Hyperandrogenism and insulin resistance modulate gravid uterine and placental ferroptosis in PCOS-like rats

in Journal of Endocrinology

Correspondence should be addressed to L R Shao: ruijin.shao@fysiologi.gu.se

*(Y Zhang and M Hu contributed equally to this work)

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Women with polycystic ovary syndrome (PCOS) have hyperandrogenism and insulin resistance and a high risk of miscarriage during pregnancy. Similarly, in rats, maternal exposure to 5α-dihydrotestosterone (DHT) and insulin from gestational day 7.5 to 13.5 leads to hyperandrogenism and insulin resistance and subsequently increased fetal loss. A variety of hormonal and metabolic stimuli are able to trigger different types of regulated cell death under physiological and pathological conditions. These include ferroptosis, apoptosis and necroptosis. We hypothesized that, in rats, maternal hyperandrogenism and insulin-resistance-induced fetal loss is mediated, at least in part, by changes in the ferroptosis, apoptosis and necroptosis pathways in the gravid uterus and placenta. Compared with controls, we found that co-exposure to DHT and insulin led to decreased levels of glutathione peroxidase 4 (GPX4) and glutathione, increased glutathione + glutathione disulfide and malondialdehyde, aberrant expression of ferroptosis-associated genes (Acsl4, Tfrc, Slc7a11, and Gclc), increased iron deposition and activated ERK/p38/JNK phosphorylation in the gravid uterus. In addition, we observed shrunken mitochondria with electron-dense cristae, which are key features of ferroptosis-related mitochondrial morphology, as well as increased expression of Dpp4, a mitochondria-encoded gene responsible for ferroptosis induction in the uteri of rats co-exposed to DHT and insulin. However, in the placenta, DHT and insulin exposure only partially altered the expression of ferroptosis-related markers (e.g. region-dependent GPX4, glutathione + glutathione disulfide, malondialdehyde, Gls2 and Slc7a11 mRNAs, and phosphorylated p38 levels). Moreover, we found decreased expression of Dpp4 mRNA and increased expression of Cisd1 mRNA in placentas of rats co-exposed to DHT and insulin. Further, DHT + insulin-exposed pregnant rats exhibited decreased apoptosis in the uterus and increased necroptosis in the placenta. Our findings suggest that maternal hyperandrogenism and insulin resistance causes the activation of ferroptosis in the gravid uterus and placenta, although this is mediated via different mechanisms operating at the molecular and cellular levels. Our data also suggest that apoptosis and necroptosis may play a role in coordinating or compensating for hyperandrogenism and insulin-resistance-induced ferroptosis when the gravid uterus and placenta are dysfunctional.

Supplementary Materials

    • Supplementary Materials and Methods
    • Supplemental Figure 1. Analyses of body weight variations in pregnant rats exposed to DHT and/or insulin. Values are expressed as means ± SEM. Group differences across GDs in overall maternal body weight were evaluated using repeated measures ANOVA, with different treatments (Control, DHT+INS, DHT, INS) as the between-subjects factor and gestation days (GD 0.5-14.5) as the within-subjects factor. GD, gestational day. DHT, 5α-dihydrotestosterone; INS, insulin.
    • Supplemental Figure 2. Tissue expression of Gpx4 protein in the adult rats. Protein samples (left) were isolated from selective tissues (testis, epididymis, and ovary; n = 6/tissue). The level of Gpx4 protein expression was determined by Western blot analysis (right). Each lane corresponds to the different rat samples. Relative mobilities of molecular mass standards (MW) is shown (in kilodaltons) on the left.
    • Supplemental Figure 3. Tissue expression of Gpx4 protein in the adult rat uterus. The level and localization of uterine Gpx4 protein expression in the diestrus stage in rats were determined by Western blot (A) and immunohistochemical analyses (B). In the Western blot analysis, each lane corresponds to the different rat samples. Relative mobilities of molecular mass standards (MW) are shown (in kilodaltons) on the left. Immunohistochemical images are representative of 3−5 tissue replicates per group. Detailed views of the boxed areas are shown in the inset (B). L, lumen; Le, luminal epithelial cells; Ge, glandular epithelial cells; Str, stromal cells; Myo, myometrium. Scale bars (100 μm) are indicated in the photomicrographs.
    • Supplemental Figure 4. Iron deposition in the uterine sections from pregnant rats at GD 6 (A, A1-2) and pregnant rats exposed to vehicle and DHT+INS at GD 14.5 (B, B1-4). The sections were stained by DAB-enhanced Perls’ staining for iron accumulation. Images are representative of 5−8 tissue replicates per group. Yellow arrowheads indicate punctate cytoplasmic, and granular iron-positive staining. MD, mesometrial decidua; AD, antimesometrial decidua; Myo, myometrium. Scale bars (100 μm) are indicated in the photomicrographs. DHT, 5α-dihydrotestosterone; INS, insulin.
    • Supplemental Figure 5. Mitochondrial ultrastructural defects in mesometrial decidua, and basal and labyrinth zones in pregnant rats exposed to DHT and/or insulin at GD 14.5. Scale bars (2 μm) are indicated in the photomicrographs. DHT, 5α-dihydrotestosterone; INS, insulin.

