RESUMO
Male and female reproductive tracts develop from anterior intermediate mesoderm with similar differentiation processes. The anterior intermediate mesoderm develops into the mesonephros, and the Wolffian duct initiates by epithelialization in the mesonephros. The Müllerian duct invaginates from the coelomic epithelium of the cranial mesonephros for ductal formation and is then regionalized into proximal to caudal female reproductive tracts. In this study, we focused on the epithelialization of the Wolffian duct, initiation of the Müllerian duct, and the regionalization step of the Müllerian ducts as a continuous process. By using intermediate mesodermal cells from mouse pluripotent stem cells, we identified that inhibition of SMAD2/3 signaling might be involved in the differentiation into mesenchymal cells, after which mesonephric cells might be then epithelialized during differentiation of the Wolffian duct. Aggregation of coelomic epithelial cells might be related to initiation of the Müllerian duct. Transcriptomic analysis predicted that consensus sequences of SMAD3/4 were enriched among highly expressed genes in the proximal Müllerian duct. SMAD2/3 signaling to regulate differentiation of the Wolffian duct was continuously activated in the proximal Müllerian duct and was involved in proximal and oviductal regionalization. Therefore, SMAD2/3 signaling may be finely tuned to regulate differentiation from initiation to regionalization steps.
Assuntos
Ductos Paramesonéfricos , Ductos Mesonéfricos , Camundongos , Animais , Masculino , Feminino , Ductos Mesonéfricos/fisiologia , Ductos Paramesonéfricos/fisiologia , Diferenciação Celular , Células Epiteliais , Transdução de SinaisRESUMO
The Wolffian duct (WD) is an embryonic tissue that undergoes androgen-induced morphological changes to become the epididymis. Toll-like receptor 4 (TLR4)- and nuclear factor kB (NFKB)-induced effectors are expressed in the adult epididymis and represent important players in epididymal innate immune responses. TLR4/NFKB signaling pathway is evolutionarily conserved and plays a critical morphogenetic role in several species; however, its function during WD morphogenesis is unknown. We hypothesized that TLR4/NFKB pathway plays a role during WD development. Here we examined TLR4 expression and regulation of TLR4-target genes during rat WD morphogenesis between embryonic days (e) 17.5-20.5. The functionality of TLR4/NFKB signaling was examined using WD organotypic cultures treated with lipopolysaccharide (LPS) from E. coli (TLR4 agonist) and PDTC (NFKB inhibitor). TLR4 was detected at mRNA level in e17.5 (uncoiled duct) and e20.5 (coiled duct) WDs, and spatio-temporal changes in TLR4 immunoreactivity were observed between these two time points. Expression level analysis of a subset of TLR4-regulated genes showed that TLR4/NFKB pathway was activated after exposure of cultured WD to LPS (4 h), an event that was abrogated by PDTC. Long-term exposure of cultured WDs to LPS (96 h) resulted in dysregulations of morphogenetic events and LAMA1 immunodistribution changes, suggesting the extracellular matrix at the intersection between WD morphogenesis and balance of innate immune components. Our results unveil the epididymal morphogenesis as an event equipped with TLR4/NFKB signaling components that may serve developmental functions, and eventually transition to host defense function when the fetus is exposed to an infectious or noninfectious threat.
Assuntos
Epididimo/fisiologia , Morfogênese/fisiologia , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Ductos Mesonéfricos/fisiologia , Animais , Células Cultivadas , Desenvolvimento Embrionário , Feminino , Imunidade Inata , Lipopolissacarídeos/imunologia , Masculino , Técnicas de Cultura de Órgãos , Gravidez , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.
Assuntos
Gônadas/fisiologia , Morfogênese/fisiologia , Ductos Mesonéfricos/fisiologia , Animais , Embrião de Mamíferos/fisiologia , Feminino , Idade Gestacional , Rim/fisiologia , Masculino , Mesonefro/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Diferenciação Sexual/fisiologiaRESUMO
The bladder's remarkable regenerative capacity had been thought to derive exclusively from its own progenitors. While examining consequences of DNA methyltransferase 1 (Dnmt1) inactivation in mouse embryonic bladder epithelium, we made the surprising discovery that Wolffian duct epithelial cells can support bladder regeneration. Conditional Dnmt1 inactivation in mouse urethral and bladder epithelium triggers widespread apoptosis, depletes basal and intermediate bladder cells, and disrupts uroplakin protein expression. These events coincide with Wolffian duct epithelial cell recruitment into Dnmt1 mutant urethra and bladder where they are reprogrammed to express bladder markers, including FOXA1, keratin 5, P63, and uroplakin. This is evidence that Wolffian duct epithelial cells are summoned in vivo to replace damaged bladder epithelium and function as a reservoir of cells for bladder regeneration.
