RESUMO
Mutations in Foxn1 and Prkdc genes lead to nude and severe combined immunodeficiency (scid) phenotypes, respectively. Besides being immunodeficient, previous reports have shown that nude mice have lower gonadotropins and testosterone levels, while scid mice present increased pachytene spermatocyte (PS) apoptosis. Therefore, these specific features make them important experimental models for understanding Foxn1 and Prkdc roles in reproduction. Hence, we conducted an investigation of the testicular function in nude and scid BALB/c adult male mice and significant differences were observed, especially in Leydig cell (LC) parameters. Although the differences were more pronounced in nude mice, both immunodeficient strains presented a larger number of LC, whereas its cellular volume was smaller in comparison to the wild type. Besides these alterations in LC, we also observed differences in androgen receptor and steroidogenic enzyme expression in nude and scid mice, suggesting the importance of Foxn1 and Prkdc genes in androgen synthesis. Specifically in scid mice, we found a smaller meiotic index, which represents the number of round spermatids per PS, indicating a greater cell loss during meiosis, as previously described in the literature. In addition and for the first time, Foxn1 was identified in the testis, being expressed in LC, whereas DNA-PKc (the protein produced by Prkdc) was observed in LC and Sertoli cells. Taken together, our results show that the changes in LC composition added to the higher expression of steroidogenesis-related genes in nude mice and imply that Foxn1 transcription factor may be associated to androgen production regulation, while Prkdc expression is also important for the meiotic process.
Assuntos
Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Células Intersticiais do Testículo/fisiologia , Células de Sertoli/fisiologia , Animais , Células Intersticiais do Testículo/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Receptores Androgênicos/metabolismo , Células de Sertoli/citologiaRESUMO
In vitro gamete derivation based on differentiation of germ cells (GC) from stem cells has emerged as a potential new strategy for the treatment of male infertility. This technology also has potential applications in animal reproduction as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to differentiate into mesodermal lineages. Under the effect of selected bioactive factors, MSC upregulate expression of pluripotent and GC specific-markers revealing their potential for GC differentiation. In addition to the effect of trophic factors, cell-to-cell interaction with Sertoli cells (SC) may be required to guide the sequential differentiation of MSC into GC. Thus, the aim of the present study was to investigate the effect of coculture with SC on the potential for in vitro GC differentiation of bovine fetal MSC (bfMSC) derived from bone marrow (BM-MSC) and adipose tissue (AT-MSC). bfMSC were isolated from male bovine fetuses and SC were collected from adult bull testes. The effect of SC interaction with BM-MSC or AT-MSC was analyzed on the expression of pluripotent factors OCT4 and NANOG, GC genes FRAGILLIS, STELLA and VASA and male GC markers DAZL, PIWIL2, STRA8 and SCP3 at Day 14 of coculture. Flow cytometry analyses detected that the majority (95,5%⯱â¯2.5; Pâ¯<â¯0.05) of the isolated population of SC cultures were positive for SC-specific marker WT1. Levels of mRNA of WT1 in BM-MSC and AT-MSC were lower (Pâ¯<â¯0.05) compared to SC; whereas, WT1 expression was not detected in bovine fetal fibroblasts (FB). Cocultures of BM-MSC and AT-MSC with SC had higher (Pâ¯<â¯0.05) OCT4 mRNA levels compared to monocultures of BM-MSC, AT-MSC and SC. Moreover, cocultures of BM-MSC with SC had higher (Pâ¯<â¯0.05) proportion of cells positive for Oct4 and Nanog compared to monocultures of BM-MSC and SC. Levels of mRNA of DAZL, PIWIL2 and SCP3 were upregulated in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Accordingly, the proportion of cells positive for Dazl were higher (Pâ¯<â¯0.05) in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Changes in gene expression profiles during coculture of SC with AT-MSC suggest that cell-to-cell interaction or bioactive factors provided by SC may induce progression of AT-MSC into early stages of GC differentiation.
