Gonadotrophin-Inhibitory Hormone in the Cichlid Fish Cichlasoma dimerus: Structure, Brain Distribution and Differential Effects on the Secretion of Gonadotrophins and Growth Hormone.

The role of gonadotrophin-inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well-established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH-immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto-retinalis, with no differences between the sexes. GnIH-immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed in vitro studies on intact pituitary cultures to evaluate the effect of two doses (10(-6)  m and 10(-8)  m) of synthetic C. dimerus (cd-) LPQRFa-1 and LPQRFa-2 on the release of gonadotrophins and GH. We observed that cd-LPQRFa-1 decreased ß-luteinising hormone (LH) and ß-follicle-stimulating hormone (FSH) and also increased GH release to the culture medium. The release of ß-FSH was increased only when it was stimulated with the higher cd-LPQRFa-2 dose. The results of the present study indicate that cd-LPQRFa-1, the cichlid fish GnIH, inhibits ß-LH and ß-FSH release and stimulates GH release in intact pituitary cultures of C. dimerus. The results also show that cd-LPQRF-2 could act as an ß-FSH-releasing factor in this fish species.

Differential expression of RFamide-related peptide, a mammalian gonadotrophin-inhibitory hormone orthologue, and kisspeptin in the hypothalamus of Abadeh ecotype does during breeding and anoestrous seasons.

Gonadotrophin-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that was discovered in birds as an inhibitory factor for gonadotrophin release. RFamide-related peptide (RFRP) is a mammalian GnIH orthologue that inhibits gonadotrophin synthesis and release in mammals through actions on gonadotrophin-releasing hormone (GnRH) neurones and gonadotrophs, mediated via the GnIH receptor (GnIH-R), GPR147. On the other hand, hypothalamic kisspeptin provokes the release of GnRH from the hypothalamus. The present study aimed to compare the expression of RFRP in the dorsomedial hypothalamus and paraventricular nucleus (DMH/PVN) and that of kisspeptin in the arcuate nucleus (ARC) of the female goat hypothalamus during anoestrous and breeding seasons. Mature female Abadeh does were used during anoestrus, as well as the follicular and luteal phases of the cycle. The number of RFRP-immunoreactive (-IR) neurones in the follicular phase was lower than in the luteal and anoestrous stages. Irrespective of the ovarian stage, the number of RFRP-IR neurones in the rostral and middle regions of the DMH/PVN was higher than in the caudal region. By contrast, the number of kisspeptin-IR neurones in the follicular stage was greater than in the luteal stage and during the anoestrous stage. Irrespective of the stage of the ovarian cycle, the number of kisspeptin-IR neurones in the caudal region of the ARC was greater than in the middle and rostral regions. In conclusion, RFRP-IR cells were more abundant in the rostral region of the DMH/PVN nuclei of the hypothalamus, with a greater number being found during the luteal and anoestrous stages compared to the follicular stage. On the other hand, kisspeptin-IR neurones were more abundant in the caudal part of the ARC, with a greater number recorded in the follicular stage compared to the luteal and anoestrous stages.

Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin: new key neuropeptides controlling reproduction.

Gonadotrophin-releasing hormone (GnRH) is the primary hypothalamic factor responsible for the control of gonadotrophin secretion in vertebrates. However, within the last decade, two other hypothalamic neuropeptides have been found to play key roles in the control of reproductive functions: gonadotrophin-inhibitory hormone (GnIH) and kisspeptin. In 2000, we discovered GnIH in the quail hypothalamus. GnIH inhibits gonadotrophin synthesis and release in birds through actions on GnRH neurones and gonadotrophs, mediated via GPR147. Subsequently, GnIH orthologues were identified in other vertebrate species from fish to humans. As in birds, mammalian and fish GnIH orthologues inhibit gonadotrophin release, indicating a conserved role for this neuropeptide in the control of the hypothalamic-pituitary-gonadal axis across species. Subsequent to the discovery of GnIH, kisspeptin, encoded by the KiSS-1 gene, was discovered in mammals. By contrast to GnIH, kisspeptin has a direct stimulatory effect on GnRH neurones via GPR54. GPR54 is also expressed in pituitary cells, but whether gonadotrophs are targets for kisspeptin remains unresolved. The KiSS-1 gene is also highly conserved and has been identified in mammals, amphibians and fish. We have recently found a second isoform of KiSS-1, designated KiSS-2, in several vertebrates, but not birds, rodents or primates. In this review, we highlight the discovery, mechanisms of action, and functional significance of these two chief regulators of the reproductive axis.

