wild-type males. Although estrogen receptor activation has been shown to be vital for masculinization of the rodent brain, our results indicate that ARs also contribute to this process in several brain regions.     

Age‐related changes in hypothalamic androgen receptor and estrogen receptor α in male rats

The control of reproductive function involves actions of sex steroids upon their nuclear receptors in the hypothalamus and preoptic area (POA). Whether hypothalamic hormone receptors change their expression in aging male mammals has not been extensively pursued, although such changes may underlie functional losses in reproductive physiology occurring with aging. We performed a stereologic analysis of immunoreactive androgen receptor (AR) and estrogen receptor alpha (ERα) cells in three POA nuclei of male Sprague‐Dawley rats (anteroventral periventricular nucleus [AVPV], median preoptic area [MePO], and medial preoptic nucleus [MPN]), at young (3 months), middle‐aged (12 months), and old (20 months) ages. Serum testosterone and estradiol levels were assayed. Testosterone concentrations decreased significantly and progressively with aging. Estradiol concentrations were significantly higher in middle‐aged than either young or old rats. Stereologic analyses of the POA demonstrated that AR‐immunoreactive cell numbers and density in the AVPV, MePO, and MPN were significantly higher in old compared with young or middle‐aged rats. No change in the total number or density of ERα‐immunoreactive cells was detected with age, although when cells were subdivided by intensity of immunolabeling, the most heavily labeled ERα cells increased in number with aging in the AVPV and MePO, and in density in the AVPV. There are several interpretations to our finding of substantially increased AR cell numbers during aging, including a potential compensatory upregulation of the AR under diminished testosterone concentrations. These results provide further information about how the neural targets of steroid hormones change with advancing age. J. Comp. Neurol. 512:688–701, 2009. © 2008 Wiley‐Liss, Inc.     

Nitric oxidergic cells related to ejaculation in gerbil forebrain contain androgen receptor and respond to testosterone

Two clusters of forebrain neurons-one in the posterodorsal preoptic nucleus (PdPN) and one in the lateral part of the posterodorsal medial amygdala (MeApd)-are activated at ejaculation in male rats and gerbils as seen with Fos immunocytochemistry. To understand the functions of these cells and how they respond synchronously, it may be useful to identify their neurotransmitters. Nitric oxide (NO) was of interest because its levels in the preoptic area affect ejaculation, and it could synchronize clustered neurons through paracrine/volume transmission. Thus, we determined whether the ejaculation-related cells produce NO by assessing Fos co-localization with NO synthase (NOS) in recently mated male gerbils. We also studied NOS-Fos co-localization in the medial part of the medial preoptic nucleus (MPNm), where half of the neurons that express Fos after mating reflect ejaculation. We also quantified NOS co-localization with androgen receptor (AR) and NOS sensitivity to androgens at these sites. Without quantification, we extended these analyses throughout the hypothalamus and amygdala. Many mating-activated PdPN, lateral MeApd, and MPNm cells contained NOS (32-54%), and many NOS neurons at these sites expressed Fos (34-51%) or AR (25-69%). PdPN and MPNm NOS cells were sensitive to testosterone but not its androgenic metabolite dihydrotestosterone. The overall distribution of NOS and NOS-AR cells was similar to that in rats. These data suggest that NO may help to synchronize the activation of PdPN and lateral MeApd neurons at ejaculation and that NOS in PdPN and MPNm cells is regulated by testosterone acting via estradiol or without undergoing metabolism.     

