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Testosterone and its metabolites masculinize the brain during a critical perinatal window, including the relative volume of sexually dimorphic brain areas such as the sexually dimorphic nucleus of the preoptic area (SDN), which is larger in males than females. Serotonin (5HT) may mediate this hormone action, since 5HT given during the second week of life decreases (i.e., feminizes) SDN volume in males and testosterone-treated females. Although previous work indicates that the 5HT2A/2C receptor is sufficient to induce feminization, it is unclear whether other serotonin receptors are required and which subpopulation(s) of SDN cells are specifically organized by 5HT. Therefore, we injected male and female Sprague-Dawley rat pups with saline, a nonselective 5HTR agonist, a 5HT2A/2C agonist, or a 5HT2A/2C antagonist over several timecourses in early life, and measured the Nissl-SDN as well as a calbindin+ subdivision of the SDN, the CALB-SDN. When examined on postnatal day 18 or early adulthood, the size of the Nissl-SDN was feminized in males treated with any of the serotonergic drugs, eliminating the typical sex difference. In contrast, the sex difference in CALB-SDN size was maintained regardless of serotoninergic drug treatment. This pattern suggests that although gonadal hormones shape the whole SDN, individual cellular phenotypes respond to different intermediary signals to become sexually dimorphic. Specifically, 5HT mediates sexual differentiation of non-calbindin population(s) within the SDN. The results also caution against using measurement of the CALB-SDN in isolation, as the absence of an effect on the CALB-SDN does not preclude an effect on the overall nucleus. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1241-1253, 2016.

Gonadal steroid hormones play important roles during critical periods of development to organise brain structures that control sexually dimorphic neuroendocrine responses and behaviours. Specific receptors for androgens and oestrogens must be expressed at appropriate times during development to mediate these processes. The present study was performed to test for sex differences in the relative expression of oestrogen receptor (ER)α and androgen receptor (AR) mRNA during the window of time in gestation that is critical for behavioural masculinisation and differentiation of the ovine sexually dimorphic nucleus (oSDN) in the sheep. In addition, we examined whether ERα and AR mRNA expression is localised within the nascent oSDN and could be involved in its development. Using the quantitative real-time polymerase chain reaction, we found that females expressed more ERα mRNA than males in medial preoptic area and medial basal hypothalamus during the mid-gestational critical period for brain sexual differentiation. No sex differences were found for AR mRNA in any tissue examined or for ERα in amygdala and frontal cortex. Using radioactive in situ hybridisation, we found that the distributions of ERα and AR mRNA overlapped with aromatase mRNA, which delineates the boundaries of the developing oSDN and identifies this nucleus as a target for both androgens and oestrogens. These data demonstrate that the transcriptional machinery for synthesising gonadal steroid receptors is functional in the foetal lamb brain during the critical period for sexual differentiation and suggest that possible mechanisms for establishing dimorphisms controlled by gonadal steroids may exist at the level of steroid hormone receptor expression.

In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.

Testosterone exposure during midgestation differentiates neural circuits controlling sex-specific behaviours and patterns of gonadotrophin secretion in male sheep. Testosterone acts through androgen receptors (AR) and/or after aromatisation to oestradiol and binding to oestrogen receptors. The present study assessed the role of AR activation in male sexual differentiation. We compared rams that were exposed to the AR antagonist flutamide (Flu) throughout the critical period (i.e. days 30-90 of gestation) to control rams and ewes that received no prenatal treatments. The external genitalia of all Flu rams were phenotypically female. Testes were positioned s.c. in the inguinal region of the abdomen, exhibited seasonally impaired androgen secretion and were azospermic. Flu rams displayed male-typical precopulatory and mounting behaviours but could not intromit or ejaculate because they lacked a penis. Flu rams exhibited greater mounting behaviour than control rams and, similar to controls, showed sexual partner preferences for oestrous ewes. Neither control, nor Flu rams responded to oestradiol treatments with displays of female-typical receptive behaviour or LH surge responses, whereas all control ewes responded as expected. The ovine sexually dimorphic nucleus in Flu rams was intermediate in volume between control rams and ewes and significantly different from both. These results indicate that prenatal anti-androgen exposure is not able to block male sexual differentiation in sheep and suggest that compensatory mechanisms intervene to maintain sufficient androgen stimulation during development.

