RESUMO
Oogenesis in the armadillo Chaetophractus villosus, a representative species of a mammalian basal clade, was investigated by light microscopy, transmission electron microscopy, and immunohistochemical localization of keratin. At the beginning of the growth phase, oocyte follicles showed one, and sometimes several, large bodies composed of lamellae (multilamellar bodies [MLBs]), which entrap other cytoplasmic organelles at more advanced stages. Lamellae diameter is described in cross-section (37 nm) and tangential sections (50 nm). The MLB of early oocytes is most frequently located close to the nucleus. In large oocytes, both, this body and the free organelles are relocated at the oocyte periphery. The MLB grows from the primary follicle up to its full development at the follicular phase characterized by tall granulosa cells. Mitochondria, smooth small vesicles, and lipofuscin granules are trapped between lamellae. MLBs engage in the formation of different sets of organelles, both trapped and free ones. When oocytes are well developed and the zona pellucida is formed, the MLB is reduced to small remnants detected only by transmission electron microscopy. The MLB disintegrates when an antrum develops. Immunohistochemical localization techniques showed the presence of cytokeratin in the MLBs. This cytokeratin pool may be involved in the filament and desmosome formation found in the periphery of late oocytes.
Assuntos
Tatus , Oócitos , Animais , Núcleo Celular , Feminino , Oogênese , Folículo OvarianoRESUMO
The heteromorphic X and Y chromosomes behave in a special way in mammalian spermatocytes; they form the XY body and synapse only partially. The aim of this article was to study the origin and the role of the special differentiations in the XY pair of the domestic cat during pachytene by analyzing its fine structural characteristics and the immunolocalization of the main meiotic proteins SYCP3, SYCP1, SYCE3, SMC3, γ-H2AX, BRCA1, H3K27me3, and MLH1. The cat XY body shows particularly striking structures: an extreme degree of axial fibrillation in late pachynema and a special location of SYCP3-containing fibrils, bridging different regions of the main X axis, as well as one bridge at the inner end of the pairing region that colocalizes with the single mandatory MLH1 focus. There are sequential changes, first bullous expansions, then subdivision into fibrils, all involving axial thickening. The chromatin of the XY body presents the usual features of meiotic sex chromosome inactivation. An analysis of the XY body of many eutherians and metatherians suggests that axial thickenings are primitive features. The sequential changes in the mass and location of SYCP3-containing fibers vary among the clades because of specific processes of axial assembly/disassembly occurring in different species.
Assuntos
Gatos/genética , Proteínas Nucleares/metabolismo , Estágio Paquíteno/genética , Complexo Sinaptonêmico/metabolismo , Cromossomo X/metabolismo , Cromossomo X/ultraestrutura , Cromossomo Y/metabolismo , Cromossomo Y/ultraestrutura , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Cromatina/metabolismo , Cromatina/ultraestrutura , Histonas/genética , Histonas/metabolismo , Masculino , Microscopia de Fluorescência , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Espermatócitos/metabolismo , Complexo Sinaptonêmico/genéticaRESUMO
Androgen insensitivity syndrome (AIS) is a hereditary condition in patients with a 46,XY karyotype in which loss-of-function mutations of the androgen receptor (AR) gene are responsible for defects in virilization. The aim of this study was to investigate the consequences of the lack of AR activity on germ cell survival and the degree of testicular development reached by these patients by analyzing gonadal tissue from patients with AIS prior to Sertoli cell maturation at puberty. Twenty-three gonads from 13 patients with AIS were assessed and compared to 18 testes from 17 subjects without endocrine disorders. The study of the gonadal structure using conventional microscopy and the ultrastructural characteristics of remnant germ cells using electron microscopy, combined with the immunohistochemical analysis of specific germ cell markers (MAGE-A4 for premeiotic germ cells and of OCT3/4 for gonocytes), enabled us to carry out a thorough investigation of germ cell life in an androgen-insensitive microenvironment throughout prepuberty until young adulthood. Here, we show that germ cell degeneration starts very early, with a marked decrease in number after only 2 years of life, and we demonstrate the permanence of gonocytes in AIS testis samples until puberty, describing 2 different populations. Additionally, our results provide further evidence for the importance of AR signaling in peritubular myoid cells during prepuberty to maintain Sertoli and spermatogonial cell health and survival.
