Ethics statement

Thirty-day-old Sprague–Dawley rats were obtained from an animal care unit (Animal Care Laboratory, Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina). Animals were killed by CO₂ asphyxiation according to protocols for animal laboratory use following the principles and procedures outlined in the National Institute of Health Guide for Care and Use of Laboratory Animals. The protocol was approved by the Ethical Commitee from the Instituto de Biología y Medicina Experimental (Ref.: CE 031/2013, IByME).

Germ cell isolation and culture

Germ cells were isolated as previously described [13]. Testes were decapsulated and digested with 0.1% collagenase (C0130 Sigma-Aldrich) and 0.006% soybean trypsin inhibitor (T9003 Sigma-Aldrich) in Hanks' balanced salt solution (HBSS) for 5 minutes at room temperature. The collagenase solution was diluted 4-fold with HBBS and seminiferous tubules allowed to sediment for 2 minutes. The supernatant was discarded and the tubular pellet was washed twice with gentle shaking. Seminiferous tubules were cut into 2 mm segments and then digested with 0.05% collagenase, 0.003% soybean trypsin inhibitor and 0.003% deoxyribonuclease (DN25, Sigma-Aldrich) for 15 minutes at room temperature, while carefully transferring the suspension from one tube to another with a pipette. The suspension was diluted with one volume HBSS and material allowed to sediment for 5 minutes. The supernatant was transferred to a tube containing sufficient 2% bovine serum albumin (BSA) to make the final concentration 0.2% BSA. The suspension was allowed to settle for 10 minutes. Germ cells remaining in suspension were collected by centrifugation at 400×g for 3 minutes at 4°C. The resulting pellet was washed twice with HBSS containing 0.2% BSA and 0.003% deoxyribonuclease. The final cell pellet was resuspended in a 1∶1 mixture of Dulbecco's Modified Eagle's Medium-Ham's F-12 Medium with the addition of 15 mM NaHCO₃, 100 IU/ml penicillin, 2.5 mg/ml amphotericin B, 20 mM Hepes, pH 7.4 (DMEM-F12) and seeded on a discontinuous four-layer (20%, 25%, 32%, 37%) Percoll density gradient. The gradient was centrifuged at 800×g for 30 minutes at 4°C. The fractions at the 25%–32% interface was collected. To remove Percoll, 4 volumes of DMEM-F12 were added and centrifugation at 400×g for 5 minutes at 4°C performed. Germ cells were resuspended in DMEM-F12 supplemented with 10 µg/ml transferrin, 5 µg/ml insulin, 5 µg/ml vitamin E and 4 ng/ml hydrocortisone. Germ cell preparations were seeded at a density of 2×10⁶ cell/ml in tissue culture flasks and cultured at 34°C in a mixture of 5% CO₂∶95% air for 18 hours. During this initial period, the few Sertoli cells contaminating the germ cell preparation attached to the plastic surface. Purified germ cells were obtained by carefully removing the medium and centrifuging at 400×g for 5 minutes at 4°C, and resuspended in Minimum Essential Media without pyruvate and L-alanine (MEM) supplemented with15 mM NaHCO₃, 100 IU/ml penicillin, 2.5 µg/ml amphotericin B, 20 mM Na Hepes, pH 7.4, 10 µg/ml transferrin and 4 ng/ml hydrocortisone.

In order to characterize the germ cell types present in the suspension, the preparation was evaluated by flow cytometry to measure the DNA content as previously described [14]. Cells were resuspended in DMEM-F12 supplemented with 50% fetal bovine serum and fixed in ice-cold 70% ethanol. Propidium iodide was added to fixed cells to a final concentration of 50 mg/ml. Flow cytometry was performed using a FACS Caliber (Becton Dickinson). The preparation contained 27% tetraploid cells (spermatocytes) and 63% haploid cells (spermatids). Furthermore, the preparation contained small proportions of diploid cells (10%) that may represent contamination with somatic cells and spermatogonia.

Culture conditions

Purified germ cells were plated on 10 cm² dishes (5×10⁶ cells/condition) and treatment with lactate 10–20 mM or H₂O₂ 500 µM was performed in the absence or presence of N-acetylcysteine (1 or 5 mM), rotenone (1 µM), apocynin (500 µM), allopurinol (100 µM), α-cyano-4-hydroxy-cinnamate (10 mM) or oxamate (10 mM). Germ cells incubated for 15, 30 or 60 minutes were used for Thiobarbituric Acid Reactive Substances assay or Western blot analysis. Germ cells incubated for 24 hours were used for Northern blot analysis.

