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Taurine induces autophagy and inhibits oxidative stress in mice Leydig cells

Taurine induces autophagy and inhibits oxidative stress in mice Leydig cells JBRA Assisted Reproduction 2020;24(3):250-256 doi: 10.5935/1518-0557.20190079 Original article Taurine induces autophagy and inhibits oxidative stress in mice Leydig cells 1,2 2 2 1,2 Shokofeh Yahyavy , Armita Valizadeh , Ghasem Saki , Layasadat Khorsandi Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran ABSTRACT MATERIALS AND METHODS Objectives: This study evaluated taurine (TAU) effects Experimental design on autophagy, apoptosis and oxidative stress in mice Ley- The mice Leydig TM3 cell line was bought through dig TM3 cells. the National Center for Genetic and Biological Reserves Methods: We treated TM3 cells with TAU (100 µg/mL) in Iran. We cultured the cells in DMEM medium, comple- or 3-Methyladenine (3-MA, an autophagy inhibitor) for 24 mented with 10% fetal bovine serum and 1% penicillin/ h, and assessed cell viability, testosterone level, oxidative o streptomycin at 37 C within a moist atmosphere with stress, apoptosis, and autophagy. 5% CO . The cells were incubated with TAU (Sigma) at Results: The results showed that TAU markedly in- various concentrations for 12, 24, 48 and 72h, as a pilot creased cell viability, testosterone levels, expression of au- study (Table 1). The cells were grouped into 4 catego- tophagy-related genes and percentage of LC3-II-positive ries (Figure 1): cells. TAU significantly reduced malondialdehyde (MDA) contents and reactive oxygen species (ROS) levels and in- I. Control: received only media for 24h creased the activities of SOD (superoxide dismutase) and II. 3-MA (3-Methyladenine, an autophagy inhibitor): CAT (Catalase) enzymes in the TM3 cells. TAU in the pres- received 10 mmol 3-MA for 24h ence of autophagy inhibitor (3-MA) increased oxidative III. TAU: received 100 µg/mL TAU for 24h stress and decreased testosterone levels. IV. TAU + 3-MA: received 10 mmol 3-MA and 100 µg/ Conclusion: The results showed that autophagy might mL TAU for 24h be involved in TAU-increased testosterone levels in mice Leydig TM3 cells. Cell viability We cultured the TM3 (1 × 10 cells/well) cells in 96-well Keywords: Leydig cells, taurine, autophagy, oxidative plates. After treating them, the supernatants were dis- stress, testosterone posed of and we added 20 µL of MTT solution (5 mg/ml) to all of the wells and incubated for four hours at 37ºC. Upon completion of incubation, the medium was eliminated, and we added 100 µL of DMSO to all of the wells. We used a INTRODUCTION micro-plate reader (BioRad, USA) to measure absorbance at 570 nm. Taurine (TAU) is a free amino acid that is present in several mammalian tissues (De Luca etal., 2015; Park Testosterone assay et al., 2002; Yang et al., 2010). TAU is present in some We incubated the cell suspensions with TAU or 3-MA foodstuff, such as fish and meat and often added to in the presence of hCG (0.05IU/ml) at 37ºC for 24h. We energy drinks. TAU has several physiological functions measured the testosterone concentration in culture me- including energy storage, membrane stabilization, anti- dia via a radio-immunoassay, complying with the manu- oxidation and xenobiotic conjugation (Huxtable, 1992). facturer`s guidelines (Monobind, USA). In male reproductive organs, TAU is present in Leydig cells, and in some other cells (Yang et al., 2010). Lobo Annexin V-FITC/propidium iodide apoptosis et al. (2000) reported that TAU stimulated testosterone assay secretion in vivo and in vitro. Despite its importance, we We seeded the TM3 cells (1.0×10 /mL) in a six-well still do not know the impact of TAU in Leydig cells. culture plate, and normal, apoptotic and necrotic cells were Leydig cells produce testosterone in testicular tis- distinguished using an Annexin V-FITC/propidium iodide sues, where autophagy is active (Yi & Tang, 1999). In assay kit (Ebioscience™ Annexin V Kit, USA) according to autophagy, microtubule-associated protein-1 light chain the manufacturer’s instruction. We analyzed the samples (LC3-I) converts to a lipid form, termed LC3-II, by a using a flow cytometer (BD Facscalibur, USA). We analyzed complex of Atg5, Atg12, and Atg16 proteins to induce the data using the FlowJo V10. autophagosome. Mammalian rapamycin (mTOR) regu- lates autophagy by activating several autophagy-related Immunocytochemistry proteins, including Beclin-1, Atg1, Atg5, and Atg7 (He After treating the TM3 cells, we deposited them over & Klionsky, 2009). Autophagy regulates ROS buildup by a glass coverslip and let it stabilize for fifteen minutes in increasing the clearance of damaged organelles and ac- a 4% para-formaldehyde in PBS at 4ºC. Then, we washed tivating antioxidant enzymes in the Leydig