Assuntos
Bexiga Urinária/fisiologia , Urotélio/fisiologia , Ductos Mesonéfricos/fisiologia , Animais , Animais Recém-Nascidos , Apoptose , Linhagem da Célula , DNA (Citosina-5-)-Metiltransferase 1/fisiologia , Dano ao DNA , Metilação de DNA , Células Epiteliais/fisiologia , Camundongos , RegeneraçãoRESUMO
Mesonephric ducts regress in genotypic females, leaving behind few remnants. These vestigial structures are often recognized in the mesosalpinx and paracervical regions. We report here 3 cases of female-to-male transgenders who underwent hysterectomy following testosterone treatment. Both female and male genital structures were identified on histologic examination. Although the morphologic appearances of the specimens were unremarkable, histologically 1 case revealed a well-formed fallopian tube as well as an epididymis and 2 cases showed prostate glands to be present in the cervical squamous epithelium.
Assuntos
Colo do Útero/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Procedimentos de Readequação Sexual/métodos , Testosterona/administração & dosagem , Ductos Mesonéfricos/efeitos dos fármacos , Adolescente , Adulto , Colo do Útero/anatomia & histologia , Colo do Útero/fisiologia , Epididimo/anatomia & histologia , Epitélio/anatomia & histologia , Epitélio/fisiologia , Feminino , Humanos , Histerectomia , Masculino , Próstata/anatomia & histologia , Pessoas Transgênero , Ductos Mesonéfricos/anatomia & histologia , Ductos Mesonéfricos/fisiologiaRESUMO
Unresolved questions remain concerning the derivation of the vagina with respect to the relative contributions from the Müllerian ducts, the urogenital sinus, and the Wolffian ducts. Recent molecular and cellular studies in rodents have opened up a large gap between the level of understanding of vaginal development in mice and understanding of human vaginal development, which is based on histology. To compare the findings in mice with human vaginal development and to address this gap, we analysed molecular characteristics of the urogenital sinus, Wolffian ducts, and Müllerian ducts in 8-14-week-old human specimens using immunohistochemical methods. The monoclonal antibodies used were directed against cytokeratin (CK) 14, CK19, vimentin, laminin, p63, E-cadherin, caspase-3, Ki67, HOX A13, and BMP-4. The immunohistochemical analysis revealed that, during weeks 8-9, the epithelium of the Müllerian ducts became positive for p63 as p63-positive cells that originated from the sinus epithelium reached the caudal tip of the fused Müllerian ducts via the Wolffian ducts. The lumen of the fused Müllerian ducts was closed by an epithelial plug that contained both vimentin-positive and vimentin-negative cells. Subsequently, the resulting epithelial tube enlarged by proliferation of basal p63-positive cells. The first signs of squamous differentiation were detected during week 14, with the appearance of CK14-positive cells. According to our results, all three components, namely, the urogenital sinus, Wolffian ducts, and Müllerian ducts, interacted during the formation of the human vagina. The sinus epithelium provided p63-positive cells, the Wollfian ducts acted as a 'transporter', and the Müllerian ducts contributed the guiding structure for the vaginal anlagen. Epithelial differentiation began at the end of the period studied and extended in a caudo-cranial direction. The present study is one of the first to provide up-to-date molecular correlates for human vaginal development that can be compared with the results of cell biological studies in rodents.
