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A crucial event in the acute regulation of steroidogenesis by trophic hormones is the delivery of cholesterol into the mitochondria where it is converted to pregnenolone by the cholesterol side chain cleavage enzyme. Although considerable controversy exists regarding the exact mechanisms that underlie this acute response to hormone stimulation, recent studies suggest that the Steroidogenic Acute Regulatory (StAR) protein, a hormone-induced 30-kilodalton mitochondrial protein, plays an essential role. We now extend these studies by establishing in MA-10 mouse Leydig tumor cells a temporal relationship between levels of StAR expression and steroidogenesis in response to hormone stimulation. These data indicate that trophic hormones regulate StAR mRNA and protein within a time frame concomitant with the acute production of steroid hormones and provide the first evidence implicating changes in StAR transcription and/or mRNA stability in the functional response of steroidogenic cells to hormone action. In addition, in situ hybridization analyses of StAR expression in embryonic and adult mice demonstrated a precise spatial and temporal relationship in vivo between StAR expression and the capacity to produce steroid hormones. These experiments strengthen considerably the evidence that StAR is the key mediator of the acute induction of steroidogenesis and provide new insights into the mechanisms by which trophic hormones activate steroidogenesis in steroidogenic cells.

Targeted disruption of the mouse Ftz-F1 gene, which encodes the orphan nuclear receptors steroidogenic factor 1 (SF-1) and embryonal long terminal repeat-binding protein (ELP), established that this gene is essential for development of the primary steroidogenic tissues and for male sexual differentiation. Associated with these dramatic developmental abnormalities, all Ftz-F1-disrupted mice died in the immediate postnatal period and had very low glucocorticoid levels. In this report, we show that treatment with corticosteroids markedly prolonged survival of the Ftz-F1-disrupted mice, proving that steroid hormone deficiency causes their death. We also generated SF-1-specific knockout mice with a targeting construct that specifically disrupted the SF-1 coding sequence without impairing the ELP protein. The phenotype of the SF-1-specific knockout mice was indistinguishable from that observed in Ftz-F1-disrupted mice that lack both SF-1 and ELP. Taken together, these results indicate that SF-1 is the Ftz-F1-encoded protein that is required for multiple aspects of endocrine development and for postnatal survival.

The nuclear receptor steroidogenic factor 1 (SF-1) regulates the biosynthesis of the two essential mediators of male sexual differentiation, androgens and Müllerian-inhibiting substance, and is required for adrenal and gonadal development and gonadotropin expression. SF-1 is also expressed in the embryonic ventral diencephalon, subsequently localizing to the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene had normal numbers and location of GnRH neurons but exhibited grossly impaired ventromedial hypothalamic nucleus structure. Despite their apparently normal GnRH neurons, treatment of Ftz-F1-disrupted mice with GnRH restored pituitary gonadotropin expression. These studies define SF-1's essential role within a discrete hypothalamic nucleus previously linked to reproduction.

Two c-myc genes have been isolated from a tetraploid fish, the common carp (Cyprinus carpio). The nucleotide (nt) sequences of the two genes and their flanking regions (CAM1, 8675 bp and CAM2, 5784 bp) have been determined. Both genes contain two coding exons which are homologs of c-myc exons 2 and 3 in higher vertebrates. The nt sequence homologous to c-myc exon 1 of higher vertebrates has not been found. Carp CAM1 and CAM2 presumably encode peptides of 394 and 401 amino acids (aa), respectively. These two peptides share 55–91% as identity with those of humans, chickens, frog, rainbow trout and zebra fish. The two carp c-Myc have a Glu stretch at the beginning region of exon 3. This Glu stretch is also present in zebra fish, suggesting that it may be characteristic to c-myc genes of cyprinid fishes. The existence of two c-myc in carp is attributed to the tetraploid nature of carp. These two genes share 92.4% nt identity in the coding region and 84.2% in the intron. The two deduced peptides share 94.2% as identity, suggesting that the tetraploid event probably occurred 58 million years ago. Both of the genes' cDNA clones were obtained from a cDNA library of carp peripheral blood leukocytes, implying that both of the genes are expressed.

