OCT4 immunohistochemistry may be necessary to identify the real risk of gonadal tumors in patients with Turner syndrome and Y chromosome sequences.

BACKGROUND: The aim of this study was to investigate the frequency of gonadal tumors among patients with Turner syndrome (TS) carrying Y-derivative sequences in their chromosomal constitution. METHODS: Six out of 260 patients with TS were selected based on mosaicism of the entire Y chromosome; 10 were included because Y-derivative sequences have been detected by PCR with specific oligonucleotides (sex-determining region on the Y, testis specific-protein, Y and DYZ3) and further confirmed by FISH. The 16 patients were subjected to bilateral gonadectomy at ages varying from 8.7 to 18.2 years. Both histopathological investigation with hematoxylin and eosin (H&E) and immunohistochemical analysis with anti-octamer-binding transcription factor 4 (OCT4) antibody were performed. RESULTS: Gonadal neoplasia was not detected in any of the 32 gonads evaluated by H&E; however, four gonads (12%) from three patients (19%) had positive OCT4 staining in 50-80% of nuclei, suggesting the existence of germ cell tumors (gonadoblastoma or in situ carcinoma). CONCLUSIONS: Evaluation of the real risk of development of gonadal tumors in TS patients with Y-derivative sequences in their chromosomal constitution may require a specific histopathological study, such as immunohistochemistry with OCT4.

A naturally occurring deletion in the SRY promoter region affecting the Sp1 binding site is associated with sex reversal.

Male to female sex reversal results from failure of testis development. Mutations in the SRY gene or in other genes involved in the sexual differentiation pathway are considered to cause XY gonadal dysgenesis. The majority of the mutations in the SRY described so far are located within the SRY coding region, mainly in the HMG-box conserved domain. Comparison of 5' flanking SRY gene sequences among different species indicated the presence of several putative conserved consensus sequences for different transcription regulators. In this study, we investigated a 360 bp sequence encompassing the SRY putative core promoter, in 17 patients with variable degrees of 46,XY sex reversal, which have been previously shown not to bear mutations in the SRYcoding region. Sequencing analysis of the SRYpromoter in one patient with complete XY gonadal dysgenesis revealed a three base pair deletion in one of the Sp1 binding sites. The deletion abolished Sp1 binding in vitro. This is the first report on a naturally occurring mutation affecting the Sp1 regulatory element in the SRY promoter region, which is associated with sex reversal. Additionally, upon familial investigation the father, who had 18 genital surgeries due to severe hypospadia without cryptorchidism, was found to bear the same deletion and several relatives were referred to have sexual ambiguity.

The effect of acupuncture and moxibustion on physical performance by sedentary subjects submitted to ergospirometric test on the treadmill.

AIM: Acupuncture and moxibustion are 2 therapeutic methods used in Traditional Chinese Medicine. The aim of this study is to evaluate the effect of acupuncture and moxibustion in physical performance by an ergospirometry test. METHODS: Thirty-one subjects, sedentary, were randomized into 3 groups: Group I (Acupuncture); Group II (AcupunctureSham) and Group III (Control). They were submitted to anamnesis and an ergospirometry test. The subjects from Groups I and II were submitted to 10 acupuncture and moxibustion sessions, twice a week, for 5 weeks. The differences between the groups were that in Group I the acupuncture and moxibustion sessions were performed in acupoints and Group II in non acupoints. After this, the ergospirometry test was performed again. RESULTS: The acupuncture and moxibustion sessions did not increase maximum oxygen uptake (VO2max) significantly. Other variables presented a significant increasing: oxygen uptake in anaerobic threshold (VO2La); velocity in anaerobic threshold (Vel LA); caloric consumption (Gast Cal). The heart rate decreased when compared to velocity pre and post-treatment. The results show peripheral, afferent stimulation (acupuncture and moxibustion) could alter the functions of structures or internal organs. The decrease in heart rate and the significant improvement in oxygen uptake in anaerobic threshold are similar to adaptations to physical training. Probably, the autonomic nervous system (mainly sympathetic system) is engaged in the acupuncture and moxibustion mechanism of action. CONCLUSIONS: Acupuncture and moxibustion decreased the heart rate during the ergospirometry test; the oxygen uptake in anaerobic threshold (VO2La); velocity in anaerobic threshold (Vel LA) and caloric consumption (Gast Cal) had significantly increased.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis.