There is a transient fall in hypothalamic serotonin (5-hydroxytryptamine; 5-HT) activity in the second week post partum in male but not female rats. When this fall is masked by administration of the 5-HT(2) agonist (-) 2,5-dimethoxy-4-iodophenyl]-2-aminopropane hydrochloride [(-)DOI], over days 8-16 post partum, males are feminised in adulthood. To investigate whether the effect of 5-HT is mediated by dopamine and whether testosterone exerts its masculinising effect by reducing 5-HT and dopamine activity, male pups were treated with (-)DOI alone or together with the dopamine antagonist, haloperidol, over days 8-16 post partum, whereas females were treated with testosterone propionate on day 2 post partum. In adulthood, the volumes of the anteroventral periventricular nucleus (AVPV), sexually dimorphic nucleus of the preoptic area (SDN-POA) and arcuate nucleus (ARC) were determined, together with the number of tyrosine hydroxylase-immunoreactive (TH-ir) cells and fibres within them. The concentrations of 5-HT, dopamine and their metabolites were also measured. (-)DOI treatment increased the volume of the AVPV, decreased that of the SDN-POA and increased the number of TH-ir cells in the AVPV. These feminising effects were antagonised by concurrent haloperidol treatment. Neonatal testosterone propionate masculinised the volumes of the female AVPV and SDN-POA and reduced the number of TH-ir cells in the AVPV. Dopamine and 5-HT turnover in the AVPV was greater in female compared to male rats and neonatal testosterone propionate reduced dopamine concentration in the female AVPV. Neonatal (-)DOI had no effect on dopamine and 5-HT activity in the AVPV but increased both in the ARC. The findings that TH-ir neurone number and dopamine activity are greater in the female AVPV; the feminising effect of 5-HT is prevented by a haloperidol; and the masculinising effect of testosterone propionate is accompanied by a decrease in TH-ir neurone number and dopamine concentration in the female AVPV, suggest that dopamine is involved in hypothalamic sexual differentiation and may mediate the effect of 5-HT.

Gonadal steroids that establish sexually dimorphic characteristics of brain morphology and physiology act at a particular stage of ontogeny. Testosterone secreted by the testes during late gestational and neonatal periods causes significant brain sexual dimorphism in the rat. This results in both sex-specific behaviour and endocrinology in adults. Sexual differentiation may be due to neurogenesis, migration or survival. Each mechanism appears to be uniquely regulated in a site-specific manner. Thus, the volume of an aggregate of neurones in the rat medial preoptic area (POA), termed the sexually dimorphic nucleus of the POA (SDN-POA), is larger in males than in females. The anteroventral periventricular nucleus (AVPV) is packed with neurones containing oestrogen receptor (ER)beta in female rats but, in males, ERbeta-positive neurones scatter into the more lateral portion of the POA. POA neurones are born up to embryonic days 16-17 and not after parturition. Therefore, neurogenesis is unlikely to contribute to the larger SDN-POA in males. DNA microarray analysis for oestrogen-responsive genes and western blotting demonstrated site-specific regulation of apoptosis- and migration-related genes in the SDN-POA and AVPV.

Inactivation of the maternally imprinted, paternally expressed gene 3 (Peg3) induces deficits in olfactory function, sexual and maternal behaviors, oxytocin neuron number, metabolic homeostasis and growth. Peg3 is expressed in a number of developing hypothalamic and basal forebrain structures and is a component of the P53 apoptosis pathway. Peg3 inactivation in neuronal cell culture lines inhibits P53 mediated apoptosis, which is important in the early postnatal development and sexual differentiation of the brain. In this study, we investigated the effect of inactivating the Peg3 gene on the incidence of caspase 3 positive cells (a marker of apoptosis) in 4- and 6-day postpartum mouse brain. Inactivating the Peg3 gene resulted in an increase in the incidence of total forebrain caspase 3 positive cells at 4 and 6 days postpartum. Increases in specific neuroanatomical regions including the bed nucleus of the stria terminalis, nucleus accumbens, caudate putamen, medial pre-optic area, arcuate nucleus, medial amygdala, anterior cortical and posteriodorsal amygdaloid nuclei, were also observed. In wild-type mice, sex differences in the incidence of caspase 3 positive cells in the medial amygdala, bed nucleus of the stria terminalis, nucleus accumbens, arcuate nucleus and the M2 motor cortex, were also observed. This neural sex difference was ameliorated in the Peg-3 mutant. These findings suggest that the neuronal and behavioral deficits seen in mice lacking a functional Peg3 gene are mediated by increases in the incidence of early neonatal apoptosis in neuroanatomical regions important for reproductive behavior, olfactory and pheromonal processing, thermoregulation and reward.

