The intertwining between lead and ethanol in the model organism Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) is a model organism widely used to evaluate the mechanistic aspects of toxicants with the potential to predict responses comparable to those of mammals. We report here the consequences of developmental lead (Pb) exposure on behavioral responses to ethanol (EtOH) in C. elegans. In addition, we present data on morphological alterations in the dopamine (DA) synapse and DA-dependent behaviors aimed to dissect the neurobiological mechanisms that underlie the relationship between these neurotoxicants. Finally, the escalation to superior animals that parallels the observed effects in both experimental models with references to EtOH metabolism and oxidative stress is also discussed. Overall, the literature revised here underpins the usefulness of C. elegans to evidence behavioral responses to a combination of neurotoxicants in mechanistic-orientated studies.

Inhibidores de histidina decarboxilasa atenúan la actividad proangiogénica de células de Leydig tumorales: ¿Potencial terapia neoadyuvante para el tratamiento de leydigiomas? / Histidine decarboxylase inhibitors attenuate the pro angiogenic activity of Leydig tumor cells: Potential neoadjuvant therapy for the treatment of leydigiomas?

Resumen Los tumores de células de Leydig (TCL) son tumores endócrinos del intersticio testicular, cuya incidencia se encuentra en aumento. Los síntomas incluyen feminización o virilización en pacientes prepuberales, y pérdida de libido, disfunción eréctil, infertilidad y/o ginecomastia en adultos. Si bien son usualmente benignos, cuando malignizan en adultos no responden a radio y quimioterapia. Múltiples trabajos han reportado que la histidina decarboxilasa (HDC), enzima que cataliza la conversión de L-histidina en histamina (HA), tiene un rol importante en el desarrollo de tumores. A su vez, en nuestro laboratorio demostramos que la HA induce la proliferación de células de Leydig tumorales (CLT) murinas, mientras que la inhibición de HDC disminuye su proliferación y capacidad esteroidogénica. Además, observamos elevada expresión de HDC en TCL pediátricos vs. controles de distintos estadios de madurez sexual; y se ha descrito que ratones knock out para HDC poseen una angiogénesis incompleta. Para evaluar el rol de HDC en la modulación de la angiogénesis se empleó la línea de CLT de rata R2C, principal modelo utilizado en estudios de Leydigioma. También se realizaron estudios en TCL pediátricos. Los medios condicionados por las CLT R2C estimularon la angiogénesis tanto in vitro como in vivo (empleando HUVEC y analizando el grado de vascularización de membranas corioalantoideas de codorniz, respectivamente). El efecto in vitro se revirtió al tratar previamente las CLT R2C con α-metil-DL-histidinadihidrocloruro, inhibidor específico de HDC. A su vez, tanto la HA como los medios condicionados provenientes de TCL pediátricos, produjeron un aumento en la proliferación de las HUVEC. Nuestros resultados sugieren que las CLT producen HA y otros factores proangiogénicos, y que la inhibición selectiva de HDC atenúa la capacidad proangiogénica de las CLT. En base a estos resultados y evidencias previas del laboratorio, inhibidores específicos de HDC podrían ser utilizados como potencial terapia neoadyuvante en TCL.