cells (Li et them in PBS. We used anti-LC3-II antibody (sc-16755, al., 2011). Santa Cruz) at 1/100 dilution overnight at 4ºC. After wash- In the present study we investigated the TAU effect ing twice in PBS, we incubated the section with FITC-con- on oxidative stress, autophagy, apoptosis, and testos- jugated anti-mice secondary anti-body (sc-2356, Santa terone level in the mouse Leydig TM3 cells. Received June 04, 2019 Accepted December 10, 2019 TAU effects on autophagy and oxidative stress - Yahyavy, S. Table 1. Viability percentage in TM3 cells at different doses and duration times of TAU treatment (pilot study) Treatments 12 h 24 h 48 72 12.5µg/mL 99.4 ± 4.5 98.7 ± 4.7 98.6 ± 2.2 100.4 ± 3.2 25 µg/mL 98.3 ± 4.8 101.5 ± 3.2 102.2 ± 5.3 102.6 ± 5.1 50 µg/mL 99.2 ± 3.5 105.7 ± 6.9 105.6 ± 4.2 104.3 ± 6.4 100 µg/mL 98.6 ± 5.5 135.2 ± 11.2* 138.1 ± 9.3* 132.8 ± 9.8* 150 µg/mL 98.8 ± 4.3 136.4 ± 12.2* 137.3 ± 10.7* 134.6 ± 12.3* 200 µg/mL 99.2 ± 3.6 136.7 ± 10.7* 137.8 ± 11.3* 135.1 ± 10.7* Values are expressed as mean ± SD (n=6). *p<0.05 *symbol indicates comparison to 12h in each row Figure 1. Schematic illustration of the study design primers, and 12.5 µL of 2x SYBR Green Master Mix Cruz) for one hour at room temperature. The fluorescence [Fermentas, Canada] (Table 2). PCR was amplified in pictures were captured by a fluorescence microscope forty cycles through this protocol: 95ºC for ten min- (Olympus, Japan). utes, 95ºC for fifteen seconds, 95ºC for thirty seconds, ΔΔCT and 60ºC for thirty-four seconds. We used the 2 Real-Time Polymerase Chain Reaction method to analyze the data. The whole RNA was drawn out from the cells via the RNeasy Mini kit (Qiagen) in accordance with the Determining MDA content, ROS level and anti- Company's guidelines. We synthesized cDNA using a oxidant enzyme activity cDNA synthesis kit from total RNA regarding the man- We treated the TM3 cells with TAU or 3-MA for 24h. ufacturer's instruction. About 2 µL of cDNA boosted in Afterwards, we performed the lysis of the gathered each 25 µL PCR reaction mix, which contains 10.1 µl specimens, and the protein contents of the TM3 cells we DEPC water, 0.2 µL of each 10pM forward and reverse JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 Original article 252 Table 2. Primer sequences. Groups Forward Reverse GCTGGACATTG- ACCACTGTGACCT- Bax GACTTCCTC GCTCCA GGATGCCTTTGTG- TCACTTGTGGC- Bcl-2 GAACTGT CCAGATAGG CGGTTTTTCTGGGACAA- AAAAACGTGTCTC- Beclin-1 CAA GCCTTTC TATTCACCTCCTGCCT- TGTGATGGCTGT- mTOR CACC GAAGATCC CCAGAAAAAGACCTTCT- CAATCCCATCCA- Atg5 GCACT GAGTTGCT GATAATCAGACGGC- ACTTCGGAGATGG- LC3-II GCTTGC GAGTGGA ACCCAGAAGACTGTG- TTCTAGACGGCAG- GAPDH GATGG GTCAGGT identified via a BCA protein assay kit (Pierce Biotechnol - ogy Inc. IL). Upon having the cell lysates centrifuged, we assessed the contents of malondialdehyde (MDA), Catalase (CAT), and super-oxide dismutase (SOD) activ- ity on the basis of the related guidelines (ZellBio, GmbH, Germany). With regards to the Company's guidelines, we measured the reactive oxygen species (ROS) using a Figure 2. Morphology (above images) and viability (the dichloro-dihydrofluorescein diacetate (DCFH-DA) deter - following pictures) of TM3 cells in experimental and mination kit (Sigma, St. Louis, MO). We measured the control groups (Scale bar: 100 µm). The arrows indicate ROS via a spectrofluorometer (LS50B, Waltham, USA; apoptotic morphology. The values are expressed as Ex: 490 nm, Em: 570 nm). # $ # mean ± SD. *p<0.05, p<0.05, p<0.05; *, and the symbols respectively indicate comparison to the control, Statistical analyses 3-MA and TAU groups We used the ANOVA SPSS 21.0 (Chicago, IL, USA) to analyze the data. Then, we carried out a post-hoc pair- wise comparison using the Bonferroni technique. We con- sidered the p value ˂0.05 as statistically significant. Each experiment were done in quadruplicates. RESULTS Cell viability The cell viability percentage significantly decreased in 3-MA exposed TM3 cells, compared to the control cells (p<0.05). The cell viability significantly increased in TAU-treated cells, compared to the control cells (p<0.05). In the TAU + 3-MA group, the cell viability of TM3 cells sig- nificantly decreased in comparison with the TAU and 3-MA groups (Figure 2). Testosterone assay The testosterone level significantly decreased in Figure 3. Testosterone concentration in the control 3-MA exposed TM3 cells when compared to the control and experimental groups. The arrows indicate apoptotic cells (p<0.05). The testosterone concentration signifi - morphology. The values are expressed as mean ± SD. cantly increased in TAU-treated cells, compared to the # ## $ *p<0.05, **p<0.01, p<0.05, p<0.01, p<0.05; *, control and 3-MA groups (p<0.01). In the TAU + 3-MA # $ and the symbols respectively indicates comparison to group, the