RESUMEN
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.
Asunto(s)
Epidídimo/metabolismo , Glutatión Peroxidasa/fisiología , ARN Interferente Pequeño/metabolismo , Envejecimiento/metabolismo , Animales , Regulación hacia Abajo , Epidídimo/enzimología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Glutatión Peroxidasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A long and ever-expanding roster of small (â¼20-30 nucleotides) RNAs has emerged during the last decade, and most can be subsumed under the three main headings of microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and short interfering RNAs (siRNAs). Among the three categories, miRNAs is the most quickly expanded group. The most recent number of identified miRNAs is 16,772 (Sanger miRbase, April 2011). However, there are insufficient publications on their primary forms, and no tissue-specific small RNAs precursors have been reported in the epididymis. Here, we report the identification in rats of an epididymis-specific, chimeric, noncoding RNA that is spliced from two different chromosomes (chromosomes 5 and 19), which we named HongrES2. HongrES2 is a 1.6 kb mRNA-like precursor that gives rise to a new microRNA-like small RNA (mil-HongrES2) in rat epididymis. The generation of mil-HongrES2 is stimulated during epididymitis. An epididymis-specific carboxylesterase named CES7 had 100% cDNA sequence homology at the 3'end with HongrES2 and its protein product could be downregulated by HongrES2 via mil-HongrES2. This was confirmed in vivo by initiating mil-HongrES2 over-expression in rats and observing an effect on sperm capacitation.
Asunto(s)
ARN no Traducido/genética , Maduración del Esperma/genética , Animales , Secuencia de Bases , Células Clonales , Clonación Molecular , Regulación hacia Abajo/genética , Epidídimo/metabolismo , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/patología , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Precursores del ARN/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Capacitación Espermática/genética , Factores de TiempoRESUMEN
Identification, detection, and use of small-ribonucleic acid (RNA) molecules have been paid increasing attention in the past decades. Here we show studies of two interrelated lines of this research in a sperm maturation-related organ, the epididymis. First, by using microarray and small-RNA library screening systems, a series of spatially and temporally regulated known and novel small regulatory RNA molecules, referred to as microRNAs, has been identified and characterized. Second, small double-stranded RNAs, called small interfering RNAs, emerged as a powerful tool to knock down gene expression by a pathway known as RNA interference. It has been successfully used to silence epididymal gene expression in vivo to elucidate the function of several sperm motility- and capacitation-related proteins in the rat epididymis.