 

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  • AgarwalAAponte-MelladoAPremkumarBJShamanAGuptaS 2012 The effects of oxidative stress on female reproduction: a review. Reproductive Biology and Endocrinology 10 49. (https://doi.org/10.1186/1477-7827-10-49)

    • Search Google Scholar
    • Export Citation
  • AzzizRCarminaEChenZDunaifALavenJSLegroRSLiznevaDNatterson-HorowtizBTeedeHJYildizBO 2016 Polycystic ovary syndrome. Nature Reviews: Disease Primers 2 16057. (https://doi.org/10.1038/nrdp.2016.57)

    • Search Google Scholar
    • Export Citation
  • Bahri KhomamiMJohamAEBoyleJAPiltonenTSilagyMAroraCMissoMLTeedeHJMoranLJ 2019 Increased maternal pregnancy complications in polycystic ovary syndrome appear to be independent of obesity – a systematic review, meta-analysis, and meta-regression. Obesity Reviews 20 659674. (https://doi.org/10.1111/obr.12829)

    • Search Google Scholar
    • Export Citation
  • BaithaluRKSinghSKKumaresanAMohantyAKMohantyTKKumarSKerkettaSMaharanaBRPatbandhaTKAttupuramN 2017 Transcriptional abundance of antioxidant enzymes in endometrium and their circulating levels in Zebu cows with and without uterine infection. Animal Reproduction Science 177 7987. (https://doi.org/10.1016/j.anireprosci.2016.12.008)

    • Search Google Scholar
    • Export Citation
  • BanulsCRovira-LlopisSMartinez de MaranonAVesesSJoverAGomezMRochaMHernandez-MijaresAVictorVM 2017 Metabolic syndrome enhances endoplasmic reticulum, oxidative stress and leukocyte-endothelium interactions in PCOS. Metabolism: Clinical and Experimental 71 153162. (https://doi.org/10.1016/j.metabol.2017.02.012)

    • Search Google Scholar
    • Export Citation
  • CaoCFlemingMD 2016 The placenta: the forgotten essential organ of iron transport. Nutrition Reviews 74 421431. (https://doi.org/10.1093/nutrit/nuw009)

    • Search Google Scholar
    • Export Citation
  • ChenLHambrightWSNaRRanQ 2015 Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis. Journal of Biological Chemistry 290 2809728106. (https://doi.org/10.1074/jbc.M115.680090)

    • Search Google Scholar
    • Export Citation
  • ChoiMEPriceDRRyterSWChoiAMK 2019 Necroptosis: a crucial pathogenic mediator of human disease. JCI Insight 4 e128834. (https://doi.org/10.1172/jci.insight.128834)

    • Search Google Scholar
    • Export Citation
  • ConradMSchneiderMSeilerABornkammGW 2007 Physiological role of phospholipid hydroperoxide glutathione peroxidase in mammals. Biological Chemistry 388 10191025. (https://doi.org/10.1515/BC.2007.130)