testosterone level of the TM3 cells signifi - the control, 3-MA and TAU groups. cantly decreased in comparison with the TAU-treated cells (Figure 3). with the control and 3-MA groups (p<0.01). In the TAU Annexin V-FITC/propidium iodide apoptosis + 3-MA group, the apoptotic and necrotic indices signifi - assay cantly increased in comparison with TAU-treated cells In 3-MA exposed TM3 cells, necrotic and apoptotic (Figure 4), and significantly reduced when compared to indices were significantly higher than those from control the 3-MA-treated cells. cells. TAU decreased the late apoptosis, necrosis, and early apoptosis percentages in TM3 cells in comparison JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 TAU effects on autophagy and oxidative stress - Yahyavy, S. Figure 4. Flow cytometry of Annexin / PI staining in the experimental & control groups. Lower left quadrant: live cells; Lower right quadrant: initial apoptosis; Upper right quadrant: late apoptosis; Upper -2 left quadrant: necrotic cells (A and B). The relative level of mRNA expression of Bax and Bcl genes and -2 # ## Bax/Bcl ratio (C and D). The values are expressed as mean±SD. *p<0.05, **p<0.01, p<0.05, p<0.01, ### $ $$ # $ p<0.001, p<0.05, p<0.01; *, and the symbols respectively indicate comparison to the control, 3-MA and TAU groups. Immunocytochemistry Quantitative Real-time RT-PCR In the 3-MA exposed TM3 cells, we did not detect LC3- In 3-MA exposed TM3 cells, the expression of Bax and II. There was a significant increase of the percentage of Bcl-2 considerably changed in comparison to the control LC3-II-positive cells in comparison with control group group. In the TAU-treated cells, the expression of the Bax (p<0.05) by TAU (p<0.05). Figure 5 depicts these results. was considerably higher, while the expression of the Bcl-2 significantly diminished compared to the control and 3-MA ROS levels, MDA content and antioxidant enzyme groups. There was a significant increase in the expression activity of the Bax gene while there was a significant reduction ROS level and MDA content of 3-MA exposed cells were in the expression of the Bcl-2 gene in TAU+3-MA-treat- significantly increased in comparison with those from the ed cells, compared with the controls and TAU-treated cells control group. There was a considerable reduction in MDA (Figure 4). In 3-MA exposed TM3 cells, the Bax/Bcl-2 ra- contents and levels of ROS in the TAU-treated cells when tio significantly increased in comparison with the control compared to the 3-MA and control groups (p<0.05). In the group. In the TAU-treated cells, the Bax/Bcl-2 ratio sig- TAU+3-MA, the ROS level and MDA content significantly nificantly decreased compared to the control and 3-MA changed in comparison with the TAU and 3-MA groups. In groups. The Bax/Bcl-2 ratio significantly increased, com - the 3-MA exposed cells, CAT and SOD activity significantly pared with the control and TAU-treated cells (Figure 4). decreased in comparison with the control group. In the In 3-MA-treated cells, the LC3-II, Atg5 and Beclin1 TAU-treated cells, the antioxidant activity significantly in - genes were not expressed while the mTOR expression was creased in comparison with the 3-MA and control groups significantly higher. In TAU-treated cells, the mTOR gene (p<0.05). In the TAU+3-MA, the CAT and SOD activity sig- expression was significantly reduced while the expres - nificantly changed in comparison with the TAU and 3-MA sion of the LC3-II, Atg5 and Beclin1 genes experienced groups (Figure 6). a significant increase, compared to that of control cells. In TAU+3-MA-treated cells, the mTOR expression showed a significant increase in comparison with those from the DISCUSSION TAU-treated cells. While there was a significant reduction The present study showed that TAU increased the in mTOR expression in comparison with 3-MA-treated cells levels of testosterone, improved antioxidant activity, de- (Figure 5). creased ROS levels, inhibited apoptosis, and induced au- JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 Original article 254 In the presence of 3-MA, TAU increased the Bax/Bcl-2 ra- tio in comparison with the absence of 3-MA, indicating that TAU may suppress apoptosis by enhancing the autophagy process. 