Pluripotent Characteristics Of Porcine Ovarian Granulosa Cells

As the reproductive organ of female animals, ovary is consisting of germ cells andsomatic cells. Its generation, development and function are all involved in stem cell.Granulosa cell as the fastest dividing cell has most closely relationship with ovarian oocyte,its rapid expansion and different functional differentiation lead to folliculogenesis. Within twoweeks, the ovarian granulosa cell divide from less than500,000to more than50million anddifferentiate into specific cell types of different function. These cells form a functionalsyncytium, can involved in steroidogenesis and provide nutrition to the oocyte. This indicatesthat granulosa cells have pluripotent characteristics. However, luteinizing granulosa cells hasbeen considered to be terminally differentiated cells, until recently, some studies haverevealed their stem cell properties. The stemness of granulosa cell show in several ways: First,the expression of stem cell factors OCT4, SOX2and NANOG, followed by high telomeraseactivity, again in vitro to differentiate into other cell types, such as osteogenic cells and nervecells. Granulosa cells can also be transdifferentiation in vitro, to study some of the symptomsof ovarian pathology. Although stem cell characteristics of granulosa cells are constantlybeing revealed, but it has been questioned and challenged. In this study, after three aspectsexperiment in porcine granulosa cells: isolation and culture, detection of gene expression anddifferentiation in vitro, we got the following conclusions.1. Isolation and culture of porcine granulosa cellsExtracted follicular fluid from pig ovaries of different size follicle with a syringe, underthe stereomicroscope with a homemade suction-embryo-needle we isolated granulosa cellsmass from follicular fluid and cultured. The results show that granulosa cells in largefollicles with respect to small have more apparent diffusion and cannot be adherent culture.Studies have shown that DMEM with10%FBS have a good effect on granulosa cellproliferation. Adherent cultured granulosa cells have certain positive alkaline phosphataseand other characteristics such as the formation of agglomerates clone. Culturing cumuluscomplexes have revealed the interaction between cumulus granulosa cells and oocytes. Onthe one hand oocyte stimulates the rapid proliferation of granulosa cells, On the other handgranulosa cells stimulate oocyte mature and activated to cleavage. 2. Detection and analysis of gene expression in porcine granulosa cellsGranulosa cells are collected, extracted RNA and detected by RT-PCR. Results showedthat granulosa cells express not only stem cell factor OCT4, SOX2, KLF4, CMYC, NANOGbut also germ cell specific expression gene FOXL2, ESRRB and FSHR. As the cellssubcultured in vitro, the amount of gene expression about stem cells factor will be reduced.Immunofluorescences staining on freshly isolated and adherent cultured granulosa cellsshowed cells express OCT4and SOX2, but the expression have difference. Western blothybridization can also detect the expression of OCT4.3. Reprogramming porcine granulosa cells and differentiation in vitroBy retroviral granulosa cells were transfected with pluripotent stem cell factor and foundthat fibroblast-like cells can be reprogrammed to the epithelioid. Since the induction time isshorter, only granulosa cells reprogrammed to form atypical cell clones, iPS-like cell clonehas not yet formed. Granulosa cells transferred with GFP protein in normal culture mediumcan be differentiated into neuron-like cells.