Efecto de la Vitamina E en Células Peritubulares de Testículos de Ratones Mus musculus Tratados con Ácido Valproico Durante el Estado Fetal y Pubertad / Effect of Vitamin E on Peritubular Cells from Testis of Mus musculus Mice Treated with Valproic Acid During the Fetal Stage and Puberty

El ácido valproico (VPA) es un fármaco antiepiléptico teratógenico que, al ser administrado durante etapas tempranas del embarazo, puede producir alteraciones en el desarrollo embriofetal, las que se manifiestan tanto a nivel del sistema nervioso como del testículo. No obstante, se ha reportado que la administración de vitamina E (VE) podría revertir dichas alteraciones. El objetivo del presente estudio fue determinar el efecto protector de la VE a nivel testicular en fetos y ratones púberes expuestos a VPA durante la fase embrionaria de su desarrollo. Se utilizó un total de 30 ratones hembra adultas gestantes (Mus musculus) cepa BALB/c, las cuales se dividieron en 6 grupos. El estudio contempló el análisis de fetos machos a los 17,5 días post-coital (dpc) y machos juveniles a las 6 semanas post-natal. A los grupos 1 y 4 se les administró 0,3 mL de solución fisiológica (grupos control para 17,5 dpc y 6 semanas postnatal, respectivamente). A los grupos 2 y 5 se les suministró la cantidad de 600 mg/kg de VPA (grupos VPA), en tanto que a los grupos 3 y 6 se les aplicó la misma dosis de VPA complementada con 200 UI de VE (grupos VPA+VE). Se describió la histología normal y patológica del compartimento peritubular del testículo. En los grupos VPA se evidenció una degeneración de la pared peritubular, y atrofia de túbulos seminíferos, así como exfoliación de las células germinales. Por el contrario, en los grupos VPA+VE tales signos no fueron observados y la morfología presentó aspecto normal solo con algunas alteraciones focales. Estos resultados corroboran el hecho que la administración de VE contrarresta en parte, los efectos deletéreos que ocasiona el VPA.

SUMMARY: Valproic acid (VPA) is a teratogenic antiepileptic drug that, when administered during the early stages of pregnancy, can produce alterations in embryo-fetal development, which manifest both at the level of the nervous system and the testicle. However, it has been reported that the administration of vitamin E (VE) could reverse these alterations. The study aimed to determine the protective effect of VE at the testicular level in fetuses and pubertal mice exposed to VPA during the embryonic phase of their development. 30 pregnant adult female mice (Mus musculus) BALB/c strain were used, which were divided into 6 groups. The study included the analysis of male fetuses at 17.5 days post-coital (dpc) and juvenile males at 6 weeks post-natal. Groups 1 and 4 were administered 0.3 mL of physiological solution. Groups 2 and 5 were given 600 mg/kg of VPA (VPA groups), while groups 3 and 6 were given the same dose of VPA supplemented with 200 IU of VE (VPA+VE). The normal and pathological histology of the peritubular compartment of the testis was described. In the VPA groups, degeneration of the peritubular wall, and atrophy of the seminiferous tubules, as well as exfoliation of the germ cells, were evident. On the contrary, in the VPA+VE groups such signs were not observed and the morphology presented a normal appearance with only some focal alterations. These results corroborate the fact that the administration of VE partially counteracts the deleterious effects caused by VPA.

In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions

BACKGROUND: Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. RESULTS: Testicular tissue samples from azoospermic patients were obtained and then these were freeze-thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P < 0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF, SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF, Tekt1 and TP1, the results indicated that the genes were positive in the test group, while not in the control group. CONCLUSION: These results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-dimensional testicular tissue culture.

La reserpina aumenta la expresión de BDNF y PCNA, y disminuye la de caspasa-3, en células intersticiales (células de Leydig) de ratas / Reserpine increases BDNF and PCNA expression, but decreases caspase-3, in rat interstitial cells (Leydig cells)

La reserpina es un antipsicótico e hipotensor arterial que reduce significativamente los niveles de monoaminas centrales, y también es utilizada para modelar los cuadros depresivos humanos en animales de laboratorio. Este trabajo estudió, en ratas Wistar machos adolescentes, cómo la reserpina afecta indicadores moleculares de la función testicular, la cual se ha visto alterada en humanos deprimidos. Una semana luego de finalizado el tratamiento con reserpina (4 dosis de 0,0 o 1,0 mg/Kg, cada 2 días) la respuesta ansiosa y depresiva fue evaluada en un laberinto en cruz elevado. Posteriormente, se sacrificaron los animales y disecaron los testículos, los cuales fueron fijados e incluidos en bloques de parafina de donde se obtuvieron cortes histológicos de 6 µm de espesor. Estos se utilizaron para medir el diámetro de los túbulos seminíferos y para medir por inmunohistoquímica el porcentaje de células intersticiales (células de Leydig) positivas a (1) Factor neurotrófico derivado del cerebro, (2) antígeno nuclear de células en proliferación (BDNF y PCNA, respectivamente, por sus siglas en inglés), y a (3) caspasa-3. Se obtuvo también un índice de positividad al receptor de andrógenos en las células intersticiales. La expresión del receptor de andrógeno fue evaluada utilizando una escala semicuantitativa de escores (0, 1, 2 y 3) y el resto de las moléculas por presencia o ausencia de expresión de cada antígeno investigado en 300 células por preparado. Los resultados comportamentales indicaron alteraciones en la respuesta de ansiedad y una significativa depresión motora (e.g., mayor latencia en conductas de escape del sector blanco) en los animales tratados con reserpina. No se observaron diferencias en los diámetros de los túbulos seminíferos ni en la expresión del receptor de andrógeno, mientras que sí se encontró mayor proporción de células intersticiales positivas a BDNF y PCNA, y menor proporción de células positivas a caspasa-3, en los animales tratados. Los resultados corroboran la capacidad de la reserpina para reproducir rasgos comportamentales de la depresión. La administración de la droga, sin embargo, no parece reproducir a nivel testicular los efectos deletéreos encontrados en humanos deprimidos, e incluso los resultados sugieren que la reserpina puede mejorar algunos aspectos de la funcionalidad testicular relacionadas con la actividad de las células intersticiales en ratas.

