Lipophagy Contributes to Testosterone Biosynthesis in Male Rat Leydig Cells

Lipophagy Contributes to Testosterone Biosynthesis in Male Rat Leydig Cells Abstract In recent years, autophagy was found to regulate lipid metabolism through a process termed lipophagy. Lipophagy modulates the degradation of cholesteryl esters to free cholesterol (FC), which is the substrate of testosterone biosynthesis. However, the role of lipophagy in testosterone production is unknown. To investigate this, primary rat Leydig cells and varicocele rat models were administered to inhibit or promote autophagy, and testosterone, lipid droplets (LDs), total cholesterol (TC), and FC were evaluated. The results demonstrated that inhibiting autophagy in primary rat Leydig cells reduced testosterone production. Further studies demonstrated that inhibiting autophagy increased the number and size of LDs and the level of TC, but decreased the level of FC. Furthermore, hypoxia promoted autophagy in Leydig cells. We found that short-term hypoxia stimulated testosterone secretion; however, the inhibition of autophagy abolished stimulated testosterone release. Hypoxia decreased the number and size of LDs in Leydig cells, but the changes could be largely rescued by blocking autophagy. In experimental varicocele rat models, the administration of autophagy inhibitors substantially reduced serum testosterone. These data demonstrate that autophagy contributes to testosterone biosynthesis at least partially through degrading intracellular LDs/TC. Our observations might reveal an autophagic regulatory mode regarding testosterone biosynthesis. Leydig cells, which are the interstitial cells adjacent to the seminiferous tubules of the testis, are characterized by large, round lipid droplets (LDs) composed of neutral lipid cores consisting mainly of cholesteryl esters (CEs) and triglycerides (1, 2). Leydig cells synthesize and secrete androgens, including testosterone, androstenedione, dehydroepiandrosterone, and so on. The process is strictly regulated by luteinizing hormone and is vulnerable to external disruptors. Hypoxia, toxicant, drugs, and many environmental hormones can adversely affect the function of Leydig cells and result in disorders of androgen secretion. Autophagy is an intracellular lysosomal degradation pathway that eliminates organelles and proteins. Classic macroautophagy is initiated from an isolated membrane, which is followed by the formation of a double-membrane autophagosome that is then degraded by lysosomes. Autophagy is controlled by the mammalian target of rapamycin signaling pathway and autophagy-related (ATG) family members. The degradation of autophagosome contents is an energy-providing and recycling process (3). Autophagy in prostate cancer cells can degrade LDs to supply energy and maintain cell survival (4). Recently, it has been found that autophagy regulates lipid metabolism in hepatocytes, macrophages, and many other cell types through a process termed lipophagy (1, 5, 6). Lipophagy mediates the delivery of LD-stored CE to the lysosome, where lysosomal acid lipase hydrolyzes CE into free cholesterol (FC) (7, 8). In mammalian Leydig cells, FC binds the steroidogenic acute regulatory protein (STAR), which is then transferred to the mitochondria. This is the rate-limiting step in the biosynthesis of testosterone. FC is the substrate for testosterone synthesis, and autophagy participates in CE hydrolysis and FC generation. Autophagy can be found in Leydig cells and is involved in cell survival or apoptosis under stress conditions (9, 10). However, the role of autophagy in testosterone biosynthesis in Leydig cells is still unknown. Hypoxia can induce autophagy, either as a survival or lethal mechanism (11, 12). In contrast, many hypoxic environments result in the dysregulation of testosterone synthesis in testis Leydig cells, such as testicular torsion and varicocele. However, it is still not clear whether hypoxia-induced autophagy also participates in the regulation of testosterone biosynthesis. In this study, we aimed to investigate the role of autophagy in lipid catabolism in primary rat Leydig cells, as well as the contribution of lipophagy to testosterone production in cells and the experimental varicocele rat models. Materials and Methods Isolation and culturing of Leydig cells Male Sprague–Dawley rats were purchased from Shanghai SLAC Laboratory Animal. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol of this study was reviewed and approved by the ethics committee of Ren Ji Hospital. Twelve 90-day-old rats were used to isolate the Leydig cells. The isolation method was as previously described (13). In brief, testes were decapsulated and digested with 0.5 mg/mL collagenase-I (C0130; Sigma-Aldrich, St. Louis, MO) for 25 minutes in a 34°C oscillating incubator (100 r/min). Cell suspensions were then filtered through 70-μm nylon strainers (352350; BD Biosciences, New York, NY). The filtrate was centrifuged at 350g for 20 minutes at 4°C. The pellet was washed and resuspended in M199 (11150; GIBCO, Grand Island, NY) and transferred to a step Percoll gradient (5%, 30%, 58%, and 70%). The cells were then centrifuged at 800g for 30 minutes at 4°C. Three layers of cells can be seen, and the cells at the bottom layer were collected. Isolated Leydig cells were incubated with F12/Dulbecco’s modified Eagle medium (DMEM) (11320; GIBCO) supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin (10378; GIBCO). The cells were cultured at 34°C with 5% CO2. If cells were treated with hypoxia, cells were cultured in 5% O2 with 90% N2 and 5% CO2 at 34°C. Isolation of Sertoli cells Primary Sertoli cells were isolated from a 20-day-old Sprague–Dawley rat. The isolation method was as previously described with minor modifications (14). Briefly, testes were decapsulated and cut into ∼1-mm pieces. Testis tissue was washed twice and centrifuged at 800g for 2 minutes, resuspended in F12/DMEM with 0.1% trypsin (25300; GIBCO), and then placed in a shaking water bath (60 osc/min) at 34°C for 30 min. The seminiferous tubules were then transferred, washed twice with F12/DMEM, and resuspended with F12/DMEM containing 0.1% collagenase V (C9263; Sigma-Aldrich) before being placed in a shaking water bath (60 osc/min) at 34°C for 40 min. The cells were washed twice and resuspended with F12/DMEM containing 0.1% hyaluronidase (H3506; Sigma-Aldrich) in the shaking water bath for 30 minutes. The cells were then washed again and incubated in F12/DMEM supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin at 34°C. Identification of Leydig cells Leydig cells were identified with 3β-hydroxysteroid dehydrogenase (3β-HSD) [sc-515120; Santa Cruz; Research Resource Identifier (RRID): AB_2721058] (15). Briefly, cells were washed with phosphate-buffered saline and fixed in paraformaldehyde for 15 minutes. After blocking for 20 minutes, cells were incubated in the primary antibodies (3β-HSD) at 4°C overnight and in the secondary antibodies (Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG), RRID: AB_141672) for 1 hour at room temperature; then they were washed and visualized by microscopy. Rat primary Sertoli cells and human lung adenocarcinoma HCC827 cells were used as negative controls. Chemical reagents, small interfering RNA, and in vitro transfection Chloroquine (CQ) and 3-methyladenine (3MA) were purchased from Sigma-Aldrich (C6628, M9281). The concentrations of CQ and 3MA used in this study were 50 µM and 100 µM, respectively. Methylation-modified small interfering RNA against rat ATG7 (59-GGAUACAAGCUUGGCUGCUACUUCU-39) was purchased from RiboBio (Guangzhou, China). Transfection of small interfering RNA (20 nM) was performed with the Sigma-Aldrich N-TER nanoparticle transfection system (N2913; Sigma-Aldrich) (16). Protein extraction and Western blot analysis The methods have been previously described (12). Protein from primary Leydig cells was isolated with M-PER Mammalian Protein Extraction Reagent (78501; Thermo Fisher Scientific, Waltham, MA). The protein concentration was detected using the bicinchoninic acid method (23225; Beyotime, Shanghai, China). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (10401396; GE Healthcare, OH). The membranes were blocked using blocking buffer (927-40000; Odyssey, MA) for 1 hour and then incubated in the primary antibodies at 4°C overnight. After washing with Tris-buffered saline with Tween 20, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Protein bands were visualized by an infrared imaging system (Odyssey) and quantified with Odyssey application software. Rabbit P62/sequestosome 1 (5114, RRID: AB_10624872), anti–microtubule-associated protein 1 light chain 3β (LC3B; 2775, RRID: AB_915950), ATG7 (8558, RRID: AB_10831194), and β actin (8457, RRID: AB_10950489) were purchased from Cell Signaling Technology (Table 1). Table 1. Antibody Table Peptide/Protein Target  Antibody  Manufacturer, Catalog No.; RRID  Species Raised in  Dilution  3β-HSD (A-1)  3β-HSD  Santa Cruz, sc-515120; RRID: AB_2721058  Mouse  1:200  LC3B  LC3B  Cell Signaling, 2775; RRID: AB_915950  Rabbit  1:1000  SQSTM1  SQSTM1  Cell Signaling, 5114; RRID: AB_10624872  Rabbit  1:1000  ATG7 (D12B11)  ATG7  Cell Signaling, 8558; RRID: AB_10831194  Rabbit  1:1000  β-Actin (D6A8)  β-Actin  Cell Signaling, 8457; RRID: AB_10950489  Rabbit  1:1000  MAP LC3β (G-9)  MAP LC3β /LC3B  Santa Cruz, sc-376404; RRID: AB_11150489  Mouse  1:200  Secondary antibody  Alexa Fluor 594 goat anti-mouse IgG  Thermo Fisher Scientific, A-11032; RRID: AB_141672  Goat  1:200  Peptide/Protein Target  Antibody  Manufacturer, Catalog No.; RRID  Species Raised in  Dilution  3β-HSD (A-1)  3β-HSD  Santa Cruz, sc-515120; RRID: AB_2721058  Mouse  1:200  LC3B  LC3B  Cell Signaling, 2775; RRID: AB_915950  Rabbit  1:1000  SQSTM1  SQSTM1  Cell Signaling, 5114; RRID: AB_10624872  Rabbit  1:1000  ATG7 (D12B11)  ATG7  Cell Signaling, 8558; RRID: AB_10831194  Rabbit  1:1000  β-Actin (D6A8)  β-Actin  Cell Signaling, 8457; RRID: AB_10950489  Rabbit  1:1000  MAP LC3β (G-9)  MAP LC3β /LC3B  Santa Cruz, sc-376404; RRID: AB_11150489  Mouse  1:200  Secondary antibody  Alexa Fluor 594 goat anti-mouse IgG  Thermo Fisher Scientific, A-11032; RRID: AB_141672  Goat  1:200  Abbreviation: MAP, mitogen-activated protein. View Large Testosterone assay Serum and supernatant testosterone levels were tested by enzyme-linked immunosorbent assay from R&D Systems (KGE010; R&D, Minneapolis, MN), according to the manufacturer’s instructions. The intra-assay coefficient of variation (CV) (n = 6) was 4.5%, the interassay CV (n = 5) was 6.9%, the mean recovery rate (n = 3) was 106%, and the range was 99% to 110%. Cholesterol determination Cholesterol was detected by the Amplex Red Cholesterol Assay (A12216; Molecular Probes, Eugene, OR), according to the manufacturer’s instructions. For the TC assay, the intra-assay CV (n = 5) was 3.2%, the interassay CV (n = 3) was 7.8%, the mean recovery rate (n = 3) was 101%, and the range was 97% to 105%. LD staining Bodipy 493/503 (D3922; Molecular Probes) was used to detect LDs. Cells were washed with phosphate-buffered saline, fixed with 4% formaldehyde for 15 minutes, and stained with bodipy 493/503 (stock concentration 2 mg/mL; working solution 1:800 dilution) for 15 minutes at room temperature. MitoTracker and reactive oxygen species measurements Cells were stained with 500 nM MitoTracker Red CMXRos (M7512; Invitrogen, Carlsbad, CA) for 30 minutes and fixed with 4% paraformaldehyde for 15 minutes. Then cells were counterstained with 4′,6-diamidino-2-phenylindole for 10 minutes and visualized using a fluorescence microscope. Reactive oxygen species (ROS) was tested with 2′,7′-dichlorodihydrofluorescein diacetate (C6827; Invitrogen), according to the manufacturer’s protocol. Briefly, cells were washed and incubated in 5 µM 2′,7′-dichlorodihydrofluorescein diacetate for 30 minutes at 37°C. The fluorescent signal was read at 492 nm (excitation) and 517 nm (emission). ROS levels were normalized based on the cell count. Immunofluorescence and confocal microscopy The method was completed, as previously described (14). Briefly, cells were incubated in the primary antibodies anti-LC3B (sc-376404; Santa Cruz; RRID: AB_11150489) or 3β-HSD (sc-515120; Santa Cruz; RRID: AB_2721058) at 4°C overnight. Alexa Fluor 594 goat anti-mouse IgG (A11032; Thermo Fisher Scientific; RRID: AB_141672) served as the secondary antibody. The cells were then visualized with confocal microscopy (12). Experimental varicocele models A total of 25 rats was randomly assigned to 5 groups: the negative control group, the sham group, the sham + CQ group, the varicocele group, and the varicocele + CQ group. Four rats were excluded because of death: one from the sham + CQ group, two from the varicocele group, and one from the varicocele + CQ group. One month after surgery, the intraperitoneal injection of CQ (4 mg/kg) was performed for 2 weeks every other day, after which all rats were euthanized, and testosterone was detected. Establishing an experimental left varicocele model in rats was previously described with some modifications (17). In brief, the retroperitoneum was exposed via a ventral midline incision, to identify the inlet of left vena spermatica interna on the left renal vein. The left renal vein was ligated with a 22-gauge needle at the distal of the inlet. After ligation, the needle was withdrawn, and a partial obstruction was formed in the left renal vein. The partial obstruction resulted in the increased intravenous pressure of the left renal vein and the left vena spermatica interna, which caused varicocele formation. The communicating branches between the common iliac vein and the vena spermatica interna were also ligated. Statistical analysis We used SPSS 16.0 (SPSS Inc., Chicago, IL) and GraphPad-Prism5 (GraphPad Software, San Diego, CA) to conduct data analyses. Data are expressed as the mean ± standard deviation and analyzed with analysis of variance. All tests were two tailed, and a difference was considered to be statistically significant when the P value was <0.05. Bar charts were plotted with GraphPad-Prism5. Results Inhibition of autophagy suppresses testosterone production Primary Leydig cells were isolated from 3-month-old rats and identified with anti–3β-HSD antibody, an indicator of steroidogenic activity. We found that nearly all cells were stained red (Fig. 1A-1). We observed that only 26 cells were not stained red in 10 random fields (653 cells). Primary rat Sertoli cells (Fig. 1A-2 ) and human lung adenocarcinoma HCC827 cells (Fig. 1A-3 ) were used as the negative controls. Figure 1. View largeDownload slide Inhibition of autophagy suppresses testosterone production. (A) Identification of primary rat Leydig cells. Isolated rat Leydig cells, Sertoli cells, and human lung adenocarcinoma HCC827 cells were incubated with anti–3β-HSD primary antibody, followed by Alexa Fluor 594 goat anti-mouse IgG secondary antibody (n = 3). (A-1) Rat Leydig cells. (A-2) Rat Sertoli cells. (A-3) Human lung adenocarcinoma HCC827 cells. (B) Leydig cells were cultured (B-1) without CQ or (B-2) with 50 µM CQ for 48 hours; LC3B were determined by immunofluorescence (n = 3). (C) Leydig cells were transfected with 20 nM siAtg7s, and ATG7 protein was detected with Western blots (n = 3). (D) Cells were treated with 50 µM CQ or transfected with 20 nM small interfering RNA, as indicated, for 48 hours, and testosterone concentration in supernatant was determined (n = 3, *P < 0.05). NC, negative control. Figure 1. View largeDownload slide Inhibition of autophagy suppresses testosterone production. (A) Identification of primary rat Leydig cells. Isolated rat Leydig cells, Sertoli cells, and human lung adenocarcinoma HCC827 cells were incubated with anti–3β-HSD primary antibody, followed by Alexa Fluor 594 goat anti-mouse IgG secondary antibody (n = 3). (A-1) Rat Leydig cells. (A-2) Rat Sertoli cells. (A-3) Human lung adenocarcinoma HCC827 cells. (B) Leydig cells were cultured (B-1) without CQ or (B-2) with 50 µM CQ for 48 hours; LC3B were determined by immunofluorescence (n = 3). (C) Leydig cells were transfected with 20 nM siAtg7s, and ATG7 protein was detected with Western blots (n = 3). (D) Cells were treated with 50 µM CQ or transfected with 20 nM small interfering RNA, as indicated, for 48 hours, and testosterone concentration in supernatant was determined (n = 3, *P < 0.05). NC, negative control. Figure 2. View largeDownload slide Inhibition of autophagy increases lipid droplets and TC. (A) Primary Leydig cells were treated with 100 µM 3MA or transfected with 20 nM siAtg7 for 48 hours; cell survival was assessed by CCK-8 assay (n = 6). (B–D) Cells were cultured in the presence of CQ (50 µM) or not; LDs were stained with Bodipy 493/503 (B); and the (C) average number of LDs per cell and (D) average size of LDs were calculated in 200 cells for each group (n = 3, *P < 0.05). (E and F) Cells were treated as indicated for 48 hours, and (E) TC and (F) FC in Leydig cells were determined (n = 3, *P < 0.05). NC, negative control. Figure 2. View largeDownload slide Inhibition of autophagy increases lipid droplets and TC. (A) Primary Leydig cells were treated with 100 µM 3MA or transfected with 20 nM siAtg7 for 48 hours; cell survival was assessed by CCK-8 assay (n = 6). (B–D) Cells were cultured in the presence of CQ (50 µM) or not; LDs were stained with Bodipy 493/503 (B); and the (C) average number of LDs per cell and (D) average size of LDs were calculated in 200 cells for each group (n = 3, *P < 0.05). (E and F) Cells were treated as indicated for 48 hours, and (E) TC and (F) FC in Leydig cells were determined (n = 3, *P < 0.05). NC, negative control. Figure 3. View largeDownload slide Hypoxia induces autophagy in primary