Ovarian surface epithelium stem cells: oogenesis in vitro?
Abstract
Background: The dogma that the total number of follicles and oocytes available for reproduction are determined at birth, and that one follicle and one oocyte are recruited from the existing pool to mature in each menstrual cycle still persists. There is an increasing experimental evidence that this might not be true. In the ovarian surface epithelium there might be nondifferentiated stem cells that might differentiate into oocytes also in adult life. In this research we aimed at isolating putative stem cells from the ovarian surface epithelium and at evaluating in vitro oogenesis in the ovarian cell culture in vitro in women without naturally present follicles and oocytes in their ovarian cortex – postmenopausal women and women with premature ovarian failure.
Methods: Ovarian surface epithelium was scraped from the ovaries of 20 postmenopausal women and 5 women with premature ovarian failure. We tried to find putative stem cells in ovarian scraping and to confirm them by transcription markers Oct-4, Sox-2, and Nanog, and by surface antigen SSEA-4. Cell culture was set up by scraped cells in DMEM/F-12 medium with phenol red, which shows a weak estrogenic activity. Ovarian cell culture was cultured for 20 days in a CO2-incubator at 37 °C and 5 % CO2. Development of cells in the culture was followed and the presence of oocyte-like cells was evaluated by using different methods – evaluation of morphology, transcription markers, surface antigen markers, oocyte immunohistochemical markers, and flow cytometry after propidium iodide staining.
Results: In all postmenopausal women and in 4 out of the 5 women with premature ovarian failure putative stem cells were isolated from the ovarian surface epithelium, which were positive for transcription markers of embryonic stem cells Oct-4, Sox-2, Nanog, and for surface antigen SSEA-4. In all these women ovarian cell culture was successfully established. In all cultures oocyte-like cells developed approximately on day 5 of culture, which were positive for transcription marker Oct-4 and immunohistological germ cell markers c-kit, Oct-4, ZP2, DAZL, and even meiotic marker SCP3. Marker SCP3 was expressed in oocyte-like cells developed in ovarian cell culture of postmenopausal women, but not in patients with premature ovarian failure. Flow cytometry after propidium iodide staining revealed a population of potentially haploid oocyte like cells in postmenopausal women. Stem cells developed also into some somatic cell types, including fibroblasts, neuron-, and mioblast-like cells. In patients with premature ovarian failure oocyte-like cells were developed only at the presence of autologous patient’s serum and mostly at the presence of heterologous follicular fluid serum, whereas they did not develop at the presence of fetal calf serum.
Conclusions: First results are promising as they indicate the new insight into the physiology of the adult human ovary. In the ovarian surface epithelium putative stem cells with embryonic character are present, which can develop into the oocyte-like cells and other types of cells in vitro. Further research is required to understand better these new findings.
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