Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice

Primary ovarian insufficiency (POI) is a common cause of infertility in around 1–2% of women aged <40 years. However, the mechanisms that cause POI are still poorly understood. Here we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to subfertility in female mice. The subfertility of Atg7 deletion females was caused by severe ovarian follicle loss, which is very similar to human POI patients. Further investigation revealed that germ cell-specific Atg7 knockout resulted in germ cell over-loss at the neonatal transition period. In addition, our in vitro studies also demonstrated that autophagy could protect oocytes from over-loss by apoptosis in neonatal ovaries under the starvation condition. Taken together, our results uncover a new role for autophagy in the regulation of ovarian primordial follicle reservation and hint that autophagy-related genes might be potential pathogenic genes to POI of women.Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), is an ovarian defect characterized by the premature depletion of ovarian follicles before the age of 40 years. POI is a common cause of infertility in women, affecting 1–2% of individuals aged <40 years and 0.1% of individuals aged <30 years.¹ Potential etiologies for POI are highly heterogeneous, which include iatrogenic, infectious, autoimmune, metabolic, chromosomal and genetic factors.² At present, about 25% of all forms of POF can be classified as iatrogenic and are related to cancer treatment, but >50% of the cases remain idiopathic. Though the pathogenic mechanism remains unexplained in the majority of the cases, several observations support a prevalent role of genetic mechanisms in the pathogenesis of idiopathic POI. It has been reported that mutations in FMR1, BMP-15, GDF-9, FOCL2, FSHR, LHR, INHA, GALT and AIRE are associated with POI.^{3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13} The genetic information of POI is very useful for family counseling, because it can predict the female relatives who may be at higher risk for POI and fertility loss in young age. The female carriers will be able to plan their conception before ovarian failure occurs. This requirement is becoming more and more important, because women nowadays tend to conceive ever more frequently in their thirties and forties,¹⁰ when the risk of POI in the general population is about 1–2%. However, still few genes could be identified that can explain a substantial proportion of the cases of POI.An important phenotype of POI is infertility, thus POI patients do not have large family histories, and therefore are difficult to study using traditional genetic methods, such as linkage analysis. Animal models of POI have been successfully used to identify candidate genes in this disease. The disruption of meiosis-specific genes, Bcl-2 family apoptotic-related genes, Pten-PI3K-Akt-Foxo3 pathway and Tsc1/2-mTOR signaling pathway result in POI-like phenotype in mice.^{14, 15, 16, 17} However, as a complex disorder, the genetic etiologies of POI still need to be further investigated to better understand the underlying molecular mechanisms.Macroautophagy (hereafter referred to as autophagy) is the primary intracellular catabolic mechanism for degrading and recycling long-lived proteins and organelles, which is evolutionarily conserved from yeast to mammals.¹⁸ During autophagy, isolation membrane enwraps parts of the cytoplasm and intracellular organelles, and fuse with each other forming a double membrane structure, known as the autophagosome. Then the outer membrane of the autophagosome fuses with the lysosome to form autolysosome, in which the cytoplasm-derived materials are degraded by resident hydrolases.¹⁹ The primary function of autophagy is to allow cells or organisms to survive nutrient starvation conditions by recycling either proteins or other cellular components. This process is important for cells to adapt their metabolism to starvation caused by decreased extracellular nutrients or by decreased intracellular metabolite concentrations. In addition to nutrient supply and adaptation to stress conditions, a number of observations have revealed that autophagy also functions in many physiological processes in mammalian systems, such as cell death, antiaging mechanisms, innate immunity, development and tumor suppression.^{20, 21, 22, 23, 24, 25}From the discovery of the molecular mechanism underlying autophagy, it was found that autophagy is required for the reproductive process in budding yeast.²⁶ In mammals, fertilization induces massive autophagy to degrade maternal proteins and messenger RNAs, and autophagy functions as a major nutrient-providing system for embryos before their implantation.²⁷ Our recent work indicates that autophagy is required for acrosome biogenesis during spermatogenesis in mice, thus essential to male fertility.²⁴ However, whether autophagy is involved in female gametogenesis or not is still unknown. Here, we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to POI in female mice, and the numbers of the oocytes and follicles were significantly declined in the adult mutant mice. Further investigation revealed that autophagy protected oocytes over-loss during the neonatal transition period. Our results suggest that autophagy-related genes might be pathogenic genes to POI.