RESUMEN
Flavonoids are major compounds of Aspalathus linearis and Camellia sinensis. They are classified as endocrine disruptors and some have been shown to inhibit testosterone production. TM3 Leydig cell cultures were treated with 250-5000 µg mL(-1) A. linearis (unfermented or fermented rooibos) or Camellia sinensis (green or black tea) for 24 h in the absence or presence of 6 mIU/200 µl human chorionic gonadotropin (hCG). Under nonstimulated conditions, all teas tend to decrease testosterone production (3.9-31.8%). However, under hCG-stimulation, a significant reduction in testosterone production was observed at all concentrations by both rooibos and tea (16.3-37.9%). MTT assay and phase contrast microscopy, revealed that at 250-1000 µg ml(-1) , both plants maintained the viability, proliferation and morphology of the cells, while 5000 µg ml(-1) was cytotoxic to the cells (P < 0.05). In conclusion, the results here demonstrate the anti-androgenic property of A. linearis and C. sinensis.
Asunto(s)
Aspalathus , Camellia sinensis , Células Intersticiales del Testículo/efectos de los fármacos , Extractos Vegetales/farmacología , Testosterona/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Masculino , Ratones , Microscopía de Contraste de FaseRESUMEN
Aspalathus linearis (rooibos tea) may improve sperm function owing to its antioxidant properties. To test this hypothesis, male rats were given 2% or 5% rooibos tea for 52 days. No significant alterations were observed in body and reproductive organs weight, serum antioxidant capacity and testosterone level. Seminiferous tubules displayed complete spermatogenesis. However, a significant (P < 0.05) decrease in tubule diameter and germinal epithelial height was observed. Epithelial height of caput epididymides showed a significant increase. Unfermented rooibos significantly enhanced sperm concentration, viability and motility. Fermented rooibos also significantly improved sperm vitality (P < 0.01), but caused a significant increase in spontaneous acrosome reaction (P < 0.05), whereas unfermented did not. Creatinine was significantly enhanced in all treated rats, consistent with significant higher kidney weights. Rooibos significantly reduced alanine transaminase level, while 2% fermented rooibos significantly decreased aspartate transaminase level (P < 0.01). In conclusion, treatment with rooibos improved sperm concentration, viability and motility, which might be attributed to its high level of antioxidants. However, prolonged exposure of rooibos might result in subtle structural changes in the male reproductive system and may induce acrosome reaction, which can impair fertility. Intake of large amounts of rooibos may also harm liver and kidney function.
Asunto(s)
Aspalathus , Infertilidad Masculina/prevención & control , Fitoterapia , Extractos Vegetales/uso terapéutico , Testículo/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Animales , Aspalathus/química , Peso Corporal/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Masculino , Extractos Vegetales/química , Extractos Vegetales/farmacología , Distribución Aleatoria , Ratas Wistar , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacosRESUMEN
We evaluated the effect of the methanol extract of Basella alba (MEBa) on testosterone level and fecundity/fertility in male rats exposed in utero to flutamide - an androgen receptor antagonist. For this purpose, 1.5- and 2.5 -month-old male rats exposed in utero to flutamide were treated with the MEBa (1 mg kg(-1) ) for 2 and 1 month respectively. Five days before the end of treatment, rats were housed with females to assess their fecundity/fertility. Thereafter, rats were sacrificed and blood collected for the quantification of testosterone. Flutamide-exposed male rats showed a decrease in their ano-genital distance (AGD, P < 0.05) and were infertile. In normal (methylcellulose-exposed) animals, MEBa provoked an increase in testosterone level in 1.5- (P < 0.008) and 2.5 -month-old rats (P < 0.01) concomitantly with the improvement in their fecundity by 25%. In flutamide-exposed male rats, MEBa increased testosterone level in 1.5 -month-old rats (P < 0.001) without any effect on their fecundity; while in 2.5- month-old rats, MEBa did not affect the testosterone level but improved fecundity (by 25%) and fertility (P < 0.001). This study demonstrated the positive effect of MEBa to enhance fecundity/fertility in normal male rats and in rats exposed to the antiandrogen flutamide during their foetal life.
