Elevation of autophagy rescues spermatogenesis by inhibiting apoptosis of mouse spermatocytes.

Autophagy and apoptosis are interlocked in an extensive crosstalk. Our previous study demonstrated that hypotonic hypoxia-induced marked apoptosis of a spermatocyte-derived cell line (GC-2). However, whether hypoxia-induced apoptosis is mediated by inhibition of autophagy under hypoxic conditions remains unclear. In this study, GC-2 cells were cultured in 1% O2 and harvested at different time points. Autophagy was determined by acridine orange staining, cyto-ID staining, mCherry-GFP-LC3B adenovirus transfection and Western blotting for various autophagy markers. Apoptosis was detected by TUNEL staining, flow cytometry, JC-1 staining and Western blotting of apoptosis-related proteins. We found that hypoxia-induced apoptosis of GC-2 cells through mitochondrial and death receptor pathways and inhibited autophagic flux in GC-2 cells in a time-dependent manner. However, while marked autolysosome formation was observed in GC-2 cells before 24-h culture in hypoxic conditions, apparent apoptosis was observed only after 24-h culture in hypoxic conditions. Caspase-8 siRNA treatment induced cell survival, accompanied by induction of the mature autophagosome, acidic vesicular organelle formation and autophagic flux. Furthermore, Beclin-1 overexpression markedly attenuated the impairment of spermatogenesis in mice by inhibiting apoptosis of spermatocytes. The results of this study demonstrate that hypoxia inhibits autophagy, which further enhances hypoxia-induced apoptosis of mouse spermatocytes by promoting caspase-8 activation in a time-dependent manner, suggesting that combined application of apoptosis inhibition and autophagy activation might be a therapeutic strategy for treating hypoxia-induced male infertility.

Hypoxia-induced apoptosis of mouse spermatocytes is mediated by HIF-1α through a death receptor pathway and a mitochondrial pathway.

Hypoxia in vivo induces oligozoospermia, azoospermia, and degeneration of the germinal epithelium, but the underlying molecular mechanism of this induction is not fully clarified. The aim of this study was to investigate the role of the death receptor pathway and the mitochondrial pathway in hypoxia-induced apoptosis of mouse GC-2spd (GC-2) cells and the relationship between HIF-1α and apoptosis of GC-2 cells induced by hypoxia. GC-2 cells were subjected to 1% oxygen for 48 hr. Apoptosis was detected by flow cytometry, TUNEL staining, LDH, caspase-3/8/9 in the absence and presence of HIF-1α siRNA. The protein levels of apoptosis-related markers were determined by Western blot in the presence and absence of HIF-1α siRNA. Mitochondrial transmembrane potential change was observed by in situ JC-1 staining. Cell viability was assessed upon treatment of caspase-8 and 9 inhibitors. The results indicated that hypoxia at 1% oxygen for 48 hr induced apoptosis of GC-2 cells. A prolonged exposure of GC-2 cells to hypoxic conditions caused downregulation of c-FLIP, Dc R2 and Bcl-2 and upregulation of DR5 , TRAIL, Fas, p53, and Bax, with an overproduction of caspase-3/8/9. Moreover, hypoxia at this level had an effect on mitochondrial depolarization. In addition, specific inhibitors of caspase-8/9 partially suppressed hypoxia-induced GC-2 cell apoptosis, and the anti-apoptotic effects of the caspase inhibitors were additive. Of note, HIF-1α knockdown attenuated hypoxia and induced apoptosis of GC-2 cells. In conclusion, our data suggest that the death receptor pathway and mitochondrial pathway, which are likely mediated by HIF-1α, contribute to hypoxia-induced GC-2 cell apoptosis.

Regulatory effects of autophagy on spermatogenesis.

Abnormal spermatogenesis is an important pathophysiological process underlying male infertility. Apoptosis of spermatogenic cells and disruption of ectoplasmic specialization (ES) have been characterized as the key biological events of this disorder. Under physiological and pathophysiological conditions (such as exposure to starvation, environmental chemicals, radiation), autophagy is activated in spermatogenic or Sertoli cells in order to maintain survival of the spermatogenic cells by inhibiting spermatogenic cell apoptosis and stabilizing the integrity of ES via degradation of PDZ and LIM domain 1 (PDLIM1), a negative regulator of cytoskeletal organization. Here, we review the most recent research progress towards understanding the pivotal effects of autophagy on spermatogenesis.

[Hypobaric hypoxia increases the expression of death receptor 5 and inhibits that of FLICE-like inhibitory protein in the rat testis].

OBJECTIVE: To evaluate the effects of hypobaric hypoxia on the expressions of death receptor 5 (DR5) and cellular FLICE-like inhibitory protein (c-FLIP) and the distribution of c-FLIP in the rat testis. METHODS: Forty adult male SD rats were randomly divided into four groups of equal number: normoxia control, 3 d hypoxia, 15 d hypoxia and 30 d hypoxia. The control rats were raised at 300 m above the sea level, while the latter three groups of rats in a hypobaric chamber at a simulated altitude of 4000 m for 5, 15 and 30 days, respectively. Then the expressions of DR5 and c-FLIP were detected by immunoblotting and the distribution of c-FLIP in the testis observed by immunofluorescence. RESULTS: The expressions of DR5 were 2.04 +/- 0.11, 1.97 +/- 0.12 and 2.34 +/- 0.11 in the 3 d, 15 d and 30 d hypoxia groups, respectively, significantly higher than 1.78 +/- 0.09 in the normoxia group (P < 0.05). The expressions of c-FLIP were 0.87 +/- 0.03 and 0.74 +/- 0.07 in the 15 d and 30 d hypoxia groups, respectively, significantly lower than 1.03 +/- 0.02 in the normoxia group (P < 0.05). CONCLUSION: Simulated hypobaric hypoxia at 4000 m above the sea level increased the expression of DR5 and inhibited that of c-FLIP in the rat testis.

The antioxidant effects of garlic saponins protect PC12 cells from hypoxia-induced damage.

Hypoxia frequently occurs under several different cellular circumstances. Excess reactive oxygen species that are induced by hypoxia may result in cell injury and dysfunction. Recently, garlic has been found to possess some biological and pharmacological activities. The present study examined the effects of garlic saponins (GSP) on the survival of differentiated PC12 (dPC12) cells and the oxidative-antioxidant system. dPC12 cells were exposed to 2 % O2 in order to establish a neuronal insult model. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay and lactate dehydrogenase (LDH) release assay. The expression of selected genes (catalase (CAT), p65 and neuron-specific class III ß-tubulin) was evaluated by real-time PCR and immunoblot assays. CAT activity, malondialdehyde (MDA) and 8-hydroxy-deoxyguanosine (8-OH-dG) concentrations were also determined. The data showed that hypoxia dramatically damaged dPC12 cells, while treatment with approximately 5 × 10- 2-10 ng/ml GSP improved cell viability, decreased LDH leakage and caused the cells to maintain neuronal-like characteristics in hypoxia. The production of MDA and 8-OH-dG was attenuated by GSP. CAT activity in dPC12 cells pretreated with GSP was higher than that of the hypoxic control. Moreover, GSP up-regulated CAT expression and decreased the total protein expression as well as the nuclear expression of p65 in hypoxic cells. These data indicate that GSP has antioxidant properties that can protect dPC12 cells from hypoxia-induced damage, which may be related to the up-regulation of CAT expression and activity as well as a decrease in the expression and nucleus distribution of p65 through effects on redox-sensitive signalling pathways.

Hypoxic training increases metabolic enzyme activity and composition of alpha-myosin heavy chain isoform in rat ventricular myocardium.

