RESUMEN
Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the ß-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of ß-defensins in the epididymis.
Asunto(s)
Andrógenos/farmacología , Embrión de Mamíferos/anatomía & histología , Epidídimo/crecimiento & desarrollo , Conductos Mesonéfricos/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismo , Animales , Embrión de Mamíferos/metabolismo , Epidídimo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Células Intersticiales del Testículo/metabolismo , Masculino , Músculo Liso/metabolismo , Orquiectomía , Especificidad de Órganos , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Few studies have focused on experimental testosterone deprivation in immature animals. Therefore, this study used sexually immature rats aiming to evaluate the testes and epididymis histology and proteins expression in these organs on PND50 and 75, after premature antiandrogen exposure, from PND21 to 44. Although the androgen deprivation from pre-puberty up to peripuberty did not alter the histological organization of the testes and epididymis either at puberty or at adulthood, the treatment impaired the expression of specific proteins in epididymal tissue at puberty and adulthood (androgen receptor, calmodulin, Rab11A). These changes may be related to impaired epididymal function, sperm quality and fertility capacity as observed in a previous study. Further studies are necessary to better investigate the molecular mechanisms involved in the impairment on reproductive competence of male rats after precocious hormonal injury.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Epidídimo/efectos de los fármacos , Flutamida/farmacología , Maduración Sexual/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Calmodulina/metabolismo , Epidídimo/anatomía & histología , Epidídimo/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Desglicasa DJ-1 , Ratas , Ratas Wistar , Receptores Androgénicos/metabolismo , Testículo/anatomía & histología , Testículo/metabolismo , Testosterona/antagonistas & inhibidores , Testosterona/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine-rich protein containing both Kunitz and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down-regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen-dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility.
Asunto(s)
Genitales Masculinos/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Maduración del Esperma , Espermatozoides/metabolismo , Andrógenos/metabolismo , Animales , Encéfalo/metabolismo , Castración , Células Cultivadas , Epidídimo/metabolismo , Masculino , Proteínas Inhibidoras de Proteinasas Secretoras/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteínas de Secreción de la Vesícula Seminal/genética , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Vesículas Seminales/metabolismo , Motilidad Espermática , Cola del Espermatozoide/metabolismo , Conducto Deferente/metabolismoRESUMEN
Androgens are steroid hormones that play key roles in the development and maintenance of male phenotype and reproductive function. These hormones also affect the function of several non-reproductive organs, such as bone and skeletal muscle. Endogenous androgens exert most of their effects by genomic mechanisms, which involve hormone binding to the androgen receptor (AR), a ligand-activated transcription factor, resulting in the modulation of gene expression. AR-induced non-genomic mechanisms have also been reported. A large number of steroidal and non-steroidal AR-ligands have been developed for therapeutic use, including the treatment of male hypogonadism (AR agonists) and prostate diseases (AR antagonists), among other pathological conditions. Here, the AR gene and protein structure, mechanism of action and AR gene homologous regulation were reviewed. The AR expression pattern, its in vivo regulation and physiological relevance in the developing and adult testis and epididymis, which are sites of sperm production and maturation, respectively, were also presented.
Os androgênios são hormônios esteroides com papel fundamental no desenvolvimento e na manutenção do fenótipo masculino e da função reprodutiva. Esses hormônios também afetam a função de diversos tecidos não reprodutivos, como, por exemplo, o ósseo e musculoesquelético. Os androgênios endógenos exercem a maioria de suas funções por mecanismo genômico, que envolve a ligação do hormônio ao receptor de androgênio (RA), um fator de transcrição ativado por ligante, o que resulta no controle da expressão gênica. Mecanismos não genômicos também têm sido associados aos efeitos induzidos pelo RA. Um grande número de ligantes do RA, esteroidais e não esteroidais, tem sido desenvolvido para o uso terapêutico, incluindo o tratamento do hipogonadismo masculino (agonistas do RA) e de doenças da próstata (antagonistas do RA), entre outras condições patológicas. Neste trabalho, foram discutidas as características estruturais básicas do RA (gene e proteína), os mecanismos de ação desse receptor, bem como aspectos relacionados à sua regulação homóloga. O padrão de expressão do RA, sua regulação in vivo e relevância fisiológica durante o desenvolvimento e a vida adulta na função do testículo e epidídimo, tecidos responsáveis pela produção e maturação de espermatozoides, respectivamente, também foram discutidos.