    • Search Google Scholar
    • Export Citation
  • DaiCChenXLiJComishPKangRTangD 2020 Transcription factors in ferroptotic cell death. Cancer Gene Therapy [epub]. (https://doi.org/10.1038/s41417-020-0170-2)

    • Search Google Scholar
    • Export Citation
  • DaltoDBRoyMAudetIPalinMFGuayFLapointeJMatteJJ 2015 Interaction between vitamin B6 and source of selenium on the response of the selenium-dependent glutathione peroxidase system to oxidative stress induced by oestrus in pubertal pig. Journal of Trace Elements in Medicine and Biology 32 2129. (https://doi.org/10.1016/j.jtemb.2015.05.002)

    • Search Google Scholar
    • Export Citation
  • DingYJiangZXiaBZhangLZhangCLengJ 2019 Mitochondria-targeted antioxidant therapy for an animal model of PCOS-IR. International Journal of Molecular Medicine 43 316324. (https://doi.org/10.3892/ijmm.2018.3977)

    • Search Google Scholar
    • Export Citation
  • DixonSJLembergKMLamprechtMRSkoutaRZaitsevEMGleasonCEPatelDNBauerAJCantleyAMYangWS 2012 Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149 10601072. (https://doi.org/10.1016/j.cell.2012.03.042)

    • Search Google Scholar
    • Export Citation
  • Escobar-MorrealeHF 2012 Iron metabolism and the polycystic ovary syndrome. Trends in Endocrinology & Metabolism 23 509515. (https://doi.org/10.1016/j.tem.2012.04.003)

    • Search Google Scholar
    • Export Citation
  • FassnachtMSchlenzNSchneiderSBWudySAAllolioBArltW 2003 Beyond adrenal and ovarian androgen generation: increased peripheral 5 alpha-reductase activity in women with polycystic ovary syndrome. Journal of Clinical Endocrinology & Metabolism 88 27602766. (https://doi.org/10.1210/jc.2002-021875)

    • Search Google Scholar
    • Export Citation
  • FengHSchorppKJinJYozwiakCEHoffstromBGDeckerAMRajbhandariPStokesMEBenderHGCsukaJM 2020 Transferrin receptor is a specific ferroptosis marker. Cell Reports 30 34113423.e7. (https://doi.org/10.1016/j.celrep.2020.02.049)

    • Search Google Scholar
    • Export Citation
  • ForcinaGCDixonSJ 2019 GPX4 at the crossroads of lipid homeostasis and ferroptosis. Proteomics 19 e1800311. (https://doi.org/10.1002/pmic.201800311)

    • Search Google Scholar
    • Export Citation
  • Friedmann AngeliJPSchneiderMPronethBTyurinaYYTyurinVAHammondVJHerbachNAichlerMWalchAEggenhoferE 2014 Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nature Cell Biology 16 11801191. (https://doi.org/10.1038/ncb3064)

    • Search Google Scholar
    • Export Citation
  • GalarisDBarboutiAPantopoulosK 2019 Iron homeostasis and oxidative stress: an intimate relationship. Biochimica et Biophysica Acta. Molecular Cell Research 1866 118535. (https://doi.org/10.1016/j.bbamcr.2019.118535)

    • Search Google Scholar
    • Export Citation
  • GaoMYiJZhuJMinikesAMMonianPThompsonCBJiangX 2019 Role of mitochondria in ferroptosis. Molecular Cell 73 354363.e3. (https://doi.org/10.1016/j.molcel.2018.10.042)

    • Search Google Scholar
    • Export Citation
  • GawelSWardasMNiedworokEWardasP 2004 Malondialdehyde (MDA) as a lipid peroxidation marker. Wiadomosci Lekarskie 57 453455.