ABSTRACT Leydig Cell tumors (LCT) are a rare group of endocrine tumors in the testicular interstitium. Between 1 and 3% of testicular malignances in adults and 4% in prepubertal children belong to LCT. An increasing incidence of this type of neoplasia has been reported recently all around the world. Particularly, a strong relationship between LCT and the use of anabolic steroids (which are commonly used nowadays) has been reported recently. In prepubertal boys, symptoms include feminization or virilization, depending on the major circulating steroid (estradiol or testosterone respectively). Adult patients show loss of libido, penile dysfunction, infertility and/or gynecomastia. Although the etiology still is unknown, several studies indicate that tumoral Leydig cells have an excessive production of insulin-like growth factor (IGF-1), as well as aromatase (CYP19) overexpression, which causes an enormous amount of estrogens (particularly estradiol, E2), and both factors play an important role in tumorigenesis. While usually benign, when LCT became malignant in adults they respond poorly to radio and chemotherapy. Likewise, it has been reported that both therapies increase the incidence of several tumors. All these data imply the need of new therapeutic targets to avoid the chirurgical dissection of the testes and the consequences of the hormonal therapies associated, which implicate not only the loss in reproductive function, but also psychological disorders. Several publications have reported that histidine decarboxylase (HDC), the only enzyme capable of catalyzing the conversion from L-histidine to histamine (HA) in mammals, has an important role in the development of several types of tumors, such as colorectal, breast and melanoma. At the same time, in our laboratory we have reported that HA induces cell proliferation of murine Leydig cells, and complementary, this cell proliferation decreases when inhibiting selectively HDC, as well as steroid synthesis (progesterone and E2). Also, we observed a higher expression of HDC in pediatric LCT (n = 3) than normal controls corresponding to different stages of sexual maturation (n = 9). It has been described that HDC knock out mice have an incomplete angiogenesis, and also that MA-10 Leydig cells HDC expression correlates with vascular endothelial growth factor (VEGF). The aim of this study is to improve our knowledge about the role of HDC in LCT biology, particularly, the angiogenesis modulation. We used the R2C Leydig cell line, the most used model for in vitro studies of Leydigioma, because it overexpresses CYP19 and constitutively produces high levels of IGF-1 and E2, as well as human LCT. R2C and pediatric LCT angiogenic capability was evaluated in vitro by measuring proliferation of human umbilical vein endothelial cells (HUVEC). In addition, we verified R2C cells angiogenic capability in vivo, using quail embryo vasculature (chorioallantoic membrane assay). Both models have been validated for the study of angiogenesis. Conditioned medium obtained from R2C cell culture stimulated angiogenesis in vitro (p <0.001) as well as in vivo (p <0.001). The in vitro effect was reverted with a previous treatment on the R2C cell culture using α-methyl-DL-histidine hydrochloride (α-MHD, 10 µM), a specific HDC activity inhibitor (p <0.001). Finally, human conditioned medium from pediatric LCT increased HUVEC proliferation (p <0.01). In the same way, the analyzed patients showed higher testosterone and estradiol levels than normal serum concentrations, which was in concordance to phenotypical features observed in presence of LCT. Our results indicate that tumoral Leydig cells (TLC) produce HA, as well as other angiogenic factors, and it could be stimulating the vascular endothelium. The selective inhibition of HDC attenuates the pro-angiogenic capability in TLC. Considering all these results and previous observations of our laboratory, specific inhibitors of HDC could be used, in the future, as a potential therapeutic target for the treatment of LCT.

Probiotic activity of Enterococcus faecalis CECT7121: effects on mucosal immunity and intestinal epithelial cells.

AIMS: To analyse the effect of Enterococcus faecalis CECT7121 on intestinal epithelial cells (IECs) and its effects on the mucosal immune response. METHODS AND RESULTS: Enterococcus faecalis CECT7121 showed a high adhesion capacity to completely and heterogeneously differentiated human intestinal epithelial cell line (Caco-2 cells). In addition, the contact of this bacterium with Caco-2 cells did not induce inflammatory chemokines (IL-8 and CCL-20). The presence of IgA(+) and IL-6(+) cells in the small intestine, as well as the production of inflammatory cytokines (TNFα, IL-6 and IL-12) in the gut, was determined after intragastric inoculation of Ent. faecalis CECT7121 in BALB/c mice. The administration of Ent. faecalis CECT7121 increased the number of IgA(+) cells in the intestinal lamina propria without modifying the percentage of IL-6(+) cells. No differences were observed in the cytokines measured in the intestinal extracts between probiotic-treated and control mice. CONCLUSIONS: Enterococcus faecalis CECT7121 stimulates local mucosal immunity and adheres to IECs without inducing inflammatory signals. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that, apart from its already reported systemic immune activity, Ent. faecalis CECT7121 has a modulatory effect at a local level.

Melatonin in testes of infertile men: evidence for anti-proliferative and anti-oxidant effects on local macrophage and mast cell populations.