3-MA in the presence of TAU decreased the apoptotic index of TM3 cells, in comparison with the absence of TAU. This finding indicated that TAU could also suppress apoptosis by involving multiple signaling pathways. In the current study, testosterone levels dramatical- ly increased in the TAU-treated cells, which may be as- sociated with the increasing viability of TM3 cells or the increasing biosynthesis of androgens. Yang et al. (2010) showed that TAU could stimulate testosterone generation in cultured Leydig cells. The reducing testosterone level in 3-MA- treated cells indicated autophagy involved in testos- terone production in the TM3 cells. Rapamycin (autophagy stimulator) induced an increase in testosterone concentra- tion in rat Leydig cells, whereas 3-MA had the opposite effect (Yang et al., 2017). Gao et al. (2018) revealed that autophagy increased cholesterol absorption by the Leydig cells and dysfunction of autophagy leads to insufficient testosterone production. Au - tophagy activity decreased in aged rat Leydig cells (Li et al., 2011), and testosterone levels reduced in autophagy deficien - cy in mice (Yoshii et al., 2016). The autophagy inhibition was accompanied by a massive loss of germ cells (González et al., 2018). By contrast, Zhang et al. (2012) showed that heat exposure caused autophagy-induced germ cell death and spermatogenesis defects in mice. In presence of TAU, 3-MA induced a marked increase in testosterone levels in compar- ison with the absence of TAU. Thus, other mechanisms may also activate testosterone production by TAU. TAU increases testosterone levels and raises testicular antioxidation in diabetic rats (Liu et al., 2017). In the pres- ent study, TAU suppressed oxidative stress by reducing MDA content and ROS levels, and increasing antioxidant enzyme activity. TAU could prevent lipid peroxidation in rabbit sperm (Alvarez & Storey, 1983). TAU reversed SOD activity in ox- idative stress induced by cadmium or arsenic (Sinha et al., 2008; Das et al., 2009). Higuchi et al. (2012) found that TAU Figure 5. Immunofluorescent microscopy of the TM3 enhanced SOD activity in the eel testicular tissue. TAU pre- cells, percentage of LC3-II-positive cells and expression vented apoptosis and oxidative stress induced by doxorubi- of autophagy-related genes. The bright green staining cin in rat testicles (Das et al., 2012). Reducing cell death by indicates LC3-II-positive cells (scale bars: 100 µm). The TAU may relate to inhibition of oxidative stress in TM3 cells. values are expressed as mean±SD; *p<0.05, **p<0.01, ROS generation can stimulate apoptosis signaling pathways # ## $ # $ p<0.05, p<0.01, p<0.05; *, and the symbols (Redza-Dutordoir & Averill-Bates, 2016). Oxidative stress can respectively indicate comparison to the control, 3-MA induce apoptosis in Leydig cells (Wang et al., 2019). and TAU groups. CONCLUSIONS In summary, this study showed that TAU induces auto- tophagy in TM3 Leydig cells. The results show that TAU phagy and suppresses apoptosis in TM3 cells. This increas- could increase the viability percentage of TM3 cells. The ing autophagy is accompanied by increasing testosterone increasing viability may be associated with the increas- levels and reducing oxidative stress. ing survival of TM3, but not for the proliferation of these cells. Indeed, flow cytometry and morphology evalua - ACKNOWLEDGEMENT tions showed that TAU markedly reduced apoptosis in TM3 cells. TAU decreased the Bax/Bcl-2 ratio in the TM3 A Grant (CMRC-9707) has been awarded by the re- cells, also showing suppression of apoptosis. Previous search council of Ahvaz Jundishapur. The University of studies have also reported anti-apoptotic effects of TAU Medical Sciences sponsored this study. This paper is a part in noncancerous cells (Takatani et al., 2004; Kim et al., of MSc thesis of Shokofeh Yahyavy. 2009; Das etal., 2012). Aly & Khafagy (2014) showed anti-apoptotic effects of TAU against endosulfan in adult CONFLICT OF INTEREST rat testicles. TAU inhibited apoptosis in Thiopurine-in- The authors declare no conflict of interest. duced testicular damages in rats (Ramadan et al., 2018). The expression of autophagy-related genes including Corresponding author: Beclin-1, Atg5, and LC3-II was markedly increased in Layasadat Khorsandi the TAU-treated cells. The percentage of LC3-II-positive Department of Anatomical Sciences cells also increased in the TAU-treated cells. Kaneko et Faculty of Medicine al. (2018) reported that TAU could induce autophagy in Ahvaz Jundishapur University of Medical Sciences adipocytes. Ahvaz, Iran. E-mail: khorsandi_cmrc@yahoo.com JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 TAU effects on autophagy and oxidative stress - Yahyavy, S. Figure 6. MDA contents, levels of ROS (DCF formations) and activities of the anti-oxidant enzyme in the # ## experimental and control groups. The values were expressed as mean±SD. *p<0.05, p<0.05, p<0.01, $ # $ p<0.05; *, and the symbols respectively indicate comparison to the control, 3-MA and TAU groups González CR, Muscarsel Isla ML, Vitullo AD. The balance REFERENCES between apoptosis and autophagy regulates testis regres- Alvarez JG, Storey BT. 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Taurine induces autophagy and inhibits oxidative stress in mice Leydig cells

JBRA Assisted Reproduction , Volume 24 (3) – Apr 1, 169

