RESUMEN
Four acyclic nucleoside triphosphates (derivatives of cytosine, thymine, 7-deazaadenine, and 7-deazaguanine) labeled with nonluminescent europium, terbium, dysprosium, and samarium chelates of 2,2',2'',2'''-[[4-(4-isothiocyanatophenyl)ethyl]pyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetic acid) were applied to minisequencing using two mutations (Delta F 508 and 1717-1 G to A) of cystic fibrosis as a model system. When synthetic targets were used, all four alleles involved could be analyzed in a single reaction using four terminating substrates labeled with four different lanthanide(III) chelates and DELFIA technology for detection. Blood spot samples without DNA isolations were used for PCR amplification and genotyping the target mutations by minisequencing. The single- and dual-labeled minisequencing assays were robust, while the four-label assay still requires further optimization of the multiplexed PCR amplification.
Asunto(s)
Quelantes/química , Elementos de la Serie de los Lantanoides/química , Nucleótidos/química , Nucleótidos/genética , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Genotipo , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa , Coloración y EtiquetadoRESUMEN
Our previous studies have shown that transgenic male mice expressing human P450 aromatase (AROM+) are infertile. In the present study, we followed the testis phenotype up to 15 months of age in these mice. The testes of the old AROM+ mice showed Leydig cell hypertrophy and hyperplasia, as indicated by the staining for steroidogenic enzymes and androgen and estrogen receptors. However, the Leydig cell adenomas did not show signs of malignization. In contrast, we observed a marked increase in the number of activated macrophages in the testicular interstitium of the aging AROM+ mice. The macrophages were further shown to express high levels of CD68 (a monocyte/macrophage marker) and secrete TNFalpha, indicating strong activation, presumably by estrogen exposure. The increased activity of the macrophages was associated with Leydig cell depletion (analyzed at the age of 9 and 15 months) and an increased number of mast cells and fibrosis in the testicular interstitium. Interestingly, similar findings have been made in testes of infertile men. Hence, the aging AROM+ males present with a phenocopy of inflammation-associated infertility in men, providing a model for further studies on the putative link among estrogens, orchitis, and infertility.
Asunto(s)
Aromatasa/genética , Infertilidad Masculina/genética , Inflamación/genética , Testículo/enzimología , Testículo/patología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Aromatasa/biosíntesis , Estrógenos/análisis , Fibrosis , Humanos , Hipertrofia , Inmunohistoquímica , Células Intersticiales del Testículo/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Monocitos/metabolismo , Fenotipo , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermatogénesis , Testículo/metabolismo , Testosterona/análisis , Factores de Tiempo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Heat shock transcription factors (HSFs) regulate expression of heat shock proteins (Hsps). We have previously shown that in zebrafish a unique isoform, zHSF1b, disappears concomitant with heat shock-induced Hsp70 expression. To characterize the role of zHSF1a and zHSF1b isoforms in the regulation of the stress response in vivo, we have carried out cadmium (10-100 microM) and copper (10-30 microM) exposures in order to specify whether the disappearance of HSF1b is specific for heat stress. After 4-h metal exposures we analyzed the expression of hsp70, zHSF1a, zHSF1b and metallothionein (MT) by reverse transcriptase polymerase chain reaction in zebrafish liver, gonads and gills. Although cadmium is a known inducer of Hsps, it did not affect hsp70 expression significantly in the studied tissues. Induction of hsp70 was observed upon copper exposure in liver and gonads, but not in gills. Neither metal affected the zHSF1a/b ratio. Both cadmium and copper exposure caused upregulation of MT, regulator of metal homeostasis and detoxification, confirming that the tissues were subjected to metal loads. Thus, hsp70 appears to be more weakly induced upon metal exposure than in response to heat shock and HSF1 isoforms may participate in stressor-specific regulation of hsp70.