Assuntos
Vagina/embriologia , Epitélio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Ductos Paramesonéfricos/anatomia & histologia , Ductos Paramesonéfricos/embriologia , Ductos Paramesonéfricos/fisiologia , Sistema Urogenital/anatomia & histologia , Sistema Urogenital/embriologia , Sistema Urogenital/fisiologia , Ductos Mesonéfricos/anatomia & histologia , Ductos Mesonéfricos/embriologia , Ductos Mesonéfricos/fisiologiaRESUMO
Ureteric bud (UB) emergence from the Wolffian duct (WD), the initiating step in metanephric kidney morphogenesis, is dependent on GDNF; however, GDNF by itself is generally insufficient to induce robust budding of the isolated WD in culture. Thus, additional factors, presumably peptides or polypeptide growth factors, might be involved. Microarray data from in vivo budding and non-budding conditions were analyzed using non-negative matrix factorization followed by gene ontology filtering and network analysis to identify sets of genes that are highly regulated during budding. These included the GDNF co-receptors GFRalpha1 and RET, as well as neuropeptide Y (NPY). By using ANOVA with pattern matching, NPY was also found to correlate most significantly to the budded condition with a high degree of connectedness to genes with developmental roles. Exogenous NPY [as well as its homolog, peptide YY (PYY)] augmented GDNF-dependent budding in the isolated WD culture; conversely, inhibition of NPY signaling or perturbation of NPY expression inhibited budding, confirming that NPY facilitates this process. NPY was also found to reverse the decreased budding, the downregulation of RET expression, the mislocalization of GFRalpha1, and the inhibition of AKT phosphorylation that resulted from the addition of BMP4 to the isolated WD cultures, suggesting that NPY acts through the budding pathway and is reciprocally regulated by GDNF and BMP4. Thus, the outgrowth of the UB from the WD might result from a combination of the upregulation of the GDNF receptors together with genes that support GDNF signaling in a feed-forward loop and/or counteraction of the inhibitory pathway regulated by BMP4.
Assuntos
Neuropeptídeo Y/fisiologia , Ductos Mesonéfricos/fisiologia , Animais , Proteína Morfogenética Óssea 4/fisiologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Morfogênese , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-ret/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para Cima , Ductos Mesonéfricos/embriologiaRESUMO
Androgen-mediated wolffian duct (WD) development is programmed between embryonic d 15.5 (e15.5) and 17.5 in male rats, and WD differentiation has been shown to be more susceptible to reduced androgen action than is its initial stabilization. We investigated regulation of these events by comparing fetal WD development at e15.5-postnatal d0 in male and female androgen receptor knockout mice, and in rats treated from e14.5 with flutamide (100 mg/kg/d) plus di-n(butyl) phthalate (500 mg/kg/d) to block both androgen action and production, testosterone propionate (20 mg/kg/d) to masculinize females, or vehicle control. In normal females, WD regression occurred by e15.5 in mice and e18.5 in rats, associated with a lack of epithelial cell proliferation and increased apoptosis, disintegration of the basement membrane, and reduced epithelial cell height. Exposure to testosterone masculinized female rats including stabilization and partial differentiation of WDs. Genetic or chemical ablation of androgen action in males prevented masculinization and induced WD regression via similar processes to those in normal females, except this occurred 2-3 d later than in females. These findings provide the first evidence that androgens may not be the only factor involved in determining WD fate. Other factors may promote survival of the WD in males or actively promote WD regression in females, suggesting sexually dimorphic differences in the preprogrammed setup of the WD.
Assuntos
Androgênios/fisiologia , Roedores/embriologia , Ductos Mesonéfricos/embriologia , Ductos Mesonéfricos/fisiologia , Androgênios/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Feto/fisiologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Gravidez , Ratos , Ratos Wistar , Receptores Androgênicos/genética , Roedores/metabolismo , Caracteres Sexuais , Testosterona/farmacologia , Virilismo/genética , Ductos Mesonéfricos/efeitos dos fármacos , Ductos Mesonéfricos/metabolismoRESUMO
dachshund/Dach gene family members encode transcriptional cofactors with highly conserved protein interaction domains and are expressed in the developing eyes, brains, and limbs in insects and vertebrates. These observations suggest that the developmental roles of dachshund/Dach in these tissues have been conserved since the divergence of arthropods and chordates. However, while Drosophila dachshund mutants have abnormalities in eye, brain, limbs, mouse Dach1 or Dach2 knockout mutants do not exhibit gross anatomical malformations in these tissues. In addition, Dach1/2 double homozygotes have intact eyes and limbs. Here we show that in Dach1/Dach2 double mutants, female reproductive tract (FRT) development is severely disrupted. This defect is associated with the Müllerian duct (MD) and not the Wolffian duct (WD), which normally differentiate into either the FRT or male reproductive tract (MRT), respectively. Dach1 and Dach2 are expressed in the MD, and in Dach1/2 double mutants, MD expression of Lim1 and Wnt7a is abnormal and MD development is disrupted. In contrast, WD and MRT development are not grossly affected. We propose that Dach1 and Dach2 proteins may redundantly control FRT formation by regulating the expression of target genes required for development of the MD. This vertebrate Dach1/2 function may have been conserved during arthropod evolution, as Drosophila dachshund mutants also exhibit an FRT phenotype.