To assess cytotoxic activity of carp (Cyprinus carpio), head kidney leukocytes were examined with special reference to the effects of rearing water temperature and of assay temperature. Leukocytes as effector cells were separated from head kidney using Histopaque 1077 and cytotoxic activities were measured by the release of 51Cr from target cells which were K562, human chronic myelogenous leukemia cells. Cytotoxic activity of leukocytes from carp kept at higher temperature (25 °C) was lower at lower assay temperature (10 °C) than at higher assay temperature (25 °C). However, activity from carp acclimated to low temperature (10 °C) increased on 10 °C assay and decreased on 25 °C assay. Cellular composition of effector cells changed in connection with the above phenomenon. In carp acclimated to 10 °C, small lymphocytes decreased while the others, e.g. large lymphocytes, granulocytes and macrophages, increased. Accommodation of carp natural killer-like cells to environmental temperatures may be regulated by changing the cellular composition of effector cells.

Dinucleotide–repeat DNA polymorphisms for rainbow trout, Oncorhynchus mykiss, are described. The potential applications of these markers in fisheries science are discussed.

As an initial step toward understanding its role in steroidogenesis, we studied the developmental profile of steroidogenic factor-1 (SF-1), a nuclear receptor that regulates the steroid hydroxylases. SF-1 transcripts first appear on embryonic day 9 (E9) in the urogenital ridge, the probable source of steroidogenic cells of both adrenals and gonads. By E11, after the adrenals and gonads are clearly separate, SF-1 transcripts are detected throughout the adrenal primordium. Thereafter, adrenal expression of SF-1 localizes to the cortex. Consistent with its proposed role in regulating cholesterol side-chain cleavage enzyme (SCC), SF-1 is expressed before SCC. During the sexually undifferentiated stage of gonadal development (E9-E12), all embryos express SF-1 in the genital ridge. As testicular cords form in males, SF-1 transcripts are diffusely expressed throughout the testis, whereas SCC mRNA is limited to the interstitium. These differences between SF-1 and SCC reflect SF-1 expression by Sertoli cells, as shown by Northern blotting and in situ hybridization. In contrast to its persistent expression in the embryonic testis, SF-1 transcripts disappear from the ovary between E13.5-E16.5, reappearing only during late gestation (E18.5). Thus, expression of SF-1 in the embryonic gonad is sexually dimorphic. Coupled with the demonstration of SF-1 mRNA in Sertoli cells, these data suggest that SF-1 plays a role in gonadal development distinct from regulating the steroidogenic enzymes. Additionally, SF-1 is expressed in the embryonic forebrain, implying a role in neural development.

Natural killer (NK) activity of head kidney leucocytes of rainbow trout (Oncorhynchus mykiss) against IPN virus-infected and uninfected RTG-2 cells was examined by 51Cr release assay. Fish leucocytes were classified into four groups by cytotoxic activities : Type I, cytotoxic activity against uninfected RTG-2 cells was higher than that against infected cells; Type II, cytotoxic activity against infected cells was higher than that against uninfected cells; Type III, cytotoxic activity was low against both infected and uninfected cells; Type IV, cytotoxic activity was high against both cells. These results indicate that there are individual variations in NK activity of rainbow trout against allogenic and viral antigens.

Studies were conducted to clarify the relationship between growth rate and disease progression of erythrocytic inclusion body syndrome (EIBS) in artificially infected coho salmon, Oncorhynchus kisutch (Walbaum). In the diseased state, the haematocrit values decreased and the number of erythrocytes with inclusions was higher in faster growing fish. Rapid growth was accompanied by an abundance of immature erythrocytes, which had the greatest incidence of inclusion bodies.

Characteristics of natural killer-like cells (NK-like cells) in carp (Cyprinus carpio) were studied by 51Cr-release assay. Among cells examined, K562 cells (human erythroleukemic cells) were found to be the most suitable as target cells for NK assay in carp. Pre-incubation of the leucocytes from head kidney and peripheral blood enhanced NK activity. Leucocytes from the head kidney and kidney showed the highest NK activity and those from peripheral blood, thymus and spleen followed in the order. The cold target inhibition test indicated that carp NK-like cells consist of more than one population for target recognition.

Recent investigations in fish have revealed the dietary dependence of immunosurveillance. This report describes the evidence for dietary link of non-specific cytotoxic activity of natural-killer cells in rainbow trout. Fish were reared for varying periods in different experiments designed to determine the role of protein, essential fatty acid and zinc. Later the immune response was recorded by measuring the release of 51 Cr from labelled P815 mouse mastocytoma target cells after 8h incubation. Leucocytes isolated from the head kidney of experimental fish were employed as effector cells.Dietary protein does not have any effect on the natural-killer like activity of the leucocytes. The activity was low when the diets were deficient in essential fatty acid and zinc.

