research article ovarian aging-like phenotype in the...

Research ArticleOvarian Aging-Like Phenotype inthe Hyperandrogenism-Induced Murine Model ofPolycystic Ovary

Mohammad Amin Rezvanfar12 Habib A Shojaei Saadi3 Maziar Gooshe4

Amir Hosein Abdolghaffari56 Maryam Baeeri2 and Mohammad Abdollahi12

1 Department of Toxicology and Pharmacology Faculty of Pharmacy Tehran University of Medical Sciences Tehran 1417614411 Iran2 Pharmaceutical Sciences Research Center Tehran University of Medical Sciences Tehran 1417614411 Iran3 Centre de Recherche en Biologie de la Reproduction Universite Laval Quebec City QC Canada G1V 0A64 Faculty of Medicine Tehran University of Medical Sciences Tehran 1417614411 Iran5 Pharmacology and Applied Medicine Department of Medicinal Plants Research Center Institute of Medicinal PlantsACECR Karaj Alborz 141554364 Iran

6 International Campus Tehran University of Medical Sciences (ICTUMS) Tehran 1417653861 Iran

Correspondence should be addressed to Mohammad Abdollahi mohammadtumsacir

Received 28 November 2013 Accepted 2 January 2014 Published 19 February 2014

Academic Editor Sara Mostafalou

Copyright copy 2014 Mohammad Amin Rezvanfar et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

There are prominently similar symptoms effectors and commonalities in themajority of characteristics between ovarian aging andpolycystic ovarian syndrome (PCOS) Despite the approved role of oxidative stress in the pathogenesis of PCOS and aging to ourknowledge the link between the PCO(S) and aging has not been investigated yet In this studywe investigated the possible exhibitionof ovarian aging phenotype in murine model of PCO induced by daily oral administration of letrozole (1mgkg body weight) for 21consecutive days in the female Wistar rats Hyperandrogenization showed irregular cycles and histopathological characteristicsof PCO which was associated with a significant increase in lipid peroxidation (LPO) and reactive oxygen species (ROS) anddecrease in total antioxidant capacity (TAC) in serum and ovary Moreover serum testosterone insulin and tumor necrosisfactor-alpha (TNF-120572) levels and ovarian matrix metalloproteinase-2 (MMP-2) were increased in PCO rats compared with healthycontrols while estradiol and progesterone diminished Almost all of these findings are interestingly found to be common withthe characteristics identified with (ovarian) aging showing that hyperandrogenism-induced PCO in rat is associated with ovarianaging-like phenotypes To our knowledge this is the first report that provides evidence regarding the phenomenon of aging in PCO

1 Introduction

Nowadays couples considerably postpone childbearing totheir late reproductive-age period due to increasing socioe-conomic demands Besides industrialization of communitieshas added a wide variety of environmental risk factors thatmay lead to disorders diseases and cancers in various organs

It has been shown that exposure to some environmentalfactors and chemicals during life may accelerate progres-sion towards the end of functional reproductive period [1]Accordingly several environmental factors have been con-firmed to contribute to reproductive aging [2] Female

reproductive system is unique in the fact that it displaysphysiologically much faster rate of aging and when it reachesa senescent state other organs in the body remain generallyhealthy

Ovary is one of the most important organs in the femalereproductive system aswell as an endocrine organ that under-goes aging by a continuous decreasing in the number of folli-cles diminished quality of oocytes menstrual irregularitiesovarian hormonal deficiency anovulation and subfertilityleading to menopause which is the final step in this process[3]

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 948951 10 pageshttpdxdoiorg1011552014948951

2 Oxidative Medicine and Cellular Longevity

Several mechanisms seem involved in aging which oxida-tive stress (OS) is considered as one of the most importantones [4] OS occurs due to excessive formation of oxygen-derived andor nitrogen-derived toxic products in the pres-ence of minimal antioxidant activity [5]

At moderate concentrations reactive oxygen species(ROS) regulate physiologic functions in female reproductionsuch as folliculogenesis oocyte maturation steroidogenesiscorpus luteal function and luteolysis [5] However increasedOS during ovarian aging may contribute to follicular atresiaand diminishing quantity and quality of oocytes [6] Substan-tial theories explain that a long lasting status of overproducedtoxic free radicals insufficient antioxidants and exposure toendocrine disrupting chemicalsmay be involved in reproduc-tive disorders which eventually confer acceleration of gonadssenescence and premature reproductive aging

Polycystic ovarian syndrome (PCOS) as an inflamma-tory condition is the leading cause of anovulatory infertili-ty in reproductive-aged women It is characterized byincreasing androgen secretion menstrual irregularity oligo-ovulationanovulation polycystic ovaries (PCO) infertilityand pregnancy complications [7] An ovarian cyst is devel-oped as a result of hormonal imbalance and has an atreticfluid-filled follicular structure with thin granulosa cell walls[8] Not only PCOSpatients display reproductive features butalso they develop several metabolic risks of type 2 diabetesand cardiovascular diseases including abdominal obesityinsulin resistance (IR) hyperinsulinemia glucose intoler-ance hypertension and metabolic syndrome [9] Thereare several different diagnostic criteria for PCOS Henceregardless of ethnicity PCOS has different prevalence inwomen at their functional reproductive period According toRotterdam criteria it is estimated that the average prevalenceof PCOS is approximately 18 that is too high [10]