Assuntos
Proteínas do Olho/fisiologia , Ductos Paramesonéfricos/embriologia , Proteínas Nucleares/fisiologia , Animais , Proteínas de Ligação a DNA , Drosophila/embriologia , Drosophila/genética , Drosophila/fisiologia , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Ductos Paramesonéfricos/fisiologia , Proteínas Nucleares/genética , Fatores de Transcrição , Ductos Mesonéfricos/embriologia , Ductos Mesonéfricos/fisiologiaRESUMO
In this study the development of the bovine Fallopian tube was investigated using light microscopic methods. Formation and differentiation of the Müllerian duct were studied in mesonephroi of 16 embryos and fetuses with a crown-rump lengths (CRL) of 0.9-8.4 cm. The funnel field, the rostral beginning of the Müllerian duct was first observed at a CRL of 0.9 cm. It appears as a thickening of the mesothelium on the craniolateral side of the mesonephros. During later development the Müllerian duct emerges by caudal outgrowth from the funnel field. Formation of a common basal lamina surrounding the caudal tips of Müllerian and Wolffian ducts could be observed at all stages up to CRL of 2.7 cm. The mesothelium and the epithelium of the Wolffian duct adjacent to the Müllerian duct showed a modification of epithelium height in all examined stages. Probably the Wolffian duct influences the growth of Müllerian duct by epithelio-mesenchymal interactions. Fetuses from a CRL of 12.0 to 94.0 cm were used for investigation of the prenatal differentiation of the oviductal mucosa. Folding of the oviductal mucosa started at a CRL of 29.0 cm and continued until birth. Individual primary, secondary and tertiary folds are formed in special proliferation zones and epithelium-folding buds. The cellular differentiation of the oviductal epithelium involves the formation of ciliated and secretory cells during different times of prenatal development. Ciliogenesis was first detected at a CRL of 33.0 cm. Active secretory cells could be observed in the oviductal epithelium from a CRL of 64.0 cm onwards.
Assuntos
Bovinos/anatomia & histologia , Bovinos/embriologia , Tubas Uterinas/embriologia , Animais , Tubas Uterinas/anatomia & histologia , Tubas Uterinas/fisiologia , Feminino , Masculino , Ductos Paramesonéfricos/anatomia & histologia , Ductos Paramesonéfricos/embriologia , Ductos Paramesonéfricos/fisiologia , Ductos Mesonéfricos/anatomia & histologia , Ductos Mesonéfricos/embriologia , Ductos Mesonéfricos/fisiologiaRESUMO
BACKGROUND: The androgen receptor (AR) is essential for the differentiation of male external and internal genitalia. It is normally present in two forms, a full-length form B and an N-terminal truncated form A with still unknown function. Mutations in the AR gene cause androgen insensitivity syndrome (AIS), which is divided into subgroups according to the degree of undermasculinization. Patients with completely female external genitalia are classified as complete AIS (CAIS). However, a recent study has shown that some CAIS patients have signs of internal male genital differentiation due to missense mutations that show some degree of residual function. OBJECTIVE: We aimed to study the expression of the different forms of the AR in two CAIS patients in relation to the development of male internal genital structures. One patient had a mutation (L7fsX33) that affects only the full-length AR-B form of the AR, whereas the other had a nonsense mutation (Q733X) affecting both isoforms. MEASUREMENTS AND RESULTS: We thoroughly analysed internal genitalia at surgery and by histological examination. No signs of Wolffian duct (WD) development were present in any of the patients. Western blotting of proteins from gonadal and genital skin fibroblasts was performed with AR antibodies directed against different AR epitopes. The N-terminally truncated A form was expressed in normal amounts in the patient with the L7fsX33 mutation while no AR was detected in the other patient. CONCLUSION: The presence of the AR-A form does not seem to be sufficient for WD maintenance and differentiation.
