Primary Study On Autophagy And Induction Of Autophagic Factors In Mouse Spermatogenic Cells And Sertoli Cells

It is well known that spermatogenesis in most mammalian animals is sensitive to heat. Heat shock can block the process and induce apoptosis of spermatogenic cells. In our previous study, it had been relieved that after8hours of heat shock on mouse testis using the temperature of42℃for30minutes, most spermatogenic cells could be induced into apoptosis pathway. Whereas, a small number of them had yet to undergo apoptosis and the cell cycle had been blocked into G2/M. It is still not known how these cells survive from heat shock. Considering the feature of autophagy induced by endoplasmic reticulum stress, we noticed that the sensibility of spematogenic cells to heat stress might indicate that some of these cells could survive from heat shock through autophagic pathway.The frequency of spontaneous autophagy is very high in leydig cells and then Sertoli cells in rat testis, reported by Xueming Tang et al.However, spontaneous autophagy has not been found in spermatogenous cell, primary spermatocytes or sperm cells. Considering the inducement and function of autophagy, we did a primary study on whether autophagy could occur in spermatogenic cells and found conditions which might induce autophagy.We first established the culture model of mouse convoluted seminiferous tubles in vitro. Expression levels of autophagy associated protein Hsp90, Hsp70. LC3were detected under conditions of starvation, oxygen deprivation and heat shock. Autophagosomes of spermatogenic cells and sertoli cells in convoluted seminiferous tubles produced after starvation were found using transmission electron microscopy (TEM).Our research indicated that autophagy could be induced by rising expression levels of LC3II or the ratio of LC3Ⅱ/LC3I when convoluted seminiferous tubles were in conditions of starvation, oxygen deprivation and heat shock. TEM results showed that autophagy could occur in spermatogenic cells and sertoli cells which cultured under normal conditions. The number of autophagosomes especially late autophagosomes increased relatively with serum withdrawal for6hours. In addition. expression levels of Hsp90. Hsp70also increased relatively. Autophagic inhibitor3-MA could inhibit autophagy by restrainting expression levels of LC3Ⅱ or transformation from LC3I to LC3Ⅱ.3-MA could also increase expression levels of Hsp90. but it did not have obvious effects on Hsp70. Autophagic activity increased relatively when convoluted seminiferous tubles were cultured at37℃compared to which cultured at33℃, but it did not have obvious effects on Hsp90or Hsp70. It did not have direct effects on expression of autophagy related protein Hsp90, Hsp70, LC3during oxidative stress.According to the fact that there are various cell types in mouse convoluted seminiferous tubles, sertoli cells are the only cells that can contact with spermatogenic cells and play an important role in spermatogenesis. studies on autophagy of sertoli cells and spermatogenic cells respectively would have great significance. We isolated and purified sertoli cells of mice in its infancy and the purity of these cells was examined using double staining immunofluorescence techniques. Spermatogenic cells of adult male mice were isolated using collagenase IV/trypsin.Autophagic models of spermatogenic cells and sertoli cells were established under culture conditions of serum withdrawal (starvation), oxygen deprivation (cultured with N2) and heat shock. Expression level of autophagy associated protein LC3was detected by western blotting analysis. Acridine orange staining method was used to detect the acidic vesicles. Autophagosomes were detected using transmission electron microscopy (TEM).Our research showed that autophagic levels of sertoli cells were high under normal conditions and could increase to a certain extent under conditions of serum withdrawal (starvation), oxygen deprivation (cultured with N2) and heat shock. Expression level of chaperone mediated autophagy associated protein LAMP-2increased to a certain extent under conditions of oxygen deprivation (cultured with N2) and heat shock, there was not some obvious change after serum removal but LAMP-2 was preferentially found in lysosomes surrounding the nucleus. After treating for6hours under these3conditions, the number of acidic vesicles and autophagosomes especially early autophagosomes increased ralatively.Autophagic levels of spermatogenic cells were low under normal conditions. Expression levels of LC3Ⅱ could be induced more effectively under conditions of oxygen deprivation (cultured with N2). which could be developed as an autophagic model of spermatogenic cells in vitro. Autophagy could not be induced effectively under conditions of serum withdrawal (starvation) within6hours. Expression levels of LC3Ⅱ could be induced relatively after heat shock (2hours). The number of acidic vesicles increased relatively after treating for6hours under conditions of oxygen deprivation (cultured with N2) and serum withdrawal (starvation). The number of autophagosomes increased to some extent under these3conditions. Expression level of chaperone mediated autophagy associated protein LAMP-2was very low under normal conditions and could not be induced under these3conditions, which indicated that levels of CMA might be very low in spermatogenic cells.Autophagic levels of spermatogenic cells were very low and could not be easily induced, so spermatogenic cells might be very sensitive to adverse factors. When spermatogenic cells were in conditions of heat shock and other adverse factors, a lot of them which have low autophagic levels would be induced into apoptosis pathway.