Involvement Of Macrophage Colony-stimulating Factor(M-CSF) In The Function Of Follicular Granulosa Cells And Its Molecular Mechanism

Macrophages are found in developing follicles,and they have been reported to promote the proliferation of granulosa cells(GCs)by secreting EGF and other cytokines.The proliferation,differentiation and activation of macrophages are mostly regulated by a homodimeric glycoprotein known as macrophage colony-stimulating factor(M-CSF;also called colony-stimulating factor-1 or CSF-1)which belongs to the family of hematopoietic growth factors.The receptor of M-CSF(M-CSFR)is a transmembrane receptor tyrosine kinase encoded by the c-fins proto-oncogene product.'M-CSF can affect various cell types,including cells of the female reproductive tract.Objective:To investigate the effect of macrophage colony-stimulating factor(M-CSF)in follicular granulosa and its molecular mechanism.Methods:Human luteinized granulosa cells(LCC)were isolated from follicular fluid of superovulated infertile patients(average age 30.8±2.1years old)undergoing in vitro fertilization and embryo transfer.LCC were cultured with DMEM/F12 medium plus various concentration of rhM-CSF(0,10,25,50,100ng/ml),rhM-CSF+Letrozole(10-6mol/l),rhFSH(0,10,25,50,10 OIU/ml),rhFSH+ Letrozole(10-6mol/l).Media were collected at 24 hours after culture and estradiol(E2)in media were measured by ELISA.Expression levels of FSH receptor and M-CSF receptor were deteced by quantiative RT-PCR.After cultured COV434 were treated with rhM-CSF,Cell proliferation was quantified by MTs assay and protein expression was detected by Western blot.Results:Either rhM-CSF or rhFSH stimulated the production of E2.The production of FSH receptors was enhanced by rhM-CSF or rhM-CSF+Letrozole in vitro in a dose-dependent manner.Conversely,rhFSH was able to promote the expression of the receptor of M-CSF when present in an appropriate concentration.With the increasing of rhM-CSF concentration,the cell proliferation of COV343 showed an upward trend.rhM-CSF induced phosphorylation of JNK and p38,the level of estradiol(E2)in media didn't change when cells were pretreated with the JNK inhibitor SP600125 or p38 inhibitor SB203580.Conclusions:M-CSF may play an important role in promoting the proliferation and differentiation of granulosa cell,also the synthesis and secretion of estrogen.