Glucose Metabolism of Capacitated Spermatozoa
In a first series of experiments, identification of the glucose transporter and metabolic studies were undertaken in a population of capacitated sperm. Using Western blot analysis (Fig. 1), we found that a band migrating at 45 kDa was recognized by anti-GLUT3, indicating that this transporter was expressed in mouse spermatozoa. Mouse sperm proteins did not react with anti-GLUT1, but mouse blastocysts, which were used as positive controls, reacted positively to anti-GLUT1. antibiotic levaquin
The metabolism of glucose through glycolysis and the PPP was measured in sperm after capacitation by using [5-3H]glucose and [1-14C]glucose, respectively. The production of 3H2O and 14CO2 by spermatozoa was first measured as a function of time of incubation and number of spermatozoa. 3H2O (Fig. 2A) and 14CO2 (Fig. 2B) increased linearly with time, but the best time courses were obtained with 1000 spermatozoa. It appears that the levels of glucose metabolized were high through glycolysis and were considerably lower through the PPP: the ratio of the production of 14CO2 from [1-14C]glucose to the production of 3H2O from [5-3H]glucose ranged from 0.5 to 0.7%. The incubation of spermatozoa with [6-14C]glucose lead to cpm values that were not different from the values of the sham preparations, indicating that no detectable levels of 14CO2 were produced with this radiolabel (data not shown).
FIG. 1. GLUT3 expression in mouse sperm. Western blot analysis of GLUT1 (left) and GLUT3 (right) was performed in mouse spermatozoa (0.25 X 1 06/lane). As a positive control, mouse blastocysts (30/lane) were probed for the presence of GLUT1.
FIG. 2. Time courses of glucose metabolism through A) glycolysis and B) the PPP in capacitated sperm. Glucose metabolism was measured in 100, 500, or 1000 capacitated spermatozoa for 1, 2, or 3 h. Each value represents the mean (± SD) of triplicate measurements performed in 2 different males.