  • GlintborgDJensenRCBentsenKSchmedesAVBrandslundIKyhlHBBilenbergNAndersenMS 2018 Testosterone levels in third trimester in polycystic ovary syndrome: odense child cohort. Journal of Clinical Endocrinology & Metabolism 103 38193827. (https://doi.org/10.1210/jc.2018-00889)

    • Search Google Scholar
    • Export Citation
  • GudipatySAConnerCMRosenblattJMontellDJ 2018 Unconventional ways to live and die: cell death and survival in development, homeostasis, and disease. Annual Review of Cell & Developmental Biology 34 311332. (https://doi.org/10.1146/annurev-cellbio-100616-060748)

    • Search Google Scholar
    • Export Citation
  • GuoYZhangNZhangDRenQGanzTLiuSNemethE 2019 Iron homeostasis in pregnancy and spontaneous abortion. American Journal of Hematology 94 184188. (https://doi.org/10.1002/ajh.25341)

    • Search Google Scholar
    • Export Citation
  • HuMZhangYGuoXJiaWLiuGZhangJCuiPLiJLiWWuX 2019a Perturbed ovarian and uterine glucocorticoid receptor signaling accompanies the balanced regulation of mitochondrial function and NFkappaB-mediated inflammation under conditions of hyperandrogenism and insulin resistance. Life Sciences 232 116681. (https://doi.org/10.1016/j.lfs.2019.116681)

    • Search Google Scholar
    • Export Citation
  • HuMZhangYGuoXJiaWLiuGZhangJLiJCuiPSferruzzi-PerriANHanY 2019b Hyperandrogenism and insulin resistance induce gravid uterine defects in association with mitochondrial dysfunction and aberrant ROS production. American Journal of Physiology – Endocrinology & Metabolism 316 E794E809. (https://doi.org/10.1152/ajpendo.00359.2018)

    • Search Google Scholar
    • Export Citation
  • ImaiHHiraoFSakamotoTSekineKMizukuraYSaitoMKitamotoTHayasakaMHanaokaKNakagawaY 2003 Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene. Biochemical & Biophysical Research Communications 305 278286. (https://doi.org/10.1016/s0006-291x(03)00734-4)

    • Search Google Scholar
    • Export Citation
  • KimJWKangKMYoonTKShimSHLeeWS 2014 Study of circulating hepcidin in association with iron excess, metabolic syndrome, and BMP-6 expression in granulosa cells in women with polycystic ovary syndrome. Fertility & Sterility 102 548554.e2. (https://doi.org/10.1016/j.fertnstert.2014.04.031)

    • Search Google Scholar
    • Export Citation
  • KimSJXiaoJWanJCohenPYenK 2017 Mitochondrially derived peptides as novel regulators of metabolism. Journal of Physiology 595 66136621. (https://doi.org/10.1113/JP274472)

    • Search Google Scholar
    • Export Citation
  • KovtunovychGEckhausMAGhoshMCOllivierre-WilsonHRouaultTA 2010 Dysfunction of the heme recycling system in heme oxygenase 1-deficient mice: effects on macrophage viability and tissue iron distribution. Blood 116 60546062. (https://doi.org/10.1182/blood-2010-03-272138)

    • Search Google Scholar
    • Export Citation
  • LaiQXiangWLiQZhangHLiYZhuGXiongCJinL 2018 Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome. Frontiers of Medicine 12 518524. (https://doi.org/10.1007/s11684-017-0575-y)

    • Search Google Scholar
    • Export Citation
  • LeiPBaiTSunY 2019 Mechanisms of ferroptosis and relations with regulated cell death: a review. Frontiers in Physiology 10 139. (https://doi.org/10.3389/fphys.2019.00139)

    • Search Google Scholar
    • Export Citation
  • LiJCaoFYinHLHuangZJLinZTMaoNSunBWangG 2020 Ferroptosis: past, present and future. Cell Death & Disease 11 88. (https://doi.org/10.1038/s41419-020-2298-2)

    • Search Google Scholar
    • Export Citation
  • LiznevaDSuturinaLWalkerWBraktaSGavrilova-JordanLAzzizR 2016 Criteria, prevalence, and phenotypes of polycystic ovary syndrome. Fertility & Sterility 106 615. (https://doi.org/10.1016/j.fertnstert.2016.05.003)

    • Search Google Scholar
    • Export Citation
  • LuSC 2009 Regulation of glutathione synthesis. Molecular Aspects of Medicine 30 4259. (https://doi.org/10.1016/j.mam.2008.05.005)

  • MaliqueoMLaraHESanchezFEchiburuBCrisostoNSir-PetermannT 2013 Placental steroidogenesis in pregnant women with polycystic ovary syndrome. European Journal of Obstetrics Gynecology and Reproductive Biology 166 151155. (https://doi.org/10.1016/j.ejogrb.2012.10.015)

