Apoptosis, necrosis and autophagy are influenced by metabolic energy sources in cultured rat spermatocytes

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(1)Apoptosis (2012) 17:539–550 DOI 10.1007/s10495-012-0709-2. ORIGINAL PAPER. Apoptosis, necrosis and autophagy are influenced by metabolic energy sources in cultured rat spermatocytes Ximena Bustamante-Marı́n • Clara Quiroga Sergio Lavandero • Juan G. Reyes • Ricardo D. Moreno. •. Published online: 7 April 2012 Ó Springer Science+Business Media, LLC 2012. Abstract Apoptosis, necrosis and autophagy are mechanistically related processes that control tissue homeostasis and cell survival. In the testis, germ cell death is important for controlling sperm output, but it is unknown whether or not germ cells can switch from apoptosis to necrosis, as has been reported in other tissues. Furthermore, autophagy has not been reported in spermatogenesis. Spermatocytes (meiotic cells) and spermatids (haploid cells) use lactate rather than glucose as their primary substrate for producing ATP. The metabolism of glucose, but not lactate, reduces ATP levels and increases intracellular [H?] and [Ca2?], Electronic supplementary material The online version of this article (doi:10.1007/s10495-012-0709-2) contains supplementary material, which is available to authorized users. X. Bustamante-Marı́n R. D. Moreno (&) Departamento de Fisiologı́a, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile e-mail: [email protected] Present Address: X. Bustamante-Marı́n Departamento Biomédico, Facultad de Ciencias de la Salud, Universidad de Antofagasta, Antofagasta, Chile C. Quiroga S. Lavandero Centro Estudios Moleculares de la Celula, Facultad Ciencias Quimicas y Farmaceuticas/Facultad Medicina, Universidad de Chile, Santiago, Chile C. Quiroga S. Lavandero Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, Texas, USA J. G. Reyes Instituto de Quı́mica, Pontificia Universidad Católica de Valparaı́so, Valparaı́so, Chile. both of which are associated with apoptosis and/or necrosis in somatic cells. In this work, we evaluated whether different energy sources, such as lactate or glucose, can influence spermatocyte death type and/or survival in primary cultures. Spermatocytes cultured for 12 h without an energy source died by necrosis, while spermatocytes cultured with 5 mM glucose showed a significant increase in apoptosis, as evidenced by caspase activity, TUNEL assay and phosphatidylserine exposure. Apoptosis was not observed in spermatocytes cultured with 5 mM lactate or deoxyglucose. Authophagy markers, such as LC3-II and autophagosomes, were detected after 12 h of culture, regardless the culture conditions. These results suggest that the availability of glucose and/or lactate affect the type of death or the survival of primary spermatocytes, where glucose can induce apoptosis, while lactate is a protective factor. Keywords Spermatogenesis Energetic metabolism Cell death Caspase Calcium. Introduction Successful sperm production is a delicate balance between germ cell survival, differentiation and cell death, and the upregulation of apoptosis has been associated with a decrease in sperm production in infertile patients [1–4], whereas its inhibition arrests spermatogenesis in mice [5]. Thus, apoptosis is an important mechanism regulating sperm production, but its physiological and molecular basis in male germ cells is still far from understood. Conventionally, three types of cell death have been described [6–8]. Programmed cell death type 1, or apoptosis, is characterized by the activation of caspases,. 123.

(2) 540. the externalization of phosphatidylserine, and internucleosomal DNA fragmentation. Caspases-3, -6 and -7 are called executioner caspases since they degrade many intracellular proteins such as nuclear laminin, actin and poly-(ADP-ribose) polymerase (PARP) [6, 9, 10]. Executioner caspases are proteolytically activated by initiator caspases, such as caspases-8, -9 and -10, which, in turn, are activated by the ligand-induced oligomerization of death receptors or by the release of mitochondrial cytochrome c along with dATP and APAF-1 [10, 11]. Programmed cell death type 2, or autophagy, is an evolutionarily conserved process involved in the degradation of misfolded proteins and excess or dysfunctional organelles [12, 13]. There are three types of autophagy: macro-autophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy (herein autophagy) delivers cytoplasmic cargo proteins into lysosomes by means of double-membrane vesicles or autophagosomes. This process is initiated when a class III phosphatidylinositol 3-kinase complex and autophagy-related proteins (ATG proteins) form a membrane with which to isolate organelles [8, 14]. Microtubule-binding protein light chain 3 (LC3), a protein present in autophagosomes, is synthesized in an inactive form (LC3-I) that is later converted to an active membranous form (LC3-II) by proteolytic processing and the attachment of phosphatidylethanolamine [14, 15]. The LC3-II protein binds to isolation membranes and, in collaboration with ATG5-ATG12ATG16 complexes and beclin-1 (ATG6), drives the membrane alterations required for autophagosome formation. Finally, necrotic death, or cell death type III, is characterized by cytoplasm swelling, the release of cytoplasmic enzymes, such as lactate dehydrogenase (LDH), and the breakdown of organelles, involving few nuclear changes [13, 16]. Many studies have shown that these three different types of cell death are mechanistically interconnected, and that the inhibition of one of them (i.e. apoptosis, by preventing the activation of caspases) can lead to necrosis or autophagy. Germ cells experience differentiation-related changes in their carbohydrate metabolism, where meiotic (spermatocytes) and post-meiotic cells (spermatids) use lactate, rather than glucose, as their energy source [17, 18]. Glucose and lactate were shown to modulate intracellular free calcium ([Ca2?]i) and pHi levels in pachytene spermatocytes and spermatids [19, 20]. These two intracellular parameters, [Ca2?]i and pHi, have been shown to play crucial roles in cell death and differentiation in somatic cells and during spermatogenesis [21–26]. In addition, apoptotic cells in seminiferous tubules are positive for periodic Schiff staining (PAS), which is widely used to detect glycogen and glucose, suggesting that they have a high content of sugars [27]. It is possible that the metabolic substrates supplied by Sertoli cells such as glucose or. 123. Apoptosis (2012) 17:539–550. lactate could regulate spermatogenesis, not only in terms of energy-dependent processes, but also by tilting the balance between the survival and death of germ cells. Thus, the aim of our study was to evaluate in vitro whether glucose or lactate influence cell death in isolated rat spermatocytes.. Materials and methods Animals Twenty-four-day-old male Sprague–Dawley rats were acquired from the Animal Facility of the Facultad Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile. The rats were housed under a 12 h light:12 h dark cycle, with water and rat chow provided ad libitum. The investigations were conducted in accordance with the rules laid down by the Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching and by the National Research Council. All animal protocols were endorsed by the Chilean National Fund for Science and Technology (FONDECYT). Spermatocyte isolation Prepubertal 24-day-old male rats were sacrificed by cervical dislocation and both testis were extracted, decapsulated and digested with 0.5 mg/ml of collagenase I (Sigma, St. Louis, MO, USA) and 0.4 lg/ml DNAse (Sigma, St. Louis, MO, USA) for 15 min at 30°C in KrebsHenseleit buffer (KHB) containing 110 mM NaCl, 4.2 mM KCl, 0.35 mM KH2PO4, 0.5 mM CaCl2, 1,6 mM MgCl2, 24 NHCO3 and 10 mM HEPES, supplemented with 5 mM L-lactate (KHB-Lac) (Sigma, St. Louis, MO, USA). The resulting seminiferous tubules were washed three times in KHB-Lac medium and then mechanically disrupted with a pipette in the presence of 0.4 lg/ml DNAse. The cell suspension was filtered through nylon meshes with 250 lm and then 70 lm openings (Small Parts, Inc. Miramar, FL), and were subsequently washed once in KHB-Lac medium. The spermatocytes were resuspended in 1.5 ml of KHBLac containing 0.7% (v/v) DNAse and 5% (v/v) BSA in Percoll (Sigma, St. Louis, MO, USA). Then, the spermatocytes were concentrated through Percoll gradients (adapted from Van Pelt et al. [28]). Briefly, isotonic and iso-osmotic Percoll solution was prepared containing 90% Percoll in KHB-Lac. A discontinuous density gradient was made by diluting the iso-osmotic Percoll suspension with KHB-Lac containing 0.2 lg/ml DNAse. The percentages of Percoll were, from top to bottom; 10, 20, 25, 30 and 40%. The cell suspension was layered on top. The gradient was centrifuged at 5009g for 30 min and 18°C. Cells collected at the interphase between 25% and 30% were identified as.

(3) Apoptosis (2012) 17:539–550. 541. spermatocytes with 75% purity. Contaminating cells were mostly Sertoli cells and spermatids.. % cell death ¼ ðexperimentally released LDH=. Spermatocyte culture. DNA fragmentation. The isolated spermatocytes (5 9 106 cells/ml) were plated in KHB medium supplemented with one of the following; 5 mM lactate (KHB-Lac), 5 mM glucose (KHB-Glu); 5 mM deoxyglucose (DOG) (KHB-DOG) or without substrate (KHB-w/oS), plus 10 ml/l essential and non-essential amino acids, 1 mM glutamine (Invitrogen, Carlsbad, CA) and 1 9 antibiotic–antimycotic (Invitrogen, Carlsbad, CA, USA). The cells were cultured from 0 to 30 h in a humidified chamber at 32°C with 5% CO2. The medium was replaced once after 15 h.. A TUNEL assay was used to determine the extent of DNA fragmentation (DeadEnd Fluorometric System, Promega Corporation, Madison, WI). Cultured spermatocytes recovered after 0, 12 and 24 h were attached to coverslips covered with poly-L-lysine and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. After three washes with phosphate buffered saline (PBS), the protocol was carried out following the manufacturer’s instructions. Finally, the samples were mounted with Vectashield (Vector Laboratories. Inc. Burlingam, CA) and observed under an Olympus BH2 epifluorescense microscope (Olympus, Tokyo, Japan). The images were captured using a Nikon 4500 digital camera (Nikon Coro, Tokyo, Japan). The TUNEL-positive cells in random fields were quantified as a percentage with respect to the total number of spermatocytes in the field. At least 50 cells were counted each time.. Spermatocyte cell death assays Trypan blue incorporation Trypan blue (Sigma, St. Louis, MO, USA) incorporation is widely used as a tool for