Minoru Takase

Novel brain function: biosynthesis and actions of neurosteroids in neurons.

Peripheral steroid hormones act on brain tissues through intracellular receptor-mediated mechanisms to regulate several important brain neuronal functions. Therefore, the brain is considered to be a target site of steroid hormones. However, it is now established that the brain itself also synthesizes steroids de novo from cholesterol. The pioneering discovery of Baulieu and his colleagues, using mammals, and our studies with non-mammals have opened the door of a new research field. Such steroids synthesized in the brain are called neurosteroids. Because certain structures in vertebrate brains have the capacity to produce neurosteroids, identification of neurosteroidogenic cells in the brain is essential to understand the physiological role of neurosteroids in brain functions. Glial cells are generally accepted to be the major site for neurosteroid formation, but the concept of neurosteroidogenesis in brain neurons has up to now been uncertain. We recently demonstrated neuronal neurosteroidogenesis in the brain and indicated that the Purkinje cell, a typical cerebellar neuron, actively synthesizes several neurosteroids de novo from cholesterol in both mammals and non-mammals. Pregnenolone sulfate, one of neurosteroids synthesized in the Purkinje neuron, may contribute to some important events in the cerebellum by modulating neurotransmission. Progesterone, produced as a neurosteroid in this neuron only during neonatal life, may be involved in the promotion of neuronal and glial growth and neuronal synaptic contact in the cerebellum. More recently, biosynthesis and actions of neurosteroids in pyramidal neurons of the hippocampus were also demonstrated. These serve an excellent model for the study of physiological roles of neurosteroids in the brain, because both cerebellar Purkinje neurons and hippocampal neurons play an important role in memory and learning. This paper summarizes the advances made in our understanding of neurosteroids, produced in neurons, and their actions.

The Dmrt1 expression in sex-reversed gonads of amphibians

Gonadal differentiation in amphibians is sensitive to steroids. The phenotypic sex can be changed by hormonal treatments, but the molecular mechanism for gonadal differentiation is not known. Up to date, many genes involved in gonadal differentiation have been isolated in vertebrates. Dmrt1, a gene that contains the DM-domain (Doublesex/Mab-3 DNA-binding motif), is considered to be one of the essential genes involved in the testicular differentiation cascade in mammals, birds, reptiles, and fish. However, this gene has not been isolated in amphibians yet. To elucidate its role(s) for gonadal differentiation in vertebrates, a molecular cloning of Dmrt1 in amphibians is urgent. In this study, we have successfully isolated a Dmrt1 homolog from the frog Rana rugosa testes cDNA library and examined its expression during gonadal differentiation and in sex-reversed gonads. The Dmrt1 mRNA was exclusively detected in testis among adult tissues by the RT-PCR analysis. The Dmrt1 was first expressed in the differentiating testis at stage XXV in which spermatogonia are only germ cells, and became stronger at later stages. Moreover, the Dmrt1 transcript was not detected during ovarian differentiation. However, this gene was clearly expressed in XX sex-reversed gonads caused by injection of testosterone into all-female tadpoles that have well-differentiated ovaries. Taken together, the results suggest that Dmrt1 is closely implicated in testicular, but not ovarian differentiation in amphibians.

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

Neurosteroid biosynthesis in vertebrate brains.

In mammals, neurosteroids are now known to be synthesized de novo in the brain as well as other areas of the nervous system through mechanisms at least partly independent of the peripheral steroidogenic glands. However, limited information is available on neurosteroids in non-mammalian vertebrates. We therefore have attempted to demonstrate neurosteroid biosynthesis in the brain of birds and amphibians. These vertebrate brains possessed the steroidogenic enzymes, cytochrome P450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD), and produced pregnenolone, pregnenolone sulfate ester and progesterone from cholesterol. Significant seasonal changes in neurosteroids in the brain were observed in seasonally breeding vertebrates. In addition, we attempted to identify the cell type involved in neurosteroidogenesis in mammalian and non-mammalian vertebrates in order to understand the physiological role of neurosteroids. Glial cells are generally accepted to be the primary site for neurosteroid formation, but the concept of neurosteroidogenesis in brain neurons has up to now been uncertain. We recently demonstrated neuronal neurosteroidogenesis in the brain and indicated that the Purkinje cell, a typical cerebellar neuron, actively synthesizes several neurosteroids de novo from cholesterol in both mammals and non-mammals. This paper summarizes the advances made in our understanding of neurosteroid biosynthesis, including neuronal neurosteroidogenesis, in a variety of vertebrate types.