Assuntos
Bovinos , Diferenciação Celular/fisiologia , Técnicas de Cocultura/veterinária , Células Germinativas/fisiologia , Células-Tronco Mesenquimais/fisiologia , Células de Sertoli/fisiologia , Animais , Linhagem da Célula , MasculinoRESUMO
Testicular anti-Müllerian hormone (AMH) production is inhibited by androgens around pubertal onset, as observed under normal physiological conditions and in patients with precocious puberty. In agreement, AMH downregulation is absent in patients with androgen insensitivity. The molecular mechanisms underlying the negative regulation of AMH by androgens remain unknown. Our aim was to elucidate the mechanisms through which androgens downregulate AMH expression in the testis. A direct negative effect of androgens on the transcriptional activity of the AMH promoter was found using luciferase reporter assays in the mouse prepubertal Sertoli cell line SMAT1. A strong inhibition of AMH promoter activity was seen in the presence of both testosterone and DHT and of the androgen receptor. By site-directed mutagenesis and chromatin immunoprecipitation assays, we showed that androgen-mediated inhibition involved the binding sites for steroidogenic factor 1 (SF1) present in the proximal promoter of the AMH gene. In this study, we describe for the first time the mechanism behind AMH inhibition by androgens, as seen in physiological and pathological conditions in males. Inhibition of AMH promoter activity by androgens could be due to protein-protein interactions between the ligand-bound androgen receptor and SF1 or by blockage of SF1 binding to its sites on the AMH promoter.
Assuntos
Androgênios/farmacologia , Hormônio Antimülleriano/metabolismo , Células de Sertoli/fisiologia , Fator Esteroidogênico 1/metabolismo , Animais , Hormônio Antimülleriano/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação para Baixo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Regiões Promotoras Genéticas , Receptores Androgênicos/metabolismo , Fator Esteroidogênico 1/genética , TranscriptomaRESUMO
Rodents are distributed worldwide, playing important ecological roles regarding preservation of forest areas. Thus, the study of their reproductive biology is a key for conservation initiatives that prevent extinction and/or improve species management. The present study aimed to describe the spermatogenic dynamics of the spiny rat Kannabateomys amblyonyx, an endemic species from Atlantic Rainforest areas, in Brazil. The average body weight was 418.43g with a gonadosomatic index of 0.41%. The testicular parenchyma organization followed the pattern described for other rodent species, with a large amount of seminiferous tubules occupying 93.57% of the organ, in a total of 26.04m per testicle. Stage I of the seminiferous tubule cycle was the most frequent in K. amblyonyx, while stage IV the most scarce. Each tubular section in stage I showed 0.47 type-A spermatogonia, 11.78 primary spermatocytes in pre-leptotene, 3.81 in zygotene and 14.31 in pachytene, whereas 32.19 cells were round spermatids and 6.23 were Sertoli cells. From these results it was possible to determine the sperm reserve of 274.49×106 cells per gram of testis. The mitotic and meiotic indexes were 25.06 and 2.25 cells, respectively, whereas the spermatogenic yield was 69.73 cells. Those findings are significant since this is the first study regarding the reproductive aspects of the only Echimyidae species in Brazil, which shows a monogamous mating system.
Assuntos
Roedores/fisiologia , Espermatogênese/fisiologia , Animais , Masculino , Epitélio Seminífero/fisiologia , Células de Sertoli/fisiologiaRESUMO
With the objective to assess the effect of scrotal bipartition on spermatogenesis in sheep, the testes were used from 12 crossbred rams of sheep farms in the municipality of Patos, Paraíba, Brazil, distributed into two groups: GI with six rams with scrotal bipartition, and GII with six rams without scrotal bipartition. The testicular biometry was measured and the testes were collected, fixed in Bouin and fragments were processed to obtain histological slides. The spermatogenesis yield and the Sertoli cell efficiency was estimated by counting the cells of the spermatogenetic line at stage one of the seminiferous epithelium cycle and the Sertoli cells. The results were submitted to analysis of variance with the ASSISTAT v.7.6 program and the mean values were compared by the Student-Newman-Keuls test (SNK) at 5% significance. The testicular biometric parameters did not show statistical difference (p>0.05) between the groups. The meiotic, spermatogenetic and Sertoli cell efficiency were higher in bipartitioned rams (p<0.05), while the mitotic yield did not differ (p>0.05) between GI and GII. The results indicated that there is superiority in the spermatogenetic parameters of bi-partitioned rams, suggesting that these sheep present, as reported in goats, indication of better reproductive indices.