Because of its complex pathogenesis and unrecognizedetiology no preventive measure has been implemented forPCOS to dateThe roles of OS and chronic low-grade inflam-mation in the pathogenesis of PCOS and the potential ben-efits of antioxidants have been the subjects of recent studies[11 12] High levels of androgen have been proposed as aninitial step in themajority of PCOS cases Hyperandrogenismimpairs maturation of developing follicles in ovaries andconsequently leads to developing cystogenesis [13] Extensiveanimal experimentations have indicated that prepubertal orpubertal exposure of low doses of androgens via aromatiza-tion to estrogen results in long-term reproductive consequen-ces including constant estrous cycles hyposteroidogenesisanovulation and development of cystic follicles at adulthood[14] Moreover exposure to exogenous estrogen in adulthoodhas been determined by having deleterious effects on theovarian physiology and endocrinology which may ultimatelylead to cystogenesis loss of follicle pool and early senescence[15] There are some evidences implying that there might bean association between aging and PCO For instance it hasbeen recently shown that accelerated aging inmouse inducedby D-galactose was concurrent with ovarian cystogenesis[16] However the causal link between hyperandrogenismand accelerated ovarian aging is not definitely recognizedIt is therefore essential to perceive what hormonal cellular

molecular metabolic environmental andor genetic factorscontribute to this condition and how they affect ovarian folli-cle development We have recently documented the poten-tial role of oxidativenitrosative stress and inflammatoryresponses in the pathogenesis of hyperandrogenism-inducedPCO in rat [17 18] However negligible information exists onthe hyperandrogenization effects on ovarian aging and themolecular basis of disturbed folliculogenesis and cystogen-esis

Overall it should be of considerable interest to investigatepossible development of ovarian aging in hyperandrogenicPCO rats Therefore the main objective of this study wasto investigate the association of ovarian aging with hyperan-drogenism-induced PCO through cellular molecular histo-pathological biochemical and endocrinal evaluations

2 Materials and Methods

21 Reagents Unless otherwise stated all chemicals werepurchased from Sigma-Aldrich (USA) Krebs-ringer-bicar-bonate (KRB) and ethyl acetate from Fluka (USA) rat TNF-120572 ELISA kit from Bender Med System (Austria) rat MMP-2 ELISA kitfrom Cusabio Biotech Co Ltd (China) steroidhormone radioimmunoassay kits from Neogen (USA) andletrozole from SOHA Pharmaceutical Co (Iran) were usedin this study

22 Rats Adult female albino Wistar rats (180ndash200 g) withnormal estrous cycle were used in the study Animals wererandomly divided into two groups containing 10 rats eachAll rats had ad libitum access to pelleted food and tap waterand housed under controlled temperature (22ndash25∘C) with arelative humidity of 40ndash55 and 12 h lights and dark cycleOnly females with at least three consecutive 4-5 d regularestrous cycles were used in the experiment Throughout theentire treatment animals were weighed and a vaginal smearwas done daily (to determine the stage of the reproductivecycle) up to the day of autopsy All rats received care inaccordance with the national health guidelines and the studyprotocol was approved by Tehran University of MedicalSciences (TUMS) review board

23 Sexual Cycle To study whether treatments altered theestrous cycle smears were obtained daily by vaginal washingand evaluated microscopically during the treatment periodAs determined in previous studies [17 18] the observationof cornified cells in the smears during a minimum of 10consecutive days was defined as persistent estrous indicatinganovulation and development of follicular cysts At thebeginning of the experiments all rats had regular cycles

24 Treatments The Control group of ten rats received onlyvehicle (09NaCl solution) orally once dailyThe treatmentgroup of ten rats was gavaged with letrozole once daily at aconcentration of 1mgkg orally dissolved in 09NaCl Effec-tive dose of letrozole (SOHA Pharmaceutical Co TehranIran) were selected upon previous experiments The treat-ment period was 21 days [17 18]

Oxidative Medicine and Cellular Longevity 3

25 Sampling After the last treatment all rats weighed andanesthetized with ether and blood samples were directlytaken from the heart Blood samples were centrifuged at1000timesg for 15min and collected sera stored at minus70∘C untilassayed for sex steroids (estradiol progesterone and testos-terone) as well as insulin TNF-120572 and OS markers All ratswere killed after anesthesia and freshly dissected ovarieswere weighed and divided as follows five of each groupwas immediately fixed in 4 (wv) paraformaldehyde forhistopathology whereas the remaining tissues were immedi-ately frozen at minus70∘C until used for determination of matrixmetalloproteinase 2 (MMP-2) and oxidant-antioxidantmark-ers

26 Histopathological Studies Five ovaries from each groupwere processed step by step through formalin fixationparaffin embedding and longitudinally and serially sectionedat 4 120583m with a rotary microtome placed on a glass slidestained with HampE (Hematoxylin and Eosin) and assessedmicroscopically by two persons blinded to the origin of thesections Only the follicles containing an oocyte nucleus werecounted in every ovarian section Follicles were characteris-tically divided to primordial follicles (an oocyte surroundedby a single layer of granulosa cells and a diameter lt100120583m)growing follicles (an oocyte surrounded by several layersof granulosa cells without an antrum and diameter of 100to 300 120583m) or graafian follicles (a peripheral oocyte sur-rounded by cumulus cells and several layers of granulosa cellsan antrum and diameter gt300120583m) Atretic follicles werecharacterized by scattered pyknotic nuclei in the granulosacell layer detachment of the granulosa cell layers loss ofoocyte-granulosa cell communication fragmentation andmalformation of the oocyte disruption of the zona pellucida(ZP) and presence of cellular debris in the antrum of thefollicleHistopathological changeswere also categorized from(minus) to (+++) according to their severity where (minus) was not aprominent or obvious pathological finding and scores of (+)(++) and (+++) represented pathological findings of lt33lt33ndash66 and gt66 of the atretic follicles respectively Theaverage value of histopathological findings was taken intoconsideration and when there was disagreement between thetwo observers as to the interpretation of some histologiccharacteristics the case was reexamined and conclusion wasmade by consensus

