Inductive role of sustentacular cells (Sertoli cells) conditioned medium on bone marrow derived mesenchymal stem cells / Papel inductivo del medio acondicionado de células sustentaculares (células de Sertoli) en las células madre mesenquimales derivadas de la médula ósea

RESUMEN: Las células madre de la línea germinal masculina son factores clave para la espermatogénesis masculina y la fertilidad. Las células sustentaculares (células de Sertoli) como células somáticas juegan un papel fundamental en la creación de un microambiente esencial para la auto-renovación y diferenciación de las células de la línea germinal masculina. Las células madre mesenquimales son reconocidas como células auto-renovables y multipotentes capaces de diferenciarse en múltiples tipos de células. La generación de células germinales masculinas a partir de células madre mesenquimales puede proporcionar un método terapéutico para tratar la infertilidad masculina. En este estudio, las células mesenquimales derivadas de la médula ósea (BMMSCs) se recuperaron de la médula ósea de ratones de 6-8 semanas de edad del Instituto de Investigación Médico Naval (NMRI). En el estudio se aislaron las células sustentaculares y se enrriquecieron usando placas revestidas con lectina. Se obtuvo el medio de condición celular después de diferentes intervalos de tiempo. Posteriormente se cultivaron las BMMSC con diferentes concentraciones de SCCM y medio de Eagle modificado por Dulbecco (DMEM) en diversos momentos. Se evaluaron marcadores específicos de células de línea germinal usando la reacción en cadena de polimerasa transcriptasa inversa (RT-PCR) e inmunocitoquímica. Los resultados mostraron que las BMMSCs cultivadas con SCCM durante 48h exhibieron transcritos específicos de línea germinal (Mvh, Iid4, piwil2) (p <0,05) y marcadores (Mvh, Scp3). Nuestros resultados indican que el cultivo de BMMSCs con SCCM puede conducir a la diferenciación efectiva de BMMSCs en células germinales y proporcionar una estrategia de tratamiento para la infertilidad masculina.

SUMMARY: Male germ line stem cells are key factors for male spermatogenesis and fertility. Sustentacular cells (Sertoli cells) as somatic cells play a pivotal role in creating essential microenvironment for the self-renewal and differentiation of the male germ line cells. Mesenchymal stem cells are recognized as self-renewing and multipotent cells able to differentiate into multiple cell types. The generation of male germ cells from mesenchymal stem cells may provide a therapeutic method to treat male infertility. In this study, Bone marrow derived mesenchymal cells (BMMSCs) were retrieved from the bone marrow of 6-8-week old Naval Medical Research Institute (NMRI) mice. Sustentacular cells (Sertoli cells) were isolated and made rich using lectin coated plates. Sustentacular cell condition medium (SCCM) was collected after different time intervals. Then the BMMSCs were cultured with different concentration of SCCM and Dulbecco's Modified Eagle's medium (DMEM) at various times. Specific markers of Germ line cells were evaluated by using Reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. The results showed that BMMSCs cultured with SCCM for 48h exhibited germ line specific transcripts (Mvh, Iid4, piwil2) (p< 0.05) and markers (Mvh, Scp3). Our findings represent that culturing BMMSCs with SCCM may lead to effective differentiation of BMMSCs into germline cells and provide a treatment strategy for male infertility.

The actin filament network associated to Sertoli cell ectoplasmic specializations

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.

An ultrastructural study on cytotoxic effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro

In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of metabolites of di(2-ethylhexyl) phthalate, on immature Shiba goat testes in vitro were examined. The testes of 2-month-old Shiba goats were cut into smaller pieces, and seeded in medium. At 1, 3, 6 and 9 hr after administration of MEHP at various concentrations (0, 100 nmol ml-1, 1 nmol ml-1, and 1 x 10-3 nmol ml-1, respectively), the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, the vacuolization and nuclear membrane rupture appeared in Sertoli cells. Such alterations tended to gradually increase in number in timeand dose-dependent manners. Moreover, by MEHP treatment, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still functioning cell organelles, and packed cell contents in membrane-bounded bodies), apoptotic Sertoli cells (characterized with nuclear membrane lysis, nuclear condensation), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), and necrotic Sertoli cells (characterized with marginated chromatins along the nuclear membrane, ruptured vesicles within the MNB, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, ultrastructurally the treatment with MEHP at low concentration tends to lead spermatogenic and Sertoli cells to apoptosis, whereas that at high concentration tends to lead spermatogenic and Sertoli cells to necrosis. Thus, the testicular tissue culture is advantageous for screening testicular toxicity of chemicals.

Sperm ultrastructure and spermatogenesis in the lizard, Tropidurus itambere

Spermatogenesis, with emphasis on spermiogenesis, is described for the lizard, Tropidurus itambere, using light microscopy, phase contrast and epifluorescence, as well as scanning and transmission electron microscopy. Cellular differentiation involves events of chromatin condensation, nuclear elongation and the formation of structural complexes, such as the acrosomal and axonemal ones. Other new characteristics, exclusive for this species, include various aspects of the subacrosomal granule, the insertion of the pro-acrosomal vesicle and the development of these structures to participate in the acrosomal complex. Radial projections occurjust above the nuclear shoulders, which have been recognized already from the beginning of cellular elongation. The development of the midpiece, the dense bodies, formation of the flagellum and elimination of residual cytoplasm result in the final characterization of the mature spermatozoon. Comparisons between Tropiduridae and other lizard families are made.