Assuntos
Síndrome de Resistência a Andrógenos/patologia , Puberdade/metabolismo , Puberdade/fisiologia , Síndrome de Resistência a Andrógenos/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Criança , Pré-Escolar , Células Germinativas/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Espermatogônias/metabolismo , Espermatogônias/patologia , Testículo/metabolismo , Testículo/patologiaRESUMO
The XX/XY system is the rule among mammals. However, many exceptions from this general pattern have been discovered since the last decades. One of these non-conventional sex chromosome mechanisms is the multiple sex chromosome system, which is evolutionary fixed among many bat species of the family Phyllostomidae, and has arisen by a translocation between one original gonosome (X or Y chromosome), and an autosome, giving rise to a "neo-XY body." The aim of this work is to study the synaptic behavior and the chromatin remodeling of multiple sex chromosomes in different species of phyllostomid bats using electron microscopy and molecular markers. Testicular tissues from adult males of the species Artibeus lituratus, Artibeus planirostris, Uroderma bilobatum, and Vampyrodes caraccioli from the eastern Amazonia were analyzed by optical/electron microscopy and immunofluorescence of meiotic proteins involved in synapsis (SYCP3 and SYCE3), sister-chromatid cohesion (SMC3), and chromatin silencing (BRCA1, γ-H2AX, and RNApol 2). The presence of asynaptic axes-labeled by BRCA1 and γ-H2AX-at meiotic prophase in testes that have a normal development of spermatogenesis, suggests that the basic mechanism that arrests spreading of transcriptional silencing (meiotic sex chromosome inactivation (MSCI)) to the autosomal segments may be per se the formation of a functional synaptonemal complex between homologous or non-homologous regions, and thus, this SC barrier might be probably related to the preservation of fertility in these systems.
Assuntos
Quirópteros/genética , Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Processos de Determinação Sexual/genética , Cromossomo X/genética , Cromossomo Y/genética , Animais , Pareamento Cromossômico/genética , Masculino , Estágio Paquíteno/fisiologia , Espermatócitos/metabolismo , Espermatogênese/fisiologiaRESUMO
Structural and immunohistochemical methods have been extremely useful for the characterization of the XY body (the structure formed by the XY pair during meiotic prophase) in Man and in other mammals. These methods are widely used at the present time for the detection of abnormalities leading to human infertility. The basic ultrastructural methods are spreading of pachytene spermatocytes, thin-sectioning techniques with or without 3-D reconstructions, and the monitoring of all specimens with semi-thin sections. Immunofluorescent techniques also use spreading of meiotic cells for the analysis of the XY body, and they can be combined with the fluorescence in situ hybridization (FISH) technique, in the so-called immuno-FISH. Epitope retrieval techniques are also used.
Assuntos
Biomarcadores/metabolismo , Cromatina/ultraestrutura , Imunofluorescência/métodos , Biópsia , Humanos , Hibridização in Situ Fluorescente , Masculino , Espermatócitos/ultraestrutura , Coloração e Rotulagem , Testículo/patologiaRESUMO
The XY body from spermatocytes of the rodent Galea musteloides shows progressive changes of the synaptonemal complex (SC) axes and the X-chromatin during pachynema. There is a gross thickening of the X-axis and the formation of a large X chromosome loop at mid and late pachytene stages. The SC proteins synaptonemal complex protein 3 (SYCP3), synaptonemal complex protein 1, and synaptonemal complex central element protein 3 and the proteins breast cancer 1, MutL homolog 1 (MLH1), and radiation-repair 51 (related to meiotic processes), the cohesin structural maintenance of chromosome 3, the centromeric protein (with CREST antibody), and the silenced chromatin (with phosphorylated (139ph) H2A histone family, member X (γ-H2AX) antibody) were analyzed in this XY body. The thick X-axis, including the interstitial loop, becomes formed by four to six laminae showing a cross-striation with a periodicity of about 20 nm. The whole length of the gross X-axis shows no significant changes during pachynema, but the interstitial chromatin of the X chromosome and the X centromere are included in the large loop, and it becomes separated from the SC. A conventional SC formed by the Y-axis, a central region and a thin lateral element originally corresponding to the X-axis, remains undisturbed up to the end of pachynema. A single MLH1 focus develops either at the distal or the proximal region of the loop end attached to the conventional SC. The chromatin surrounding the thickened axis is labeled with γ-H2AX. It is shown that most of the SYCP3 protein associated with the X chromosome loop is not involved in the SC maintenance, but it is located with the cohesin axis separated from the SC proper.
