Effects Of Luteinizing Hormone On Gap Junction And Apoptosis In The Preovulatory Follicles

Background and objectives:Granulosa cells(GC) are the main somatic cells in follicles, which are very important during the follicular growth and development. The ovarian oocyte-granulosa cell interactions are essential for normal follicular development. The characteristics of ultrastructure and apoptosis of luteinized granulosa cells obtained from in vitro fertilization-embryo transfer (IVF-ET) maybe the good indicators for IVF prognosis. But it is unclear on some factors that influencing the apoptosis of luteinized GC. To explore the effects of different concentration of luteinizing hormone (LH) on the apoptosis-associated factors and gap junction (GJ) in the follicle fluid and luteinized GC , we have studied the preovulatoty follicles during IVF, so that we would find one or more factors to indicate the IVF result.Materials and methods:All luteinized GC were obtained from women who undergoing IVF at IVF center, West China Second University Hospital. After 3 days primary cultures, all cells were grouped as adding FSH 3IU/ml (control),LH HU/ml(LHl group), LH 3IU/ml(LH2 group) and LH 6IU/mL(LH3 group)for 24 hours. We analyzed the mRNA and protein expression levels of caspase-3, caspase-7 and Connexin 43. We also detected the LH, soluble Fas (sFas) and the soluble Fas ligand (sFasL) in the follicular fluid to analyzed relevance between these factors and pregnancy results, as well as between the LH and sFas -sFasL system.Results:1,The advantage in primary culture of luteinized GC is to preserve the its determined properties , which making the experiments in vitro more likely with the environment in vivo.2,After adding FSH and different dosage of LH, the relative mRNA and protein expressions in LH 3 group are higher than other groups, the relative mRNA expressions have statistical difference, but relative protein expressions have not statistical difference. Moreover, the expression of mRNA and protein of caspase-3 are gradually increased along with the increased dosage of exogenous LH. The expression of mRNA and protein of caspase-7 are also gradually increased along with the increased dosage of exogenous LH, and the mRNA expression in LH 3 group are statistical higher than that in LH 1 and LH 2 group, but there are no statistical difference in protein expression between these four groups.4,The relative expressions of mRNA and protein of Cx43 in control group,LH1 group and LH2 group have no difference. However, the relative expression of mRNA in LH 3 group is lower than in other three groups, and no difference on protein expression.5,The levels of LH and sFasL in the preovulatory follicle fluid are higher inpregnancy group than that in non pregnancy group, and in these twogroups ,there is no difference in sFas level.6,Using logistic regression analysis method, we found that LH in follicle fluidhad statistical relationship with the pregnancy result, and the odds ratio was4.117. Although there were statistical difference in sFasL between pregnancygroup and non pregnancy group by using the matched t-test, there were norelevance between sFasL and pregnancy result by using logistic regressionanalysis.7,The LH level in follicular fluid had positive relevant relationship withsFasL, but no relationship with sFas, by using Spearman relevanceanalysis .Conclusions:1,Low dose of LH (1IU/ml, 3IU/ml) can mimic the function of FSH on luteinized GC, that making the expression of mRNA and protein of caspase-3and caspase-7 increased, Cx43 decreased . But the high dose of LH(6IU/ml)would make the caspase-3 and caspase-7 increasing, and induce luteinized GC apoptosis, meanwhile it also influence the expression of Cx43, which may destroy the gap junctional coupling between GCs, influence the normal message conduction within follicles and induce the apoptosis of luteinized GC,and as a result,indirectly influence the oocyte development and maturation.2,In the preovulatory follicle fluid, the levels of LH and sFasL in pregnancy group are significantly higher than that in non-pregnancy group, but through relevance analysis and logistic regression analysis, only LH level in follicle fluid was associated with pregnancy. LH may influence sFasL level in follicle fluid through other pathway, and it maybe a good indicator for IVF prognosis.