27 Evaluation of Oxidative Stress Biomarkers To assess theeffect of hyperandrogenization on ovarian oxidant-antioxi-dant balance the LPO index ROS and level of TAC wereanalyzed

271 Determination of Cellular Lipid Peroxidation (LPO)The cellular LPOwas assessed in blood and ovaries using thi-obarbituric acid reactive substances (TBARS) assay asdescribed in our previous work [19] The LPO levels wereexpressed as the extent of malondialdehyde (MDA) produc-tion during an acid-heating reaction Data were reported as120583gmg of protein

272 Determination of Reactive Oxygen Species (ROS) Theisolated ovaries were placed in a mitochondrial isolationbuffer containing 025M sucrose 20mMKCl 10mMEDTAand 50mM HEPES (pH 74) at a weight volume ratio of1 10 Tissues were minced thoroughly and homogenizedwith a manual glass homogenizer at 0ndash4∘C A portion ofthe homogenate and blood serum were used to determineROS production which was measured by use of fluorescenceDCFH with some modifications as set up in our lab Theassay buffer contained 130mM KCl 5mM MgCl

2 20mM

NaH2PO4 20mM Tris-HCl 01mM FeCl

3 17mM ADP

and 01mM NADPH and 30mM glucose (pH 74) with atotal volume of 200120583L Assay buffer contained 5 120583MDCFH-diacetate (DCFH-DA) dissolved in 125mM methanol with5 120583M final methanol concentration For homogenates 50120583L(sim1mg protein) was included in either assay medium andincubated at 37∘C for 15min This permitted DCFH-DA tobe broken by intracellular esterase to derive free DCFHThe rate of oxidation from DCFH to dichlorofluorescein(DCF) indicative of oxidant production was followed at theexcitation wavelength of 488 nm and emission wavelength of525 nm andmeasured every 6min for 60min using an ELISAF-2000 fluorescence spectrometer The rate was linear forat least 60min at various concentrations of protein presentcorrected for the autooxidation rate of DCFH [20]

273 Total Ferric Reducing Antioxidant Power Assay Totalantioxidant power of the ovaries and sera was evaluated bymeasuring the ability to reduce Fe3+ to Fe2+ To do that weused TPTZ which its interaction with Fe2+ results in theformation of a blue color with a maximum absorbance at593 nmDatawere expressed asmmolL ferric ions reduced toferrous per mg of protein as described in our previous work[21]

28 Evaluation of Serum Insulin as a Metabolic BiomarkerAfter 8 h fasting serum insulin concentration was ana-lyzed by enzyme-linked immunoassay (ELISA) technique asdescribed previously [20]

29 Evaluation of Inflammatory Biomarkers Concentrationsof TNF-120572 were assessed using a rat sandwich ELISA kit andexpressed as pgmg protein According to the procedurea color product is formed in proportion to the amount ofcytokine present in the sample After adding stop solution toterminate the reaction absorbance was measured at 450 nmas the primary wavelength and 620 nm as the referencewavelength [22] To determine the concentrations of TNF-120572per unit of protein the Bradfordmethodwas used tomeasureprotein content using concentrated Comassie blue as reagentand BSA as the standard

210 Evaluation of Matrix Metalloproteinase 2Gelatinase A(MMP-2) Ovarian samples which had been preserved atminus80∘C were warmed to minus20∘C and weighed A phosphatebuffer (pH 74) which was prepared in weight-appropriatequantities was diluted 10 times The tissue samples were


homogenized and then centrifuged in a refrigerated centri-fuge at 2000timesg for 15 minutes and their supernatants weretransferred into microtubes The MMP-2 was analyzed usingcommercial quantitative immunoassay kit (Cusabio BiotechCo Newark NJ USA) Assay was conducted according tomanufacturerrsquos guidelines

211 Evaluation of Sex Steroids Ovarian steroidogenesisfunction after induction of ovarian cystogenesis was deter-mined by competitive radioimmunoassay using commercialRIA kits (Neogen USA) as described in our previous studies[18 19]

212 Statistical Analysis Analysis of variance was used whenseveral parameters of the two groups were compared Dif-ferences between control and PCO group were calculated byStudentrsquos 119905-test All analyseswere conducted using StatsDirect3097 A 119875 lt 005 was considered as statistically significantResults are presented as means plusmn SD

3 Results

31 Body and Ovaries Weight (g) The results showed that thehyperandrogenized rats gained significantly (119875 lt 0001)more body and ovarian weights in comparison to the controls(Table 1)

32 Sexual Cycle With respect to the sexual cycle all controlrats showed regular estrous cycles with an expected time of4-5 days between two consecutive cycles However all hyper-androgenized rats were completely acyclic and exhibitedconstant estrous

33 Morphological and Histopathological Findings Histolog-ical characteristics of the ovaries are shown in Tables 1 and 2All ovaries of control females had corpora lutea (CL) confirm-ing normal ovulation and contained follicles in various stagesof development including primary and secondary folliclesgraafian follicles and recently formed CL (Figures 1(a) and1(b)) In the hyperandrogenized females a clear absence ofboth large secondary and tertiary follicles as well as CL wasevident andmultiple follicular cysts were visible as fluid-filledsacs on the ovarian surface forming PCO (Tables 1 and 2Figure 1(c)) Furthermore the number of atretic preantraland antral follicles increased and ovarian theca-interstitialtissues are hyperplastic in hyperandrogenized ovary (Table 2and Figure 1(d)) In addition granulosa cells in the majorityof atretic antral follicles were found luteinized (Figure 1(e))and many of oocytes were malformed with the signs of zonapellucida (ZP) fragmentation in the atretic follicles (Figures1(e) and 1(f))