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46,XY pure gonadal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes.

The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians. In humans mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases. We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites. One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis. The Escherichia coli expressed SRY(N65H) protein did not present DNA-binding activity in vitro. The other mutation, a G to T transversion, causes the R30I substitution. This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings. The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR. The R30I mutation is located upstream to the HMG box, within the (29)RRSSS(33) phosphorylation site. The E. coli expressed SRY(R30I) protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro.

Morphometry and histology of gonads from 13 children with dysgenetic male pseudohermaphroditism.

BACKGROUND: Dysgenetic male pseudohermaphroditism (DMP) is a sexual differentiation disorder characterized by bilateral dysgenetic testes, persistent müllerian structures, and cryptorchidism in individuals with a 46,XY karyotype. However, the histologic criteria for the diagnosis of DMP are poorly established. OBJECTIVE: To determine gonadal histology in children with DMP. PATIENTS AND METHODS: Between 1996 and 1998, 13 patients with DMP were evaluated on our service. The clinical diagnosis of DMP was based on a 46,XY karyotype, sex ambiguity, high levels of follicle-stimulating hormone and low levels of antimüllerian hormone, a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors, and the presence of müllerian structures. Molecular sequencing the HMGbox region of the SRY gene did not reveal any mutations. Biopsies were performed for 22 of 26 gonads (patient age at the time of biopsy, 16 months to 10 years). Conventional microscopy was used to evaluate mean tubular diameter, tubular fertility index, and number of Sertoli cells per tubular profile. RESULTS: All 26 gonads were located outside of the labioscrotal folds. Their histologic features varied from only a reduction in tubular size to features of a streak gonad. Five of the 22 gonads grossly resembled a streak gonad. The mean tubular diameter was severely reduced (>30% reduction relative to the normal tubular diameter for the patient's age) in 4 gonads, markedly reduced (10%-30%) in 11 gonads, slightly reduced (<10%) in one gonad, and normal in one gonad. The tubular fertililty index, expressed as the percentage of tubular profiles containing germ cells, was severely reduced (<30% of normal values) in 9 gonads, markedly reduced (50%-30%) in 2 gonads, and normal in 6 gonads. The number of Sertoli cells per tubular profile was elevated in 16 gonads and normal in one gonad. Thin tubules surrounded by fibrous tissue were occasionally observed. CONCLUSION: The histologic findings confirmed the clinical diagnosis of DMP in every patient in the present series. However, gonadal histology was variable, and careful morphometric evaluation may be necessary to establish the diagnosis.

Molecular mapping of an idic(Yp) chromosome in an Ullrich-Turner patient.

We describe a woman with Ullrich-Turner manifestations and a 45,X/46, X,+mar karyotype. Fluorescence in situ hybridization (FISH) and DNA analysis were carried out in order to determine the origin and structure of the marker. FISH showed that the marker was a Y-derived dicentric chromosome. The breakpoint at Yq11 (interval 6) was mapped using Southern blotting and polymerase chain reaction (PCR). There were no nucleotide alterations in the SRY conserved domain. Histological analysis of the gonads showed an ovarian-like stroma with no signs of testicular tissue. These findings indicate that the patient was a mosaic 45,X/46,X,idic(Yp) whose phenotypic expression, including sex determination, appeared to have had more influence from the 45,X cell line.

Recurrence of a nonsense mutation in the conserved domain of SRY in a Brazilian patient with 46,XY gonadal dysgenesis.

We tested a female patient with 46,XY karyotype and pure gonadal dysgenesis for the presence of the SRY gene and for mutations within the SRY conserved domain. A point mutation was identified at nucleotide position 209 with respect to the first ATG. The base substitution is a G-->A transition in the first nucleotide of codon 70 which changes a tryptophan (TGG) to a stop codon (TAG). Even though the father was not available for investigation we assumed that it is a de novo mutation, since it probably generates a nonfunctional truncated protein.

True hermaphrodites in the southeastern region of Brazil: a different cytogenetic and gonadal profile.

