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.

Role of somatic testicular cells during mouse spermatogenesis in three-dimensional collagen gel culture system

Spermatogonial stem cells [SSCs] are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional [3D] culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting [MACS] using GDNF family receptor alpha-1 [Gfr Alpha -1] antibody. Two experimental designs were investigated. 1. Gfr Alpha -1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfr Alpha -1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 [SCP3]-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 [TTF1] as post meiotic markers. For statistical analysis student t test was performed Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro

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.

Enzymatic digestion improves testicular sperm retrieval in non-obstructive azoospermic patients

In patients with non-obstructive azoospermia [NOA], vital spermatozoa from the tissue is obtained from testes by enzymatic treatment besides the mechanical treatment. To increase the sperm recovery success of testicular sperm extraction [TESE], with enzymatic digestion if no sperm is obtained from testis tissue by mechanical method. Tissue samples were collected from 150 men who presented with clinical and laboratory data indicating NOA by means of TESE and micro dissection TESE methods. Initially, mature spermatozoa were examined for by mechanical extraction technique shredding the biopsy fractions. In cases whom no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was followed by enzymatic digestion using DNaseI and collagenase type IV. Surgery type, pathology, AZF, karyotype, hormones and testis size were compared in patients. Of 150 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery in 13 [about 9%] patients. Comparison of parameters revealed that level of FSH and LH were significantly different [p=0.04 and 0.08 respectively] between two groups that response negative and positive to enzymatic digestion. The combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa. The benefit of the mincing combined with enzyme to sperm retrieval for NOA firstly shorten the mechanical searching time, leading to minimizing further cellular damage as well as exposure to external conditions, and secondly reduce the number of cases with sperm recovery failures. Also, the serum level of FSH and LH are factors that influence the chance of sperm retrieval

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.

Vascular differentiation of multipotent spermatogonial stem cells derived from neonatal mouse testis

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and alpha-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both alpha-SMA and SM22-alpha proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.

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 ...

Testicular cells apoptosis in opium-addicted rats: [short report]

Apoptosis is a physiological mechanism of cell death and it can be triggered by a variety of internal and external stimuli. It has been shown that some opium derivatives promote cell apoptosis. This study was designed to examine the influence of opium addiction on testicular cell apoptosis in Wistar rats. This experimental study was performed on 7 opium-addicted as case group and 7 normal rats as control group. In the case group, animals treated with peritoneal injections of opium twice a day at 8 a.m and 8 p.m for 8 days based on the following regimen; at the first day 30 mg/kg, second day 60 mg/kg, third day 90 mg/kg, fourth day 120 mg/kg, and from fifth to eighth day 150 mg/kg. The control group received only normal saline. Apoptosis was then evaluated by TUNEL and DNA fragmentation assays. The results of this study showed that the rate of testicular cells apoptosis in opium-addicted rats were significantly higher than the normal rats [p<0.001]. These results indicated that opium addiction may play an important role in testicular cells apoptosis and as a result can cause testicula dysfunction and reduced testosterone production which may culminate in infertility

Histological study of the toxic effects of solder fumes on spermatogenesis in rats

Toxic fumes generated during the soldering process contain various contaminants released at sufficient rates to cause both short- and long-term health problems. Studies have shown that these fumes change the quality and quantity of semen fluid in exposed workers. The aim of the present study was to determine the potentially toxic effects of solder fumes on spermatogenesis in seminiferous tubules of rats as an experimental model, with conditioned media in an exposed chamber. A total number of 48 male Sprague Dawley adult rats were randomly divided into experimental [n=30] and control [n=18] groups. Based on exposure time, each group was further subdivided into two, four and six subgroups. Rats in the experimental groups were exposed to solder fumes in an exposure chamber for one hour/ day. The concentrations of fumes [formaldehyde, stanurn [Sn] and lead [Pb]] were measured by a standard method via atomic absorption and spectrophotometry. According to a timetable, under deep anesthesia, the rats of both experimental and control subgroups were killed. After fixation of testes, specimens were weighed and routinely processed. Paraffin sections were stained by hematoxylin and eosin. Spermiogenesis index was calculated and data analyzed by Mann Whitney NPAR test. Analysis of air samples in the exposure chamber showed the following fume concentrations: 0.193 mg/m[3] for formaldehyde, 0.35 mg/m[3] for Sn and 3 mg/m[3] for Pb. Although there was no significant difference in testes weight between control and experimental subgroups, there was only a significant difference in spermiogenesis index between the six week experimental and control subgroups [p<0.02]. The results of this study showed that solder fumes can change the spermiogenesis index in experimental groups in a time dependent manner

Leydig cell dysfunction after bilateral efferent duct ligation in Wistar rats

The modifications of Leydig cell function after efferent duct ligation [EDL] are mainly due to local changes within the testes providing further evidence for an intratesticular control of Leydig cell function. The aim of the current study was to investigate the effect of disruption of spermatogenesis that follows bilateral EDL on the functions of the Leydig cells. Four groups of animals were studied at 3, 7, 14, and 28 days after treatment, each group consisting of 12 animals, six treated and six controls. Pairs of animals [one control and one treated] were anaesthetized after the lapse of each period; their serum was subjected to the determination of testosterone, follicle stimulating hormone [FSH] and luteinizing hormone [LH]. Both testes were removed and weighed to the nearest gram weight. Sham-operated animals were used as controls. Bilateral EDL resulted in an initial increase in weight of the testes 3 days following the treatment compared to the control group. Thereafter, the testicular weight decreased significantly from day 7, 14 and 28 following treatments compared to that of the respective controls. The decrease in testicular weights was markedly obvious approaching up to 50% of the control groups on the day 14 and 28. There were no differences between serum testosterone concentration in the peripheral circulation on days 3 and 7 following treatments. However, testosterone level started to rise slightly on days 14 and 28 but was not significantly different from that of the controls. The serum LH remained normal until day 3 after the operation, followed by a gradual rise from days 7, 14 and 28 after treatment compared with that of the control groups, whereas FSH showed a sustain rise from day 7 and 14 onward reaching a highly significant levels on day 28 post-treatment. The bilateral EDL resulted in severe spermatogenic damage accompanied by a Leydig cell dysfunction. It is not possible to determine whether disordered androgen production is a manifestation of bilateral EDL or results from disruption of local control mechanisms between the seminiferous tubules and Leydig cells. The nature of the signals that mediate between seminiferous tubules and Leydig cells still remain elusive, hence this possibility needs further investigation to be elucidated

Involvement of the Fas and Fas ligand in testicular germ cell apoptosis by zearalenone in rat

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause testicular toxicity in animals. In the present study, the effects of ZEA on spermatogenesis and possible mechanisms involved in germ cell injury were examined in rats. Ten-week-old Sprague-Dawley rats were treated with 5 mg/kg i.p. of ZEA and euthanized 3, 6, 12, 24 or 48 h after treatment. Histopathologically, spermatogonia and spermatocytes were found to be affected selectively. They were TUNEL-positive and found to be primarily in spermatogenic stages I-VI tubules from 6 h after dosing, increasing gradually until 12 h and then gradually decreasing. Western blot analysis revealed an increase in Fas and Fas ligand (Fas-L) protein levels in the ZEA-treated rats. However, the estrogen receptor (ER)alpha expression was not changed during the study. Collectively, our data suggest that acute exposure of ZEA induces apoptosis in germ cells of male rats and that this toxicity of ZEA is partially mediated through modulation of Fas and Fas-L systems, though ERalpha may not play a significant role.

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.