Reserpine, a drug that depletes central monoamines, has been used as an antipsychotic and arterial hypotensive, and to model depression in animals. The present study analyzed, in adolescent male rats, the effects of chronic reserpine treatment on molecular indexes of testicular function. A week after termination of the treatment (4 doses of 0,0 or 1,0 mg/Kg/every 48 h) the animals were tested for anxiety response and depression patterns in an elevated plus maze. They were then euthanized, their testes dissected, fixed and embedded in paraffin to obtain blocks. Histological sections (6 µm) were obtained and used to measure the diameter of seminiferous tubules and the expression in Leydig cells of Brain-derived neurotrophic factor (BDNF), Proliferating cell nuclear antigen (PCNA), Caspase-3 and androgen receptors, by immunohistochemistry. Behavioral results indicated significant alterations in anxiety responses and a significant motor depression (e.g., greater latency to escape from the white sector). There were no differences between groups in the diameter of seminiferous tubules nor in the androgen receptors positivity. Reserpine-treated animals, however, exhibited more BDNF and PCNA positive cells, and less positive Caspase-3 cells in Leydig cells, than control animals. The results corroborate the efficacy of reserpine to reproduce some of the behavioral components of depression. The drug, however, does not seem to exert in rats the same effects on testicular function that have been found in humans diagnosed with depression. Furthermore the drug seems to enhance some aspects of testicular function related to Leydig cells function in rats.

Morphology of the interstitial tissue of active and resting testis of the Guinea fowl / Morfología del tejido intersticial del testículo activo y en reposo de la gallina de Guinea

SUMMARY: The morphology of the interstitial tissue of sexually active and resting testis of the guinea fowl were studied. Six adult health birds of active and resting phases of reproductive cycle were used for this study. The interstitial tissue consisted of loose connective tissue, interstitial cells (Leydig cells), few connective cells, blood vessels and adrenergic nerve fibres in the present study in both active and resting testes. The interstitial tissue was compact in sexually active testis whereas, the volume of the tissue was found to be increased in resting testis. The loose connective tissue of the interstitial tissue composed of mainly of collagen fibres and few reticular fibres whereas elastic fibres were absent in both groups studied. The interstitial cells appeared in clusters of a few cells and were relatively less in the active testis than the resting testis. The interstitial cells were pale staining or polygonal cells with euchromatic nuclei with few large lipid droplets in the active testis whereas the cells were flat and highly heterochromatic with numerous small lipid droplets in resting testis. Few macrophages were found only in resting testis. Interstitial cells showed negative reaction to alkaline, acid phosphatases and PAS in both groups studied but positive for lipids. The interstitial tissue was well vascularised with centrally located blood vessels in the active testis whereas the blood vessels were small and inconspicuous in the resting testis. The lymphatic vessels were not identified in both groups studied.

RESUMEN: Se estudió la morfología del tejido conectivo intersticial en testículos sexualmente activos y en reposo de la gallina de Guinea (Numida meleagris). Se utilizaron seis gallinas de Guinea machos adultos sanos, en fase activa y de reposo del ciclo reproductivo. El tejido conectivo intersticial estaba formado por tejido conectivo laxo, células endocrinas intersticiales, pocas células conectivas, vasos sanguíneos y fibras nerviosas adrenérgicas, tanto en testículos activos como en reposo. El espacio intertubular en los testículos sexualmente activos era menor en comparación a los del testículos en reposo. El tejido conectivo laxo estaba compuesto principalmente de fibras colágenas y en menor cantidad de fibras reticulares. Las fibras elásticas estaban ausentes en ambos grupos. Las células endocrinas intersticiales se organizaban en racimos de pocas células y se observaban con menor frecuencia en los testículos sexualmente activos. Las células endocrinas intersticiales de los testículos activos presentaban forma poligonal, citoplasma levemente eosinofílico con algunas gotas lipídicas de gran tamaño en su interior y nucleos redondos con cromatina laxa. Las células intersticiales de los testículos en reposo eran planas y altamente heterocromáticas, con numerosas gotas lipídicas pequeñas en su citoplasma. Las células intersticiales mostraron una reacción negativa a las fosfatasas ácidas, alcalinas y PAS en ambos grupos, Sin embargo presentaron reacción positivas para los lípidos. El tejido conectivo intersticial estaba bien vascularizado con vasos sanguíneos situados centralmente en el testículo activo y vasos sanguíneos pequeños y discretos en el testículo en reposo. Los vasos linfáticos no fueron identificados en los dos grupos estudiados.Los macrófagos fueron observados solo en los testículos en reposo, aunque en escasa cantidad.