Com o objetivo de avaliar o efeito da bipartição escrotal sobre a espermatogênese em ovinos, foram utilizados os testículos de 12 ovinos sem raça definida oriundos de criadouros do município de Patos-PB, Brasil, distribuídos em dois grupos, GI de seis animais com bipartição escrotal e o GII de seis animais sem bipartição escrotal. Realizou-se a aferição da biometria testicular, em seguida, os testículos foram coletados, fixados em Bouin e fragmentos foram processados para obtenção de lâminas histológicas. Foi estimado o rendimento da espermatogênese e eficiência das células de Sertoli contando-se as células da linhagem espermatogênica no estádio I do Ciclo do Epitélio Seminífero, bem como as células de Sertoli. Os resultados foram submetidos à análise de variância pelo programa ASSISTAT v.7.6 e os valores médios foram comparados pelo teste Student-Newman-Keuls (SNK) a 5% de significância. Os parâmetros de biometria testicular não apresentaram diferença estatística (p>0,05) entre os grupos. Os rendimentos meiótico, espermatogênico e a eficiência das células de Sertoli mostraram-se superiores em animais bipartidos (p<0,05), enquanto o rendimento mitótico não diferiu (p>0,05) entre GI e GII. Os resultados indicaram existir superioridade nos parâmetros espermatogênicos de ovinos bipartidos, sugerindo que estes animais apresentam, assim como constatado em caprinos, indicativo de melhores índices reprodutivos.
Assuntos
Animais , Masculino , Células de Sertoli/fisiologia , Escroto/anatomia & histologia , Escroto/fisiologia , Espermatogênese/fisiologia , Ovinos/fisiologia , Biometria , Testículo/fisiologiaRESUMO
Nicotine is largely consumed in the world as a component of cigarettes. It can cross the placenta and reach the milk of smoking mothers. This drug induces apoptosis, affects sex hormone secretion, and leads to male infertility. To investigate the exposure to nicotine during the whole intrauterine and lactation phases in Sertoli cells, pregnant rats received nicotine (2 mg/kg per day) through osmotic minipumps. Male offsprings (30, 60, and 90 days old) had blood collected for hormonal analysis (FSH and LH) and their testes submitted for histophatological study, analysis of the frequency of the stages of seminiferous epithelium cycle, immunolabeling of apoptotic epithelial cells (TUNEL and Fas/FasL), analysis of the function and structure of Sertoli cells (respectively using transferrin and vimentin immunolabeling), and analysis of Sertoli-germ cell junctional molecule (ß-catenin immunolabeling). The exposure to nicotine increased the FSH and LH plasmatic levels in adult rats. Although nicotine had not changed the number of apoptotic cells, neither in Fas nor FasL expression, it provoked an intense sloughing of epithelial cells and also altered the frequency of some stages of the seminiferous epithelium cycle. Transferrin and ß-catenin expressions were not changed, but vimentin was significantly reduced in the early stages of the seminiferous cycle of the nicotine-exposed adult rats. Thus, we concluded that nicotine exposure during all gestational and lactation periods affects the structure of Sertoli cells by events causing intense germ cell sloughing observed in the tubular lumen and can compromise the fertility of the offspring.
Assuntos
Gonadotropinas/sangue , Lactação/efeitos dos fármacos , Nicotina/toxicidade , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/patologia , Células de Sertoli/efeitos dos fármacos , Animais , Feminino , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Ratos , Células de Sertoli/patologia , Células de Sertoli/fisiologia , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Testículo/efeitos dos fármacos , Testículo/patologiaRESUMO
With the objective to assess the effect of scrotal bipartition on spermatogenesis in sheep, the testes were used from 12 crossbred rams of sheep farms in the municipality of Patos, Paraíba, Brazil, distributed into two groups: GI with six rams with scrotal bipartition, and GII with six rams without scrotal bipartition. The testicular biometry was measured and the testes were collected, fixed in Bouin and fragments were processed to obtain histological slides. The spermatogenesis yield and the Sertoli cell efficiency was estimated by counting the cells of the spermatogenetic line at stage one of the seminiferous epithelium cycle and the Sertoli cells. The results were submitted to analysis of variance with the ASSISTAT v.7.6 program and the mean values were compared by the Student-Newman-Keuls test (SNK) at 5% significance. The testicular biometric parameters did not show statistical difference (p>0.05) between the groups. The meiotic, spermatogenetic and Sertoli cell efficiency were higher in bipartitioned rams (p<0.05), while the mitotic yield did not differ (p>0.05) between GI and GII. The results indicated that there is superiority in the spermatogenetic parameters of bi-partitioned rams, suggesting that these sheep present, as reported in goats, indication of better reproductive indices.(AU)