Octopus gonadotrophin-releasing hormone: a multifunctional peptide in the endocrine and nervous systems of the cephalopod.

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris. In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotrophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix. Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.

Effects of cyclic tensile forces on the expression of vascular endothelial growth factor (VEGF) and macrophage-colony-stimulating factor (M-CSF) in murine osteoblastic MC3T3-E1 cells.

It has been reported that vascular endothelial growth factor (VEGF), expressed by osteoblasts, can induce osteoclast recruitment and thus affects bone remodeling. The purpose of this study was to investigate the effects of cyclic tensile forces on the expression of VEGF and macrophage-colony-stimulating factor (M-CSF) in osteoblastic MC3T3-E1 cells. VEGF and M-CSF gene expression and protein concentration were determined by real-time PCR and enzyme-linked immunoassay. The expression of VEGF and M-CSF mRNA in the experimental group was higher than in the control group. The increase in the concentration of VEGF and M-CSF protein in the experimental group was time-dependent. Moreover, gadolinium (an S-A channel inhibitor), but not nifedipine (L-Type Ca2+ channel blocker), treatment reduced the concentration of VEGF and M-CSF mRNA and protein in the experimental groups. These findings suggest that cyclic tensile forces increase the expression of VEGF and M-CSF in osteoblastic MC3T3-E1 cells via a stretch-activated channel (S-A channel).

Gonadotropin-inhibitory hormone in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments.

The neuropeptide control of gonadotropin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, GnRH. We recently identified a novel hypothalamic dodecapeptide with a C-terminal LeuPro-Leu-Arg-Phe-NH2 sequence in the domestic bird, Japanese quail (Coturnix japonica). This novel peptide inhibited gonadotropin release in vitro from the quail anterior pituitary; thus it was named gonadotropin-inhibitory hormone (GnIH). GnIH may be an important factor regulating reproductive activity not only in domesticated birds but also in wild, seasonally breeding birds. Thus, we tested synthetic quail GnIH in seasonally breeding wild bird species. In an in vivo experiment, chicken gonadotropin-releasing hormone-I (cGnRH-I) alone or a cGnRH-I/quail GnIH cocktail was injected i.v. into non-breeding song sparrows (Melospiza melodia). Quail GnIH rapidly (within 2 min) attenuated the GnRH-induced rise in plasma LH. Furthermore, we tested the effects of quail GnIH in castrated, photostimulated Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), using quail GnIH or saline for injection. Again, quail GnIH rapidly reduced plasma LH (within 3 min) compared with controls. To characterize fully the action of GnIH in wild birds, the identification of their endogenous GnIH is essential. Therefore, in the present study a cDNA encoding GnIH in the brain of Gambel's white-crowned sparrow was cloned by a combination of 3' and 5' rapid amplification of cDNA ends and compared with the quail GnIH cDNA previously identified. The deduced sparrow GnIH precursor consisted of 173 amino acid residues, encoding one sparrow GnIH and two sparrow GnIH-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2) that included Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Although the homology of sparrow and quail GnIH precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in two species. In situ hybridization revealed the cellular localization of sparrow GnIH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemical analysis also showed that sparrow GnIH-like immunoreactive cell bodies and terminals were localized in the PVN and median eminence respectively. Thus, only the sparrow PVN expresses GnIH, which appears to be a hypothalamic inhibitory factor for LH release, as evident from our field injections of GnIH into free-living breeding white-crowned sparrows. Sparrow GnIH rapidly (within 2 min) reduced plasma LH when injected into free-living Gambel's white-crowned sparrows on their breeding grounds in northern Alaska. Taken together, our results indicate that, despite amino acid sequence differences, quail GnIH and sparrow GnIH have similar inhibitory effects on the reproductive axis in wild sparrow species. Thus, GnIH appears to be a modulator of gonadotropin release.

