RESUMO
Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Fenômenos Biofísicos , Lectinas de Plantas/química , Triticum/química , Aglutininas do Germe de Trigo/química , Peptídeos Catiônicos Antimicrobianos/genética , Células Germinativas/química , Lectinas de Plantas/genética , Triticum/genética , Aglutininas do Germe de Trigo/genéticaRESUMO
BACKGROUND: Endometriosis is a gynaecological disorder that affects 6-10 % of female population. It is characterized by the presence of endometrial tissue outside the uterus, most often in the pelvic peritoneum or ovaries. Recent studies have indicated that mesenchymal endometrial stem cells might get involved in endometriosis progression. Although germ line stem cells have been proved to exist in the ovary, their involvement in ovarian endometriosis has not been investigated. In this preliminary report we aimed to identify germinal stem cell markers in ovarian endometriosis. FINDINGS: Ten paraffin-embedded ovarian endometriosis samples were screened for germ cell-specific proteins DDX4 (VASA) and IFITM3, and its relation with stem cell marker OCT4, proliferation marker PCNA and estrogen receptor alpha (ESR1), by immunohistochemistry, immunofluorescence and PCR. DDX4 and IFITM3 proteins were expressed in isolated cells and clusters of cells in the cortical region of ovarian endometriotic cysts. DDX4 and IFITM3 co-localized in cells from endometriotic stroma, and DDX4/IFITM3-expressing cells were positive for ESR1, OCT4 and PCNA. No cells expressing neither DDX4 nor IFITM3 were detected in normal endometrial tissue. CONCLUSION: The identification of germ cell-specific proteins DDX4 and IFITM3 provides the first evidence of ovarian-sourced cells in ovarian endometriotic lesions and opens up new directions towards understanding the still confusing pathogenesis of endometriosis.
Assuntos
RNA Helicases DEAD-box/metabolismo , Endometriose/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adolescente , Adulto , Biomarcadores Tumorais/metabolismo , Feminino , Células Germinativas/química , Humanos , Pessoa de Meia-Idade , Células-Tronco/química , Adulto JovemRESUMO
Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre-spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21-25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti-human OCT-4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT-4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre-spermatogonia would be observed at later stages of canine foetus development. We also showed that anti-human OCT-4 antibody can be useful to identify canine PGC in vivo.
Assuntos
Cães/embriologia , Desenvolvimento Embrionário , Células Germinativas , Animais , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Feminino , Células Germinativas/química , Células Germinativas/ultraestrutura , Idade Gestacional , Imuno-Histoquímica , Masculino , Fator 2 de Transcrição de Octâmero/análise , Fator 3 de Transcrição de Octâmero/análise , Testículo/citologia , Testículo/embriologiaRESUMO
A conservação da variabilidade genética de animais silvestres pode ser garantida pela proteção dos animais em seus habitats naturais (in situ), pela manutenção destes em cativeiro, em criatórios conservacionistas ou de pesquisa (ex situ vivos), bem como pela formação de bancos de germoplasma animal, nos quais gametas, embriões e células somáticas podem ser utilizados em programas de biotecnologia da reprodução. A conservação in situ consiste em uma estratégia de conservação elementar. Entretanto, é necessário considerar que a perda de material genético ocorre diariamente de forma acelerada, fazendo-se necessário lançar mão de técnicas para recuperar e preservar o potencial genético de animais mesmo após a sua morte. Desta forma, as biotecnologias de reprodução consistem em auxílio para projetos de conservação in situ.
Conservation of genetic material can be guaranteed by animal and natural habitat protection, i.e. in situ, by animal captivity in conservationists or research centers, i.e. ex situ in life, or by germplasm bank formation. Such a genetic bank can preserve gametes, embryos or somatic cells, which can be applied in reproductive programs. Conservation in situ consists in an elementary strategy to preserve biodiversity. However, we must bear in mind that genetic material losses occurs rapid and daily, which demands the urgent application of techniques to recover and preserve genetic material even after animal death. Consequently, reproductive biotechnologies will help to conserve genetic material from endangered non-domestic mammals in situ.