Sex ambiguity may be due to several disorders of gonadal differentiation, including true hermaphroditism (TH), as well as male and female pseudohermaphroditism. Although TH is a rare cause of intersex in Europe and North America, in Africa it presents one of the highest frequencies. The 46,XX karyotype has been found in the majority of the reported patients (70.6%), and aberrations in the sex chromosomes have been observed in about 22% of the cases. The 46,XY karyotype has been described as less frequent. Herein we describe ten cases of TH which have been diagnosed over the last 7 years, six lateral TH, two unilateral TH, and two cases of ovotestes with absent contralateral gonad. From a total of 18 gonads analyzed, there were 8 testes, 6 ovaries and 4 ovotestes. Nine subjects had originally a male sex assignment, and in three cases this was reverted to female. Four cases had a 46,XY karyotype. Additional sex chromosome aberrations had been found in four different cases [two 46,XX/46,XY, one 45,X/47,XYY, one 46,X,del(Yq)]. A 46,XX karyotype was found in only two individuals, and both were SRY negative. Our preliminary data, especially on the constitution of chromosomes and gonads, indicate marked differences from those in the literature.

Evidence for neuronal release of isotopically labelled glycine from the rat ventral medullary surface in vivo.

Spontaneous and stimulus-induced release of isotopically labelled glycine was studied in the superfused rat dorsal or ventral medullary surface in vivo. Superfusion of the ventral medullary surface of anesthetized (urethane, 1.2 g/kg, ip) male adult Wistar rats (250-350 g) with high K+ (40 mM) surrogate cerebrospinal fluid (CSF) produced an average increase of 45% over the spontaneous efflux of exogeneously applied glycine (N = 5, P < 0.01). In experiments in which the calcium of the CSF was replaced by an equimolar amount of magnesium, the increase in glycine efflux in response to high K+ was reduced to 15%, a value not statistically different from that observed in control experiments (N = 6). Veratridine stimulation evoked a large (80%) increase in glycine efflux (N = 5, P < 0.001), which was inhibited by tetrodotoxin. High potassium or veratridine failed to modify spontaneous release of glycine on the dorsal medullary surface. Results obtained in control experiments showed that neither high K+ nor veratridine is effective in modifying spontaneous efflux of [3H]-leucine or [3H]-inulin on the ventral or dorsal medullary surface. These data support the hypothesis that glycine is a neurotransmitter on the ventral medullary surface and that it may be part of neural pathways involved in cardiorespiratory regulation present in this region.

Evidence for neuronal release of isotopically labelled glycine from the rat ventral medullary surface in vivo

Spontaneous and stimulus-induced release of isotopically labelled glycine was studied in the superfused rat dorsal or ventral medullary surface in vivo. Superfusion of the ventral medullary surface of anesthetized (urethane, 1.2 g/kg, ip) male adult Wistar rats (250-350 g) with high K+ (40 mM) surrogate cerebrospinal fluid (CSF) produced an average increase of 45 per cent over the spontaneous efflux of exogenously applied glycine (N = 5, P<0.01). In experiments in which the calcium of the CSF was replaced by an equimolar amount of magnesium, the increase in glycine efflux in response to high K+ was reduced to 15 per cent, a value not statistically different from that observed in control experiments (N = 6). Veratridine stimulation evoked a large (80 per cent) increase in glycine efflux (N = 5, P<0.001), which was inhibited by tetrodotoxin. High potassium or veratridine failed to modify spontaneous release of glycine on the dorsal medullary surface. Results obtained in control experiments showed that neither high K+ nor veratridine is effective in modifying spontaneous efflux of [(3)H]-leucine or [(3)H]-inulin on the ventral or dorsal medullary surface. These data support the hypothesis that glycine is a neurotransmitter on the ventral medullary surface and that it may be part of neural pathways involved in cardiorespiratory regulation present in this region.

Release of labelled taurine from the rat dorsal medulla and cerebellum in vivo.

