| Backgound: Polycystic ovary syndrome (PCOS) is one of the most commonheterogeneous endocrine disorders, which primarily characterized by biochemical orclinical hyperandrogenism, polycystic ovaries on ultrasound, and oligo-ovulation oranovulation. Besides, PCOS patients often accompanied with insulin resistance orpancreatic dysfunction, which resulted in an increased risk of type2diabetes,metabolic syndrome and cardiovascular disorders. Hyperandrogenism is the mostcommon characteristic of PCOS, which is of both ovarian and adrenal origin andprimarily from the ovary. Until now, the etiology and pathogenesis of PCOS are notclear. To further explore the molecular mechanism of PCOS, cDNA microarray wasperformed between PCOS ovaries and normal ones. SET was expressed wildly invarious tissues including the sterodgenic cells from nervous system, adrenal glands,and sexual glands. And it is found that SET in PCOS ovaries was twice higher thanthat in normal ones. It is found that in human NT2neuronal precursor cells andMA-10cells, SET activates its basal and hormone-stimulated transcription. In manycells SET inhibited PP2A activity and in NCI-H295A cells PP2A inhibiteddephosphorylation of P450c17and increased its17,20-lyase activity. So it ishypothesized that SET may be involved in the steroidogenesis of ovaries and thepotential pathologic mechanism of hyperandrogenemia observed in PCOS patients.The study examined the localization and differential expression of SET in PCOSovaries and normal ovaries. By SET overexpression and knockdown usingrecombinant adenoviruses, the effect of SET on androgen production and itsmechanism had also been studied. This study provide basis for us to further explorethe mechanism of SET on the pathophysiology development of PCOS.Materials and MethodsPart One: The localization and differential expression of SET in PCOS ovariesand normal ovaries1. Examine the localization of SET in PCOS ovaries and normal ovaries byimmunohistochemistry.2. Compare the expression of SET between PCOS ovaries and normal ovaries by Western blot.Part Two: The effect of SET on steroidogenesis in theca cells1. Examine the localization of SET in mouse ovaries by immunohistochemistry.2. After48hours of post-infection with AdCMV-SET or AdH1-SiRNA/SETadenoviruses, validate the efficiency of SET overexpression and knockdown byWestern blot, and examine the testosterone concentration by EIA.3. After48hours of post-infection with AdCMV-SET or AdH1-SiRNA/SETadenoviruses, the mRNA expression of StAR, CYP11A1, CYP17A1and HSD3B2were analyzed by real-time RT-PCR.Part Three: SET regulated steroidogenesis by PP2A in theca cells1. After24hours of post-infection with AdCMV-SET or AdH1-SiRNA/SETadenoviruses, the P450c17lyase activity was analyzed by UPLC–ESI MS/MS andthe P450c17expression was analyzed by Western blot.2. After48hours of post-infection with AdCMV-SET or AdH1-SiRNA/SETadenoviruses, examine the effect of SET overexpression and knockdown on POR andCYB5by real-time RT-PCR.3. Examine the localization of SET in mouse ovaries by immunofluorescence.4. Examine the localization of SET and PP2A in mouse ovaries byco-immunofluorescence.5. Examine the localization of PP2A and P450c17in mouse ovaries byco-immunofluorescence and the interaction of them by co-immunoprecipitation..6. After24hours of post-infection with AdCMV-SET or AdH1-SiRNA/SETadenoviruses, examine the effect of SET on PP2A activity by PP2Aimmunoprecipitation phosphatase assay kit and PP2A expression by Western blot.7. After48hours treatment of PP2A inhibitor OA and activators1,9-dideoxyforskolin and FTY720, examine the testosterone concentration by EIA.After48hours of post-infection with AdH1-SiRNA/PP2A adenoviruses, examine thetestosterone concentration by EIA, and analyze the P450c17lyase activity byUPLC–ESI MS/MS, P450c17expression by Western blot.8. The AdH1-SiRNA/SET-infected and AdH1-SiRNA/NS-infected follicles were treated with OA for24h. Media were collected for testosterone assay by EIA.ResultsPart One: The localization and differential expression of SET in PCOS ovariesand normal ovaries1. SET is predominantly localized in theca cells and oocytes of PCOS ovaries andnormal ovaries. In theca cells SET is localized in both cytoplasm and nucleus.2. The level of SET protein was three times higher in ovaries of PCOS patients thanin normal ovaries.Part Two: The effect of SET on steroidogenesis in theca cells1. SET is predominantly localized in theca cells and oocytes of mouse ovaries. Intheca cells of primary follicle, preantral follicle, and antral follicle SET is expressedand localized in both cytoplasm and nucleus.2. After48hours of post-infection with AdCMV-SET adenoviruses, SET expressionwas increased compared with AdCMV-GFP infected group (p<0.05), and testosteroneproduction was increased compared with AdCMV-GFP infected group (177.42±54.94pg/ml vs79.43±25.23pg/ml)(p<0.05). After48hours of post-infection withAdH1-SiRNA/SET adenoviruses, SET expression was decreased compared withAdH1-SiRNA/NS infected group (p<0.05), and testosterone production wasdecreased compared with AdH1-SiRNA/NS infected group (73.59±21.34pg/ml vs33.11±6.64pg/ml)(p<0.05).3. After24hours of post-infection with AdCMV-SET adenoviruses, the mRNA levelof SET was increased1.66times than AdCMV-GFP infected group (p<0.05), and themRNA level of CYP17A1, HSD3B2mRNA was also increased2.46and3.36timesthan the AdCMV-GFP infected group (p<0.05). After24hours of post-infection withAdH1-SiRNA/SET adenoviruses, the mRNA level of SET was decreased comparedwith AdH1-SiRNA/NS infected group (p<0.05), and the mRNA level of CYP17A1,HSD3B2mRNA was also decreased compared with AdH1-SiRNA/NS infected group(p<0.05).Part Three: SET regulated steroidogenesis by PP2A in theca cells1. After24hours of post-infection with AdCMV-SET adenoviruses, the level of P450c17protein was not altered compared with AdCMV-GFP infected group(p>0.05)), and the P450c17lyase activity was increased compared with AdCMV-GFPinfected group (p<0.05). After24hours of post-infection with AdH1-SiRNA/SETadenoviruses, the level of P450c17protein was not altered compared withAdH1-SiRNA/NS infected group (p>0.05)), and the P450c17lyase activity wasdecreased compared with AdCMV-GFP infected group (p<0.05).2. After24hours of post-infection with AdCMV-SET or AdH1-SiRNA/SETadenoviruses, the mRNA level of POR and CYB5was not altered compared withcontrol group (p>0.05)).3. In mouse ovaries PP2A was widely expressed and detected primarily in theca cells,granulosa cells, interstitial cells and so on.4. In theca-interstitial cells SET localized in both cytoplasm and nucleus, and mainlyin nucleus. While PP2A localized mainly in cytoplasm, which partially colocalizedwith SET.5. In theca-interstitial cells PP2A colocalized and binded with P450c17.6. After24hours of post-infection with AdCMV-SET adenoviruses, the level ofPP2A protein was not altered compared with AdCMV-GFP infected group (p>0.05)),and the PP2A activity was decreased compared with AdCMV-GFP infected group(p<0.05). After24hours of post-infection with AdH1-SiRNA/SET adenoviruses, thelevel of PP2A protein was not altered compared with AdH1-SiRNA/NS infectedgroup (p>0.05)), and the PP2A activity was increased compared withAdH1-SiRNA/NS infected group (p<0.05).7. After48hours of OA treatment, testosterone production was increased comparedwith control group (252.13±92.83pg/ml vs93.46±2.91pg/ml)(p<0.05). After48hours of1,9-dideoxy-forskolin or FTY720treatment, testosterone production wasdecreased compared with control group (74.84±13.85pg/ml vs65.63±22.10pg/ml；104.77±15.80pg/ml vs65.63±22.10pg/ml)(p<0.05). After48hours of post-infectionwith AdH1-SiRNA/PP2A adenoviruses, the level of PP2A protein was decreasedcompared with AdH1-SiRNA/NS infected group (p<0.05), and testosteroneproduction was increased (p<0.05). After48hours of post-infection with AdH1-SiRNA/PP2A adenoviruses, the level of P450c17protein was not alteredcompared with AdH1-SiRNA/NS infected group (p>0.05), while the P450c17lyaseactivity was increased (p<0.05).8. The AdH1-SiRNA/SET-infected and AdH1-SiRNA/NS-infected follicles weretreated with OA for24h. The testosterone level in the AdH1-SiRNA/SET infected andDMSO incubated follicles decreased when compared to the AdH1-SiRNA/NSinfected and DMSO incubated follicles (41.55±10.30pg/ml vs111.19±14.17pg/ml)(p<0.05). The testosterone level in the AdH1-SiRNA/SET infected and OA incubatedfollicles increased when compared to the AdH1-SiRNA/SET infected and DMSOincubated follicles (41.55±10.30pg/ml vs115.47±23.23pg/ml)(p<0.05). Thetestosterone level in the AdH1-SiRNA/NS infected and DMSO incubated folliclesand the AdH1-SiRNA/SET infected and OA incubated follicles had no significantdifference (111.19±14.17pg/ml vs115.47±23.23pg/ml)(p>0.05).Conclusion1. SET protein was higher in ovaries of PCOS patients compared to normal ovaries,and is predominantly localized in theca cells and oocytes of PCOS ovaries andnormal ovaries. In theca cells of primary follicle, preantral follicle, and antral follicleSET is expressed and localized in both cytoplasm and nucleus, which implied SETmay regulate some biological processes of theca cells.2. SET regulates testosterone production positively, and promotes the mRNAexpression of CYP17A1and HSD3B2. SET also promotes the P450c17lyase activityand does not affect the mRNA expression of POR and CYB5. So SET regulates thesteroidogenesis of theca cells.3. In mouse ovaries PP2A was widely expressed and detected primarily in theca cells,granulosa cells, interstitial cells and so on. In theca-interstitial cells PP2A localizedmainly in cytoplasm, which partially colocalized with SET. Besides, intheca-interstitial cells PP2A colocalized and binded with P450c17. PP2A inhibited thetestosterone production and P450c17lyase activity. SET promotes testosteronebiosynthesis by inhibition of PP2A activity and further activation of the17,20-lyaseactivity of P450c17, which provides a pathologic pathway leading to hyperandrogenism of PCOS.4. Our study indicates that in ovaries SET regulates P450c17lyase activity by PP2A,and participate in the regulation of ovarian steroidogenesis. The overexpression ofSET in PCOS ovaries may be one of the pathogenesis of PCOS. |
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