Study On The Mechanism Of Mouse Germ Cell Specific Transcription Factor NOBOX Regulating Primary Follicle Development

Objective:Follicles are the basic unit of ovarian structure and function.According to the developmental period or physiological state of follicles,follicles can be divided into primordial follicles,primary follicles,secondary follicles,tertiary follicles and mature follicles,and finally ovulate.Among them,primary follicles,secondary follicles and tertiary follicles are called growth follicles.The stage where follicles develop from primitive to secondary is called the precavity or non-gonadotropin-dependent stage,when the growth of follicles depends on the regulation of germ cell-specific transcription factors and the material exchange and signal exchange between oocytes and granulosa cells.Nobox is a gene specifically expressed in mouse germ cells and encodes a transcription factor,which is mainly expressed in ovarian tissue.Its knockout mice show premature ovarian failure,and female mice are not fertile.Nobox is expressed throughout the follicular development stage and participates in the formation and activation of primordial follicles.Follicle development is a continuous and dynamic process.Germ cell-specific genes have staged characteristics.At present,the widespread knockout of Nobox can only explore its mechanism of action in primordial follicles.Its role in primary follicles and beyond has not been studied.As a widely expressed gene,Kit also plays an important role in germ cells.Kit is expressed in all stages of follicular development,and runs through the entire process of oogenesis.It affects the proliferation and differentiation of PGCs and can participate in PI3 K / AKT and KITL / KIT pathways to affect follicular development.During primitive follicle activation,germ cell-specific transcription factor SOHLH1 can directly transcribe and activate Kit expression,and participate in primitive follicle activation through the PI3 K / AKT pathway.Different from the NOBOX expression pattern,SOHLH1 is only expressed in ovarian cells and primordial follicles,and the expression gradually disappears in the primary follicles,and is no longer expressed in the secondary follicles and subsequent stages.When the original follicle develops into a primary follicle,the expression of SOHLH1 gradually disappears,and Kit continues to express a large amount.At this time,Kit should also be regulated by other transcription factors.As a germ cell-specific transcription factor,NOBOX is expressed throughout the developmental stage of follicles.Through software analysis,it is found that there are multiple NOBOX binding sites in the Kit promoter,and there is the possibility of transcriptional regulation.In addition,during the development of primary follicles to secondary follicles,the TGF-? / SMADs signaling pathway,as a landmark pathway for oocytes and granulosa cells to communicate,began to regulate follicular development.As a member of the TGF-? superfamily,growth factor GDF9 begins to be expressed in primary follicles.Studies have shown that NOBOX can directly transcribe Gdf9 and participate in the GDF9 / SMAD pathway to affect the proliferation and differentiation of granulosa cells.This study intends to explore the role of transcription factor NOBOX in regulating the transcription of Kit during the development of primary follicles.By isolating and cultivating primary follicles in vitro,using microinjection of Nobox-si RNA to interfere with Nobox in primary follicles and observe primary follicle Developmental changes,through a series of molecular biology experiments to explore the effect of the transcription factor NOBOX in the primary follicles of mice on the follicle development through Kit and GDF9 / SMAD pathway,and improve the mechanism of the transcription factor NOBOX in the development of follicles.Methods:In this study,the Nobox-pc DNA3.0 eukaryotic expression plasmid was constructed and co-transfected with Nobox-si RNA into HEK293 T cells to verify the interference efficiency of Nobox-si RNA;primary follicles were isolated and cultured in vitro to observe the non-injected group,DEPC water injection and injection,the morphological changes of the three groups of Nobox-si RNA from primary follicles to secondary follicles were counted and the follicular development time,survival rate and the time for the three groups of primary follicles to develop to secondary follicles were counted;Real-time PCR was used to detect the non-injected group and DEPC injection The three groups of water and Nobox-si RNA injection of Nobox-si RNA microinjection of Nobox,Kit,Gdf9 m RNA expression changes in primary follicles;Western Blot test to detect the injection of DEPC water and Nobox-si RNA group microinjection of Nobox-si RNA primary The protein expression changes of NOBOX,KIT,P-SMAD in follicles;use JASPAR software to predict and analyze the NBE site upstream of the Kit gene start site(-6177 bp ?