Yayoi Ikeda

Regional expression of a gene encoding a neuron-specific Na(+)-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain.

We have analyzed expression of a gene encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter (DNPI), which was recently cloned from human brain, in rat forebrain using in situ hybridization. The expression of DNPI mRNA showed a widespread but highly heterogeneous pattern of distribution in the forebrain, where hybridization signals were observed in neurons but not in any other types of cells. Neurons expressing the mRNA were far more numerous in the diencephalon than in the telencephalon. In the thalamus, a number of neurons with high levels of signals were localized to all nuclei of the dorsal thalamus, habenular nuclei and subthalamic nucleus, but not the reticular nucleus and zona incerta. Moderate signal levels were seen in many neurons throughout the hypothalamus, particularly the ventromedial, paraventricular, supraoptic and arcuate nuclei, lateral hypothalamic area and mammillary complex. In contrast, expression of DNPI mRNA in the telencephalon was generally at a low level and occurred locally in some restricted regions within the neocortex, retrosplenial cortex, piriform cortex, olfactory regions, hippocampal formation and medial amygdaloid nucleus. The present results suggest that DNPI functions in heterogeneous neuron populations as a neuron-specific Na(+)-dependent inorganic phosphate cotransport system predominantly expressed in the diencephalon of the rat.

Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse

reelin has recently been isolated as a candidate gene, the mutation of which gives rise to the reeler phenotype in mice. In this study, we analyzed the expression of reelin during embryonic development in the mouse and in adult mouse tissues, by in situ hybridization. reelin transcripts were present on embryonic day (E) 8.5 in the somite, foregut, yolk sac, and unclosed neural plate. reelin was expressed in the brain, spinal cord, liver, and kidney throughout embryonic development, and transiently in many developing organs such as the optic cup, blood vessels, precartilage, stomach, pituitary, vibrissae, tooth germ, and in cells along growing nerve fibers. These observations indicate a role for reelin in development of organs in addition to that in neuronal migration. Furthermore, we demonstrated the existence of reelin mRNA and its cellular distribution in the adult brain, spinal cord, liver, kidney, testis, and ovary, suggesting additional roles for reelin in stabilizing the cytoarchitecture and in remolding in adult organs. However, we detected no obvious phenotype of the reelin‐expressing organs except for the brain in the reeler mouse, indicating the functional redundancy of this gene during the development of these organs. Dev. Dyn. 1997;210:157–172.

Comparative localization of Dax‐1 and Ad4BP/SF‐1 during development of the hypothalamic‐pituitary‐gonadal axis suggests their closely related and distinct functions

Two nuclear receptors, Ad4BP/SF‐1 and Dax‐1, are essential regulators for development and function of the mammalian reproductive system. Similarity in expression sites, such as adrenal glands, gonads, pituitary, and hypothalamus, suggests a functional interaction, and the phenotype similarities were manifested in Ad4BP/SF‐1‐deficient mice and in cases of natural human mutations of Dax‐1. In this study, quantitative reverse transcriptase polymerase chain reaction analyses revealed that expression profiles of Dax‐1 in embryonic gonads are different between the two sexes and also from those of Ad4BP/SF‐1. Immunohistochemical analyses clarified the spatial and temporal expressions of the Dax‐1 protein during development of tissues composing the hypothalamic‐pituitary‐gonadal axis. During gonadal development, Dax‐1 occurred after Ad4BP/SF‐1 exhibiting a sexually dimorphic expression pattern at indifferent stages, indicating a possibility of Dax‐1 involvement in earliest sex differentiation. When cord formation begins in the testis at embryonic day 12.5 (E12.5), Dax‐1 was expressed strongly in Sertoli cells, but its expression level markedly decreased in Sertoli cells and increased in interstitial cells between E13.5 and E17.5. In the female, Dax‐1 was strongly expressed in the entire ovarian primordium from E12.5 until E14.5, and then its expression level was decreased and limited to cells near the surface epithelium between E17.5 and postnatal day 0 (P0). During postnatal development of the testis, the variable staining of Dax‐1 in Sertoli cells was detected as early as P7 and Dax‐1‐expressing Leydig cells became rare. In the postnatal ovary, Dax‐1 expression was detected in granulosa cells with variable staining intensity, and occasionally in interstitial cells. During pituitary organogenesis, Dax‐1 but not Ad4BP/SF‐1 was expressed in the dorsal part of Rathkes pouch from E9.5. Later in development after E14.5, the distribution of Dax‐1 overlapped with that of Ad4BP/SF‐1, being restricted to gonadotropic cells in the anterior pituitary. In the ventromedial hypothalamus (VMH), Dax‐1 and Ad4BP/SF‐1 were mostly colocalized throughout the embryonic and postnatal development. Thus, the coexpression of Dax‐1 and Ad4BP/SF‐1 indicates their closely related functions in the development of the reproductive system. Furthermore, we noticed the presence of cells that express Dax‐1 but not Ad4BP/SF‐1, further indicating additional functions of Dax‐1 in an Ad4BP/SF‐1‐independent molecular mechanism.

