RESUMO
Temperature-dependent sex determination (TSD) was first discovered in reptiles. Since then, a great diversity of sex-determining responses to temperature has been reported. Higher temperatures can produce either males or females, and the temperature ranges and lengths of exposure that influence TSD are remarkably variable among species. In addition, transitory gene regulatory networks leading to gonadal TSD have evolved. Although most genes involved in gonadal development are conserved in vertebrates, including TSD species, temporal and spatial gene expression patterns vary among species. Despite variation in TSD pattern and gene expression heterochrony, the structural framework, the medullary cords, and cortex of the bipotential gonad have been strongly conserved. Aromatase (CYP19), which regulates gonadal estrogen levels, is proposed to be the main target of a putative thermosensitive factor for TSD. However, manipulation of estrogen levels rarely mimics the precise timing of temperature effects on expression of gonadal genes, as occurs with TSD. Estrogen levels may influence sex determination or gonad differentiation depending on the species. Furthermore, the process leading to sex determination under the influence of temperature poses problems that are not encountered by species with genetic sex determination. Yolk steroids of maternal origin and steroids produced by the embryonic nervous system should also be considered as sources of hormones that may play a role in TSD.
Assuntos
Meio Ambiente , Répteis/genética , Processos de Determinação Sexual/genética , Animais , Estrogênios/metabolismo , Feminino , Redes Reguladoras de Genes/genética , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Masculino , TemperaturaRESUMO
Echeveria gibbiflora is a plant widely used for its contraceptive activity in traditional Mexican medicine. Data on calcium crystals in plants are not outstanding. In the case of the Echeveria gibbiflora leaves, however, its quality, quantity, and salt type are quite surprising; one striking result of its X-ray crystallographic data shows the presence of calcium bis (hydrogen-1-malate) hexahydrate [2(C4H5O(5)1), Ca(1)2+, 6(H2O1)]. This highly soluble compound might explain the rapid shape changes of calcium crystals. Because SEM-EDS analysis shows that calcium malate crystals were obtained in a highly pure state and the immobilization and agglutination pattern that OBACE show on human and bull spermatozoa are not found even when high concentrations of calcium bis (hydrogen-1-malate) hexahydrate salt are present it is not feasible to involucrate molecules as calcium malate as part of the OBACE contraceptive activity.
Assuntos
Crassulaceae/química , Malatos/farmacologia , Extratos Vegetais/farmacologia , Espermatozoides/efeitos dos fármacos , Animais , Bovinos , Cristalização , Humanos , Masculino , Microscopia de Contraste de Fase , Folhas de Planta/química , Aglutinação Espermática/efeitos dos fármacos , Imobilizantes dos Espermatozoides/farmacologiaRESUMO
SOX9 is expressed at the onset of the genital ridge formation in both sexes. It is assumed that SRY, the testis determining gene, turns SOX9 on in male embryos because it is turned off in female embryos. Spatial expression of SRY follows a cranio-caudal pattern. Here, we asked if SOX9 is expressed in the same cell lineage and with a similar pattern as SRY. A correlative study between the structural changes in the genital ridge and the immunocytochemical localization of SOX9-positive cells was undertaken. We used a transgenic strain expressing the green fluorescent protein (GFP) that considerably enhanced the cell context where the first SOX9-positive cells appear. Although SOX9-positive cells are located among loose mesenchymal cells by stages of 8-14 tail somites (ts) in both sexes, they are absent in the thickening coelomic epithelium of females. At 15 ts the first SOX9-positive cells appear within the core of the condensed cells only in male genital ridges. At 17 ts, a gradient of SOX9-positive cells in males is apparent, closely following the cranio-caudal pattern of cell aggregation seen in genital ridges of both sexes. Hence, our results suggest that SOX9 is expressed only in loose mesenchymal cells in both sexes and that expression of SOX9 in males requires the prior aggregation of cells in the genital ridges. The correspondence of SOX9 and SRY pattern of expression supports that both genes are expressed in the preSertoli cell lineage in the core of the genital ridges.
Assuntos
Genitália/citologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Células de Sertoli/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Agregação Celular , Linhagem da Célula , Feminino , Genitália/embriologia , Genitália/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOX9 , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Sex determination is controlled either by genetic or environmental factors. In mammals Sry initiates determination but no homologue of this gene exists in non-mammalian species. Other genes of the mammalian sex-determining pathway have been identified in gonads of different vertebrates. Sox9, Dax1, and Dmrt1 are expressed at the onset of gonadal development in birds and reptiles. In the sea turtle Lepidochelys olivacea, a species with temperature sex determination (TSD), Sox9 is expressed in undifferentiated gonads at male- (MPT) or female-promoting temperatures (FPT). At MPT, Sox9 remains expressed in male gonads, but at FPT it is downregulated coinciding with the onset of the ovarian morphologic differentiation and female sex determination. At MPT however, male sex is determined early than at FPT in still undifferentiated gonads suggesting that other genes maintain Sox9 expression in testis. Here we used RT-PCR to study the expression profiles of Dax1, Dmrt1, and Sox9 in gonads of embryos of L. olivacea incubated at MPT or at FPT. The profiles were correlated with sex determination during and after the temperature-sensitive period (TSP). Dax1 maintained similar levels at both temperatures during the TSP. The Dax1 expression level increased significantly in ovaries compared to testes at stage 27, once they were morphologically distinct. The expression levels of Dmrt1 were higher at MPT than at FPT at all stages, in contrast with Sox9 levels which were similar at both temperatures at stages 23-25. Together, current results suggest that, whereas Dax1 is not involved in TSD in L. olivacea, upregulation of Dmrt1 and downregulation of Sox9 may play a role in male and female sex determination, respectively.
Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Grupo de Alta Mobilidade/análise , Receptores do Ácido Retinoico/análise , Proteínas Repressoras , Processos de Determinação Sexual , Diferenciação Sexual/fisiologia , Fatores de Transcrição/análise , Tartarugas/genética , Sequência de Aminoácidos , Animais , Temperatura Corporal/fisiologia , Receptor Nuclear Órfão DAX-1 , Proteínas de Ligação a DNA/genética , Embrião não Mamífero , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Grupo de Alta Mobilidade/genética , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores do Ácido Retinoico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9 , Homologia de Sequência de Aminoácidos , Testículo/fisiologia , Fatores de Transcrição/genética , Tartarugas/embriologiaRESUMO
Sperm obtained from bull epididymes were used to validate in vitro the effect of heparin and reduced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and Triton X-100 in the presence of propidium iodide (IP) and diacetate fluorescein (FDA). The metabolic activities of treated sperm were qualitatively monitored using an alamar Blue Redox fluorescence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at 26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage. Results validated by electron microscopy of thin sections of treated sperm showed complete lack of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100. Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus, leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic activity was supported over 52 h of incubation in any of the incubation systems tested, including Triton X-100, which showed a spermaticide effect. The authors propose that membrane damage does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining can be used as a fluorescent marker of sperm plasmatic membrane permeabilization and nuclear swelling.
Assuntos
Membrana Celular/efeitos dos fármacos , Glutationa/farmacologia , Heparina/farmacologia , Espermatozoides/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Acrossomo/ultraestrutura , Animais , Bovinos , Membrana Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Fluoresceínas/farmacologia , Fluorescência , Técnicas In Vitro , Masculino , Octoxinol/farmacologia , Propídio/farmacologia , Dodecilsulfato de Sódio/farmacologia , Espermatozoides/ultraestrutura , Fatores de TempoRESUMO
During the late 1940s, Alfred Jost demonstrated that mammalian sex differentiation begins in fetal testis, producing two factors necessary for the establishment of phenotypic males. Castrated embryos prior to testis differentiation led to phenotypic female differentiation. Jost proposed the existence of a testis-determining factor (TDF), elucidated in 1990 and named SRY for humans and Sry for mice. Thereafter, an increasing list of genes expressed in the genital ridges of mouse embryos at the onset of gonad differentiation has appeared. To date, it is clear that complete understanding of the mechanisms underlying gonadal sex differentiation in mammals requires identification of key cell lineages in which gonadal-specific genes are expressed. Here, a correlation between known gene expression and gonadal morphologic changes is attempted.
Assuntos
Proteínas Nucleares , Processos de Determinação Sexual , Diferenciação Sexual/fisiologia , Fatores de Transcrição , Animais , Castração , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Feminino , Proteínas Fetais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes sry , Células Germinativas/citologia , Proteínas de Homeodomínio/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like , Células Intersticiais do Testículo/citologia , Masculino , Mamíferos/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Ductos Paramesonéfricos/citologia , Ductos Paramesonéfricos/embriologia , Ovário/citologia , Ovário/embriologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/fisiologia , Epitélio Seminífero/citologia , Células de Sertoli/citologia , Diferenciação Sexual/genética , Proteína da Região Y Determinante do Sexo , Fator de Células-Tronco/fisiologia , Células Estromais/fisiologia , Testículo/citologia , Testículo/embriologia , Testículo/fisiologia , Ductos Mesonéfricos/citologia , Ductos Mesonéfricos/embriologia , Cromossomo Y/genéticaRESUMO
Although sex determination starts in the gonads, this may not be the case for species with temperature sex determination (TSD). Since temperature affects the whole embryo, extragonadal thermosensitive cells may produce factors that induce gonadal sex determination as a secondary event. To establish if gonads of a species with TSD respond directly to temperature, pairs of gonads were cultured, one at female-promoting temperature (FPT) and the contralateral at male-promoting temperature (MPT). Histological and immunohistochemical detection of SOX9 revealed that the response to temperature of isolated gonads was similar to that of the gonads of whole embryos. While gonads cultured at MPT maintained SOX9 expression, it was downregulated in gonads at FPT. Downregulation of SOX9 took longer in gonads cultured at stage 23 than in gonads cultured at stage 24, suggesting that a developmental clock was already established at the onset of culture. To find out if sex commitment occurs in vitro, gonads were switched from FPT to MPT at different days. Results showed that the ovarian pathway was established after 4 days of culture. The present demonstration that gonads have an autonomous temperature detector that regulates SOX9 expression provides a useful starting point from which the molecular pathways underlying TSD can be elucidated.