Assuntos
Síndrome de Resistência a Andrógenos/genética , Mutação de Sentido Incorreto/fisiologia , Mutação Puntual/fisiologia , Receptores Androgênicos/fisiologia , Adulto , Síndrome de Resistência a Andrógenos/embriologia , Síndrome de Resistência a Andrógenos/metabolismo , Western Blotting , Castração , Fibroblastos , Genitália Masculina/anormalidades , Cabelo/crescimento & desenvolvimento , Humanos , Cariotipagem , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/análise , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ductos Mesonéfricos/fisiologiaRESUMO
The development of a vagina as a separate outlet of the birth canal evolves at the transition of egg laying species to eutherian mammals. The derivation of the vagina from the Wolffian and Müllerian ducts and the contribution of the urogenital sinus are still open questions. Here experiments with the complete androgen receptor defect in the testicular feminisation (Tfm) mouse are reported which show that the vagina is formed by caudal migration of Wolffian and Müllerian ducts. The cranial ends of the Wolffian ducts successively regress while the Müllerian ducts fuse to form the vagina. Immunohistochemistry of the androgen receptor reveals that the caudal ends of the Wolffian ducts remain in the indifferent stage and therefore have been mistaken as sinuvaginal bulbs. The Wolffian ducts do not contribute to the vagina itself but have a helper function during downward movement of the vaginal bud in the female. In the male the caudal ends serve as androgen operated switch for the negative control of vaginal development. The results indicate that the rudimentary vagina in the complete androgen insensitivity syndrome (CAIS) corresponds to non obliterated caudal ends of the Müllerian ducts. Selective atresia of the vagina in the MRKH (Mayer-Rokitansky-Kuster-Hauser) syndrome may be explained by the failure of Wolffian and Müllerian ducts to descend caudally.
Assuntos
Androgênios/fisiologia , Vagina/anatomia & histologia , Ductos Mesonéfricos/fisiologia , Androgênios/metabolismo , Animais , Feminino , Heterozigoto , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Mutantes , Modelos Biológicos , Ductos Paramesonéfricos/anatomia & histologia , Ductos Paramesonéfricos/fisiologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Vagina/embriologia , Vagina/metabolismo , Ductos Mesonéfricos/anatomia & histologiaRESUMO
Fibroblast growth factor 9 (FGF9) signal has a role in organogenesis of the mammalian testis by controlling migration of mesonephric cells to the XY gonad, but neither it nor the FGF receptors is expressed sex-specifically. Of the Sprouty genes encoding antagonists of receptor tyrosine kinases including FGFr, mSprouty2 expression was confined to the developing testis and mesonephros. Gain of SPROUTY2 function in the male genital ridge and mesonephros malformed the vas deferens and epididymis, and diminished the number of seminiferous tubules and interstitium associating with reduced mesonephric cell migration and Fgf9 expression in embryonic testis, whereas exogenous FGF9 signaling recovered mesonephric cell migration inhibited by SPROUTY2. These phenotypes associated also with the decreased expression of Sox9, Desert hedgehog, Hsd3beta, Platelet/endothelial cell adhesion molecule, and alpha-smooth muscle actin, which are markers of the Sertoli, Leydig, endothelial, and peritubular myoid cells of the developing testis. Based on these data, we propose that the Sprouty proteins are involved normally in mediating the sexually dimorphic signaling of FGF9 and controlling cell migration from the mesonephros during testis development.