Interacciones epitelio-mesenquimáticas en el desarrollo testicular / Epithelial-mesenchymal transitions in the development of testis

RESUMEN: La espermatogénesis es un proceso continuo que se inicia durante el desarrollo embriofetal. Las relaciones auto, para y yuxtacrinas indican la interdependencia de las células intersticiales (de Leydig) con las células peritubulares (lamina propia) y células sustentaculares (de Sertoli). Ciertos morfógenos son fundamentales en este proceso. Las células sustentaculares son capaces de regular la diferenciación y función de las células peritubulares e intersticiales a través de la producción de IGF1, TGFA, TGFB y DHH. Las células peritubulares son capaces de producir P-Mod-S, regulando la diferenciación de las células sustentaculares, y a través de FGF2 y FGF9 modulan las transiciones epitelio-mesenquimática entre células sustentaculares y mesonefros. También remodelan la membrana basal del condón testicular y regulan la diferenciación y función de las células intersticiales por medio de IGF1, TGFA y TGFB. Las células intersticiales son las reponsables de la producción de testosterona e INSL3, influyendo en la diferenciación sexual masculina. Se plantea que provienen de células mesenquimales del epitelio celómico y mesonefros. Sin embargo, otros autores proponen su origen a partir de células de la cresta neural. Estas influyen a través de mecanismos paracrinos en la proliferación de las células sutentaculares por medio de activina A, teniendo como resultado la expansión del cordón testicular. Las interacciones entre las distintas poblaciones celulares a través de morfógenos inducen una transición epitelio-mesénquima fundamental en la formación y diferenciación de la gónada masculina.

SUMMARY: Spermatogenesis is a continuous process which starts during the embryo-fetal development. Auto, para and juxtacrine relations indicate the interdependence of the interstitial cells (Leydig) with the peritubular cells (lamina propria) and sustentacular cells (Sertoli). Certain morphogens are fundamental in this process. Sustentacular cells are able to regulate differentiation and function and peritubular interstitial cells through production of IGF1, TGFA, TGFB and DHH. Peritubular cells are able to produce P-Mod-S regulating differentiation sustentacular cells and through FGF2 and FGF9 modulate epithelial-mesenchymal transitions between sustentacular cells and mesonephros. They also remodel the basal membrane of the testicular condom and regulate the differentiation and function of the interstitial cells by means of IGF1, TGFA and TGFB. Interstitial cells are responsible for the production of testosterone and INSL3, influencing male sexual differentiation. It is suggested that they come from mesenchymal cells of the coelomic epithelium and mesonephros. However, other authors propose their origin from cells of the neural crest. These influence through paracrine mechanisms proliferation sutentaculares cells by activin A, resulting in the expansion of cord testicular. The interactions between the different cell populations through morphogens induce a fundamental epithelial-mesenchymal transition in the formation and differentiation of the male gonad.

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.

Preparation of infertile male rabbits by local electron beam irradiation for intratesticular transplantation of autologous bone marrow stem cells

PURPOSE: To explore an efficient and safe protocol for the preparation of infertile male rabbits from which bone marrow stem cells (BMSCs) could be isolated and cultured. METHODS: Autologous BMSCs could be used for intratesticular transplantation and male infertility research. For this model, various doses (e.g., 6, 8, 10, or 12 Gy) of electron beam irradiation from a linear accelerator were locally applied to the scrotum of 5-month-old male New Zealand white rabbits. The effects of irradiation were compared between treatment groups, and with age-matched normal controls. Both morphology and hollow ratios of seminiferous tubules (HRST) were examined two, four, six, eight and 12-weeks post-irradiation. RESULTS: The seminiferous epithelium showed varying degrees of damage in all treatment groups compared with unirradiated controls, yet Sertoli and Leydig cells appeared unaffected. A dose-dependent response in spermatogenesis was also observed. BMSCs that were isolated and cultured from rabbits of the normal control group and the 12 Gy treatment group were compared with respect to morphology and growth. Starting at 6 weeks, HRST of the 12 Gy-treatment group were stable, and were the highest among all the groups. BMSCs from rabbits treated with 12 Gy also exhibited similar growth as the control group. CONCLUSION: Local dose of 12 Gy to the testes of 5-month-old male New Zealand rabbits is a protocol with which to obtain autologous bone marrow stem cells.

