Abstract
IntroductionFollicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is not only rich in proteins but cells. Besides oocytes, the follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. However, epithelial cells in follicular fluid are poorly studied. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program.MethodsFollicular fluid samples were collected from 20 women in the assisted reproduction program. Epithelial cells were characterized by flow cytometry assay, immunofluorescence staining, real-time PCR, and time lapse photography.ResultsEpithelial cell cultures were established from 18 samples. A small population of epithelial cells expresses germ-line stem cell markers, such as octamer-binding transcription factor 4 (OCT4), NANOG, and DEAD box polypeptide 4 (DDX4). In the epithelial cell culture system, oocyte-like cells formed spontaneously in vitro and expressed the following transcription markers: deleted in azoospermia-like (DAZL), developmental pluripotency associated protein 3 stella-related protein (STELLA), zona pellucida gene family C (ZPC), Syntaptonemal complex protein (SCP), and growth and differentiation factor 9 (GDF9). Some of the oocyte-like cells developed a zona pellucida-like structure. Both the symmetric and asymmetric division split of epithelial cells and early developing oocytes were observed using time lapse photography. Cell colonies were formed during epithelial culturing, which maintained and proliferated in an undifferentiated way on the feeder layer and expressed some pluripotency markers. These colonies differentiated in vitro into various somatic cell types in all three germ layers, but did not form teratoma when injected into immunodeficient mice. Furthermore, these epithelial cells could be differentiated directly to functional hepatocyte-like cells, which do not exist in ovarian tissues.ConclusionsThe epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/multipotent character for safe application in oogenesis and regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0004-6) contains supplementary material, which is available to authorized users.
Highlights
Follicular fluid is important for follicular development and oocyte maturation
The epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/ multipotent character for safe application in oogenesis and regenerative medicine
There are three ovarian functional somatic cell types involved in folliculogenesis remodeling: the ovarian surface epithelium (OSE) that surrounds the ovary, the theca cells, and the granulosa cells (GCs), which essentially reside within the ovarian follicle’s avascular space [2] (Additional file 1)
Summary
Follicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is rich in proteins but cells. The follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program. In human-assisted reproduction programs, follicular fluid fills the antrum and surrounds the oocyte. The aspirated follicular fluid contains GCs, thecal cells, and ovarian surface epithelial cells. Kossowska-Tomaszczuk and colleagues first indicated that GCs collected from the follicular fluid had stem cell potential multipotency They demonstrated that luteinizing GCs isolated from the ovarian follicles of infertile patients included in the assisted reproduction program can be differentiated into three germ cell types, including neurons, chondrocytes, and osteoblasts [9]. Scant attention has been shown to epithelial cells in follicular fluid
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