rat Leydig cells. (A) Cells were incubated with or without hypoxia (5% O2) for 24 hours; CQ (50 µM) was used to block autophagy; LC3B was determined by immunofluorescence (n = 3). (B) Cells were cultured as indicated for 1 or 6 days; LC3B and P62 were detected with Western blots (n = 3). NC, negative control. Figure 3. View largeDownload slide Hypoxia induces autophagy in primary rat Leydig cells. (A) Cells were incubated with or without hypoxia (5% O2) for 24 hours; CQ (50 µM) was used to block autophagy; LC3B was determined by immunofluorescence (n = 3). (B) Cells were cultured as indicated for 1 or 6 days; LC3B and P62 were detected with Western blots (n = 3). NC, negative control. Previous studies have found that autophagy was active in Leydig cells (18). We further found that, in the presence of CQ, an inhibitor of fusion of autophagosomes and lysosomes, the prominent accumulation of autophagosomes, was detected (Fig. 1B-2 ). However, without CQ, autophagosomes were rarely detected (Fig. 1B-1). The results indicated that the degradation of autophagosomes via lysosomal turnover is rapid in primary Leydig cells. To investigate whether autophagy regulates testosterone production, we inhibited autophagic flux with CQ or siAtg7, and the concentration of testosterone in the supernatant was determined. The efficacies of three siAtg7s were verified with Western blot analysis (Fig. 1C; Supplemental Fig. 1); the one with the highest efficacy was used. The results demonstrated that inhibiting autophagy substantially reduced testosterone production (P < 0.05) in Leydig cells (Fig. 1D). Inhibition of autophagy increases lipid droplets and TC in Leydig cells It has been reported that autophagy contributes to Leydig cell survival or apoptosis under stress conditions (9, 10). To elucidate the mechanism underlying autophagy and testosterone production, we first examined cell survival. However, we found that inhibiting autophagy (3MA/siAtg7) did not affect cell viability (Fig. 2A). Autophagy can also modulate lipid metabolism (1). Autophagy might degrade LDs to provide substrates for testosterone production, so we hypothesized that autophagy degrades LDs in Leydig cells. LDs were detected (Fig. 2B), and the results showed that inhibiting autophagy raised their sizes and numbers (P < 0.05; Fig. 2B, 2C, and 2D). In addition, the blockage of autophagy in Leydig cells substantially increased the TC (P < 0.05; Fig. 2E) while decreasing FC (P < 0.05; Fig. 2F). The results indicated that autophagy might regulate testosterone production by modulating lipid catabolism. Hypoxia-induced autophagy regulates testosterone biosynthesis Hypoxic testicles can be found in some circumstances, such as testicular torsion, high altitude hypoxia, and varicoceles (19). Hypoxia can induce autophagy in most cells. To investigate whether hypoxia induces autophagy in Leydig cells, LC3 was determined by immunofluorescence (Fig. 3A). We found that cells exposed to hypoxia for 24 hours exhibited LC3 puncta, and the number of LC3 puncta increased when cells were further treated with CQ. LC3B and P62 were also assessed by Western blot analysis (Fig. 3B; Supplemental Figs. 2 and 3). Cells were exposed to hypoxia for 1 day or 6 days, and we found that hypoxia decreased P62 and increased the ratio of LC3II/LC3I (P < 0.05). The lack of a prominent increase in LC3II under hypoxia is probably the consequence of an increased autophagic flux, as similar results have also been reported in rat Sertoli cells (14). CQ can suppress autophagy flux, thereby increasing the expression levels of both LC3II and LC3I (20). The results demonstrated that the increased LC3II and LC3I levels with CQ under hypoxic conditions were less than those in normal conditions (P < 0.05; Fig. 3B), suggesting that hypoxia enhanced autophagy flux in Leydig cells. To investigate whether hypoxia-induced autophagy regulates testosterone production, Leydig cells were cultured in short- or long-term hypoxia (5% O2) for 1 day or 6 days, and the resulting concentration of testosterone in the supernatant was detected. The results demonstrated that short-term hypoxia promoted testosterone production (P < 0.05), but that the inhibition of autophagy abolished stimulated testosterone release (Fig. 4A). Although long-term hypoxia decreased testosterone production (P < 0.05), the inhibition of autophagy led to more substantial decline in the testosterone level compared with normoxia (Fig. 4B and Fig. 1D ), suggesting that hypoxia-induced autophagy may also participate in testosterone synthesis. We hypothesized that long-term hypoxia induced a decline in testosterone that might be due to the more notable mitochondrial dysfunction (Supplemental Figs. 4 and 5). Figure 4. View largeDownload slide Hypoxia-induced autophagy regulates testosterone production. (A, B) Primary Leydig cells were treated as indicated for (A) 1 day or (B) 6 days; CQ (50 µM) or siAtg7 (20 nM) was used to inhibit autophagy, and testosterone in supernatant was assayed (n = 3, *P < 0.05). (C) Cells were cultured with or without hypoxia (5% O2) for 6 days; CQ (50 µM) was used to suppress autophagy; and LDs were detected with Bodipy 493/503 (n = 3). (D and E) Cells were treated as indicated under hypoxia (5% O2) for (D) 1 day or (E) 6 days; TC was detected (n = 3, *P < 0.05). (F and G) Cells were treated as indicated in hypoxia (5% O2) for (F) 1 day or (G) 6 days; FC was determined (n = 3, *P < 0.05). (H) Cells were treated as indicated; STAR expression was determined by Western blots (n = 3). (I) Cells were treated with hypoxia (5% O2) for 48 hours; CQ (50 µM) or 3MA (100 µM) was used to suppress autophagy; cell viability was tested by CCK-8 assay (n = 6). NC, negative control. Figure 4. View largeDownload slide Hypoxia-induced autophagy regulates testosterone production. (A, B) Primary Leydig cells were treated as indicated for (A) 1 day or (B) 6 days; CQ (50 µM) or siAtg7 (20 nM) was used to inhibit autophagy, and testosterone in supernatant was assayed (n = 3, *P < 0.05). (C) Cells were cultured with or without hypoxia (5% O2) for 6 days; CQ (50 µM) was used to suppress autophagy; and LDs were detected with Bodipy 493/503 (n = 3). (D and E) Cells were treated as indicated under hypoxia (5% O2) for (D) 1 day or (E) 6 days; TC was detected (n = 3, *P < 0.05). (F and G) Cells were treated as indicated in hypoxia (5% O2) for (F) 1 day or (G) 6 days; FC was determined (n = 3, *P < 0.05). (H) Cells were treated as indicated; STAR expression was determined by Western blots (n = 3). (I) Cells were treated with hypoxia (5% O2) for 48 hours; CQ (50 µM) or 3MA (100 µM) was used to suppress autophagy; cell viability was tested by CCK-8 assay (n = 6). NC, negative control. We found that hypoxia (5% O2) decreased the size and number of LDs (P < 0.05), but that blocking autophagy with CQ largely rescued these changes (Fig. 4C; Supplemental Figs. 6 and 7). Furthermore, the inhibition of autophagy under hypoxia led to a significant accumulation of TC (P < 0.05; Fig. 4D and 4E). The blockage of autophagy decreased FC levels when cells were incubated in short-term hypoxia (P < 0.05; Fig. 4F). However, blocking autophagy under long-term hypoxic conditions increased FC levels (P < 0.05; Fig. 4G), which may also be associated with mitochondrial dysfunction. Autophagy may protect the STAR protein against ROS in rat Leydig cells (21). However, the results demonstrated that suppressing autophagy with CQ did not influence STAR expression under either normal or hypoxic conditions (Fig. 4H; Supplemental Fig. 8). In addition, like the results in normoxia, the inhibition of autophagy (CQ/3MA) under hypoxic conditions did not affect cell viability (Fig. 4I). To investigate how autophagy regulates LD metabolism, we used anti-LC3 antibodies and Bodipy 493/503 to identify their positions, and found LC3 colocalized with LDs in Leydig cells (Fig. 5), indicating that LDs may be engulfed by autophagosomes and degraded by lysosomes. We also found the colocalizations were promoted by treatments with hypoxia (P < 0.05), suggesting that LD degradation can be regulated by the autophagic flux (Fig. 5; Supplemental Fig. 9). Figure 5. View largeDownload slide Hypoxia promotes the colocalizations of LC3 and LDs. Primary Leydig cells were cultured in normoxia or hypoxia for 48 hours; CQ (50 µM) was used to inhibit autophagic flux; LC3 and LDs were assessed with double immunofluorescence (n = 3). Arrow: the colocalizations of LC3 and LDs. Figure 5. View largeDownload slide Hypoxia promotes the colocalizations of LC3 and LDs. Primary Leydig cells were cultured in normoxia or hypoxia for 48 hours; CQ (50 µM) was used to inhibit autophagic flux; LC3 and LDs were assessed with double immunofluorescence (n = 3). Arrow: the colocalizations of LC3 and LDs. Autophagy inhibitor suppresses testosterone secretion in rat varicocele models Rats were