    • Search Google Scholar
    • Export Citation
  • MistryHDKurlakLOWilliamsPJRamsayMMSymondsMEBroughton PipkinF 2010 Differential expression and distribution of placental glutathione peroxidases 1, 3 and 4 in normal and preeclamptic pregnancy. Placenta 31 401408. (https://doi.org/10.1016/j.placenta.2010.02.011)

    • Search Google Scholar
    • Export Citation
  • MistryHDWilsonVRamsayMMSymondsMEBroughton PipkinF 2008 Reduced selenium concentrations and glutathione peroxidase activity in preeclamptic pregnancies. Hypertension 52 881888. (https://doi.org/10.1161/HYPERTENSIONAHA.108.116103)

    • Search Google Scholar
    • Export Citation
  • NgSWNorwitzSGNorwitzER 2019 The impact of iron overload and ferroptosis on reproductive disorders in humans: implications for preeclampsia. International Journal of Molecular Sciences 20 3283. (https://doi.org/10.3390/ijms20133283)

    • Search Google Scholar
    • Export Citation
  • PossKDTonegawaS 1997 Heme oxygenase 1 is required for mammalian iron reutilization. PNAS 94 1091910924. (https://doi.org/10.1073/pnas.94.20.10919)

    • Search Google Scholar
    • Export Citation
  • RamosRSOliveiraMLIzaguirryAPVargasLMSoaresMBMesquitaFSSantosFWBinelliM 2015 The periovulatory endocrine milieu affects the uterine redox environment in beef cows. Reproductive Biology and Endocrinology 13 39. (https://doi.org/10.1186/s12958-015-0036-x)

    • Search Google Scholar
    • Export Citation
  • RiegmanMBradburyMSOverholtzerM 2019 Population dynamics in cell death: mechanisms of propagation. Trends in Cancer 5 558568. (https://doi.org/10.1016/j.trecan.2019.07.008)

    • Search Google Scholar
    • Export Citation
  • RosenfieldRLEhrmannDA 2016 The pathogenesis of polycystic ovary syndrome (PCOS): the hypothesis of PCOS as functional ovarian hyperandrogenism revisited. Endocrine Reviews 37 467520. (https://doi.org/10.1210/er.2015-1104)

    • Search Google Scholar
    • Export Citation
  • SchatzFGuzeloglu-KayisliOArlierSKayisliUALockwoodCJ 2016 The role of decidual cells in uterine hemostasis, menstruation, inflammation, adverse pregnancy outcomes and abnormal uterine bleeding. Human Reproduction Update 22 497515. (https://doi.org/10.1093/humupd/dmw004)

    • Search Google Scholar
    • Export Citation
  • SchneiderMForsterHBoersmaASeilerAWehnesHSinowatzFNeumullerCDeutschMJWalchAHrabe de AngelisM 2009 Mitochondrial glutathione peroxidase 4 disruption causes male infertility. FASEB Journal 23 32333242. (https://doi.org/10.1096/fj.09-132795)

    • Search Google Scholar
    • Export Citation
  • SchootsMHGordijnSJScherjonSAvan GoorHHillebrandsJL 2018 Oxidative stress in placental pathology. Placenta 69 153161. (https://doi.org/10.1016/j.placenta.2018.03.003)

    • Search Google Scholar
    • Export Citation
  • SharmaSGodboleGModiD 2016 Decidual control of trophoblast invasion. American Journal of Reproductive Immunology 75 341350. (https://doi.org/10.1111/aji.12466)

    • Search Google Scholar
    • Export Citation
  • SharpANHeazellAECrockerIPMorG 2010 Placental apoptosis in health and disease. American Journal of Reproductive Immunology 64 159169. (https://doi.org/10.1111/j.1600-0897.2010.00837.x)

    • Search Google Scholar
    • Export Citation
  • SilfenMEDenburgMRManiboAMLoboRAJaffeRFerinMLevineLSOberfieldSE 2003 Early endocrine, metabolic, and sonographic characteristics of polycystic ovary syndrome (PCOS): comparison between nonobese and obese adolescents. Journal of Clinical Endocrinology & Metabolism 88 46824688. (https://doi.org/10.1210/jc.2003-030617)