Com o objetivo de avaliar o efeito da bipartição escrotal sobre a espermatogênese em ovinos, foram utilizados os testículos de 12 ovinos sem raça definida oriundos de criadouros do município de Patos-PB, Brasil, distribuídos em dois grupos, GI de seis animais com bipartição escrotal e o GII de seis animais sem bipartição escrotal. Realizou-se a aferição da biometria testicular, em seguida, os testículos foram coletados, fixados em Bouin e fragmentos foram processados para obtenção de lâminas histológicas. Foi estimado o rendimento da espermatogênese e eficiência das células de Sertoli contando-se as células da linhagem espermatogênica no estádio I do Ciclo do Epitélio Seminífero, bem como as células de Sertoli. Os resultados foram submetidos à análise de variância pelo programa ASSISTAT v.7.6 e os valores médios foram comparados pelo teste Student-Newman-Keuls (SNK) a 5% de significância. Os parâmetros de biometria testicular não apresentaram diferença estatística (p>0,05) entre os grupos. Os rendimentos meiótico, espermatogênico e a eficiência das células de Sertoli mostraram-se superiores em animais bipartidos (p<0,05), enquanto o rendimento mitótico não diferiu (p>0,05) entre GI e GII. Os resultados indicaram existir superioridade nos parâmetros espermatogênicos de ovinos bipartidos, sugerindo que estes animais apresentam, assim como constatado em caprinos, indicativo de melhores índices reprodutivos.(AU)
Assuntos
Animais , Masculino , Escroto/anatomia & histologia , Escroto/fisiologia , Espermatogênese/fisiologia , Células de Sertoli/fisiologia , Ovinos/fisiologia , Biometria , Testículo/fisiologiaRESUMO
The intratesticular testosterone concentration is high during the early postnatal period although the intracellular androgen receptor expression (iAR) is still absent in Sertoli cells (SCs). This study aimed to evaluate the non-classical effects of testosterone and epitestosterone on calcium uptake and the electrophysiological effects of testosterone (1µM) on SCs from rats on postnatal day (pnd) 3 and 4 with lack of expression of the iAR. In addition, crosstalk on the electrophysiological effects of testosterone and epitestosterone with follicle stimulating hormone (FSH) in SCs from 15-day-old rats was evaluated. The isotope (45)Ca(2+) was utilized to evaluate the effects of testosterone and epitestosterone in calcium uptake. The membrane potential of SCs was recorded using a standard single microelectrode technique. No immunoreaction concerning the iAR was observed in SCs on pnd 3 and 4. At this age, both testosterone and epitestosterone increased the (45)Ca(2+) uptake. Testosterone promoted membrane potential depolarization of SCs on pnd 4. FSH application followed by testosterone and epitestosterone reduced the depolarization of the two hormones. Application of epitestosterone 5 min after FSH resulted in a delay of epitestosterone-promoted depolarization. The cell resistance was also reduced. Thus, in SCs from neonatal Wistar rats, both testosterone and epitestosterone act through a non-classical mechanism stimulating calcium uptake in whole testes, and testosterone produces a depolarizing effect on SC membranes. Testosterone and epitestosterone stimulates non-classical actions via a membrane mechanism, which is independent of iAR. FSH and testosterone/epitestosterone affect each other's electrophysiological responses suggesting crosstalk between the intracellular signaling pathways.
Assuntos
Androgênios/farmacologia , Epitestosterona/farmacologia , Células de Sertoli/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/fisiologia , Masculino , Potenciais da Membrana , Ratos Wistar , Células de Sertoli/efeitos dos fármacos , Testículo/citologia , Testículo/efeitos dos fármacosRESUMO
Testis differentiation from representatives of the Otophysi (Cyprinus carpio), Percomorpha (Amatitlania nigrofasciata), and Atherinomorpha (Poecilia reticulata) was comparatively described. In the undifferentiated gonad of C. carpio, the primordial germ cells (PGCs) are scattered throughout the gonads while in A. nigrofasciata and P. reticulata the PGCs are restricted to the ventral periphery. In the dorsal region of the developing gonads, with the exception of C. carpio, somatic cell rearrangements result in the differentiation of the sperm duct. Pre-Sertoli cells wrap around single spermatogonia forming cysts that proliferate forming acinar-clusters. In C. carpio and A. nigrofasciata, the cysts in each acinar-cluster move away from each other, creating a central lumen. In C. carpio, the acinar-clusters then fuse to each other forming tubules that become lined by the germinal epithelium. Subsequently, the tubules anastomose dorsally and create the sperm duct. In A. nigrofasciata, the acinar-clusters elongate, forming lobules that individually connect to the sperm duct. These are lined by the germinal epithelium. In P. reticulata, the spermatogonial cysts remain in the acinar-cluster organization. Subsequently, developing ducts connect each cluster to the sperm duct and lobules subsequently develop. In the differentiated testis of C. carpio and A. nigrofasciata, spermatogonia are distributed along the lengths of the anastomosing tubules or lobules, respectively. However, in P. reticulata, the spermatogonia remain restricted to the terminal end of the lobules. Considering testis ontogeny, the spermatogonial acinar-cluster is the adult characteristic of more derived taxa that approximate the early gonad developmental stages of the basal taxa.
