Table 1.
Rat-specific primers sets for RT-PCR analysis.
Figure 1.
Effect of lactate on MCT2, LDH C, MCT4 and LDH A expression in germ cells.
Male germ cells were incubated for 24(10 or 20 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 (A), LDH C (B), MCT4 (C) and LDH A (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., *P<0.05 versus basal.
Figure 2.
Effect of lactate on ROS production in male germ cells.
Male germ cells were incubated for variable periods of time (15, 30 or 60 minutes) in the absence or presence of lactate 20 mM or H2O2 500 µM (positive control) (A), or incubated for 30 minutes in the absence or presence of different doses of lactate (10 or 20 mM) (B). Cell extracts were prepared at the designated intervals and utilized for TBARS assay. Values are expressed as means±S.D. of triplicate incubations in one representative experiment out of three. *P<0.05 versus basal (B).
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).
Table 2.
H2DCFDA positive cells quantification.
Figure 4.
Effect of lactate on P-Akt, P-p38-MAPK and P-Erk1/2 levels in male germ cells.
Male germ cells were incubated for variable periods of time (15, 30 or 60 minutes) in the absence or presence of lactate 10 mM with or without NAC 5 mM or incubated for 15 minutes with H2O2 500 µM. Cell extracts were prepared at the designated intervals and utilized for Western blot analysis using specific antibodies for P-Akt and Akt (A, D), P-p38-MAPK and p38-MAPK (B, E) or P-Erk½ and Erk½ (C). The autoradiographies show a representative experiment out of three. The lower panels show pooled data of three independent experiments indicating the fold variation in phosphorylation (ratio of P-Akt, P-p38-MAPK and P-Erk1/2 to Akt, p38-MAPK and Erk1/2 respectively in each sample) relative to basal (B). Results are expressed as means ± S.D. *P<0.05 versus basal. Different letters indicate statistically significant differences among treatment groups (P<0.05).
Figure 5.
(A) and (B) Mechanisms involved in lactate regulation of ROS levels in male germ cells.
Male germ cells were incubated for 30α-cyano-4-hydroxy-cinnamate (CHC) 10 mM, oxamate (OXA) 10 mM (A), apocynin (APO) 500 µM, allopurinol (ALL) 100 µM or rotenone (ROT) 1 µM (B). Cell extracts were prepared and utilized for TBARS assay. 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). (C) Expression of NOX family members in male germ cells. Total RNA of rat colon (C), skeletal muscle (M), kidney (K), testis (T), Sertoli cells isolated as previously described [44] (SC) and male germ cells (GC) were extracted and analyzed by RT-PCR and visualized by ethidium bromide staining. NT is no template control.
Figure 6.
Proposed mechanism of lactate effects on male germ cell function.
See “Discussion” for details.