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Pubmed Central
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1517-5693
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1518-0557
DOI
10.5935/1518-0557.20190079
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Abstract

JBRA Assisted Reproduction 2020;24(3):250-256 doi: 10.5935/1518-0557.20190079 Original article Taurine induces autophagy and inhibits oxidative stress in mice Leydig cells 1,2 2 2 1,2 Shokofeh Yahyavy , Armita Valizadeh , Ghasem Saki , Layasadat Khorsandi Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran ABSTRACT MATERIALS AND METHODS Objectives: This study evaluated taurine (TAU) effects Experimental design on autophagy, apoptosis and oxidative stress in mice Ley- The mice Leydig TM3 cell line was bought through dig TM3 cells. the National Center for Genetic and Biological Reserves Methods: We treated TM3 cells with TAU (100 µg/mL) in Iran. We cultured the cells in DMEM medium, comple- or 3-Methyladenine (3-MA, an autophagy inhibitor) for 24 mented with 10% fetal bovine serum and 1% penicillin/ h, and assessed cell viability, testosterone level, oxidative o streptomycin at 37 C within a moist atmosphere with stress, apoptosis, and autophagy. 5% CO . The cells were incubated with TAU (Sigma) at Results: The results showed that TAU markedly in- various concentrations for 12, 24, 48 and 72h, as a pilot creased cell viability, testosterone levels, expression of au- study (Table 1). The cells were grouped into 4 catego- tophagy-related genes and percentage of LC3-II-positive ries (Figure 1): cells. TAU significantly reduced malondialdehyde (MDA) contents and reactive oxygen species (ROS) levels and in- I. Control: received only media for 24h creased the activities of SOD (superoxide dismutase) and II. 3-MA (3-Methyladenine, an autophagy inhibitor): CAT (Catalase) enzymes in the TM3 cells. TAU in the pres- received 10 mmol 3-MA for 24h ence of autophagy inhibitor (3-MA) increased oxidative III. TAU: received 100 µg/mL TAU for 24h stress and decreased testosterone levels. IV. TAU + 3-MA: received 10 mmol 3-MA and 100 µg/ Conclusion: The results showed that autophagy might mL TAU for 24h be involved in TAU-increased testosterone levels in mice Leydig TM3 cells. Cell viability We cultured the TM3 (1 × 10 cells/well) cells in 96-well Keywords: Leydig cells, taurine, autophagy, oxidative plates. After treating them, the supernatants were dis- stress, testosterone posed of and we added 20 µL of MTT solution (5 mg/ml) to all of the wells and incubated for four hours at 37ºC. Upon completion of incubation, the medium was eliminated, and we added 100 µL of DMSO to all of the wells. We used a INTRODUCTION micro-plate reader (BioRad, USA) to measure absorbance at 570 nm. Taurine (TAU) is a free amino acid that is present in several mammalian tissues (De Luca etal., 2015; Park Testosterone assay et al., 2002; Yang et al., 2010). TAU is present in some We incubated the cell suspensions with TAU or 3-MA foodstuff, such as fish and meat and often added to in the presence of hCG (0.05IU/ml) at 37ºC for 24h. We energy drinks. TAU has several physiological functions measured the testosterone concentration in culture me- including energy storage, membrane stabilization, anti- dia via a radio-immunoassay, complying with the manu- oxidation and xenobiotic conjugation (Huxtable, 1992). facturer`s guidelines (Monobind, USA). In male reproductive organs, TAU is present in Leydig cells, and in some other cells (Yang et al., 2010). Lobo Annexin V-FITC/propidium iodide apoptosis et al. (2000) reported that TAU stimulated testosterone assay secretion in vivo and in vitro. Despite its importance, we We seeded the TM3 cells (1.0×10 /mL) in a six-well still do not know the impact of TAU in Leydig cells. culture plate, and normal, apoptotic and necrotic cells were Leydig cells produce testosterone in testicular tis- distinguished using an Annexin V-FITC/propidium iodide sues, where autophagy is active (Yi & Tang, 1999). In assay kit (Ebioscience™ Annexin V Kit, USA) according to autophagy, microtubule-associated protein-1 light chain the manufacturer’s instruction. We analyzed the samples (LC3-I) converts to a lipid form, termed LC3-II, by a using a flow cytometer (BD Facscalibur, USA). We analyzed complex of Atg5, Atg12, and Atg16 proteins to induce the data using the FlowJo V10. autophagosome. Mammalian rapamycin (mTOR) regu- lates autophagy by activating several autophagy-related Immunocytochemistry proteins, including Beclin-1, Atg1, Atg5, and Atg7 (He After treating the TM3 cells, we deposited them over & Klionsky, 2009). Autophagy regulates ROS buildup by a glass coverslip and let it stabilize for fifteen minutes in increasing the clearance of damaged organelles and ac- a 4% para-formaldehyde in PBS