PMC

Addition of creatine increases ATP levels in sperm and enhances sperm motility in vitro. (A) Changes in ATP levels of sperm after culture in HTF medium with/without creatine. Sperm were collected from the murine epididymis and then treated with 0 (control), 200, 500 or 1000 μM creatine. The experiment was repeated four times using a total of four male mice. Values represent the mean ± SD of four replicates. *P < 0.05 compared with the level of the control. (B) Effects of creatine antagonist GPA (0, 50, or 500 μM) on ATP levels in sperm after culture in HTF medium containing 500 μM creatine. Sperm were collected from the mouse epididymis and cultured in each treatment group. The experiment was repeated four times using sperm from a total of four male mice. Values represent the mean ± SD of four replicates. Different superscripts denote significant differences among the GPA concentrations at each time point (P < 0.05). Control: Sperm were cultured without creatine and GPA. (C) Percentage of motile sperm in HTF medium with/without creatine. Sperm were collected from the murine epididymis and incubated in HTF medium with/without creatine for a maximum of 6 h. The numbers of motile and total sperm were counted at each time point. The percentage of motile sperm was then calculated. The experiment was repeated four times using a total of four male mice. Values represent the mean ± SD of four replicates. (D) Percentages of motile sperm in HTF medium containing creatine with/without creatine antagonist GPA. Sperm were collected from the mouse epididymis and cultured in HTF medium containing 500 μM creatine with various doses of creatine antagonist GPA (0, 50 or 500 μM) for a maximum of 6 h. The numbers of motile and total sperm were counted at each time point. The percentage of motile sperm was then calculated. The experiment was repeated four times using a total of four male mice. Values represent the mean ± SD of four replicates. (E) Percentages of swim-up sperm in HTF medium with/without creatine. Sperm were collected from the murine epididymis and cultured in HTF medium with/without creatine for a maximum of 6 h. The numbers of sperm in the upper layer and total sperm were counted at each time point. The percent of swim-up sperm was then calculated. The experiment was repeated four times using a total of four male mice. Values represent the mean ± SD of four replicates. (F) Percentages of swim-up sperm in HTF medium containing creatine with/without creatine antagonist GPA. Sperm were collected from the mouse epididymis and cultured in HTF medium containing 500 μM creatine with various doses of creatine antagonist GPA (0, 50 or 500 μM) for a maximum of 6 h. The numbers of sperm in the upper layer and total sperm were counted at each time point. The percentage of swim-up sperm was then calculated. The experiment was repeated four times using a total of four male mice. Values represent the mean ± SD of four replicates. Different superscripts denote significant differences among the GPA concentrations at each time point (P < 0.05).

Effects of Tat, mutant-Tat and creatine on levels of creatine and phosphocreatine. (A) Creatine (Cr, 3 mM) application resulted in significantly increased cellular levels of creatine in the absence (p<0.01) or presence of Tat (p<0.001). Tat and mutant-Tat (mTat) at concentrations of 100 nM did not significantly affect levels of creatine. (B) Treatment of cortical neurons with creatine, Tat and mutant-Tat did not produce statistically significant changes in levels of phosphocreatine (PCr). (C) Ratios of phosphocreatine to creatine were decreased significantly by creatine (Cr, p<0.01), Tat (p<0.05), and creatine plus Tat (p<0.001). Co-application of creatine and Tat decreased significantly (p<0.001) the ratio of phosphocreatine to creatine to a level significantly (p<0.05) less than that observed with Tat alone (* p < 0.05, ** p < 0.01, *** p ≤ 0.001 versus control).

ATP consumption by creatine kinases. 1 μM of each of the four purified isoforms of creatine kinase were incubated for 30 min with (ATP+/Cr+) or without (ATP−/Cr-) 50 μM of ATP and 5 mM of creatine. Luminescence was measured using red-luciferase and normalized by positive control (without creatine kinase, with 50 μM of ATP and 5 mM creatine). Standard deviation represent three independent experiments. Ø – without creatine kinase; CKB – brain creatine kinase; CKM – muscle creatine kinase; uMtCK – ubiquitous mitochondrial creatine kinase; sMtCK – sarcomeric mitochondrial creatine kinase

The creatine kinase circuit. Creatine can be produced endogenously by some cells or taken up from the circulation via the creatine transporter (SLC6A8). Creatine is then phosphorylated from adenosine triphosphate (ATP) to form phosphocreatine and adenosine diphosphate (ADP) in a reaction catalyzed by ubiquitous mitochondrial creatine kinase (uMt-CK). Isoforms of creatine kinase in the cell cytosol (mainly brain-type creatine kinase CKBB) are also linked to glycolytic enzymes (G) to generate phosphocreatine and ADP from glycolytic ATP. Then, when required for energy-dependent cellular processes, cytosolic isoforms of creatine kinase (CKBB) hydrolyze the bond between creatine and the phosphate group stored as phosphocreatine, thus regenerating ATP and creatine. Note: muscle-type cytosolic creatine kinase (CKMM) expression has also been detected in mouse oocytes.