Lactate Regulates Rat Male Germ Cell Function through Reactive Oxygen Species
Figure 3
Participation of ROS in lactate actions in male germ cells.
Male germ cells were incubated for 30(A). Values are expressed as means±S.D. of triplicate incubations in one representative experiment out of three. Different letters indicate statistically significant differences among treatment groups (p<0.05). Male germ cells pre-loaded with 10 µM 2′,7′-dichlorofluorescin diacetate (H2DCFDA) were incubated for 30 minutes without (Basal) or with lactate 10 mM in the absence or presence of N-acetylcysteine (5 mM). After incubation, cells were centrifuged and then placed on a glass slide for observation at ×400 magnification. A positive control was included (H2O2 500 µM for 15 minutes) (B). Images are representative of three independent experiments. Scale bar: 50 µm. Male germ cells were incubated for 24 hours in the absence or presence of lactate 10 mM without or with NAC 1 or 5 mM. Total cellular RNA was then extracted and Northern blot analysis was performed using 10 µg RNA per lane. Membranes were hybridized with labeled cDNA probes for MCT2 (C) and LDH C (D). The upper panels show a representative experiment out of three. The lower panels show pooled data of three independent experiments performed indicating the fold variation in mRNA levels (ratio of mRNA to 18S in each sample) relative to basal (B). Results are expressed as means±S.D. Different letters indicate statistically significant differences among treatment groups (P<0.05).