Expression of Dmrt1 protein in developing and in sex-reversed gonads of amphibians

Many genes are known to be involved in gonadal differentiation in vertebrates. Dmrt1, a gene that encodes a transcription factor with a DM-domain, is considered to be one of the essential genes controlling testicular differentiation in mammals, birds, reptiles, amphibians and fish. However, it still remains unknown which testicular cells of animals other than mice and chicks express Dmrt1 protein. For an explanation of its role(s) in testicular differentiation in vertebrates, the expression of the Dmrt1 protein needs to be studied. For this purpose, we conducted an immunohistochemical study of this protein in an amphibian by using an antibody specific for Dmrt1. No positive signal was found in the indifferent gonad of tadpoles of Rana rugosa at early stages. However, in the testis of tadpoles at later stages (XV–XXV) and in frogs one month after metamorphosis, this protein was expressed in interstitial cells and Sertoli cells. In the testis of adult frogs, germ cells also expressed Dmrt1 protein. RT-PCR analysis revealed that the gene for this protein was not transcribed at any time during ovarian development, but was expressed in the female to male sex-reversed gonad. This was true when immunohistological studies were performed. In addition, Southern blot analysis showed Dmrt1 to be an autosomal gene. Taken together, our findings indicate that Dmrt1 protein is expressed by interstitial cells, Seroli cells and germ cells in the testis of R. rugosa. Dmrt1 may thus be very involved in the testicular differentiation of amphibians.

Expression of Dax-1 during gonadal development of the frog

Dax-1, a member of the nuclear hormone receptor superfamily of transcription factors, is known to be involved in gonadal development in mammals. To date, Dax-1 has only been isolated in reptiles, birds and mammals. The expression of Dax-1 is down-regulated in the developing testis, but persists in the ovary of mice (Swain et al., Nat. Genet. 12 (1996) 404) and chicken (Smith et al., J. Mol. Endocrinol. 24 (2000) 23). Curiously, there is no sex difference in the expression patterns of Dax-1 in the American alligator (Western et al., Gene 241 (2000) 223). To understand its role(s) in gonadal development in vertebrates, molecular cloning of Dax-1 in amphibians is required. In this study, we cloned an amphibian Dax-1 homologue of the frog Rana rugosa and examined its expression profile during gonadal development. Cloned Dax-1 cDNA encoded a protein of 287 amino acids. Unlike mammalians that possess the three and one half repeat elements representing the putative DNA binding domain in the predicted sequence of Dax-1 protein, the frog had a single poorly conserved copy of the repeat unit. By RT-PCR analysis, the Dax-1 mRNA was detected in the liver and pancreas, but not in the testis and ovary of adult frogs. However, Dax-1 expression was seen first in the embryo at stage 12 and became stronger in tadpoles until stage X. The Dax-1 was transcribed in the testis stronger than in the ovary of frogs at stage XXV (just after completion of metamorphosis). In the gonad of frogs 2 months after metamorphosis (at this stage postmeiotic cells can be seen in the seminiferous tubules), the Dax-1 was expressed only in males. In addition, the Dax-1 transcription declined gradually as ovarian development proceeded, but its expression was down-regulated and then up-regulated rapidly when female-to-male sex reversal was caused by administration of testosterone into female tadpoles. Taken together, the results suggest that the Dax-1 may be closely involved in testicular development of amphibians.

COMPARISON OF CATALASE IN DIPLOID AND HAPLOID RANA RUGOSA USING HEAT AND CHEMICAL INACTIVATION TECHNIQUES

The present study examines differences in the hydrogen peroxide (H2O2) detoxifying enzyme, catalase, found in the tails and livers of diploid and haploid Rana rugosa. Investigative techniques include measurement of catalase activity and tests for temperature stability and chemical inhibition. Catalase from the tails of pre-climactic (stage XXIII) haploids was found to be over three times as H2O2 destructive as catalase from similar tails of diploids. Catalase from the livers of newly metamorphosed (stage XXV) froglets, on the other hand, displayed only one third the activity seen in diploid livers. The catalase in haploid tail and liver proved to be more heat resistant, retaining 40-60% of its original activity after 5 min of treatment at 55 degrees C, whereas diploid catalase was totally inactivated under the same conditions. Haploid and diploid catalase also responded differently to inhibition using urea and aminotriazole. These differences suggest that haploid catalase has diverged from normal diploid catalase through molecular modification, resulting in abnormal systems for H2O2 metabolism, which in turn are thought to be responsible for organ dysfunction and early death seen in haploid individuals.