Assuntos
Roedores/genética , Complexo Sinaptonêmico/ultraestrutura , Cromossomo X/ultraestrutura , Animais , Centrômero/genética , Centrômero/ultraestrutura , Cromatina/genética , Cromatina/ultraestrutura , Evolução Molecular , Inativação Gênica , Cobaias , Masculino , Membrana Nuclear , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estágio Paquíteno , Análise de Sequência de DNA , Complexo Sinaptonêmico/genética , Cromossomo X/genéticaRESUMO
Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.
Assuntos
Tatus/fisiologia , Epitélio Seminífero/citologia , Espermatogênese , Animais , Argentina , Forma do Núcleo Celular , Montagem e Desmontagem da Cromatina , Masculino , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Tamanho do Órgão , Estações do Ano , Epitélio Seminífero/crescimento & desenvolvimento , Epitélio Seminífero/metabolismo , Epitélio Seminífero/ultraestrutura , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Células de Sertoli/ultraestrutura , Espermátides/citologia , Espermátides/crescimento & desenvolvimento , Espermátides/metabolismo , Espermátides/ultraestrutura , Espermatócitos/citologia , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo , Espermatócitos/ultraestrutura , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/metabolismo , Espermatogônias/ultraestrutura , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Testículo/ultraestrutura , Testosterona/sangue , Testosterona/metabolismoRESUMO
Three xenarthrans species Chaetophractus villosus, Chaetophractus vellerosus, and Zaedyus pichiy have been used for the analysis of the structure, behavior, and immunochemical features of the XY body during pachytene. In all these species, the sex chromosomes form an XY body easily identifiable in thin sections by the special and regular packing of the chromatin fibers of the internal region of the XY body ("differential" regions) and those of the peripheral region (synaptic region). Spermatocyte spreads show a complete synapsis between the X- and the Y-axis, which lasts up to the end of pachytene. From the early pachytene substages to the late ones, the X-axis develops prominent branches, which in late pachytene span the synaptic region. Synapsis is regular as shown by SYCP1 labeling. Axial development is followed by SYCP3 labeling and in the asynaptic region of the X-axis by BRCA1. Gamma-H2AX labels exclusively the differential (asynaptic) region of the X chromosome. A single focus is labeled by MLH1 in the synaptic region. The location of this MLH1 focus spans from 0.3 to 1.6 µm from the telomere in the analyzed xenarthrans, covering approximately half of the Y-axis length. It is concluded that xenarthrans, as basal placental mammals, harbor the largest pseudoautosomal regions of presently analyzed mammals, and shows the typical features of meiotic sex chromosome inactivation (MSCI).
Assuntos
Tatus/genética , Montagem e Desmontagem da Cromatina , Pareamento Cromossômico , Recombinação Genética , Animais , Cromatina/ultraestrutura , Masculino , Cromossomos Sexuais , Espermatócitos/metabolismo , Cromossomo X/ultraestrutura , Cromossomo Y/ultraestruturaRESUMO
Giardia lamblia is a medically important protozoan parasite with a basal position in the eukaryotic lineage and is an interesting model to explain the evolution of biochemical events in eukaryotic cells. G. lamblia trophozoites undergo significant changes in order to survive outside the intestine of their host by differentiating into infective cysts. In the present study, we characterize the previously identified Orf-C4 (G. lamblia open reading frame C4) gene, which is considered to be specific to G. lamblia. It encodes a 22 kDa protein that assembles into high-molecular-mass complexes during the entire life cycle of the parasite. ORF-C4 localizes to the cytoplasm of trophozoites and cysts, and forms large spherical aggregates when overexpressed. ORF-C4 overexpression and down-regulation do not affect trophozoite viability; however, differentiation into cysts is slightly delayed when the expression of ORF-C4 is down-regulated. In addition, ORF-C4 protein expression is modified under specific stress-inducing conditions. Neither orthologous proteins nor conserved domains are found in databases by conventional sequence analysis of the predicted protein. However, ORF-C4 contains a region which is similar structurally to the alpha-crystallin domain of sHsps (small heat-shock proteins). In the present study, we show the potential role of ORF-C4 as a small chaperone which is involved in the response to stress (including encystation) in G. lamblia.
Assuntos
Giardia lamblia/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Regulação da Expressão Gênica , Giardia lamblia/genética , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/metabolismo , Dados de Sequência Molecular , Análise de Sequência de DNA , Estresse Fisiológico , alfa-Cristalinas/genética , alfa-Cristalinas/metabolismoRESUMO
The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.