Masculinization of the brain is dependent upon a perinatal surge in testosterone. It also requires a transient decrease in hypothalamic 5-HT concentration and turnover and an increase in androgen receptor (AR) expression during the second postnatal week. We have previously shown that increasing 5-HT activity over this period in male or androgenized female rats feminizes their adult behaviour and also feminizes the size of their anteroventral periventricular nucleus (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA). To investigate the role of 5-HT in sexual differentiation of the brain, 5-HT activity was raised over postnatal days 8-16 in male, female and androgenized female rats by daily administration of the 5-HT 2 receptor agonist (-)[2,5 dimethoxy-4-iodophenyl]-2-amino propane hydrochloride [(-)DOI]. By postnatal day 18, the size of the AVPV and SDN-POA was sexually dimorphic; their sizes were feminized by (-)DOI treatment. In the absence of (-)DOI treatment, there were significantly more AR-immunoreactive cells in the AVPV of males, and in the SDN-POA of males and androgenized females, than in those of females on postnatal day 18. (-)DOI treatment reduced the number of AR-immunoreactive cells in the AVPV and SDN-POA of males and androgenized females, but not of females, by postnatal day 18. These results suggest that 5-HT 2 receptor activation can influence sexual differentiation of the brain by controlling AR expression.

In vitro, the organophosphate insecticide fenitrothion is a potent competitive androgen receptor antagonist, whereas in vivo it affects the development of the male rat reproductive system. The purpose of this pilot study was to determine whether prenatal exposure to fenitrothion affects development of the rat sexually dimorphic nucleus of the medial preoptic area (SDN-POA). Pregnant rats (n = 5–6 litters/group) were orally dosed with corn oil (vehicle) or fenitrothion (20 or 25 mg kg−1 day−1) from gestation day (GD) 12–21. Offspring were euthanized after reaching sexual maturity (females 60–65 days old and males 96–105 days old) and the SDN-POA volumes determined for two rats/sex/litter. Tremors, increased lacrimation and decreased body weight gain were observed in dams from both fenitrothion exposure groups. Reproductive effects in male offspring, including reduced anogenital distance on postnatal day (PND) 1 and increased retention of areolae (PND 13) were observed following fenitrothion exposure at these dose levels. These effects did not persist into adulthood. There was a dose-related increase in the SDN-POA volume in males and a dose-related decrease in SDN-POA volume in females exposed to fenitrothion. These SDN-POA volume changes contrast with those seen with flutamide, another potent anti-androgen, and suggest that fenitrothion may have mixed endocrine effects on the developing brain. Copyright © 2007 John Wiley & Sons, Ltd.

Previous studies on polytocous rodents have revealed that the fetal intrauterine position influences its later anatomy, physiology, reproductive performance and behavior. To investigate whether the position of a fetus in the uterus modifies the development of the brain, we examined whether the structure of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of rat brains accorded to their intrauterine positions. Brain sections of adult rats gestated between two male fetuses (2M) and between two female fetuses (2F) in the uterus were analysed for their immunoreactivity to calbindin-D28k, which is a marker of the SDN-POA. The SDN-POA volume of the 2M adult males was greater than that of the 2F adult males, whereas the SDN-POA volume of the 2M and 2F adult females showed no significant difference. This result indicated that contiguous male fetuses have a masculinizing effect on the SDN-POA volume of the male. To further examine whether the increment of SDN-POA volume in adulthood was due to exposure to elevated steroid hormones during fetal life, concentrations of testosterone and 17beta-estradiol in the brain were measured with 2M and 2F fetuses during gestation, respectively. On gestation day 21, the concentrations of testosterone and 17beta-estradiol in the brain were significantly higher in the 2M male rats as compared with the 2F male rats. The results suggested that there was a relationship between the fetal intrauterine position, hormone transfer from adjacent fetuses and the SDN-POA volume in adult rat brains.