assigned to five groups, and left varicocele models were established (Fig. 6A–6C). One month later, rats were administered CQ for 2 weeks and euthanized. Testis hypoxia-inducible factor 1A (HIF1A) was detected to confirm the hypoxic environment in the varicocele models (Fig. 6D). The results demonstrated that the HIF1A protein was nearly absent in the negative control and sham groups, and the HIF1A expression in the varicocele models was threefold higher than that in the negative control or sham groups (P < 0.05; Fig. 6D and 6E). The concentration of rat serum testosterone was also assayed for each group. We found that either CQ or varicocele decreased serum testosterone, but the declines were not prominent. However, varicocele + CQ led to a significant decrease in serum testosterone (P < 0.05; Fig. 6F). Figure 6. View largeDownload slide Autophagy inhibitor suppresses testosterone secretion in rat varicocele models. (A–C) Varicocele models were established. Left vena spermatica interna (A) before, (B) during, and (C) after operation. (D and E) Left testis HIF1A was detected with Western blots in the negative control, sham, and varicocele groups (n = 3, *P < 0.05). (F) Serum testosterone in the sham group (n = 5), sham + CQ group (n = 4), varicocele group (n = 3), and varicocele + CQ group (n = 4) was determined (*P < 0.05). NC, negative control. Figure 6. View largeDownload slide Autophagy inhibitor suppresses testosterone secretion in rat varicocele models. (A–C) Varicocele models were established. Left vena spermatica interna (A) before, (B) during, and (C) after operation. (D and E) Left testis HIF1A was detected with Western blots in the negative control, sham, and varicocele groups (n = 3, *P < 0.05). (F) Serum testosterone in the sham group (n = 5), sham + CQ group (n = 4), varicocele group (n = 3), and varicocele + CQ group (n = 4) was determined (*P < 0.05). NC, negative control. Discussion We found that autophagy could degrade LDs to provide substrates for testosterone synthesis, and hypoxia-induced autophagy might also participate in lipid metabolism and testosterone production. Our observation might represent the discovery of a regulatory mode by which autophagy modulates testosterone synthesis and reveal a mechanism regarding lipid metabolism under hypoxic conditions. In this study, we found that blocking autophagy in vitro by pharmacological or genetic means led to increased LDs and TC, in comparison with decreased levels of FC and testosterone. FC, known as the substrate for testosterone production, is generated from intracellular biosynthesis and degradation of LDs or extracellular high-density lipoprotein/low-density lipoprotein. In this study, primary Leydig cells were incubated in F12/DMEM without animal serum. Serum starvation inhibited cholesterol biosynthesis and the degradation of extracellular high-density lipoprotein/low-density lipoprotein; therefore, FC mainly originates from the degradation of intracellular LDs. Lipophagy can degrade LDs. Thus, the blockage of lipophagy under serum starvation prominently decreased the FC levels. Lipophagy can also maintain energy homeostasis and protect cells during serum starvation (22). However, in Leydig cells, we found that the inhibition of autophagy did not affect cell survival. When lipophagy was suppressed in Leydig cells, FC decreased. We therefore hypothesized that the decline of the FC level resulted in a reduction in the testosterone. Although the transfer of FC to mitochondria by STAR is the rate-limiting step in the biosynthesis of testosterone, a substantial decrease in the substrate may also have a negative effect on this process. Furthermore, we did not observe any changes in the STAR expression levels when autophagy was suppressed. We used hypoxia to promote autophagy in Leydig cells. Although rapamycin also stimulates autophagy in Leydig cells, it substantially suppresses cell viability (data not shown). Hypoxia-induced autophagy is a selective degradation process (23). In this study, we found that hypoxia reduced the size and number of LDs, and a blockage in autophagy under hypoxia led to a more substantial accumulation of TC and more colocalizations when compared with normoxia, which indicated that hypoxia-induced autophagy could also regulate lipid metabolism. This study demonstrates that hypoxia-induced autophagy can participate in lipid metabolism. It has been long recognized that severe hypoxia (<1% O2) causes an accumulation of LD and triglycerides (24–27). However, a recent study has demonstrated that modest hypoxia (5% O2) could substantially reduce LD size and number, even while lipolysis-associated proteins remain unchanged (28). We hypothesize that hypoxia-induced autophagy participates in LD degradation. Although either severe or modest hypoxia can increase lipolysis (28, 29), severe hypoxia may also lead to mitochondrial dysfunction (24). Rotenone, an inhibitor of mitochondrial complex I, can interrupt lipolysis and increase lipid accumulation (24). In addition, treatments of mitochondrial respiratory inhibitors resulted in the notable elevation of free fatty acids and the intracellular accumulation of LDs (30). However, in modest hypoxia, part of the mitochondrial function may be maintained, such that the process of lipolysis can continue. That may be the reason that training under hypoxia results in a greater reduction in body fat compared with working under normoxia (31). In this study, we found that short-term hypoxia (5% O2) raised the level of testosterone, which has also been reported by previous studies (32–35); however, the mechanism by which this occurs is unclear. We hypothesized that it might be due to the enhanced lipophagy under hypoxia. Although hypoxia-induced autophagy provided substrates for testosterone production, long-term hypoxia also caused more substantial mitochondrial dysfunction (as demonstrated in Fig. 4H; Supplemental Figs. 4 and 5) and the apoptosis of Leydig cells, finally resulting in a decrease in the testosterone levels. In patients with varicoceles, a decline in the testosterone along with an increment of cholesterol esters, total lipids, and glycerides has been observed (36–38). In this study, we found that either the administration of CQ (2 weeks) or experimental varicocele (6 weeks) decreased the serum testosterone, but the decline was not prominent. These results suggest that the effects of short-term administration of CQ or experimental varicocele on testosterone production can be compensated in vivo, which was also reported by previous studies (39, 40). However, the administration of CQ in varicocele models led to a substantial decrease in serum testosterone in rats. There may be other reasons for the decline of testosterone production when autophagy is suppressed. Hypoxia leads to ROS accumulation, which in turn results in endoplasmic reticulum stress, unfolded protein response, lipid peroxidation, and cell apoptosis (41, 42). Autophagy can regulate ROS formation, thus protecting the cells against oxidative stress and maintaining redox homeostasis (41, 43). Autophagy can also maintain energy homeostasis, inhibiting autophagy under starvation-induced cell death (41). In addition, some approaches used to promote or inhibit autophagy in this study (CQ, hypoxia, and varicocele) are not strictly specific, and they thus might have other effects on testosterone production. Therefore, further studies are required to understand the role of autophagy in testosterone biosynthesis. In conclusion, we determined that, in primary rat Leydig cells, autophagy can degrade LDs to provide substrates for testosterone biosynthesis. Of note, the level of testosterone can be regulated through modulating autophagy. In addition, we observed hypoxia-induced autophagy in Leydig cells, which also participate in lipid catabolism and testosterone production. Although further studies are required, our results have revealed an autophagic regulatory mode regarding testosterone production. Abbreviations: 3MA 3-methyladenine 3β-HSD 3β-hydroxysteroid dehydrogenase ATG autophagy-related CE cholesteryl ester CQ chloroquine CV coefficient of variation DMEM Dulbecco’s modified Eagle medium FC free cholesterol HIF1A hypoxia-inducible factor 1A IgG immunoglobulin G LC3B microtubule-associated protein 1 light chain 3β LD lipid droplet ROS reactive oxygen species RRID Research Resource Identifier STAR steroidogenic acute regulatory protein TC total cholesterol. Acknowledgments We thank Long-Mei Xu and Hao-Zheng Yang for establishing animal models. Financial Support: This work was supported by National Natural Science Foundation of China Grants 81501310, 81502602, and 81671511. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo AM, Czaja MJ. 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Lipophagy Contributes to Testosterone Biosynthesis in Male Rat Leydig Cells

