Conditional disruption of Nr5a1 directed by Sox9-Cre impairs adrenal development.

The current study aimed to investigate the effect of Sox9-Cre-directed Nr5a1-conditional knockout (Sox9-Cre;Nr5a1flox/flox) on adrenal development. We showed that SOX9 is expressed by adrenocortical cells at E10.5-E11.5 but is extinguished no later than E12.5. The number of adrenocortical cells significantly reduced in Sox9-Cre;Nr5a1flox/flox mice while the number of cleaved caspase 3-positive cells increased compared to that in the controls at E11.5-E12.5, when the adrenal primordium (AP) is about to expand. This indicated that fetal adrenocortical cells are lost via apoptosis due to Nr5a1 ablation by E12.5. Both medulla formation and encapsulation were perturbed, accompanied by a smaller AP size, in Sox9-Cre;Nr5a1flox/flox mice during embryonic development. Adult Sox9-Cre;Nr5a1flox/flox adrenals were hypoplastic and exhibited irregular organization of the medulla with aberrant sex differentiation in the X zone. Additionally, there were histologically eosin-negative vacuolated cells, which were negative for both the X-zone marker 20αHSD and the steroidogenesis marker 3ßHSD at the innermost cortex of Sox9-Cre;Nr5a1flox/flox adrenals. Although Nr5a1+/- adrenals were hypoplastic, a small number of chromaffin cells were properly located in the center, having normal sex differences in the X-zone. The results collectively provided in-vivo evidence that Nr5a1 plays a critical role in AP expansion and subsequent adrenal development.

Hyaluronic acid hydrogels support to generate integrated bone formation through endochondral ossification in vivo using mesenchymal stem cells.

Engineered cartilage tissue from differentiated mesenchymal stem cells (MSCs) can generate bone in vivo through endochondral ossification (ECO). This ECO-mediated approach has the potential to circumvent the severe problems associated with conventional MSC-based bone tissue engineering techniques that lack mechanisms to induce angiogenesis. Hyaluronic acid (HA) is a key component in the cartilage extracellular matrix. However, the ECO-supporting properties of HA remain largely unclear. This study aimed to compare the ability of HA and collagen hydrogels to support in vitro differentiation of MSC-based hypertrophic cartilage tissues and to promote endochondral bone formation in vivo. Following the chondrogenic and hypertrophic differentiation in vitro, both HA and collagen constructs accumulated sulfated glycosaminoglycan (sGAG) and type 1, type II, and type X collagen. However, HA hydrogels exhibited a more uniform distribution of sGAG, type 1 collagen, type X collagen, and osteocalcin proteins; in addition, the cells embedded in the hydrogels had more rounded cell morphologies than those in the collagen constructs. At week 5 of in vitro culture, two to three constructs were implanted into a subcutaneous pocket in nude mice and harvested after 4 and 8 weeks. Both HA and collagen constructs promoted endochondral bone formation with vascularization and bone marrow development; however, the HA constructs fused to form integrated bone tissues and the bone marrow developed along the space between the two adhered grafts in all implanted pockets (n = 5). In the collagen constructs, the integration was observed in 40% of the pockets (n = 5). Microcomputer CT analysis revealed that the bone volume of HA constructs was larger than that of collagen constructs. In conclusion, compared to collagen hydrogels, HA hydrogels had superior potential to generate integrated bone with vascularization and bone marrow development. This study provides valuable insights for applying ECO-mediated bone tissue engineering approaches for the repair of critical-sized bone defects.

Anatomical characteristics of two cases of aberrant right subclavian artery.

An aberrant right subclavian artery is a branching variation of the aortic arch. We encountered two female cadavers with an aberrant right subclavian artery during routine student dissection at our school. In both cases, the right subclavian artery was not a branch of the brachiocephalic trunk but originated directly from the distal part of the aortic arch as the last branch and ran between the esophagus and vertebral column, traveling to the upper limb. The right recurrent laryngeal nerve was absent, but a non-recurrent inferior laryngeal nerve branching from the vagus and traveling directly toward the larynx was observed. In the first case, the right and left common carotid arteries originated solely from the aortic arch as the first and second branches, respectively, whereas the right and left common carotid arteries formed a bicarotid trunk at their origin in the second case. A Kommerell diverticulum was present at the base of the aberrant right subclavian artery in the second case, but not in the first case. We analyzed the anatomical differences between the two cases and discussed the developmental aspects and potential clinical risks.