Melatonin acting through the hypothalamus and pituitary regulates testicular function. In addition, direct actions of melatonin at the testicular level have been recently suggested. We have described that melatonin inhibits androgen production in hamster Leydig cells via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. The initial events of the melatonin/CRH signalling pathway have also been established. Melatonin and all components of the melatonergic/CRH system were also detected in Leydig cells of infertile men. This study attempted to search for additional targets of melatonin in the human testis, and to investigate the effects of melatonin on proliferation and the oxidative state in these novel target cells. To this aim, evaluation of human testicular biopsies of patients suffering from hypospermatogenesis or Sertoli cell only syndrome and cell culture studies were performed. Melatonergic receptors were found in macrophages (MACs) and mast cells (MCs) of the human testis. In biopsies of patients suffering idiopathic infertility, melatonin testicular concentrations were negatively correlated with MAC number per mm(2) and TNFα, IL1ß and COX2 expression, but positively correlated with the expression of the anti-oxidant enzymes SOD1, peroxiredoxin 1 and catalase. Melatonin inhibited proliferation and the expression of pro-inflammatory cytokines and cyclooxygenase 2 (COX2) in both the human non-testicular THP-1 MAC cell line and primary cell cultures of hamster testicular MACs. In the human HMC-1 MC line, melatonin increased the expression of anti-oxidant enzymes and decreased reactive oxygen species (ROS) generation. The results reveal new testicular targets of melatonin and describe anti-proliferative and anti-inflammatory effects of this hormone on testicular MACs. Furthermore, melatonin might provide protective effects against oxidative stress in testicular MCs.

Abnormal assembly of annulate lamellae and nuclear pore complexes coincides with fertilization arrest at the pronuclear stage of human zygotic development.

BACKGROUND: The assembly of nuclear pore complexes (NPC) and their cytoplasmic stacks, annulate lamellae (AL), promote normal nucleocytoplasmic trafficking and accompany pronuclear development within the mammalian zygote. Previous studies showed that a percentage of human oocytes fertilized in vitro failed to develop normal pronuclei and cleave within 40-48 h post insemination. We hypothesized that an aberrant recruitment of NPC proteins, nucleoporins and/or NPC preassembled into AL, might accompany human fertilization arrest. METHODS AND RESULTS: We explored NPC and AL assembly in unfertilized human oocytes, and fertilized and arrested zygotes by immunofluorescence with an NPC- and AL-specific antibody, mAb 414, and by transmission electron microscopy. Major NPC or AL assembly was not observed in the unfertilized human oocytes. Once fertilization took place, the formation of AL was observed throughout the cytoplasm and near the developing pronuclei with NPC. On the contrary, NPC assembly was disrupted in the arrested zygotes, whereas AL were clustered into large sheaths. This was accompanied by the lack of NPC incorporation into the nuclear envelopes. CONCLUSIONS: We conclude that the aberrant assembly of NPC and AL coincides with early developmental failure in humans.

Controversies in the evolution of paediatric-adolescent varicocele: clinical, biochemical and histological studies.

OBJECTIVE: To study hormonal and histological parameters of paediatric-adolescent varicocele in order to know certain aspects of its natural history, in an attempt to find prognostic markers of testicular damage. DESIGN AND METHODS: In a prospective cross-sectional study, we evaluated 93 children and adolescents with left unilateral varicocele and 29 healthy males as control group. All of them were classified according to Tanner stage. Scrotal Doppler in both testes and GnRH and human chorionic gonadotrophin (hCG) tests were performed in all subjects. Surgery was performed in 28 patients and homolateral testicular biopsy in 18. RESULTS: Hormonal measurements of patients with varicocele were compared with a control group for each Tanner stage. Testicular biopsy specimens were analysed by light and electron microscopy. We only observed statistical differences in Tanner III patients in basal FSH (median and range) controls=1.70 (1.10-3.70) IU/l vs varicocele=4.20 (1.00-7.50) IU/l, P<0.05 and in Tanner IV patients in LH post-GnRH: controls=11.0 (7.50-15.0) IU/l vs varicocele=18.0 (5.10-29.0) IU/l, P<0.05 and in testosterone post-hCG: controls=9.50 (7.7-10.0) ng/ml vs varicocele=12.0 (6.2-23.0) ng/ml, P<0.01. No correlation was found between the various clinical grades of varicocele and hormonal measurements for each Tanner stage. No statistically significant differences were found between pre- and post-operative hormonal findings, either in basal levels or in maximal responses. On the other hand, no morphological abnormalities were observed by electron microscopy in germ cells, tubular wall and interstice. CONCLUSIONS: There appears to be no reliable biochemical marker in children and adolescents that may predict impaired testicular function. A significant size discrepancy between both testes, testicular pain and a hyperresponse to GnRH stimulation should continue to be, for the time being, the indications for surgery.

