Chitose Orikasa

DISTRIBUTION AND HORMONE REGULATION OF ESTROGEN RECEPTOR IMMUNOREACTIVE CELLS IN THE HIPPOCAMPUS OF MALE AND FEMALE RATS

Estrogen regulates the synaptic plasticity and physiology of the hippocampus as well as learning behaviors that are mediated by the hippocampus. The density of dendritic spines and synapses, the number of N‐methyl‐D‐aspartate (NMDA) binding sites, the levels of NMDA receptor subunit NR1 protein, muscimol binding to the γ‐amino butyric acid (GABA)A receptor, and levels of glutamic acid decarboxylase message in the CA1 region of the hippocampus are altered with estrogen treatment. In addition, some of these parameters exhibit sex differences in their response to estrogen treatment. To establish that estrogen can have a direct effect on the hippocampus and to determine whether or not sex differences in estrogen responsiveness are due to sex differences in estrogen receptor (ER) levels, we used immunocytochemistry with the AS409 antibody to map the location of ER‐immunoreactive (ER‐ir) cells in the hippocampus of male and female rats. We found that (1) the ERs appear to be in interneurons rather than pyramidal or granule cell neurons, (2) ER‐ir cells are located in greatest concentration in the hilus of the dentate gyrus and the stratum radiatum of the CA1 region, (3) the density of ER‐ir cells exhibits a rostral to caudal gradient in the hilus and the CA1 regions, (4) there are no sex differences in either the number or immunostaining intensity of ER‐ir cells in the hippocampus, (5) the ER levels are down‐regulated by estrogen in both male and female rats, and (6) the mean intensity of staining for the ER‐ir cells in the hippocampus is about 25% of that in the ER‐ir cells of the hypothalamus. From this, we can conclude that estrogen can have a direct effect on hippocampal neurons and that any sex differences in estrogen responsiveness is due to something other than sex differences in ER levels or function in the hippocampus. J. Comp. Neurol. 388:603–612, 1997.

Sexually dimorphic expression of estrogen receptor β in the anteroventral periventricular nucleus of the rat preoptic area: Implication in luteinizing hormone surge

Striking sex difference was detected in the expression of estrogen receptor (ER) β mRNA and protein by nonisotopic in situ hybridization and immunohistochemistry in the anteroventral periventricular nucleus (AVPV) of the rat preoptic area. In females more than in males, a significantly larger number of ERβ mRNA-positive cells were visualized in the medial-most portion of the AVPV within 50 μm from the ependymal lining of the third ventricle. Rats of 7, 14, 21, 35, and 60 days of age (d 1 = day of birth) showed the sex difference. Orchidectomy of male neonates or estrogen treatment of female pups reversed the brain phenotype when examined on d 14. In the AVPV of adult females, ERα immunoreactivity colocalized in 83% of ERβ mRNA-positive cells. Tyrosine hydroxylase immunoreactivity colocalized in 18% of ERβ immunoreactive cells in d 21 females. Infusion of an ERβ antisense oligonucleotide into the third ventricle in the vicinity of the AVPV resulted in significantly longer days of successive estrus and a 50% reduction in the number of ERβ-immunoreactive cells in the AVPV. These findings provide support for the hypothesis that activation of ERβ in the AVPV is an important regulatory event in the female-typical induction of luteinizing hormone surge by estrogen.

Estrogen configures sexual dimorphism in the preoptic area of C57BL/6J and ddN strains of mice

Immunohistochemistry using a calbindin D28k antibody revealed a marked sex difference in neuronal distribution in the central portion of the medial preoptic area in C57BL/6J and ddN strains of mice when the animals were sacrificed on D65 (D1 = the day of birth). Male mice had a distinct ellipsoidal cell aggregate, whereas females lacked such a structure. This sex difference was not observed in Nissl‐stained sections. Co‐localization of calbindin D28k and the neuron‐specific nuclear protein NeuN confrmed that the cells in the aggregate were neurons. The aggregates were larger in males than in females in both strains. When observed on D65, males orchidectomized on D1 had smaller aggregates. However, daily injections of 2 μg estradiol benzoate through D1–D5 as well as a single injection of 100 μg testosterone propionate on D1 enlarged the aggregates in females, but a single injection of 100 μg dihydrotestosterone on D1 had no effect on the female phenotype. Similar endocrine manipulations had no effects in adult animals of both sexes. Thus, the calbindin‐immunoreactive cell aggregates in the preoptic area of C57BL/6J and ddN mice are homologous to the sexually dimorphic nucleus of the rat preoptic area in terms of the morphology and sex steroid‐dependent organization. J. Comp. Neurol. 518:3618–3629, 2010.

Estrogen receptor alpha, but not beta, is expressed in the interneurons of the hippocampus in prepubertal rats: an in situ hybridization study.