Assuntos
Animais , Biotecnologia , Callithrix/embriologia , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/química , Espécies em Perigo de Extinção , Embrião de Mamíferos/químicaRESUMO
A conservação da variabilidade genética de animais silvestres pode ser garantida pela proteção dos animais em seus habitats naturais (in situ), pela manutenção destes em cativeiro, em criatórios conservacionistas ou de pesquisa (ex situ vivos), bem como pela formação de bancos de germoplasma animal, nos quais gametas, embriões e células somáticas podem ser utilizados em programas de biotecnologia da reprodução. A conservação in situ consiste em uma estratégia de conservação elementar. Entretanto, é necessário considerar que a perda de material genético ocorre diariamente de forma acelerada, fazendo-se necessário lançar mão de técnicas para recuperar e preservar o potencial genético de animais mesmo após a sua morte. Desta forma, as biotecnologias de reprodução consistem em auxílio para projetos de conservação in situ.(AU)
Conservation of genetic material can be guaranteed by animal and natural habitat protection, i.e. in situ, by animal captivity in conservationists or research centers, i.e. ex situ in life, or by germplasm bank formation. Such a genetic bank can preserve gametes, embryos or somatic cells, which can be applied in reproductive programs. Conservation in situ consists in an elementary strategy to preserve biodiversity. However, we must bear in mind that genetic material losses occurs rapid and daily, which demands the urgent application of techniques to recover and preserve genetic material even after animal death. Consequently, reproductive biotechnologies will help to conserve genetic material from endangered non-domestic mammals in situ.(AU)
Assuntos
Animais , Células Germinativas/química , Células Germinativas/crescimento & desenvolvimento , Callithrix/embriologia , Espécies em Perigo de Extinção , Biotecnologia , Embrião de Mamíferos/químicaAssuntos
Humanos , Masculino , Animais , Feminino , Andrologia/educação , Andrologia/métodos , Medicina Reprodutiva/educação , Medicina Reprodutiva/métodos , Células Germinativas/citologia , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/fisiologia , Células Germinativas/química , Síndrome do Ovário Policístico/prevenção & controle , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/terapiaRESUMO
BACKGROUND: Germ cell number during ovarian organogenesis is regulated through programmed cell death. We investigated the expression of germ-cell-specific VASA protein, apoptosis-related proteins BAX and BCL-2 and DNA fragmentation in developing human ovaries from gestation week 12 to term. METHODS: Human fetal ovaries from 13 women undergoing spontaneous abortion were fixed, paraffin-embedded and processed for immunohistochemistry to analyse temporal and cellular localization of VASA, BCL-2 and BAX, and to detect apoptosis by TUNEL assay. RESULTS: VASA showed a differential pattern of expression throughout the differentiation and proliferative phase and prophase I to finally associate with Balbiani's body in primordial and primary follicles. BCL-2 was detected from week 12 to 17 and became undetectable thereafter. Strong BAX signal was detected in oogonia and oocytes from week 12 to term. Low levels (Assuntos
Apoptose/fisiologia
, RNA Helicases DEAD-box/metabolismo
, Células Germinativas/química
, Ovário/embriologia
, Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
, Proteína X Associada a bcl-2/metabolismo
, Aborto Espontâneo
, Fragmentação do DNA
, Feminino
, Humanos
, Imuno-Histoquímica
, Marcação In Situ das Extremidades Cortadas
, Ovário/citologia
, Gravidez
RESUMO
We studied the expression and function of the IL (interleukin)-3 and IL-5 family of receptors in male germ cells. RT (reverse transcription)-PCR showed expression of mRNAs encoding the alpha and beta subunits of the IL-3 and IL-5 receptors in human testis, and the presence of IL-3 and IL-5 receptors alpha and beta proteins was confirmed by immunoblotting with anti-alpha and anti-beta antibodies. The immunolocalization studies showed expression of these receptors in the germ line in the human testis and in human and bovine ejaculated spermatozoa. Functional studies with bull spermatozoa indicated that IL-3 signalled for increased uptake of hexoses in these cells at picomolar concentrations compatible with expression of functional high-affinity IL-3 receptors in these cells. In contrast, IL-5 failed to induce increased hexose uptake in bull spermatozoa. Experiments using HL-60 eosinophils that express functional IL-3 and IL-5 receptors confirmed that IL-3, but not IL-5, signalled for increased hexose uptake. Our findings suggest that differential signalling for increased hexose uptake by heteromeric high-affinity IL-3 and IL-5 receptors in mammalian spermatozoa is a property that depends on the identity of the alpha-subunit forming part of the alphabeta-complex and is not a property specific to the germ cells.