evaluating cell death in cultured cells because it is only incorporated into cells after plasma membrane damage. A volume of 10 ll (0.1% w/v) trypan blue was added to each plate at different times throughout culture (0–30 h) and incubated for an additional 10 min. Trypan blue incorporation was recorded in five random fields using an Olympus digital camera (model C-5060, Japan) in a light field microscope (Olympus CKX41, Center Valley, PA). The percentage of blue cells with respect to the total number of spermatocytes present in the field was determined using the Image J 4.0 program. At least 150 cells were counted each time. Lactate dehydrogenase (LDH) activity The release of LDH was used as another method for determining the percentage of cell death (mainly necrosis). To this end, 5 9 105 spermatocytes/ml, counted in an haemocytometer chamber, were cultured for 0, 3, 6, 12, 24 and 30 h, and the supernatants were collected and centrifuged at 2509g for 4 min. The LDH activity in the supernatant was determined using the CytoTox 96Ò Non-Radioactive Cytotoxicity Assay (Promega Corporation, Madison, WI), measuring the absorbance at 490 nm in an ELISA reader (model EL 310 Boots CellTech Diagnostics Inc., Slough, UK). Lactate dehydrogenase activity was used to determine the percentage of cell death according to the manufacturer’s instructions. In order to determine the maximum liberation of LDH, 5 9 105 cells/ml were exposed to 30 ll of lysis buffer and the maximum liberation of LDH was determined. The results are expressed as the percentage of cell death, as described in the following equation:. maximum released LDHÞ 100. Annexin V-PI assay Phosphatidylserine exposure and membrane rupture in cultured spermatocytes were evaluated by flow cytometry using the Annexin V-FITC and propidium iodide (PI) kit following the manufacturer’s recommendations (Invitrogen, Carlsbad, CA), with some modifications. Spermatocytes (1 9 106 cells/ml) were recovered by centrifugation at 1009g at 4°C after 0, 12 and 24 h of culture, and carefully resuspended in 400 ll of annexin binding buffer. The suspension was split into four tubes, of 100 ll each. One tube was used to evaluate autofluorescence, the second was incubated with 0.5 lM propidium iodide (PI), another with annexin V-FITC, and the fourth with Annexin V-FITC plus 0.5 lM PI. The samples were incubated for 5 min at 4°C. The fluorescence was evaluated in a Coulter Epics XL cytometer (GMI Inc. Ramsey, MN), and 10,000 gated events were acquired for each condition. All data were analysed using FCS express V2.0 software (De Novo Software, Los Angeles, CA, USA). Caspase activity Isolated spermatocyte suspensions were homogenized in RIPA buffer containing 150 mM NaCl, 1 mM EDTA, 10 lg/ml PMSF, 1% Triton X-100 and 20 mM Tris–HCl, pH 7.4. The colourimetric substrates for caspase-3 (Ac-DEVD-pNA), caspase-8 (Ac-IETD-pNA) and caspase9 (Ac-LEHD-pNA) were purchased from Merck (Darmstadt, Germany). Upon caspase activity, pNA was released. 123.

(4) 542. and produced a yellow colour, which was measured by a spectrophotometer at 405 nm. The amount of product generated was calculated by extrapolation in a standard curve of free pNA. One international unit (IU) was defined as the amount of caspase required to hydrolyse 1 lmol of pNA/min at 25°C. The results of specific activity are expressed in international units per milligram of protein (IU/mg protein). Protein extraction and Western blot assay Protein extraction was performed by homogenizing a suspension of 5 9 106 spermatocytes/ml in RIPA buffer and centrifuging it for 10 min at 16,0009g at 4°C. The samples were run on a 7% polyacrylamide gel (SDS-PAGE) under reducing and denaturing conditions and then transferred to a nitrocellulose sheet at 400 mA for 2 h. The nitrocellulose membrane was blocked with 5% (w/v) non-fat milk, 0.1% Tween in TBS, pH 7.4, and then incubated overnight at 4°C with the following primary antibodies: 0.2 lg/ml anti-PARP (Santa Cruz, Biotechnology, CA, USA) and 0.6 lg/ml anti b-actin (Sigma, St. Louis, MO, USA). The membranes were then incubated with a secondary antibody conjugated with horseradish peroxidise, obtained from KPL (Gaithersburg, MD), diluted 1:3000 in blocking solution, for 1 h at room temperature, and antigen–antibody complexes were detected using the Super Signal West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). In order to detect LC3-I, LC3-II and Beclin-1, 50 lg protein was resolved on a 15% SDS-PAGE at 60 mV for 5 h. The proteins were then transferred to a PVDF membrane. We used a polyclonal rabbit anti-LC3 for LC3-I and LC3-II detection (1:1,000, Cell Signaling, Beverly, MA, USA), whilst the levels of Beclin-1 were evaluated using 0.2 lg/ml polyclonal goat anti-beclin antibody (Santa Cruz Biotechnology, CA, USA). We used b-actin as a loading control (0.6 lg/ml, Sigma, St. Louis, MO). Densitometric analysis was performed using Image J 4.0 software, and the results were expressed as the ratio between LC3-II and LC3-I to b-actin band intensities. Transmission electron microscopy In order to evaluate the presence of autophagosomes, 106 spermatocytes/ml, cultured for 0, 6 and 24 h, were recovered by centrifugation at 809g for 3 min. The supernatant was then discarded and the pellet was fixed for 6 h in 2% glutaraldehyde prepared in 0.136 M sodium cacodilate buffer, pH 7.2. Then, the cells were washed three times for 30 min in sodium cacodilate buffer. Post-fixation was carried out using 1% osmium tetroxide diluted in sodium cacodilate buffer for 90 min. The pellet was washed three times with distilled H2O for 30 min, incubated with 1%. 