Study Of Granulosa Stem Cells In The Ovary

The ovarian follicle represents the basic functional unit of the ovary which consists of an oocyte,granulosa cells(GCs),theca cells and follicular antrum.In the process of follicular growth and development,GCs play an important role.They connect to each other and to the oocyte through gap junctions and paracrine signals.Cross talk between oocytes and granulosa cells is mainly involved in follicle growth.The state of GCs has a major influence on developmental competence of the oocyte and embryo.99.9% of all follicles in the ovary become atresic at each stages of developing follicles.During Folliculogenesis,the atresia of follicles was closed related with the apoptosis of granulosa cells.The one of the first important mechanism of follicles falling into atresia is the apoptosis of granulosa cells.Therefore,it is thought that if the proliferation or differentiation of granulosa cells does not occur appropriately,follicles will fall into atresia.To the end of 21 Century,infertility will be the third disease after tumor and cardiovascular diseases,the main causes of infertility were premature ovarian failure(POF)and bad quality of oocyte.The factors including Chemotherapy,Radiotherapy,Poison,Environmental Pollution and Smoking can hurt GCs and lead to the apoptosis of GCs in the primordial and primary follicles,decrease the number of ovarian primordial follicles,affect the reproductive ability,the ovarian function and quality of oocyte.The stem cell is characterized by its ability to self-renew,its ability to proliferate indefinitely and its ability to differentiate along multiple lineage pathways,they are considered as a potential cell source for regenerative medicine.Somatic stem cells present in many adult tissues and organs,which have the ability to differentiate into terminally differentiated cells which can repair failing organs,regenerate missing parts of tissue.GCs can provide essential nutrients for oocyte development and accumulation of oocyte secreted metabolites,oocyte communicating with the body only rely on GCs through gap junctions and paracrine signals,whether granulosa stem cells(GSCs)can originate from GCs in preovulatory follicular fluid from oocyte retrieval during IVF?If it is true,it may play a crucial role in the treatments for infertility,particularly in poor quality of oocyte,diminished ovarian reserve,regeneration of ovarian function after chemotherapy,radiotherapy,and fertility preservation.OBJECTIVE: In the current study,we are going to establish a culture system that could long-termly culture human luteinizing GCs,to isolate stem cell population from GCs and to test if GCs can be a potential source of stem cells.Subsequently we are going to establish a granulosa stem cell lines(GSCs),to characterize and investigate stem cell characteristics and steroidogenic performance of GSCs.METHODS: Human GCs were collected from aspirated follicular fluid of infertile patients in vitro fertilization programs and cultured on bacterial-culture dishes with MEF conditional culture Medium,subsequently we are going to establish a granulosa stem cell lines(GSCs),and observe cell growth curve,doubling time(DT),the changes in the morphology,proliferation,and function of GSCs,.After 10 passages,GSCs were characterized by immunofluorescent staining and the cell surface markers were analyzed by flow cytometry.RT-PCR analyzed expression of reproduction-related genes and stem cell-related genes,such as: oct4,nanog,Sox-2,Rex-1 and GDF-3.The numbers and structure of the chromosomes were analyzed.GSCs functional experiments were performed in vivo and in vitro during the 10 th to 80 th passages:GSCs passage 13 was stimulated by diferent gonadotrophins,analyzed production of c AMP by ELISA,the concentrations of estradiol and progesterone by a chemiluminescent immunoassay;We transfect e GFP gene to GSCs through lentiviral,then e GFP tagged GSCs were transplanted into the ovary of SCID mice to observe the capacity of growth of GSCs by fluorescence microscope and HE staining,analyzed the level of gonadal hormone in SCID mice.To detect stem cell characteristics of GSCs: cells at passage 10 and passage 60 were analyzed under adipogenic and osteogenic induction,Specific staining and RT-PCR were employed to evaluate the adipogenic,osteogenic differentiation.RESULTS: We have established a protocol that could culture human luteinizing GCs to passages 8 or 9 with MEF conditional culture Medium,GSCs can survive to 90 days in vitro,the estimated doubling time of the cells was approximately 22 h in vitro.Furthermore we have established a putative granulosa stem cell lines(GSCs)to 140 passages,GSCs can retain their cell morphology,ability of proliferation and colony formation.The cells express cell surface markers CD29,CD49 f,CD166 and HLA-ABC,which were similar to BMSCs(bone marrow mesenchymal stem cells),not express CD45,CD31,CD34,CD90,CD105;Immunohistochemical staining demonstrated the expression of Mesoderm marker vimentin,Gonadotropin receptors FSHR,LH,Aromatase P450(CYP450);RT-PCR analysis revealed the expression of gonadotropin receptors FSHR,LHR,sex steroids hormonal receptor ERα,ERβ,AR,PR;Chromosomes analysis revealed that chromosomes numbers and structure of GSCs was abnormal.GSCs can synthetic c AMP,E2 and P with the stimulation of different gonadotrophins in vitro.Immunohistochemical staining show the expression of SSEA1(stage-specific embryonic antigen-1),RT-PCR analysis revealed GSCs expressed stem-related cell markers such as Oct-4,Nanog,Sox-2,REX-1,GDF-3,not expressed germ cell specific markers Dazl,Vasa;Cells were positive for alkaline phosphatase staining and Alizarin Red staining after osteogenic induction,which was confirmed by RT-PCR analyses of BSP and Cbfa-1expression.Cells were positive for Oil Red O staining after adipogenic induction,and expression of leptin were observed by RT-PCR analysis.It was demonstrated that GSCs can be differentiated into other cell types such as osteoblasts and lipocytes.The e GFP-GSCs was successfully xenotransplanted into the ovary of nude mice,The GSCs could survive and proliferate in the ovary,and spontaneously differentiated into adipocyte-like cells,granulosa-like cells and follicular-like structure.CONCLUSION: In conclusion,we have established a protocol that could long-termly culture human luteinizing GCs.Moreover we have established a granulosa stem cell line(GSCs)which have the ability of cell proliferation and colony formation,but with abnormal chromosomes.The cells express granulosa cell surface receptor markers,marker of pluripotency and reproduction-related hormones receptor genes which were revealed by RT-PCR analysis and Immunohistochemistry analysis,showing stem cell characteristics.The GSCs express functional FSHR,LHR,after stimulation GSCs can secrete sex steroids in vitro;GSCs possess osteogenic and adipogenic differentiation potential;After GSCs was transplanted into the ovary of nude mice,it can proliferate and spontaneously differentiate into adipocyte-like cells,granulosa-like cells and follicle-like structures.