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.

Gonadal development in the freshwater crab Sylviocarcinus pictus (H. Milne Edwards, 1853) (Brachyura: Trichodactylidae) from the Guamá river, state of Pará, Brazil

Os estádios de desenvolvimento das gônadas de machos e fêmeas de caranguejos Sylviocarcinus pictus (H. Milne Eduards, 1853) foram descritos por meio de observações macroscópicas e microscópicas (técnica histológica). A descrição histológica foi baseada em 40 espécimes (20 de cada sexo). Foram identificados quatro estádios de desenvolvimento para as fêmeas: imaturo, em maturação, maturo e em reabsorção. As seguintes células foram encontradas: ovogônias, ovócitos em vitelogênese inicial, ovócitos em vitelogênese avançada, células foliculares e folículos pós-ovulatórios. Três estádios de desenvolvimento foram encontrados para os machos: imaturo, em maturação e maturo, com indicação de: espermatogônias, espermatócitos, espermátides, espermatozóides e espermatóforos. Tais dados sugerem o padrão descrito na literatura. O tamanho da maturidade sexual foi de 32,3 mm de largura da carapaça para machos e 31,5 mm para fêmeas. Os estádios gonadais observados macroscopicamente por meio do volume e da coloração das gônadas foram validados pela análise histológica, sendo um critério útil e ágil para a identificação da maturidade sexual para a espécie. O presente estudo oferece informações inéditas sobre a biologia reprodutiva de Sylviocarcinus pictus.

Bioactivation of Müllerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease.

During male gonadal development Müllerian duct regression is mediated by the actions of the hormone Müllerian inhibiting substance (MIS), a member of the transforming growth factor beta superfamily. MIS is considered to be unique among members of this superfamily because bioactivation of MIS via proteolytic processing is hypothesized to occur at its target organ, the Müllerian duct. We find instead that the majority of MIS is processed and secreted from the embryonic testes as a complex in which the mature region remains noncovalently associated with the prodomain. In addition, we have identified two candidate endoproteases that are expressed in the testes and that may be capable of processing MIS in vivo. These kex2/subtilisin-like enzymes, PC5 and furin, are members of the proprotein convertase family that have been implicated in hormone bioactivation via proteolytic processing after dibasic amino acid cleavage recognition sites. Coexpression of PC5 and MIS in transfected mammalian cells results in efficient processing and bioactivation of MIS. Our results suggest that MIS is a natural substrate for PC5, thereby supporting a role for prohormone convertases in the activation of transforming growth factor beta-related hormones during development.

The Role of SRY Mutations in the Etiology of Gonadal Dysgenesis in Patients with 45,X/46,XY Disorder of Sex Development and Variants

Background: The potential involvement of SRY in abnormal gonadal development in 45,X/46,X,der(Y) patients was proposed following the identification of SRY mutations in a few patients with Turner syndrome (TS). However, its exact etiological role in gonadal dysgenesis in patients with Y chromosome mosaicisms has not yet been clarified. Aims: It was the aim of this study to screen for allelic variation in SRY in a large cohort of patients with disorders of sex development due to chromosomal abnormalities with 45, X/46, X, der(Y) karyotype. Patients: Twenty-seven patients, 14 with TS and 13 with mixed gonadal dysgenesis (MGD), harboring 45, X/46, X, der(Y) karyotypes were selected. Methods: Genomic DNA was extracted from peripheral blood leukocytes of all patients and from gonadal tissue in 4 cases. The SRY coding region was PCR amplified and sequenced. Results: We identified only 1 polymorphism (c.561C -> T) in a 45,X/46,XY MGD patient, which was detected in blood and in gonadal tissue. Conclusion: Our results indicate that mutations in SRY are rare findings in patients with Y chromosome mosaicisms. Therefore, a significant role of mutated SRY in the etiology of gonadal dysgenesis in patients harboring 45, X/46, XY karyotype and variants seems very unlikely. Copyright (C) 2010 S. Karger AG, Basel

Regulation of male sexual development by Sry and Sox9

Sry, a gene from the Y chromosome, is known to initiate testis formation and subsequent male differentiation in mammals. A related gene, Sox9, also plays a critical role in testis determination, possibly in all vertebrates. A number of models have been presented regarding the molecular modes of action of these two genes. However, details regarding their regulation, regulatory target genes, and interacting protein factors and co-factors have not been established with any certainty. In this review, we examine new evidence and re-examine existing evidence bearing on these issues, in an effort to build up an integrative model of the network of gene activity centred around Sry and Sox9. J. Exp. Zool. 290:463-474, 2001. (C) 2001 Wiley-Liss, Inc.