123. Apoptosis (2012) 17:539–550. aqueous uranil acetate for 1 h, and then dehydrated with acetone. The cellular pellet was pre-included over night with a 1/1 mixture of acetone and epoxy resin. The following day the mixture was replaced by pure resin and was allowed to include for 4 h. After the addition of fresh resin, it was left to polymerize in a stove at 60°C for 24 h. Slices (60–90 nm of thickness) were cut using a Sorvall MT 5000 ultra microtome (Thermo Scientific, Waltham, MA, USA). The slices were dyed with 4% uranil acetate in methanol for 1 min and then exposed to lead citrate for 5 min, according to the protocol described by Reynolds [29]. The slices were observed under a Phillips Tecnai 12 electron microscope (FEI, Hillsboro, OR, USA). The images were acquired using Kodak electron image film SO-163 and digitalized with a Cannon 9950F scanner at 300 dpi. Intracellular Ca2? measurements in spermatocytes in suspension Cells were cultured for 0, 3 and 24 h with different metabolic substrates followed by incubation (3 9 106 cells/ml) with 5 lM acetoxymethyl Fura-2 (Invitrogen, Carlsbad, CA, USA) for 1 h at 32°C under a 95% O2 and 5% CO2 atmosphere. Next, the cells were washed three times in KHB and maintained at 4°C until used (within the next 15-30 min). The measurements were performed in a Fluoromax-2 fluorometer (Jobin Ivon-Spex, Edison, NJ) using a ratiometric method described by Grynkiewicz [30]. Following this, Fura-2 was calibrated by lysing cells with 20 mg/ml digitonin in a medium containing 0.5 mM Ca2? (Fmax), with the subsequent addition of a final concentration of 5 mM EGTA (pH 7.4) (Fmin). The Kd value for the fura-2/Ca2? to be used (250 nM) was interpolated from data in a study by Larsson et al. [31]. The [Ca2?]i for 0, 3 and 24 h incubation times were obtained as the mean of a continuous 8 min [Ca2?]i measurement. In order to determine the [Ca2?] released from intracellular stores (ICa2?S), 1 lM of cyclopiazonic acid (CPA, Sigma, St. Louis, MO), an inhibitor of the SERCA pump, was added to the spermatocytes and the levels of [Ca2?]i were quantified as described; [ICa2?S] was determined by subtracting the [Ca2?]i determined in the presence and absence of CPA. Statistical analysis We used analysis of variance (ANOVA) and a Tukey posttest for comparisons of the mean. Significance between the slopes was evaluated through ZAR’s statistical analysis [32]. The values of n correspond to the number of independent experiments. The cells were obtained from the testis of three rats and each determination was carrying out in duplicate. The results are presented as the mean ± SEM..

(5) Apoptosis (2012) 17:539–550. Results Glucose induces cell death in a concentrationdependent manner The freshly isolated rat spermatocytes (t = 0) were round with a diameter of 18.0 ± 0.5 lm, and only a small percentage of them (4.1 ± 0.1%) incorporated trypan blue (Fig. 1a–b; Table 1). The morphology of the spermatocytes cultured for 24 h in KHB-Lac was similar to that observed at t = 0, and 26.6 ± 3.8% of cell incorporated trypan blue. In contrast, about 40% of cells cultured in KHB-w/oS or KHB-Glucose showed trypan blue incorporation, along with vesicles inside the cytoplasm (Fig. 1a, b, arrow head). However, although the percentage of cell death was similar under both conditions, debris and fragmentation were more. 543. apparent in the cells cultured in KHB-w/oS compared to those cultured in KHB-Glucose. There was a rapid increase in the percentage of dead cells (about 30%) during the first 3 h of culture under all experimental conditions, and this remained similar throughout for those incubated in KHB-Lac (Fig. 1b). Spermatocytes in KHB-Glu, KHB-DOG or KHB-w/oS showed a significantly higher percentage of cell death (about 40%) than those in KHB-Lac, starting at 6 h, which remained similar throughout the entire study period (Fig. 1b, asterisks). Regardless of the culture conditions, the spermatocytes showed a rapid increase in cell death within the first 3 h of culture and, afterwards, the rate of apoptosis changed between the different conditions. This initial and non-specific cell death could have been a consequence of the initial stress conditions that the cells were subjected to. Fig. 1 The effect of metabolic energy sources on spermatocyte cell death. Spermatocytes were cultivated between 0 and 30 h in KHB-Lac, -Glu, -DOG or w/oS. a Evaluation of cellular death with trypan blue at time zero (t = 0) and after 30 h. Bar 20 lm, 9400. Dead spermatocytes (blue cells, arrow heads) were quantified and the percentage of cell death was determined at different times. The insert shows a dead (blue) cell. b The death kinetics of spermatocytes cultured in medium supplemented with Lac (circles), Glu (squares), DOG (rhombuses) or w/oS (triangles). Each point corresponds to the mean ± S EM (n = 6), *p \ 0.05 (DOG, Glu and w/oS compared to Lac). Asterisks indicate that the rate of cell death in spermatocytes cultured in Lac was significantly lower than in other experimental conditions (Color figure online). 123.