Passive immunization with anti-laminin immunoglobulin G modifies the integrity of the seminiferous epithelium and induces arrest of spermatogenesis in the guinea pig.

In the testis, the base of the Sertoli cells is in contact with the basement membrane matrix, in which the laminins constitute the major noncollagenous components. We have previously demonstrated that antibodies against a preparation enriched in basement membranes of seminiferous tubules (STBM) or a noncollagenous fraction of STBM passively transferred induced modifications to the basement membranes and focal sloughing of the seminiferous epithelium in the rat. In the present report, we tested the effect of passive immunization with anti-laminin IgG on the limiting membrane of the seminiferous tubules, spermatogenesis, and maintenance of the blood-testis barrier in the adult guinea pig. Rabbit antibodies to laminin 1 (IgG fraction) were injected in adult male guinea pigs (GP). Nonimmunized GP and GP immunized with normal rabbit serum IgG were used as controls. Measurements of variations in the diameter and lumen of the tubules and in the size of individual components of the tubular limiting membrane showed that the highest percentage of tubules with reduced lumen occurred 30 days after passive immunization with anti-laminin, when the limiting membrane was thickest and lesions to the seminiferous epithelium were most severe. The lesions included thickening of the limiting membrane, infolding in the basal lamina, deposits of immune complexes coincident with sloughing of pachytene spermatocytes and spermatids, and vacuolization of the Sertoli cells. Mononuclear cell infiltration of the tubules was rare. Permeability tracer studies revealed that Sertoli cell tight junctions remained impermeable. Fifty and 80 days after treatment, the basement membrane of the tubules and the progression of the spermatogenesis were normal. Passive immunization with anti-laminin IgG provided a valuable experimental model for the in vivo study of the influence of the basement membrane on the issue of spermatogenesis and the integrity of the seminiferous epithelium.

Atypical cilia in testicular cells from normal rats.

During our observations solitary cilia have been noted in Sertoli cells, fibroblasts and myoid cells of the rat testes. They mostly presented a 9 + 0 pattern and appeared similar to those previously reported in other cell types. In the present report, we describe their structure and analyze their putative functional significance.

The abnormal development of male sex organs in the rat using a pure antiandrogen and a 5 alpha-reductase inhibitor during gestation.

The effects of a pure antiandrogen flutamide and the 5 alpha-reductase inhibitor finestaride on both prenatal differentiation of prostate and external genitalia were studied in the rat. In control male offspring the mean value of anogenital distance was 3.1 +/- 0.24 mm, and in control female was 1.2 +/- 0.10 mm. In control male newborn rats, histological sections at cranial portion of the urethra revealed prostate bud formation. Male offspring prenatally exposed to 6 mg/Kg/day of flutamide had a significant decrease in anogenital distance, but no alteration in prostate bud formation. At higher dosages of flutamide, the external genitalia were virtually feminizated and the prostatic budding was completely inhibited. In male offspring treated 'in utero' with doses of finestaride of 2, 8 and 16 mg/Kg/day, the anogenital distance became progressively reduced, but complete abolition of prostate development never occurred. However, in male offspring given finestaride at a dose of 2 mg/Kg/day concomitantly with flutamide at a dose of 6 mg/Kg/day, prostate differentiation was completely abolished.

The abnormal development of male sex organs in the rat using a pure antiandrogen and a 5 alpha-reductase inhibitor during gestation.

