Protein Prenylation Regulates The Oocyte Quality And Primary-secondary Follicle Transition

Ovarian follicles are the basic units of a mammalian ovary, each follicle contains an oocyte and its surrounding somatic (granulosa) cells. The oocyte arrested in the diplotene stage of meiotic prophase I is enveloped by flattened granulosa cells, thereby producing primordial follicles during birth. The dormant primordial follicles get together in ovarian cortex and form primordial follicle pool. Under a progressive and highly regulated process, a limited number of primordial follicles are recruited from the resting follicle pool and formed primary follicle, then undergo secondary follicle, antral follicle and finally ovulation. The abnomal activation and dvelopment of follicle will influence the ovary function and female reproductive life, such as premature ovarian failure(POF). Therefore, studying the mechanism of follicle activation and development have important clinical significance in the prevention and treatment of POF.Protein prenylation which is crucial for the cell membrane location and activation of several small G protein contains farnesylation modified by FPP and geranylgeranylation modified by GGPP. Geranylgeranyl diphosphate synthase 1 (GGPPS), a synthetase of GGPP from FPP for protein prenylation modification, expresses in the oocyte of different stage of follicle, but decreases in the oocyte with aging. It was suggested that GGPPS regulated protein prenylation is improtant for follicle develoment.In order to understand the role of GGPPS in the follicular development, we knock out GGPPS in oocyte by using Cre-loxp system. After koncking out GGPPS in PGC through DDX4-Cre system, we found out that the formation of primordial follicles was normal, while the develpoment of primary follicles are retarded, eventually leading to premature ovarian failure(POF) and subfertility. Further study showed that knocking out GGPPS could reduce the geranylgeranyl modification of Racl, which can influence the phosphorylation of STAT3 Y705. After that, Stat3 nuclear import was blocked, then inhibited the transcription of oocyte-specific expression GDF9 (growth differentiation factor 9), causing less GDF-9 recepted by granulosa cells. Then Smad2 signaling pathway and proliferation of granulosa cells were blocked. Taken together, knockout of GGPPS leads to abnomal Racl-STAT3-GDF9 pathway and influences the communication between oocytes and granulosa cells, eventually resultes in retardation of primary follicular development.On the other side,80% of mitochondria in the oocytes of primary follicles showed the tipical autophagy form of double membrane package in GGPPS kockout mice. We verified such phenomenon through LC3 immunostaining and found the quantity of mitochondria was also decreased. As we know, protein prenylation controlled by GGPPS is closely related to the progress of protiens combining to the cell membrane. So we conjectured knockout of GGPPS could directly influence the orientation to the mitochondria of some protein, which resulted in both the quality and quantity of mitochondria decrease. Subsequently, mitochondria could not meet the huge energy demand during the rapid growth of oocytes, finally resulted in retardation of primary follicular development.The internal molecular signals changed in oocytes during primordial follicles activation. On the one hand, the genome of oocyte activated and synthesized large number of molecule, such as GDF9. GDF9 could promote granulosa cells proliferation by binding to the receptor of granulosa cells. On the other hand, oocytes synthetized a large number of cell contents and the demand for energy increase dramatically. These two aspects determined the quality and the development competence of oocytes. Consequently, knockout of GGPPS influenced the change of molecular signals during primordial follicles activation:GDF 9 and mitochondrial function, then the quality and the development competence of oocyte was decreased and finally resulted in retardation of primary follicular development.Our findings showed that the protein prenyltion controlled by GGPPS played an important role in the early stage of follicular development. On the one hand, this progress influences communication between oocytes and granulosa cells through Racl-STAT3-GDF9 pathway. On the other hand, GGPPS influenced the follicular development through the regulation of the quality and quantity of mitochondria in oocyte.