Histological and Immunohistochemical Studies to Determine the Mechanism of Cleft Palate Induction after Palatal Fusion in Mice Exposed to TCDD.

Rupture of the basement membrane in fused palate tissue can cause the palate to separate after fusion in mice, leading to the development of cleft palate. Here, we further elucidate the mechanism of palatal separation after palatal fusion in 8-10-week-old ICR female mice. On day 12 of gestation, 40 µg/kg of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), sufficient to cause cleft palate in 100% of mice, was dissolved in 0.4 mL of olive oil containing toluene and administered as a single dose via a gastric tube. Fetal palatine frontal sections were observed by H&E staining, and epithelial cell adhesion factors, apoptosis, and cell proliferation were observed from the anterior to posterior palate. TUNEL-positive cells and Ki67-positive cells were observed around the posterior palatal dissection area of the TCDD-treated group. Moreover, in fetal mice exposed to TCDD, some fetuses exhibited cleft palate dehiscence during fusion. The results suggest that palatal dehiscence may be caused by abnormal cell proliferation in epithelial tissues, decreased intercellular adhesion, and inhibition of mesenchymal cell proliferation. By elucidating the mechanism of cleavage after palatal fusion, this research can contribute to establishing methods for the prevention of cleft palate development.

The conditional deletion of steroidogenic factor 1 (Nr5a1) in Sox9-Cre mice compromises testis differentiation.

Steroidogenic factor 1 (NR5A1) is essential for gonadal development. To study the importance of NR5A1 during early gonadal sex differentiation, we generated Sox9-Cre-Nr5a1 conditional knockout (cKO) mice: Sox9-Cre;Nr5a1flox/flox and Sox9-Cre;Nr5a1flox/- mice. Double-immunostaining for NR5A1 and AMH revealed silenced NR5A1 in Sertoli cells and reduced AMH+ cells in the gonads of XY Sox9-Cre-Nr5a1 cKO mice between embryonic days 12.5 (E12.5) and E14.5. Double-immunostaining for SOX9 and FOXL2 further indicated an early block in Sertoli cells and ectopic granulosa cell differentiation. The number of cells expressing the Leydig cell marker 3ßHSD obviously reduced in the gonads of XY Sox9-Cre;Nr5a1flox/- but not Sox9-Cre;Nr5a1flox/flox mice at E15.5. The presence of STRA8+ cells indicated that germ cells entered meiosis in the gonads of XY Sox9-Cre-Nr5a1 cKO mice. The results of qRT-PCR revealed remarkably reduced and elevated levels of testis and ovary markers, respectively, in the gonads of XY Sox9-Cre-Nr5a1 cKO mice at E12.5‒E13.5. These data suggested that the loss of Nr5a1 abrogates the testicular pathway and induces the ectopic ovarian pathway, resulting in postnatal partial/complete male-to-female gonadal sex reversal. Our findings provide evidence for the critical role of NR5A1 in murine gonadal sex determination in vivo.

A rare case of hyperostosis frontalis interna in an 86-year-old Japanese female cadaver.

Hyperostosis frontalis interna (HFI) is a condition characterized by abnormal bone outgrowth on the inner surface of the frontal bone. Most HFI cases occur in post-menopausal elderly women. The pathology of HFI development is uncertain. The estimated incidence of HFI ranges from 5 to 12% in Western countries, but few cases have been reported in the Japanese population. Here, we report a case of HFI in an 86-year-old Japanese female cadaver. Macroscopically, the internal surface of the frontal bone exhibited bilateral nodular protrusion with sparing of the midline, while the external surface was normal. According to the morphological classification of HFI proposed by Hershkovitz et al. this case belongs to type D, the most severe type. Using computed tomography (CT), we defined five layers, designated as I-V from the inner to the outer layer, in the nodular region of HFI; however, the normal frontal bone is composed of three layers. Histological results demonstrated that layers I, III, and V consisted of the cortical bone, and layers II and IV consisted of the trabecular bone. We also observed increases in the numbers of lamellar bone and blood vessels on the dural side of layer I, indicating increased vascularization and active osteogenesis. These results indicate that layer II represents a new diploe within the inner table, which split into layers I and III, suggesting that diploization within the inner table by activated remodeling may be involved in the development of hyperostosis in this case.