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Grupo de Alta Mobilidade/genética , Ovário/embriologia , Diferenciação Sexual , Testículo/embriologia , Fatores de Transcrição/genética , Tartarugas/embriologia , Sequência de Aminoácidos , Animais , Epitopos/química , Feminino , Proteínas de Grupo de Alta Mobilidade/análise , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOX9 , Temperatura , Fatores de Transcrição/análiseRESUMO
When the Y chromosome of Mus musculus domesticus (Y(TIR)) was introduced onto the C57BL/6J (B6) mouse background, testis development was impaired and half of the XY progeny (Y(TIR).B6) developed a female phenotype. Y(TIR).B6 fetal ovaries showed massive death of medullary oocytes and, after birth, produced abnormal levels of steroid hormones, exhibited irregular estrous cycles, and failed to become fertile. In this study we examined whether alterations during perinatal development observed in Y(TIR).B6 ovaries permanently impaired the establishment of the hypothalamus-pituitary-ovary axis (HPOa). B6 fetal and postnatal ovaries at different stages (fetal, infantile, or adult) were transplanted orthotopically (to the ovarian bursa) to either ovariectomized B6 normal females or Y(TIR).B6 sex-reversal females. Percentage of pregnancy, litter size, and capacity to feed pups were recorded. Reciprocally, XY(TIR).B6 ovaries were orthotopically transplanted into B6 females. After crossing with fertile males, several Y(TIR).B6 sex-reversal females with B6 ovarian transplants at all ages became pregnant, had offspring, and fed their pups. On the other hand, none of the B6 female hosts with XY(TIR) ovaries became pregnant. Results demonstrated that Y(TIR).B6 sex-reversal females maintain a functional HPOa and that their failure to reproduce is primarily due to an ovarian defect.
Assuntos
Fertilidade , Infertilidade Feminina/genética , Ovário/transplante , Cromossomo Y , Animais , Morte Celular , Transtornos do Desenvolvimento Sexual , Feminino , Infertilidade Feminina/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Ovário/anatomia & histologia , Ovário/crescimento & desenvolvimento , Fenótipo , GravidezRESUMO
The nucleon, a highly organized chromatin structure, was studied to learn if its swelling takes place by the action of heparin/GSH, without the participation of any mechanism provided by sperm membranes, subcellular organelles, or other proteins foreign to the sperm nucleus. Sperm suspensions of guinea pigs and rats were incubated with 9 mM DTT and 1% CTAB. The nucleons obtained from washed epididymal spermatozoa appear under a phase-contrast microscope to preserve their original nucleus shape and to completely lack the acrosome, middle piece, and tail. In an electron microscope, nucleon thin sections show a slight nuclear chromatin decompressed from the periphery toward the center. An outstanding result was that the nucleon swelling pattern by heparin/GSH showed the same classic organization into hub-like nuclear bodies joined by a network of chromatin fibers ranging in thickness from 25 to 1.5 nm. Under the conditions of this study there was no need of any membrane or subcellular structure. At stage IV, all the thick fibers disappear, leaving only thin bead fibers on a string. With respect to nuclear swelling there is no doubt that the sperm chromatin is organized in a special form that decides a specific required pattern of unpacking.
Assuntos
Núcleo Celular/fisiologia , Cromatina/fisiologia , Modelos Biológicos , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Epididimo , Cobaias , Masculino , Microscopia Eletrônica , RatosRESUMO
Guinea-pig spermatozoa in the presence of a purified fraction from Echeveria gibbiflora aqueous crude extract suffer a hypotonic-like effect. The phenomena exhibited included a distension of the plasma membrane over the acrosome region, inducing the formation of a huge 'head-bubble'. The agglutination effect was so enhanced that instead of inducing sperm clusters, it produced cane-like 'stalk' structures. The immobilizing activity was induced instantaneously after the addition of the purified fraction. At electron microscope level it was possible to observe a heavy amount of electron dense material of the purified fraction embedded or intercalated along the plasma membrane. It was also possible to corroborate the dispersion of the acrosomal content and the disappearance of the external acrosome membrane. The purified fraction induced loosening of the plasma membrane all along the sperm cell, however, the distension of the membrane was only produced in the apical portion of the sperm head and not in the post equatorial region. The results suggest that the plant may yield a compound suitable for use as a vaginal barrier or male contraceptive agent.