Assuntos
Evolução Biológica , Peixes/crescimento & desenvolvimento , Diferenciação Sexual/fisiologia , Testículo/crescimento & desenvolvimento , Animais , Masculino , Organogênese/fisiologia , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Especificidade da Espécie , Espermatogênese/fisiologia , Espermatogônias/citologia , Espermatogônias/fisiologia , Testículo/citologiaRESUMO
The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis.
Assuntos
Tatus/fisiologia , Adesão Celular/fisiologia , Células Germinativas/fisiologia , Epitélio Seminífero/fisiologia , Células de Sertoli/fisiologia , Animais , Apoptose/fisiologia , Caderinas/metabolismo , Caspase 3/metabolismo , Moléculas de Adesão Celular/metabolismo , Citocromos c/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Junções Intercelulares/fisiologia , Masculino , Meiose/fisiologia , Microscopia Eletrônica de Transmissão , Nectinas , Fagocitose/fisiologia , Estações do Ano , beta Catenina/metabolismoRESUMO
Kinosternon scorpioides is a Brazilian freshwater turtle that belongs to the class Reptilia, encompassing almost 10,000 species. Nevertheless, very little is known about the testicular quantitative parameters, particularly those related to spermatogenesis, in this vertebrate class. Our main objectives were to investigate in detail the structure and function of the testis in K. scorpioides, particularly the aspects related to spermatogenic cycle length and Sertoli cell (SC) and spermatogenic efficiencies. Nine sexually mature turtles were examined, and intraperitoneal bromodeoxyuridine injections were administered to estimate duration of spermatogenesis. Based on the acrosome development in spermatids and the overall germ cell associations, 10 stages of the seminiferous epithelium cycle were characterized. Similar to birds, humans, and some primate species, several stages were observed per seminiferous tubule cross-sections. One spermatogenic cycle and the entire spermatogenic process lasted, respectively, 12 and 53 days. The SC efficiency (number of round spermatids per SC) and daily sperm production per gram of testis were, respectively, 20 and 40 million spermatids. As established for mammals, our findings suggest that SC efficiency is also a critical determinant of sperm production in reptiles. To our knowledge, this is the first study to investigate the kinetics of spermatogenesis and testis function in any reptilian species. Besides allowing a better understanding of reproductive biology in reptiles, these data will be useful in comparative studies. Moreover, these results could provide the basis for investigations related to the evaluation of spermatogonial stem cell physiology niche in Kinosternon scorpioides.
Assuntos
Espermatogênese/fisiologia , Espermatozoides/fisiologia , Tartarugas/fisiologia , Animais , Água Doce , Masculino , Epitélio Seminífero/citologia , Epitélio Seminífero/fisiologia , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Espermatogônias/citologia , Espermatogônias/fisiologia , Testículo/citologia , Fatores de TempoRESUMO
Insectivorous bats play a very important role in the regulation of tropical ecosystems, but information about their reproductive cycle is lacking. Thus, male Molossus molossus were captured over the four seasons, and morphometric analyses of their testes were conducted to infer on the gonadal dynamics and the reproductive capacity of the species. Testes were immersed in Karnovsky fixative, and fragments were embedded in methacrylate and paraplast for morphometric and TUNEL assay respectively. The least gonadosomatic index (0.3%), tubulesomatic index (0.2%) and tubular diameter (133.2µm) occurred in summer. An adult M. molossus showed a total average of 48.9m of seminiferous tubules per gram of testis. Primary spermatocytes were observed in the zygotene at Stage 1 of the seminiferous epithelium cycle. The greatest meiotic index was obtained in winter (3.8 cells), and the general yield of spermatogenesis was higher in winter (64.5 cells) than in summer (19.1 cells). There was no difference in the apoptotic cells count among seasons. The Sertoli cell index was less in summer (5.9) than in fall (11.6), while the number of Sertoli cells per gram of testis did not vary significantly among the seasons (28.0×10(7)). The spermatic reserve per gram of testis was greater in the fall (63.9×10(7)) and winter (69.8×10(7)) than summer (37.1×10(7)). We conclude that M. molossus males show a continuous reproductive cycle, featuring greater spermatogenic activity during the fall and winter, a tubular length above the average of other mammals and a less support capacity of the Sertoli cells.