Asunto(s)
Fertilidad/efectos de los fármacos , Magnoliopsida , Testosterona/sangre , Antagonistas de Receptores Androgénicos/toxicidad , Animales , Femenino , Feto/efectos de los fármacos , Flutamida/toxicidad , Masculino , Medicinas Tradicionales Africanas , Metanol , Fitoterapia , Extractos Vegetales/administración & dosificación , Embarazo , Ratas , Ratas Wistar , Testículo/efectos de los fármacos , Testículo/patologíaRESUMEN
To improve the biocompatibility of polyurethane (PUR), we modified the surface by irradiation with different ions (Carbon; C, Oxygen; O, Nitrogen; N, or Argon; Ar) at 0.3-50 keV energy and doses of 1,00E+13 - 1,00E+15 ions/cm(2). The effects of ion implantation using different ion energies and densities were observed on adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVECs). The long-term in vitro stability of ion-implanted PUR was also investigated. Ion irradiation moderately affected the surface roughness (R(a)), but strongly enhanced the work of adhesion (W(a)). Cell adhesion was markedly improved on O-, N-, and Ar-, but not on C-implanted PUR surfaces. Medium ion energies and lower ion doses produced the best HUVEC attachment and proliferation, indicating the importance of choosing the proper range of energy applied during ion irradiation. In addition, apoptosis rates were significantly reduced when compared with unmodified PUR (uPUR). N implantation significantly protected the surface, although C implantation led to stronger surface erosions than on uPUR. In total, ion implantation on flexible PUR surfaces strongly improved the material surface characteristics and biocompatibility. Electron beam ion implantation within an appropriate energy window is thus a key to improving flexible PUR surfaces for clinical use to support endothelial cell performance. Thus, it can contribute to designing small-diameter grafts, which are in great demand, towards vascular tissue engineering applications.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Implantes Experimentales , Docilidad/efectos de los fármacos , Poliuretanos/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Células Endoteliales/ultraestructura , Humanos , Iones , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Propiedades de Superficie/efectos de los fármacos , TermodinámicaRESUMEN
The aim of this study was to develop and characterize novel metal-polymer constructs to improve the biocompatibility of flexible but hydrophobic polyurethane (PUR) implants. Using a physical vapor deposition (PVD) technique, thin films (< or =100 nm) of zirconium (Zr) or titanium (Ti) were deposited on the polyurethane surface. Both coatings displayed good stability when subjected to cross-cutting test and especially Zr showed only minor and superficial cracks in the scanning electron microscopy analysis. PVD coating resulted in significantly lowered contact angles and the standard surface free energy of wetting (Delta(wet)G degrees ) turned to more favorable negative values (Ti: -40; Zr: -30; untreated PUR (uPUR): +10.1 mN/m). This may lead to the highly enhanced adhesion and proliferation properties observed with human umbilical vein endothelial cells (HUVECs). In addition, the novel coatings had no toxic effect and even drastically reduced apoptosis rates of HUVECs. Cell morphology, nitric oxide production, and mitochondrial membrane potential--both at static and flow conditions--were superior compared with uPUR, thus demonstrating intact physiological functions. Therefore, we suggest that combining PUR as a flexible material with a thin coating of Zr or Ti as the improved biocompatible surface may have advantages for use, for example, vascular graft material.