A cDNA clone encoding the c-myc gene has been isolated from the common carp (Cyprinus carpio). This clone consisted of 1334 base pairs (bp) and contained the whole exonIII and most of exonII of c-myc gene of carp. The derived amino acid sequence showed higher homology to that of rainbow trout (71.5%) and lower to humans (55.0%). The significance of the use of the clone as a probe for the analysis of fish tumors was discussed.

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.

Epizootics attributable to erythrocyytc inclusion body syndrome (EIBS) occurred among populations of coho salmon Oncorhynchus kisutch cultured in seawater in Japan. Onset of the disease was correlated with water temperatures declining to below 10°C. Symptoms of EIBS were severe anemia with hematocrits of less than 20% and corresponding changes in erythrocyte counts, hemoglobin concentration, and mean corpuscular hemoglobin concentration. Erythrocytes had characteristic inclusion bodies that contained enveloped viral particles with a diameter of approximately 77 nm. The disease was reproduced in artificially induced infections.

Acquired resistance to erythrocytic inclusion body syndrome (EIBS) was investigated in coho salmon (Oncorhynchus kisutch).Fish which were experimentally infected with EIBS and maintained at 15°C for 242 days were challenged by EIBS virus and kept at 8°C. The fish survived the challenge, though they showed a slight decrease in Ht values which recovered presently. This result indicates that acquired resistance to EISB continues at least as 242 days. Follow-up examination of coho salmon which survived EIBS in freshwater farm ponds revealed that they maintained acquired resistance after being transferred to marine farms.

1986年以来， 毎年5-7月の低水温期に宮城県下の淡水養魚場のギンザケに集団発生している貧血症について， 赤血球内封入体の検出， 電顕観察によるウイルスの確認と人工感染の成立ならびに本病耐過魚が示したEIBS抵抗性から， 本病がEIBSウイルスに起因する疾病であることが確かめられた。死亡率の上昇に伴い， 病魚はミズカビの寄生を受けるものが多く見られたので， ギンザケの水カビ病の一次因の一つとしてEIBSが疑われた。本報は淡水ギンザケのEIBSの日本での最初の記載である。

Erythrocytic inclusion body syndrome (EIBS) is a major contributor to mortality of coho salmon reared in seawater net-pen culture in Japan. In this paper, a study of disease progression after artificial infection was carried out to better understand the disease and its causative agent.The five stages described by Piacentini et al. were recognized. Stage II and III were divided into two substages, respectively. A substage (Stage II-a) in which inclusions appeared only in immature erythrocytes was characterized. Stage III which was characterized by appearence of lysed erythrocytes was divided into two substages; Stage III-a (18-21 days postinjection), during which the prevalence of inclusions in mature erythrocytes increased rapidly and incidence of immature erythrocytes decreased rapidly, and Stage III-b (by 24 days postinjection), when the Ht values were lowest and no inclusions were observed in erythrocytes.Our result showed that a viral agent which causes high mortality in sea-cultured coho salmon in Japan has the same characteristic disease progression as the EIBS virus of North America.

EIBS(rythrocytic inclusion body syndrome,赤血球封入体症候群)病魚由来の血液， 脾臓， 頭腎および腸内容物のホモジネート濾液を用いて， 健康なギンザケ稚魚を種々の条件下で人工感染させた。人工感染用生体材料としては封入体を有する軽症魚赤血球が最も強い感染性を示した。この魚の全血ホモジネート濾液を腹腔注射またはその希釈液に浸漬すると水温8～10℃で， 赤血球封入体はそれぞれ3または4週目に最も高率に出現し， Ht値は4または5週目に最低となった。その感染性は腹腔注射で10,000倍希釈， 浸漬で1,000倍希釈まで保持された。

