RESUMO
Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoplasma/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Barreira Hematotesticular/metabolismo , Células Cultivadas , Técnicas Imunoenzimáticas , Masculino , Ratos , Ratos Wistar , Epitélio Seminífero/citologia , Epitélio Seminífero/metabolismo , Epitélio Seminífero/ultraestrutura , Células de Sertoli/ultraestrutura , Testículo/citologia , Testículo/ultraestruturaRESUMO
In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.
Assuntos
Corpo Lúteo/citologia , Técnica de Fratura por Congelamento/métodos , Poro Nuclear/ultraestrutura , Parto , Prenhez , Animais , Apoptose , Feminino , Marcação In Situ das Extremidades Cortadas , Masculino , Gravidez , Ratos , Ratos WistarRESUMO
In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.
Assuntos
Masculino , Animais , Feminino , Gravidez , Ratos , Corpo Lúteo/citologia , Poro Nuclear/ultraestrutura , Técnica de Fratura por Congelamento/métodos , Marcação In Situ das Extremidades Cortadas , Parto , Prenhez , Ratos WistarRESUMO
In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe en face the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.(AU)
Assuntos
Masculino , Animais , Feminino , Gravidez , Ratos , Corpo Lúteo/citologia , Técnica de Fratura por Congelamento/métodos , Poro Nuclear/ultraestrutura , Marcação In Situ das Extremidades Cortadas , Parto , Prenhez , Ratos WistarRESUMO
The male gonad receives nerve fibres from the autonomic ganglionic system. These fibres converge on the testis along two pathways, the superior and the inferior spermatic nerves. The superior spermatic nerve runs from the superior mesenteric ganglion alongside the testicular artery, whereas the inferior spermatic nerve originates in inferior mesenteric ganglion, accompanies the vas deferens and penetrates the inferior pole of the testis. The aim of this work was to evaluate androgen release after the addition of noradrenaline or adrenoreceptor antagonists (propranolol or phentolamine) to the ganglionic compartment. An ex vivo system used in a previous work was incubated in two separate containers, one for the testis and the other for the ganglion. Both organs remain interconnected (as in vivo) by the respective spermatic nerve. When noradrenaline was added to the inferior mesenteric ganglion, testosterone release in the gonad container underwent a progressive and significant increment. Propranolol diminishes and phentolamine increases the androgen release. When using the superior mesenteric ganglion, no changes were observed. These results indicate that the ganglionic stimulation of the autonomic system clearly participates in testosterone release from the testis. This effect depends on the ganglion involved. These results make it evident that not only the classical and well-known hypothalamus-hypophysial axis, but also the peripheral nervous system, via the autonomic ganglia, are directly involved in the endocrine control of the testis.
Assuntos
Gânglios Autônomos/metabolismo , Norepinefrina/metabolismo , Testículo/inervação , Testículo/metabolismo , Testosterona/metabolismo , Antagonistas Adrenérgicos alfa/administração & dosagem , Antagonistas Adrenérgicos beta/administração & dosagem , Animais , Gânglios Autônomos/efeitos dos fármacos , Técnicas In Vitro , Masculino , Norepinefrina/administração & dosagem , Fentolamina/administração & dosagem , Propranolol/administração & dosagem , Ratos , Ratos Wistar , Testículo/efeitos dos fármacos , Fatores de TempoRESUMO
This research explores the initial assembly of the blood-testis barrier (BTB) during puberty, when a massive physiological apoptosis in the first spermatogenic wave takes place. Fragments of testis from 14- to 20-day-old rats were studied by conventional transmission electron microscopic techniques. Lanthanum hydroxide was used as an intercellular tracer. Light microscopy was used to confirm apoptotic death when paraffin-embedded sections were studied by TUNEL analysis. When the seminiferous cords reached the zygotene-pachytene spermatocyte level, they exhibited abundant apoptotic figures, whereas the remaining segments showed sporadic apoptosis. We found a BTB not yet assembled in the cords with zygotene-pachytene spermatocytes and abundant apoptosis. The observed apoptosis frequency diminished drastically when BTB was organized, as confirmed by the use of the tracer. Our conclusion is that the massive apoptosis found in the zygotene-pachytene spermatocytes between days 14 and 20 coincides with an open BTB. The absence of BTB could be one of the factors causing massive apoptosis of zygotene-pachytene spermatocytes, at least within the time span analyzed. The zygotene-pachytene spermatocytes are left exposed in an open environment instead of being isolated in the adluminal compartment to which they are destined.
Assuntos
Apoptose , Barreira Hematotesticular/ultraestrutura , Maturidade Sexual , Espermatócitos/ultraestrutura , Espermatogênese , Animais , Barreira Hematotesticular/crescimento & desenvolvimento , Masculino , Prófase Meiótica I , Microscopia Eletrônica de Transmissão , Estágio Paquíteno , Ratos , Ratos Wistar , Espermatócitos/crescimento & desenvolvimento , Fatores de TempoRESUMO
Dendritic cells (DCs) are the professional antigen-presenting cells responsible for initiating of the immune response. Langerhans cells (LCs) are a type of DC that is a permanent resident of the oral epithelium. LCs are organized conforming a network in such a way as to maximize their surface area for efficient apprehension of antigens. To detect age-related changes in the LCs network, fragments of gingival epithelium spontaneously accompanying dental removals were processed by immunohistochemistry. Monoclonal antibody CD1a followed by biotinized immunoglobulin-streptoavidin peroxidase were used to identify the LCs with the light microscope. LC density and LC types were analyzed according to their morphology and intraepithelial distribution. In the older age group (61-74 years) the density was significantly lower than in the younger age groups. Morphologically, LCs showed fewer dendritic-branching processes and had a rounded shape in the older age group. Present observations indicate that the LC network changes markedly with aging. These results suggest that immunological defense of the oral tissue might be compromised in old age.