Abstract：Granulosa cells play an important biological role in ovary development. To reveal the characteristics of porcine (Sus scrofa) ovarian granulosa cells during the development, granulosa cells from porcine follicular fluid were isolated and cultured in vitro.The results of cell morphology and genes expression pattern showed that porcine granulosa cells retained some features as stem cells. The interaction between cumulus granulosa cells and oocytes inhibited the differentiation of cumulus granulosa cells. When granulosa cells and oocytes were cultured together, granulosa cells were under the undifferentiated state, but losing the inhibition of oocytes, the growth of granulosa cells were rapidly, and quickly became fibrosis and apoptosis. On the other hand, cumulus granulosa cells could promote oocyte maturation. Under the regular cell culture conditions, oocytes cultured in vitro could be activated by cumulus granulosa cells and developed to the morula stage. In this study, the primary cultured porcine granulosa cells were found to be able to form cell aggregates spontaneously, and showed the weak alkaline phosphatase staining. The result of culturing granulosa cells in the medium that was used for culturing pluripotent stem cells showed that granulosa cells could be well maintained and present the epithelial-like state. After several passages, granulosa cells still maintained a high growth efficiency. RT-PCR analysis showed that the cultured granulosa cells were expressed multiple pluripotent genes, including ctamer-binding transcription factor 4 (OCT4), SRY (sex determining region Y)-box 2(SOX2), Kruppel-like factor 4 (KLF4), cellular myelocytomatosis viral oncogene (c-MYC), and Tir na n-Og (NANOG), as well as granulosa cells pecific marker gene follicle-stimulating hormone receptor (FSHR). The results showed that OCT4 was highly expressed and showed the 2 spliceosomes. After low-voltage, long-time agarose gel electrophoresis, RT-PCR products of OCT4 were found to have the 2 bands, indicating that the granulosa cells were expressed OCT4 spliceosome OCT4A and OCT4B. Western blot analysis and immunofluorescence staining experiments further proved that granulosa cells were expressed OCT4 and retained OCT4 spliceosomes, and the translocations of OCT4 in granulosa cells were varied. In the primary cultured granulosa cells, OCT4 protein was found in the cytoplasm, however, in the passaged granulosacells, OCT4 protein was translocated into the nucleus. Western blot analysis showed that cytoplasmic proteins forming primary cultured granulosa cells had OCT4 protein, which was not detected in nuclear proteins. The mechanism of OCT4 distribution in granulosa cells remained to be investigated. In summary, this study showed that porcine granulosa cells retained certain characteristics to express pluripotency factors, which lays the foundation for the development of the ovaries.
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