Asunto(s)
Adhesión Celular/fisiología , Materiales Biocompatibles Revestidos/química , Células Endoteliales/fisiología , Poliuretanos/química , Titanio/química , Circonio/química , Apoptosis/fisiología , Proliferación Celular , Forma de la Célula , Células Endoteliales/citología , Humanos , Ensayo de Materiales , Resistencia al Corte , Propiedades de SuperficieRESUMEN
The setting of a local tissue kallikrein kinin system (tKKS) within the reproductive organs of the male rat was investigated by analysing bradykinin subtype 2 receptor (B2R) gene expression and cellular distribution of B2R protein and the kinin-liberating protease tissue kallikrein (tK). Reverse transcription-polymerase chain reaction showed B2R expression in testis, epididymis and prostate from prepubertal and sexually mature rats. In mature testis, in situ hybridization and immunohistochemistry localized B2R mRNA and protein besides endothelial cells of blood vessels exclusively on pachytene spermatocytes and round and elongated spermatids. B2R expression within the seminiferous tubules was found to be dependent on the stage of the spermatogenic cycle. In pre-pubertal rat testis, B2R mRNA and protein were additionally located in peritubular cells. In the testis, specific staining for tK occurred in addition to endothelial cells of blood vessels on the acrosomal cap of round and elongated spermatids. This immunostaining was also stage-dependent. In the epididymis, tK was detected on epithelial cells near the apical surface. The stage-dependent specific expression of tK and bradykinin B2Rs in developing germ cells and peritubular cells suggests a potential role of the tKKS in the local regulation of spermatogenesis and seminiferous tubule function.
Asunto(s)
Genitales Masculinos/química , Calicreínas/análisis , Receptores de Bradiquinina/análisis , Acrosoma/química , Animales , Células Cultivadas , Endotelio Vascular/química , Epidídimo/química , Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Masculino , Próstata/química , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túbulos Seminíferos/química , Maduración Sexual , Espermátides/química , Espermatogénesis , Espermatozoides/química , Testículo/químicaRESUMEN
Male fertility can be impaired by various toxicants. Some of them are known to target mainly Sertoli cells, which play an essential role in spermatogenesis. In this study, the in vitro response of immature rat Sertoli cells to various environmental pollutants, including pesticides, oestrogenic compounds and heavy metals, has been investigated. Mitochondrial dehydrogenase activity has been used to measure Sertoli cell viability, while production of lactate and secretion of inhibin B have been used as general and specific cell markers. Sertoli cell viability was not affected after 24-h exposure to lindane, DDT, ethinyloestradiol or bisphenol A in the concentration range analysed (up to 100, 25 or 50 microM, respectively). In contrast, mercury(II) (EC50 = 31 microM) and cisplatin (15% decrease in viability at 100 microM) induced some cytotoxic effect. With the exception of the pesticide DDT, all chemicals investigated induced a significant dose-dependent increase in lactate production after 24-h exposure to Sertoli cells. Owing to the cytotoxic effect of mercury(II), lactate levels dropped again at concentrations above 20 microM. The pesticide lindane (but not DDT) and both oestrogens significantly increased the production of the Sertoli cell specific hormone inhibin B without affecting cell viability. In contrast, the heavy metals mercury(II) and platinum(II) markedly decreased inhibin B levels. This sharp decrease was already significant at metal concentrations that reduced Sertoli cell viability only moderately (10-15%). In conclusion, the secretion of lactate and inhibin B by immature rat Sertoli cells seems to be a useful and sensitive marker with which to explore potential Sertoli cell toxicants.