Charting the course of ovarian development in vertebrates

The decision of the embryonic gonad to differentiate as either a testis or an ovary is a critical step in vertebrate development. The molecular basis of this decision has been the focus of much study, particularly over the past decade. Here we contrast the knowledge of early gonadal development and the switch to testis differentiation with the lack of molecular understanding of ovarian development at early stages. We review current knowledge regarding mechanisms of ovarian morphogenesis and propose a model for the hierarchical control of development of the fetal ovary, incorporating the few genes already known to be important and several signals or factors that are hypothesised to exist in the early ovary.

Estágios de desenvolvimento gonadal de fêmeas do camarão-barba-ruça (Artemesia longinaris - Decapoda: Penaeidae)

The stages of gonadal development for the female of "barba-ruça" shrimp (Artemesia longinaris Bate, 1888) were characterized based on histological analysis. Four stages (immature, almost mature, ripe and spawned) were determined according to the structure and arrangement of cells in the ovary. Each stage corresponds macroscopically to a characteristic color, except stages I (immature) and IV (spawned), in which colors are very similar and can be distinguished only microscopically. The chromatic scale varies from white/translucent (stage I), neutral green (almost mature) to dark green (ripe). The mean size of cells was 56.9 µm (±3.5) (stage I), 127 µm (±2.6) (stage II) and 183 µm (±1.91) (stage III). The size frequency of cells was polimodal, and different cell stages were observed in ripe ovary, suggesting the occurrence of multiple spawning. The chromatic scale developed is an important tool for laboratory analysis, and can be easily used to identify the gonadal stages.

Analyses of the development and glycoproteins present in the ovarian follicles of Poecilia vivipara (Cyprinodontiformes, Poeciliidae)

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

Crescimento, desenvolvimento gonadal e composição muscular de matrinxãs (Brycon cephalus) submetidos à restrição alimentar e realimentação durante um ano

Neste trabalho, avaliou-se o efeito da restrição de ração alternada com realimentação no crescimento, desenvolvimento gonadal e composição muscular de matrinxãs (Brycon cephalus) adultos, de ambos os sexos, durante um ano (janeiro de 1998 a janeiro de 1999). Foram utilizados 135 peixes, separados em dois grupos: controle, alimentado diariamente até aparente saciação, e experimental, submetido ininterruptamente a ciclos de três dias de alimentação/2 dias de restrição de ração (40% de restrição ao mês). Foram realizadas 7 amostragens, nas quais foram utilizados 8 a 10 peixes por grupo. Após anestesia, os peixes foram pesados e as gônadas foram retiradas para determinação do IGS, sexo e fase do ciclo reprodutivo. Porções dos músculos branco e vermelho foram retiradas para determinação da porcentagem de lipídio total, proteína bruta, matéria seca e umidade. Os resultados mostraram que a estratégia alimentar utilizada não afetou o crescimento, o desenvolvimento gonadal e a composição muscular do matrinxã. A restrição de ração seguida por realimentação parece ter desencadeado mecanismos de ajuste metabólico para melhor utilização do alimento e aporte suficiente de energia para o crescimento, processo de maturação gonadal e composição corporal. É possível estabelecer formas de manejo alimentar mais econômicas para o matrinxã sem que processos fisiológicos importantes sejam afetados.

Desenvolvimento gonadal de fêmeas de matrinxã, Brycon amazonicus, submetidas a restrição alimentar