Assuntos
Quirópteros/fisiologia , Reprodução/fisiologia , Células de Sertoli/fisiologia , Espermatogênese/fisiologia , Testículo/fisiologia , Animais , Apoptose/fisiologia , Brasil , Quirópteros/anatomia & histologia , Histocitoquímica/veterinária , Marcação In Situ das Extremidades Cortadas/veterinária , Masculino , Tamanho do Órgão/fisiologia , Estações do Ano , Testículo/citologiaRESUMO
To assess the testicular Sertoli cell function according to inhibin B levels in ankylosing spondylitis (AS) patients and the possible effect of anti-TNF therapy on this hormone production, 20 consecutive AS patients and 24 healthy controls were evaluated. At study entry, AS patients were not receiving sulfasalazine/methotrexate and never have used biological/cytotoxic agents. They were assessed by serum inhibin B levels, hormone profile, urological examination, testicular ultrasound, seminal parameters, and clinical features. Ten of these patients received anti-TNF treatment and they were reevaluated for Sertoli function and disease parameters at 6 months. Four of them agreed to repeat sperm analysis. At study entry, the median of inhibin B (68 vs. 112.9 pg/mL, p = 0.111), follicle-stimulating hormone levels (3.45 vs. 3.65 IU/L, p = 0.795), and the other hormones was comparable in AS patients and controls (p > 0.05). Sperm analysis was similar in AS patients and controls (p > 0.05) with one AS patient presenting borderline low inhibin B levels. Further analysis at 6 months of the 10 patients referred for anti-TNF therapy, including one with borderline inhibin B, revealed that median inhibin B levels remained stable (116.5 vs. 126.5 pg/mL, p = 0.431) with a significant improvement in C-reactive protein (27.8 vs. 2.27 mg/L, p = 0.039). Sperm motility and concentration were preserved in the four patients who repeated this analysis after TNF blockage. In conclusion, this was the first study to report, using a specific marker, a normal testicular Sertoli cell function in AS patients with mild to moderate disease activity.
Assuntos
Inibinas/sangue , Células de Sertoli/fisiologia , Espermatozoides/patologia , Espondilite Anquilosante/fisiopatologia , Adolescente , Adulto , Sedimentação Sanguínea , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Fertilidade , Humanos , Masculino , Pessoa de Meia-Idade , Sêmen/metabolismo , Células de Sertoli/diagnóstico por imagem , Espondilite Anquilosante/sangue , Testículo/diagnóstico por imagem , Testículo/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Ultrassonografia , Adulto JovemRESUMO
BACKGROUND: The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [14C]-MeAIB (alpha-(methyl-amino)isobutyric acid) accumulation stimulated by T4 and the role of the integrin receptor in this event, and also to clarify whether the T4 effect on MeAIB accumulation and on Ca2+ influx culminates in cell secretion. METHODS: We have studied the rapid and plasma membrane initiated effects of T4 by using 45Ca2+ uptake and [45C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC. RESULTS: The stimulation of MeAIB accumulation byT4 appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T4 increases extracellular Ca2+ uptake and Ca2+ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T4. Also, the cytoskeleton and CIC-3 chloride channel contribute to the membrane-associated responses of SC. CONCLUSIONS: T4 integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone. GENERAL SIGNIFICANCE: The integrin receptor activation by T4 may take a role in plasma membrane processes involved in the male reproductive system.
Assuntos
Membrana Celular/efeitos dos fármacos , Integrina alfaVbeta3/genética , Receptores de Superfície Celular/genética , Células de Sertoli/efeitos dos fármacos , Espermatogênese/fisiologia , Tiroxina/farmacologia , Aminoácidos/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Radioisótopos de Carbono , Canais de Cloreto/metabolismo , Citoesqueleto/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Integrina alfaVbeta3/metabolismo , Masculino , Oligopeptídeos/farmacologia , Ratos , Ratos Wistar , Receptores de Superfície Celular/metabolismo , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Transdução de Sinais/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Timidina/metabolismo , Tiroxina/análogos & derivadosRESUMO
Epitestosterone is the 17α-epimer of testosterone. This steroid possesses antiandrogenic activities. The mechanism of action of epitestosterone has not been elucidated. The aim of this study was to investigate the nonclassical effect of epitestosterone on the membrane of Sertoli cells in proliferative phase (rats aged 15 days) and in nonproliferative phase (rats aged 21 and 35 days). The membrane potential of Sertoli cells was recorded using a standard single microelectrode technique. Epitestosterone (0.5, 1, and 2 µM) or testosterone (1 µM) was administered alone and after infusion with flutamide (1 µM), verapamil (100 µM), or U-73122 (2 µM). The testes of rats aged 12-15 days were preincubated with 45Ca2+ with or without flutamide (1 µM) and incubated with epitestosterone (1 µM) or testosterone (1 µM). Epitestosterone and testosterone produced a depolarization in the membrane potential and increased the membrane input resistance on Sertoli cells from rats of all 3 ages. The effect of epitestosterone did not change after perfusion with flutamide. Epitestosterone increased 45Ca2+ uptake within 5 min and this effect was not inhibited by flutamide. The absence of an effect by flutamide suggests that epitestosterone acts independently of the intracellular androgen receptor. The depolarizing effect was inhibited by verapamil, a voltage-dependent calcium channel blocker, and by U-73122, a phospholipase C inhibitor. These results indicate that epitestosterone acts on the membrane via a nonclassical signaling pathway; the effect was similar to the testosterone action on membrane of Sertoli cells in whole seminiferous tubules from rat testes.