Sex and region-specific regulation of oestrogen receptor beta in the rat hypothalamus

Sexual dimorphism in the expression of oestrogen receptor (ER)beta mRNA and protein was characterized in the rostral forebrain of the rat and its dependence on the neonatal endocrine environment was revealed. We present novel data demonstrating, in gonadectomized adult rats, that the amount of oestrogen caused a significant reduction in the number of ERbeta messages and protein in the ventromedial nucleus in both sexes, but no such effects were detected in the preoptic area or the amygdala. In gonadectomized females, more so than in males, the ventromedial nucleus of the adult rat contained a significantly larger number of ERbeta-positive neurones both in terms of ERbeta mRNA and protein. In the juvenile rat on day 14, sex difference in ERbeta expression was already observed in the ventromedial nucleus. Treatment of neonatal females with oestrogen from days 1-10 or neonatal orchidectomy of males reversed the sex difference in the ventromedial nucleus when observed on day 14, showing that the neonatal presence of oestrogen had caused irreversible masculinization of this structure. Our results suggest that sex-specific expression of ERbeta is patterned by perinatal hormone exposure: down-regulation of ERbeta caused by oestrogen in a region-specific manner.     

The sexually dimorphic region of the preoptic area in rats contains denser opiate receptor binding sites in females

Quantitative autoradiographic analysis of opiate receptor binding using [³H]naloxone shows higher levels in the sexually dimorphic region of the medial preoptic area in female rats than in males. Opiate receptor density varies across the estrous cycle being densest in diestrous females. The sexually dimorphic nucleus of the preoptic are lies within the opiate receptor-rich region. Endogenous opiates in the medial preoptic region acting at opiate receptors which are of differential density in males and females could influence sex-specific behavior mediated by the region.     

Tyrosine hydroxylase neurons in the male hamster chemosensory pathway contain androgen receptors and are influenced by gonadal hormones

Chemosensory and hormonal signals, both of which are essential for mating in the male Syrian hamster, are relayed through a distinct forebrain circuit. Immunocytochemistry for tyrosine hydroxylase, a catecholamine biosynthetic enzyme, previously revealed immunoreactive neurons in the anterior and posterior medial amygdaloid nucleus, one of the nuclei within this pathway. In addition, dopamine-immunoreactive neurons were located in the posterior, but not hte anterior, medial amygdala. In the present study, tyrosine hydroxylase-immunostained neurons were also observed in other areas of the chemosensory pathway, including the posteromedial bed nucleus of the stria terminalis and the posterior, lateral part of the medial preoptic area, while dopamine immunostaining was only seen in the posteromedial bed nucleus of the stria terminalis. The colocalization of tyrosine hydroxylase and androgen receptors was examined in these four tyrosine hydroxylase cell groups by a double immunoperoxidase technique. The percentage of tyrosine hydroxylase-immunolabeled neurons that were also androgen receptor-immunoreactive was highest in the posterior medial amygdaloid nucleus (74%) and the bed nucleus of the stria terminalis (79%). Fewer tyrosine hydroxylase-immunostained neurons in the anterior medial amygdala (33%) and the medial preoptic area (4%) contained androgen receptors. Surprisingly, castration resulted in a significant decrease in the number of tyrosine hydroxylase-immunoreactive neurons only in the anterior medial amygdaloid nucleus, and this effect was transient. Six weeks after castratio, the anterior medial amygdala contained 61% fewer tyrosine hydroxylase-immunolabeled neurons, but 12 weeks after gonadectomy, immunostaining returned to intact values. The number of immunostained neurons in testosterone-replaced, castrated hamsters was not significantly different from that of intact or castrated animals at any time. The results of this study indicate that a substantial number of tyrosine hydroxylase-immunostained neurons in the chemosensory pathway are influenced by androgens; the majority of these neurons in the posterior medial amygdala and the posteromedial bed nucleus of the stria terminalis produce androgen receptors, and tyrosine hydroxylase immunoreactivity is altered by castration in the anterior medial amygdala. © 1993 Wiley-Liss, Inc.     