Assuntos
Fator 9 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas/metabolismo , Testículo/embriologia , Testículo/fisiologia , 3-Hidroxiesteroide Desidrogenases/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Movimento Celular/fisiologia , Células Endoteliais/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 9 de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Homeodomínio LIM , Laminina/genética , Células Intersticiais do Testículo/fisiologia , Masculino , Proteínas de Membrana , Mesonefro/citologia , Mesonefro/embriologia , Mesonefro/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Miócitos de Músculo Liso/fisiologia , Técnicas de Cultura de Órgãos , Fator de Transcrição PAX2/genética , Gravidez , Regiões Promotoras Genéticas/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Diferenciação Sexual/fisiologia , Testículo/citologia , Fatores de Transcrição , Proteínas Wnt/genética , Proteína Wnt4 , Ductos Mesonéfricos/citologia , Ductos Mesonéfricos/embriologia , Ductos Mesonéfricos/fisiologiaRESUMO
BACKGROUND: Wolffian ducts (WDs) are the embryonic precursors of the male reproductive tract. Their development is induced by testosterone, which interacts with the androgen receptor (AR). The molecular pathways underlying androgen-dependent WD development are largely unknown. We aimed to identify AR target genes important in this process. METHODS: RNA was isolated from rat WDs at E17.5 and E20.5. Affymetrix GeneChip expression arrays were used to identify transcripts up- or downregulated more than 2-fold. Regulation of seven transcripts was confirmed using quantitative PCR. RESULTS: Transcripts from 76 known genes were regulated, including modulators of insulin-like growth factor and transforming growth factor-beta signalling. By controlling these modulators, androgens may indirectly affect growth factor signalling pathways important in epithelial-mesenchymal interactions and organ development. Caveolin-1, also upregulated, may play a role in modifying as well as mediating AR signalling. Differentiation of WD epithelium and smooth muscle, innervation and extracellular matrix synthesis were reflected in regulation of other transcripts. Several genes were previously suggested to be regulated by androgens or contained functional or putative androgen/glucocorticoid response elements, indicating they may be direct targets of androgen signalling. CONCLUSION: Our results suggest novel cohorts of signals that may contribute to androgen-dependent WD development and provide hypotheses that can be tested by future studies.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Receptores Androgênicos/fisiologia , Ductos Mesonéfricos/crescimento & desenvolvimento , Ductos Mesonéfricos/fisiologia , Androgênios/fisiologia , Animais , Caveolina 1/genética , Caveolina 1/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Cistatinas , DNA/análise , DNA/genética , Feminino , Masculino , Músculo Liso/citologia , Músculo Liso/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Proteínas/genética , Proteínas/fisiologia , RNA/análise , Ratos , Ratos Wistar , Receptores Androgênicos/genética , Elementos de Resposta/genética , Elementos de Resposta/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Somatomedinas/genética , Somatomedinas/fisiologia , Testosterona/fisiologia , Transcrição Gênica , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia , Ductos Mesonéfricos/química , Ductos Mesonéfricos/citologiaRESUMO
Glial-Cell-Line-Derived Neurotrophic Factor (GDNF) is the major mesenchyme-derived regulator of ureteric budding and branching during nephrogenesis. The ligand activates on the ureteric bud epithelium a receptor complex composed of Ret and GFRalpha1. The upstream regulators of the GDNF receptors are poorly known. A Notch ligand, Jagged1 (Jag1), co-localises with GDNF and its receptors during early kidney morphogenesis. In this study we utilized both in vitro and in vivo models to study the possible regulatory relationship of Ret and Notch pathways. Urogenital blocks were exposed to exogenous GDNF, which promotes supernumerary ureteric budding from the Wolffian duct. GDNF-induced ectopic buds expressed Jag1, which suggests that GDNF can, directly or indirectly, up-regulate Jag1 through Ret/GFRalpha1 signalling. We then studied the role of Jag1 in nephrogenesis by transgenic mice constitutively expressing human Jag1 in Wolffian duct and its derivatives under HoxB7 promoter. Jag1 transgenic mice showed a spectrum of renal defects ranging from aplasia to hypoplasia. Ret and GFRalpha1 are normally downregulated in the Wolffian duct, but they were persistently expressed in the entire transgenic duct. Simultaneously, GDNF expression remained unexpectedly low in the metanephric mesenchyme. In vitro, exogenous GDNF restored the budding and branching defects in transgenic urogenital blocks. Renal differentiation apparently failed because of perturbed stimulation of primary ureteric budding and subsequent branching. Thus, the data provide evidence for a novel crosstalk between Notch and Ret/GFRalpha1 signalling during early nephrogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Proteínas de Membrana/fisiologia , Fatores de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Ureter/embriologia , Animais , Proteínas de Ligação ao Cálcio , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-1 , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-ret , Receptores Notch , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Serrate-Jagged , Transdução de Sinais , Fatores de Tempo , Transgenes , Regulação para Cima , Ductos Mesonéfricos/fisiologiaRESUMO
In the human male fetus, testes develop by the 7th week and begin to secrete two hormones: anti-müllerian hormone (AMH) induces the regression of müllerian ducts, the anlagen of the uterus, fallopian tubes and upper vagina, upon binding to a specific membrane receptor, whereas testosterone induces the differentiation of the wolffian ducts into the epididymes, vasa deferentia and seminal vesicles. In some target tissues, testosterone is converted to dihydrotestosterone, which is responsible for masculinization of the urogenital sinus and external genitalia. Both androgens act upon binding to the same nuclear receptor. In the absence of AMH and androgen action, or example in the female or in abnormal male differentiation, the internal and external genital primordia differentiate following the female pathway, even in the absence of ovaries. In males, an impaired function of the AMH-dependent pathway results in the persistent müllerian duct syndrome, a disorder characterized by the presence of uterus and fallopian tubes in otherwise normally virilized boys. Several mutations found in the AMH and AMH-receptor genes explain the pathophysiology of this syndrome.