Stimulus-induced release of labelled taurine has been studied in the superfused rat cerebellar cortex and dorsal medulla in vivo. In the cerebellum both elevated potassium and electrically induced depolarization consistently produced marked increases in the efflux of exogenously applied taurine in a calcium-dependent fashion. Veratridine-stimulation evoked a large Ca2+-independent taurine efflux which was, however, prevented by tetrodotoxin. In the dorsal medulla, both high K+ and veratridine induced a clear Ca2+-independent increase in taurine efflux. Electrical stimulation was always ineffective in changing taurine efflux from the dorsal medulla. These data strongly support a possible neurohumoral role for taurine in the cerebellum but not in the dorsal medulla.

Efluxo de amino-acidos inibidores no cortex cerebelar do rato. / Amins acids inhibitor efflux in cerebellar cortex of the rat

Estudou-se a liberacao in vivo de GABA, taurina e glicina radiativos, induzida por diferentes agentes despolarizadores aplicados diretamente ao cortex cerebelar exposto de ratos antestesiados. Os resultados obtidos indicam que a glicina provavelmente nao e neurotransmissora no cortex cerebelar, enquanto que o GABA e a taurina devem estar envolvidos na transmissao sinaptica naquela estrutura, como neurotransmissores e talvez, tambem, como moduladores da excitabilidade neuronal

Efluxo de provaveis amino-acidos neurotransmissores inibidores da regiao do nucleo cuneiforme do rato. / Efflux of the inhibitory transmitter probable amino acids of the cuneiform nucleus region of the rat

GABA, taurina e glicina isotopicamente marcdos, em preparacoes in vivo, induzida marcados, em preparacoes in vitro induzida por diferentes agentes despolarizadores aplicados diretamente sobre a superficie pial da regiao dorsal da medula (MD). Os resultados obtidos permitem concluir que o GABA e, provavelmente, o principal neurotransmissor inibidor nessa regiao, a taurina provavelmente esta envolvida como moduladora da excitabilidade neuronal, enquanto que a glicina nao deve ter acao neurotransmissora da MD

Selective antagonism by benzodiazepines of neuronal responses to excitatory amino acids in the cerebral cortex.

1 The recently discovered benzodiazepine receptor exists in high concentration in the cerebral cortex. We have, therefore, examined the effects of diazepam and chlordiazepoxide on cortical neurone responses to excitatory and inhibitory amino acids and acetylcholine, in the cortex of rats anaesthetized with urethane.2 Chlordiazepoxide applied by microiontophoresis reduced the responses to glutamate and aspartate but acetylcholine responses were unaffected on most cells even by much higher doses of benzodiazepine. gamma-Aminobutyric acid (GABA) and taurine responses were unaffected on most cells, but were reduced on 4 of 25 units. After intravenous diazepam, responses to GABA and taurine were reduced on 3 cells and unchanged on 11.3 On Purkinje cells in the cerebellum a number of cells (5 of 16) exhibited a substantial increase in responses to GABA and taurine following intravenous or iontophoretic application of benzodiazepines.4 It is suggested that the highly selective reduction of excitatory amino acid responses in the cerebral cortex may be of particular relevance to the behavioural effects of benzodiazepines.

A possible neuronal release of [14C] GABA from the rat cerebellum in vivo.

Release patterns for exogenously applied [14C] labelled alpha-amino-n-butyric-acid (GABA) have been investigated in rat cerebellar cortex in vivo. An increase in [14C] GABA release could be evoked by stimulating with high (40 mM) K+ or veratridine (10(-4)M) but not with direct electrical stimulation. Biphasic patterns for high K+ and possibly veratridine stimulated release of GABA suggest the existence of two separate anatomical sources of isotope which are sensitive to these depolarising stimuli. Both K+ and veratridine-evoked GABA release are calcium dependent. Studies involving partial replacement of Na+ with HEPES, (N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid), sucrose or choline chloride also reveal a sodium dependency of [14C] GABA release. These studies collectively indicate a neuronal source for evoked GABA release, a criterion for transmitter identification not previously satisfied in the cerebellar cortex.

Evidence for a neuronal release of isotopically labelled gamma-amino-n-butyric acid (GABA) from the rat dorsal medulla in vivo.

High potassium and electrical stimulation consistently increase efflux of labelled GABA from the in vivo superfused rat dorsal medulla in a calcium-dependent fashion. The depolarizing alkaloid, veratridine, also evokes a large increase in efflux of labelled GABA. These data strongly suggest release from a neurotransmitter pool in this region.