Mesostriatal and mesolimbic projections of midbrain neurons immunoreactive for estrogen receptor beta or androgen receptors in rats

The dopamine (DA) inputs to the caudate putamen, the nucleus accumbens, and the amygdala in rats are sensitive to circulating estrogens and androgens. One mechanism for the hormone modulation of these systems may be via actions at cognate intracellular estrogen and androgen receptors. However, although it is known that specific subsets of midbrain DA neurons are immunopositive for estrogen receptor beta (ERbeta) or androgen receptors (ARs), it is not known where these receptor-bearing cells project. To address this issue, we combined double-label immunocytochemistry with retrograde tract tracing to identify the forebrain projections of ERbeta- or AR-immunoreactive (IR) midbrain neurons. Specifically, Fluoro-Gold and/or cholera toxin were injected into discrete subregions of the caudate-putamen, the nucleus accumbens, or the amygdala. Evaluations of the resultant midbrain labeling revealed that ERbeta-IR neurons sent collateral projections mainly to both the ventral caudate-putamen and the amygdala, but not to the dorsal caudate or nucleus accumbens. In contrast, AR-IR neurons projected either to the amygdala or the nucleus accumbens but not to the caudate-putamen. The organization of these forebrain projections concurs with some of the known hormone sensitivities of mesostriatal and mesolimbic DA systems in rats and provides an anatomical model that predicts separate influences for androgens and estrogens over mesostriatal and mesolimbic DA systems.     

Bisphenol A increases progesterone receptor immunoreactivity in the hypothalamus in a dose-dependent manner and affects sexual behaviour in adult ovariectomized rats

Recently, we reported that bisphenol A (BPA), an endocrine disrupter, increased progesterone receptor (PR) mRNA in the preoptic area (POA) in adult ovariectomized rats. In the present study, we examined whether BPA also induced expression of PR proteins in both the POA and the ventromedial hypothalamic nucleus (VMH), and whether those proteins were involved in the induction of sexual behaviour. Two weeks after ovariectomy, rats received a subcutaneous (s.c.) injection of BPA, 17 beta-oestradiol or vehicle. Twenty-four hours after the injection, the rats were killed and their tissues were examined by immunocytochemistry. Some rats that received a s.c. injection of BPA, E2 or vehicle alone on the day before were injected with progesterone at 15.00 h and examined for sexual behaviour 5-7 h later. As expected, injection of 10 microg E2 significantly increased the number of PR immunoreactive cells in both the POA and the VMH compared to the number after injection of vehicle alone. In both the POA and the VMH, injection of BPA at a dose of 10 mg also significantly increased the number of PR immunoreactive cells compared to the number after injection of sesame oil alone. Furthermore, BPA induced a dose-dependent increase in the number of PR immunoreactive cells in both the POA and the VMH, demonstrating that the number of PR cells was significantly increased by as little as 100 microg of BPA. Ovariectomized (OVX) rats that were primed with 10 mg of BPA, followed by 1 mg of progesterone, displayed mainly rejection behaviour, but not lordosis as typically observed in OVX rats primed with E2 followed by progesterone. The present study suggests that BPA influences reproductive functions, including sexual behaviour even in adulthood, by altering the PR system in the hypothalamus.     

Co‐Expression of Leptin and Oestrogen Receptors in the Preoptic‐Hypothalamic Area

The interaction between the reproductive axis and energy balance suggests that leptin acts as a possible mediator. This hormone acts in the regulation of metabolism, feeding behaviour and reproduction. Animals homozygous for the gene ‘ob’ (ob/ob) are obese and infertile, and these effects are reversed after systemic administration of leptin. Thus, the present study aimed to determine: (i) whether cells that express leptin also express oestrogen receptors of type‐α (ER‐α) or ‐β (ER‐β) in the medial preoptic area (MPOA) and in the arcuate (ARC), dorsomedial (DMH) and ventromedial hypothalamic nucleus and (ii) whether there is change in the gene and protein expression of leptin in these brain areas in ovariectomised (OVX) animals when oestrogen‐primed. Wistar female rats with normal oestrous cycles or ovariectomised oestrogen‐primed or vehicle (oil)‐primed were utilised. To determine whether there was a co‐expression, immunofluorescence was utilised for double staining. Confocal microscopy was used to confirm the co‐expression. The technique of real‐time polymerase chain reaction and western blotting were employed to analyse gene and protein expression, respectively. The results obtained showed co‐expression of leptin and ER‐α in the MPOA and in the DMH, as well as leptin and ER‐β in the MPOA, DMH and ARC. However, we did not detect leptin in the MPOA, ARC and DMH using western blotting and there was no statistical difference in leptin gene expression in the MPOA, DMH, ARC, pituitary or adipose tissue between OVX rats treated with oestrogen or vehicle. In conclusion, the results obtained in the present study confirm that the brain is also a source of leptin and reveal co‐expression of oestrogen receptors and leptin in the same cells from areas related to reproductive function and feeding behaviour. Although these data corroborate the previous evidence obtained concerning the interaction between the action of brain leptin and reproductive function, the physiological relevance of this interaction remains uncertain and additional studies are necessary to elucidate the exact role of central leptin.     