Assuntos
Hexoses/metabolismo , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Transdução de Sinais/genética , Espermatozoides/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/química , Células Germinativas/metabolismo , Células HL-60/química , Células HL-60/metabolismo , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-3/genética , Interleucina-3/farmacologia , Interleucina-3/fisiologia , Interleucina-5/genética , Interleucina-5/farmacologia , Interleucina-5/fisiologia , Masculino , Subunidades Proteicas/genética , RNA Mensageiro/genética , Receptores de Interleucina/genética , Receptores de Interleucina-3/genética , Receptores de Interleucina-5 , Sêmen/citologia , Espermatozoides/química , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Testículo/química , Testículo/metabolismoRESUMO
In the present report we followed the distribution of hyaluronan during the phases of separation, migration, and colonization of the primordial germ cell migratory process. Hyaluronan was detected by the use of two cytochemical methods: (1) ruthenium hexammine trichloride (RHT) associated with enzymatic treatment with hyaluronate lyase and (2) a binding specific probe for hyaluronan. After RHT treatment the proteoglycans and/or glycosaminoglycans were observed as a meshwork formed by electron-dense granules connected by thin filaments. After enzymatic digestion, no filaments could be detected in the migratory pathway. Quantitative analysis showed a close correlation between cell migration and the concentration of RHT-positive filaments. It was also shown that high amounts of hyaluronan were expressed in the separation phase and migration phases whereas during the colonization phase the amount of hyaluronan was clearly diminished. This study showed that the presence of primordial germ cells in each compartment of the migratory pathway was always accompanied by a high expression of hyaluronan. These results indicate that hyaluronan is an important molecule in the migratory process, providing the primordial germ cells with a hydrated environment that facilitates their movement toward the genital ridges.
Assuntos
Movimento Celular/fisiologia , Células Germinativas/química , Ácido Hialurônico/análise , Animais , Embrião de Mamíferos/química , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Feminino , Células Germinativas/citologia , Células Germinativas/ultraestrutura , Histocitoquímica/métodos , Ácido Hialurônico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Microscopia Eletrônica , Polissacarídeo-Liases/metabolismo , Compostos de Rutênio , Fatores de TempoRESUMO
In this paper we present an in situ ultrastructural cytochemical study on the distribution and spatial-temporal expression of proteoglycans (PGs) in the extracellular matrix of the migratory pathway of mouse primordial germ cells (PGCs) during the different phases of migration, by the use of the cationic dye ruthenium hexammine trichloride (RHT). Embryos of 9, 10, 11 and 12 days of development were used. The treatment with RHT revealed PGs as electron dense layers, granules, and filaments. Whereas granules prevailed in the extracellular spaces of the migratory route during the whole migratory process, the amount of filamentous structures increased during the migration phase of PGCs. At the end of the migratory process the surface of the PGCs lost its reaction by RHT. There were differences in the size of the granules of PGs at the initial migratory period (9-day-old embryos) as compared with the other days of gestation. There was a strong reaction for PGs in the extracellular spaces, expressed as a meshwork of granules interconnected by filaments, as well as reaction on the basement membranes during the peak of the PGCs migration in 10-day-old embryos. These results support the hypothesis that these molecules may have an important role in the migration of PGCs, although the precise mechanism involved in this process is not yet clear.