Morphological aspects of spermatogenic cells at different stagesof sexual maturation in black isogenic C57BL/6/Uni mice / Aspectos morfológicos de células de la espermatogénesis en diferentes etapas de maduración sexual del ratón negro isogénico C57BL/6/Uni

In order to study the morphological changes that occur in cells of the testes of isogenic black mouse C57BL/6/Uni into three periods during spermatogenetic used 15 mice divided into 3 groups of 5 animals with 40, 50 and 60 days of age. The mice were sacrificed and weighed. Testicles were weighed and measured, and histologically processed and stained with HE, PAS and Masson Massom-H and evaluated under light microscopy. It was observed that group I with 40 days of age in the seminiferous tubules had a lumen with sparse small amount of interstitial tubular cells. In the seminiferous epithelium type A spermatogonia, intermediate and B were identified, which occupied the compartment adbasal and intermingled with these cells in spermatocytes I in Pachytene and leptotene was observed, whereas in the adluminal compartment Golgi phase spermatids we observed the presence of acrosomal granule. In group II, the cells of the seminiferous epithelium were developed and it was observed in round spermatids cephalic hood phase plus many elongated spermatids in acrosome phase and Sertoli cells. In group III, 60 days old, it was found that seminiferous epithelium which was of the tubules had elongated spermatids in acrosome phase and maturation, with elongated nuclei and acrosomal system typical of spermiation in the presence of sperm and residual bodies near the tubular lumen. Therefore morphological evolution of germ cell testicular spermatids can be checked and recognized in its four phases: golgi, cap, acrosome and maturation over the age of the animal.

Con el fin de estudiar los cambios morfológicos que ocurren en las células de los testículos del ratón negro isogénico C57BL/6/Uni en tres períodos durante el proceso espermatogenético, se utilizaron 15 ratones divididos en 3 grupos de 5 animales con 40, 50 y 60 días de edad. Los ratones fueron sacrificados y pesados. Luego se pesaron y midieron los testículos, para ser finalmete procesados histológicamente y teñidos con HE, PAS y tricrómico Massom-H y evaluados bajo microscopía de luz. Se observó en el grupo I con 40 días de edad que los túbulos seminíferos tenían un lumen pequeño con escaza cantidad de células tubulares intersticiales. En el epitelio seminífero se identificaron espermatogonias tipo A, intermedio y B, que ocuparon el compartimiento adbasal y entremezcladas con estas células, se observó los espermatocitos I en paquiteno y leptoteno, mientras que en el compartimiento adluminal se observaron espermátidas de fase Golgi en presencia de gránulos acrosomales. En el grupo II, las células del epitelio seminífero se desarrollaron y fue posible observar espermátidas redondas en fase de capuz cefálico además de numerosas espermátidas elongadas en fase acrosómica y células sustentaculares. En el grupo III, de 60 días de edad, se encontró que el epitelio seminífero que revestía los túbulos presentaban espermátidas alargadas en fase acrosómica y de maduración, con sus núcleos alargados y el sistema acrosomal típico de la espermiación, con la presencia de espermatozoides y cuerpos residuales cerca del lumen tubular. Así, se puede comprobar con el paso de la edad del animal, la evolución morfológica de las células germinativas testiculares y reconocer las espermátidas en sus cuatro fases: golgi, capuchón, acrosomal y de maduración.

Morphological aspects of spermatogenic cells at different stages of sexual maturation in black isogenic C57BL/6/Uni Mice / Aspectos morfológicos de las células de la espermatogénesis en diferentesetapas de maduración sexual del ratón negro C57BL/6/Uni

In order to study the morphological changes that occur in cells of the testes of isogenic black mouse C57BL/6/Uni into three periods during spermatogenetic used 15 mice divided into 3 groups of 5 animals with 40, 50 and 60 days of age. The mice were sacrificed and weighed. Then weighed and measured the testicles, to be processed finalmete histologically and stained with HE, PAS and Masson Massom-H and evaluated under light microscopy. Was observed in group I with 40 days of age in the seminiferous tubules had a lumen with sparse small amount of interstitial tubular cells. In the seminiferous epithelium were identified type A spermatogonia, intermediate and B, which occupied the compartment adbasal and intermingled with these cells was observed in spermatocytes I in Pachytene and leptotene, whereas in the adluminal compartment Golgi phase spermatids observed in the presence of acrosomal granule. In group II, the cells of the seminiferous epithelium were developed and it was observed in round spermatids cephalic hood phase plus many elongated spermatids in acrosome phase and Sertoli cells. In Group III, 60 days old, it was found that the seminiferous epithelium which was of the tubules had elongated spermatids in acrosome phase and maturation, with elongated nuclei and acrosomal system typical of spermiation in the presence of sperm and residual bodies near the tubular lumen. So you can check the morphological evolution of germ cell testicular spermatids and recognize its four phases: Golgi, cap, acrosome and maturation over the age of the animal.