Expression of progesterone receptor, estrogen receptors α and ß, and kisspeptin in the hypothalamus during perinatal development of gonad-lacking steroidogenic factor-1 knockout mice.

Gonadal hormones contribute to brain sexual differentiation. We analyzed expression of progesterone receptor (PR), estrogen receptor-α (ERα), ERß, and kisspeptin, in the preoptic area (POA) and/or the arcuate nucleus (ARC), in gonad-lacking steroidogenic factor-1 knockout (KO) mice during perinatal development. At postnatal-day (P) 0-P7, POA PR levels were higher in wild-type (WT) males compared with WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females. At P14-P21, PR levels in all groups increased similarly. POA ERα levels were similar in all groups at embryonic-day (E) 15.5-P14. Those in WT but not KO males reduced during postnatal development to be significantly lower compared with females at P21. POA ERß levels were higher in WT males than in WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females at P0-P21. POA kisspeptin expression was female-biased in WT mice, while levels in KO females were lower compared with WT females and similar to those in WT and KO males. ARC kisspeptin levels were equivalent among groups at E15.5-P0. At P7-P21, ARC levels in WT but not KO males became lower compared with WT females. Diethylstilbestrol exposure during P0-P6 and P7-P13 increased POA PR and ERß, and decreased POA ERα and ARC kisspeptin levels at P7 and/or P14 in both sexes of KO mice. These data further understanding of gonadal hormone action on neuronal marker expression during brain sexual development.

Cleft palate formation after palatal fusion occurs due to the rupture of epithelial basement membranes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and hydronephrosis in the mouse embryo. Cleft palate occurs due to failure in palatal grow, but the underlying mechanisms are unclear. We investigated the mechanisms of cleft palate development in TCDD-exposed mouse embryos. We administered olive oil (control group) or TCDD diluted in olive oil (40 µg/kg) via gastric tubes to pregnant mice on gestational day (GD) 12. Embryos of control and TCDD-exposed groups were removed from pregnant mice on GD 14 and GD 15, respectively. One mouse embryo from the control group had anteroposterior palatal fusion. Palatal fusion was observed in three TCDD-exposed mouse embryos. Palates of TCDD-exposed mice fused from the interior to the middle of the palates, while the palates were separated in the posterior region. The middle of the embryonic palatal shelves in TCDD-exposed animals was narrow and split at the fusional position. At this position, palatal and blood cells were dispersed from the palatal tissue and the epithelium was split, with a discontinuous basement membrane. The results suggest that decreased intercellular adhesion or insufficient tissue strength of the palatal shelves may be involved in the development of cleft palate following palatal fusion.

Bisphenol A exposure induces increased microglia and microglial related factors in the murine embryonic dorsal telencephalon and hypothalamus.

Bisphenol A (BPA) is a widely used compound in the food packaging industry. Prenatal exposure to BPA induces histological abnormalities in the neocortex and hypothalamus in association with abnormal behaviors. Yet, the molecular and cellular neurodevelopmental toxicological mechanisms of BPA are incompletely characterized on neuroinflammatory-related endopoints. To evaluate the neurodevelopmental effects of BPA exposure in mouse embryos, we examined microglial numbers as well as the expression of microglial-related factors in the E15.5 embryonic brain. BPA-exposed embryos exhibited significant increases in Iba1-immunoreactive microglial numbers in the dorsal telencephalon and the hypothalamus compared to control embryos. Further, the expression levels of microglial markers (Iba1, CD16, iNOS, and CD206), inflammatory factors (TNFα and IL4), signal transducing molecules (Cx3Cr1 and Cx3Cl1), and neurotrophic factor (IGF1) were altered in BPA-exposed embryos. These findings suggest that BPA exposure increases microglial numbers in the brain and alters the neuroinflammatory status at a transcriptional level. Together, these changes may represent a novel target for neurodevelopmental toxicity assessment after BPA exposure.

A case of double inferior vena cava with renal, ovarian and iliac vein variation.