Estradiol is involved in the differentiation and plasticity of hippocampal neurons. In the CA1 region, estrogen treatment increases dendritic spines and synapse density on pyramidal cells. In the adult hippocampus, immunoreactivity for estrogen receptor alpha (ERalpha) has been reported in inhibitory interneurons, but neither in the pyramidal neurons nor in granule cells. Estrogens also mediate aspects of sexual differentiation of the hippocampus. To examine the possibility that an alteration in expression of the two types of estrogen receptors (ERalpha and ERbeta) in the hippocampus underlies different roles of estrogen and/or ERs during development and in adult life, we applied non-isotopic, digoxigenin (dig)-labeled, in situ hybridization histochemistry (ISHH) for the both ER forms and examined the distribution pattern of their messages in serial, frontal sections over the postnatal period and in the adult. ERalpha mRNA expression was found scattered throughout the hippocampus especially in the hilar region of the dentate gyrus, and in the strata radiatum and pyramidale in the cornus ammonis at postnatal days (PND) 14, 21 and 35. In the hilus of the dorsal hippocampus, the density of ERalpha-labelled cells was greater in the rostro-medial aspect, while less in the lateral and the caudal region. In the ventral hippocampus the signals for ERalpha mRNA were also found in relatively high density in the hilus. No significant sex difference in distribution and intensity of the ERalpha mRNA positive cells were detected. The hippocampal distribution of ERalpha mRNA expression at PND 14 remained the same on PND 21 and 35 and in adulthood. As reported for adults, ERalpha mRNA signals appear to be in interneurons of the hippocampus but neither in the pyramidal cells nor in the dentate granular cells based on their size and location. In contrast to the result of ERalpha, no clear signals for ERbeta mRNA were detected in the hippocampus across all ages examined, whereas they were clearly detected in the hypothalamus.

Exogenous estrogen acts differently on production of estrogen receptor in the preoptic area and the mediobasal hypothalamic nuclei in the newborn rat

Expression of the estrogen receptor (ER) in the preoptic area (POA) and the mediobasal hypothalamus (MBH) in newborn female rats was studied by immunohistochemistry (IHC) and in situ hybridization histochemistry (ISHH). The number of ER immunoreactive (ER-IR) cells decreased and expression of ER mRNA was suppressed in the arcuate (ARH) and the ventromedial (VMH) hypothalamic nuclei by daily injections of estradiol benzoate (EB) for ten consecutive days. In contrast, in the POA, expression of ER mRNA was not suppressed by EB treatment, while the ER immunoreactivity and the number of ER-IR cells was decreased by EB treatment. Results of quantification of ER mRNA by reverse transcription-polymerase chain reaction correlated well with results from ISHH: that is, ER mRNA expression decreased in the MBH but not in the POA. Thus, estrogen affects ER gene expression differently in these two brain regions.

Ontogeny of gender-specific responsiveness to stress and glucocorticoids in the rat and its determination by the neonatal gonadal steroid environment.

The neuroendocrine response to stress in the rat displays gender-specific characteristics resulting from both sex hormone-dependent organization of neuroendocrine regulatory mechanisms and the modulatory action of circulating gonadal steroids. To define the role of gonadal steroid-mediated brain differentiation in the emergence of sex-specific differences in pituitary-adrenal function, and the necessity of physiological gonadal secretions for the manifestation of these differences, we examined the ontogeny of diurnal and stress-induced corticosterone (B) secretion, and suppressibility of the latter by dexamethasone (DEX) in intact male and female rats, and in animals that were subject to neonatal manipulations of the gonadal steroid environment (orchidectomy in males and neonatal estrogenization in females). Further, gene expression of corticosteroid receptors (MR and GR), corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP) under basal conditions, and following adrenalectomy (ADX) and chronic supplementation with high doses of B, were investigated in adult male and female rats, and individuals of both sexes which have been exposed to alterations of the gonadal steroid milieu during early development. The results demonstrate that: i) gender-specific differences in basal and stress-induced adrenocortical secretion are present at birth, but are still maleable by neonatal alterations of the gonadal steroid environment; ii) gender-specific dichotomy in the sensitivity of the secretory stress response to glucocorticoid feedback becomes fully manifest in adulthood; iii) sex differences in basal adrenocortical secretion become fully expressed only in the presence of intact gonads, whereas, once established by the neonatal hormonal milieu, differential sensitivity of the stress response to glucocorticoids persists in the absence of functioning gonads; iv) neonatal hormone manipulations alter sex-specific characteristics of CRH, AVP, MR and GR gene expression in the brain, and the changes persist in adulthood independently of gonadal secretions; v) regulation of CRH gene expression by glucocorticoids displays gender-specific patterns which are probably established during the period of sex hormone-dependent brain organization and their manifestation does not require physiological gonadal secretions in adulthood.

