3.1. Cell culture results:
Granulosa cells after isolation and during the first day of culture were roughly spherical shape, but gradually attached to the plastic culture flask and, began to cytoplasmic extension that get the fibroblastic- like shape after 7 days. (Fig1.A) cell line prepared after cell volume reach to about 80% of confluent. (Fig1.B)
Flowcytometry analysis results:
After cell gating (R region), and use of control isotopes to remove cellular debris fragments, the stem cells had a different percentage of expression of each surface marker. The results showed that the percentage of cell markers Stro-1, CD19, CD105, CD146, CD90, CD106, CD44, CD166 and CD45 are respectively 34.08, 41.99, 21.47, 26.82, 13.47, 25.73, 35.90, 20.23 and 8.84. (Fig2)
Induction experimental results:
Morphological analysis of four experimental groups showed that, during the first week of induction, cells began to lose their fibroblastic- like shape. Nucleus of some cells was placed in the middle of the cell and two extensions on both sides of the cell were observed (Fig3.A). During the second week of induction, in each of differentiated cells, the nucleus wascompletelydrawninto one side of thecell and a small extension was observed on the other side of the cell. The cell was formed almost like a club (Fig3.B). At the end of the third week the extension at the end of the cell was longer, head of the cell also became longer, get a short neck and the nucleus of the cell was placed in the middle of it. (Fig3.C) At the end of induction time, 28 days culturing cells in sperm inducing medium, tail and head got more clear and cells were more similar to sperm morphology. (Fig3.D)
Western blotting analysis
Western blotting results of GPR125 protein that was used as a marker protein for spermatogonial cells, showed no expression of this protein in the first week of culture in terms of induction, but expression started from the second week of induction and showed a slight increase in the next weeks (Fig4.A). Results for two other proteins, VASA (Fig4.B) and DAZL (Fig4.C), were similar. In the control group GCs without treatment for induction were used as negative control and,testis extractwas considered as positive control.
Image j software analysis
In order to compare documents obtained by Western blotting, Image J software was used. Comparison of protein expression in different weeks showed that the expression of GPR125protein during four weeks was 0.125%, 12.96%, 17.08%, 25.07% respectively (Fig5.A). And the VASA protein expression in 4 different times was respectively 0.73%, 17.924%, 19.378%, 27.501% (Fig5.B). The results for DAZL protein were 0.79, 17.10, 13.79, 22.69 respectively
(Fig 5.C). Numbers do not indicate the exact expression level of proteins and the basis for comparison between weeks in this software was considered the widths of the bands obtained from the western blotting analysis.