34 OS Biomarkers In ovarian tissues and serum LPO andROS were significantly higher in the PCO group than in thecontrols (Table 3) Moreover the ovarian and serum antiox-idants activities were decreased significantly (119875 lt 0001) inthe PCO rats compared to the controls

35 Inflammatory and Metabolic Biomarkers The hyperan-drogenized PCO rats showed a considerable increase (119875 lt0001) in serum TNF-120572 concentrations compared to thecontrols (682 plusmn 257 versus 419 plusmn 042 resp) The seruminsulin concentration was notably (119875 lt 0001) increased afterhyperandrogenization in PCO rats as compared with thecontrols (11 plusmn 014 versus 032 plusmn 0047 Table 3)

36 MMP-2 Activity The mean tissue levels of MMP-2 inthe ovaries are presented in Table 3 Statistically significantdifference (119875 lt 0001) was found between the MMP-2 valuesof control and hyperandrogenized rats (239 plusmn 6 versus 655 plusmn1557 resp)

37 Sex Steroid Concentrations As seen in Table 4 hyper-androgenization significantly increased serum testosteroneconcentrations compared with the controls (119875 lt 00018022 plusmn 1102 versus 2158 plusmn 223 resp) Meanwhile serumconcentrations of estradiol (119875 lt 0001 1242plusmn114 versus 60plusmn376) and progesterone (119875 lt 0001 1186plusmn136 versus 2934plusmn313) were significantly reduced in the hyperandrogenizedrats in comparison with the controls

4 Discussion

Aging and reproduction biology are two rapidly growingfields of modern biomedical researches Aging process is ofinterest not only to scientists but also to public health henceit has been the research question of many studies includingthose in the field of reproductive biology However the asso-ciation of aging with reproductive disorders has not been wellstudied and documented In the present studywe investigatedthe evidences of the ovarian aging characteristicswith PCOasone of the most prevalent female reproductive disorder

In this study we showed that treatment with letrozole-induced hyperandrogenismdue to blocking the conversion ofandrogens to estradiol which leads to higher serum testos-terone concentrations (hyperandrogenemia) Hyperandro-genemia was associated with ovarian cystogenesis and sig-nificantly increased LPO and total ROS (markers of OS) anddecreasedTAC (marker of antioxidant potential) in PCO ratsIn addition synthesis or release of inflammatory mediatorslike TNF-120572 and concentrations of insulin andMMP-2 activitywere significantly increased by hyperandrogenization Thesemolecular and biochemical alterations were consistent withhistological evidence of significant disruption in microscopiccharacters of folliculogenesis when compared to the con-trol group Histopathology revealed a large increase in thenumber of atretic and cystic follicles lack of CL formationanovulation and sexual acyclicity in hyperandrogenized ratswhich was due to aberrant folliculogenesis Increased ovarianweight seemed to be a result of increased number of fluid-filled cysts and large atretic follicles within ovary of PCOcases Almost all of these findings were interestingly found tobe common with the characteristics identified with (ovarian)aging as discussed Interestingly we observed that there was aclose association between identified symptoms and effectorsregarding ovarian aging in hyperandrogenized PCO rats so


Table 1 Comparative assessment of weight and histopathological changes of ovary in experimental and control groups

Control PCOBody weight (g) 2312 plusmn 111 2498 plusmn 081

lowastlowastlowast

Ovary weight (g) 0044 plusmn 0002 006 plusmn 0002lowastlowastlowast

Luteinization of follicular wall and granulosa cells mdash +++Vascularization of follicular wall mdash +++Degenerated and deformed oocyte mdash +++Pyknosis and chromatinization of granulosa cells mdash +++Disintegration and dispersion of granulosa cells mdash +++Hyperplasia of theca cells mdash +++Breakdown and fragmentation of ZP mdash ++lowastrepresents a significant difference between control and PCO groupsThe symbols represent statistical significance lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001Intensity of the histopathological changes (mdash) no change (+) slight change (++) marked change (+++) sever change

Table 2 Comparison of mean number of atretic and cystic primordial growing and graafian follicles and corpus luteum in PCO and controlgroups

Control PCOMean number of atretic primordial follicles 3581 plusmn 134 5227 plusmn 101

lowastlowastlowast

Mean number of atretic growing follicles 5245 plusmn 057 8118 plusmn 091lowastlowastlowast

Mean number of atretic graafian follicles 872 plusmn 033 2736 plusmn 038lowastlowastlowast

Mean number of cystic primordial follicles 0 0Mean number of cystic growing follicles 018 plusmn 012 772 plusmn 038

lowastlowastlowast

Mean number of cystic graafian follicles 082 plusmn 018 109 plusmn 056lowastlowastlowast

Mean number of corpus luteum (CL) 527 plusmn 019 036 plusmn 015lowastlowastlowast

lowastrepresents a significant difference between control and PCO groupThe symbols represent statistical significance lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

Table 3 Comparative assessment of OS and inflammation parameters in blood and ovary of PCO and control groups

