Abstract: BACKGROUND: Ovarian granulosa cells, as the main functional cells in ovarian follicles, communicate with oocytes through complex gap junctions, and regulate the growth and maturation of oocytes. With the deepening of exosome research, it is found that it actively participates in the communication between cells, and carries a variety of genetic materials, which can be absorbed by cells through autocrine or paracrine, regulate cell proliferation, and participate in the physiological and pathological processes of diseases. It can be seen that the exosome secretion capacity and extraction and identification of ovarian granulosa cells are of great significance for the study of female reproduction.  
OBJECTIVE: To explore the ability of human ovarian granulosa cells to isolate exosomes and the differences between exosomes from human ovarian granulosa cells of different origins.
METHODS:  SVOG (human ovarian granulosa cell line), KGN (human ovarian granulosa cell tumor cells) and human primary ovarian granulosa cells were cultured in vitro. Exosomes were isolated from the supernatants of the three different cells by ultra-high speed centrifugation. The morphological structures, particle sizes, and the expression of CD63, tumor susceptibility gene (TSG101) and calnexin were measured by transmission electron microscopy, nanoparticle tracer technique, and western blot assay, respectively.  
RESULTS AND CONCLUSION: (1) The nuclei of SVOG cells, KGN cells and human primary ovarian granulosa cells were round or oval with large nuclei, blue-stained nucleosomes and light red cytoplasm. SVOG cells and KGN cells were long spindle-shaped or polygonal with irregular morphology, and human primary ovarian granulosa cells were mostly protruding or spindle-shaped with elongated pseudopodia and closely intertwined with each other. (2) Immunofluorescence from three different sources of human ovarian granulosa cells was positive for FSHR expression, and the positive expression rate was greater than 95%. (3) The morphology of the exosomes isolated from the supernatants of the three different cells presented a cup-like bilayer structure. (4) Nanoparticle tracer technique showed that the mean particle sizes of exosomes from SVOG cells, KGN cells and human primary ovarian granulosa cells were (183.5±4.9) nm, (125.3±1.0) nm, and (171.1±2.0) nm, respectively. Among them, human primary ovarian granulosa cell-derived exosomes had the highest secretion levels. The exosomes from KGN cells had the smallest particle size, and the exosomes from SVOG cells were relatively similar in size to those from human primary ovarian granulosa cells. (5) Western blot assasy results showed that the exosomes from three different sources of ovarian granulosa cells were positive for the specific protein CD63 and the tumor susceptibility gene (TSG101) but negative for Calnexin. (6) These findings confirm that the ultra-high speed centrifugation method can successfully isolate and extract ovarian granulosa cell exosomes, among which KGN cell-derived exosomes have the smallest particle size; the particle sizes of exosomes derived from SVOG cells and human primary ovarian granulosa cells are similar; human primary ovarian granulosa cells have the strongest ability to secrete exosomes.

Key words: granulosa cell, exosome, transmission electron microscopy, nanoparticle tracer analysis, western blot assay