Effects of sex hormone disturbances on craniofacial growth in newborn mice.

It is well-known that sex hormones influence bone metabolism. However, it remains unclear as to how sex hormones affect bone growth in newborn mice. In this study, we performed orchiectomy (ORX) and ovariectomy (OVX) on newborn mice, and examined the effects on craniofacial growth morphometrically. ORX and OVX were performed on five-day-old C57BL/6J mice. Four weeks after surgery, lateral cephalograms were taken of all of the mice, with the use of a rat and mouse cephalometer. Cephalometric analysis of the craniofacial skeleton was performed by means of a personal computer. Inhibition of craniofacial growth was found in the experimental groups but not in the sham-operated groups. In the nasomaxillary bone and mandible, the amount of growth was significantly reduced. These results suggest that craniofacial growth is inhibited by sex hormone disturbances not only in puberty but also immediately after birth.

Gonadotrophin inhibitory hormone depresses gonadotrophin alpha and follicle-stimulating hormone beta subunit expression in the pituitary of the domestic chicken.

Studies performed in vitro suggest that a novel 12 amino acid RF amide peptide, isolated from the quail hypothalamus, is a gonadotrophin inhibitory hormone (GnIH). The aim of the present study was to investigate this hypothesis in the domestic chicken. Injections of GnIH into nest-deprived incubating hens failed to depress the concentration of plasma luteinizing hormone (LH). Addition of GnIH to short-term (120 min) cultures of diced pituitary glands from adult cockerels depressed follicle-stimulating hormone (FSH) and LH release and depressed common alpha and FSHbeta gonadotrophin subunit mRNAs, with no effect on LHbeta subunit mRNA. Hypothalamic GnIH mRNA was higher in incubating (out-of-lay) than in laying hens, but there was no significant difference in the amount of hypothalamic GnIH mRNA in out-of-lay and laying broiler breeder hens at the end of a laying year. It is concluded that avian GnIH may play a role in controlling gonadotrophin synthesis and associated constitutive release in the domestic chicken.

A novel p63 sterile alpha motif (SAM) domain mutation in a Japanese patient with ankyloblepharon, ectodermal defects and cleft lip and palate (AEC) syndrome without ankyloblepharon.

We present a sporadic case of a Japanese female infant with ectodermal dysplasia, complete cleft lip and palate, severe skin erosions at birth and recurrent scalp infection. She had typical clinical features of ankyloblepharon, ectodermal defects and cleft lip and palate (AEC) syndrome without ankyloblepharon. Histological and immunohistochemical analyses showed reduced granular cell layers and aberrant expression of the p63 protein in the suprabasal keratinocytes. Mutation analysis of the exon 13 of p63 gene revealed a heterozygous in-frame 3-bp insert (c. 538-539 ins TTC) encoding additional amino acid residues (F). This is the first report of sterile alpha motif domain mutation except for single nucleotide transitions.

Developmental changes in gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica) hypothalamo-hypophysial system.