Con el fin de estudiar los cambios morfológicos que ocurren en las células del testículo del ratón negro isogénico C57BL/6/Uni en tres períodos diferentes del proceso espermatogenético; fueron utilizados 15 ratones divididos en 3 grupos (n=5) con 40, 50 y 60 días de edad respectivamente. Todos los animales fueron sacrificados y pesados. Posteriormente sus testículos se pesaron, midieron y procesaron histológicamente para HE, PAS y tricrómico Massom-H. Las muestras obtenidas fueron evaluados con microscopía de luz. En el grupo I con 40 días se observaron túbulos seminíferos con un lúmen pequeño y escaza cantidad de células intersticiales. En el epitelio seminífero se identificaron espermatogonias tipo A, intermedio y B, quienes ocuparon el compartimiento basal entremezclándose con espermatocitos I en paquiteno y leptoteno. En el compartimiento adluminal se observaron espermatidas de fase Golgi y presencia de gránulos acrosomales. En el grupo II de 50 días, se observaron células del epitelio seminífero desarrolladas, espermatidas en fase de capuz cefálico, muchas espermatidas elongadas en fase acrosomica y células sustentaculares. En el Grupo III de 60 días se observó el epitelio del túbulo seminífero con espermátidas alargadas en fase acrosómica y de maduración, con núcleos alargados y un sistema acrosomal típico de la espermiación, con presencia de espermatozoides y cuerpos residuales cerca del lúmen tubular. En conclusión se observa la evolución morfológica de las células germinativas testiculares y se reconocen las espermatides en sus cuatro fases: Golgi, capuchón, acrosomal y de maduración en las diferentes edades del animal.

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.

Protective effects of melatonin on testicular torsion and detorsion damage in sprague-dawley rats / Efectos protectores de la melatonina en el daño por torsión y destorsión testicular en ratas prague-dawley

In this study, we evaluated the ultrastructural findings of testis with systemic administration of different doses of melatonin during ischemic period in a rat model of testicular torsion/detorsion (T/D). Testis ischemia-reperfusion (I/R) injury was induced by torsion of the left testis, with a 720 degrees twisting of the spermatic cord so as to produce a total occlusion of testis for 2.5 hours. Subsequently, the same testis was then detorsioned. According to surgical procedure in each group, unilateral orchiectomies were performed for histopathologic examination. The groups were labelled as control group, torsion group (T), torsion and detorsion group (T/D), torsion-detorsion and melatonin group (T/D+20,50 and 100 mg/kg melatonin). For the histological examination, testicular tissues were fixed in 2.5 percent glutheraldehyde and postfixation 1 percent osmic acid solutions. They were examined under transmission electron microscopy after application of contrast stained. In torsion group testis cross-sections, cytoplasm residues of mature sperms and large vacuole-like structures were noticeable. In detorsion group testis cross-sections, dissociations in spermatocide nuclei, many vacuoles and residual particles resulting from organelle degeneration, local voids in cytoplasms of spermatogonia, dilatation in granulated endoplasmic reticulum, large lipid droplets, chromatid particles, along with mitochondrial crystalisis were determined. In the testis cross-sections of the group of T/D+50 mg/kg melatonin administration, sertoli and spermatogonia cells that showed membrane-like structures and cytoplasmic voids were observed. Testis cross-sections of rats that were administered with T/D+50 mg/kg melatonin showed small mitochondrions and vacuole-like structures placed on the edge. Testis cross-sections of rats that were administered with T/D+100 mg/kg melatonin resulted in views similar to those of controls in the microstructural level. As a result, the most effective...

Se evaluaron, en un modelo de torsión/detorsión (T/D) testicular en rata, los cambios ultraestructurales producidos en los testículos, posterior a la administración sistémica de diferentes dosis de melatonina, durante el período de isquemia. La lesión de isquemia-reperfusión (I/R) testicular fue inducida por la torsión del testículo izquierdo, con un giro de 720 grados del cordón espermático con el fin de producir una oclusión total de los testículos durante 2,5 horas. Posteriormente, los mismos testículos fueron detorsionados. De acuerdo con el procedimiento quirúrgico en cada grupo, fueron realizados exámenes histopatológicos de las orquiectomías unilaterales. Los grupos fueron divididos en grupo control, grupo torsión (T), grupo torsión/destorsión (T/D), y grupo torción/destorsión con melatonina (T/D +20, 50 y 100 mg/kg de la melatonina). Para el examen histológico, los tejidos testiculares fueron fijados en soluciones de glutaraldehído al 2,5 por ciento y postfijados al 1 por ciento en ácido ósmico. Luego fueron examinados, después de la aplicación de contrastes de colores, a través de microscopía electrónica de transmisión. En las secciones transversales del grupo con torsión testicular, fueron visibles residuos citoplas-máticos de espermatozoides maduros y grandes estructuras vacuolares. En las secciones transversales del grupo con destorsión testicular, se observaron disociaciones en los núcleos espermáticos, numerosas vacuolas y partículas residuales derivadas de la degeneración de organelos; además de espacios localizados en el citoplasma de las espermatogonias, dilatación en el retículo endoplasmático rugoso, grandes gotas de lípidos y partículas de cromátidas, junto con cristálisis mitocondrial. En las secciones transversales del grupo T/D +50 mg/kg de administración de melatonina, células sustentaculares y espermatogonias mostraron estructuras tipo membrana y vacíos citoplasmáticos. Las secciones transversales del grupo con torsión en la que fue ...

In vitro amplification of BVDV field strains isolated in Argentina: effect of cell line and culture conditions / Amplificación in vitro de cepas de campo de virus de la diarrea viral bovina (VDVB) aisladas en Argentina: efecto de la línea celular y las condicioes de cultivo

The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD- 420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.