at 4ºC. Then, we washed tivating antioxidant enzymes in the Leydig cells (Li et them in PBS. We used anti-LC3-II antibody (sc-16755, al., 2011). Santa Cruz) at 1/100 dilution overnight at 4ºC. After wash- In the present study we investigated the TAU effect ing twice in PBS, we incubated the section with FITC-con- on oxidative stress, autophagy, apoptosis, and testos- jugated anti-mice secondary anti-body (sc-2356, Santa terone level in the mouse Leydig TM3 cells. Received June 04, 2019 Accepted December 10, 2019 TAU effects on autophagy and oxidative stress - Yahyavy, S. Table 1. Viability percentage in TM3 cells at different doses and duration times of TAU treatment (pilot study) Treatments 12 h 24 h 48 72 12.5µg/mL 99.4 ± 4.5 98.7 ± 4.7 98.6 ± 2.2 100.4 ± 3.2 25 µg/mL 98.3 ± 4.8 101.5 ± 3.2 102.2 ± 5.3 102.6 ± 5.1 50 µg/mL 99.2 ± 3.5 105.7 ± 6.9 105.6 ± 4.2 104.3 ± 6.4 100 µg/mL 98.6 ± 5.5 135.2 ± 11.2* 138.1 ± 9.3* 132.8 ± 9.8* 150 µg/mL 98.8 ± 4.3 136.4 ± 12.2* 137.3 ± 10.7* 134.6 ± 12.3* 200 µg/mL 99.2 ± 3.6 136.7 ± 10.7* 137.8 ± 11.3* 135.1 ± 10.7* Values are expressed as mean ± SD (n=6). *p<0.05 *symbol indicates comparison to 12h in each row Figure 1. Schematic illustration of the study design primers, and 12.5 µL of 2x SYBR Green Master Mix Cruz) for one hour at room temperature. The fluorescence [Fermentas, Canada] (Table 2). PCR was amplified in pictures were captured by a fluorescence microscope forty cycles through this protocol: 95ºC for ten min- (Olympus, Japan). utes, 95ºC for fifteen seconds, 95ºC for thirty seconds, ΔΔCT and 60ºC for thirty-four seconds. We used the 2 Real-Time Polymerase Chain Reaction method to analyze the data. The whole RNA was drawn out from the cells via the RNeasy Mini kit (Qiagen) in accordance with the Determining MDA content, ROS level and anti- Company's guidelines. We synthesized cDNA using a oxidant enzyme activity cDNA synthesis kit from total RNA regarding the man- We treated the TM3 cells with TAU or 3-MA for 24h. ufacturer's instruction. About 2 µL of cDNA boosted in Afterwards, we performed the lysis of the gathered each 25 µL PCR reaction mix, which contains 10.1 µl specimens, and the protein contents of the TM3 cells we DEPC water, 0.2 µL of each 10pM forward and reverse JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 Original article 252 Table 2. Primer sequences. Groups Forward Reverse GCTGGACATTG- ACCACTGTGACCT- Bax GACTTCCTC GCTCCA GGATGCCTTTGTG- TCACTTGTGGC- Bcl-2 GAACTGT CCAGATAGG CGGTTTTTCTGGGACAA- AAAAACGTGTCTC- Beclin-1 CAA GCCTTTC TATTCACCTCCTGCCT- TGTGATGGCTGT- mTOR CACC GAAGATCC CCAGAAAAAGACCTTCT- CAATCCCATCCA- Atg5 GCACT GAGTTGCT GATAATCAGACGGC- ACTTCGGAGATGG- LC3-II GCTTGC GAGTGGA ACCCAGAAGACTGTG- TTCTAGACGGCAG- GAPDH GATGG GTCAGGT identified via a BCA protein assay kit (Pierce Biotechnol - ogy Inc. IL). Upon having the cell lysates centrifuged, we assessed the contents of malondialdehyde (MDA), Catalase (CAT), and super-oxide dismutase (SOD) activ- ity on the basis of the related guidelines (ZellBio, GmbH, Germany). With regards to the Company's guidelines, we measured the reactive oxygen species (ROS) using a Figure 2. Morphology (above images) and viability (the dichloro-dihydrofluorescein diacetate (DCFH-DA) deter - following pictures) of TM3 cells in experimental and mination kit (Sigma, St. Louis, MO). We measured the control groups (Scale bar: 100 µm). The arrows indicate ROS via a spectrofluorometer (LS50B, Waltham, USA; apoptotic morphology. The values are expressed as Ex: 490 nm, Em: 570 nm). # $ # mean ± SD. *p<0.05, p<0.05, p<0.05; *, and the symbols respectively indicate comparison to the control, Statistical analyses 3-MA and TAU groups We used the ANOVA SPSS 21.0 (Chicago, IL, USA) to analyze the data. Then, we carried out a post-hoc pair- wise comparison using the Bonferroni technique. We con- sidered the p value ˂0.05 as statistically significant. Each experiment were done in quadruplicates. RESULTS Cell viability The cell viability percentage significantly decreased in 3-MA exposed TM3 cells, compared to the control cells (p<0.05). The cell viability significantly increased in TAU-treated cells, compared to the control cells (p<0.05). In the TAU + 3-MA group, the cell viability of TM3 cells sig- nificantly decreased in comparison with the TAU and 3-MA groups (Figure 2). Testosterone assay The testosterone level significantly decreased in Figure 3. Testosterone concentration in the control 3-MA exposed TM3 cells when compared to the control and experimental groups. The arrows indicate apoptotic cells (p<0.05). The testosterone concentration signifi - morphology. The values are expressed as mean ± SD. cantly increased in TAU-treated cells, compared to the # ## $ *p<0.05, **p<0.01, p<0.05, p<0.01, p<0.05; *, control and 3-MA groups (p<0.01). In the TAU + 