Asunto(s)
Contaminantes Ambientales/toxicidad , Células de Sertoli/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Estrógenos no Esteroides/toxicidad , Técnicas In Vitro , Inhibinas/biosíntesis , Ácido Láctico/biosíntesis , Masculino , Metales Pesados/toxicidad , Oxidorreductasas/metabolismo , Plaguicidas/toxicidad , Ratas , Células de Sertoli/citología , Células de Sertoli/fisiologíaRESUMEN
Peptide hormones are involved in the paracrine regulation of several physiological processes. A possible function of the kallikrein-kinin system (KKS) in mammalian reproduction has been discussed. To evaluate its putative role in spermatogenesis, we searched for components of the KKS (kallikrein, kininases, kinin receptor) in the rat testis. Specific immunostaining demonstrated that the kininogenase tissue kallikrein was present in round and elongated spermatids. Leydig cells, Sertoli cells, peritubular cells, spermatogonia and spermatocytes were not stained. Bradykinin in the supernatant of Sertoli cell cultures was effectively degraded. The resulting metabolites were analysed by high-performance liquid chromatography (HPLC). Specific protease inhibition in the degrading experiments confirmed the occurrence of several metalloproteases on Sertoli cell membranes, including neutral metalloendopeptidases (NEP 24.11 and NEP 24.15), kininase type II (angiotensin converting enzyme, ACE), and kininase type I (metallocarboxypeptidase). Northern blots hybridized with a bradykinin B2 receptor probe showed the presence of B2 receptor mRNA in testis homogenate and Sertoli cell extract. All components of the kallikrein-kinin system are present within the seminiferous epithelium of the rat. Therefore, this paracrine peptide system may play a role in the regulation of Sertoli cell function or in the Sertoli cell-germ cell crosstalk.
Asunto(s)
Sistema Calicreína-Quinina , Epitelio Seminífero/química , Animales , Northern Blotting , Células Cultivadas , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Bradiquinina/análisis , Epitelio Seminífero/enzimología , Calicreínas de Tejido/análisisRESUMEN
Proteolytic enzymes, which are synthesized and secreted by cells of the seminiferous tubule of the testis, have important functions in spermatogenesis. We performed metabolic studies using small peptide hormones as a substrate to investigate the activity of proteases in cultured Sertoli cells of the rat. High-performance liquid chromatographic analysis of the cell culture supernatants showed cleavage of met- and leu-enkephalin, substance P, and bradykinin. No peptidolysis was observed for the cyclic peptide oxytocin. The hormone cleavage pattern and the use of specific protease inhibitors in peptide degradation experiments demonstrated activities of several proteases in Sertoli cells. These are mainly metalloproteinases including neutral metalloendopeptidases, angiotensin-converting enzyme and aminopeptidases. In addition, activities of serine and aspartic proteases were detected. Only marginal proteolytic activities were observed in Sertoli cell conditioned supernatants, indicating that the investigated proteases are mainly located on Sertoli cell membranes. The peptide hormones used in this study have been found to play a potential role in the endocrine, paracrine or autocrine regulation of testicular cells. The membrane-associated proteases reported here may therefore be involved in the metabolism and inactivation of these peptides.
Asunto(s)
Endopeptidasas/metabolismo , Células de Sertoli/enzimología , Espermatogénesis/fisiología , Animales , Bradiquinina/metabolismo , Células Cultivadas , Encefalina Leucina/metabolismo , Encefalina Metionina/metabolismo , Masculino , Péptidos/metabolismo , Inhibidores de Proteasas/farmacología , Ratas , Ratas Sprague-Dawley , Sustancia P/metabolismoRESUMEN
Cottonseed gossypol is a potent male contraceptive in several mammalian species including man. Sertoli cells play a crucial role in spermatogenesis. Therefore, the antifertility competence of gossypol may reflect a change in Sertoli cell function. Rat primary cultures were used to examine the effect of gossypol on cell viability, mitochondrial dehydrogenase function, lactate production and secretion of the Sertoli cell-specific protein inhibin. Exposure for 24 h to gossypol (3-6 microM) significantly enhance secretion of lactate but reduce secretion of inhibin without affecting cell viability. At 9-15 microM, the observed decrease of both lactate and inhibin accumulation apparently resulted from Sertoli cell degeneration and death, because viability and mitochondrial function were also reduced. The results suggest that mitochondria of Sertoli cells are a possible target for gossypol-induced infertility.