Hypothalamic 5HT concentrations are transiently lower in male compared to female Wistar rats in the second week post partum (pp) and our previous findings have shown that pharmacologically potentiating 5HT activity over this period feminizes certain aspects of sexually differentiated behaviours in adult males and androgenized females. In order to investigate whether neonatal testosterone and 5HT interact to influence physiological and morphological brain sexual differences, females, androgenized females and males were treated with the 5HT2 agonist (-) [2,5 dimethoxy-4-iodophenyl]-2-amino propane HCl [(-) DOI], over days 8-16 pp. In androgenized females (250 microg testosterone proprionate, day 2 pp) (-) DOI prevented the delay in vaginal opening, but did not prevent the androgen-induced constant oestrus in females treated with 100 microg TP, day 2 pp. (-) DOI overcame the neonatal androgen effect in suppressing the positive feedback of ovarian steroids in a few males and androgenized females. (-) DOI had a feminizing effect on the volume of the anteroventral periventricular nucleus (normally smaller in males), by significantly increasing its volume in male and androgenized females. It also had a significant antagonistic effect on the testosterone-induced increase in the volume of the sexually dimorphic nucleus of the preoptic area in males and androgenized females. These findings support the view that raised 5HT activity in the second week of life antagonizes the masculinizing effect of neonatal testosterone.

Male green anoles possess two copulatory organs (hemipenes), which are independently controlled by bilateral muscles: the transversus penis (TPN) and retractor penis magnus (RPM). Adult females do not possess hemipenes or either of the two related muscles. Motoneurons projecting to the TPN lie in spinal segments trunk 17 and sacral 1 (T17-S1). Overall, motoneurons in this region are larger and more numerous in males than females. The present studies were designed to determine 1) whether motoneurons projecting to the RPM are located in the same sexually dimorphic nucleus, 2) other targets of T17-S1 motoneurons, and 3) the approximate proportion of motoneurons projecting to each muscle. In Study 1, unilateral injection of the retrograde tracer Fast Blue (FB) into RPMs and simultaneous unilateral injection of either Cholera Toxin-fluorescein (CT-FITC) or Diamidino Yellow into TPNs revealed that RPM and TPN motoneurons are indeed interdigitated in T17-S1. In Study 2, FB was used to characterize other targets of this nucleus in both males and females. In adult males, projections to four muscles accounted for 96% of the T17-S1 motoneurons: the TPN, RPM, caudifemoralis (CF), and cloacal sphincter (SC). In adult females, projections to the CF and SC comprised 70% of this nucleus. These data demonstrate that the T17-S1 nucleus is a mixed spinal nucleus that has projections to muscles present in both sexes, as well as those present only in males and specialized for male copulatory behavior.

Using quantitative in situ hybridization histochemistry (ISHH), we determined the effect of castration on single cell levels of glutamic acid decarboxylase (GAD) mRNA in discrete hypothalamic regions of the male rat brain associated with the control of gonadotropin secretion. A 48-base oligodeoxynucleotide probe was used to detect with equal affinity the two isoforms of GAD message, GAD65 and GAD67. GAD message also was quantitated in a number of selected areas of the brain to contrast GAD gene expression amongst several populations of GABAergic neurons. Comparison of 11 brain regions demonstrated a 9.3-fold range in the quantity of single cell GAD mRNA with levels being highest in the amygdala and the diagonal band of Broca, moderate in the piriform cortex, caudate nucleus, substantia innominata, globus pallidus, cingulate cortex and medial septal nucleus, and lowest in the lateral septal nucleus and the medial preoptic nucleus (MPN). Castration markedly reduced single cell GAD mRNA levels in the DBB and the MPN, two discrete hypothalamic structures known to contain dendritic fields, cell bodies, and axons of GnRH neurons projecting to the median eminence. A striking finding was a dense core of steroid-sensitive GABAergic neurons within the MPN comprising the sexually dimorphic nucleus of the preoptic area (SDN-POA). Similar to the MPN as a whole, the amount of GAD mRNA expressed by cells in the SDN-POA of sham operated control rats was greater than in castrated animals. GAD mRNA levels were inversely related to serum LH titers, suggesting a role for these neurons in the mechanism controlling gonadal steroid negative feedback on LH secretion. This report provides the basis for future work to determine if GAD65, GAD67 or whether both isoforms are affected by gonadal steroid input.