We encountered a rare case of an anatomic variant of inferior vena cava (IVC) duplication with renal, ovarian and iliac vein variation in an 81-year-old Japanese female cadaver during a student dissection course of anatomy at Aichi Gakuin University School of Dentistry. The two IVCs ran upwards bilaterally to the abdominal aorta. The left IVC joined with the left renal vein (RV) to form a common trunk that crossed anterior to the aorta and ended at the right IVC. We detected a vein [interiliac vein (IiV)] connecting the two IVCs at the level of the aortic bifurcation. The IiV was formed by the union of two tributaries from the left IVC and a tributary from the left internal iliac vein (IIV) and ran obliquely upwards from left to right. Two right ovarian veins, arising separately from the ipsilateral pampiniform plexus, ran vertically in parallel to each other, and each one independently terminated at the right IVC and the right RV. Two right IIVs, connecting each other with small branches, ascended and separately joined the right external iliac vein. The right and left IIVs were connected to each other. These variations cause abnormal drainage, which could lead to clinical symptoms associated with the dysfunction of the vascular and urogenital systems. Here we describe the detailed anatomical features of the area and discuss the related anatomical and developmental aspects.

Mechanisms underlying neuro-inflammation and neurodevelopmental toxicity in the mouse neocortex following prenatal exposure to ethanol.

Fetal alcohol spectrum disorders (FASD) constitute a wide range of disorders that arise from prenatal exposure to ethanol (EtOH). However, detailed reports regarding the adverse effects of prenatal EtOH exposure on neocortical morphology and its underlying pathogenic mechanisms are limited. In the present study, we aimed to characterize the anatomical abnormalities of neocortical development and their correlation with microglial properties and neuro-inflammation in a mouse model of FASD. We evaluated the development and maturation of the neocortex in ICR mice prenatally exposed to 25% (w/v) EtOH using histological and molecular analyses. Reduced proliferation and excessive cell death were observed in the dorsal telencephalon. Abnormal neuronal distribution, layer formation, and dopaminergic neuronal projections were observed in the neocortex. Disruption of microglial differentiation (M1/M2 microglial ratio) and abnormal expression of pro-inflammatory and neurotrophic factors were induced, and these abnormalities were ameliorated by co-treatment with an anti-inflammatory drug (pioglitazone). FASD model mice displayed histological abnormalities, microglial abnormalities, and neuro-inflammation in both the embryonic and newborn stages. Thus, anti-inflammatory therapeutics may provide a novel preventive approach for the treatment of FASD.

Expression of Kisspeptin in Gonadotrope Precursors in the Mouse Pituitary during Embryonic and Postnatal Development and in Adulthood.

BACKGROUND: Kisspeptins are important regulators of the development and function of the hypothalamic-pituitary-gonadal axis. However, the importance of kisspeptin at the pituitary level is unclear. METHODS: We examined the expression profile of kisspeptin in the mouse pituitary during development and in adulthood using RT-PCR, quantitative PCR and immunohistochemistry. RESULTS: Kiss1 mRNA was detected in both embryonic and postnatal pituitaries. Kisspeptin-immunoreactive (+) cells were detected from embryonic day (E) 13.5 throughout adulthood, being localized to the rostroventral portion in the anterior pituitary (AP) in embryos, and also to the dorsocaudal AP postnatally. A large proportion of kisspeptin+ cells were double-labeled with gonadotrope markers including Foxl2, SF-1, and LHß, and the percentage of LHß+ cells in kisspeptin+ cells increased during development. No kisspeptin+ cells were positive for the proliferating cell marker MCM7 (minichromosome maintenance protein 7), but a few kisspeptin+ cells co-expressed the stem/progenitor cell marker Sox2. Kisspeptin expression was similar between sexes and between agonadal SF-1 knockout embryos and wild-type littermates. Kiss1 mRNA levels were not significantly different between sexes or during early postnatal development, but levels in females increased when puberty began and were significantly higher than in males at postpubertal ages. CONCLUSIONS: These results suggest that kisspeptin is expressed in gonadotrope precursors during gonadotrope differentiation, and that kisspeptin expression begins soon after the initiation of αGSU production and is extinguished soon after the initiation of LH production. Furthermore, pituitary kisspeptin expression may be regulated in a gonad-independent manner during development, but may be associated with gonadotrope function in adulthood.