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-03020
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Abstract

Abstract In recent years, autophagy was found to regulate lipid metabolism through a process termed lipophagy. Lipophagy modulates the degradation of cholesteryl esters to free cholesterol (FC), which is the substrate of testosterone biosynthesis. However, the role of lipophagy in testosterone production is unknown. To investigate this, primary rat Leydig cells and varicocele rat models were administered to inhibit or promote autophagy, and testosterone, lipid droplets (LDs), total cholesterol (TC), and FC were evaluated. The results demonstrated that inhibiting autophagy in primary rat Leydig cells reduced testosterone production. Further studies demonstrated that inhibiting autophagy increased the number and size of LDs and the level of TC, but decreased the level of FC. Furthermore, hypoxia promoted autophagy in Leydig cells. We found that short-term hypoxia stimulated testosterone secretion; however, the inhibition of autophagy abolished stimulated testosterone release. Hypoxia decreased the number and size of LDs in Leydig cells, but the changes could be largely rescued by blocking autophagy. In experimental varicocele rat models, the administration of autophagy inhibitors substantially reduced serum testosterone. These data demonstrate that autophagy contributes to testosterone biosynthesis at least partially through degrading intracellular LDs/TC. Our observations might reveal an autophagic regulatory mode regarding testosterone biosynthesis. Leydig cells, which are the interstitial cells adjacent to the seminiferous tubules of the testis, are characterized by large, round lipid droplets (LDs) composed of neutral lipid cores consisting mainly of cholesteryl esters (CEs) and triglycerides (1, 2). Leydig cells synthesize and secrete androgens, including testosterone, androstenedione, dehydroepiandrosterone, and so on. The process is strictly regulated by luteinizing hormone and is vulnerable to external disruptors. Hypoxia, toxicant, drugs, and many environmental hormones can adversely affect the function of Leydig cells and result in disorders of androgen secretion. Autophagy is an intracellular lysosomal degradation pathway that eliminates organelles and proteins. Classic macroautophagy is initiated from an isolated membrane, which is followed by the formation of a double-membrane autophagosome that is then degraded by lysosomes. Autophagy is controlled by the mammalian target of rapamycin signaling pathway and autophagy-related (ATG) family members. The degradation of autophagosome contents is an energy-providing and recycling process (3). Autophagy in prostate cancer cells can degrade LDs to supply energy and maintain cell survival (4). Recently, it has been found that autophagy regulates lipid metabolism in hepatocytes, macrophages, and many other cell types through a process termed lipophagy (1, 5, 6). Lipophagy mediates the delivery of LD-stored CE to the lysosome, where lysosomal acid lipase hydrolyzes CE into free cholesterol (FC) (7, 8). In mammalian Leydig cells, FC binds the steroidogenic acute regulatory protein (STAR), which is then transferred to the mitochondria. This is the rate-limiting step in the biosynthesis of testosterone. FC is the substrate for testosterone synthesis, and autophagy participates in CE hydrolysis and FC generation. Autophagy can be found in Leydig cells and is involved in cell survival or apoptosis under stress conditions (9, 10). However, the role of autophagy in testosterone biosynthesis in Leydig cells is still unknown. Hypoxia can induce autophagy, either as a survival or lethal mechanism (11, 12). In contrast, many hypoxic environments result in the dysregulation of testosterone synthesis in testis Leydig cells, such as testicular torsion and varicocele. However, it is still not clear whether hypoxia-induced autophagy also participates in the regulation of testosterone biosynthesis. In this study, we aimed to investigate the role of autophagy in lipid catabolism in primary rat Leydig cells, as well as the contribution of lipophagy to testosterone production in cells and the experimental varicocele rat models. Materials and Methods Isolation and culturing of Leydig cells Male Sprague–Dawley rats were purchased from Shanghai SLAC Laboratory Animal. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol of this study was reviewed and approved by the ethics committee of Ren Ji Hospital. Twelve 90-day-old rats were used to isolate the Leydig cells. The isolation method was as previously described (13). In brief, testes were decapsulated and digested with 0.5 mg/mL collagenase-I (C0130; Sigma-Aldrich, St. Louis, MO) for 25 minutes in a 34°C oscillating incubator (100 r/min). Cell suspensions were then filtered through 70-μm nylon strainers (352350; BD Biosciences, New York, NY). The filtrate was centrifuged at 350g for 20 minutes at 4°C. The pellet was washed and resuspended in M199 (11150; GIBCO, Grand Island, NY) and transferred to a step Percoll gradient (5%, 30%, 58%, and 70%). The cells were then centrifuged at 800g for 30 minutes at 4°C. Three layers of cells can be seen, and the cells at the bottom layer were collected. Isolated Leydig cells were incubated with F12/Dulbecco’s modified Eagle medium (DMEM) (11320; GIBCO) supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin (10378; GIBCO). The cells were cultured at 34°C with 5% CO2. If cells were treated with hypoxia, cells were cultured in 5% O2 with 90% N2 and 5% CO2 at 34°C. Isolation of Sertoli cells Primary Sertoli cells were isolated from a 20-day-old Sprague–Dawley rat. The isolation method was as previously described with minor modifications (14). Briefly, testes were decapsulated and cut into ∼1-mm pieces. Testis tissue was washed twice and centrifuged at 800g for 2 minutes, resuspended in F12/DMEM with 0.1% trypsin (25300; GIBCO), and then placed in a shaking water bath (60 osc/min) at 34°C for 30 min. The seminiferous tubules were then transferred, washed twice with F12/DMEM, and resuspended with F12/DMEM containing 0.1% collagenase V (C9263; Sigma-Aldrich) before being placed in a shaking water bath (60 osc/min) at 34°C for 40 min. The cells were washed twice and resuspended with F12/DMEM containing 0.1% hyaluronidase (H3506; Sigma-Aldrich) in the shaking water bath for 30 minutes. The cells were then washed again and incubated in F12/DMEM supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin at 34°C. Identification of Leydig cells Leydig cells were identified with 3β-hydroxysteroid dehydrogenase (3β-HSD) [sc-515120; Santa Cruz; Research Resource Identifier (RRID): AB_2721058] (15). Briefly, cells were washed with phosphate-buffered saline and fixed in paraformaldehyde for 15 minutes. After blocking for 20 minutes, cells were incubated in the primary antibodies (3β-HSD) at 4°C overnight and in the secondary antibodies (Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG), RRID: AB_141672) for 1 hour at room temperature; then they were washed and visualized by microscopy. Rat primary Sertoli cells and human lung adenocarcinoma HCC827 cells were used as negative controls. Chemical reagents, small interfering RNA, and in vitro transfection Chloroquine (CQ) and 3-methyladenine (3MA) were purchased from Sigma-Aldrich (C6628, M9281). The concentrations of CQ and 3MA used in this study were 50 µM and 100 µM, respectively. Methylation-modified small interfering RNA against rat ATG7 (59-GGAUACAAGCUUGGCUGCUACUUCU-39) was purchased from RiboBio (Guangzhou, China). Transfection of small interfering RNA (20 nM) was performed with the Sigma-Aldrich N-TER nanoparticle transfection system (N2913; Sigma-Aldrich) (16). Protein extraction and Western blot analysis The methods have been previously described (12). Protein from primary Leydig cells was isolated with M-PER Mammalian Protein Extraction Reagent (78501; Thermo Fisher Scientific, Waltham, MA). The protein concentration was detected using the bicinchoninic acid method (23225; Beyotime, Shanghai, China). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (10401396; GE Healthcare, OH). The membranes were blocked using blocking buffer (927-40000; Odyssey, MA) for 1 hour and then incubated in the primary antibodies at 4°C overnight. After washing with Tris-buffered saline with Tween 20, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Protein bands were visualized by an infrared imaging system (Odyssey) and quantified with Odyssey application software. Rabbit P62/sequestosome 1 (5114, RRID: AB_10624872), anti–microtubule-associated protein 1 light chain 3β (LC3B; 2775, RRID: AB_915950), ATG7 (8558, RRID: AB_10831194), and β actin (8457, RRID: AB_10950489) were purchased from Cell Signaling Technology (Table 1). Table 1. Antibody Table Peptide/Protein Target  Antibody  Manufacturer, Catalog No.; RRID  Species Raised in  Dilution  3β-HSD (A-1)  3β-HSD  Santa Cruz, sc-515120; RRID: AB_2721058  Mouse  1:200  LC3B  LC3B  Cell Signaling, 2775; RRID: AB_915950  Rabbit  1:1000  SQSTM1  SQSTM1  Cell Signaling, 5114; RRID: AB_10624872  Rabbit  1:1000  ATG7 (D12B11)  ATG7  Cell Signaling, 8558; RRID: AB_10831194  Rabbit  1:1000  β-Actin (D6A8)  β-Actin  Cell Signaling, 8457; RRID: AB_10950489  Rabbit  1:1000  MAP LC3β (G-9)  MAP LC3β /LC3B  Santa Cruz, sc-376404; RRID: AB_11150489  Mouse  1:200  Secondary antibody  Alexa Fluor 594 goat anti-mouse IgG  Thermo Fisher Scientific, A-11032; RRID: AB_141672  Goat  1:200  Peptide/Protein Target  Antibody  Manufacturer, Catalog No.; RRID  Species Raised in  Dilution  3β-HSD (A-1)  3β-HSD  Santa Cruz, sc-515120; RRID: AB_2721058  Mouse  1:200  LC3B  LC3B  Cell Signaling, 2775; RRID: AB_915950  Rabbit  1:1000  SQSTM1  SQSTM1  Cell Signaling, 5114; RRID: AB_10624872  Rabbit  1:1000  ATG7 (D12B11)  ATG7  Cell Signaling, 8558; RRID: AB_10831194  Rabbit  1:1000  β-Actin (D6A8)  β-Actin  Cell Signaling, 8457; RRID: AB_10950489  Rabbit  1:1000  MAP LC3β (G-9)  MAP LC3β /LC3B  Santa Cruz, sc-376404; RRID: AB_11150489  Mouse  1:200  Secondary antibody  Alexa Fluor 594 goat anti-mouse IgG  Thermo Fisher Scientific, A-11032; RRID: AB_141672  Goat  1:200  Abbreviation: MAP, mitogen-activated protein. View Large Testosterone assay Serum and supernatant testosterone levels were tested by enzyme-linked immunosorbent assay from R&D Systems (KGE010; R&D, Minneapolis, MN), according to the manufacturer’s instructions. The intra-assay coefficient of variation (CV) (n = 6) was 4.5%, the interassay CV (n = 5) was 6.9%, the mean recovery rate (n = 3) was 106%, and the range was 99% to 110%. Cholesterol determination Cholesterol was detected by the Amplex Red Cholesterol Assay (A12216; Molecular Probes, Eugene, OR), according to the manufacturer’s instructions. For the TC assay, the intra-assay CV (n = 5) was 3.2%, the interassay CV (n = 3) was 7.8%, the mean recovery rate (n = 3) was 101%, and the range was 97% to 105%. LD staining Bodipy 493/503 (D3922; Molecular Probes) was used to detect LDs. Cells were washed with phosphate-buffered saline, fixed with 4% formaldehyde for 15 minutes, and stained with bodipy 493/503 (stock concentration 2 mg/mL; working solution 1:800 dilution) for 15 minutes at room temperature. MitoTracker and reactive oxygen species measurements Cells were stained with 500 nM MitoTracker Red CMXRos (M7512; Invitrogen, Carlsbad, CA) for 30 minutes and fixed with 4% paraformaldehyde for 15 minutes. Then cells were counterstained with 4′,6-diamidino-2-phenylindole for 10 minutes and visualized using a fluorescence microscope. Reactive oxygen species (ROS) was tested with 2′,7′-dichlorodihydrofluorescein diacetate (C6827; Invitrogen), according to the manufacturer’s protocol. Briefly, cells were washed and incubated in 5 µM 2′,7′-dichlorodihydrofluorescein diacetate for 30 minutes at 37°C. The fluorescent signal was read at 492 nm (excitation) and 517 nm (emission). ROS levels were normalized based on the cell count. Immunofluorescence and confocal microscopy The method was completed, as previously described (14). Briefly, cells were incubated in the primary antibodies anti-LC3B (sc-376404; Santa Cruz; RRID: AB_11150489) or 3β-HSD (sc-515120; Santa Cruz; RRID: AB_2721058) at 4°C overnight. Alexa Fluor 594 goat anti-mouse IgG (A11032; Thermo Fisher Scientific; RRID: AB_141672) served as the secondary antibody. The cells were then visualized with confocal microscopy (12). Experimental varicocele models A total of 25 rats was randomly assigned to 5 groups: the negative control group, the sham group, the sham + CQ group, the varicocele group, and the varicocele + CQ group. Four rats were excluded because of death: one from the sham + CQ group, two from the varicocele group, and one from the varicocele + CQ group. One month after surgery, the intraperitoneal injection of CQ (4 mg/kg) was performed for 2 weeks every other day, after which all rats were euthanized, and testosterone was detected. Establishing an experimental left varicocele model in rats was previously described with some modifications (17). In brief, the retroperitoneum was exposed via a ventral midline incision, to identify the inlet of left vena spermatica interna on the left renal vein. The left renal vein was ligated with a 22-gauge needle at the distal of the inlet. After ligation, the needle was withdrawn, and a partial obstruction was formed in the left renal vein. The partial obstruction resulted in the increased intravenous pressure of the left renal vein and the left vena spermatica interna, which caused varicocele formation. The communicating branches between the common iliac vein and the vena spermatica interna were also ligated. Statistical analysis We used SPSS 16.0 (SPSS Inc., Chicago, IL) and GraphPad-Prism5 (GraphPad Software, San Diego, CA) to conduct data analyses. Data are expressed as the mean ± standard deviation and analyzed with analysis of variance. All tests were two tailed, and a difference was considered to be statistically significant when the P value was <0.05. Bar charts were plotted with GraphPad-Prism5. Results Inhibition of autophagy suppresses testosterone production Primary Leydig cells were isolated from 3-month-old rats and identified with anti–3β-HSD antibody, an indicator of steroidogenic activity. We found that nearly all cells were stained red (Fig. 1A-1). We observed that only 26 cells were not stained red in 10 random fields (653 cells). Primary rat Sertoli cells (Fig. 1A-2 ) and human lung adenocarcinoma HCC827 cells (Fig. 1A-3 ) were used as the negative controls. Figure 1. View largeDownload slide Inhibition of autophagy suppresses testosterone production. (A) Identification of primary rat Leydig cells. Isolated rat Leydig cells, Sertoli cells, and human lung adenocarcinoma HCC827 cells were incubated with anti–3β-HSD primary antibody, followed by Alexa Fluor 594 goat anti-mouse IgG secondary antibody (n = 3). (A-1) Rat Leydig cells. (A-2) Rat Sertoli cells. (A-3) Human lung adenocarcinoma HCC827 cells. (B) Leydig cells were cultured (B-1) without CQ or (B-2) with 50 µM CQ for 48 hours; LC3B were determined by immunofluorescence (n = 3). (C) Leydig cells were transfected with 20 nM siAtg7s, and ATG7 protein was detected with Western blots (n = 3). (D) Cells were treated with 50 µM CQ or transfected with 20 nM small interfering RNA, as indicated, for 48 hours, and testosterone concentration in supernatant was determined (n = 3, *P < 0.05). NC, negative control. Figure 1. View largeDownload slide Inhibition of autophagy suppresses testosterone production. (A) Identification of primary rat Leydig cells. Isolated rat Leydig cells, Sertoli cells, and human lung adenocarcinoma HCC827 cells were incubated with anti–3β-HSD primary antibody, followed by Alexa Fluor 594 goat anti-mouse IgG secondary antibody (n = 3). (A-1) Rat Leydig cells. (A-2) Rat Sertoli cells. (A-3) Human lung adenocarcinoma HCC827 cells. (B) Leydig cells were cultured (B-1) without CQ or (B-2) with 50 µM CQ for 48 hours; LC3B were determined by immunofluorescence (n = 3). (C) Leydig cells were transfected with 20 nM siAtg7s, and ATG7 protein was detected with Western blots (n = 3). (D) Cells were treated with 50 µM CQ or transfected with 20 nM small interfering RNA, as indicated, for 48 hours, and testosterone concentration in supernatant was determined (n = 3, *P < 0.05). NC, negative control. Figure 2. View largeDownload slide Inhibition of autophagy increases lipid droplets and TC. (A) Primary Leydig cells were treated with 100 µM 3MA or transfected with 20 nM siAtg7 for 48 hours; cell survival was assessed by CCK-8 assay (n = 6). (B–D) Cells were cultured in the presence of CQ (50 µM) or not; LDs were stained with Bodipy 493/503 (B); and the (C) average number of LDs per cell and (D) average size of LDs were calculated in 200 cells for each group (n = 3, *P < 0.05). (E and F) Cells were treated as indicated for 48 hours, and (E) TC and (F) FC in Leydig cells were determined (n = 3, *P < 0.05). NC, negative control. Figure 2. View largeDownload slide Inhibition of autophagy increases lipid droplets and TC. (A) Primary Leydig cells were treated with 100 µM 3MA or transfected with 20 nM siAtg7 for 48 hours; cell survival was assessed by CCK-8 assay (n = 6). (B–D) Cells were cultured in the presence of CQ (50 µM) or not; LDs were stained with Bodipy 493/503 (B); and the (C) average number of LDs per cell and (D) average size of LDs were calculated in 200 cells for each group (n = 3, *P < 0.05). (E and F) Cells were treated as indicated for 48 hours, and (E) TC and (F) FC in Leydig cells were determined (n = 3, *P < 0.05). NC, negative control. Figure 3. View largeDownload slide Hypoxia induces autophagy in primary rat Leydig cells. (A) Cells were