Optimizing a Male Reproductive Aging Mouse Model by D-Galactose Injection

Abstract

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8-10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging.

Keywords: aging; male infertility; mouse model.

Figure 1

Model of male reproductive aging optimized through d-Galactose (d-Gal) injections. (A) Mice divided into five groups: the vehicle group (Control: Phosphate-buffered saline, PBS, administered over eight weeks) and the d-Gal-injected groups, namely G1 (d-Gal dosage of 100 mg/kg over six weeks), G2 (d-Gal dosage of 100 mg/kg over eight weeks), G3 (d-Gal dosage of 200 mg/kg over six weeks) and G4 (d-Gal dosage of 200 mg/kg over eight weeks). The mice were injected intraperitoneally daily for six or eight weeks. The bars indicate the ages of the mice in weeks; (B) The serum of mice injected with d-Gal showed lower superoxide dismutase) SOD activity (SOD is a critical antioxidant enzyme) than did the serum of those administered PBS. One-way Analysis of variance (ANOVA) test: “ns” denotes nonsignificant; bars represent the mean ± SD; * p < 0.05; ** p < 0.01.

Figure 1

Model of male reproductive aging optimized through d-Galactose (d-Gal) injections. (A) Mice divided into five groups: the vehicle group (Control: Phosphate-buffered saline, PBS, administered over eight weeks) and the d-Gal-injected groups, namely G1 (d-Gal dosage of 100 mg/kg over six weeks), G2 (d-Gal dosage of 100 mg/kg over eight weeks), G3 (d-Gal dosage of 200 mg/kg over six weeks) and G4 (d-Gal dosage of 200 mg/kg over eight weeks). The mice were injected intraperitoneally daily for six or eight weeks. The bars indicate the ages of the mice in weeks; (B) The serum of mice injected with d-Gal showed lower superoxide dismutase) SOD activity (SOD is a critical antioxidant enzyme) than did the serum of those administered PBS. One-way Analysis of variance (ANOVA) test: “ns” denotes nonsignificant; bars represent the mean ± SD; * p < 0.05; ** p < 0.01.

Figure 2

Effects of superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) in testis from mice treated with d-Gal. (A) Mice injected with d-Gal l showed decreased SOD activity in testis compared to those administered PBS; (B) mice injected with d-Gal showed increased TBARS activity in testis (TBARS is a type of lipid peroxidation) compared to those administered PBS. One-way ANOVA test: bars represent the mean ± SD; Student’s t-test: “ns” denotes nonsignificant; * p < 0.05.

Figure 3

Effects of d-Gal injection on mice (I). (A) Body weights from the different groups are similar; (B) testis weights/body weights from the d-Gal-injected groups show decreases compared to those of the control group; (C) d-Gal-injected groups showed a trend of decreasing sperm counts (×10⁶) compared to the control group; (D) the d-Gal-injected mice exhibited an increase in immotile sperm (%) compared to those administered PBS. One-way ANOVA test: bars represent the mean ± SD; “ns” denotes nonsignificant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Effects of d-Gal injection on mice (II) (A) d-Gal-injected mice showed increased abnormal sperm morphology (%) compared to the control group. One-way ANOVA test: bars represent the mean ± SD; ** p < 0.01; *** p < 0.001; **** p < 0.0001; (B) Increased abnormal sperm morphology (%) (arrow: sperm with tail defects; arrowhead: head defects) from mice injected with d-Gal (bottom) compared to those administered PBS. 4,6-diamidino-2-phenylindole (DAPI): blue; Mito Tracker: green (magnification: ×400).

Figure 5

Mouse model of male reproductive aging induced by d-Galactose injection. The d-Gal-injected mice demonstrated decreased SOD activity and increased lipid peroxidation amount. Further, SOD accumulation affects the expression of spermatogenic genes (e.g., Cycl2 and Katnb1) and results in the decreased sperm count and increased ratio of immotile and abnormal sperm morphology.