This study reports the first systematic rostrocaudal distribution of estrogen receptor beta immunoreactivity (ERbeta-ir) in the human hypothalamus and adjacent areas in five males and five females between 20-39 years of age and compares its distribution to previously reported ERalpha in the same patients. ERbeta-ir was generally observed more frequently in the cytoplasm than in the nucleus and appeared to be stronger in women. Basket-like fiber stainings, suggestive for ERbeta-ir in synaptic terminals, were additionally observed in various areas. Men showed more robust nuclear ERbeta-ir than women in the medial part of the bed nucleus of the stria terminalis, paraventricular and paratenial nucleus of the thalamus, while less intense, but more nuclear, ERbeta-ir appeared to be present in, e.g., the BSTc, sexually dimorphic nucleus of the medial preoptic area, diagonal band of Broca and ventromedial nucleus. Women revealed more nuclear ERbeta-ir than men of a low to intermediate level, e.g., in the suprachiasmatic, supraoptic, paraventricular, infundibular, and medial mamillary nucleus. These data indicate potential sex differences in ERbeta expression. ERbeta-ir expression patterns in subjects with abnormal hormone levels suggests that there may be sex differences in ERbeta-ir that are "activational" rather than "organizational" in nature. Similarities, differences, potential functional, and clinical implications of the observed ERalpha and ERbeta distributions are discussed in relation to reproduction, autonomic-function, mood, cognition, and neuroprotection in health and disease. (C) 2003 Wiley-Liss, Inc

Calbindin-D28k (calbindin) is a 28 kilodalton calcium binding protein which potentially plays a role in neuroprotection. We report here the normal development and gonadal steroid modulation of a sexually dimorphic group of calbindin immunoreactive cells within the sexually dimorphic nucleus of the preoptic area (SDN) which we call the calbindin-immunoreactive SDN or CALB-SDN. Beginning on PN2, a faintly immunoreactive CALB-SDN is present, however, the volume is not sexually dimorphic. On PN4, the staining of the CALB-SDN appears more robust but the volume is still not sexually dimorphic. By PN8 and extending through PN12 and PN26, the latest age analysed, the volume of the CALB-SDN is larger in males by two- to fourfold. Cresyl violet counterstain reveals a similar developmental profile of the SDN as well as clusters of darkly staining calbindin immunonegative cells which lie around the CALB-SDN. Castration of males on PN0 decreases the volume of the CALB-SDN by PN12 and administration on the day of birth and PN1 of either testosterone propionate or oestradiol benzoate, but not dihydrotestosterone propionate to females increases the volume of the CALB-SDN by PN12. By demonstrating the sexual dimorphism and gonadal steroid modulation of the CALB-SDN, we hereby establish that calbindin is a specific marker of a subdivision of the SDN and can be used as such in future studies.

Sprague-Dawley rats were treated intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU) at 0, 12.5 or 50 mg/kg/day on days 9 through 15 of gestation to evaluate the effects on development of the brain of offspring. Prenatal exposure to BrdU induced abnormal development of the brain; dilatation of the lateral ventricles in male offspring in the postnatal period. The ratio of the length of the longitudinal fissure to that of the cerebral cortex decreased in a dose-dependent manner in the embryonic period and thereafter. In 14-week-old male offspring exposed prenatally to BrdU at 50 mg/kg, the cortex layer of the cerebrum was thinner than that of the controls. Masculine sexual behavior was markedly impaired and the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was decreased in the 50 mg/kg group as compared with the controls. These results demonstrate that prenatal exposure to BrdU affected the development of the brain in the prenatal and postnatal stages and reduced the volume of SDN-POA after puberty, resulting in a disruption of reproductive ability in male rats.