Control PCOBlood LPO (120583molmL) 409 plusmn 032 1272 plusmn 027

lowastlowastlowast

Ovary LPO (120583molmL) 345 plusmn 028 1421 plusmn 056lowastlowastlowast

Blood ROS (nmolminmg protein) 078 plusmn 018 276 plusmn 032lowastlowastlowast

Ovary ROS (nmolminmg protein) 004 plusmn 000 014 plusmn 001lowastlowastlowast

Blood TAC (nmolL per mg protein) 20952 plusmn 466 11660 plusmn 421lowastlowastlowast

Ovary TAC (umg) 768 plusmn 049 291 plusmn 016lowastlowastlowast

Serum TNF-120572 (pgmg protein) 4190 plusmn 042 6820 plusmn 257lowastlowastlowast

MMP-2 (ngmg protein) 2390 plusmn 600 6550 plusmn 1557lowastlowastlowast

Insulin (ngmL) 032 plusmn 005 110 plusmn 014lowastlowastlowast


Table 4 Sex hormone levels in PCO and control groups

Control PCOTestosterone (ngdL) 2158 plusmn 223 8022 plusmn 1102

lowastlowastlowast

Progesterone (ngmL) 2934 plusmn 313 1186 plusmn 136lowastlowastlowast

Estradiol (pgmL) 6000 plusmn 376 1242 plusmn 114lowastlowastlowast



(a)

(b)

(c)

(d)

(e)

(F)

Control PCO

CL CL

CL

CL

CC

CC

C

C

C CCCCC

CC

C

C

PAFAPF

HTC

AAF

TZP

OS

ZPBPGC

LGC

LGC Malformedoocyte

Figure 1 (a) Lower magnification (40x) of ovarian section showing normal folliculogenesis as well as several CL from the control rat (b)Higher magnification (400x) of control ovary showing fresh CL (c) Section of ovary from PCO rats showingmultiple fluid-filled subcapsularcysts (40x) (d) Another section of ovary from PCO rats with two preantral and antral cystic follicles having hyperplastic theca cells alongwith thin granulosa cell layers Two preantral and antral degenerating atretic follicles are also seen in this section (100x) (e) The completelyluteinized granulosa cells are clear in an atretic follicle with a malformed oocyte (400x) (f) Higher magnification of a segmented oocyte in anatretic follicle with typical ZP thickness and also ZP breakdown and fragmentation (1000x) AAF atretic antral follicle APF atretic preantralfollicle PAF precocious antrum formation C cystic follicle CL corpora lutea DGC degenerated granulosa cells HTC hyperplasia of thecacells LGC luteinized granulosa cells MO malformed oocyte OS oocyte segmentation PAF precocious antrum formation TZP thickenzona pellucida ZPB zona pellucida breakdown

that all of these characteristics have been related to (ovarian)aging in several recent studies [3 4 6 7 16]

41 Low Estradiol Levels and High Concentrations of Andro-gen (Hyperandrogenemia) Leading to Accelerated FollicularAtresia Letrozole blocks aromatization of testosterone toestradiol leading to a significant reduction in serum estradiolconcentration and consequently accumulation of nonaroma-tizable androgens [17 18] Furthermore due to the developingof CL or decreased number of mature CL as a consequence ofanovulation decreased concentration of serum progesteronewas predictable in PCO cases These hormonal disturbancesare involved in constant estrous manifestation Estradiol isknown to play important roles in preventing OS so thatlower estradiol concentrations are associatedwith an increasein follicular OS [23] A meaningful depletion in estradiollevels causes apoptosis and oxidative DNA damage As wellelevated OS resulting from reduced estradiol concentrationswould predispose granulosa cells to apoptosis in preovulatoryfollicles [24] Therefore it is likely that the decreased levels

of estradiol at least in part stimulates progressive follicularatresia and predisposes the ovary to be senescent prematurely

42 High Concentrations of Insulin (Hyperinsulinemia) Lead-ing to Hyperandrogenemia and Accelerated Follicular AtresiaPCOS is associated with hyperinsulinemia Hyperinsuline-mia has been determined to stimulate the ovarian cystogene-sis causing an increase in the size of the cystic follicles and inthe size of the ovary [25] Insulin excess stimulates androgenproduction by theca cells and elevates serum free testosteronelevels thereby perpetuating ovarian hyperandrogenism [26]In the present study ovarian theca-interstitial cell growthwas consistent with high concentrations of testosterone andinsulin in comparison with controls Ovarian mesenchyma(theca-interstitial tissues) are a major source of androgensand normal ovarian function including folliculogenesis andsteroidogenesis which requires effective mechanisms regu-lating theca-interstitial growth and function However inPCOS ovarian theca-interstitial tissues are hyperplastic dueto increased cellular proliferation andor reduced apoptosiswhich is concomitant with androgens oversynthesis [27]


In this regard androgens might enhance apoptosis of gran-ulosa cells leading to increased rate of follicular atresia [28]

43 Increased TNF-120572 Activity Leading to Sustained Inflamma-tory Status Progressive Ovarian Cystogenesis and FollicularAtresia In the present study PCO rats had higher serumTNF-120572 concentrations than controls There is a positiverelationship between hyperandrogenism and serum TNF-120572 excessive concentration as hallmarks in patients withPCOS [11 12] TNF-120572 is considered as an initiator of theinflammatory cascade Furthermore higher TNF-120572 produc-tion is involved in OS [14 29] which arrests folliculardevelopment [30] and correlates with poor oocyte quality[31] A similar situation has been determined in female ratswhere the levels of TNF-120572 and LPO were elevated in oldovariectomized animals as an experimental model of aging[32]Therefore in hyperandrogenism condition the crosstalkbetween OS and proinflammatory cytokines particularlyTNF-120572 seem to play a pivotal role in the progression ofovarian cystogenesis follicular atresia and premature ovariandysfunction as was evident in the present study

