Denervation-induced mitochondrial dysfunction and autophagy in skeletal muscle of apoptosis-deficient animals

Skeletal muscle undergoes remarkable adaptations in response to chronic decreases in contractile activity, such as a loss of muscle mass, decreases in both mitochondrial content and function, as well as the activation of apoptosis. Although these adaptations are well known, questions remain regarding the signaling pathways that mediated these changes. Autophagy is an organelle turnover pathway that could contribute to these adaptations. The purpose of this study was to determine whether denervation-induced muscle disuse would result in the activation of autophagy gene expression in both wild-type (WT) and Bax/Bak double knockout (DKO) animals, which display an attenuated apoptotic response. Denervation caused a reduction in muscle mass for WT and DKO animals; however, there was a 40% attenuation in muscle atrophy in DKO animals. Mitochondrial state 3 respiration was significantly reduced, and reactive oxygen species production was increased by two- to threefold in both WT and DKO animals. Apoptotic markers, including cytosolic AIF and DNA fragmentation, were elevated in WT, but not in DKO animals following denervation. Autophagy proteins including LC3II, ULK1, ATG7, p62, and Beclin1 were increased similarly following denervation for both WT and DKO. Interestingly, denervation markedly increased the localization of LC3II to subsarcolemmal mitochondria, and this was more pronounced in the DKO animals. Thus denervation-induced muscle disuse activates both apoptotic and autophagic signaling pathways in muscle, and autophagic protein expression does not exhibit a compensatory increase in the presence of attenuated apoptosis. However, the absence of Bax and Bak may represent a potential signal to trigger mitophagy in muscle.     

Lactate accelerates calcification in VSMCs through suppression of BNIP3-mediated mitophagy

Arterial media calcification is one of the major complications of diabetes mellitus, which is related to oxidative stress and apoptosis. Mitophagy is a special regulation of mitochondrial homeostasis and takes control of intracellular ROS generation and apoptotic pathways. High circulating levels of lactate usually accompanies diabetes. The potential link between lactate, mitophagy and vascular calcification is investigated in this study. Lactate treatment accelerated VSMC calcification, evaluated by measuring the calcium content, ALP activity, RUNX2, BMP-2 protein levels, and Alizarin red S staining. Lactate exposure caused excessive intracellular ROS generation and VSMC apoptosis. Lactate also impaired mitochondrial function, determined by mPTP opening rate, mitochondrial membrane potential and mitochondrial biogenesis markers. Western blot analysis of LC3-II and p62 and mRFP-GFP-LC3 adenovirus detection for autophagy flux revealed that lactate blocked autophagy flux. LC3-II co-staining with LAMP-1 and autophagosome quantification revealed lactate inhibited autophagy. Furthermore, lactate inhibited mitophagy, which was confirmed by TOMM20 and BNIP3 protein levels, LC3-II colocalization with BNIP3 and TEM assays. In addition, BNIP3-mediated mitophagy played a protective role against VSMC calcification in the presence of lactate. This study suggests that lactate accelerates osteoblastic phenotype transition of VSMC and calcium deposition partly through the BNIP3-mediated mitophagy deficiency induced oxidative stress and apoptosis.     

Quantitation of “autophagic flux” in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure “autophagic flux” in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the “autophagometer.” In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. the addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a “colchicine block.” Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an “in vivo autophagometer” study using colchicine in skeletal muscle.Key words: autophagy, rapamycin, skeletal muscle     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.     

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.     