Tamoxifen is an antiestrogen widely used for the treatment of breast cancer. Current evolutions in preventive strategies to include healthy premenopausal women warrant the study of its developmental toxicity. Perinatal treatment of male rodents with tamoxifen caused reproductive tract lesions and sexual behavior deficits similar to those induced by diethylstilbestrol (DES). Those abnormalities could originate, at least in part, from lesions of the hypothalamic-pituitary axis. The initial alterations caused by tamoxifen in the hypothalamic medial preoptic area (MPOA) and sexually dimorphic nucleus of the preoptic area (SDN-POA) were studied in 6-day-old male rat pups treated with 100 μg tamoxifen (group 1), 1 μg DES (group 2) or vehicle (group 3) from PN1 to 5. In situ hybridization was performed to analyze the expression of the GAP-43 gene, a marker of neuronal differentiation, and morphometry was used to study the neuronal density in the SDN-POA and MPOA and the volume and number of neurons in the SDN-POA. Tamoxifen reduced severely the volume and neuron numbers in the SDN-POA (46.1% and 47.8% of controls, respectively). The neuronal density of the MPOA was not modified. GAP-43 gene expression was decreased as demonstrated by a greater percentage of unlabeled neurons (grade 0) mirrored by a lesser percentage of intensely labeled ones (grade 2) in the SDN-POA and MPOA. In contrast to the effects of the antiestrogen, DES did not affect the above endpoints. These data indicated that developmental exposure of male rat pups to tamoxifen-induced immediate neuronal loss in one and altered differentiation in two hypothalamic areas crucial to reproduction. How those initial alterations contribute to the pathogenesis of the reproductive disorders observed in the adult male needs further investigation. Teratology 56:220–228, 1997. © 1997 Wiley-Liss, Inc.

The anteroventral periventricular nucleus of the hypothalamus (AVPV) is a sexually dimorphic nucleus in the preoptic region that appears to be a nodal point in forebrain circuits, mediating hormonal feedback on gonadotropin secretion. The results of anterograde transport experiments indicate that the AVPV sends ascending projections to the ventral part of the lateral septal nucleus, the parastrial nucleus, and the region adjacent to the vascular organ of the lamina terminalis (OVLT) that contains a subpopulation of gonadotropin releasing hormone (GnRH)-containing neurons. The majority of projections from the AVPV pass caudally through the periventricular zone of the hypothalamus and form dense terminal fields in the periventricular nuclei, parvicellular parts of the paraventricular nucleus, and in the arcuate nucleus. Inputs to medial zone nuclei are more limited, with substantial projections to only the medial preoptic and dorsomedial nuclei. The AVPV sends few projections to the caudal brainstem, but terminals were observed reliably in the periaqueductal gray and medial part of the nucleus of the solitary tract. Anterograde double-labeling experiments demonstrate terminals derived from neurons in the AVPV in close apposition to GnRH-containing neurons in the preoptic region, and to dopaminergic neurons in the arcuate nucleus. Thus, the organization of projections from the AVPV in female rats suggests that neurons in this nucleus may influence the secretion of luteinizing hormone and prolactin through direct projections to GnRH neurons and tuberoinfundibular dopaminergic neurons.

The sexually dimorphic nucleus of the preoptic area (SDN-POA) and the anteroventral periventricular nucleus of the preoptic area (AVPvN-POA) are sexually dimorphic regions in the rat brain. These regions are sensitive to reproductive function-related drugs, and thus applied for aromatase inhibitor evaluation in the present study. Effects of NKS01 (an aromatase inhibitor) and tamoxifen on the SDN-POA and AVPvN-POA were examined. Five mg of NKS01 or its vehicle was injected to SD male and female rats from days 1 to 14 (the day of birth = day 0). Other 10 males and 10 females received a single injection of 100 μg tamoxifen on day 1. Rats in each group were sacrificed at day 30 or 60. The SDN-POA was examined at days 30 and 60, and the AVPvN-POA at day 60. When NKS01 was given to male rats for the first 14 days of life, a significant reduction of SDN-POA size was observed on day 60. When tamoxifen was given, SDN-POA reduction in size was also observed at days 30 and 60 in male rats. As for the AVPvN-POA, tamoxifen markedly reduced volume in female rats, resulting in polycystic ovaries at day 60. This may be due to an estrogenic property of tamoxifen. On the other hand, NKS01 was not significantly effective in the AVPvN-POA, although the volume of the nucleus in NKS01 males was inclined to increase. From these results, as well as the SDN-POA, the AVPvN-POA may be useful to evaluate the side effect of reproductive function-related drug.

Summary The medial preoptic nucleus of the porcine hypothalamus is characterized by an age-dependent sexual dimorphism in the postnatal period of the development. It manifests itself as sexual differences in the density and volume of the perikarya. A transient concentration of perikarya in the centre of the medial preoptic nucleus, especially distinctive in male piglets, is somewhat similar to the ‘sexually dimorphic nucleus of the preoptic area’ of Gorski, as characterized in rats. Zusammenfassung Der Nucleus praeopticus medialis hypothalami des Schweines zeichnet sich in der postnatalen Entwicklungsphase durch einen altersbezogenen neuronalen Sexualdimorphismus aus. Er tritt in Form uberaus deutlicher Geschlechtsunterschiede der Perikaryenpackungsdichte und des Perikaryenvolumens in Erscheinung. Eine vorubergehend bei mannlichen Ferkeln auffallende Perikarienkonzentration im Zentrum des Nucleus praeopticus medialis erinnert entfernt an den fur die Ratte charakteristischen ‘sexually dimorphic nucleus of the preoptic area’ Gorskis.

D1 dopamine receptors were pharmacologically characterized and localized by quantitative autoradiography in the basal ganglia of male and female European starlings (Sturnus vulgaris). The D1 selective antagonist SCH 23390 was used to label this receptor subtype. Starlings are songbirds and possess a neural circuit implicated in the learning and production of song. This circuit includes a sexually dimorphic nucleus, area X, that is a subregion of the parolfactory lobe of the basal ganglia and is known from work on zebra finches to receive dopaminergic input from the area ventralis of Tsai. We focused our investigation on the D1-like receptor subtype because they are abundant in the basal ganglia. Competition studies indicate that a variety of dopaminergic ligands compete with [3H] SCH 23390 for the binding site in an order of potency characteristic of a D1-like receptor. Autoradiographic studies of the basal ganglia revealed high D1 receptor densities in the avian homologues of the caudate-putamen and relatively low-receptor densities were observed in the avian homologue of the globus pallidus. In male starlings, area X could be reliably discerned on the autoradiograms by the higher density of D1 receptors compared to the surrounding parolfactory lobe (LPO). This was also true for females, though nt as reliably as in males. When we compared the mean D1 receptor density in area X for males and females we did not find a significant sex difference. However, we also analyzed the data by comparing sex differences in the degree to which area X has a higher receptor density in comparison with the surrounding LPO. When we normalized D1 receptor density in area X relative to the LPO, we did find a significant sex difference. This sex difference in relative receptor density represents another neural sex difference in the song circuit that may mediate sex differences in the learning and production of song in starlings and other songbirds. 1994 John Wiley & Sons, Inc.

Sex differences in the growth and dendritic development of neurons in the sexually dimorphic nucleus of the preoptic area were examined with quantitative Golgi techniques during early postnatal life in rats. Neuronal size and dendritic extent were found to increase more in males than in females during the first 10 postnatal days, while the numbers of primary and terminal dendrites were similar in the two sexes. The onset of greater dendritic growth in males occurs just after the volume of the nucleus begins to exhibit sexual dimorphism, between 24 and 26 days after fertilization. Growth of dendrites in this region may be related to the presence of sex hormones during the critical period of sexual brain differentiation.

The sexually dimorphic nucleus of the preoptic area (SDN-POA) is larger in volume in males, is responsive to steroids developmentally, and contains a subpopulation of late-arising neurons that can be specificially labeled with 3H-thymidine on embryonic day 18 (E18). The cytoarchitecture of this region has been described, and one component, the central part of the medial preoptic nucleus (MPNc), shows considerable overlap with the SDN-POA. One goal of the present study was to relate the two by determining if testosterone propionate (TP) exposure perinatally increases MPNc volume and neuronal number, and by characterizing the distribution of the late arising neurons of the SDN-POA with respect to the MPNc. A second goal was to determine if these late-arising neurons are a representative, hormone-sensitive population. Finally, TP exposure was delayed past the time of the endogeneous testosterone surge in males and after the neurons have become postmitotic, to determine if female brain structure could still be sex-reversed under these conditions. Pregnant rats were injected on E18 with 3H-thymidine. Daily injections of 2.0 mg TP were given to the mothers starting on either E16 or E20 and continued through birth. The pups were injected daily with 100 μg TP from birth through postnatal day 10. Control rats, from mothers given oil from E16 until birth, were injected with oil from birth through postnatal day 10. Rats were sacrificed at 30 days of age and their brains processed for autoradiography. Neurons and labeled cells were counted in three areas: (1) the MPNc, (2) the region surrounding the MPNc, and (3) a region anterior to the MPNc within the SDN-POA. Labeled cells composed approximately 16% of the neurons in the MPNc and only 3 to 4% in the control regions. Within the MPNc there were more neurons and labeled cells in males than in females. Testosterone treatment, regardless of the starting day, increased MPNc volume and its number of neurons and labeled cells to those of males. These data demonstrate that there is a sex difference in the number of late-arising neurons at 30 days of age, that they are sensitive to steroids during development, and that they are localized to the MPNc. In addition, TP exposure is effective beyond the time of the endogeneous surge in males. Steroid injections in one group of animals were not started until neurons in this region had become postmitotic but still resulted in a sex-reversal, suggesting that testosterone does not modify neurogenesis.

The volume of an intensely staining component of the preoptic area of the male rat is markedly larger than that of the female. Moreover, its volume in both sexes is altered by perinatal hormone exposure consistent with the view that this brain region undergoes hormone dependent sexual differentiation. The present study was carried out to determine if this sexually dimorphic area of the brain has a greater cell density than that of the surround, and if a unique population or distribution of cells, either within one sex or between males and females, characterized this region. A single coronal paraffin section (10 micrometer) through the approximate center of this sexually dimorphic area in four adult gonadectomized rats of each sex was evaluated systematically. Each cell was labelled as being inside or outside of the sexually dimorphic area. In addition to cell density per unit area the following parameters were evaluated through a closed-circuit video system: cell size, staining intensity, shape, and the presence of processes and of a nucleolus. The presence of a nucleolus was further used to identify neurons within the total population of almost 5000 cells that was evaluated. In both sexes, the sexually dimorphic area was characterized by a significantly increased cell density per unit area compared to that of the surround. On this basis, the term, the Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA) is proposed, for this region. Moreover, the SDN-POA of the male was characterized by increased neuronal density per unit area. The SDN-POA in the male was also found to contain larger cells and neurons, as determined by direct measurement of their greatest diameter, as well as a greater percentage of cells and neurons rated large on a three-point scale (small, medium, and large). No consistent differences in frequency distribution by stain intensity, shape, or the presence of cell processes were found to characterize the SDN-POA or contribute to the sexual dimorphism. It is concluded that the marked sex difference in the volume of the SDN-POA is due principally to an increase in the male of the total area of higher cell and neuronal density. However, the present results do not eliminate the possibility that more subtle differences in neuronal characteristics may exist in the SDN-POA.

Opiate receptor labeling was examined throughout the early postnatal period using autoradiography to localize and quantify [ 3 H]naloxone binding to μ-type opiate receptors in the medial preoptic area (MPOA). This region begins to exhibit sexual dimorphism of volume and dendritic growth shortly after birth. A distinct concentration of opiate receptor labeling appears on postnatal day 3 in females: this labeling is directly associated with the sexually dimorphic nucleus of the preoptic area (SDN-POA). SDN-POA labeling becomes denser through postnatal day 10 in females and the densely labeled area increases in size to encompass and surround the SDN-POA. These changes in opiate receptor labeling occur only in females, since males show relatively uniform labeling across the region throughout the early postnatal period. The critical time of formation of dense MPOA opiate receptor labeling may be related to endogenous MPOA opioid function and to the critical period of dendritic growth of SDN-POA neurons. The timing of these critical periods and their focus in the SDN-POA are coincident. The possible role of MPOA opiate receptors in modulating growth of MPOA neurons is discussed.