Differential localization and colocalization of two neuron-types of sodium-dependent inorganic phosphate cotransporters in rat forebrain.

We studied by immunohistochemistry the distribution of differentiation-associated sodium-dependent inorganic phosphate (Pi) cotransporter (DNPI) in the rat forebrain, in comparison with brain-specific cotransporter (BNPI). DNPI-staining was principally seen in axonal synaptic terminals which showed a widespread but discrete pattern of distribution different from that of the BNPI-staining. In the diencephalon, marked DNPI-staining was seen in the dorsal lateral geniculate, medial geniculate, ventral posterolateral, ventral posteromedial, anterior, and reticular thalamic nuclei without the colocalization with BNPI-staining. DNPI-staining showed a strong mosaical pattern and overlapped well the BNPI-staining in the medial habenular nucleus. DNPI-staining was moderate over the hypothalamus and notably localized in neurosecretory terminals containing corticotropin-releasing hormone in the median eminence. In contrast, the BNPI-staining was region-related and strong in the ventromedial and mammillary nuclei. In the telencephalon, laminar DNPI-staining was seen over the neocortex, corresponding to the thalamocortical termination, and also found in the retrosplenial cortex and the striatum, with the highest intensity in the accumbens nucleus shell. The present results suggest that DNPI serves as a dominant Pi transport system in synaptic terminals of diencephalic neurons including thalamocortical and thalamostriatal pathways as well as the hypothalamic neuroendocrine system in the rat forebrain.

Impaired Follicle Development and Infertility in Female Mice Lacking Steroidogenic Factor 1 in Ovarian Granulosa Cells

Abstract The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.

Neonatal estrogen exposure inhibits steroidogenesis in the developing rat ovary

Treatment of newborn female rats with estrogens significantly inhibits the growth and differentiation of the ovary. To understand the molecular mechanism of estrogen action in the induction of abnormal ovary, we examined the expression profiles of steroidogenic factor 1 (SF‐1) and several of its target genes in the developing ovaries after neonatal exposure to synthetic estrogen, estradiol benzoate (EB) by using reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. Morphologic examination indicated inhibitory effects of estrogen on the stratification of follicles and development of theca and interstitial gland during postnatal ovarian differentiation. The expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side‐chain cleavage cytochrome P450 (P450SCC), which are both essential for steroid biosynthesis, markedly decreased in theca and interstitial cells throughout the postnatal development of the EB‐treated ovary. However, expression of the transcriptional activator of the two genes, SF‐1 was unaffected in theca and interstitial cells, although the number of these cells was lower in the EB‐treated ovary than in the control ovary. The expression of the estrogen mediator, estrogen receptor‐α (ER‐α), diminished specifically in theca cells at P6 and recovered by P14 in the EB‐treated ovary. These results indicate that the effect of estrogens is mediated by means of ER‐α resulting in the down‐regulation of StAR and P450SCC genes during early postnatal development of the ovary. These results suggest that the abnormal ovarian development by neonatal estrogen treatment is closely correlated with the reduced steroidogenic activity, and the data obtained by using this animal model may account in part the mechanism for aberrant development and function of the ovary in prenatally estrogen‐exposed humans.

