| Objective and significance: Immature spermatogonial stem cells (SSCs) are kinds of special somatic stem cells which can differentiate into sperm or transforme into pluripotent stem cells. However, little is known about the mechanism of its development and apoptosis in vitro and in vivo. Sertoli cells, which are the only cells that adhere to SSCs during development process, provide niche environment for SSCs with distinct structure and function. Stem cell factor (SCF), secreted by Sertoli cells which was affected by growth factors and hormones, acts as a potent regulator for SSCs proliferation and apoptosis. The aim of the study was to establish a method of isolation, in vitro culture and identification for human fetal Sertoli cells and examine the role ofβ-estradiol in the regulation of SCF secretion of fetal Sertoli cells at different dose and time. Then we investigated the effect ofβ-estradiol on in vitro proliferation of immature spermatogonial stem cells by employing a Sertoli-germ cell co-culture system at cellular and molecular level.Material and methods:Section One: To establish an in vitro cell culture system, Sertoli cells were purified in a modified culture system by two-step enzymatic digestion and differential adhesion method. The cultured cells were maintained for at least 12 passages in vitro and confirmed to be Sertoli cells with biological function by morphological observation, cell marker detection and function assay.Section Two: To examine the role ofβ-estradiol in the regulation of SCF secretion of fetal Sertoli cells, RT-PCR assay and immunofluorescence staining were performed to detect the expression of estrogen receptor in Sertoli cells. Then dose and time effects ofβ-estradiol on SCF expression in Sertoli cells were analyzed by Real-time PCR and immunofluorescence assay.Section Three: To investigate the effect ofβ-estradiol on in vitro proliferation of SSCs. Immature spermatogonial stem cells, co-cultured with fetal Sertoli cells, were evaluated by detection of specific germ cell and pluripotent markers. Also, 5-Bromodeoxyuridine (BrdU) labeling was used to determine the cell proliferation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for the anti-apoptotic effect ofβ-estradiol in the co-culture system.Result: The purified fetal Sertoli cells were characterized by the expression of MIS+/c-Kit-/AP-. There was a dramatic increase in the SCF induction from fetal Sertoli cells at 100 ng/mlβ-estradiol in our experiments and it began to exert effect in 15 min, reach peak value after 24h and maintained for more hours. B-estradiol also up-regulated the protein level of SCF. The spermatogonial stem cells, cultured in our co-culture system, were confirmed to be immature spermatogonial stem cells which maintained the pluripotent potential by the detection of c-Kit+/SSEA-4+/Oct-4+. To induce the production of SCF,β-estradiol was added at suitable concentration in the culture medium. Consistent with theβ-estradiol- withdrawal group, the incorporation index of BrdU was significantly higher in theβ-estradiol-treated cultures, which demonstrate thatβ-estradiol have remarkable effects on enhancing the proliferation ability of immature spermatogonial stem cells indirectly. The apoptotic index was dramatically decreased in theβ-estradiol-present cultures in contrast to theβ-estradiol-withdrawal and inhibitor-treated groups, indicating thatβ-estradiol played a role in suppressing the apoptosis of human immature spermatogonial stem cells through indirect actions.Conclusion: We established a system of isolation, in vitro culture and subculturing for human fetal Sertoli cells. For the first time, we found thatβ-estradiol could induce SCF secretion of fetal Sertoli cells in a dose- and age-dependent manner. We also showed thatβ-estradiol played a role in the regulation of human immature spermatogonial stem cells proliferation and apoptosis through SCF induction.
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