Sirtuin 1 (SIRT1), the most conserved nicotinamide adenine dinucleotide-dependent protein deacetylase, is involved in the regulation of energy metabolism, genomic stability, and development. SIRT1 knockout (SIRT1) mice exhibit decreased energy expenditure and hypersensitivity to a high-fat diet (HFD). SIRT1 deficiency in the testis has also been shown to cause male infertility in animal models. Therefore, the present study was conducted to examine the alteration of the testicular function of SIRT1 mice on HFD. Six-week-old mice were fed ad libitum as wild type (WT) and SIRT1 male mice with either a control diet or with HFD for 32 weeks and then were sacrificed. The levels of biomarkers for hepatotoxicity, sex hormones, and cytokines were analyzed in the serum and blood-testis barrier, and the sperm morphology was examined in the testis and epididymal spermatozoa. Interestingly, an enlargement of seminal vesicles was observed in the SIRT1 mice fed with HFD. A significantly higher level of hepatotoxicity was also seen in these mice. The concentration of serum testosterone increased in HFD-fed SIRT1 mice compared to the controls. The levels of interleukin-1β and TNF-α increased in both HFD-fed WT and SIRT1 mice. In RT-PCR, the m RNA expression of tight junction protein 2 and claudin 3 significantly decreased in HFD-fed SIRT1 compared to those of the controls. Degenerative spermatocytes and spermatids were detected in the HFD-fed SIRT1 mice testicular section. Sperm motility decreased in WT and SIRT1 with HFD feeding, and sperm concentration decreased significantly in WT-HFD and SIRT1 mice with or without HFD feeding. Taken together, HFD can alter energy and steroid metabolism in SIRT1-deficient mice, which can lead to imbalances in motility and production of sperm and testosterone that can result in male reproductive disorders.

This study investigated the characteristics of obesity induced by a high-fat diet (HD) over 13 weeks in Rhbdf2 gene knockout (KO) mice. Forty 7-week-old Rhbdf2 wild and KO mice were used and the mice were divided into 4 groups: Wild-ND (n=10, Rhbdf2 wild mice, normal diet (ND)), Wild-HD (n=10, Rhbdf2 wild mice, HD), KO-ND (n=10, Rhbdf2 KO mice, ND) and KO-HD (n=10, Rhbdf2 KO mice, HD). The relative epididymal fat weight in KO-HD was significantly increased compared with that in KO-ND (P<0.01). The relative liver and spleen weights in KO-HD were decreased compared with those in Wild-HD (p < 0.05) and KO-ND (p < 0.01). The mRNA expression of SOD1 in KO-ND was significantly reduced compared with that in Wild-ND (p < 0.05). In Wild-ND and HD, the mRNA expressions of TNF-α and IL-6 in epididymal fat were significantly increased compared with those in KO-ND and HD (p < 0.01). A significant increase of TNF- α and IL-6 mRNA expression was observed in KO-HD compared with KO-ND (p < 0.01). These results indicated that Rhbdf2 genes may regulate high fat diet-induced obesity damage by anti-inflammatory and anti-oxidative roles in fat tissue of mice.

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.