(6) 544. Apoptosis (2012) 17:539–550. Table 1 Percentages of cell death of spermatocytes cultures at t = 0 and for 24 h Annexin V? (%). Condition. Annexin V?-IP? (%). LDH (%). Trypan Blue (%). TUNEL? (%). t=0. 4.6 ± 0.6. 19.3 ± 10.6. 3.7 ± 0.5. 4.1 ± 0.1. 6.4 ± 3.9. Lac. 5.6 ± 2.7. 16.1 ± 5.1. 11.8 ± 0.3. 26.6 ± 3.8. 5.5 ± 6.6. Gluc. 10.2 ± 2.3. 29.4 ± 9.1. 21.1 ± 1.0. 43.1 ± 3.0. 22.2 ± 5.5. Gluc/Lac. 4.0 ± 1.5. 22.8 ± 11.6. ND. 29.6 ± 2.1. ND. w/oS. 12.4 ± 4.2. 14.8 ± 11.4. 33.0 ± 1.0. 44.5 ± 4.8. ND. DOG. 2.1 ± 0.4. 14.6 ± 0.8. ND. 49.5 ± 7.3. 9.3 ± 3.1. ND not determined. Table 2 Influence of metabolic energy sources on cell death rate Condition. Velocity (% death/h). SD. Significant respect to 0 (p \ 0.05). Lac. 0.32. 0.16. No. Glu. 0.69. 0.23. Yes. w/oS. 0.94. 0.26. Yes. DOG. 1.10. 0.25. Yes. throughout the purification procedure, thus requiring an ‘‘acclimatization period’’ of 3 h in culture in order to ensure that any response was specifically due to the different experimental conditions. Thus, the specific cell death rate (the slope of cell death versus time) was calculated between 3 and 24 h of culture for each condition (Fig. 1b; Table 2). Our results showed that the slope for spermatocytes cultured in KHB-Lac was not statistically different from zero, which means that the proportion of dead cells did not increase during the culture period (Table 2). In contrast, all of the other conditions (KHB-Glu, -w/oS and -DOG) showed slopes significantly greater than zero, which means that there was a progressive increase in the proportion of dead cells between 3 and 24 h of culture (Table 2). Next, we studied the effect of glucose concentration on spermatocyte death (Fig. 2a). Spermatocytes cultured with 0.1 mM Glu for 24 h showed a percentage of dead cells (20 ± 6%) that was similar to that observed in 5 or 10 mM Lac (26 ± 4% or 26 ± 7%, respectively). The rate of cell death tended to increase when the spermatocytes were cultured with 0.5 or 1 mM Glu, but the values were not significantly different from those cultured in 5 or 10 mM Lac (Fig. 2a). However, 5 or 10 mM Glu caused a significant increase in cell death (44 ± 11% and 42 ± 9%, respectively), similar to the cultures w/oS (46 ± 5%, Fig. 2a). In order to further establish whether or not spermatocyte death was due to the presence of Glu or the absence of Lac, we cultured spermatocytes in KHB supplemented with different Glu and Lac concentration combinations (Lac/Gluc) for 24 h and then cell death was assayed by trypan blue exclusion. Our results showed that when the concentration of Glu and Lac was the same. 123. Fig. 2 Lactate is necessary for spermatocyte survival. Freshly isolated spermatocytes were cultured for 0 and 24 h. a Dose–response curve. Spermatocytes were cultured with different concentrations (mM) of Lac, Glu or w/oS (n = 4). b Spermatocytes were cultured with different Glu/Lac ratios: R1 (10/10 mM), R2 (5/10 mM), R4 (2.5/10 mM), R10 (1/10 mM) and in the presence of 10 mM lactate (Lac10) or glucose (Glu10) and w/oS (n = 3). Cell death was evaluated by trypan blue incorporation. Each bar shows the mean ± S EM *p \ 0.05. (10 mM Glu and 10 mM Lac, R1), and when Glu was 10 times less concentrated than Lac (1 mM Glu and 10 mM Lac, R10), the percentage of dead cells was lower than in cultures with Glu alone or w/oS (Fig. 2b). These results suggest that concentrations of 5 mM Glu or higher are only harmful to cultured spermatocytes at low concentrations or in the absence of Lac. Apoptosis and necrosis in cultured spermatocytes In order to evaluate plasma membrane damage and possible necrosis, the release of LDH was evaluated in the supernatant after 3, 6, 12, 24 and 30 h of culture in KHBLac, KHB-Glu or KHB-w/oS. The results showed that after 6 h of culture a significant increase in cell death, was.