Effects of organisational oestradiol on adult immunoreactive oestrogen receptors (alpha and beta) in the male mouse brain

Steroid hormones act on developing neural circuits that regulate the hypothalamic-pituitary-gonadal axis and are involved in hormone-sensitive behaviours. To test the hypothesis that developmental exposure to oestradiol (E(2)) organises the quantity of adult oestrogen receptors (ERalpha and ERbeta), we used male mice with a targeted mutation of the aromatase enzyme gene (ArKO) and their wild-type (WT) littermates. These mice are unable to aromatise testosterone to E(2), but still express both ERalpha and beta. To evaluate adult responsiveness to E(2), gonadectomised males were implanted with Silastic capsules containing E(2), or an empty implant, 5 days prior to sacrifice. Immunoreactivity for ERalpha and ERbeta was quantified in the caudal ventromedial nucleus (VMN) and the medial preoptic area (POA). Regardless of genotype, adult treatment with E(2) reduced ERalpha-immunoreactive (ir) and ERbeta-ir cell numbers in the POA, as well as ERbeta-ir, but not ERalpha-ir, cell numbers in the VMN. Genotype, and thus endogenous exposure to E(2), produced opposite effects on ER expression in the two brain areas. In the VMN, ArKO males had more ERalpha-ir and ERbeta-ir cells than did WT males. In the POA, ArKO males had fewer ERalpha-ir and ERbeta-ir cells than did WT males. Thus, numbers of immunoreactive neurones containing both ERs in the adult ArKO male were enhanced in the POA, but decreased in the VMN, and most likely these patterns were established during the developmental critical period. Furthermore, although both ERalpha and beta-ir cell numbers are altered by the disruption of the aromatase gene, ERbeta is altered in a more robust and region-specific manner.     

Ovarian steroids alter dopamine receptor populations in the medial preoptic area of female rats: implications for sexual motivation,desire, and behaviour

Dopamine (DA) transmission in the medial preoptic area (mPOA) plays a critical role in the control of appetitive sexual behaviour in the female rat. We have shown previously that a DA D1 receptor (D1R)‐mediated excitatory state appears to occur in females primed with estradiol benzoate (EB) and progesterone (P), whereas a DA D2 receptor (D2R)‐mediated inhibitory state appears to occur in females primed only with EB. The present experiment employed three techniques to better understand what changes occur to DA receptors (DARs) in the mPOA under different hormonal profiles. Ovariectomized females were randomly assigned to one of three steroid treatment groups: EB + P (10 and 500 μg, respectively), EB + Oil, or the control (Oil + Oil), with hormone injections administered at 48 and 4 h prior to euthanizing. First, the number of neurons in the mPOA that contained D1R or D2R was assessed using immunohistochemistry. Second, the mPOA and two control areas (the prelimbic cortex and caudate putamen) were analysed for DAR protein levels using western blot, and DAR functional binding levels using autoradiography. Ovarian steroid hormones affected the two DAR subtypes in opposite ways in the mPOA. All three techniques supported previous behavioural findings that females primed with EB have a lower D1R : D2R ratio, and thus a D2R‐mediated system, and females primed with EB + P have a higher D1R : D2R ratio, and thus a D1R‐mediated system. This provides strong evidence for a DA‐driven pathway of female sexual motivation, desire, and behaviour that is modified by different hormone priming regimens.     