To study the effects of gestational exposure to estrogen on early gonadal differentiation, pregnant mice were treated by sc injection of diethylstilbestrol (DES) or vehicle from embryonic day (E) 8.5 to E14.5, and gonads at E11.5, E12.5, and E14.5 were examined. Quantitative real-time RT-PCR and in situ hybridization revealed that mRNA levels of steroidogenic factor 1 (SF-1), a key regulator of gonadal differentiation, and several male gonad-specific genes, including Mullerian-inhibiting substance (MIS), steroidogenic acute regulatory protein, cholesterol side-chain cleavage cytochrome P450, and Cerebellin 1 precursor protein, were significantly decreased in the DES-treated testis, compared with the control testis at E12.5 and/or E14.5. Immunohistochemistry demonstrated that the staining intensities for SF-1 and MIS in Sertoli cells were apparently reduced in the DES-treated testis, compared with those of the controls, at E12.5 and E14.5. Because MIS, steroidogenic acute regulatory protein, cholesterol side-chain cleavage cytochrome P450, and Cerebellin 1 precursor protein are activated under the regulation of SF-1, the down-regulation of these factors may be due to reduced SF-1 expression. Immunohistochemistry for laminin-1 demonstrated that ovigerous cords in the DES-treated ovary were smaller than those in controls at E14.5. Moreover, the number of 5-bromo-2′deoxyuridine-5-monophosphate-labeled cells in the DES-treated testis was significantly reduced at E12.5 and E14.5, compared with controls, and that in the DES-treated ovary remained higher than that in the control ovary at E14.5. The results suggest that exogenous estrogens can alter sex-specific genetic pathways governing early differentiation and cell proliferation of both male and female gonads.

GPT or GPT sensor was prepared by a combination of pyruvate oxidase or pyruvate oxidase-oxaloacetate decarboxylase and an oxygen electrode. The optimum condition for each sensor was established, then GPT activity in fish serum was determined by the proposed GPT sensor. GPT activities in fish serum obtained by the proposed sensor system were in good agreement with those determined by a conventional method. One assay could be completed within 20min. Good results were obtained for continuous determination during 10h (30 assays). The conditions for the determination of GPT activities in fish serum were as follows: pH, 7.0; temperature, 33°C; flow rate of sample solution, 0.1ml/min; flow rate of substrate solution, 0.08ml/min; and sample volume, 10μl.

The ventromedial hypothalamus (VMH) is a sexually dimorphic region of the brain related to female reproductive behavior. The effect of estrogen in the adult rat VMH is thought to be mediated predominantly via estrogen receptor (ER)alpha, because this receptor is expressed at considerably higher levels than ER beta. The present study revealed, using in situ hybridization and immunohistochemistry, that both ER beta mRNA and protein were expressed in the ventrolateral portion of the caudal VMH, at remarkably higher levels during early postnatal development than in adulthood. In addition, the expression was sexually dimorphic, with females having significantly more ER beta-immunoreactive (-ir) cells than males, between postnatal d 5 (P5) and P14, although the sex difference was not significant by P21. Double-label immunofluorescence revealed that 66% of ER beta-ir cells coexpressed ER alpha in the caudal VMH of the P5 female rat. Furthermore, neonatal treatment with E2 benzoate down-regulated ER beta mRNA in the female rat VMH at P5 and decreased VMH ER beta-ir cells during the period between P5 and P14. In contrast to females, no differences in expression of ER beta mRNA or protein were detected between control and E2 benzoate-treated males. These results suggest that estrogen is involved in regulating the sexually dimorphic expression of ER beta in the VMH during early postnatal development of the rat.

Exposing neonatal rats with the synthetic estrogen, diethylstilbestrol (DES), induces morphological and functional abnormalities in the adult ovary. We examined the events that lead to this condition using female rats that were exposed to DES for the first five days after birth. The expression of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), which are both required for steroidogenesis in the theca/interstitial region was markedly reduced. The expression of Mullerian inhibiting substance (MIS) was transiently increased in small growing follicles in the ovary of DES-treated rats at postnatal day 7 (P7), and the expression profile in the ovary differed between DES- and vehicle oil-treated rats at P14 and P21. The expression of the transcription factor, steroidogenic factor-1 (SF-1), reduced in theca/interstitial cells, but increased in granulosa cells of primary follicles. These results indicate that altered steroidogenesis and MIS production are mechanisms through which DES induces abnormal ovarian development, and support the notion that androgens and MIS are both critical factors in regulating early ovarian differentiation.

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)

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.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) was identified originally as a key regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases. Hints at considerably broader roles for SF-1 came from analyses of its expression pattern in mouse embryos. As anticipated, SF-1 was expressed in the adrenal glands and gonads from their early stages of development. Surprisingly, SF-1 also was expressed outside of the primary steroidogenic tissues in the anterior pituitary and hypothalamus. SF-1 knockout mice dramatically confirmed its multiple essential roles in vivo. These mice lacked adrenal glands and gonads, leading to adrenocortical insufficiency and male-to-female sex reversal of their internal and external genitalia. SF-1 knockout mice also had impaired pituitary expression of gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-gonadal axis. With some focus on the ovary, this review summarizes experiments that have defined essential roles of SF-1 in endocrine development, and highlights important areas for future studies.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) has emerged as a critical regulator of steroidogenic cell function within the adrenal cortex and gonads. SF-1 also contributes to endocrine development and function at all three levels of the hypothalamic-pituitary-gonadal axis. First identified as a regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases, SF-1 subsequently was shown to play considerably broader roles in endocrine function. Knockout mice deficient in SF-1 lacked adrenal glands and gonads and exhibited male-to-female sex reversal of their internal and external genitalia; moreover, they had impaired gonadotrope function and agenesis of the ventromedial hypothalamic nucleus (VMH). These studies delineated multiple essential roles of SF- 1 in regulating endocrine differentiation and function. However, the molecular basis underlying these profound consequences of the SF-1 knockout remains to be determined. Moreover, with respect to the biosynthesis of steroids in sites other than the adrenal cortex and gonads, including the neurosteroids, an important goal for further studies is to identify the mechanisms that regulate expression of the steroidogenic enzymes in the absence of SF-1.

Steroidogenic acute regulatory protein (StAR) delivers cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroid hormone biosynthesis. StAR expression is restricted to steroidogenic cells and is rapidly induced by treatment with trophic hormones or cAMP. We analyzed the 5'-flanking region of the mouse StAR gene to elucidate the mechanisms that regulate its cell-specific and hormone-induced expression. In transient transfection assays, a luciferase reporter gene driven by the StAR 5'-flanking region was preferentially expressed by steroidogenic Y1 adrenocortical and MA-10 Leydig cells in a cAMP-responsive manner. 5'-Deletion and site-directed mutagenesis studies identified a region between -254 and -113 that is essential for full levels of promoter activity. This region contains a binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) that, although not required for hormone induction, is critical for basal promoter activity, thus implicating SF-1 in StAR expression. Analyses of knockout mice deficient in SF-1 further supported an important role for SF-1 in StAR gene expression. These studies provide novel insights into the mechanisms that regulate StAR gene expression and extend our understanding of SF-1's global roles within steroidogenic cells.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) was isolated as a transcription factor expressed specifically in the mouse primary steroidogenic tissues. SF-1 expression occurs at the earliest stages of adrenal and gonadal development and the expression pattern is sexually dimorphic in gonads during sexual differentiation. The two hormones required for male differentiation, testosterone and Mullerian-inhibiting substance, are regulated by SF-1. Analyses of knockout mice lacking SF-1 by gene targeting disruption demonstrated that the SF-1-disrupted mice lack adrenal glands and gonads, supporting the suggestion that SF-1 is an essential regulator of the endocrine development and differentiation. Additionally, SF-1 is expressed in the pituitary gonadotropes and the ventrolateral hypothalamic nucleus, which are higher levels of the reproductive regulatory axis, of both adults and embryos. These tissues are also affected in SF-1 knockout mice, indicating that SF-1 plays extended roles at all levels of the reproductive axis, by regulating more genes involved in reproductive function and development.

Steroid hormone biosynthesis requires the cytochrome P-450 steroid hydroxylases, which act sequentially to convert cholesterol to physiologically active steroid hormones (reviewed in 1). Expression of these enzymes is generally limited to steroidogenic cells and, within these cells, is coordinated induced by trophic hormones, raising the possibility that their expression is controlled by a shared transcriptional regulator. Studies analyzing promoter activity of the 5′-flanking regions of these genes in cell lines derived from steroidogenic cells showed that multiple promoter elements were needed for full levels of expression. Notably, each gene was regulated by elements that matched the AGGTCA “half-site” motif for nuclear receptor family members (2, 3). Recent studies described here indicate that these elements interact with a cell-selective nuclear receptor, steroidogenic factor-1 (SF-1), that is essential for adrenal and gonadal development and that also plays key roles at the hypothalamic and pituitary levels of the reproductive axis.

Recent analyses of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1), in their coordinate, cell-selective expression. SF-1 was proposed to regulate the steroid hydroxylases by interacting with shared promoter elements in their 5′-flanking regions. During mouse embryonic development, SF-1 was expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting a key role in steroidogenic cell differentiation. Finally, disruption of the gene encoding SF-1 revealed its essential function in the development of the adrenal glands and gonads and in pituitary gonadotrope function. These studies suggest that SF-1 acts at multiple levels of the reproductive axis to maintain reproductive competence.

Recent studies of the gene regulation of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1). SF-1 binds to shared promoter elements upstream of the steroid hydroxylases to mediate their coordinate expression in steroidogenic cells. Analyses of SF-1 expression during mouse embryonic development showed that SF-1 is expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting an intimate link between SF-1 and steroidogenic cell differentiation. Finally, in gene disruption experiments, the gene encoding SF-1 was shown to be essential for development of the adrenal glands and gonads. These results establish the essential role of this orphan nuclear receptor in the development and function of the primary steroidogenic tissues.

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, regulates the enzymes that produce sex steroids, and disruption of the Ftz-F1 gene encoding SF-1 precludes adrenal and gonadal development. We now study the role of SF-1 at other levels of the hypothalamic/pituitary/gonadal axis. In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones showed a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. In situ hybridization analyses confirmed these results; pituitaries from Ftz-F1-disrupted mice lacked transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibited decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first became detectable at embryonic day 13.5-14.5, preceded the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalized with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacted with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice. These studies establish novel roles of this orphan nuclear receptor in reproductive function.

Normal male sex differentiation requires that Sertoll cells in the embryonic testes produce Mullerian Inhibiting substance (MIS), a TGFβ-like hormone that causes Mullerian duct regression. In primary Sertoli cells, the orphan nuclear receptor, steroidogenic factor 1 (SF-1), regulates the MIS gene by binding to a conserved upstream regulatory element. In heterologous (HeLa) cells, MIS gene activation by SF-1 requires removal of the SF-1 ligand-binding domain, implicating a Sertoll cell-specific ligand or cofactor. Finally, the sexually dimorphic expression of SF-1 during development coincides with MIS expression and Mullerian duct regression. We propose that SF-1 regulates MIS in vivo and participates directly in the process of mammalian sex determination.

Studies in adrenocortical cells have implicated the orphan nuclear receptor SF-1 in the gene regulation of the steroid hydroxylases. We used targeted disruption of the Ftz-F1 gene, which encodes SF-1, to examine its role in intact mice. Despite normal survival in utero, all Ftz-F1 null animals died by postnatal day 8; these animals lacked adrenal glands and gonads and were severely deficient in corticosterone, supporting adrenocortical insufficiency as the probable cause of death. Male and female Ftz-F1 null mice had female internal genitalia, despite complete gonadal agenesis. These studies establish that the Ftz-F1 gene is essential for sexual differentiation and formation of the primary steroidogenic tissues.

The cytochrome P450 steroid hydroxylases are coordinately regulated by steroidogenic factor 1 (SF-1), a protein expressed selectively in steroidogenic cells. Based on its expression in steroidogenic tissues and DNA-binding specificity, we isolated a putative SF-1 cDNA from an adrenocortical cDNA library. As evidence that this cDNA encodes SF-1, we now show that it is selectively expressed in steroidogenic cells, that an antiserum against its protein product specifically abolishes the SF-1-related gel-shift complex, and that its coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. Sequence analyses of the SF-1 cDNA revealed that it is the mouse homolog of fushi tarazu factor I (FTZ-F1), a nuclear receptor that regulates the fushi tarazu homeobox gene in Drosophila. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contained both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defined shared exons encoding common regions and alternative promoters and 3'-exons leading to differences between the two FTZ-F1 transcripts. We used in situ hybridization with transcript-specific probes to study the ontogeny of SF-1 and ELP expression. ELP transcripts were not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

プラスチックシャーレの底に Edwardsiella tarda を付着させて作った細菌薄層にコイおよびニジマスの血液をのせ, 薄層上に形成された食菌プラークを画像処理し, プラークの数および面積を測定した。10, 20, 30℃の反応温度における貧食能を比較した結果, コイでは30℃で最大の貧食能を示したが, ニジマスではプラーク数および全プラークの面積は20℃で, 1個当りのプラーク面積は30℃で最大であった。