(7) Apoptosis (2012) 17:539–550. 545. detected for spermatocytes cultured in KHB-Glu (15.0 ± 1.0%) and KHB-w/oS (29.5 ± 0.5%) compared to those cultured with KHB-Lac (5.5 ± 0.9%). These differences were even higher after 30 h of culture (Fig. 3a). In order to determine whether or not spermatocytes undergo apoptosis, we first evaluated DNA fragmentation by TUNEL under the different culture conditions. Our results showed that freshly isolated spermatocytes had 6.4 ± 1.7% of TUNEL(?) cells. After 24 h of culture in KHB-Lac, this value did not change significantly (Fig. 3b, c; Table 1). However, when the spermatocytes were cultured for the same period of time with KHB-Glu (5 mM), the percentage of TUNEL(?) cells reached 22 ± 2% (p \ 0.05) (Fig. 3b, c; Table 1). Surprisingly, the percentage of TUNEL(?) cells was only 9 ± 2% in the spermatocytes cultured for 24 h with KHB-DOG. In order to determine whether or not this DNA fragmentation was associated with apoptosis, we assessed phosphatidylserine externalization using the binding of Annexin V along with PI incorporation in live spermatocytes by flow cytometry. The percentage of Annexin V-FITC(?) cells was similar in those cultured in the presence of KHB-Glu (10 ± 2%) or KHB-w/oS (12 ± 4%) (Fig. 3d; Table 1). In both cases, the percentages of Annexin V-FITC(?) cells were significantly higher than those cultured in KHB-Lac (6 ± 3%, Table 1). In agreement with our previous results using TUNEL, only 2.1 ± 0.4% of cells cultured in KHB-DOG. were Annexin V-FITC(?) (Fig. 3d; Table 1). The percentage of double-positive cells (secondary apoptosis) was similar in the spermatocytes cultured in KHB-Glu or KHBLac/Glu, and significantly higher than those cultured in KHB-Lac, -w/oS or -DOG (Table 1). Since caspase activation is an early event in apoptosis, we evaluated caspase activity at different times throughout culture. Our results showed that after 12 h of culture the specific activities of caspase-3 and caspase-8 in spermatocytes cultured in KHB-Glu were significantly higher than in freshly purified spermatocytes or in spermatocytes cultured for 12 h with KHB-Lac (Fig. 4a). Caspase-3 activity was also significantly higher in spermatocytes cultured in KHB-w/oS. Spermatocytes cultured with KHB-DOG showed an increase in the activities of caspase-3 and 8, but this was not significantly different from the freshly isolated spermatocytes. Caspase-9 activity was low and showed no difference between the different conditions studied. Furthermore, the irreversible caspase inhibitor zVAD-fmk was able to prevent cell death in spermatocytes cultured with KHB-Glu (Fig. 4b). Finally, the cleaved form of PARP (a caspase-3 substrate) was significantly greater in spermatocytes cultured in KHB-Glu than in those cultured in DOG or w/oS, as shown by the densitometric analysis of Western blots (Fig. 4c). Therefore, our results clearly show that in the absence of Lac, spermatocytes cultured with 5 mM Glu. Fig. 3 Glucose induces apoptosis in cultured spermatocytes. a Necrosis was determined by measuring LDH activity between 0 and 30 h in the supernatant of each culture supplemented with 5 mM Lac (circles, red line), 5 mM Glu (squares, blue line) or w/oS (triangles, black line). Each point corresponds to the mean ± SEM, (n = 3). *Statistical. differences with respect to the lactate condition (p \ 0.05). Spermatocytes were cultivated for 0 and 24 h in the presence of 5 mM Lac, Glu or DOG. Apoptosis was evaluated by TUNEL (b, c) and d Annexin V (?) cells evaluated by flow cytometry. Each bar shows the mean ± SEM, n = 3, *p \ 0.05. Bar 20 lm (Color figure online). 123.