-5218 bp and-4092 bp ?-2228bp);by dual luciferase Reporter gene experiment research analyzes NOBOX's transcriptional regulation effect on Kit promoter(-6177bp?-5218 bp and-4092bp?-2228bp);Ch IP experiment is used to verify the binding site of transcription factor NOBOX on Kit gene promoter.Results:1.The Nobox-pc DNA3.0 eukaryotic expression plasmid was successfully constructed.The Nobox-pc DNA3.0 eukaryotic expression plasmid and Nobox-si RNA were co-transfected into HEK293 T cells,and Negativl Control si RNA was transfected as a negative control and DEPC water was used as a blank control.There was no significant difference between the negative control and the blank control,and the interference efficiency of Nobox-si RNA-3 was the highest,and the inhibition rate was 77.1%.2.Real-time PCR and Western Blot experiments detected the interference of Nobox-si RNA in the primary follicle oocytes.Compared with the DEPC water group,the Nobox m RNA and protein expression in the primary follicles decreased significantly.Microinjection of si RNA can effectively cause Nobox gene silencing in primary follicles.3.By isolating and culturing primary follicles in vitro and performing statistical analysis,it is found that primary follicles can develop into secondary follicles on the 5th day of in vitro culture;the three groups were separated and cultured without injection,DEPC water injection and Nobox-si RNA injection.Primary follicles,after statistical analysis,the blank control group compared with the negative control group showed that there was no statistical difference in the The rate of the three groups of follicles,the microinjection did not affect the primary follicles,and the non-injected group and DEPC water injected two groups for 2 days survival They all began to develop gradually and developed to secondary follicles on the 5th day.Compared with the negative control group injected with DEPC water,the Nobox-si RNA group: after Nobox-si RNA interfered with the primary follicles,the follicles delayed the development of secondary follicles for 2 days.4.It was detected by Real-time PCR and Western Blot experiments that after Nobox-si RNA was injected into primary follicle oocytes,compared with DEPC water injection group,the expression of Kit and Gdf9 m RNA in primary follicles was significantly down-regulated,KIT,P-SMAD2/3Reduced protein expression.5.Use JASPAR software to predict and analyze the NBE sites upstream of the Kit gene's starting site(-6177bp?-5218 bp and-4092bp?-2228bp),and use 5high-scoring NBE sites as the main detection sites(-5638bp?-5634 bp,-5432bp?-5425 bp,-2933bp?-2926 bp,-2874bp?-2867 bp and-2676bp?-2669bp);5.The results of the dual luciferase experiment showed that the Nobox-pc DNA3.0 eukaryotic expression plasmid was combined with When the Kit promoter fluorescent expression vector(-6177bp?-5218 bp and-4092bp?-2228bp)was co-transfected,the fluorescence activity in the-4092bp?-2228 bp region upstream of the start site of the Kit gene was significantly enhanced,and the fluorescence in the region from-6177bp?-5218 bp There is no significant difference in activity.NOBOX can directly activate the transcription of Kit gene at-4092 bp ?-2228 bp upstream of the start site of Kit gene.The fluorescence activity is significantly enhanced,and NOBOX can directly activate the transcription of Kit gene.6.Ch IP experiments proved that the transcription factor NOBOX can bind to the NBE sites upstream of the Kit gene transcription start site(-2933 bp ?-2926bp),(-2874 bp ?-2867bp)and(-2676 bp ?-2669bp).Conclusion: 1.The transcription factor NOBOX is a key gene for the development of primary follicles to secondary follicles.2.The transcription factor NOBOX can be directly combined with the NBE sites upstream of the Kit gene transcription start site(-2933 bp ?-2926bp),(-2874 bp ?-2867bp)and(-2676 bp ?-2669bp),presuming that NOBOX can be directlytranscribed Regulate the expression of Kit gene.3.Nobox gene silencing in primary follicles can inhibit the KIT / KITL and TGF-? /SMAD pathways between oocytes and granulosa cells.