GABA and glutamate in mating-activated cells in the preoptic area and medial amygdala of male gerbils

The posterodorsal medial amygdala (MeApd), the posterodorsal preoptic nucleus (PdPN), and the medial cell group of the sexually dimorphic preoptic area (mSDA) contain cells that are activated specifically at ejaculation as assessed by Fos expression. The mSDA also expresses Fos early in the mating context. Because little is known about the neurotransmitters of these activated cells, the possibility that they use gamma-aminobutyric acid (GABA) or glutamate was assessed. Putative glutamatergic cells were visualized with immunocytochemistry (ICC) for glutamate and its neuron-specific transporter. Their distributions were compared with those of GABAergic cells visualized with ICC for the 67-kDa form of glutamic acid decarboxylase (GAD(67)) and in situ hybridization for GAD(67) messenger RNA (mRNA). Colocalization of Fos and GAD(67) mRNA in recently mated males indicated that half of the activated cells in the PdPN, mSDA, and lateral MeApd are GABAergic. Colocalization of Fos and glutamate suggested that a quarter of the activated mSDA and lateral MeApd cells are glutamatergic. The PdPN does not appear to have glutamatergic cells. In the lateral MeApd, the percentage of activated cells that are GABAergic (45%) matches the percentage that project to the principal part of the bed nucleus of the stria terminalis (BST; 43%), and the percentage likely to be glutamatergic (27%) matches the percentage projecting to the mSDA (27%). The latter could help to trigger ejaculation. The distribution of GABAergic and putative glutamatergic cells in the caudal preoptic area, caudal BST, and medial amygdala of male gerbils is also described.     

Characterisation and origins of melanin‐concentrating hormone immunoreactive fibres of the posterior lobe of the pituitary and median eminence during lactation in the Long‐Evans rat

Although the melanin‐concentrating hormone (MCH) and its coding mRNA are predominantly found in the tuberal hypothalamus, there is detectable synthesis of MCH in the preoptic hypothalamus exclusively in lactating dams, suggesting a participation of MCH in the alterations that take place after parturition. Also implicated in the dam physiology is oxytocin, a neurohormone released from the posterior pituitary that is necessary for milk ejection. Because the projection fields from oxytocin‐immunoreactive (‐IR) neurones and the mediobasal preoptic hypothalamus overlap and MCH‐IR neurones are found in proximity to oxytocin neurones, we investigated the spatial relationship between MCH and oxytocin fibres. Accordingly, we employed multiple immunohistochemistry labelling for MCH and oxytocin for light and electron microscopy techniques, in addition to i.v. tracer injection combined with in situ hybridisation to identify MCH neurones that project to neurosecretory areas. As described for other strains, lactating Long‐Evans dams also display immunoreactivity for MCH in the preoptic hypothalamus on days 12 and 19 of lactation. The appearance of these neurones is contemporaneous with an increase in MCH‐IR fibres in both the internal layer of the median eminence and the posterior pituitary. In both regions, MCH‐ and oxytocin‐IR fibres were found in great proximity, although there was no evidence for synaptic interaction between these two populations at the ultrastructural level. The tracer injection revealed that only mediobasal preoptic MCH neurones project to the posterior pituitary, suggesting a neuroendocrine‐modulatory role for this population. When taken together, the results obtained in the present study indicate that neuroplasticity events at the mediobasal preoptic hypothalamus that occur during late lactation may be part of a neuroendocrinology control loop involving both MCH and oxytocin.     