(8) 546. Apoptosis (2012) 17:539–550. Fig. 4 Caspase activation in spermatocytes cultured without lactate. Caspase activity was evaluated by colorimetric assays. a Activity of caspases-8, -3 and -9 in spermatocytes cultured in the presence of different experimental conditions for 0 and 12 h. b Caspase inhibitor z-VAD-fmk dose–response curve. Spermatocytes were cultured in KHB-Glu for 30 h in the presence or absence of different concentrations of z-VAD-fmk, and cell death was assessed by trypan blue (n = 3). c PARP cleavage detection in spermatocytes cultured for 12 h. Densitometric analysis of three independent experiments. Each bar represents the mean ± SEM, *p \ 0.05. undergo apoptosis and necrosis, whereas those incubated w/oS primarily die by necrosis. Autophagy in cultured spermatocytes Autophagy in cultured spermatocytes was studied via the detection of LC3-I and its proteolytically active fragment (LC3-II) by Western blots (Fig. 5). The results showed that in freshly purified spermatocytes (t = 0) or after 2 and 4 h of culture, there was no cleavage of LC3-I into LC3-II (Suppl. Fig. 1). The presence of LC3-I and LC3-II was detected under all study conditions after 12 and 24 h (Fig. 5a–b). The levels of LC3-II were greater after 24 h than 12 h of culture under all study conditions, except in the spermatocytes cultured in KHB-DOG (Fig. 5a). In order to corroborate these results, the autophagosomes were visualized by transmission electron microscopy in cultured spermatocytes. The results showed that at 24 h of culture, double membrane vesicles, indicative of autophagy vesicles, were visualized in spermatocytes cultured in KHB-Lac and KHB-Glu, in agreement with our results obtained from the Western blots (Fig. 5c). These results showed that the spermatocytes underwent autophagy under all of the culture conditions tested. Intracellular calcium levels in cultured spermatocytes The levels of [Ca2?]i in spermatocytes cultured for 3 h with KHB-Glu (47 ± 2 nM), KHB-DOG (92 ± 4 nM) or KHB-w/oS (72 ± 2 nM) were significantly higher than those cultured with KHB-Lac (22 ± 1 nM) or t = 0. 123. (28 ± 1 nM,) (Fig. 6a). Spermatocytes cultured for 24 h showed a [Ca2?]i higher than at 3 h under all conditions(Fig. 6b). However, the level of [Ca2?]i in spermatocytes cultured with KHB-Lac was significantly lower (53 ± 2 nM) than in those cultured with KHB-Glu (140 ± 3 nM), DOG (230 ± 6 nM) or KHB-w/oS (200 ± 5 nM). In order to determine the amount of [Ca2?] released from intracellular stores ([ICa2?S]), we used CPA, an inhibitor of the SERCA calcium pump. Normally, Ca2? leaks from intracellular stores into the cytoplasm, and the SERCA pump re-incorporates these ions into intracellular stores [33]. Thus, the addition of CPA prevents Ca2? re-incorporation and leads to a depletion in intracellular stores. This method has been used in other models in order to quantify the extent of intracellular stores of Ca2? under different conditions [13]. The addition of 1 lM CPA to freshly isolated spermatocytes provided an estimation of the size of the intracellular store from the amount of Ca2? released from this compartment (Fig. 6c–d). The level of [ICa2?S] was similar under all conditions, and lower than for the freshly isolated spermatocytes, suggesting that the depletion of CPA-sensitive stores occurs to a similar extent under all conditions (Fig. 6c). In contrast, after 24 h, a similar decrease in the level of [ICa2?S] was found in spermatocytes cultured in KHB-Lac or KHB-Glu, although this was not statically significant (Fig. 6d). Interestingly, this parameter was near zero in spermatocytes incubated with DOG or w/oS, suggesting an almost complete depletion of Ca2? from CPA-sensitive stores (Fig. 6d). These two conditions (DOG and w/oS) are most likely to energetically deplete the cells to a greater degree during long-term incubation..

(9) Apoptosis (2012) 17:539–550. Fig. 5 Effect of metabolic energy sources on spermatocyte autophagy. Spermatocytes were cultivated for 0, 12 and 24 h in the presence of KHB-Lac, -Glu, -DOG, -Glu/Lac (1:1) or -w/oS. a Representative Western blots (n = 4). Upper panel Evaluation of LC3-I and LC3-II. Middle panel Beclin-1. Lower Panel b-actin. b Densitometric analysis of LC3-II levels normalized by LC3-I after 12 and 24 h of culture. Each bar is the average ± SEM. c Presence of autophagosomes in. 547. spermatocytes cultured for 24 h in the presence of 5 mM lactate and glucose. The left panels show microphotographs of whole spermatocytes with normal nuclei, and the right panels show the amplification of cellular cytoplasm, as indicated by arrow heads, clearly showing the autophagosome. Cell deterioration and debris were only observed under the condition without substrate (data not shown). Fig. 6 Evaluation of intracellular Ca2? levels. Spermatocytes were cultured for 3 (upper panel) and 24 h (lower panel) with KHB-Lac, -Glu, -DOG, or -w/oS. a, b [Ca2?]i intracellular and c, d Ca2? released from the ER after the addition of CPA ([ICa2?S]). Each bar shows the mean ± SEM, n = 3, *p \ 0.05. Discussion Previous evidence has suggested a protective role of lactate in male germ cell survival [34]. However, these studies used whole seminiferous tubules and could not determine. whether the effect occurred directly on germ cells or indirectly through Sertoli cells. The present study is the first to show that physiological concentrations of lactate and low concentrations of glucose protect rat spermatocytes from apoptosis. We showed that glucose promotes. 123.