3-MA # $ and the symbols respectively indicates comparison to group, the testosterone level of the TM3 cells signifi - the control, 3-MA and TAU groups. cantly decreased in comparison with the TAU-treated cells (Figure 3). with the control and 3-MA groups (p<0.01). In the TAU Annexin V-FITC/propidium iodide apoptosis + 3-MA group, the apoptotic and necrotic indices signifi - assay cantly increased in comparison with TAU-treated cells In 3-MA exposed TM3 cells, necrotic and apoptotic (Figure 4), and significantly reduced when compared to indices were significantly higher than those from control the 3-MA-treated cells. cells. TAU decreased the late apoptosis, necrosis, and early apoptosis percentages in TM3 cells in comparison JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 TAU effects on autophagy and oxidative stress - Yahyavy, S. Figure 4. Flow cytometry of Annexin / PI staining in the experimental & control groups. Lower left quadrant: live cells; Lower right quadrant: initial apoptosis; Upper right quadrant: late apoptosis; Upper -2 left quadrant: necrotic cells (A and B). The relative level of mRNA expression of Bax and Bcl genes and -2 # ## Bax/Bcl ratio (C and D). The values are expressed as mean±SD. *p<0.05, **p<0.01, p<0.05, p<0.01, ### $ $$ # $ p<0.001, p<0.05, p<0.01; *, and the symbols respectively indicate comparison to the control, 3-MA and TAU groups. Immunocytochemistry Quantitative Real-time RT-PCR In the 3-MA exposed TM3 cells, we did not detect LC3- In 3-MA exposed TM3 cells, the expression of Bax and II. There was a significant increase of the percentage of Bcl-2 considerably changed in comparison to the control LC3-II-positive cells in comparison with control group group. In the TAU-treated cells, the expression of the Bax (p<0.05) by TAU (p<0.05). Figure 5 depicts these results. was considerably higher, while the expression of the Bcl-2 significantly diminished compared to the control and 3-MA ROS levels, MDA content and antioxidant enzyme groups. There was a significant increase in the expression activity of the Bax gene while there was a significant reduction ROS level and MDA content of 3-MA exposed cells were in the expression of the Bcl-2 gene in TAU+3-MA-treat- significantly increased in comparison with those from the ed cells, compared with the controls and TAU-treated cells control group. There was a considerable reduction in MDA (Figure 4). In 3-MA exposed TM3 cells, the Bax/Bcl-2 ra- contents and levels of ROS in the TAU-treated cells when tio significantly increased in comparison with the control compared to the 3-MA and control groups (p<0.05). In the group. In the TAU-treated cells, the Bax/Bcl-2 ratio sig- TAU+3-MA, the ROS level and MDA content significantly nificantly decreased compared to the control and 3-MA changed in comparison with the TAU and 3-MA groups. In groups. The Bax/Bcl-2 ratio significantly increased, com - the 3-MA exposed cells, CAT and SOD activity significantly pared with the control and TAU-treated cells (Figure 4). decreased in comparison with the control group. In the In 3-MA-treated cells, the LC3-II, Atg5 and Beclin1 TAU-treated cells, the antioxidant activity significantly in - genes were not expressed while the mTOR expression was creased in comparison with the 3-MA and control groups significantly higher. In TAU-treated cells, the mTOR gene (p<0.05). In the TAU+3-MA, the CAT and SOD activity sig- expression was significantly reduced while the expres - nificantly changed in comparison with the TAU and 3-MA sion of the LC3-II, Atg5 and Beclin1 genes experienced groups (Figure 6). a significant increase, compared to that of control cells. In TAU+3-MA-treated cells, the mTOR expression showed a significant increase in comparison with those from the DISCUSSION TAU-treated cells. While there was a significant reduction The present study showed that TAU increased the in mTOR expression in comparison with 3-MA-treated cells levels of testosterone, improved antioxidant activity, de- (Figure 5). creased ROS levels, inhibited apoptosis, and induced au- JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 Original article 254 In the presence of 3-MA, TAU increased the Bax/Bcl-2 ra- tio in comparison with the absence of 3-MA, indicating that TAU may suppress apoptosis by enhancing the autophagy process. 