Glucose metabolism was measured in the presence of phloretin and cytochalasin B (Fig. 3), which are inhibitors of facilitative glucose transport. Blocking glucose uptake by 50 ^M cytochalasin B induced an inhibition of both glycolysis and PPP activity by 36% and 46%, respectively. The PPP activity was diminished by 80% in the presence of 200 ^M cytochalasin B (data not shown). In contrast, 0.5 mM phloretin, which induced a glycolysis inhibition of 80%, was unable to induce a decrease in the PPP activity. Iodoacetate was a strong inhibitor of sperm glycolysis at 10 ^M.
Involvement of the PPP in the Fusion Capacity of Spermatozoa
To determine whether the glucose metabolism of the sperm was involved in gamete fusion, the fusing capacity of the sperm was assessed after it had been depressed for 1 h with glucose uptake or glycolysis inhibitors prior to insemination (Table 1). When 0.5 mM phloretin and 10 ^M iodoacetate were used to inhibit glycolysis but not PPP activity, the percentage of penetration and the number of decondensed sperm per oocyte were similar to those of the control. When both glycolysis and the PPP activities were decreased by using 50 ^M cytochalasin B, the fusing capacity of sperm was significantly reduced. A 1-h exposure of sperm to cytochalasin B was required to induce an inhibition of fertilization; when the preincubation time was reduced to 30 min or 0 min, spermatozoa fused with the oocytes as in the control medium. antibiotics levaquin
In the next series of experiments, NADPH, the main product generated by the PPP, was substituted for glucose. Spermatozoa were capacitated in glucose-containing medium, washed to remove glucose, and incubated in glucose-free medium for 1 h to eliminate their ability to fuse with the oocytes. Glucose (5.5 mM) or NADPH (5 mM) was then added to these sperm suspensions before insemination of the zona-free oocytes and was continuously present in the medium during sperm penetration and decondensation. Table 2 shows that the fertilizing capacity of the mouse sperm was rapidly restored by the addition of NADPH or glucose. However, longer incubation (1 h) of the sperm in the presence of NADPH tended to diminish its beneficial effect, and a 2-h preincubation did not result in successful gamete fusion (data not shown).
FIG. 3. Inhibition of the metabolism of glucose through A) glycolysis and B) the PPP in capacitated spermatozoa. Glucose metabolism was measured in 1000 capacitated spermatozoa for 3 h, in the absence or presence of the different inhibitors. Each bar represents the mean levels of metabolized glucose, expressed as a percentage of the control (± SD), the latter being fixed at 100%. Controls were normalized to 100% because of their high variability (± 32%). Triplicate measurements were performed in 4 different males. CytoB, cytochalasin B; IA, iodoacetate. *Significantly different from the control (p < 0.05).
|Treatment||Sperm preincubation time (min)||No. of oocytes||No. of replicates||No. of bound sperm per oocytea||No. of decondensed sperm per penetrated oocytea||Penetration rate (%)a|
|Control||—||163||12||9.0 ± 2.9||1.3 ± 0.3||93 ± 6|
|Cytochalasin B||0||58||4||1.9 ± 1.7b||1.8 ± 0.6||81 ± 29|
|30||65||4||1.5 ± 0.5b||1.5 ± 0.5||76 ± 21|
|60||118||8||6.3 ± 4.9||1.1 ± 0.2||28.7 ± 10b|
|Phloretin||60||55||4||6.8 ± 3.7||2.0 ± 0.9||100 ± 0|
|Iodoacetate||60||64||5||11.9 ± 3.3||1.5 ± 0.5||78 ± 29|
a Mean ± SD.
b Significantly different from the control (p < 0.05).
TABLE 2. Restoration of the fertilizing ability of sperm by NADPH and glucose after preincubation in glucose-free medium.
|Treatmenta||Sperm preincubation time (min)||No. of oocytes||No. of replicates||No. of bound sperm per oocyteb||No. of decondensed sperm per penetrated oocyteb||Penetration rate (%)b|
|No glucose||60||78||5||16.3 ± 6.4||1.0 ± 0||7 ± 4|
|NADPH||15||77||5||8.7 ± 3.8||1.5 ± 0.7||78 ± 33c|
|60||71||5||7.6 ± 2.0||1.2 ± 0.4||53 ± 38|
|Glucose||60||73||5||8.0 ± 3.6||1.9 ± 0.9||94 ± 5c|
a All sperm suspensions were incubated for 1 h in glucose-free medium before the addition of NADPH or glucose. b Mean ± SD.
c Significantly different from the control without glucose (p < 0.05).