With respect to aging and TNF-120572 age-dependent inc-rease in TNF-120572 was found in senescence-accelerated mice[33] It has been shown that aging is mechanistically associ-ated with the gene expression of pro-inflammatory cytokinessuch as TNF-120572 leading to inflammation and apoptosis [34]Moreover it has been well determined that TNF-120572 is able tomarkedly increase cellular senescence through stimulation ofprolonged inflammation [35]

44 Overproduction of Intraovarian Toxic Free Radicals alongwith a Depletion of TAC The role of ROS in the pathogenesisof PCOShas been the subject of numerous studies and emerg-ing evidences that show that both ROS and the activation ofinflammatory pathways play a central role in PCOS [5 11 12]In the present study we showed that hyperandrogenizationsignificantly increased LPO and total ROS (markers of OS)and decreased TAC (marker of antioxidant potential) in PCOrats Aging has been shown to be mechanistically associatedwith impaired mitochondrial function which is related totoxic free radicals overproduction and decreased antioxidantactivity [36]

45 The Alternation in Extracellular Matrix (ECM) One ofthe main findings reported in this study is that hyperandro-genization increased MMP-2 activation in rats with respectto controls Matrix metalloproteinases (MMPs) which are agroup of matrix-degrading enzymes are involved in thebreakdownof all kinds of ECMproteins and so play an impor-tant role in various physiological processes as well as patho-logical states of disease It has been exhibited that follicularrupture requires focal degradation of the apical ECM thatis controlled in part by ovarian MMPs The MMP-2 hasbeen proposed to play an important role in separation of thegranulosa cells from the theca cells [37] Increased level ofMMP-2 in our experiment was in agreement with a previousreport showing elevated MMP-2 levels in obese womenwith PCOS [38] The imbalances in circulating MMPs are

associatedwith excess circulating androgens in PCOSwomen[39] In this regard MMP-2 expression has been shown tobe stimulated by androgen via androgen receptor transactiva-tion [40] There is also a correlation between MMPs and thequality of the developing follicles [41] Moreover luteinizedgranulosa cells (as an atresia characteristic) from womenwith PCOS have been approved to exhibit greater MMP-2activity [41] In terms of aging the overexpression of severalmetalloproteinases in senescent cells has been reported [42]Therefore it is reasonable to speculate that MMP-2 may beassociated with inappropriate follicular atresia in PCOS caseswhich eventually leads ovary to be senescent faster than thenormal condition

46 Ovarian Aging-Like Phenotype in the Hyperandrogenism-Induced PCO Ovarian aging process is characterized by thedisappearance of the preovulatory follicle pool in the ovaryand ceases in the female fertility due to lack of CL formationanovulation and discontinued activity of sexual cycling [43]It has been proved that progressive follicular atresia byapoptosis at any stage could accelerate follicular depletionfrom the ovary and result in premature reproductive senes-cence [44] Furthermore hyperandrogenization as one ofthe common models of PCO seems to exhibit some aging-specific characteristics Supporting this subject postnatalandrogenization has been shown to induce accelerated agingof ovarian interstitial glands and transformation of largeantral into cystic follicles as common similarities betweenhyperandrogenized and aging rats [45] It is believed thatmenopause associated with various diseases is mediatedthrough depression of estradiol levels [46] Interestingly con-dition of chronic estradiol deficiency and higher androgenlevels (as seen in our results) would lead to many age-relatedchanges like obesity metabolic syndrome diabetes skeletalabnormalities and cardiovascular disturbances [47] whereall of these diseases and disorders can be observed in PCOSwomen [7] Considering the facts and results of this studyit would not be surprising to consider that the PCO(S) andovarian aging notably have similar symptoms effectors andcommonality in majority of their characteristics Althoughnot investigated in this study there are several findings whichsupport this idea For instance recently in mouse model ofaccelerated aging irregular estrous cycles and ovarian cystsas common signs of ovarian failure were identified [16]Another good example is advanced glycation end products(AGEs) Accumulation of AGEs is well known among manycauses of aging [48] AGEs contribute directly to proteindamage toxic stress and increasing the inflammatory reac-tions [49] Recent studies have shown a clear connectionbetween PCOS and increased AGEs in the body so that AGEsand its receptor were highly expressed in granulosa cellsof the PCOS ovary [50] The AGEs seem associated withanovulation and hyperandrogenism [51]

Altogether according to above discussion the first con-cept coming tomind is that during PCOS condition constantexposure of oocytes and other ovarian cells to OS andhormonal alternations predispose the ovarian follicles toprogressive rate of atresia leading to exhausted ovaries


with reduced number of intact follicles Therefore ovarianoocyte reserved will be washed up earlier in hyperandro-genism condition in accordance with premature onset ofacyclicity and constant estrous Moreover supraphysiologiclong-lasting androgenic microenvironment may eventuallyaffect process of follicular recruitment growth and ovulationleading to early ovarian senescence [15] via disruption ofintraovarian milieu This is a phenomena resembling prema-tureaccelerated ovarian aging According to recent studiesincluding our reports and based on current data we give theidea that hyperandrogenism triggers ovarian senescence inPCOS cases through the following mechanisms (1) pivotaldecrease in estradiol levels and high concentrations of andro-gen leading to accelerated follicular atresia (2) increasedTNF-120572 activity leading to OS and sustained inflammatorystatus (3) overproduction of intraovarian toxic free radicalsalong with a depletion of TAC