(10) 548. cell death in the absence of lactate. Interestingly, low concentrations of glucose (0.1–1 mM) did not trigger apoptosis, suggesting that higher concentrations are required to induce spermatocyte apoptosis. Since a physiological concentration of this molecule (5 mM) is harmful for cultured germ cells, it seems that the threshold of glucose toxicity is lower for spermatocytes than somatic cells (see also ref. [18]). In mammals, and under normal conditions, blood glucose levels range between 3.6 and 5.8 mM. For somatic cells, a high glucose level is considered to be around 25–40 mM in culture [35–37]. Growing evidence suggests that high extracellular glucose concentrations induce apoptosis via activation of the extrinsic pathway of apoptosis, involving upregulation of the Fas receptor and increased caspase-8 activity [36, 38]. In the present study, we showed that spermatocytes cultured in 5 mM glucose, which should be considered as a high level of glucose for this cell type, undergo apoptosis, as evaluated by the TUNEL, caspase activity and phosphatidylserine exposure assays. In addition, our results suggest that the extrinsic pathway of apoptosis is activated under these conditions since we observed the activation of caspase-8 and caspase3 but not caspase-9, in agreement with our previous findings showing that the extrinsic pathway of apoptosis seems to have a major role in germ cells during the first wave of spermatogenesis [22, 27, 39]. Interestingly, the cells incubated with KHB-w/oS released more LDH than the cells incubated with KHBGlu, despite the fact that both showed a similar percentage of cell death, as evaluated by trypan blue and Annexin V assays. The simplest explanation for this is that under both conditions the amount of dead cells is similar, but in those cultured with KHB-w/oS the rate of LDH release is faster than in those cultured with KHB-Glu, probably due to greater plasma membrane and/or mitochondrial damage in these cells. In fact, the pictures showed more cell debris and fragmented cells in KHB-w/oS than in KHB-Glu. Annexin V is a protein that binds phosphatidylserine, a phospholipid that normally faces the cytoplasmic leaf of the plasma membrane. Upon apoptosis it becomes externalized, and this event has been used as a reliable apoptosis marker. However, during necrosis the plasma membrane is broken, allowing the release and access of large molecules such as LDH and dextrane. Thus, we hypothesized that under this last condition Annexin V could cross the plasma membranes and bind to phosphatidylserine on the cytoplasmic side of the membrane. Therefore, Annexin V may bind to cells undergoing necrosis, thus it would not be a reliable parameter of apoptosis by itself. This proposition is reinforced by the finding that with KHB-w/oS, we did not detect PARP cleavage, even though caspase-3 activity was found at a similar level as with KHB-Glu. It is possible that. 123. Apoptosis (2012) 17:539–550. plasma membrane integrity is a requirement for proceeding with bona fide apoptosis; otherwise, the apoptosis programme will shift to necrosis. This explanation is supported by reports of caspase-3 activation without PARP cleavage in cultured neurons following trophic factor starvation, and the fact that cells expressing an uncleavable form of PARP undergo necrosis while the wild type undergo apoptosis [40, 41]. In this study, we showed for the first time that, in vitro, rat spermatocytes are able to undergo autophagy, which was detected after 12 h of culture. Basal autophagy occurs at low levels because it is a reparative and cellular cleaning process [8, 42]. Our data show that male germ cell activate the autophagy machinery under certain metabolic conditions, that lead to cell death. It is possible that in vivo, during spermatogenesis, autophagy may functions as a cell survival mechanism, similar to what has been found in the ovary [8, 42]. Previous studies have shown that lactate and glucose can modulate intracellular [Ca2?]i levels in cultured spermatocytes [19, 20]. Our results showed that lactate prevented an increase in intracellular [Ca2?]i with glucose, DOG or w/oS, and that it acted as a protective agent in spermatocytes (Fig. 6). Interestingly, the levels of [Ca2?]i in spermatocytes cultured for 24 h with glucose and without substrate were related to the levels of necrosis observed in each condition [24]. Our experimental evidence suggests that the intracellular Ca2? probably comes from intracellular stores of Ca2?, such as the endoplasmic reticulum (ER), but we cannot rule out the possible contribution of extracellular calcium. The loss of ER luminal Ca2? causes ER stress and activates the unfolded protein response (UPR), which, depending on the duration and severity of the stress, can lead to cell death [26, 43]. Calcium is a widespread intracellular signal that can also induce apoptosis by regulating the level of antiapoptotic signals in germ cells [22, 23]; thus, the levels of [Ca2?]i may be related to whether the cells enter into autophagy, necrosis or apoptosis. The results of studies with a non-metabolizable glucose analogue, together with the different rates of glucose utilization by spermatogenic and Sertoli cells, strongly suggest that the glucose concentration in the adluminal compartment is controlled by Sertoli cells [17, 18, 44–46]. Our data suggest that Sertoli cells control the number of germ cells by controlling the adluminal concentration of glucose. In this model, the spermatocytes that do not undergo cell-to-cell interactions with Sertoli cells are possible candidates for apoptosis because they would be exposed to high adluminal glucose levels instead of lactate. Interestingly, spermatocyte apoptosis coincides with opening of the Blood-Tesis-Barrier [47], a condition where spermatocytes are exposed to plasma glucose concentrations. Therefore, we propose that glucose could be an in.

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