We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH(2) sequence, SIKPSAYLPLRF-NH(2) (RFamide peptide), from the Japanese quail (Coturnix japonica) brain. This novel quail peptide was shown to be located in neurons of the paraventricular nucleus (PVN) and their terminals in the median eminence (ME), and to decrease gonadotropin release from cultured anterior pituitary in adult birds. We therefore designated this peptide gonadotropin-inhibitory hormone (GnIH). Furthermore, a cDNA encoding the GnIH precursor polypeptide has been characterized. To understand the physiological roles of this peptide, in the present study we analyzed developmental changes in the expressions of GnIH precursor mRNA and the mature peptide GnIH during embryonic and posthatch ages in the quail diencephalon including the PVN and ME. GnIH precursor mRNA was expressed in the diencephalon on embryonic day 10 (E10) and showed a significant increase on E17, just before hatch. GnIH was also detected in the diencephalon on E10 and increased significantly around hatch. Subsequently, the diencephalic GnIH content decreased temporarily, and again increased progressively until adulthood. GnIH-like immunoreactive (GnIH-ir) neurons were localized in the PVN on E10, but GnIH-ir fibers did not extend to the ME. However, GnIH-ir neurons increased in the PVN on E17, just before hatch, and GnIH-ir fibers extended to the external layer of the ME, as in adulthood. These results suggest that GnIH begins its function around hatch and acts as a hypothalamic factor to regulate gonadotropin release in the bird.

Gonadotropin-inhibitory peptide in song sparrows (Melospiza melodia) in different reproductive conditions, and in house sparrows (Passer domesticus) relative to chicken-gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) regulates reproduction in all vertebrates. Until recently, an antagonistic neuropeptide for gonadotropin was unknown. The discovery of an RFamide peptide in quail that inhibits gonadotropin release in vitro raised the possibility of direct hypothalamic inhibition of gonadotropin release. This peptide has now been named gonadotropin-inhibitory hormone (GnIH). We investigated GnIH presence in the hypothalamus of two seasonally breeding songbird species, house sparrows (Passer domesticus) and song sparrows (Melospiza melodia). Using immunocytochemistry (ICC), GnIH-containing neurones were localized in both species in the paraventricular nucleus, with GnIH-containing fibres visible in multiple brain locations, including the median eminence and brainstem. Double-label ICC with light microscopy and fluorescent ICC with confocal microscopy indicate a high probability of colocalization of GnIH with GnRH neurones and fibres within the avian brain. It is plausible that GnIH could be acting at the level of the hypothalamus to regulate gonadotropin release as well as at the pituitary gland. In a photoperiod manipulation experiment, GnIH-containing neurones were larger in birds at the termination of the breeding season than at other times, consistent with a role for this neuropeptide in the regulation of seasonal breeding. We have yet to elucidate the dynamics of GnIH synthesis and release at different times of year, but the data imply temporal regulation of this peptide. In summary, GnIH has the potential to regulate gonadotropin release at more than one level, and its distribution is suggestive of multiple regulatory functions in the central nervous system.

Expression of vascular endothelial growth factor and the effects on bone remodeling during experimental tooth movement.

Vascular endothelial growth factor (VEGF) has an ability to induce functional osteoclasts as well as neovascularization. We recently reported that the number of osteoclasts was enhanced by the injection of recombinant human VEGF (rhVEGF) with the application of mechanical force for experimental tooth movement. In this study, the expression of VEGF was detected in osteoblasts on the tension side of the alveolar bone. Moreover, the rate of tooth movement was significantly increased in the rhVEGF injection groups compared with the controls. These results suggested that VEGF, highly expressed by mechanical stimuli, enhances the number of osteoclasts as a paracrine factor, and that the amount of tooth movement is accelerated by both endogenous VEGF and injected rhVEGF.

A case of gallbladder carcinoma diagnosed preoperatively by the detection of human telomerase reverse transcriptase mRNA in endoscopically obtained gallbladder bile.

It is difficult to accurately diagnose the nature of polypoid lesions in the gallbladder. To increase diagnostic accuracy, we have developed an endoscopic technique for obtaining gallbladder bile, termed endoscopic transpapillary catheterization into the gallbladder (ETCG). We describe here a case of gallbladder carcinoma diagnosed preoperatively by the detection of human telomerase reverse transcriptase (hTERT) mRNA, a catalytic subunit of telomerase, in gallbladder bile obtained using the ETCG technique. A patient with a polypoid lesion 15 mm in diameter in the gallbladder was admitted to our hospital for further examinations. Gallbladder bile collected by the ETCG technique was evaluated cytologically and also analyzed for telomerase activity and hTERT mRNA. Although the results for cytology and telomerase activity were negative, that for hTERT mRNA was positive. Open surgery was carried out and it was confirmed that the lesion was an adenocarcinoma invading the subserosa. The molecular biological analysis of gallbladder bile collected using the ETCG technique was shown to be effective for diagnosing the nature of the polypoid lesion in the gallbladder.