Although a close association with the symptoms andeffectors of hyperandrogenemia-induced PCO and (ovarian)agingwas observed in our study a number of important ques-tions may be raisedWhat is the mechanistically precise asso-ciation of PCOS with ovarian aging Is it logical to considerPCOS as one of the contributors of accelerated developmentof ovarian senescence in risky individuals And in general isPCOS a unique and sophisticated type of accelerated ovarianaging phenomenonThis is an ideahypothesis which will bewell scrutinized and discussed appropriately as a researchquestion in our upcoming project

5 Conclusion

To our knowledge this is the first report that provides evi-dence on phenomenon of aging in PCO Our results showedthat PCO(S) which is coupled with premature onset ofconstant estrous OS and proinflammatory condition maylead to premature transition to reproductive senescence viaendocrine disruption and develops as one of the features ofaccelerated aging at the level of the ovary Further studies arerequired to clarify precisemechanisms explaining similaritiesof hyperandrogenism-induced PCO and ovarian aging

Conflict of Interests

The authors have no conflict of interests to disclose

Authorsrsquo Contribution

The work presented here was carried out in collaborationbetween all authors Mohammad Amin Rezvanfar definedthe research theme designed methods and experiments anddrafted the paper Habib A Shojaei Saadi participated in theidea and editing the manuscript Maziar Gooshe and AmirHosein Abdolghaffari cocarried out experiments MaryamBaeeri carried out the laboratory experiments and analysis ofdata Mohammad Abdollahi gave the idea conceived thestudy and edited the manuscript

Acknowledgment

The authors acknowledge partial support from TUMS

References

[1] A C Gore D M Walker A M Zama A E Armenti andM Uzumcu ldquoEarly life exposure to endocrine-disruptingchemicals causes lifelong molecular reprogramming of thehypothalamus and premature reproductive agingrdquo MolecularEndocrinology vol 25 no 12 pp 2157ndash2168 2011

[2] M A Ottinger ldquoMechanisms of reproductive aging conservedmechanisms and environmental factorsrdquoAnnals of theNewYorkAcademy of Sciences vol 1204 pp 73ndash81 2010

[3] Q Li X Geng W Zheng J Tang B Xu and Q Shi ldquoCurrentunderstanding of ovarian agingrdquo Science China Life Sciencesvol 55 no 8 pp 659ndash669 2012

[4] A B Salmon A Richardson and V I Perez ldquoUpdate on theoxidative stress theory of aging does oxidative stress play a rolein aging or healthy agingrdquo Free Radical Biology and Medicinevol 48 no 5 pp 642ndash655 2010

[5] A Agarwal A Aponte-Mellado B J Premkumar A Shamanand S Gupta ldquoThe effects of oxidative stress on female repro-duction a reviewrdquo Reproductive Biology and Endocrinology vol10 no 49 pp 1477ndash7827 2012

[6] C Tatone F Amicarelli M C Carbone et al ldquoCellular andmolecular aspects of ovarian follicle ageingrdquoHuman Reproduc-tion Update vol 14 no 2 pp 131ndash142 2008

[7] C K Welt and E Carmina ldquoLifecycle of polycystic ovary syn-drome (PCOS) from in utero to menopauserdquo The Journal ofClinical Endocrinology amp Metabolism vol 98 no 12 pp 4629ndash4638 2013

[8] V De Leo A La Marca and F Petraglia ldquoInsulin-loweringagents in themanagement of polycystic ovary syndromerdquoEndo-crine Reviews vol 24 no 5 pp 633ndash667 2003

[9] L J Moran M L Misso R A Wild and R J NormanldquoImpaired glucose tolerance type 2 diabetes andmetabolic syn-drome in polycystic ovary syndrome a systematic review andmeta-analysisrdquo Human Reproduction Update vol 16 no 4 pp347ndash363 2010

[10] W A March V M Moore K J Willson D I W Phillips R JNorman and M J Davies ldquoThe prevalence of polycystic ovarysyndrome in a community sample assessed under contrastingdiagnostic criteriardquo Human Reproduction vol 25 no 2 pp544ndash551 2010

[11] M Murri M Luque-Ramirez M Insenser M Ojeda-Ojedaand H F Escobar-Morreale ldquoCirculating markers of oxidativestress and polycystic ovary syndrome (PCOS) a systematicreview andmeta-analysisrdquoHumanReproductionUpdate vol 19no 3 pp 268ndash288 2013

[12] H F Escobar-Morreale M Luque-Ramırez and F GonzalezldquoCirculating inflammatory markers in polycystic ovary syn-drome a systematic review and metaanalysisrdquo Fertility andSterility vol 95 no 3 pp 1048ndashe2 2011

[13] M O Goodarzi D A Dumesic G Chazenbalk and R AzzizldquoPolycystic ovary syndrome etiology pathogenesis and diag-nosisrdquo Nature Reviews Endocrinology vol 7 no 4 pp 219ndash2312011

[14] A B Motta ldquoDehydroepiandrosterone to induce murine mod-els for the study of polycystic ovary syndromerdquo Journal ofSteroid Biochemistry andMolecular Biology vol 119 no 3ndash5 pp105ndash111 2010


[15] H Shirwalkar D N Modi and A Maitra ldquoExposure of adultrats to estradiol valerate induces ovarian cyst with early senes-cence of folliclesrdquo Molecular and Cellular Endocrinology vol272 no 1-2 pp 22ndash37 2007

[16] J H Park and T S Choi ldquoPolycystic ovary syndrome (PCOS)-like phenotypes in the d-galactose-induced aging mousemodelrdquo Biochemical and Biophysical Research Communicationsvol 427 no 4 pp 701ndash704 2012