Assuntos
Membrana Celular/fisiologia , Epitestosterona/farmacologia , Túbulos Seminíferos/citologia , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Testosterona/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Membrana Celular/efeitos dos fármacos , Flutamida/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Androgênicos/metabolismo , Células de Sertoli/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
BACKGROUND: Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats. METHODS: Prepubertal rats received the dose of 5 mg/Kg of doxorubicin, which was fractioned in two doses: 3 mg/Kg at 15dpp and 2 mg/Kg at 22 dpp. The testes were collected at 40, 64 and 127 dpp, fixed in Bouin's liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS + H-stained sections. RESULTS: The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64 dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats. CONCLUSIONS AND DISCUSSION: These data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , Células de Sertoli/efeitos dos fármacos , Maturidade Sexual , Animais , Doxorrubicina/administração & dosagem , Masculino , Ratos , Ratos Wistar , Epitélio Seminífero/química , Epitélio Seminífero/patologia , Células de Sertoli/patologia , Células de Sertoli/fisiologia , Espermatogênese , Testículo/patologia , Transferrina/análiseRESUMO
The objective of this research study was to evaluate the proliferation of somatic and germ cells in the seminiferous epithelium of Alpine goats. A total of 47 goats reared in semi-intensive conditions were used, divided into age groups from birth up to 12 months of age. Following castration, testis fragments were included in plastic resin and were mounted in Entellan(®) for histometric evaluations. In general, the total number of germ cells per seminiferous cord transverse section was low until 4 months of age. An increase was observed between 4 and 5 months, lasting until 9 months of age. From that age on, this number tended to stabilize, until 12 months. The population of support cells (undifferentiated support cells and Sertoli cells) remained constant from birth to the first month, when it peaked. This was followed by a reduction until the fifth month of age. From that age on, there was differentiation in mature Sertoli cells, and its population remained constant until 12 months of age. It can be concluded that Alpine goats were in the impuberal phase from birth to 3 months of age, prepuberal phase during the fourth month, reached puberty at 5 months of age, postpuberal phase from 6 to 8 months, and reached sexual maturity at 9 months of age. Overall yield of spermatogenesis and Sertoli cell index increased from puberty up to the 12th month of age.
Assuntos
Células Germinativas/fisiologia , Cabras/fisiologia , Epitélio Seminífero/fisiologia , Células de Sertoli/fisiologia , Maturidade Sexual/fisiologia , Espermatogênese/fisiologia , Fatores Etários , Animais , Processos de Crescimento Celular/fisiologia , Células Germinativas/citologia , Histocitoquímica/veterinária , Masculino , Epitélio Seminífero/citologia , Células de Sertoli/citologiaRESUMO
Bipotential gonads represent the structural framework from which alternative molecular sex determination networks have evolved. Maintenance of Sox9 expression in Sertoli cells is required for the structural and functional integrity of male gonads in mammals and probably in most amniote vertebrates. However, spatial and temporal patterns of Sox9 expression have diversified along evolution. Species with temperature sex determination are an interesting predictive model since one of two alternative developmental outcomes, either ovary or testis occurs under controlled laboratory conditions. In the sea turtle Lepidochelys olivacea, Sox9 is expressed in the medullary cords of bipotential gonads when incubated at both female- or male-promoting temperature (FT or MT). Sox9 is then turned off in presumptive ovaries, while it remains turned on in testes. In the current study, Sox9 was used as a marker of the medullary cell lineage to investigate if the medullary cords originate from mesothelial cells at the genital ridges where Sox9 is upregulated, or, if they derive from a cell population specified at an earlier developmental stage, which maintains Sox9 expression. Using immunofluorescence and in situ hybridization, embryos were analyzed prior to, during and after gonadal sex determination. A T-shaped domain (T-Dom) formed by cytokeratin (CK), N-cadherin (Ncad) and SOX9-expressing cells was found at the upper part of the hindgut dorsal mesentery. The arms of the T-Dom were extended to both sides towards the ventromedial mesonephric ridge before the thickening of the genital ridges, indicating that they contained gonadal epithelial cell precursors. Thereafter, expression of Sox9 was maintained in medullary cords while it was downregulated at the surface epithelium of bipotential gonads in both FT and MT. This result contrasts with observations in mammals and birds, in which Sox9 upregulation starts at a later stage in the inner cells underlying the Sox9-negative surface epithelium, suggesting that the establishment of a self-regulatory Sox9 loop required for Sertoli cell determination has evolved. The T-shaped domain at the upper part of the hindgut dorsal mesentery found in the current study may represent the earliest precursor of the genital ridges, previously unnoticed in amniote vertebrates.