The effects of a pure antiandrogen flutamide and the 5 alpha-reductase inhibitor finestaride on both prenatal differentiation of prostate and external genitalia were studied in the rat. In control male offspring the mean value of anogenital distance was 3.1 +/- 0.24 mm, and in control female was 1.2 +/- 0.10 mm. In control male newborn rats, histological sections at cranial portion of the urethra revealed prostate bud formation. Male offspring prenatally exposed to 6 mg/Kg/day of flutamide had a significant decrease in anogenital distance, but no alteration in prostate bud formation. At higher dosages of flutamide, the external genitalia were virtually feminizated and the prostatic budding was completely inhibited. In male offspring treated in utero with doses of finestaride of 2, 8 and 16 mg/Kg/day, the anogenital distance became progressively reduced, but complete abolition of prostate development never occurred. However, in male offspring given finestaride at a dose of 2 mg/Kg/day concomitantly with flutamide at a dose of 6 mg/Kg/day, prostate differentiation was completely abolished.

Serum luteinizing hormone pulsatility and intratesticular testosterone and oestradiol concentrations in idiopathic infertile men with high and normal follicle stimulating hormone serum concentrations.

The purpose of the study was to evaluate pulsatile luteinizing hormone (LH) release and intratesticular concentrations of testosterone and oestradiol in infertile men, to determine if alterations in gonadotrophin secretion are associated with changes in the testicular concentrations of steroids. Patients with idiopathic oligo/azoospermia were divided into a high follicle stimulating hormone (FSH) group (n = 5) and a normal FSH group (n = 6). Blood samples were taken every 15 min for 6 h to determine LH, FSH, testosterone, oestradiol, sex hormone binding globulin, bioactive LH and bioavailable testosterone. The patients underwent a bilateral testicular biopsy for histological assessment and to determine testosterone and oestradiol concentrations. Serum measurements were compared with those of seven fertile men. The high FSH group had a higher concentration of serum LH and oestradiol than normal men (P < 0.01) and showed a lower frequency of LH pulses than the normal FSH group and control men (P < 0.01). Intratesticular oestradiol was higher in the high FSH group (P < 0.001), with a lower testosterone/oestradiol ratio (P < 0.01). Patients showed a negative correlation between the serum testosterone/LH ratio and FSH (r = -0.75; P < 0.01) and a positive correlation between the testicular oestradiol concentration and serum FSH (r = 0.86; P < 0.01). The histopathological examination only showed a smaller tube diameter in the high FSH group (P < 0.05). These data seem to indicate that a higher intratesticular concentration of oestradiol with a lower testosterone/oestradiol ratio in the high FSH group could have a deleterious effect on spermatogenesis.

Synaptonemal complex karyotyping in an oligospermic patient with heterochromatin duplication in chromosome n§ 9

Se ha realizado el análisis de complejos sinaptonépticos en espermatocitos de un paciente con oligospermia severa de etiologéa desconocida y portador de un polimorfismo de la heterocromatina paracentrométrica del cromosoma 9. La espermatogénesis del paciente está disminuida en las espermátidas, que presenta anormalidades ultraestructurales en especial en la condensación de la cromatina. En los espermatocitos en paquitene temprano hay un lazo muy visible y asimétrico en el bivalente 9, que en paquitene tardío desaparece frecuentemente, probablemente por un proceso de reajuste sináptico. El lazo es debido a que uno de los elementos es en promedio 7.02% mayor que el otro y que la media de los elementos 9 normales. Esta diferencia indica la presencia de un reordenamiento cromosómico en estado heterocigótico en la región paracentromérica del brazo largo del par 9, que corresponde probablemente a una duplicación en tánden de alrededor del 50% de la zona heterocromática normal. La extensión del lazo sugiere que puede sobrepasar el centrómero y ocupar un segmento del brazo corto. Si bien no es posible asegurar la existencia de una asociación causal entre la anomalía y la hipoespermatogénesis, esta observación se suma a otras sobre inversiones pericéntricas con lazos asinápticos en paquitene, en las cuales hay severa oligospermia o azoospermia. Sobre esta base se sugiere un estudio análogo en otros pacientes portadores de polimorfismos del 9

Synaptonemal complex karyotyping in an oligospermic patient with heterochromatin duplication in chromosome nº 9

Se ha realizado el análisis de complejos sinaptonépticos en espermatocitos de un paciente con oligospermia severa de etiologéa desconocida y portador de un polimorfismo de la heterocromatina paracentrométrica del cromosoma 9. La espermatogénesis del paciente está disminuida en las espermátidas, que presenta anormalidades ultraestructurales en especial en la condensación de la cromatina. En los espermatocitos en paquitene temprano hay un lazo muy visible y asimétrico en el bivalente 9, que en paquitene tardío desaparece frecuentemente, probablemente por un proceso de reajuste sináptico. El lazo es debido a que uno de los elementos es en promedio 7.02% mayor que el otro y que la media de los elementos 9 normales. Esta diferencia indica la presencia de un reordenamiento cromosómico en estado heterocigótico en la región paracentromérica del brazo largo del par 9, que corresponde probablemente a una duplicación en tánden de alrededor del 50% de la zona heterocromática normal. La extensión del lazo sugiere que puede sobrepasar el centrómero y ocupar un segmento del brazo corto. Si bien no es posible asegurar la existencia de una asociación causal entre la anomalía y la hipoespermatogénesis, esta observación se suma a otras sobre inversiones pericéntricas con lazos asinápticos en paquitene, en las cuales hay severa oligospermia o azoospermia. Sobre esta base se sugiere un estudio análogo en otros pacientes portadores de polimorfismos del 9 (AU)

Synaptonemal complex karyotyping in an oligospermic patient with heterochromatin duplication in chromosome n. 9.

Synaptonemal complex karyotyping has been performed in an oligospermia of unknown etiology in a patient carrying a 9qh+ chromosomal polymorphism. The testicular histology showed hypospermatogenesis at the spermatid level and an abnormal pattern of chromatin condensation. Spermatocytes at early pachytene showed a large, asymmetric loop in SC #9, which disappeared at late pachytene, probably because of synaptic adjustment. The loop was formed by a lateral element 7.02% longer than the average normal one. The loop exceeded the centromere towards the short arm, and it is interpreted as a tandem duplication of about 50% of the paracentromeric heterochromatin. The present observation and the previously reported asynaptic loops in carriers of pericentric inversions and showing severe oligospermia suggest that chromosomal variants producing asynaptic loops may be associated with germ cell loss. Further meiotic studies in infertile carriers of such variants are indicated.

Synaptonemal complex karyotyping in an oligospermic patient with heterochromatin duplication in chromosome n. 9.

Synaptonemal complex karyotyping has been performed in an oligospermia of unknown etiology in a patient carrying a 9qh+ chromosomal polymorphism. The testicular histology showed hypospermatogenesis at the spermatid level and an abnormal pattern of chromatin condensation. Spermatocytes at early pachytene showed a large, asymmetric loop in SC #9, which disappeared at late pachytene, probably because of synaptic adjustment. The loop was formed by a lateral element 7.02

longer than the average normal one. The loop exceeded the centromere towards the short arm, and it is interpreted as a tandem duplication of about 50

of the paracentromeric heterochromatin. The present observation and the previously reported asynaptic loops in carriers of pericentric inversions and showing severe oligospermia suggest that chromosomal variants producing asynaptic loops may be associated with germ cell loss. Further meiotic studies in infertile carriers of such variants are indicated.

In vivo selection and characterization of a murine mammary tumor subline with high potential for spontaneous lymph node metastasis.

A transplantable mammary adenocarcinoma, grown in Balb/c mice, with a marked enhancement in its draining lymph node metastatic ability (MM3LN), was obtained through an in vivo procedure from a variant tumor moderately metastatic to lymph nodes (MM3). Both MM3 and MM3LN presented a similar latency and tumor growth rate and reached the same tumor mean diameter at death. MM3LN tumor-bearing mice exhibited a larger mean survival time. The new variant showed a 2.5-fold higher incidence of tumor-draining lymph node metastases than MM3 line, with no differences in the incidence of lung metastases. Morphology as well as cytogenetic and in vitro adhesion properties were studied in order to characterize the new subline. This murine tumor model has potential application in the study of the metastatic process in lymphoid tissue.