Asunto(s)
Gosipol/toxicidad , Células de Sertoli/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Aceite de Semillas de Algodón , Relación Dosis-Respuesta a Droga , Inhibinas/metabolismo , Ácido Láctico/metabolismo , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidorreductasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Peritubular cells, Sertoli cells, and germ cells of the seminiferous tubule synthesize and secrete several proteases and protease inhibitors. Experimental evidence suggests that the complex network of proteolytic enzyme activity and their regulation by protease inhibitors play an important role in male reproduction. Interaction between protease and protease inhibitors seems to play an important role in remodeling and restructuring of the seminiferous tubule during spermatogenesis. Controlled proteolytic activity is also involved in the migration of germ cells from the basal compartment to the lumen of the seminiferous epithelium, and in the release of spermatids during spermiation. The recently reported occurrence of Sertoli cell membrane-associated proteases indicate the possible involvement of regulatory peptide systems within the testis. This view is supported by the detection of all components of one of these paracrine systems, the kallikrein-kinin system, in cells of the seminiferous tubule.
Asunto(s)
Metaloendopeptidasas/metabolismo , Inhibidores de Proteasas/metabolismo , Células de Sertoli/enzimología , Espermatozoides/metabolismo , Animales , Comunicación Celular , Cromatografía Líquida de Alta Presión , Humanos , Masculino , Metaloendopeptidasas/antagonistas & inhibidores , Túbulos Seminíferos/enzimología , Espermatogénesis/fisiologíaRESUMEN
Sertoli cells play a key role in spermatogenesis. To study the involvement of the kallikrein-kinin system in the testis, the pattern of bradykinin-inactivating kininases in rat Sertoli cells was investigated. Exogenous bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is cleaved at Pro7-Phe8, Phe5-Ser6, and Gly4-Phe5, as demonstrated by high performance liquid chromatography analysis. Degradation of bradykinin was strongly inhibited by phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase-24.11. The kininase type II-specific inhibitors, captopril and enalapril, were only partially effective in preventing peptidolysis. This indicates that the main kininases responsible for rapid bradykinin inactivation are neutral metalloendopeptidase and, to a lesser extent, kininase type II. Neutral metalloendopeptidase and kininase type II were shown to be located on Sertoli cell membranes. A low degree of bradykinin degradation was detected by simultaneous inhibition of neutral metalloendopeptidase-24.11 and kininase II, pointing out the involvement of further peptidases with minor activities. This remaining activity is probably not due to the action of kininase type I or cysteine proteases, as shown by specific inhibitors. The data presented indicate the occurrence of membrane-bound kininases, which are an important part of the kallikrein-kinin system, in rat Sertoli cell cultures.
Asunto(s)
Bradiquinina/metabolismo , Células de Sertoli/metabolismo , Secuencia de Aminoácidos , Animales , Bradiquinina/efectos de los fármacos , Células Cultivadas , Masculino , Peptidil-Dipeptidasa A/metabolismo , Inhibidores de Proteasas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Kininases are an important part of the kallikrein-kinin system. We investigated the pattern of kininases in rat Sertoli cells. Sertoli cells are found in the seminifereous tubule of the testis and play a key role in spermatogenesis. Bradykinin was actively cleaved by cultivated Sertoli cells at the Pro7-Phe8, Phe5-Ser6 and Gly4-Phe5 bonds as demonstrated by high performance liquid chromatography analysis. Addition of phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase 3.4.24.11 (NEP), strongly inhibited the degradation of bradykinin. In contrast, the kininase type II-specific inhibitors captopril and enalapril were only partially effective in preventing peptidolysis. NEP and kininase type II were shown to be located in Sertoli cell membranes. The action of kininase type I leads to the formation of the metabolite bradykinin (1-8) which could be detected in small amounts by HPLC analysis. Cleavage of the Ph5-Ser6 bond might be caused by the action of the endopeptidases 24.15 and 24.16, which are phosphoramidon-insensitive. Our results indicate that neutral metalloendopeptidase 24.11 is the main kininase responsible for rapid bradykinin inactivation in Sertoli cells. Further kininases with minor activities are the kininases type I and II and probably the metalloendopeptidases 24.15 and 24.16.