| FHL2(four and a half LIM domains protein 2), a member of Four and a Half LIM-only protein family, can interact with several proteins to regulate gene expression, signal transduction, cell proliferation and differentiation,and cell immigration and apoptosis mainly as a co-regulator or co-repressor. FHL2, which is expressed in ovarian tissue, can lead primitive gonad into the ovary in the embryonic period and also regulate the expression of INHα. But only a little is known about the localization and roles of FHL2 on mouse follicle granulosa cells in cell proliferation, apoptosis and steroid hormone secretion.In order to clarify the localization and roles of FHL2 on mouse follicle granulosa cells, we study the roles of FHL2 on mouse granulosa cells with the ways of gene mapping, expression, subcellular localization, function study etc. Following are the experimental results: 1. Expression and localization of FHL2 in mouse ovary(1) In the different ages mouse ovary(7d, 14 d, 21 d, 28d), we proved that FHL2 can express in these ovaries with the RT-PCR, and there was no expression difference in each group.(2) Immunohistochemistry results showed that FHL2 was mainly localized in the follicle granulosa cells and stroma cells. Analysis of expression of FHL2 in different developmental stages of the follicles, no significant difference was found.(3) Immunofluorescence results showed that FHL2 was mainly localized in the nucleus of follicle granulosa cells. 2. Effects on the growth and apoptosis of ovarian GCs and secretion after RNAi(1) Granulose cells were transfected with a plasmid encoding a silencer sequence directed against FHL2 for 72 h.To detect the cell apoptosis,we found that the proportion of normal cells in the experimental group were significantly higher than that in control group(91.52 ± 0.78 vs 85.78 ± 0.14,P <0.05),the late apoptosis rate was significantly lower than that of the control group(4.18 ± 0.16 vs 10.18 ± 1.81, P <0.05),using flow cytometry. We also detected the protein related to apoptosis, suggesting that the Bax and Casepase-3 was up-regulating and the Bcl-2 was down-regulating and the survival rate of granulose cells was increasing after interference.(2) Analysis of the cell cycle of transfected group, the result showed that most cells in the experimental group were in S- phrase(36.83 ± 0.40 vs 24.02 ± 0.33, P <0.05,the cells in G1-phase were significantly lower than that of the control group(48.31 ± 1.02 vs 62.96 ± 0.72,P <0.05). The detection of protein related to cell cycle, suggesting that the Cyclin E and Cyclin B was up-regulating and the p21 expression was down-regulating. So FHL2 could regulate the cell cycle after RNAi.(3) Cell proliferation assays using the MTT assay, revealed that knockdown the FHL2 can increase the cell activity(53.60% vs 15.43%, 48 h, P<0.05; 109.86% vs 33.33%, 72 h, P<0.05).(4) Compared with control group, treatment of granuloma cells with FHL2 sh RNA to knockdown FHL2 protein significantly increased the section of both estrogen and progesterone(8.19 ± 0.19 vs 2.53 ± 0.41, ng/L, Estradiol,; 13.96 ± 1.07 vs 1.64 ± 0.08, ng/m L, Progesterone; P <0.01).This research explores the expression and location of FHL2 in mouse ovarian tissue and granulose cells. Our data demonstrates that FHL2 can express in diffrent ages ovaries with no expression difference in each age, and mainly localized in the nucleus of follicle granulosa cells with no significant difference in all developmental stages of the follicles. Further study the roles of FHL2 on m GCs, it shows that knockout the endogenous FHL2 can inhibit cell apoptosis, promote cell proliferation and secretion of steroids, also influent cell cycle. These results indicate that the FHL2 affects mouse follicle development significantly, providing a foundation on the regulatory mechanism and signal pathway of ovarian follicle development for the further research. |
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