Single bout of running exercise changes LC3-II expression in rat cardiac muscle

Macroautophagy (autophagy) is an intracellular catalytic process. We examined the effect of running exercise, which stimulates cardiac work physiologically, on the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, an indicator of autophagy, as well as some autophagy-related proteins in rat cardiac muscle. The left ventricles were taken from rats immediately (0 h), and at 0.5 h, 1 h or 3 h after a single bout of running exercise on a treadmill for 30 min and also from rats in a rest condition. In these samples, we evaluated the level of LC3-II and p62, and the phosphorylation level of mammalian target of rapamycin (mTOR), Akt and AMP-activated protein kinase alpha (AMPKα) by Western blotting. The exercise produced a biphasic change in LC3-II, with an initial decrease observed immediately after the exercise and a subsequent increase 1 h thereafter. LC3-II then returned to the rest level at 3 h after the exercise. A negative correlation was found between the LC3-II expression and mTOR phosphorylation, which plays a role in inhibiting autophagy. The exercise increased phosphorylation of AMPKα, which stimulates autophagy via suppression of mTOR phosphorylation, immediately after exercise. The level of p62 and phosphorylated Akt was not altered significantly by the exercise. These results suggest for the first time that a single bout of running exercise induces a biphasic change in autophagy in the cardiac muscle. The exercise-induced change in autophagy might be partially mediated by mTOR in the cardiac muscle.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

The TWEAK-Fn14 system: breaking the silence of cytokine-induced skeletal muscle wasting

The occurrence of skeletal muscle atrophy, a devastating complication of a large number of disease states and inactivity/disuse conditions, provides a never ending quest to identify novel targets for its therapy. Proinflammatory cytokines are considered the mediators of muscle wasting in chronic diseases; however, their role in disuse atrophy has just begun to be elucidated. An inflammatory cytokine, tumor necrosis factor (TNF)- like weak inducer of apoptosis (TWEAK), has recently been identified as a potent inducer of skeletal muscle wasting. TWEAK activates various proteolytic pathways and stimulates the degradation of myofibril protein both in vitro and in vivo. Moreover, TWEAK mediates the loss of skeletal muscle mass and function in response to denervation, a model of disuse atrophy. Adult skeletal muscle express very low to minimal levels of TWEAK receptor, Fn14. Specific catabolic conditions such as denervation, immobilization, or unloading rapidly increase the expression of Fn14 in skeletal muscle which in turn stimulates the TWEAK activation of various catabolic pathways leading to muscle atrophy. In this article, we have discussed the emerging roles and the mechanisms of action of TWEAK-Fn14 system in skeletal muscle with particular reference to different models of muscle atrophy and injury and its potential to be used as a therapeutic target for prevention of muscle loss.     

HMGB1 Promotes Mitochondrial Dysfunction–Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation

Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP–induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP–induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.     

The role of autophagy in corpus luteum regression in the rat

Autophagy is associated with luteal cells death during regression of the corpus luteum (CL) in some species. However, the involvement of autophagy or the association between autophagy and apoptosis in CL regression are largely unknown. Therefore, we investigated the role of autophagy in CL regression and its association with apoptosis. Ovaries were obtained from pseudopregnant rats at Days 2 (early), 7 (mid-), and 14 and 20 (late-luteal stage) of the pseudopregnancy; autophagy-associated protein (microtuble-associated protein light chain 3 [LC3]) was immunolocalized and its expression level was measured. Luteal cell apoptosis was evaluated by measuring cleaved caspase 3 expression. LC3 expression increased slightly from early to mid-luteal stage, with maximal levels detected at the late-luteal stage in steroidogenic luteal cells. The expression level of the membrane form of LC3 (LC3-II) also increased during luteal stage progression, and reached a maximum at the end point of late-luteal stage (Day 20). This pattern coincided with cleaved caspase 3 expression. Furthermore, LC3-II expression increased, as did levels of cleaved caspase 3 in luteal cells cultured with prostaglandin F(2alpha) known to induce CL regression. These findings suggest that luteal cell autophagy is directly involved in CL regression, and is correlated with increased apoptosis. In addition, autophagic processes were inhibited using 3-methyladenine or bafilomycin A1 to evaluate the role of autophagy in apoptosis induction. Inhibition of autophagosome degradation by fusion with lysosomes (bafilomycin A1) increased apoptosis and cell death. Furthermore, inhibition of autophagosome formation (3-methyladenine) decreased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces luteal cell apoptosis. In conclusion, these results indicate that autophagy is involved in rat luteal cell death through apoptosis, and is most prominent during CL regression.     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2^−/− MEFs but not JNK1^−/− MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.     

Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment

Kaempferol, a dietary cancer chemopreventive polyphenol, has been reported to trigger apoptosis in several tumor histotypes, but the mechanism underlying this phenomenon is not fully understood. Here, we demonstrate that, in HeLa cells, kaempferol induces energetic failure due to inhibition of both glucose uptake and Complex I of the mitochondrial respiratory chain. As adaptive response, cells activate autophagy, the occurrence of which was established cytofluorometrically, upon acridine orange staining, and immunochemically, by following the increase of the autophagolysosome-associated form of the microtubule-associated protein light chain 3 (LC3-II). Autophagy is an early and reversible process occurring as survival mechanisms against apoptosis. Indeed, chemical inhibition of autophagy, by incubations with monensin, wortmannin, 3-methyladenine, or by silencing Atg5, significantly increases the extent of apoptosis, which takes place via the mitochondrial pathway, and shortens the time at which the apoptotic markers are detectable. We also demonstrate that autophagy depends on the early activation of the AMP-activated protein kinase (AMPK)/mTOR-mediated pathway. The over-expression of dominant negative AMPK results in a decrease of autophagic cells, a decrement of LC3-II levels, and a significant increase of apoptosis. Experiments performed with another carcinoma cell line yielded the same results, suggesting for kaempferol a unique mechanism of action.     

Regulation of muscle protein degradation,not synthesis,by dietary leucine in rats fed a protein-deficient diet

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.     

The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction

Autophagy is a membrane trafficking process involved in intracellular degradation and recycling in eukaryotic cells. DRAM2 (damage-regulated autophagy modulator 2) is a homologue of DRAM that regulates p53-mediated cell death. As its name implies, DRAM expression induces autophagy in a p53-dependent manner; however, the role of DRAM2 in autophagy is not clear. In this study, we report that DRAM2 expression contributes to autophagy induction. Overexpression of DRAM2 induces cytoplasmic GFP-LC3 punctuates, and increases the level of endogenous LC3-II. Moreover, the silencing of endogenous DRAM2 interferes with starvation-induced autophagy. Thus, we propose that DRAM2 as well as DRAM are involved in autophagy.     

p53 mediates mitochondria dysfunction-triggered autophagy activation and cell death in rat striatum

In vivo administration of the mitochondrial inhibitor 3-nitropropionic acid (3-NP) produces striatal pathology mimicking Huntington disease (HD). However, the mechanisms of cell death induced by metabolic impairment are not fully understood. The present study investigated contributions of p53 signaling pathway to autophagy activation and cell death induced by 3-NP. Rat striatum was intoxicated with 3-NP by stereotaxic injection. Morphological and biochemical analyses demonstrated activation of autophagy in striatal cells as evidenced by increased the formation of autophagosomes, the expression of active lysosomal cathepsin B and D, microtubule associate protein light chain 3 (LC3) and conversion of LC3-I to LC3-II. 3-NP upregulated the expression of tumor suppressor protein 53 (p53) and its target genes including Bax, p53-upregulated modulator of apoptosis (PUMA) and damage-regulated autophagy modulator (DRAM). 3-NP-induced elevations in pro-apoptotic proteins Bax and PUMA, autophagic proteins LC3-II and DRAM were significantly reduced by the p53 specific inhibitor pifithrin-α (PFT). PFT also significantly inhibited 3-NP-induced striatal damage. Similarly, 3-NP-induced DNA fragmentation and striatal cell death were robustly attenuated by the autophagy inhibitor 3-methyladenine (3-MA) and bafilomycin A1 (BFA). These results suggest that p53 plays roles in signaling both autophagy and apoptosis. Autophagy, at least partially, contributes to neurodegeneration induced by mitochondria dysfunction.     

Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice

Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis.