Research On Human Ovarian Granulosa Cells Apoptosis Induced By2,5-Hexanedione And The Control Mechanism Of Sonic Hedgehog Signaling Pathways

n-Hexane (CAS:110-54-3), alias hexane, are straight-chain saturated fattyhydrocarbon. As the main active metabolite of n-hexane in the human body, thetoxicity of2,5-hexanedione (2,5-HD) and n-hexane are related. n-Hexane is widelyused as an organic solvent. n-Hexane toxicity causes serious harm in workers,particularly in women. The toxicity of n-hexane and2,5-HD has been extensivelyresearched, but research has primarily concentrated on neural toxicity. Damage to thereproductive system, especially for female reproductive function, has been lessfrequently reported. In2005, some scholars had the first report of the Hedgehogsignaling pathway derived from mouse ovarian granulosa cells, in which sonichedgehog (SHH) signaling pathway was more researched. SHH signaling pathwaydrives cell proliferation, directs cell differentiation and promotes cell away fromapoptosis. The emerging roles for SHH signaling pathway in the gonads (testis, ovary),uterus and accessory sex glands such as the prostate and mammary gland are beingfocused on in recent years. Therefore, this study established the efficient and stableprimary culture in vitro and identification methods of human ovarian granulosa cells.We used human ovarian granulosa cells as target cells to study whether the2,5-HDinduced cell apoptosis and in which the mechanism of SHH signaling pathway. In theconclusion, we could clarify the toxic effects and mechanism of2,5-HD on femalereproductive function, as well as the regulation mechanism involved in which, so could provide an important reference about how to ease or blocked2,5-HD toxicitydamage to ovarian.Objective:To establish the efficient and stable primary culture in vitro and identificationmethods of human ovarian granulosa cells, so to lay the foundation for in vitroreproductive toxicology studies. The other purpose was to investigate the mechanismsof human ovarian toxicity induced by2,5-HD through the morphological changes andapoptosis in human ovary granulosa cells and BCL-2family (BCL-2,BAX) andCASPASE family (CASPASE-3) expression changes in which. And to investigate theSHH signaling pathway in2,5-HD induced apoptosis in human ovarian granulosacells and its mechanism, in order to provide new ideas about how to ease or blocked2,5-HD toxicity damage to ovarian.Methods:1. Primary granulosa cells were obtained from women who were meeting theinclusion criteria and undergoing artificial reproductive technology (ART). Humangranulosa cells were collected by gradient centrifugation, red blood cell lysis withlysis of red blood cells and trypsin digestion. The hematoxylin-eosin (HE) stainingand follicle-stimulating hormone (FSHR) immunohistochemistry methods were usedto identify ovarian granulosa cells. CCK-8method was used to assay the cellproliferation. Meanwhile, in vitro granulosa cell secretion of estradiol (E2) andprogesterone (P) were determined.2. The study used cultured human granulosa cells as target cells, which wereexposed to0,16μmol/L,64μmol/L, and256μmol/L2,5-HD in vitro for24h. The HEstaining, Hoechst33342staining, transmission electron microscopy, and flowcytometry using FITC-Annexin V/PI double staining were used to prove that2,5-HDcould lead to apoptosis in human ovarian granulosa cells. Realtime quantitative PCRand Western blot analysis were used to detect changes in the expression of theapoptosis-related BCL-2family (BCL-2, BAX) and CASPASE family (CASPASE-3) with increasing2,5-HD concentration.3. The study used cultured human granulosa cells as target cells, which wereexposed to0and256μmol/L2,5-HD in vitro for24h. Realtime PCR and Western blotanalysis were used to detect the endogenous expression of SHH signaling in theprocess of2,5-HD-induced granulosa cells apoptosis. At the same time, the cellapoptosis were determined when the activation or blockade of SHH signaling pathwayby the recombinant SHH and cyclopamine, as well as the impact on anti-apoptoticgene BCL-2and apoptotic gene BAX.Results:1. Separaton, Primary culture and identification of human ovarian granulosa cells:(1) trypan blue staining showed that the granulosa cell viability over90%by thismethod. Cell purity was above95%by FSHR immunohistochemical identification.(2)By the light microscopy, cells in vitro were slow to develop, and two days later thecells proliferated significantly. At the6-7d cells gradually deformed and degradated.(3) CCK-8curve showed cultured human ovarian granulosa cells in24h at adherentgrowing season. And cultured cells during the first3-5d were to reach the splittingpeak, then began to degenerate at6-7d.(4) Cultured human granulosa cells under theeffect of recombinant FSH (rFSH) could significantly promote the secretion of E2, butthe secretion of P ability had nothing to do with the role of rFSH. Primary culturedhuman ovarian granulosa cells were maintaining the original cell characteristics.2. The mechanism of2,5-hexanedione inducing human ovarian granulosa cellapoptosis: this study established a human ovarian granulosa cell apoptosis modelinduced by2,5-HD.2,5-HD was demonstrated to cause significant apoptosis of humanovarian granulosa cells in a dose-dependent manner. The results showed that withincreasing2,5-HD doses, the expression of BCL-2decreased. However, a markeddose-dependent increase in the expression of BAX and active CASPASE-3(p17) wasobserved in human ovarian granulosa cells.3. The regulation mechanism of Sonic Hedgehog signaling pathways in the processof2,5-hexanedione inducing apoptosis in human ovarian granulosa cells:(1) Control group of human ovarian granulosa cells in vitro culture, the expression of a smallamount of SHH signaling pathway components, but no significant difference inchange over time. In the2,5-HD-induced apoptosis in human ovarian granulosa cells,SHH, PTCH1, SMO, and GLI1mRNA levels in3h,6h, respectively compared withthe control group, were significantly different. SHH,SMO and GLI1mRNA levels in24h were lower. GLI2and GLI3mRNA at different times compared with the controlgroup were not statistically significant.(2) Results were consistent with the realtimePCR, SHH, GLI1protein of human ovarian granulosa cells in3h and6h wereelevated, and decreased in24h.(3) The exogenous SHH could increase the survivalrate of human ovarian granulosa cells and reduce cells apoptosis. The cyclopaminecould increase the cells apoptosis by blocking SHH signaling pathway.(4) In the2,5-HD-induced apoptosis in human ovarian granulosa cells, exogenous recombinantSHH could lead to increase expression of BCL-2and decrease expression of BAX.Conclusions:1. Good growth activity, cell purity and more stable human ovarian granulosa cellscould be obtained by the method used in this study. Human ovarian granulosa cellscould be simply and rapidly identified by HE staining and FSHR immuno-histochemistry methods.2. These results suggest that the mechanisms of2,5-HD causing increased apoptosisin human ovarian granulosa cells might be a marked dose-dependent on the expressionof BCL-2decreased and the expression of BAX and active CASPASE-3(p17)increased.3. Cultured human granulosa cells expressed a small amount of SHH signalingpathway components. SHH signaling pathway was activated and could reduce theapoptosis rate of granulosa cells during the2,5-HD-induced granulosa cells apoptosis.Its mechanism might be by regulating anti-apoptotic gene BCL-2and apoptosis geneBAX to prevent or alleviate the2,5-HD-induced granulosa cells apoptosis.