Abstract
In mammals, Sirt1, a member of the sirtuin family of proteins, functions as a nicotinamide adenine dinucleotide-dependent protein deactylase, and has important physiological roles, including the regulation of glucose metabolism, cell survival, and mitochondrial respiration. The initial investigations of Sirt1 deficient mice have revealed a phenotype that includes a reduced lifespan, small size, and an increased frequency of abnormal sperm. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (−/−) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Numbers of mature sperm and spermatogenic precursors, as early as d15.5 of development, are significantly reduced (∼2-10-fold less; P≤0.004) in numbers in Sirt1−/− mice, whereas Sirt1 deficiency did not effect the efficiency oocyte production following superovulation of female mice. Furthermore, the proportion of mature sperm with elevated DNA damage (∼7.5% of total epididymal sperm; P = 0.02) was significantly increased in adult Sirt1−/− males. Analysis of global gene expression by microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and viable offspring in in vitro fertilization (IVF) experiments using gametes from Sirt1−/− and sibling Sirt1+/− mice. While viable animals were derived in both Sirt1−/− X wild type and Sirt1−/− X Sirt1−/− crosses, the efficiency of producing both 2-cell zygotes and viable offspring was diminished when IVF was performed with Sirt1−/− sperm and/or oocytes. Together, these data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function.
Highlights
The Sir (Silent Information Regulator) genes were originally discovered in yeast (Saccharomyces cerevisiae) where they were shown to affect expression of genes, due to their ability to promote heterochromatin, at the HML and HMR mating type loci [1]
Using in vitro fertilization (IVF), we show that both male and female gametes from Sirt1 deficient mice have a reduced efficiency at generating viable zygotes, these zygotes are fully capable of developing to term following embryo transfer to pseudo-pregnant females
To assess the affect of Sirt1 deficiency on germ cell development, we initially examined germ cell number in sibling Sirt12/2 and Sirt1+/2 male and female mice
Summary
The Sir (Silent Information Regulator) genes were originally discovered in yeast (Saccharomyces cerevisiae) where they were shown to affect expression of genes, due to their ability to promote heterochromatin, at the HML and HMR mating type loci [1]. While the exact nature of the neuro-protective effects of Sirt on neurons is presently unknown, it likely involve inhibition of apoptosis by the interaction of Sirt with a number of different proteins involved in the survival response to stress, including p53 [6] FOXO transcription factors [7] NF-KB[15], E2F1 [16], and Ku70 [17]. In mouse embryonic fibroblasts derived from Sirt knock-out mice, Sirt deficiency promotes extension of replicative lifespan, and survival following exposure to genotoxic stress [21]. Together these data suggest that the effect of Sirt deficiency is pleiotropic and dependent on cell type and/or stage of development. Using in vitro fertilization (IVF), we show that both male and female gametes from Sirt deficient mice have a reduced efficiency at generating viable zygotes, these zygotes are fully capable of developing to term following embryo transfer to pseudo-pregnant females
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