Se estudió la interacción de aislamientos de campo de Argentina del VDVB (Pestivirus, Flaviridae) en las líneas celulares continuas MDBK, BoTur y BHK-21. Se utilizaron los virus de campo genotipo 1b, 99/134, 00/693 (casos compatibles con enfermedad de las mucosas) y 04P7016 (cuadro respiratorio) y las cepas de referencia genotipo 1a Singer y NADL. Además se evaluó la interacción de VDVB-NADL con las líneas celulares experimentales de bovino RD-420 y NCL-1 y de riñón porcino (PKZ). Se usaron 2 protocolos de infección. Los títulos virales observados dependieron del virus y del tiempo de infección y no así del modo de infección. Mientras que MDBK y BoTur resultaron susceptibles a la infección, BHK-21 y PKZ no lo fueron. Los virus NADL, 00/693 y 04/89 incrementaron su título entre las 24 y las 48 h p.i. en BoTur para mantenerlo posteriormente; los virus 99/134 y 04P7016 no presentaron variaciones y la cepa Singer presentó título máximo a las 24 h p.i para luego descender. La cinética del virus NADL en las células MDBK, RD-420 y NCL-1 tuvo un incremento de título para MDBK y NCL-1 entre las 24 y 48 h p.i que descendió a las 72 h p.i. La interacción virus-línea celular no estaría relacionada con el sub-genotipo del virus (1a o 1b), ni con el cuadro clínico; las células MDBK y NCL-1 serían más susceptibles a la replicación del VDVB.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae): [review]

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.

Histological and histochemical analysis of the gonadal development of males and females of Armases rubripes (Rathbun 1897) (Crustacea, Brachyura, Sesarmidae) / Análise histológica e histoquímica do desenvolvimento gonadal de machos e fêmeas de Armases rubripes (Rathbun, 1897) (Crustacea, Brachyura, Sesarmidae)

The objective of this study was to provide information on the histological characteristics of the gonads of male and female Armases rubripes crabs, and to try to establish a relationship between the microscopic and macroscopic stages previously identified. Thirty-six crabs were collected by hand between February 2003 and January 2004 in banks of Spartina alterniflora on Sahy Beach in Mangaratiba, Rio de Janeiro state, Brazil. The histological analysis of the ovaries of A. rubripes demonstrated a gradual process of development of the oocytes. According to their cellular characteristics, five types of cells were distinguished: oogonia, oocyte I, oocyte II, oocyte III and oocyte IV. The ovaries showed four stages during gonadal activity: stage I (rudimentary), stage II (developing or maturing), stage III (developed or mature) and stage IV (resting). The results of the histochemical analyses showed that the ovaries vary according to the gonad development stage. The histological aspect of one section of the male gonad was always the same in all of the seminiferous tubules, where the lumen of these tubules always contained spermatozoa and/or spermatids. It was not possible to characterize the three stages of gonad development in the males. This agrees with previous reports in the literature. However, in the females there was a relationship between the gonad stages distinguished macroscopically and the results obtained through the histological and histochemical analysis, due to the presence of different cell types, as well as the lysis process and reabsorption of the oocytes in spent females.

O objetivo deste estudo foi fornecer informações sobre as características histológicas das gônadas de machos e de fêmeas de Armases rubripes, tentando estabelecer uma relação entre os estágios microscópicos e os macroscópicos anteriormente identificados. Foram coletados manualmente 36 caranguejos, durante o período de fevereiro de 2003 a janeiro de 2004, em bancos de Spartina alterniflora na praia do Sahy Mangaratiba, Estado do Rio de Janeiro. A análise histológica dos ovários de A. rubripes demonstrou um processo gradual de desenvolvimento dos oócitos. De acordo com sua característica celular, cinco tipos de células foram distinguidos: ovogônias, oócito I, oócito II, oócito III, oócito IV. Os ovários revelaram quatro estágios de atividade gonadal: estágio I (rudimentar), estágio II (em desenvolvimento ou em maturação), estágio III (desenvolvido ou maduro), estágio IV (desovada). Os resultados das análises histoquímicas permitem afirmar que os ovários variam de acordo com o estágio de desenvolvimento gonadal. O aspecto histológico de uma sessão de gônada masculina é sempre o mesmo em todos os túbulos seminíferos, onde o lúmen deste túbulo sempre contém espermatozóides e/ou espermátides. Não foi possível a caracterização de três estágios de desenvolvimento gonadal em machos, conforme descrito previamente na literatura. Entretanto, em fêmeas, houve uma relação entre os estágios gonadais distinguidos macroscopicamente e os resultados obtidos através da análise histológica e histoquímica, devido à presença de diferentes tipos celulares, assim como processo de lise e reabsorção dos oócitos em fêmeas desovadas.