incubated with or without hypoxia (5% O2) for 24 hours; CQ (50 µM) was used to block autophagy; LC3B was determined by immunofluorescence (n = 3). (B) Cells were cultured as indicated for 1 or 6 days; LC3B and P62 were detected with Western blots (n = 3). NC, negative control. Figure 3. View largeDownload slide Hypoxia induces autophagy in primary rat Leydig cells. (A) Cells were incubated with or without hypoxia (5% O2) for 24 hours; CQ (50 µM) was used to block autophagy; LC3B was determined by immunofluorescence (n = 3). (B) Cells were cultured as indicated for 1 or 6 days; LC3B and P62 were detected with Western blots (n = 3). NC, negative control. Previous studies have found that autophagy was active in Leydig cells (18). We further found that, in the presence of CQ, an inhibitor of fusion of autophagosomes and lysosomes, the prominent accumulation of autophagosomes, was detected (Fig. 1B-2 ). However, without CQ, autophagosomes were rarely detected (Fig. 1B-1). The results indicated that the degradation of autophagosomes via lysosomal turnover is rapid in primary Leydig cells. To investigate whether autophagy regulates testosterone production, we inhibited autophagic flux with CQ or siAtg7, and the concentration of testosterone in the supernatant was determined. The efficacies of three siAtg7s were verified with Western blot analysis (Fig. 1C; Supplemental Fig. 1); the one with the highest efficacy was used. The results demonstrated that inhibiting autophagy substantially reduced testosterone production (P < 0.05) in Leydig cells (Fig. 1D). Inhibition of autophagy increases lipid droplets and TC in Leydig cells It has been reported that autophagy contributes to Leydig cell survival or apoptosis under stress conditions (9, 10). To elucidate the mechanism underlying autophagy and testosterone production, we first examined cell survival. However, we found that inhibiting autophagy (3MA/siAtg7) did not affect cell viability (Fig. 2A). Autophagy can also modulate lipid metabolism (1). Autophagy might degrade LDs to provide substrates for testosterone production, so we hypothesized that autophagy degrades LDs in Leydig cells. LDs were detected (Fig. 2B), and the results showed that inhibiting autophagy raised their sizes and numbers (P < 0.05; Fig. 2B, 2C, and 2D). In addition, the blockage of autophagy in Leydig cells substantially increased the TC (P < 0.05; Fig. 2E) while decreasing FC (P < 0.05; Fig. 2F). The results indicated that autophagy might regulate testosterone production by modulating lipid catabolism. Hypoxia-induced autophagy regulates testosterone biosynthesis Hypoxic testicles can be found in some circumstances, such as testicular torsion, high altitude hypoxia, and varicoceles (19). Hypoxia can induce autophagy in most cells. To investigate whether hypoxia induces autophagy in Leydig cells, LC3 was determined by immunofluorescence (Fig. 3A). We found that cells exposed to hypoxia for 24 hours exhibited LC3 puncta, and the number of LC3 puncta increased when cells were further treated with CQ. LC3B and P62 were also assessed by Western blot analysis (Fig. 3B; Supplemental Figs. 2 and 3). Cells were exposed to hypoxia for 1 day or 6 days, and we found that hypoxia decreased P62 and increased the ratio of LC3II/LC3I (P < 0.05). The lack of a prominent increase in LC3II under hypoxia is probably the consequence of an increased autophagic flux, as similar results have also been reported in rat Sertoli cells (14). CQ can suppress autophagy flux, thereby increasing the expression levels of both LC3II and LC3I (20). The results demonstrated that the increased LC3II and LC3I levels with CQ under hypoxic conditions were less than those in normal conditions (P < 0.05; Fig. 3B), suggesting that hypoxia enhanced autophagy flux in Leydig cells. To investigate whether hypoxia-induced autophagy regulates testosterone production, Leydig cells were cultured in short- or long-term hypoxia (5% O2) for 1 day or 6 days, and the resulting concentration of testosterone in the supernatant was detected. The results demonstrated that short-term hypoxia promoted testosterone production (P < 0.05), but that the inhibition of autophagy abolished stimulated testosterone release (Fig. 4A). Although long-term hypoxia decreased testosterone production (P < 0.05), the inhibition of autophagy led to more substantial decline in the testosterone level compared with normoxia (Fig. 4B and Fig. 1D ), suggesting that hypoxia-induced autophagy may also participate in testosterone synthesis. We hypothesized that long-term hypoxia induced a decline in testosterone that might be due to the more notable mitochondrial dysfunction (Supplemental Figs. 4 and 5). Figure 4. View largeDownload slide Hypoxia-induced autophagy regulates testosterone production. (A, B) Primary Leydig cells were treated as indicated for (A) 1 day or (B) 6 days; CQ (50 µM) or siAtg7 (20 nM) was used to inhibit autophagy, and testosterone in supernatant was assayed (n = 3, *P < 0.05). (C) Cells were cultured with or without hypoxia (5% O2) for 6 days; CQ (50 µM) was used to suppress autophagy; and LDs were detected with Bodipy 493/503 (n = 3). (D and E) Cells were treated as indicated under hypoxia (5% O2) for (D) 1 day or (E) 6 days; TC was detected (n = 3, *P < 0.05). (F and G) Cells were treated as indicated in hypoxia (5% O2) for (F) 1 day or (G) 6 days; FC was determined (n = 3, *P < 0.05). (H) Cells were treated as indicated; STAR expression was determined by Western blots (n = 3). (I) Cells were treated with hypoxia (5% O2) for 48 hours; CQ (50 µM) or 3MA (100 µM) was used to suppress autophagy; cell viability was tested by CCK-8 assay (n = 6). NC, negative control. Figure 4. View largeDownload slide Hypoxia-induced autophagy regulates testosterone production. (A, B) Primary Leydig cells were treated as indicated for (A) 1 day or (B) 6 days; CQ (50 µM) or siAtg7 (20 nM) was used to inhibit autophagy, and testosterone in supernatant was assayed (n = 3, *P < 0.05). (C) Cells were cultured with or without hypoxia (5% O2) for 6 days; CQ (50 µM) was used to suppress autophagy; and LDs were detected with Bodipy 493/503 (n = 3). (D and E) Cells were treated as indicated under hypoxia (5% O2) for (D) 1 day or (E) 6 days; TC was detected (n = 3, *P < 0.05). (F and G) Cells were treated as indicated in hypoxia (5% O2) for (F) 1 day or (G) 6 days; FC was determined (n = 3, *P < 0.05). (H) Cells were treated as indicated; STAR expression was determined by Western blots (n = 3). (I) Cells were treated with hypoxia (5% O2) for 48 hours; CQ (50 µM) or 3MA (100 µM) was used to suppress autophagy; cell viability was tested by CCK-8 assay (n = 6). NC, negative control. We found that hypoxia (5% O2) decreased the size and number of LDs (P < 0.05), but that blocking autophagy with CQ largely rescued these changes (Fig. 4C; Supplemental Figs. 6 and 7). Furthermore, the inhibition of autophagy under hypoxia led to a significant accumulation of TC (P < 0.05; Fig. 4D and 4E). The blockage of autophagy decreased FC levels when cells were incubated in short-term hypoxia (P < 0.05; Fig. 4F). However, blocking autophagy under long-term hypoxic conditions increased FC levels (P < 0.05; Fig. 4G), which may also be associated with mitochondrial dysfunction. Autophagy may protect the STAR protein against ROS in rat Leydig cells (21). However, the results demonstrated that suppressing autophagy with CQ did not influence STAR expression under either normal or hypoxic conditions (Fig. 4H; Supplemental Fig. 8). In addition, like the results in normoxia, the inhibition of autophagy (CQ/3MA) under hypoxic conditions did not affect cell viability (Fig. 4I). To investigate how autophagy regulates LD metabolism, we used anti-LC3 antibodies and Bodipy 493/503 to identify their positions, and found LC3 colocalized with LDs in Leydig cells (Fig. 5), indicating that LDs may be engulfed by autophagosomes and degraded by lysosomes. We also found the colocalizations were promoted by treatments with hypoxia (P < 0.05), suggesting that LD degradation can be regulated by the autophagic flux (Fig. 5; Supplemental Fig. 9). Figure 5. View largeDownload slide Hypoxia promotes the colocalizations of LC3 and LDs. Primary Leydig cells were cultured in normoxia or hypoxia for 48 hours; CQ (50 µM) was used to inhibit autophagic flux; LC3 and LDs were assessed with double immunofluorescence (n = 3). Arrow: the colocalizations of LC3 and LDs. Figure 5. View largeDownload slide Hypoxia promotes the colocalizations of LC3 and LDs. Primary Leydig cells were cultured in normoxia or hypoxia for 48 hours; CQ (50 µM) was used to inhibit autophagic flux; LC3 and LDs were assessed with double immunofluorescence (n = 3). Arrow: the colocalizations of LC3 and LDs. Autophagy inhibitor suppresses testosterone secretion in rat varicocele models Rats were assigned to five groups, and left