Assuntos
Genitália Feminina/embriologia , Genitália Masculina/embriologia , Glicoproteínas , Diferenciação Sexual/genética , Hormônio Antimülleriano , Criança , Pré-Escolar , Proteínas de Ligação a DNA/genética , Feminino , Fatores de Transcrição Fushi Tarazu , Genitália Feminina/fisiologia , Genitália Masculina/fisiologia , Disgenesia Gonadal/embriologia , Disgenesia Gonadal/fisiopatologia , Inibidores do Crescimento/metabolismo , Proteínas de Homeodomínio , Humanos , Lactente , Masculino , Ductos Paramesonéfricos/embriologia , Ductos Paramesonéfricos/fisiologia , Receptores Citoplasmáticos e Nucleares , Fator Esteroidogênico 1 , Hormônios Testiculares/metabolismo , Testosterona/metabolismo , Fatores de Transcrição/genética , Ductos Mesonéfricos/embriologia , Ductos Mesonéfricos/fisiologiaRESUMO
Recently, we identified a protein in the developing male reproductive tract of the fetal mouse which induced Wolffian duct differentiation in vitro in the absence of testis or testosterone. In this paper, we further evaluated the masculinizing role of this protein using an immunological approach. Thus, we purified a 72K protein from the 18-day-old male fetal reproductive tracts, prepared an antibody against the protein, and determined the role of this protein in producing masculine differentiation of the Wolffian duct using the polyclonal antibody against the protein. We demonstrate that the antibody reacted with the purified 72K protein, producing only one immunoreactive band around the 72K region in Western blot analysis. However, it reacted with 72K and 63K proteins present in the 18-day-old male reproductive tract. Both of these immunoreactive bands disappeared when the antibody was pretreated with the purified antigen. Nonimmune immunoglobulin G (IgG) produced no reactive bands with the extract of the male reproductive tracts. The IgG preparation of the immune serum specifically prevented Wolffian duct differentiation when tested in organ culture containing 13-day-old fetal male or female reproductive tracts in the presence of testis or testosterone (1 micrograms/ml) in a dose-dependent manner. No effect was found on the development of other organs, namely testis, Mullerian duct, and urogenital sinus, that were included in the organ culture. Nonimmune IgG, as expected, produced no effect on Wolffian duct differentiation. Western blot analysis of male and female reproductive tracts indicated a difference in their reactivities with this antibody. In females, the reactivity to the proteins around the 72K and 63K regions was very weak compared to that found with male proteins in those regions. Both of the above bands of the female reproductive tract disappeared when the antibody was pretreated with an excess of female reproductive tract extract, but in males, only the 63K band disappeared, whereas its 72K band remained. In conclusion, it appears that a 72K protein of the male reproductive tract plays a role in the Wolffian duct differentiation of the fetal mouse.
Assuntos
Western Blotting , Feto/fisiologia , Proteínas/fisiologia , Diferenciação Sexual , Ductos Mesonéfricos/fisiologia , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Feminino , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Técnicas Imunológicas , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Proteínas/imunologia , Proteínas/isolamento & purificaçãoRESUMO
Sperm maturation occurs in the mammalian spermatozoon during its passage through the epididymis. Maturation comprises a series of morpho-physiological changes, which includes the acquisition of the fertilizing capacity in the gamete. This maturative process has been particularly studied in mammals, but different data reveal that birds, reptiles and some kind of fish have a similar characteristic. Anatomical and histological analyses of mammalian epididymis and of Wolffian ducts of some birds and reptiles show the predominance of a secretory cell system. Proteins secreted by the male ducts seem to be an important factor involved in the acquisition of motility, as well as in the changes in the molecular organization of the plasma membrane. Changes occurring in the plasma membrane of the mammalian spermatozoon are related to the acquisition of foreign proteins (of epididymal origin). Some of these membrane changes seem to be connected with the capacitation phenomena and also with gamete interaction during fertilization. The use of antibodies against Wolffian duct proteins has shown that spermatozoa birds and reptiles also acquire proteins during their passage through the male duct. Nevertheless, in birds, and probably in reptiles, capacitation is not a pre-requisite for fertilization and some testicular spermatozoa are able to fertilize the egg. Then, what is the real significance of the membrane maturative changes in these subtherian vertebrates? Proteins acquired during maturation in such species must have different functions from those in mammals, to support spermatozoon survival and/or transport in the female tract, where spermatozoa are stored for a long time. Surface changes in mammals would possibly have similar roles when the gametes are in the female tract.