Histological and functional organization in human testicle: expression of receptors c-kit and androgens / Organización histológica y funcional en el testículo humano: expresión de los receptores c-kit y andrógenos

The objective of this work was identify the presence of interstitial cells of Cajal, muscle cells, nerves and androgen receptor positive cells in adult human testicle, using immunohistochemical detection for c-kit/CD-117, actin smooth muscle specific (ASMS), neurofilament (N) and androgen receptor (AR), respectively. The samples were obtained from patients (n= 10) with diagnosis of prostate cancer, with surgery of orchiectomy. Subsequently were processed by histology and for immunohistochemistry using specific antibodies. It showed the presence of cells c-kit/CD-117, with diverse degrees of positivity, distributed mainly in the interstitial peritubular area of the human testicle. The peritubular myoides cells were positive to the presence of the actin smooth muscle and androgen receptor. The neurofilaments elements (+) only were observed in the vascular tunic. The specific immunohistochemistry describe the presence of the interstitial cells of Cajal in human testicular interstitium, opening a new perspective for the functional interpretation of the testicular cellularity and tubular motility. Possibly associated functionally to peribubulars cells of smooth muscle to regulate the mobility of the seminiferous tabules, whose integration and function would be androgen dependent. The cells that express the c-kit receptor, were found exclusively in the interstitial compartment. This cellular type in addition of the muscular cells of peritubules and the absence of nervous fibers to the interior of the testicle, could be responsible for the regulation of tubular mobility, as it happens in the gastrointestinal apparatus.

El objetivo de este trabajo fue identificar la presencia de células interticiales de Cajal, células musculares lisas, células nerviosas y células que expresan receptores de andrógeno en el testículo de humano adulto, usando inmunohistoquímica específica para: c-kit/CD-117, músculo liso actina específico (ASMS), neurofilamentos (N) y para receptores de andrógenos (AR). Las muestras fueron obtenidas de pacientes (n=10) con diagnóstico de cáncer prostático sometidos a cirugía de orquiectomía. Las biopsias se procesaron para histología e inmunohistoquímica usando anticuerpos específicos. Se muestra la presencia de células c-kit/CD-117, con diversos grados de positividad y distribuidas en el compartimento interticial del testículo. Las células peritubulares mioides fueron positivas para la presencia de músculo liso actina específico y para receptor de andrógenos. La marcación de neurofilamentos positivos, sólo fueron observados en la túnica vascular. Conclusiones: La inmunohistoquímica específica describe la presencia de células interticiales de Cajal en los interticios testiculares humanos, abriendo una nueva visión en la interpretación funcional de la celularidad testicular y la motilidad tubular. Lo anterior asociado a la funcionalidad de las células peritubulares (músculo liso) regularían la motilidad de los túbulos seminíferos. Este proceso posiblemente es andrógeno dependiente. Las células que expresan receptores c-kit se encuentran exclusivamente en los compartimentos interticiales, estas células en conjunto con las células musculares peritubulares agregado a la ausencia de fibras nerviosas al interior del testículo, podrían ser los responsables de la regulación de la motilidad tubular, similar a como se informa para el tracto gastrointestinal.

Morphologic and morphometric analysis of testis of Pseudis limellum (Cope, 1862) (Anura, Hylidae) during the reproductive cycle in the Pantanal, Brazil

The spermatogenesis of Pseudis limellum, from the Southern Pantanal, Brazil, was studied from July 1995 to May 1996, through histological sections of the testis. The cells could be differentiated as: primary spermatogonia, large cells, generally with bilobed nucleus; secondary spermatogonia, smaller cells, with darker cytoplasm, chromatin of radial form; primary and secondary spermatocytes, differentiated according to the different stages of the nucleus during the successive cells divisions. Furthermore, we observed cells in process of morphologic differentiation: rounded spermatids much smaller, with nucleus containing chromatin in compacting process and cytoplasm reduction; elongated spermatids, generally parallel organized in well defined bundles, with the anterior region directed toward the periphery of the seminiferous tubule and the tail directed toward the lumen. Spermatozoa are free in the lumen of the seminiferous tubule. All the cells are organized as cysts, which are supported by a large amount of Sertoli cells. The spermatogenesis in P. limellum is very similar to that of other anurans, but peculiarities were observed regarding the organization of the germ cells, the great amount of free Sertoli cells in the lumen of testis collected in May, and the long cytoplasmatic extensions of the cells bearing pigments and involving the seminiferous tubule. The diameter of the seminiferous tubule (SD) exhibited an annual mean of 251.79 +/- 37.57 microm. Spermatozoa number by seminiferous tubule (SN) exhibited an annual mean of 306.66 +/- 39.83, also with higher and lower values at each month. Variations in SD and SN were not significantly correlated with climatic variables. In this species, reproduction occurs throughout the year in ponds and flooded areas, despite the seasonal climate of the Pantanal. Although males varied in their annual reproductive activity, they were considered potentially reproductive in all months throughout the year.

Pattern of silver nitrate-staining during meiosis and spermiogenesis in testicular lobes of Antiteuchus tripterus (Heteroptera: Pentatomidae)

The pattern of silver nitrate (Ag)-staining differed among testicular lobes of Antiteuchus tripterus. In general, these differences are in regard to the number, size, shape, coloring intensity, and location of the stained bodies or masses, observed during meiosis and spermiogenesis. These characteristics were similar in lobes 1-3. Lobes 4-6, however, differed from each other and from lobes 1-3 as well. Because the Ag-staining method is specific for nucleolar organizing regions and nucleolar material, the observations in meiosis of lobes 1-3 suggested the presence of a single pair of nucleolar organizing region-bearing chromosomes in A. tripterus, as previously found in other Pentatomidae species. In general, the amount of Ag-stained material seen in meiosis of the testicular lobes 1-3 of A. tripterus is smaller than in the other lobes. The differences among Ag-staining in testicular lobes of Antiteuchus tripterus lobes observed during spermiogenesis included a striking variation in morphology of the Ag-stained material found in the head and tail of the spermatids. Given that the key role of the nucleolar material is to participate in protein synthesis, interlobular variations seem to be related to the different functions attributed to each lobe (reproduction to lobes 1-3 and basically nutrition to lobes 4-6). To our knowledge, this is the first time that the nucleolar material was studied in each testicular lobe during spermatogenesis. The present observations encourage further studies since, in addition to being of basic biological interest, several Pentatomidae species are agricultural pests and added knowledge of their biology, mainly in reproduction, may be important for the development of control strategies.