Extended Culture Conditions for Multipotent Bone Marrow-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) offer a promising source of cells for musculoskeletal regeneration because of their potential to differentiate into bone, cartilage and fat. However, their proliferation and multilineage differentiation potential decreases with aging or increased time in in vitro culture. To determine culture conditions capable of enabling maintenance of MSCs for extended periods of time, human bone marrow-derived MSCs (BM-MSCs) were cultured in growth medium containing various combinations of growth factors and small chemical compounds. Upon reaching confluence, MSCs were subcultured continuously and then tested for differentiation capacity. After screening various growth factors and small chemical compounds, we found a combination capable of maintaining the proliferation potential of BM-MSCs obtained from a 19-year-old donor (young MSCs) up to passage 13 (P13). In contrast, unsupplemented MSCs reached senescence at P10. Total population doublings of control (P10) and supplemented MSCs (P12) were estimated at 20.4 and 42, respectively. Young MSCs cultured with supplements maintained osteogenic, adipogenic and chondrogenic differentiation capacities at P12 as confirmed by expression of lineage-specific differentiation markers. Furthermore, the supplementation of to BM-MSCs obtained from 65- and 79-year-old donors (aged MSCs) also continued to proliferate until P12, and maintained osteogenic and adipogenic differentiation capacity until P7 and P8, respectively, whereas, unsupplemented aged MSCs stopped proliferating at P8. These results indicate that our extended culture conditions maintained the proliferative capacity of young MSCs while retaining their multipotent differentiation potential, and improved both proliferation and differentiation of aged MSCs.

Cyclin E marks quiescent neural stem cells and caspase-3-positive newborn cells during adult hippocampal neurogenesis in mice.

Cyclin E is a key regulator of progression through the G1-phase of the cell cycle. Recently, a cell cycle-independent role for cyclin E in the adult mouse central nervous system has been suggested. In the present study, we examined expression of cyclin E in the mouse hippocampal dentate gyrus (DG), a region of neurogenesis in adulthood, using immunofluorescence. In the adult DG, cyclin E-immunoreactive (cyclin E+) cells was limited to postmitotic cells. In the subgranular zone, cyclin E was detected in the vertical process of radial glia-like cells, which were marked by the neural stem cell markers nestin and GFAP. Cyclin E was also detected in the nucleus of cells, which were labeled with stage-specific neuronal cell markers, including Pax6, Sox2, NeuroD, doublecortin, and NeuN. The densities of cyclin E+ cells in the DG reduced and increased with age and running, respectively. Furthermore, the majority of cyclin E+ cells co-expressed active caspase-3, a marker of apoptosis. Together, the results indicate that cyclin E is expressed in the process of quiescent neural stem cells and in the nucleus of active caspase-3+ cells during neuronal cell differentiation, suggesting that cyclin E has a Cdk-independent function, which might be important for the mechanisms regulating adult hippocampal neurogenesis.

Involvement of SF-1 in neurogenesis and neuronal migration in the developing neocortex.

The nuclear receptor steroidogenic factor-1 (SF-1) plays essential roles in the development and function of the endocrine and reproductive systems. During embryogenesis, SF-1 is expressed in the ventromedial hypothalamic nucleus (VMH) and regulates the migration and terminal differentiation of the VMH neurons. Additionally, in situ hybridization data indicated SF-1 expression in the dorsal telencephalon at embryonic day (E) 13.5. In this study, we investigated the neocortical development in SF-1 knockout (KO) mouse embryos. The number of neurons was increased in the intermediate/subventricular zones and decreased in the cortical plate in the SF-1 KO embryos. SF-1 KO embryos produced more neural stem/progenitor cells, especially apical progenitor cells, and showed abnormal radial glial fiber morphology. The increase in neural stem/progenitor cells was caused by an increased S-phase fraction in the proliferative cells and the inhibition of cell cycle exit in these cells. The mRNA expression of the estrogen receptor ESRα was up-regulated and that of the estrogen synthetase Cyp19a1 was down-regulated in the dorsal telencephalon of SF-1 KO embryos. We showed that SF-1 is expressed in the dorsal telencephalon at E15.5 and E18.5, but not in adult animals. Our data demonstrated that SF-1 is involved in cell cycle regulation, neurogenesis, and neuronal migration via controlling the estrogen signaling for proper neocortical development.

Newborn mice exposed prenatally to bisphenol A show hyperactivity and defective neocortical development.