varicocele models were established (Fig. 6A–6C). One month later, rats were administered CQ for 2 weeks and euthanized. Testis hypoxia-inducible factor 1A (HIF1A) was detected to confirm the hypoxic environment in the varicocele models (Fig. 6D). The results demonstrated that the HIF1A protein was nearly absent in the negative control and sham groups, and the HIF1A expression in the varicocele models was threefold higher than that in the negative control or sham groups (P < 0.05; Fig. 6D and 6E). The concentration of rat serum testosterone was also assayed for each group. We found that either CQ or varicocele decreased serum testosterone, but the declines were not prominent. However, varicocele + CQ led to a significant decrease in serum testosterone (P < 0.05; Fig. 6F). Figure 6. View largeDownload slide Autophagy inhibitor suppresses testosterone secretion in rat varicocele models. (A–C) Varicocele models were established. Left vena spermatica interna (A) before, (B) during, and (C) after operation. (D and E) Left testis HIF1A was detected with Western blots in the negative control, sham, and varicocele groups (n = 3, *P < 0.05). (F) Serum testosterone in the sham group (n = 5), sham + CQ group (n = 4), varicocele group (n = 3), and varicocele + CQ group (n = 4) was determined (*P < 0.05). NC, negative control. Figure 6. View largeDownload slide Autophagy inhibitor suppresses testosterone secretion in rat varicocele models. (A–C) Varicocele models were established. Left vena spermatica interna (A) before, (B) during, and (C) after operation. (D and E) Left testis HIF1A was detected with Western blots in the negative control, sham, and varicocele groups (n = 3, *P < 0.05). (F) Serum testosterone in the sham group (n = 5), sham + CQ group (n = 4), varicocele group (n = 3), and varicocele + CQ group (n = 4) was determined (*P < 0.05). NC, negative control. Discussion We found that autophagy could degrade LDs to provide substrates for testosterone synthesis, and hypoxia-induced autophagy might also participate in lipid metabolism and testosterone production. Our observation might represent the discovery of a regulatory mode by which autophagy modulates testosterone synthesis and reveal a mechanism regarding lipid metabolism under hypoxic conditions. In this study, we found that blocking autophagy in vitro by pharmacological or genetic means led to increased LDs and TC, in comparison with decreased levels of FC and testosterone. FC, known as the substrate for testosterone production, is generated from intracellular biosynthesis and degradation of LDs or extracellular high-density lipoprotein/low-density lipoprotein. In this study, primary Leydig cells were incubated in F12/DMEM without animal serum. Serum starvation inhibited cholesterol biosynthesis and the degradation of extracellular high-density lipoprotein/low-density lipoprotein; therefore, FC mainly originates from the degradation of intracellular LDs. Lipophagy can degrade LDs. Thus, the blockage of lipophagy under serum starvation prominently decreased the FC levels. Lipophagy can also maintain energy homeostasis and protect cells during serum starvation (22). However, in Leydig cells, we found that the inhibition of autophagy did not affect cell survival. When lipophagy was suppressed in Leydig cells, FC decreased. We therefore hypothesized that the decline of the FC level resulted in a reduction in the testosterone. Although the transfer of FC to mitochondria by STAR is the rate-limiting step in the biosynthesis of testosterone, a substantial decrease in the substrate may also have a negative effect on this process. Furthermore, we did not observe any changes in the STAR expression levels when autophagy was suppressed. We used hypoxia to promote autophagy in Leydig cells. Although rapamycin also stimulates autophagy in Leydig cells, it substantially suppresses cell viability (data not shown). Hypoxia-induced autophagy is a selective degradation process (23). In this study, we found that hypoxia reduced the size and number of LDs, and a blockage in autophagy under hypoxia led to a more substantial accumulation of TC and more colocalizations when compared with normoxia, which indicated that hypoxia-induced autophagy could also regulate lipid metabolism. This study demonstrates that hypoxia-induced autophagy can participate in lipid metabolism. It has been long recognized that severe hypoxia (<1% O2) causes an accumulation of LD and triglycerides (24–27). However, a recent study has demonstrated that modest hypoxia (5% O2) could substantially reduce LD size and number, even while lipolysis-associated proteins remain unchanged (28). We hypothesize that hypoxia-induced autophagy participates in LD degradation. Although either severe or modest hypoxia can increase lipolysis (28, 29), severe hypoxia may also lead to mitochondrial dysfunction (24). Rotenone, an inhibitor of mitochondrial complex I, can interrupt lipolysis and increase lipid accumulation (24). In addition, treatments of mitochondrial respiratory inhibitors resulted in the notable elevation of free fatty acids and the intracellular accumulation of LDs (30). However, in modest hypoxia, part of the mitochondrial function may be maintained, such that the process of lipolysis can continue. That may be the reason that training under hypoxia results in a greater reduction in body fat compared with working under normoxia (31). In this study, we found that short-term hypoxia (5% O2) raised the level of testosterone, which has also been reported by previous studies (32–35); however, the mechanism by which this occurs is unclear. We hypothesized that it might be due to the enhanced lipophagy under hypoxia. Although hypoxia-induced autophagy provided substrates for testosterone production, long-term hypoxia also caused more substantial mitochondrial dysfunction (as demonstrated in Fig. 4H; Supplemental Figs. 4 and 5) and the apoptosis of Leydig cells, finally resulting in a decrease in the testosterone levels. In patients with varicoceles, a decline in the testosterone along with an increment of cholesterol esters, total lipids, and glycerides has been observed (36–38). In this study, we found that either the administration of CQ (2 weeks) or experimental varicocele (6 weeks) decreased the serum testosterone, but the decline was not prominent. These results suggest that the effects of short-term administration of CQ or experimental varicocele on testosterone production can be compensated in vivo, which was also reported by previous studies (39, 40). However, the administration of CQ in varicocele models led to a substantial decrease in serum testosterone in rats. There may be other reasons for the decline of testosterone production when autophagy is suppressed. Hypoxia leads to ROS accumulation, which in turn results in endoplasmic reticulum stress, unfolded protein response, lipid peroxidation, and cell apoptosis (41, 42). Autophagy can regulate ROS formation, thus protecting the cells against oxidative stress and maintaining redox homeostasis (41, 43). Autophagy can also maintain energy homeostasis, inhibiting autophagy under starvation-induced cell death (41). In addition, some approaches used to promote or inhibit autophagy in this study (CQ, hypoxia, and varicocele) are not strictly specific, and they thus might have other effects on testosterone production. Therefore, further studies are required to understand the role of autophagy in testosterone biosynthesis. In conclusion, we determined that, in primary rat Leydig cells, autophagy can degrade LDs to provide substrates for testosterone biosynthesis. Of note, the level of testosterone can be regulated through modulating autophagy. In addition, we observed hypoxia-induced autophagy in Leydig cells, which also participate in lipid catabolism and testosterone production. Although further studies are required, our results have revealed an autophagic regulatory mode regarding testosterone production. Abbreviations: 3MA 3-methyladenine 3β-HSD 3β-hydroxysteroid dehydrogenase ATG autophagy-related CE cholesteryl ester CQ chloroquine CV coefficient of variation DMEM Dulbecco’s modified Eagle medium FC free cholesterol HIF1A hypoxia-inducible factor 1A IgG immunoglobulin G LC3B microtubule-associated protein 1 light chain 3β LD lipid droplet ROS reactive oxygen species RRID Research Resource Identifier STAR steroidogenic acute regulatory protein TC total cholesterol. Acknowledgments We thank Long-Mei Xu and Hao-Zheng Yang for establishing animal models. Financial Support: This work was supported by National Natural Science Foundation of China Grants 81501310, 81502602, and 81671511. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo AM, Czaja MJ. 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EndocrinologyOxford University Press

Published: Feb 1, 2018

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