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods us for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll(). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll( gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ñ 8.2 per cent, mean ñ SEM) and spermatids (84 ñ 2.6 per cent) using centrifugal elutriation followed by continuous Percoll( gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll( gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis.

Características ultra-estruturais das células de Sertoli do morcego hematófago (Desmodus rotundus rotundus, Geoffrey, 1810) / Ultrastructural characteristics of Sertoli cells of the vampire bat (Desmodus rotundus rotundus, Geoffrey, 1810)

Este trabalho apresenta estudo ultra-estrutural sobre as células de Sertoli (CS) e suas interrelaçöes com outras células em diferentes estágios de diferenciaçäo no epitélio seminífero do Desmodus rotundus ("Desmodus"), um microquiróptero hematófago da fauna brasileira. No epitélio seminífero do "Desmodus" ocorrem diferentes tipos de especializaçöes juncionias entre as CS adjacentes e entre as CS e diferentes germinativas testiculares. Destacam-se as junçöes oclusivas e do tipo "desmosômic" bem como as junçöes ectocitoplasmáticas, envolvendo inclusive o retículo endoplasmático (RE) liso. Estas interaçöes celulares e seu papel citofisiológico säo aqui discutidos. Também säo relatadas algumas caradterísticas ultra-estruturais sobre o núcleo, núcléolo, orgânulos citoplasmáticos e inclusöes lipídicas nas CS do "Desmodus"

A célula de Sertoli de Oreochromis niloticus (Peixe, Teleósteo) / The Sertoli cell of Oreochromis niloticus (Fish, Teleost)

Nos túbulos seminíferos de tilápia nilótica säo encontrados cistos, os quais contêm as células espermatogênicas. Em cada cisto, as células estäo na mesma fase da espermatogênese. As paredes dos cistos säo formadas por células de Sertoli. Ao microscópio óptico, o citoplasma destas células é delgado e de difícil visualizaçäo. Seu núcleo é fusiforme ou triangular. Ao microsópio eletrônicos, o citoplasma pe abundante ao redor do núcleo e em áreas onde ocorre digestäo de material fagocitado. O segmento de sua membrana citplasmática voltada para a lâmina basal exibe vesículas depinocitose. As áreas de contato entre as células de Sertoli nos cistos de espermatogônias e de espermatócitos primários com complexos sinaptonêmicos, näo apresentam especializaçöes de membrana. Junçöes de oclusäo e desmosomas ocorrem em cistos cujas células estäo em fase mais avançada da espermatogênese. Tais complexos juncionais pdem estar correlacionados com funçöes de compartimentaçäo do testículo

Barreira hemotesticular em Oreochromis niloticus (Peixe, Teleósteo) / Blood-testis barrier in Oreochromis niloticus (Fish, Teleost)

As células de Sertoli da tilápia nilótica constituem a parede de cistos nos quais as células espermatogênicas se desenvolvem de modo sincrônico. Cistos em diferentes fases da espermatogênese säo encontrados ao longo da parede dos túbulos seminíferos. O lantânio, empregado como traçador de espaços intercelulares, aparece no interior de cistos de espermatogônias e de espermatocitos em fase inicial da prífse meiótica, sendo excluído de cistos de espermátides e do lume dos túbulos seminíferos. A penetraçäo do traçador é interrompida ao nível de complexos juncionais do tipo junçöes de oclusäo. Estes complexos juncionais constituem a barreira cuja presença parece estar relacionada com o estádio de desenvolvimento espermatogênico do cisto

The blood-testis barrier in lizards during the annual spermatogenic cycle

La barrera hemato-testicular, primero descripta en los mamíferos, ha sido recientemente demostrada en la mayoría de los vertebrados y en muchos invertebrados. Nosotros hemos utilizado trazadores electrónicamente densos (hidróxido de lantano) y técnicas de criofractura para estudiar las características morfológicas y de permeabilidad de la barrera hemato-testicular en lagartos que muestran ciclos espermatogénicos anuales (Liolaemus bibroni, ruibali y elongatus y Phymaturus palluma). Durante la actividad de espermatogénesis completa la barera hemato-testicular aisla a todas las células germinales, desde la formación de los complejos sinaptonémicos (espermatocitos cigoténicos), en el compartimiento adluminal del epitelio seminífero. Las características especializaciones de uniones interstolianas, equivalente morfológico de la barrera de permeabilidad, desorganizan durante los períodos estacionales de involución de células germinales concomitantemente con la libre percolación de hidróxido de lantano dentro del compartimiento adluminal del epitelio seminífero. La formación y desorganización ciclica de la barrera hemato-testicular en lagartos refuerza la hipótesis de un control local de las células germinales sobre las especialziaciones de uniones intersertorianas