Mild Heat Stress-induced Autophagy Regulates Lactate Secretion In Cultured Immature Sertoli Cell In Vitro

Lactate secreted by Sertoli cell is utilized as energetic substrates to developing germ cells in seminiferous tubule, and has an anti-apoptotic effect on those cells. There are three proteins involved in lactate secretion: Glucose transporters(GLUTs), Lactate dehydrogenases(LDHs) and Monocarboxylate transporters(MCTs). Developing germ cells are susceptible to high temperature and undergo apoptosis in response to heat stress(HS). However, autophagy has an effect of maintaining cytoplasmic homeostasis and cellular function. It could remove denatured proteins, regenerate some small molecular substances if it was triggered by stress. Therefore, heat stress-induced autophagy has a deep relation with lactate secretion and the expression of those proteins. Heat stress not only induced autophagy, but also apoptosis, so we also analysis the relationship between autophagy and apoptosis by their level after heat stress. The objective of this study is to determine whether autophagy could be induced by heat stress in Sertoli cells, and whether HS-induced autophagy could regulate Sertoli cell lactate secretion and apoptosis.The immature boar(3 weeks) Sertoli cells were cultured in vitro, and heat-treated in 43 °C incubator for 30 min and recovered in 32 °C incubator for different time(0 h, 12 h, 24 h, 36 h and 48 h). The autolysosome was detected by Immunofluorescence, the LC3B-II/LC3B-I(LC3B), Cleaved-Caspase-3(CASP3), GLUT3(SLC2A3), LDHA(LDHA) and MCT1(SLC16A1) were examined by western blotting and quantitative real-time PCR.Results:(1) Compared to controls, the ratio of LC3B-II/LC3B-I and expression of LC3 B m RNA had a time-dependence tendency at 0-48 h after HS, and the peak was at 24 h, increased by 30.25%, 257%(P< 0.01), respectively. The intensity of autolysosomes detected by observing the intensity of red fluorescent was higher at 24 h than these at other time points.(2) In addition, lactate secretion, and expressions of SLC2A3(glucose transporter 3, GLUT3), LDHA(lactate dehydrogenase A, LDHA) and SLC16A1(monocarboxylate transporter 1, MCT1) showed the peak at 24 h(P< 0.01).(3) the apoptosis rate, cleaved-caspase-3 and CASP3(caspase-3) m RNA also had a time-dependence tendency at 0-48 h after HS, and highest level of were observed at 12 h, increased by 49%, 65.63% and 43.27%, respectively(P< 0.01).(4) SCs with pretreatment of LY294002(20 μM), significantly inhibited the formation of autolysosomes and reduced the expression of LC3B-II/LC3B-I(LC3B)(P< 0.01).(5) LY294002(20 μM) significantly enhanced apoptosis rate and the expression of caspase-3(CASP3)(P< 0.01).(6) LY294002(20 μM) significantly increased lactate secretion and expressions of, GLUT3(SLC2A3), LDHA(LDHA) and MCT1(SLC16A), compared to the heat stress alone(P< 0.01-0.05).These results showed that heat stress-induced autophagy by forming autolysosomes and inducing the expression of LC3B-II/LC3B-I(LC3B), inhibited cell apoptosis by regulating apoptosis rate and the expression of caspase-3(CASP3), and promoting lactate secretion via increasing expressions of, GLUT3(SLC2A3), LDHA(LDHA) and MCT1(SLC16A). These findings also indicated that autophagy may be a target for improving quality and quantity of sperm after heat stress.