The Function Of Atg7 And Involved Autophagy In Hyperthermia Induced Mice Suppresion Of Spermatogenesis

Mild Testicular heating suppresses spermatogenesis in several mammalian species. This process is characterized by increased germ cell death and loss. Recently, it is reported that autophagy also can be induced by heat stress in somatic cells. Autophagy is a process of self-degradation whereby cellular organelles and proteins are phagocytosed during metabolic stress. It is an evolutionarily conserved physiological process that is thought to promote cell survival. On the other hand, autophagy has also been shown to promote cell death under certain conditions and stimuli. In fact, autophagy is often referred to as type II programmed cell death. The relationship between autophagy and apoptosis is very complicated, there are still controversial. Currently, there is still no report about the role of autophagy in the process of spermatogenesis. The overall goal of this study is to clarify the role of autophagy in the impairment of spermatogenesis after heat stress, by which we can further understand the molecular mechanism of spermatogenesis and might indentify new targets for infertile diagnosis and male contraceptive development.In this study, we use mouse as a model, studied from both in vivo and in vitro. First, the status of autophagy in germ cells after heat stress, as well as the partnership between autophagy and apoptosis in these cells was investigated. The tails and the scrotums containing the testes of heat-treated mice were immersed in a thermostatically controlled water bath at 42°C for 15 min, then sacrificed these animals at 0.5 h, 2 h, 6 h, 12 h and 2 d after heat exposure (3 animals per time point). The results suggested that apoptotic rate and the level of autophagy increased compared with that in the untreated testes. Heat-treated GC2-spd cells were incubated at 42°C for 3h and then returned to the incubation at 37°C, the results suggested that apoptotic rate and the level of autophagy also increased compared with that of untreated cells. The results demonstrated that besides initiating apoptotic pathways, hyperthermia also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process.Furthermore, to investigated weather Ubiquitin-like protein conjugation pathways participate in heat-induced autophagy in germ cell, we studied from two aspects. (1) the expression of Ubiquitin-like protein conjugation pathways in testis. Atg genes were originally described in yeast, and in some cases their orthologs have been isolated and functionally,characterized in mammals. These include genes that regulate autophagosome formation, which requires two evolutionarily conserved ubiquitin-like conjugation systems—the Atg12-Atg5 and the Atg8 (LC3)-PE (phosphatidylethanolamine) systems. In this study, the expression of Atg12-Atg5 and Atg8 (LC3)-PE systems in mouse testis was demonstrated by RT-PCR. We further detected the expression of Atg7, a gene upstream to both these two systems, in the mouse testes on the protein level by using Western blotting, and immunohistochemical results showed that Atg7 protein was expressed in Leydig cells, Sertoli cells and all kinds of spermatogenic cells. These results confirm that the ubiquitin-like conjugation systems exist in the testis, and may be responsible for heat-induced autophagy in germ cells. (2) The expression level of Atg7 was positively correlated with the level of autophagy. We examined the relativity of Atg7 level with autophagy in germ cells after heat stress both in vivo and in vitro. Results showed that the expression of Atg7 in the testes and in GC2-spd cells increased after heat treatment and the increase was significant at 12 h following heat treatment, which coincided with the increased levels of autophagy. The above results indicated that Atg7 protein might participate in regulating autophagy and apoptosis in germ cells. (3) Atg7 knockdown conferred significant protection against heat-induced autophagy in germ cells. To further study the role of Atg7 on heat-induced autophagy in germ cells, we selected Atg7 as the investigative target, which represented autophagy involving the ubiquitin-like conjugation pathway. We knockdown the expression of Atg7 in vivo and in vitro. Atg7 knockdown confers marked protection against hyperthermia-mediated conversion of LC3-I to LC3-II. These results demonstrated a critical role of Atg7 protein and the ubiquitin-like conjugation pathway in regulation of hyperthermia-mediated autophagy in germ cells.Finally, we further investigated the function of Atg7 and involved autophagy in hyperthermia-mediated apoptosis of germ cell. Hyperthermia-mediated apoptosis in vitro and vivo is also significantly suppressed by knockdown of Atg7 protein level. These results demonstrate that autophagy participate in the process of apoptosis induced by hyperthermia.In conclusion, the present study indicates that heat treatment selectively triggers autophagy in germ cells, which participates in germ ell apoptosis and lead to destruction of spermatogenesis eventually. Atg7 and its attended autophagy pathway are critically involved in regulation of heat-mediated autophagic and apoptotic cell death.