Development of Vaginal Adenosis-like Lesions and Uterine Epithelial Stratification in Mice Exposed Perinatally to Diethylstilbestrol

Abstract Pregnant ICR/JCL mice were given either four daily subcutaneous injections of 20-2000 μg diethylstilbestrol (DES)/day or a 4-day intravenous infusion of 20-200 μg DES/day starting on Day 15 of gestation. Female mice were injected with 0.01-50 μg DES/day for 5 days starting on the day of birth. Females given oil injections during the neonatal period and offspring of mothers given injections of an infusion of respective vehicles alone served as controls when corresponding ages were attained. Incidence of adenosis-like lesions (ADL) in the vaginal epithelium and stratification of the uterine epithelium (UST) were determined in both offspring and neonatally exposed mice at 30 days of age. Injections of 0.1-50 μg DES/day during the neonatal period induced ADL in the fornical epithelium of vagina (36-70%), but not UST. A high incidence of ADL (80-88%) was found in the fornical and upper-vaginal epithelia in offspring of mothers given injections of 200-2000 μg DES/day, and UST was encountered in 38-70% of these mice. Offspring of mothers given an infusion of 20 μg DES/day had low incidences of ADL (11%) and UST (22%), whereas offspring of mothers infused with 200 μg DES/day exhibited high incidences of ADL (71%) and UST (86%). In offspring of mothers given injections of 2000 μg DES/day, ADL appeared in the fornical and upper-vaginal epithelia as early as 15 days of age. Thus, the present study revealed that ADL and UST occur at a high incidence in infantile mice exposed prenatally to high doses of DES. Previous studies failed to detect such effects perhaps due to the strain of mice, methods, duration, and/or dose of DES used.

Two Sox9 messenger RNA isoforms : isolation of cDNAs and their expression during gonadal development in the frog Rana rugosa

Sox is a family of SRY‐related testis‐determining genes. We have isolated two different mRNA isoforms of the frog Sox9 gene from adult frog testis cDNAs. One form (Sox9 α) encodes a 482 amino acid protein containing the HMG box, whereas the other form (Sox9 β), which completely lacks the HMG box, is a truncated 265 amino acid protein of Sox9 α. Sox9 α is 82% similar to mouse, 86% to chicken, and 77% to trout Sox9 at the amino acid level. Sox9 expression was up‐regulated in embryos after stage 16, and was seen in both developing testes and ovaries. The size of Sox9 transcripts was determined to be 7.8 knt by Northern blot analysis. In addition, Sox9 α expression was found prominently in the testis and brain among various tissues of adult frogs examined, and was considerably higher than Sox9 β. The fact that Sox9 is expressed in both sexes suggests that this gene is involved in gonadal development of male and female frogs. This is dissimilar to the pattern in birds and mammals, in which Sox9 expression is male‐specific.

Two isoforms of FTZ-F1 messenger RNA: molecular cloning and their expression in the frog testis

FTZ-F1, a member of the orphan nuclear receptors, is a transcriptional factor regulating the expression of the fushi tarazu gene in Drosophila (Lavorgna et al., 1991. Science 252, 848-851). Previously, we cloned a frog homologue of FTZ-F1 (rrFTZ-F1alpha; GenBank Accession No. AB035498). In this study, we isolated the rrFTZ-F1beta cDNA encoding a protein of 469 amino acids. Then, expressions of two types (alpha and beta) of rrFTZ-F1 mRNAs were examined during development of embryos and gonads in the frog Rana rugosa. They were expressed in the embryo at stage 12. Expressions of both the alpha and beta mRNAs became stronger in the testis of frogs at stage XXV and were most prominent in that of frogs 2months after metamorphosis. In the former testis, spermatogonia were the only germ cells in the seminiferous tubules, whilst postmeiotic cells were observed in the latter testis. Expression of the typealpha mRNA was more prominent. In addition, we cloned the regions with either exon I or II of the rrFTZ-F1 gene. Genomic structure analysis revealed that rrFTZ-F1beta is a partial exon I-truncated variant of rrFTZ-F1alpha. The results suggest that rrFTZ-F1alpha and -beta are expressed from the same gene by alternative splicing and that they may play an important role(s) in differentiation of premeiotic germ cells in the testis of the frog R. rugosa.