Improvement Of Mitochondrial Function In Somatic Cells Preserves Follicle Reserve In Mice

In mammalian ovary, follicle development is a continuous process during the female reproductive life span. Follicle is the basic functional unit in reproductive system, consisting of an oocyte surrounded by granulosa cell. Ovarian folliculogenesis is the process in which the recruited primordial follicles develop into preantral follicles, characterized by proliferation and differentiation of granulosa cells and oocyte enlargement. The oocytes take months to complete its growth phase from primordial follicle to mature oocytes as a result of instructive paracrine of oocyte and granulosa. In fact, only several oocytes (about 1%) are released into oviduct and capable to be fertilized, multiple follicles undergo atresia through apoptosis, autophagy and necrosis. Rapidly dividing and the growing follicles require favorable energetic and substrates to grow and divide that constitute a requisite gateway to the follicle development. It is presumable that a mitochondrial adaptation would be necessary during the ovarian cycle to match the highly increased energy demands. The other is that mitochondrial biogenesis increases cell vitality and is being pursued as a strategy to combat age-related disease. The reproductive senescence is primarily due to an age-related reduced ovarian reserve and may be associated with mitochondrial dysfunction and consequentially cellular viability.Energy metabolism cooperation between oocytes and granulosa cells in follicle plays essential roles for substrate provision for growing oocytes. Although many target genes for controlling mitochondrial function have been identified by functional genomic analysis (Mootha et al.,2004, PNAS, Schreiber et al 2004, PNAS), there is largely paucity of information regarding the switching of mitochondrial function in the follicle growth. Therefore, we focus on energy metabolism in follicles, especially in granulosa cells supporting follicle development, to validate the mechanism of this energy metabolic cooperation.The mitochondria of mammalian cells support cellular function and survival through several important processes, including oxidative phosphorylation (OXPHOS), various metabolic processes and cell apoptosis in response to cellular and extracellular stress. However, the genetic basis of mitochondrial modulation in the ovary is poorly understood. A major breakthrough in the understanding how primordial follicles are activated from the dormant pool came with the discovery of PTEN (Reddy, Science,2008) and mTORC/Tsc1/2, revealing that deletion of Pten or Tsc1/2 in mice ovary causes activation of the primordial follicle pool. PTEN and TSC1/2 were identified as tumor suppressor genes in preventing the onset and progression of numerous cancers, suggesting that activation of primordial follicles and the growing follicle share some common characters with tumor cells. These tumor suppressor genes may act as "brakes" to stop primordial follicle in their track before they can take the road to develop oocyte mature.CRISPR/Cas9 system has been widely utilized for genome editing with the high efficacy and specificity. And it has been used for pooled genome-scale functional screening. Here, we performed a CRISPR/Cas9-mediated targeting for the "tumor suppressor genes" to search genes conferring mitochondrial biogenesis in human GCT-derived KGN cells. This study uncovered previously unrecognized roles including TP53, BRCA1, PTEN, CTNNA2, PTCH1, HBP1 in augmentation of mitochondrial function. Furthermore, specific analysis of Hbpl by generating Hbpl conditional knockout mice, found that improved mitochondrial function in granulosa cells (GCs) is accompanied by decreased follicle apoptosis in Hbp1-/- and Hbp1f/f; Amhr2-cre, but not for Hbp1f/f; Gdf9-icre or Hbp1f/f; Zp3-cre mice, suggesting that reduced HBP1 in surrounding GCs play critical node in control follicle survival, while Hbpl transgenic mice showed impaired mitochondria function and increased follicle apoptosis. Furthermore, Hbp1-/- and Hbp1f/f; Amhr2-cre female exhibit increased growing follicles, oocyte production and follicle reserve. Transcriptome analysis revealed that the expression levels of some mitochondrial function related genes are up-regulated in Hbp1-/- ovaries. Our results thus identify a key component of the mitochondrial regulatory pathway that involves in follicle development and provide a critical place to look for the control of reproductive lifespan and fertility.