Thiobarbituric Acid Reactive Substances (TBARS) assay

Phospholipid oxidation was determined by the colorimetric assay of TBARS [15]. Treated germ cells (5×10⁶ cells) collected by centrifugation at 400×g for 5 minutes at 4°C were resuspended with 80 µl PBS containing 0.4% w/v butylated hydroxytoluene on ice and then disrupted by ultrasonic irradiation. An aliquot (25 µl) of total cell extract (corresponding to 450 µg protein) was added to 175 µl mixed reaction solution (0.15% w/v SDS, 0.5 N HCl, 0.75% w/v phosphotungstic acid and 0.175% w/v 2-thiobarbituric acid). The mixture was heated in a boiling water bath for 45 minutes. TBARS were extracted with 200 µl of n-butanol. After a centrifugation at 10000×g for 5 minutes at 4°C, the absorbance at 532 nm of the butanolic phase was measured. A calibration curve was performed using malondialdehyde (MDA), generated from 1,1,3,3-tetramethoxypropane (0.4–8 µM), as standard to express the absorbance changes as nmol MDA/µg protein.

Reactive oxygen species (ROS) assay

Germ cells (2×10⁶/condition) were loaded with the dye 2′,7′-dichlorodihydrofluorescin diacetate (H₂DCFDA; Sigma-Aldrich) (10 µM, 15 minutes) and then treated for 30 minutes with lactate 10 mM in the absence or presence of N-acetylcysteine (5 mM)or for 15 minutes with H₂O₂ 500 µM in phenol red-free Minimum Essential Media without pyruvate and L-alanine. After incubation, cells were centrifuged at 400×g for 5 minutes, resuspended in 75 µl phenol red-free Minimum Essential Media without pyruvate and L-alanine, placed on a glass slide and visualized and photographed under a fluorescent light microscope (Axiophot; Carl Zeiss, Jena, Germany) [16], [17]. The percentage H₂DCFDA-positive germ cells was calculated as (H₂DCFDA-positive cells/total germ cells)×100.

Western blot analysis

Treated germ cells were collected by centrifugation at 400×g for 5 minutes at 4°C and resuspended in 200 µl lysis buffer (Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% w/v Triton X100, 100 mM NaF, 10 mM Na₄P₂O₇, 10 mM Na₃VO₄ and complete Mini, EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany)) and incubated for 30 minutes on ice. Cell extracts were obtained by centrifugation at 16000×g for 30 minutes at 4°C. An appropriate volume of 4× Laemmli buffer (8% w/v SDS, 40% v/v glycerol, 20% v/v 2-mercaptoethanol, 0.008% w/v bromophenol blue and 250 mM Tris-HCl, pH 6.8) was added and thoroughly mixed. Samples were immersed in a boiling water bath for 5 minutes and then immediately settled on ice. Proteins (150 µg per lane seeded) were resolved in 10% SDS-PAGE (10% acrylamide/bisacrylamide for the resolving gel and 4.3% acrylamide/bisacrylamide for the stacking gel) in a Mini Protean 3 Cell (Bio-Rad, Hercules, CA, USA). After SDS-PAGE, gels were equilibrated in transfer buffer (25 mM Tris pH 8.3, 192 mM glycine, 20% v/v methanol) for 10 minutes and electrotransferred at 100 V for 60 minutes onto polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Bucks, UK) using a Mini Trans-Blot Cell (Bio-Rad). Membranes were probed with commercial antibodies (Phospho-Akt (Ser473) Antibody, Akt Antibody, Phospho-p38-MAPK (Thr180/Tyr182) Antibody, p38-MAPK Antibody, Phospho-Erk1/2 (Thr202/Tyr204) Antibody and Erk1/2 Antibody; Cell Signaling Technology, Inc., Danvers, MA, USA) that allow specific recognition of phosphorylated (P-Akt, P-p38-MAPK and P-Erk1/2) and total (Akt, p38-MAPK and Erk1/2) proteins. The intensities of autoradiographic bands were estimated by densitometric scanning using NIH Image software (Scion Corporation, Frederick, MD, USA).