Cardiac muscle adaptation is essential for maintaining physical capacity after ascending to high altitude. This study examines the effects of high altitude training on myocardial metabolic enzyme activity and composition of alpha-myosin heavy chain (MHC). Rats were randomly divided into normobaric sedentary (NS) and training (NT) groups, and hypobaric sedentary (HS) and training (HT) groups. HS and HT rats were exposed to hypobaric hypoxia (simulated 4,000-5,000 m) for 5 weeks (24 h/day), and HT rats simultaneously received swim training. Hypoxia exposure for 5 weeks led to a decrease in succinate dehydrogenase (SDH) and citrate synthase (CS) activities in the left ventricle (LV), and a decrease in CS, hexokinase (HK) and total lactate dehydrogenase (LDH) activities in the right ventricle (RV) (p < 0.05, HS vs. NS). Furthermore, 1 h/day swim training during hypoxia exposure enhanced the CS activity in LV and the SDH and CS activities in RV (p < 0.05, HT vs. HS). The percentages of alpha-MHC in both ventricles in HT were higher than those in HS (p < 0.05). We conclude that exercise training at high altitude is beneficial for cardiac muscle adaptation to hypoxia by increasing activities of enzymes and percentage of alpha-MHC. This may contribute to improved cardiac function and work capacity at high altitude.

[Hypoxia promotes apoptosis of germ cells in rat testes].

OBJECTIVE: To explore the effects of hypobaric hypoxia on the apoptosis of germ cells in male rats. METHODS: Adult male Wistar rats were randomly divided into four groups: a control group raised at sea level; a 5 d, a 15 d and a 30 d hypoxic group raised in a hypobaric chamber simulating 5000 m altitude for 5 days, 15 days and 30 days respectively. Flow cytometry and TUNEL were used to evaluate the apoptosis of germ cells in the testis. Bax and Bcl-2 in the testis were measured by Western blot. RESULTS: Seminiferous tubules with apoptotic germ cells were significantly more in the hypoxic groups than in the control (P < 0.01). Most apoptotic germ cells were spermatogonia and spermatocytes. Compared with the control group, apoptotic germ cells detected by PI flow cytometry were significantly increased in the hypoxic 15 d and 30 d groups (P < 0.05); Bax was significantly higher (P < 0.05), and so was the ratio of Bax to Bcl-2 in the hypoxic 30 d group (P < 0.01). CONCLUSION: Hypoxia promotes apoptosis of testicular germ cells in male rats. Chronic hypoxia increases Bax expression in the rat testis.

[Hypoxia upregulates the expression of cytoglobin in lung cancer cells].

OBJECTIVE: To explore the characters of expression of cytoglobin in tumor cells after hypoxia. METHODS: Human pulmonary tumor cells of the line A549 were cultured and divided into 4 groups to be cultured under 5% CO2 and 95% air and exposed to 2% O2, 5% CO2, and 93% N(2) for 4, 12, or 24 hours respectively. The distribution of cytoglobin was examined by immunohistochemistry and the expression of cytoglobin was detected by Western blotting. The mRNA level of cytoglobin in the A549 cells was assayed by reverse-transcription PCR. RESULTS: Immunohistochemistry showed that cytoglobin was located in the plasma of the A549 cells, and the staining strength of cytoglobin was enhanced in the hypoxic groups in comparison with the normoxic group. Western blotting showed significantly stronger expression of protein of cytoglobin in the 3 hypoxic groups than in the normoxic group (all P < 0.05). The expression of cytoglobin was upregulated significantly in the hypoxic 12- and 24-hour groups than in the hypoxic 4-hour group (both P < 0.05). The cytoglobin mRNA levels of the 3 hypoxic groups were all significantly higher than that of the normoxic group (all P < 0.05). The cytoglobin mRNA levels of the hypoxic 12- and 24-hour groups were significantly higher than that of the hypoxic 4-hour group (both P < 0.05), however, without a significant difference between the hypoxic 12- and 24-hour groups (P > 0.05). CONCLUSION: Hypoxia upregulates the expression of cytoglobin in tumor cells.