Sex difference in the expression and regulation of nitric oxide synthase gene in the rat preoptic area

Neuronal nitric oxide synthase (nNOS) mRNA-positive cells were visualized by non-isotopic in situ hybridization histochemistry in the organum vasculosum of the lamina terminalis (OVLT) and the preoptic area (POA) in gonadectomized juvenile female and male rats. In the rostral POA (rPOA) at the level of the anteroventral periventricular nucleus, nNOS mRNA-positive cells were distributed in an inverted V-shaped area over the third ventricle and were in close proximity to cell bodies of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons. In the caudal POA (cPOA) at the level of the medial preoptic nucleus, no topological association existed between GnRH and nNOS. Throughout the rPOA, both the number and the area of nNOS mRNA positive cells were significantly larger in the gonadectomized females than in the gonadectomized males. Treatment with estradiol for 2 days, followed by progesterone in the next morning, which caused an increase in serum luteinizing hormone 6 h later, induced a significant reduction of the nNOS mRNA expression in the rPOA in the female but not in the male rat at the time of sacrifice. In the OVLT and the cPOA, ovarian steroids had no effect on nNOS mRNA expression of both sexes. The results indicate that nNOS mRNA expression in the rPOA is sexually dimorphic and regulated by ovarian steroids in a sex specific manner.

ESTROGEN RECEPTOR FOUND IN THE FACIAL NUCLEUS OF THE NEWBORN RAT IS SUPPRESSED BY EXOGENOUS ESTROGEN : IMMUNO- AND IN SITU HYBRIDIZATION HISTOCHEMICAL STUDIES

Expression of the messenger RNA coding estrogen receptor (ER-mRNA) was detected in the ventromedial subnucleus of the facial nucleus of the newborn rat by in situ hybridization histochemistry (ISHH). The hybridization signal in this subnucleus increased from 1 to 6 days of age, then decreased at 11 days. By immunohistochemistry (IHC) using an antiserum which detects estrogen receptor (ER) specifically, immunopositive signals were also detected in the same subnucleus of the adjacent sections. On the other hand, neither of these signals were encountered in the same subnucleus of the adult rat. Thus, the present result extend our previous work (Yokosuka and Hayashi, 1992) showing that the expression of the ER in the facial nucleus is transient. A sex difference in the expression of ER molecules was not apparent by ISHH and IHC. Moreover, daily injections of estradiol from the day of birth suppressed the expression of ER in the subnucleus at 6 and 11 days of age. Thus, as has been detected in the mediobasal hypothalamus, ER-mRNA was revealed to be down-regulated by estrogen.

Expression of estrogen receptor in the facial nucleus is suppressed by estradiol, but not by testosterone, indicating a lack of requirement for aromatization

The transient expression of estrogen receptor (ER) in the ventromedial subnucleus of the facial nucleus was previously detected in the newborn rat, and the expression of ER molecules was down-regulated by daily injections of estradiol. Here we examined possible involvement of aromatization in this process. ER molecules were measured by immunohistochemistry and in situ hybridization histochemistry after daily injections of testosterone propionate (TP; 100 micrograms/0.02 ml) and estradiol benzoate (EB; 10 micrograms/0.02 ml) in the male pups castrated within 24 h of birth. Daily injections of TP for 5 consecutive days did not suppress ER and ER mRNA in the facial nucleus, while they were both suppressed by daily injections of EB. Moreover, aromatase immunoreactivity was not detected in the facial nucleus of both castrated, TP injected and intact control males at 6 days of age. The present findings therefore suggest that ER molecules expressed transiently in the facial nucleus are not directly involved in masculine sexual differentiation of the brain in newborn rat.

Similar numbers of neurons are generated in the male and female rat preoptic area in utero

The birth date of neurons comprising the sexually dimorphic nucleus of the rat preoptic area (SDN-POA) was determined by bromodeoxyuridine (BrdU) injections at a prescribed time during the embryonic period. Calbindin immunostaining was used as a marker to identity the SDN-POA. The animals were bred from dams injected with BrdU on days 14, 16 or 18 of pregnancy (fertilization defined as day 1). On day 15 after birth (PD), all offspring were euthanized and brain sections were prepared for histology. Neurogenesis in the SDN-POA began around embryonic day (ED) 14 and culminated on ED 18, whereas the preoptic neurons surrounding the SDN-POA generated earlier than did those of the SDN-POA. Although the SDN-POA was significantly larger in males than in females at PD15, the total numbers of neurons comprising the SDN-POA were not significantly different between sexes. Similar aggregates of somatostatin mRNA-positive cells in the central portion of the SDN-POA were observed in both sexes at PD8. On PD15, the aggregates became scattered in males, whereas the aggregates in females remained congested. These data suggest that sexual dimorphism in the SDN-POA results from male-specific postnatal radial spreading of cells rather than cell proliferation during embryonic neurogenesis.