Assuntos
Evolução Biológica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Gônadas/citologia , Gônadas/embriologia , Fatores de Transcrição SOX9/metabolismo , Tartarugas/embriologia , Animais , Bromodesoxiuridina , Caderinas/metabolismo , Primers do DNA/genética , Feminino , Imunofluorescência , Gônadas/metabolismo , Hibridização In Situ , Queratinas/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Células de Sertoli/metabolismo , Células de Sertoli/fisiologia , Processos de Determinação Sexual/fisiologia , Fatores Sexuais , TemperaturaRESUMO
The blood-testis barrier (BTB) is known for its ability to create an immune privilege site in the seminiferous epithelium, but less is known of the blood-epididymal barrier (BEB). It is already established that the fully functional BTB and BEB are much more complex and consist of anatomical/physical (tight junctions, basolateral and apical membranes), physiological and immunological components, which are all necessary to make a functioning barrier in the testis and epididymis. However, comparative data for metazoans suggest that an effective Sertoli cell barrier is not entirely necessary for the development of germ cells during spermatogenesis or that our knowledge about the barrier structure/function in metazoans is still immature. This chapter compares the unique barrier formed by the Sertoli cells of the testis to that formed by the apical junctional complexes of the epididymal epithelium.
Assuntos
Barreira Hematotesticular/imunologia , Epididimo/imunologia , Testículo/imunologia , Animais , Barreira Hematotesticular/anatomia & histologia , Barreira Hematotesticular/fisiologia , Diferenciação Celular , Permeabilidade da Membrana Celular , Epididimo/anatomia & histologia , Epididimo/fisiologia , Sobrevivência de Enxerto/imunologia , Humanos , Tolerância Imunológica , Masculino , Filogenia , Epitélio Seminífero/imunologia , Epitélio Seminífero/fisiologia , Células de Sertoli/imunologia , Células de Sertoli/fisiologia , Células de Sertoli/ultraestrutura , Maturação do Esperma , Espermatogênese , Espermatozoides/imunologia , Espermatozoides/fisiologia , Testículo/anatomia & histologia , Testículo/fisiologia , Junções Íntimas/imunologia , Junções Íntimas/fisiologia , Junções Íntimas/ultraestrutura , Imunologia de TransplantesRESUMO
Studies show a mechanism of action of testosterone, nandrolone and catechin as agonists of the membrane androgen receptor. The aim of this work is to investigate the non-classical effect of androgens and catechin in Sertoli cells from immature rats. The membrane potential of Sertoli cells in whole seminiferous tubules was recorded using a standard single microelectrode technique. It was performed a topical application of testosterone (1 µM), nandrolone (0.1, 0.5 and 1 µM) and the flavonoid catechin (0.1, 0.5 and 1 µM) alone and also after infusion with flutamide (1 µM), diazoxide (100 µM) or U73122 (1 µM). The immature testes were incubated for 5 min in KRb with (45)Ca(2+), with or without nandrolone (1 µM). The results were given as mean±SEM. The data were analyzed using ANOVA for repeated measures with Bonferroni post-test. Testosterone produces a depolarization in the membrane potential at 120 s after application. Catechin (1 µM) and nandrolone (1 µM) have shown a similar response to testosterone: depolarization at 120 s after the application. The same response of catechin and nandrolone was observed at different doses. The effects of testosterone, catechin and nandrolone were not affected after perfusion with flutamide. Perfusion with diazoxide and U73122 nullified the effect of nandrolone (1 µM) and catechin (1 µM). Nandrolone and testosterone increased (45)Ca(2+) uptake with or without flutamide within 5min. These results indicate that nandrolone and catechin act through a receptor on the plasmatic membrane, as well as testosterone, showing a non-classical pathway in Sertoli cells from immature rat testes.