The central nervous system is especially susceptible to toxic insults during development. Prenatal administration of bisphenol A (BPA) induces histologic anomalies in the dorsal telencephalon of the embryo. Whether these anomalies affect the morphogenesis and maturation of neuronal function of the newborn neocortex, however, is unknown. To evaluate the neurodevelopmental and behavioral effects of prenatal BPA exposure at 20 and 200µg/kg/day in newborn mice, we performed a detailed histologic analysis of the neocortex and tested for the presence of behavioral abnormalities in newborn mice prenatally exposed to BPA using our newly developed behavioral test. Observations of newborn mice prenatally exposed to BPA revealed abnormal neuronal distribution and layer formation, hypoplasia of layer 6b, and abnormal dopaminergic neuronal projections in the neocortex. Further, the newborn mice exhibited hyperactivity. These findings suggest that prenatal BPA exposure induces neurobehavioral toxicity associated with abnormal dopaminergic neuronal projections, and abnormal corticogenesis and lamination. Histologic and behavioral analyses of newborn mice are considered useful for assessing the neurodevelopmental and behavioral toxicity of chemicals.

NR5A1 is required for functional maturation of Sertoli cells during postnatal development.

The orphan nuclear receptor steroidogenic factor 1 (NR5A1 (SF-1)) is expressed in both Sertoli and Leydig cells in the testes. This study investigates the postnatal development of the testes of a gonad-specific Nr5a1 knockout (KO) mouse, in which Nr5a1 was specifically inactivated. The KO testes appeared histologically normal from postnatal day 0 (P0) until P7. However, disorganized germ cells, vacuoles, and giant cells appeared by P14 in the seminiferous tubules of KO but not control mice. Expression of NR5A1 and various factors was examined by immunohistochemistry (IHC). The number of NR5A1-positive Sertoli cells in the KO testes was lower compared with controls at all the developmental stages and decreased to nearly undetectable levels by P21. IHC for anti-Müllerian hormone and p27, immature and mature Sertoli cell markers, respectively, indicated a delay in Sertoli cell maturation in the KO testes. The number of Sertoli cell-expressing factors involved in Sertoli cell differentiation including WT1, SOX9, GATA4, and androgen receptor were lower in the KO testes compared with controls. Furthermore, fewer proliferating cell nuclear antigen-positive proliferative germ cells were observed, and the number of TUNEL-labeled cells was significantly higher in the KO testes compared with controls at P14 and P21, indicating impaired spermatogenesis. IHC for CYP11A1 (SCC) indicated the presence of steroidogenic Leydig cells in the interstitium of the KO testes at all stages examined. These results suggest that NR5A1 is essential for Sertoli cell maturation and therefore spermatogenesis, during postnatal testis development.

Coactivation of SF-1-mediated transcription of steroidogenic enzymes by Ubc9 and PIAS1.

Steroidogenic factor-1 (SF-1) is a nuclear orphan receptor, which is essential for adrenal development and regulation of steroidogenic enzyme expression. SF-1 is posttranslationally modified by small ubiquitin-related modifier-1 (SUMO-1), thus mostly resulting in attenuation of transcription. We investigated the role of sumoylation enzymes, Ubc9 and protein inhibitors of activated STAT1 (PIAS1), in SF-1-mediated transcription of steroidogenic enzyme genes in the adrenal cortex. Coimmunoprecipitation assays showed that both Ubc9 and PIAS1 interacted with SF-1. Transient transfection assays in adrenocortical H295R cells showed Ubc9 and PIAS1 potentiated SF-1-mediated transactivation of reporter constructs containing human CYP17, CYP11A1, and CYP11B1 but not CYP11B2 promoters. Reduction of endogenous Ubc9 and PIAS1 by introducing corresponding small interfering RNA significantly reduced endogenous CYP17, CYP11A1, and CYP11B1 mRNA levels, indicating that they normally function as coactivators of SF-1. Wild type and sumoylation-inactive mutants of Ubc9 and PIAS1 can similarly enhance the SF-1-mediated transactivation of the CYP17 gene, indicating that the coactivation potency of Ubc9 and PIAS1 is independent of sumoylation activity. Chromatin immunoprecipitation assays demonstrated that SF-1, Ubc9, and PIAS1 were recruited to an endogenous CYP17 gene promoter in the context of chromatin in vivo. Immunohistochemistry and Western blotting showed that SF-1, Ubc9, and PIAS1 were expressed in the nuclei of the human adrenal cortex. In cortisol-producing adenomas, the expression pattern of SF-1 and Ubc9 were markedly increased, whereas that of PIAS1 was decreased compared with adjacent normal adrenals. These results showed the physiological roles of Ubc9 and PIAS1 as SF-1 coactivators beyond sumoylation enzymes in adrenocortical steroidogenesis and suggested their possible pathophysiological roles in human cortisol-producing adenomas.

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain.

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.

Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells.

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.