O presente estudo avaliou o efeito de ciclos de restrição alimentar e realimentação (2/3 dias), aplicados durante seis meses antes da desova, no desenvolvimento gonadal de matrinxã. Na ocasião da desova, fêmeas alimentadas diariamente e submetidas ao regime alimentar experimental, selecionadas para a indução hormonal, foram sacrificadas para retirada das gônadas e do fígado, com os quais se calculou o IGS (índice gonadossomático) e o IHS (índice hepatossomático), sendo os ovários processados para análise histológica. Não houve alteração no peso relativo dos ovários e fígado, e o desenvolvimento gonadal não foi afetado pelo esquema alimentar. Os valores de IGS foram de 5,09±4,98% e 9,79±4,17% e os de IHS foram de 0,84±0,07% e 0,91±0,11%, para as fêmeas controle e experimentais, respectivamente, sem diferenças significativas entre os grupos. Os ovários de peixes dos dois grupos apresentaram as mesmas características do estádio maduro, com predominância de ovócitos na fase final de maturação, repletos de vitelo. O estudo indica que a restrição alimentar não afetou a preparação das fêmeas para a reprodução e que ciclos adequados de restrição e realimentação poderão ser aplicados na criação do matrinxã, assegurando menores custos de produção.

First gonadal maturation of Pinirampus pirinampu (Siluriformes: Pimelodidae) in the Pantanal, Mato Grosso do Sul State, Brazil

Informações sobre o ciclo reprodutivo de espécies exploradas pela pesca são imprescindíveis para o seu manejo adequado. O tamanho de início da atividade reprodutiva do Pinirampus pirinampu (Spix, 1829) foi determinado. Os exemplares foram capturados em setembro, outubro e dezembro de 1997 e fevereiro e março de 1998. O estádio de desenvolvimento gonadal foi identificado macroscopicamente. O índice gonadossomático médio (IGS), calculado mensalmente, foi utilizado como indicador da época de desova. Para determinação do comprimento da primeira maturação gonadal agruparam-se, separadamente, machos e fêmeas por classes de comprimento em imaturos e adultos. Os resultados referentes aos indivíduos adultos foram lançados em gráficos e a mediana correspondeu à estimativa do comprimento no qual 50% dos indivíduos atingem a maturidade (L50). Foi também determinado o L100, estimativa do comprimento em que todos os indivíduos estão aptos à reprodução. Machos e fêmeas em processo de maturação gonadal foram encontrados a partir de outubro, com maior freqüência em fevereiro, e, somente a partir deste mês, foram encontrados indivíduos com gônadas esvaziadas. O índice gonadossomático mostrou que a partir de setembro inicia-se o processo de desenvolvimento gonadal, com seu valor máximo em fevereiro. O L50 para fêmeas foi 574 mm e para machos foi 536 mm. O L100 para fêmeas foi 590 mm e para os machos, 580 mm.

Desenvolvimento gonadal inicial e reversão sexual em Astyanax altiparanae (Teleostei, characidae)

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Ontogenia do trato digestório e desenvolvimento gonadal de Betta splendens: aspectos morfológicos

Pós-graduação em Aquicultura - FCAV

The basement membrane and the sex establishment in the juvenile hermaphroditism during gonadal differentiation of the Gymnocorymbus ternetzi (Teleostei: Characiformes: Characidae)

Although there are several studies on morphogenesis in Teleostei, until now there is no research describing the role of the basement membrane in the establishment of the germinal epithelium during gonadal differentiation in Characiformes. In attempt to study these events that result in the formation of ovarian and testicular structures, gonads of Gymnocorymbus ternetzi were prepared for light microscopy. During gonadal development in G. ternetzi, all individuals first developed ovarian tissue. The undifferentiated gonad was formed by somatic cells (SC) and primordial germ cells (PGCs). After successive mitosis, the PGCs became oogonia, which entered into meiosis originating oocytes. An interstitial tissue developed. In half of the individuals, presumptive female, prefollicle cells synthesized a basement membrane around oocyte forming a follicle. Along the ventral region of the ovary, the tissue invaginated to form the ovigerous lamellae, bordered by the germinal epithelium. Stroma developed and the follicle complexes were formed. The gonadal aromatase was detected in interstitial cells in the early steps of the gonadal differentiation in both sexes. In another half of the individuals, presumptive male, there was no synthesis of basement membrane. The interstitium was invaded by numerous granulocytes. Pre-Leydig cells proliferated. Apoptotic oocytes were observed and afterward degenerated. Spermatogonia appeared near the degenerating oocytes and associated to SCs, forming testicular tubules. Germinal epithelium developed and the basement membrane was synthesized. Concomitantly, there was decrease of the gonadal aromatase and increase in the 3β-HSD enzyme expression. Thus, the testis was organized on an ovary previously developed, constituting an indirect gonochoristic differentiation.