Asunto(s)
Adulto , Humanos , Masculino , Genitales Masculinos/metabolismo , Receptores Androgénicos/fisiología , Factores de Edad , Epidídimo/metabolismo , Regulación de la Expresión Génica , Receptores Androgénicos/genética , Testículo/metabolismoRESUMEN
Androgens are steroid hormones that play key roles in the development and maintenance of male phenotype and reproductive function. These hormones also affect the function of several non-reproductive organs, such as bone and skeletal muscle. Endogenous androgens exert most of their effects by genomic mechanisms, which involve hormone binding to the androgen receptor (AR), a ligand-activated transcription factor, resulting in the modulation of gene expression. AR-induced non-genomic mechanisms have also been reported. A large number of steroidal and non-steroidal AR-ligands have been developed for therapeutic use, including the treatment of male hypogonadism (AR agonists) and prostate diseases (AR antagonists), among other pathological conditions. Here, the AR gene and protein structure, mechanism of action and AR gene homologous regulation were reviewed. The AR expression pattern, its in vivo regulation and physiological relevance in the developing and adult testis and epididymis, which are sites of sperm production and maturation, respectively, were also presented.
Asunto(s)
Genitales Masculinos/metabolismo , Receptores Androgénicos/fisiología , Adulto , Factores de Edad , Epidídimo/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Receptores Androgénicos/genética , Testículo/metabolismoRESUMEN
This study reports the genomic organization of the rhesus alpha(1A)-adrenoceptor gene (ADRA1A). Full-length cloning of rhesus ADRA1A splice variants was achieved by combining PCR screening of a seminal vesicle cDNA library and 5'-RACE assays with total RNA from seminal vesicle. The classical ADRA1A mRNA (ADRA1A_v1) and six full-length ADRA1A splice variants were identified representing transcripts that code for functional (ADRA1A_v1, ADRA1A_v2a, ADRA1A_v3a, ADRA1A_v3d, ADRA1A_v3e) and truncated (ADRA1A_v2c and ADRA1A_v3c) receptor isoforms. Comparative analysis of the deduced amino acid sequence indicated that rhesus ADRA1A_i1 isoform (corresponding to the ADRA1A_v1 transcript) shares high identity to the amino acid sequence present in the classical alpha(1A)-adrenoceptor from human and other mammalian species. Partial nucleotide sequences for rhesus alpha(1B)-(ADRA1B) and alpha(1D)-adrenoceptor (ADRA1D) transcripts were also characterized. RT-PCR studies indicated differential distribution of all ADRA1A-related splice variants as well as ADRA1B and ADRA1D mRNAs, in tissues from rhesus and human male reproductive tract. Immunohistochemistry revealed alpha(1A)-adrenoceptor (ADRA1A_i1) immunostaining in smooth muscle cells and epithelial cells of rhesus efferent ductules, epididymis and seminal vesicle. Taken together the present results demonstrate that the complexity of the splicing mechanisms involved in the regulation of the ADRA1A gene is not restricted to human and is a common characteristic among Old World monkeys.
Asunto(s)
Perfilación de la Expresión Génica , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Epidídimo/metabolismo , Humanos , Inmunohistoquímica , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Próstata/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Seminales/metabolismo , Testículo/metabolismoRESUMEN
The biological effects of catecholamines in mammalian pigment cells are poorly understood. Our previous results showed the presence of alpha(1)-adrenoceptors in SK-Mel 23 human melanoma cells. The aims of this work were to (1) characterize catecholamine effects on proliferation, tyrosinase activity and expression, (2) identify the alpha(1)-adrenoceptor subtypes, and (3) verify whether chronic norepinephrine (NE) treatment modified the types and/or pharmacological characteristics of adrenoceptors present in SK-Mel 23 human melanoma cells. Cells treated with the alpha(1)-adrenergic agonist, phenylephrine (PHE, 10(-5) or 10(-4) M), for 24-72 h, exhibited decreased cell proliferation and enhanced tyrosinase activity, but unaltered tyrosinase expression as compared with the control. The proliferation and tyrosinase activity responses were inhibited by the alpha(1)-adrenergic antagonist prazosin, suggesting they were evoked by alpha(1)-adrenoceptors. The presence of actinomycin D, a transcription inhibitor, did not diminish PHE-induced effects. RT-PCR assays, followed by cloning and sequencing, demonstrated the presence of alpha(1A)- and alpha(1B)-adrenoceptor subtypes. NE-treated cells (24 or 72 h) were used in competition assays, and showed no significant change in the competition curves of alpha(1)-adrenoceptors as compared with control curves. Other adrenoceptor subtypes were not identified in these cells, and NE pretreatment did not induce their expression. In conclusion, the activation of SK-Mel 23 human melanoma alpha(1)-adrenoceptors elicit biological effects, such as proliferation decrease and tyrosinase activity increase. Desensitization or expression of other adrenoceptor subtypes after chronic NE treatment were not observed.