3-MA in the presence of TAU decreased the apoptotic index of TM3 cells, in comparison with the absence of TAU. This finding indicated that TAU could also suppress apoptosis by involving multiple signaling pathways. In the current study, testosterone levels dramatical- ly increased in the TAU-treated cells, which may be as- sociated with the increasing viability of TM3 cells or the increasing biosynthesis of androgens. Yang et al. (2010) showed that TAU could stimulate testosterone generation in cultured Leydig cells. The reducing testosterone level in 3-MA- treated cells indicated autophagy involved in testos- terone production in the TM3 cells. Rapamycin (autophagy stimulator) induced an increase in testosterone concentra- tion in rat Leydig cells, whereas 3-MA had the opposite effect (Yang et al., 2017). Gao et al. (2018) revealed that autophagy increased cholesterol absorption by the Leydig cells and dysfunction of autophagy leads to insufficient testosterone production. Au - tophagy activity decreased in aged rat Leydig cells (Li et al., 2011), and testosterone levels reduced in autophagy deficien - cy in mice (Yoshii et al., 2016). The autophagy inhibition was accompanied by a massive loss of germ cells (González et al., 2018). By contrast, Zhang et al. (2012) showed that heat exposure caused autophagy-induced germ cell death and spermatogenesis defects in mice. In presence of TAU, 3-MA induced a marked increase in testosterone levels in compar- ison with the absence of TAU. Thus, other mechanisms may also activate testosterone production by TAU. TAU increases testosterone levels and raises testicular antioxidation in diabetic rats (Liu et al., 2017). In the pres- ent study, TAU suppressed oxidative stress by reducing MDA content and ROS levels, and increasing antioxidant enzyme activity. TAU could prevent lipid peroxidation in rabbit sperm (Alvarez & Storey, 1983). TAU reversed SOD activity in ox- idative stress induced by cadmium or arsenic (Sinha et al., 2008; Das et al., 2009). Higuchi et al. (2012) found that TAU Figure 5. Immunofluorescent microscopy of the TM3 enhanced SOD activity in the eel testicular tissue. TAU pre- cells, percentage of LC3-II-positive cells and expression vented apoptosis and oxidative stress induced by doxorubi- of autophagy-related genes. The bright green staining cin in rat testicles (Das et al., 2012). Reducing cell death by indicates LC3-II-positive cells (scale bars: 100 µm). The TAU may relate to inhibition of oxidative stress in TM3 cells. values are expressed as mean±SD; *p<0.05, **p<0.01, ROS generation can stimulate apoptosis signaling pathways # ## $ # $ p<0.05, p<0.01, p<0.05; *, and the symbols (Redza-Dutordoir & Averill-Bates, 2016). Oxidative stress can respectively indicate comparison to the control, 3-MA induce apoptosis in Leydig cells (Wang et al., 2019). and TAU groups. CONCLUSIONS In summary, this study showed that TAU induces auto- tophagy in TM3 Leydig cells. The results show that TAU phagy and suppresses apoptosis in TM3 cells. This increas- could increase the viability percentage of TM3 cells. The ing autophagy is accompanied by increasing testosterone increasing viability may be associated with the increas- levels and reducing oxidative stress. ing survival of TM3, but not for the proliferation of these cells. Indeed, flow cytometry and morphology evalua - ACKNOWLEDGEMENT tions showed that TAU markedly reduced apoptosis in TM3 cells. TAU decreased the Bax/Bcl-2 ratio in the TM3 A Grant (CMRC-9707) has been awarded by the re- cells, also showing suppression of apoptosis. Previous search council of Ahvaz Jundishapur. The University of studies have also reported anti-apoptotic effects of TAU Medical Sciences sponsored this study. This paper is a part in noncancerous cells (Takatani et al., 2004; Kim et al., of MSc thesis of Shokofeh Yahyavy. 2009; Das etal., 2012). Aly & Khafagy (2014) showed anti-apoptotic effects of TAU against endosulfan in adult CONFLICT OF INTEREST rat testicles. TAU inhibited apoptosis in Thiopurine-in- The authors declare no conflict of interest. duced testicular damages in rats (Ramadan et al., 2018). The expression of autophagy-related genes including Corresponding author: Beclin-1, Atg5, and LC3-II was markedly increased in Layasadat Khorsandi the TAU-treated cells. The percentage of LC3-II-positive Department of Anatomical Sciences cells also increased in the TAU-treated cells. Kaneko et Faculty of Medicine al. (2018) reported that TAU could induce autophagy in Ahvaz Jundishapur University of Medical Sciences adipocytes. Ahvaz, Iran. E-mail: khorsandi_cmrc@yahoo.com JBRA Assist. Reprod. | v.24 | nº3 | Apr-May-Jun/ 2020 TAU effects on autophagy and oxidative stress - Yahyavy, S. Figure 6. MDA contents, levels of ROS (DCF formations) and activities of the anti-oxidant enzyme in the # ## experimental and control groups. The values were expressed as mean±SD. *p<0.05, p<0.05, p<0.01, $ # $ p<0.05; *, and the symbols respectively indicate comparison to the control, 3-MA and TAU groups González CR, Muscarsel Isla ML, Vitullo AD. The balance REFERENCES between apoptosis and autophagy regulates testis regres- Alvarez JG, Storey BT. 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Published: Apr 1, 169

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