Effects of vascular endothelial growth factor on osteoclast induction during tooth movement in mice.

UNLABELLED: For orthodontic tooth movement, remodeling of the alveolar bone is maintained by a repeated process of bone resorption and new bone formation, controlled, respectively, by osteoclasts and osteoblasts. Recently, we have found that recombinant human vascular endothelial growth factor (rhVEGF) acts as a macrophage colony-stimulating factor in osteoclast induction in osteopetrotic (op/op) mice. The purpose of this study was to investigate whether rhVEGF stimulates osteoclast differentiation during experimental tooth movement. Purified rhVEGF was injected once into the buccal gingival groove around the incisors. An experimental appliance with a helical loop was bonded onto the upper incisors, and an initial force of 1.0 g was applied for three days. The number of osteoclasts appearing in the periodontal ligament space on the pressure side of the alveolar bone was increased markedly. These results suggest that local administration of rhVEGF enhances the number of osteoclasts, and may increase the rate of orthodontic tooth movement. ABBREVIATIONS: recombinant human vascular endothelial growth factor (rhVEGF), macrophage colony-stimulating factor (M-CSF), osteopetrotic mice (op/op mice),fms-like tyrosine kinase (Flt-1), periodontal ligament (PDL), phosphate-buffered saline (PBS), tartrate-resistant acid phosphatase (TRAP), and analysis of variance (ANOVA).

Plasmids with a mammalian replication origin and a matrix attachment region initiate the event similar to gene amplification.

Gene amplification plays a crucial role in the development of many human malignancies. Amplified genes are frequently localized on double minutes (DMs). We show here that plasmids bearing both a mammalian replication origin and a nuclear matrix attachment region were able to integrate into DMs if transfected to cells having DMs (COLO 320DM). Furthermore, these plasmids triggered the events leading to the de novo formation of the structure similar to DMs if transfected to the cells without DMs (COLO 320HSR or HeLa). Autonomous replication of these plasmids was suggested to be a prerequisite for these events to occur, because the presence of the origin sequences in the plasmids was required. The presence of matrix attachment region in the plasmids is also required for these events to occur, suggesting that matrix attachment plays an indispensable role in extrachromosomal replication. This model system will allow us to investigate the mechanism of gene amplification as well as to analyze the autonomous replication of the plasmid with mammalian replication origins.

Activity and localization of 3beta-hydroxysteroid dehydrogenase/ Delta5-Delta4-isomerase in the zebrafish central nervous system.

Little information is available for neurosteroidogenesis in the central nervous system (CNS) of lower vertebrates. Therefore, in the present study, we examined the enzymatic activity and localization of 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3betaHSD), a key steroidogenic enzyme, in the CNS of adult male zebrafish to clarify central progesterone biosynthesis. Biochemical studies together with HPLC analysis revealed that the zebrafish brain converted pregnenolone to progesterone, suggesting the enzymatic activity of 3betaHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3betaHSD. By using Western immunoblotting with the polyclonal antiserum directed against purified bovine adrenal 3betaHSD, a 3betaHSD-like substance was found in homogenates of the zebrafish brain. Immunocytochemical analysis was then undertaken to investigate the localization of the 3betaHSD-like substance in the zebrafish brain and spinal cord. Clusters of immunoreactive cell bodies were localized in the dorsal telencephalic areas (D), central posterior thalamic nucleus (CP), preoptic nuclei (NPO), posterior tuberal nucleus (PTN), paraventricular organ (PVO), and nucleus of medial longitudinal fascicle (NMLF). 3betaHSD-like immunoreactivity was also observed in somata of cerebellar Purkinje neurons. A widespread distribution of immunoreactive fibers was found throughout the brain and spinal cord. In addition, positively stained cells were restricted to other organs, such as the pituitary and retina. Preabsorbing the antiserum with purified bovine adrenal microsome resulted in a complete absence of 3betaHSD-like immunoreactivity. These results suggest that the fish CNS possesses steroidogenic enzyme 3betaHSD and produces progesterone. The present study further provides the first immunocytochemical mapping of the site of 3betaHSD expression in the fish CNS.

Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state.

There is evidence that Matrix Attachment Region (MAR)-binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR-binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin kappa gene (Ig kappa MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA-binding proteins were identified as SAF-A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF-A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [1987] Nature 326:720-722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF-A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR-binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments.

The tumor-preventing effect of a mixture of several lactic acid bacteria on 1,2-dimethylhydrazine-induced colon carcinogenesis in mice.

The anti-tumor effect of a dietary supplement obtained from mixed cultures of several lactic acid bacteria was examined in the colon of tumor-inducing ICR male mice by use of a carcinogen, 1,2-dimethylhydrazine (DMH, 20 mg/kg body weight, 1 intra-muscular injection per week for 10 weeks). The animals were sacrificed either 15 weeks or 24-26 weeks after the first carcinogen injection. Macroscopically, the incidence of colon tumors at a 24-26 week period of tumor induction was apparently lower in mice treated with both the DMH and dietary supplement (76%) than in those treated with DMH alone (100%). Histologically, microadenomas were induced predominantly in the anal half of the total colon, and large lymphoid aggregates were often associated with dysplastic crypts in the distal colon. Apoptotic cell masses were shed into the distended lumen of the involved crypts. The statistical analysis at a 15-week period of tumor induction indicated that the incidence of microadenomas per tumor-induced mouse was lowered significantly by use of the dietary supplement. From the present results, it is suggested that the intake of the dietary supplement inhibits the early development of colon adenomas, and the inhibition of microadenomas results in a reduction of subsequent polyp and tumor yield in the mouse colon.

Effects of progesterone synthesized de novo in the developing Purkinje cell on its dendritic growth and synaptogenesis.

De novo steroidogenesis from cholesterol is a conserved property of vertebrate brains, and such steroids synthesized de novo in the brain are called neurosteroids. The identification of neurosteroidogenic cells is essential to the understanding of the physiological role of neurosteroids in the brain. We have demonstrated recently that neuronal neurosteroidogenesis occurs in the brain and indicated that the Purkinje cell actively synthesizes several neurosteroids de novo from cholesterol in vertebrates. Interestingly, in the rat, this neuron actively synthesizes progesterone de novo from cholesterol only during neonatal life, when cerebellar cortical formation occurs most markedly. Therefore, in this study, the possible organizing actions of progesterone during cerebellar development have been examined. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone promotes dose-dependent dendritic outgrowth of Purkinje cells but dose not affect their somata. This effect was blocked by the anti-progestin RU 486 [mifepristone; 17beta-hydroxy-11beta-(4-methylaminophenyl)-17alpha-(1-propynyl) estra-4,9-dien-3 one-6-7]. In vivo administration of progesterone to pups further revealed an increase in the density of Purkinje spine synapses electron microscopically. In contrast to progesterone, there was no significant effect of 3alpha,5alpha-tetrahydroprogesterone, a progesterone metabolite, on Purkinje cell development. Reverse transcription-PCR-Southern and immunocytochemical analyses showed that intranuclear progesterone receptors were expressed in Purkinje cells. These results suggest that progesterone promotes both dendritic outgrowth and synaptogenesis in Purkinje cells through intranuclear receptor-mediated mechanisms during cerebellar development. Such organizing actions may contribute to the formation of the cerebellar neuronal circuit.