RESUMEN
BACKGROUND: The majority of beta-defensin family members are exclusively expressed in the epididymis, and some members have been shown to play essential roles in sperm maturation and fertility in rats, mice and humans. Therefore, beta-defensins are hypothesized to be potential targets for contraception and infertility diagnosis and treatment. Clarifying the regulatory mechanisms for the expression of these genes is necessary. Androgen/androgen receptor (AR) signaling plays an important regulatory role in epididymal structure and function. However, very little is known about the androgenic regulation on the production and secretion of the epididymal beta-defensins. METHODS: The expression of beta-defensins was detected by quantitative RT-PCR. The androgen dependence of beta-defensins was determined by bilateral orchiectomy and androgen supplementation. The androgen response elements (AREs) in the promoters of beta-defensins were identified using the MatInspector software. The binding of AR to AREs was assayed by ChIP-PCR/qPCR. RESULTS: We demonstrated that 23 mouse caput epididymal beta-defensins were differentially regulated by androgen/androgen receptor. Six genes, Defb18, 19, 20, 39, 41, and 42, showed full regulation by androgens. Ten genes, Defb15, 30, 34, 37, 40, 45, 51, 52, 22 and Spag11a, were partially regulated by androgens. Defb15, 18, 19, 20, 30, 34, 37, 39, 41, 42, 22 and Spag11a were associated with androgen receptor binding sites in their promoter or intronic regions, indicating direct regulation of AR. Six genes, Defb1, 12, 13, 29, 35, and spag11b/c, exhibited an androgen-independent expression pattern. One gene, Defb25, was highly dependent on testicular factors rather on androgens. CONCLUSIONS: The present study provides novel insights into the mechanisms of androgen regulation on epididymal beta-defensins, enabling a better understanding of the function of beta-defensins in sperm maturation and fertility.
Asunto(s)
Andrógenos/farmacología , Epidídimo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Receptores Androgénicos/metabolismo , Elementos de Respuesta/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , beta-Defensinas/metabolismo , Andrógenos/administración & dosificación , Andrógenos/química , Andrógenos/metabolismo , Animales , Sitios de Unión , Castración , Inmunoprecipitación de Cromatina , Biología Computacional , Epidídimo/metabolismo , Inyecciones Intraperitoneales , Intrones/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/efectos de los fármacos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgénicos/química , Espermatogénesis/efectos de los fármacos , Propionato de Testosterona/administración & dosificación , Propionato de Testosterona/química , Propionato de Testosterona/metabolismo , Propionato de Testosterona/farmacología , beta-Defensinas/agonistas , beta-Defensinas/antagonistas & inhibidores , beta-Defensinas/genéticaRESUMEN
The α-(1,2) fucosyltransferases (Fut1 and Fut2) and α-(1,3) fucosyltransferases (Fut4, Fut9) are responsible for the synthesis of Lewis X (LeX) and Lewis Y (LeY) conjugated to glycoproteins. We recently reported that these fucosyltransferases were differentially expressed in the reproductive tract of male mouse. Here, we studied the effect of androgen on fucosyltransferase expression through the use of mouse castration models. We found that Fut1 mRNA and Fut4 mRNA were upregulated, while Fut2 mRNA and Fut9 mRNA were downregulated by androgen in the caput epididymis. However, in the vas deferens and prostate, only Fut4 mRNA and Fut2 mRNA were respectively upregulated following exposure to androgen. In the seminal vesicle, all fucosyltransferases, with the exception of Fut9, were upregulated. We identified the androgen receptor binding sites (ARBSs) of Fut2, Fut4 and Fut9 in the caput epididymis. Luciferase assay for these ARBSs is able to provide an indication as to why Fut4 and Fut9 are differently expressed and regulated by androgen, although they catalyze the same α-(1,3) fucose linkage. Our study showed that androgen could differentially regulate the expression of these fucosyltransferases and provided an insight into the characteristic distribution of each fucosyltransferase responsible for LeX/LeY biosynthesis in the male reproductive tract.
Asunto(s)
Andrógenos/genética , Fucosiltransferasas/biosíntesis , Andrógenos/metabolismo , Animales , Sitios de Unión , Epidídimo/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Unión Proteica , ARN Mensajero/biosíntesis , Reproducción/genética , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
The mammalian epididymis not only plays a fundamental role in the maturation of spermatozoa, but also provides protection against various stressors. The foremost among these is the threat posed by oxidative stress, which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids, proteins, and nucleic acids. In mice, the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5 (GPX5) as a major luminal scavenger in the proximal caput epididymidal segment. Accordingly, the loss of GPX5-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx5-/- mice. To explore the underlying mechanism, we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged (13 months old) Gpx5-/- mice. This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts, including the downregulation of a subgroup of piRNA pathway genes, in aged Gpx5-/- mice. In agreement with these findings, we also observed the loss of piRNAs, which potentially bind to the P-element-induced wimpy testis (PIWI)-like proteins PIWIL1 and PIWIL2. The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5-/- mice. Importantly, the oxidative stress response genes tend to have more targeting piRNAs, and many of them were among the top increased genes upon the loss of GPX5. Taken together, our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible involvement in the aging and oxidative stress-mediated responses.