    • Search Google Scholar
    • Export Citation
  • Sir-PetermannTMaliqueoMAngelBLaraHEPerez-BravoFRecabarrenSE 2002 Maternal serum androgens in pregnant women with polycystic ovarian syndrome: possible implications in prenatal androgenization. Human Reproduction 17 25732579. (https://doi.org/10.1093/humrep/17.10.2573)

    • Search Google Scholar
    • Export Citation
  • SpencerSJCataldoNAJaffeRB 1996 Apoptosis in the human female reproductive tract. Obstetrical & Gynecological Survey 51 314323. (https://doi.org/10.1097/00006254-199605000-00023)

    • Search Google Scholar
    • Export Citation
  • StockwellBRFriedmann AngeliJPBayirHBushAIConradMDixonSJFuldaSGasconSHatziosSKKaganVE 2017 Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171 273285. (https://doi.org/10.1016/j.cell.2017.09.021)

    • Search Google Scholar
    • Export Citation
  • SunXNiuXChenRHeWChenDKangRTangD 2016 Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology 64 488500. (https://doi.org/10.1002/hep.28574)

    • Search Google Scholar
    • Export Citation
  • TangDKangRBergheTVVandenabeelePKroemerG 2019 The molecular machinery of regulated cell death. Cell Research 29 347364. (https://doi.org/10.1038/s41422-019-0164-5)

    • Search Google Scholar
    • Export Citation
  • WelshAO 1993 Uterine cell death during implantation and early placentation. Microscopy Research & Technique 25 223245. (https://doi.org/10.1002/jemt.1070250305)

    • Search Google Scholar
    • Export Citation
  • XieYHouWSongXYuYHuangJSunXKangRTangD 2016 Ferroptosis: process and function. Cell Death & Differentiation 23 369379. (https://doi.org/10.1038/cdd.2015.158)

    • Search Google Scholar
    • Export Citation
  • ZhangHHeYWangJXChenMHXuJJJiangMHFengYLGuYF 2020 miR-30-5p-mediated ferroptosis of trophoblasts is implicated in the pathogenesis of preeclampsia. Redox Biology 29 101402. (https://doi.org/10.1016/j.redox.2019.101402)

    • Search Google Scholar
    • Export Citation
  • ZhangJBaoYZhouXZhengL 2019a Polycystic ovary syndrome and mitochondrial dysfunction. Reproductive Biology and Endocrinology: RB&E 17 67. (https://doi.org/10.1186/s12958-019-0509-4)

    • Search Google Scholar
    • Export Citation
  • ZhangYMengFSunXSunXHuMCuiPVestinELiXLiWWuXK 2018 Hyperandrogenism and insulin resistance contribute to hepatic steatosis and inflammation in female rat liver. Oncotarget 9 1818018197. (https://doi.org/10.18632/oncotarget.24477)

    • Search Google Scholar
    • Export Citation
  • ZhangYSunXSunXMengFHuMLiXLiWWuXKBrännströmMShaoR 2016 Molecular characterization of insulin resistance and glycolytic metabolism in the rat uterus. Scientific Reports 6 30679. (https://doi.org/10.1038/srep30679)

    • Search Google Scholar
    • Export Citation
  • ZhangYZhaoWXuHHuMGuoXJiaWLiuGLiJCuiPLagerS 2019 Hyperandrogenism and insulin resistance-induced fetal loss: evidence for placental mitochondrial abnormalities and elevated reactive oxygen species production in pregnant rats that mimic the clinical features of polycystic ovary syndrome. Journal of Physiology 597 39273950. (https://doi.org/10.1113/JP277879)

    • Search Google Scholar
    • Export Citation
  • ZhuLJBagchiMKBagchiIC 1995 Ferritin heavy chain is a progesterone-inducible marker in the uterus during pregnancy. Endocrinology 136 41064115. (https://doi.org/10.1210/endo.136.9.7649119)

    • Search Google Scholar
    • Export Citation