Assuntos
Fertilização , Maturação do Esperma , Vertebrados/fisiologia , Animais , Epididimo/fisiologia , Epididimo/ultraestrutura , Genitália Masculina/anatomia & histologia , Genitália Masculina/fisiologia , Masculino , Glicoproteínas de Membrana/fisiologia , Microscopia Eletrônica , Especificidade da Espécie , Capacitação Espermática , Espermatozoides/ultraestrutura , Ductos Mesonéfricos/fisiologiaRESUMO
La maduración del espermatozoide de los mamíferos ocurre durante su tránsito a través del epidídimo. La maduración comprende una serie de cambios morfológicos y fisiológicos que comprenden la adquisición de la capacidad fertilizante de la gameta. Este proceso de maduración ha sido estudiado principalmente en los mamíferos, pero existen datos que revelan que los pájaros, reptiles y ciertos tipos de peces poseen características similares. El análisis anatómico e histológico del epidídimo de los mamíferos y de los conductos de Wolff de algunos pájaros y reptiles muestra el predominio de un sistema de células secretoras. Las proteínas secretadas por los conductos de los machos parece ser un factor importante en la adquisición de la motilidad del espermatozoide, así como en los cambios que ocurren en la organización molecular de la membrana plasmática. Los cambios que ocurren en la membrana plasmática de los espermatozoides de mamíferos se relacionan con la adquisición de proteínas foráneas (de origen epididimario). Algunos de estos cambios de la membrana parecen conectarse con el fenómeno de capacitación y también con la interacción de las gametas durante la fertilización. El uso de anticuerpos contra las proteínas del conducto de Wolff ha mostrado que los espermatozoides de los pájaros y reptiles también incorporan proteínas durante su pasaje a través de este conducto. Sin embargo, en los pájaros y también en los reptiles, la capacitación no es un prerequisito para fertilizar y algunos son capaces de fertilizar con espermatozoides del testículo. Por consiguiente se plantea la cuestión acerca del real significado de los cambios de maduración en estos vertebrados. Las proteínas adquiridas durante la maduración en tales especies tendrían funciones diferentes a las de los mamíferos, probablemente como apoyo para la sobrevivencia de los espermatozoides durante el transporte en el tracto reproductor femenino, donde as veces son depositados por largo tiempo. Posiblemente un rol similar podrían tener los cambios de superficie en las gametas de los mamíferos cuando están en el tracto reproductor femenino
Assuntos
Animais , Masculino , Fertilização , Maturação do Esperma , Vertebrados/fisiologia , Epididimo/fisiologia , Epididimo/ultraestrutura , Especificidade da Espécie , Genitália Masculina/anatomia & histologia , Genitália Masculina/fisiologia , Glicoproteínas de Membrana/fisiologia , Microscopia Eletrônica , Capacitação Espermática , Espermatozoides/ultraestrutura , Ductos Mesonéfricos/fisiologiaRESUMO
At the 1.6-1.8 cm stages in both male and female bovine fetuses, the anlage of the Müllerian duct was first observed as an elevation of mesothelium on the cranial tip of the mesonephros. The duct extended toward the urogenital sinus in parallel with the Wolffian duct. Its caudal end was solid and was fused with the Wolffian duct at about the level of the middle of the gonadal anlage. Thereafter, the Müllerian duct became gradually independent of the Wolffian duct throughout its whole length. Therefore, it may be that the main part of the Müllerian duct is established by budding from the Wolffian duct. At the 4.3-4.5 cm stages the male Müllerian duct began to degenerate and the female one continued to grow. This suggests that the male Müllerian duct is affected by the Müllerian inhibiting substance at these stages. At the 12.0 cm stage, the female Wolffian duct disappeared. The male Wolffian duct was also much reduced in diameter at this stage, but began to grow again from the 14 cm stage onward.