Central Nervous System-Specific Knockout of Steroidogenic Factor 1 Results in Increased Anxiety-Like Behavior

Steroidogenic factor 1 (SF-1) plays key roles in adrenal and gonadal development, expression of pituitary gonadotropins, and development of the ventromedial hypothalamic nucleus (VMH). If kept alive by adrenal transplants, global knockout (KO) mice lacking SF-1 exhibit delayed-onset obesity and decreased locomotor activity. To define specific roles of SF-1 in the VMH, we used the Cre-loxP system to inactivate SF-1 in a central nervous system (CNS)-specific manner. These mice largely recapitulated the VMH structural defect seen in mice lacking SF-1 in all tissues. In multiple behavioral tests, mice with CNS-specific KO of SF-1 had significantly more anxiety-like behavior than wild-type littermates. The CNS-specific SF-1 KO mice had diminished expression or altered distribution in the mediobasal hypothalamus of several genes whose expression has been linked to stress and anxiety-like behavior, including brain-derived neurotrophic factor, the type 2 receptor for CRH (Crhr2), and Ucn 3. Moreover, transfection and EMSAs support a direct role of SF-1 in Crhr2 regulation. These findings reveal important roles of SF-1 in the hypothalamic expression of key regulators of anxiety-like behavior, providing a plausible molecular basis for the behavioral effect of CNS-specific KO of this nuclear receptor.

Increased Expression of Müllerian-Inhibiting Substance Correlates with Inhibition of Follicular Growth in the Developing Ovary of Rats Treated with E2 Benzoate

Mullerian-inhibiting substance (MIS) is an essential factor for male sexual differentiation. In the present study, we exam- ined whether the expression of MIS and several of its related transcription factors is altered in the ovaries of rats treated with the synthetic estrogen, E2 benzoate (EB; 10 g/0.02 ml), from postnatal day 1 (P1) to P5. The EB-treated rats had a significantly reduced number of layered follicles at P6 in com- parison with the control rats that were treated with vehicle alone. The expression levels of both MIS mRNA and protein in the granulosa cells of small growing follicles in the ovary at P6 were higher in the EB-treated rats than in the controls. These results indicate that the inhibitory effect of EB on the follic- ular stratification may correlate with the inappropriately in- creased expression level of MIS. Furthermore, the expression levels of one of its transcriptional activators, steroidogenic factor 1, and ER- in granulosa cells of small growing follicles were higher in EB-treated ovaries than in the control ovaries. These results suggest the role of MIS in the regulation of follicular growth and the possible involvement of steroido- genic factor 1and/or ER- in this molecular cascade may con- tribute to postnatal ovarian development. (Endocrinology 143: 304 -312, 2002)

Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the –129/–114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.

Differential expression of the estrogen receptors alpha and beta during postnatal development of the rat cerebellum.

Estrogen receptor (ER) beta is a dominant ER subtype in the adult cerebellum. However, it is not known if this is also the case for the developing cerebellum. In the present study, quantitative real-time RT-PCR demonstrated that levels of cerebellar ERalpha mRNA in neonatal pups were significantly higher than in adults. In contrast, expression levels of cerebellar ERbeta mRNA remained significantly unchanged during postnatal development. In situ hybridization and immunohistochemistry demonstrated that ERalpha mRNA and protein were predominantly expressed by Purkinje cells at all ages examined. ERalpha-expressing Purkinje cells were confined to the anterior lobes at postnatal day 7 (P7) but distributed in most lobes at P14 and P21. In the adult cerebellum, however, only a few ERalpha-immunoreactive Purkinje cells were observed. Thus, ERalpha expression was transiently increased during the time when Purkinje cell dendritic growth and synapse formation proceed, suggesting that a role for ERalpha in Purkinje cell differentiation. ERbeta expression occurred in Golgi type neurons in the granular layer at P7, Purkinje cells at P14, and basket cells in the molecular layer at P21 and was detected in all the cell types in the adult cerebellum, suggesting a role for ERbeta associated with neuronal differentiation and maintenance. Furthermore, double-labeled immunofluorescence for ERalpha and ERbeta demonstrated their colocalization in Purkinje cells at P14, suggesting a possibility of their interaction. The discrete expression profiles for ERalpha and ERbeta in the developing cerebellum suggest the two ERs play distinct roles in cerebellar development.