Cytogenetic analysis in the spermatogenesis of Triatoma melanosoma (Reduviidae; Heteroptera)

Triatomines are of great concern in public health because they are vectors of Chagas’ disease. This study presents an analysis of the species Triatoma melanosoma. The cytogenetic characteristics of triatomines include holocentric chromosomes, post-reductional meiosis in the sex chromosomes and nucleolar fragmentation in the meiotic cycle. The methodology utilized consisted of the techniques of lacto-acetic orcein staining and silver ion impregnation. The organs analyzed were adult testicles. The results enabled to classify the chromosomes by number and size, being three large, eight medium and one small heterochromosome. The three largest chromosomes and the heterochromosomes showed heteropyknotic chromatin in meiosis. The heterochromosomes in 8.05% of the cells in metaphase I behaved as pseudobivalents, contrasting with 91.95% of the cells with individualized sex chromosomes, confirming the achiasmatic nature of these chromosomes. However, the pseudobivalents occurred prominently in metaphase II (78.38%), this fact probably is related to the post-reductional nature of the sex chromosomes. The nucleolus in T. melanosoma persisted until the diplotene phase after which it began to fragment. Nucleolar corpuscles were observed in metaphases I and II and during anaphases I and II, these characteristics being related to the phenomenon of nucleolar persistence. In the initial spermatids, peripheral silver ion impregnation occurred, which could be analogous to the pre-nucleolar corpuscles observed after fragmentation. Thus, this study extends our knowledge of the characteristics of triatomines, in particular, heteropyknotic degree, kinetic activity, formation of sex chromosome achiasmatic pseudobivalency, confirmation of the fragmentation phenomenon, and post-meiotic nucleolar reactivation.

Embarazos producto de inyección intracitoplasmática de espermatozoides testiculares frescos y congelados obtenidos de biopsia testicular / Pregnancies obtained by intracitoplasmatic sperm injection from fresh and frozen specimens retrieved with testicular biopsy

Objetivo: Evaluar los resultados de ICSI realizadas con espermatozoides frescos y congelados obtenidos desde biopsias testiculares en pacientes azoospermicos desde junio 2003 a julio 2005 en el Hospital Clínico de la Universidad de Chile. Pacientes y Método: Catorce pacientes portadores de azoospermia fueron sometidos a biopsia testiculares con fines diagnósticos y terapéuticos. Las biopsias de 11 pacientes fueron realizadas el día previo a la aspiración folicular femenina y en 3 casos se utilizaron espermatozoides móviles criopreservados meses antes del procedimiento ICSI. Se cuantifico el número de pacientes en los que rescataron espermatozoides; y las tasas de fecundación y embarazo en los grupos de espermatozoides frescos y criopreservados. Resultados: En los 14 pacientes evaluados se recuperaron espermatozoides. En 3 casos se utilizaron espermatozoides congelados obtenidos de biopsias previas, y en 11 casos espermatozoides frescos para ICSI. Se obtuvieron 6 embarazos en el grupo de espermatozoides frescos y dos embarazos en el grupo de espermatozoides congelados. La tasa de fecundación en ambos grupos fue similar, 64,3 por ciento y 60,7 por ciento respectivamente. Conclusión: Nuestro trabajo demuestra que es posible obtener tasas de fecundación y embarazo semejantes con el uso de espermatozoides frescos y congelados obtenidos a partir de biopsias testiculares en pacientes azoospermicos. Recomendamos la criopreservación de espermatozoides testiculares cada vez que se realice una biopsia testicular y se encuentren espermatozoides, puesto que esto permitirá su uso posterior sin tener que someter al paciente a nuevas cirugías.

Objective: To report eight pregnancies obtained by intracitoplasmic sperm injection (ICSI) using fresh and frozen spermatozoa obtained by testicular sperm extraction (TESE) from 14 azoospermic patients. Patients and Methods: Fourteen azoospermic patients underwent TESE for ICSI. Eleven TESE were made the day previous the oocyte retrieval and three were made the previous months in association with sperm cryopreservation. We evaluated the fertilization and pregnancy rate in both groups: fresh and frozen spermatozoa. Results: Fertilization rate in both groups of patients was similar 64.3 percent and 60.7 percent respectively. Six pregnancies were obtained in patients with fresh spermatozoa and two in the frozen spermatozoa group. Conclusions: We demonstrate that is possible to obtain similar fertilization and pregnancy rates using fresh and frozen spermatozoa from TESE. To decrease the number of testicular biopsies and aborted ICSI cycles we recommend spermatozoa cryopreservation every time a diagnosis testicular biopsy is made in patients with azoospermia.