Northern blot analysis

Northern blot analysis was carried out in total RNA samples isolated from treated germ cells. Extraction was performed using TRI Reagent (Sigma-Aldrich) according to the manufacturer's recommendations. The amount of RNA was estimated by spectrophotometry at 260 nm. About 10 µg total RNA was electrophoresed on a 1% agarose-10% formaldehyde gel. After migration, RNAs were transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Bucks, UK) by capillary transfer with 10× SSC (10× stock solution: 1.5 M NaCl and 0.15 M sodium citrate, pH 7.4) and fixed with U.V. Stratalinker (Stratagene Cloning Systems, La Jolla, CA, USA). cDNA probes were labeled with [α-³²P]deoxy-CTP (NEN, Perkin Elmer Life and Analytical Sciences, Boston, MA, USA) using a random-primed labeling kit (Prime-a-Gene Labeling System, Promega Corporation, Madison, USA). The cDNA probes used were the following: rat LDH C, a 141b probe previously obtained using a RT-PCR technique with specific primes (5′-ACGGTCATCCTTGTTTCTTAAC-3′ and 5′-TTCATCTGGAGCTAGGTTCTGA-3′, Accession Number: "type":"entrez-nucleotide","attrs":"text":"NM_017266","term_id":"51036642","term_text":"NM_017266"NM_017266); rat MCT2 1.5 Kb insert, HindIII-BamHI [18], and rat MCT4 1.7 Kb insert, HindIII-BamHI [19] (kindly gifted by Dr. Bröer, Canberra Australia); LDH A, a rat 3′UTR 0.4 kb insert, Pst I-Bgl II [20] (kindly gifted by Dr. Jungmann, Chicago USA) and a 18S oligonucleotide. Blots were prehybridized for 3 hours at 42°C in 50% w/v formamide, 0.75 M NaCl, 20 mM sodium phosphate (pH 7.5), 1 mM EDTA, 5× Denhart's solution, 10% w/v dextran sulfate, 0.5% w/v SDS and 100 µg/ml herring sperm DNA. Hybridization was then performed overnight at 42°C in the same hybridization buffer containing 1–4×10⁶ c.p.m./ml ³²P-labeled probe. Membranes were washed utilizing different astringency conditions depending on the probe utilized. Membranes were exposed to Hiperfilm ECL (GE Healthcare UK Limited, Buckinghamshire, UK). The 18S signal was used to standardize mRNA contents. The intensities of autoradiographic bands were estimated by densitometric scanning using NIH Image software (Scion Corporation, Frederick, MD, USA).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Colon, skeletal muscle, kidney and testicular tissue, as well as, purified Sertoli and germ cell from thirty day-old rat were utilized to isolate total RNA using TRI Reagent (Sigma-Aldrich) according to the manufacturer's recommendations. The amount of RNA was estimated by spectrophotometry at 260 nm. Reverse transcription (RT) was performed on 2 µg RNA at 42°C for 50 minutes using 200 U SuperScript II reverse transcriptase enzyme (Invitrogen, Carlsbad, CA, USA) containing 125 ng random primer and 0.5 mM dNTP Mix. The cDNAs enconding NOX1, NOX2 and NOX4 were amplified from 1 µl of the cDNA reaction mixture using specific gene primers (table 1). PCR was performed with GoTaq DNA polymerase (Promega Corporation, Madison, USA) under the following conditions: initial denaturation at 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds; 55°C, 57°C and 56°C (NOX1, NOX2 and NOX4 respectively) for 30 seconds and extension at 72°C for 50 seconds followed by 10 minutes at 72°C. The PCR products were resolved by 2% agarose gel and stained with ethidium bromide.

Other assays

Protein content in male germ cells lysates was determined by Lowry's assay [21].

ROS mediate lactate regulation of Akt- and p38-MAPK-signalling pathways

As mentioned in the introduction, recent findings suggest that the initiation and/or proper functioning of several signal transduction pathways, such as PI3K/Akt, p38-MAPK and Erk1/2, may be involved in the mechanism of action of ROS [7], [8], [9]. In this context, the redox-dependent protein modification has been recognized as an important mechanism in signal transduction [34]. Considering these observations, we designed experiments to analyze the ability of lactate to regulate cell signaling pathways and a possible participation of ROS in this regulation. For this purpose, male germ cells were incubated in the absence or presence of 10 mM lactate for variable periods of time and the levels of phosphorylated Akt (P-Akt), p38-MAPK (P-p38-MAPK) and Erk1/2 (P-Erk1/2) were determined. Figures 4A and 4B show that lactate promoted a time-dependent increase in P-Akt and in P-p38-MAPK levels. On the other hand, Figure 4C shows that lactate did not modify P-Erk1/2 levels. These figures also show that incubation of germ cells with 500 µM H₂O₂ for 15 minutes increased P-Akt, P-p38-MAPK and P-Erk1/2 levels.

Object name is pone.0088024.g004.jpg" title="Click on image to zoom" class="tileshop" src="/pmc/articles/PMC3909278/bin/pone.0088024.g004.jpg"/>

Open in a separate window

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).

In order to analyze a possible role of ROS in lactate regulation of P- Akt and P-p38-MAPK levels we combined lactate treatment with NAC. Figure 4D and 4E show that NAC partially inhibited the ability of lactate to raise P-Akt and P-p38-MAPK levels. The fact that H₂O₂ but not lactate activated Erk1/2 may be related to different levels of ROS attained by both treatments or alternatively to the specificity of the response. Even though H₂O₂ is widely used as an experimental exogenous ROS, it has to be kept in mind that treatment of a cell with H₂O₂ may not adequately reflect endogenous ROS signaling. For instance, exogenous H₂O₂ produces broad signaling responses in endothelial cells, which include the activation of Erk1/2, JNK and p38-MAPKs. In contrast, the EGF-stimulated signaling response in the above-mentioned cell type, which is known to be mediated by ROS, is restricted to the mitogenic Erk1/2 pathway [35].

The authors express their gratitude to Dr Bröer (Canberra, Australia) for providing MCT-2 and MCT-4 cDNA, and to Dr Jungmann (Chicago, USA) for providing LDH A cDNA. The technical help of Mercedes Astarloa is gratefully acknowledged. We also thank Celia Nieto for revising our English usage. MNG, MFR, SBM, and SBC are established investigators of CONICET. Besides, MNG is a teaching assistant at the Departamento de Bioquímica Humana, Facultad de Medicina, UBA.