Dopaminergic control of male sex behavior in rats: Effects of an intracerebrally-infused agonist

Systemically-administered dopaminergic drugs have been found to facilitate sexual behavior of men and male rats. The present experiments investigated the localization within the brain of dopaminergic effects on copulation of male rats. Apomorphine, a dopamine agonist, was microinfused into the medial preoptic area, caudate-putamen, nucleus accumbens, lateral septum and lateral ventricle. The lowest dose of apomorphine (0.2 microgram) infused into the ventricle reduced the number of ejaculations, slowed the rate of intromitting and decreased the percentage of mounts on which the male gained vaginal intromission. The higher two doses (0.5 and 2.0 micrograms) infused into the medial preoptic area and, in some cases, the ventricle, increased the number of ejaculations and the percentage of mounts with vaginal intromission, increased the rate of intromitting and decreased the latency to ejaculate and the postejaculatory interval before resuming copulation. Infusions into the caudate-putamen and lateral septum were without effect. Those into nucleus accumbens produced only a slight dose-related decrease in latency to begin copulating. The copulatory impairments associated with infusions of the lowest dose into the ventricle may have resulted from stimulation of autoreceptors, or from preferential stimulation by low doses of an undetermined area. The facilitative effects of the two higher doses into the medial preoptic area and lateral ventricle may have been due to stimulation of dopaminergic postsynaptic receptors.     

Localisation of oestrogen receptors in stem cells and in stem cell-derived neurons of the mouse

Oestrogen receptors (ER) transduce the effects of the endogenous ligand, 17β-estradiol in cells to regulate a number of important processes such as reproduction, neuroprotection, learning and memory and anxiety. The ERα or ERβ are classical intracellular nuclear hormone receptors while some of their variants or novel proteins such as the G-protein coupled receptor (GPCR), GPER1/GPR30 are reported to localise in intracellular as well as plasma membrane locations. Although the brain is an important target for oestrogen with oestrogen receptors expressed differentially in various nuclei, subcellular organisation and crosstalk between these receptors is under-explored. Using an adapted protocol that is rapid, we first generated neurons from mouse embryonic stem cells. Our immunocytochemistry approach shows that the full length ERα (ERα-66) and for the first time, that an ERα variant, ERα-36, as well as GPER1 is present in embryonic stem cells. In addition, these receptors typically decrease their nuclear localisation as neuronal maturation proceeds. Finally, although these ERs are present in many subcellular compartments such as the nucleus and plasma membrane, we show that they are specifically not colocalised with each other, suggesting that they initiate distinct signalling pathways.     

Immunocytochemical detection of progesterone receptor in the female rabbit forebrain: distribution and regulation by oestradiol and progesterone

There is no information on the neuroanatomical distribution of the progesterone receptor (PR) in the rabbit. Therefore, we mapped the distribution of PR-immunoreactive cells in the forebrain of ovariectomized female rabbits. Vehicle-injected ovariectomized rabbits showed PR-immunoreactive cells only in the infundibular nucleus (IN) and nucleus X (lateral to the ventromedial hypothalamic nucleus). The injection of oestradiol benzoate (EB; 5 micro g/day for 5 days) increased the number of PR-immunoreactive cells in the IN and in three nuclei of the preoptic region (periventricular, medial, and principal). Abundant PR were also found in the paraventricular nucleus and nucleus X. Administration of progesterone (10 mg/day) for 3 days to EB-treated rabbits (a treatment that induces digging behaviour for the maternal nest and suppresses sexual receptivity and scent-marking) eliminated PR-immunoreactivity from all brain areas analysed except the IN. Thus, one-third of the number of cells seen in the ovariectomized + EB condition persisted in this region despite progesterone injections. Withdrawal of progesterone (and continuation of EB) for 5 (but not for 2) days (in a schedule similar to the one that induces straw-carrying and hair-pulling for the maternal nest) increased the number of PR-immunoreactive cells in all regions analysed. These results show that restricted regions of the female rabbit forebrain express abundant PR which are either: (i). up-regulated by oestradiol and down-regulated by progesterone; (ii). oestradiol-insensitive and down-regulated by progesterone; or (iii). insensitive to both oestradiol and progesterone.