[17] M A Rezvanfar M A Rezvanfar A Ahmadi H A ShojaeiSaadi M Baeeri andM Abdollahi ldquoMechanistic links betweenoxidativenitrosative stress and tumor necrosis factor alpha inletrozole-induced murine polycystic ovary Biochemical andpathological evidences for beneficial effect of pioglitazonerdquoHuman amp Experimental Toxicology vol 31 no 9 pp 887ndash8972011

[18] M A Rezvanfar M A Rezvanfar A Ahmadi H A Shojaei-Saadi M Baeeri and M Abdollahi ldquoMolecular mechanisms ofa novel selenium-based complementary medicine which con-fers protection against hyperandrogenism-induced polycysticovaryrdquoTheriogenology vol 78 no 3 pp 620ndash631 2012

[19] M A Rezvanfar M A Rezvanfar A R Shahverdi et al ldquoPro-tection of cisplatin-induced spermatotoxicity DNA damageand chromatin abnormality by selenium nano-particlesrdquo Tox-icology and Applied Pharmacology vol 266 no 3 pp 356ndash3652013

[20] M Pakzad S Fouladdel A Nili-Ahmadabadi et al ldquoSublethalexposures of diazinon alters glucose homostasis in Wistar ratsbiochemical and molecular evidences of oxidative stress inadipose tissuesrdquo Pesticide Biochemistry and Physiology vol 105pp 57ndash5[ 2012

[21] P Hasani N Yasa S Vosough-Ghanbari A MohammadiradGDehghan andMAbdollahi ldquoIn vivo antioxidant potential ofTeucrium polium as compared to 120572-tocopherolrdquoActa Pharma-ceutica vol 57 no 1 pp 123ndash129 2007

[22] A Hosseini-Tabatabaei H Esmaily R Rahimian et al ldquoBenefitof nicorandil using an immunologic murine model of experi-mental colitisrdquo Central European Journal of Biology vol 4 no 1pp 74ndash85 2009

[23] T Seino H Saito T Kaneko T Takahashi S Kawachiya andH Kurachi ldquoEight-hydroxy-21015840-deoxyguanosine in granulosacells is correlated with the quality of oocytes and embryos inan in vitro fertilization-embryo transfer programrdquo Fertility andSterility vol 77 no 6 pp 1184ndash1190 2002

[24] S A Lund J Murdoch E A Van Kirk and W J MurdochldquoMitogenic and antioxidant mechanisms of estradiol actionin preovulatory ovine follicles relevance to luteal functionrdquoBiology of Reproduction vol 61 no 2 pp 388ndash392 1999

[25] L Poretsky J Clemons and K Bogovich ldquoHyperinsulinemiaand human chorionic gonadotropin synergistically promote thegrowth of ovarian follicular cysts in ratsrdquo Metabolism vol 41no 8 pp 903ndash910 1992

[26] D A Dumesic and J S Richards ldquoOntogeny of the ovary inpolycystic ovary syndromerdquo Fertility Sterility vol 100 no 1 pp23ndash38 2013

[27] P EHughesdon ldquoMorphology andmorphogenesis of the Stein-Leventhal ovary and of so-called rsquohyperthecosisrsquordquo Obstetricaland Gynecological Survey vol 37 no 2 pp 59ndash77 1982

[28] H Billig I Furuta and A J W Hsueh ldquoEstrogens inhibitand androgens enhance ovarian granulosa cell apoptosisrdquoEndocrinology vol 133 no 5 pp 2204ndash2212 1993

[29] D Belgorosky VAnalıa SanderM PDi Yorio AG Faletti andA BMotta ldquoHyperandrogenism alters intraovarian parameters

during early folliculogenesis inmicerdquoReproductive BioMedicineOnline vol 20 no 6 pp 797ndash807 2010

[30] S Jonard and D Dewailly ldquoThe follicular excess in polycysticovaries due to intra-ovarian hyperandrogenism may be themain culprit for the follicular arrestrdquo Human ReproductionUpdate vol 10 no 2 pp 107ndash117 2004

[31] J Y Lee C K Baw S Gupta N Aziz and A Agarwal ldquoRole ofoxidative stress in polycystic ovary syndromerdquoCurrentWomenrsquosHealth Reviews vol 6 pp 96ndash107 2010

[32] R A Kireev A C F Tresguerres C Garcia et al ldquoHormonalregulation of pro-inflammatory and lipid peroxidation pro-cesses in liver of old ovariectomizedfemale ratsrdquoBiogerontologyvol 11 no 2 pp 229ndash243 2010

[33] G F Oxenkrug ldquoMetabolic syndrome age-associated neu-roendocrine disorders and dysregulation of tryptophanmdashkynurenine metabolismrdquo Annals of the New York Academy ofSciences vol 1199 pp 1ndash14 2010

[34] D D Haines B Juhasz and A Tosaki ldquoManagement of multi-cellular senescence and oxidative stressrdquo Journal of Cellular andMolecular Medicine pp 1ndash22 2013

[35] Y Zhang B-S Herbert G Rajashekhar et al ldquoPrematuresenescence of highly proliferative endothelial progenitor cellsis induced by tumor necrosis factor-120572 via the p38 mitogen-activated protein kinase pathwayrdquo FASEB Journal vol 23 no5 pp 1358ndash1365 2009

[36] HCui Y Kong andH Zhang ldquoOxidative stressmitochondrialdysfunction and agingrdquo Journal of Signal Transduction vol2012 Article ID 646354 13 pages 2012

[37] K Rosewell L Al-Alem F Li B Kelty andT E Curry Jr ldquoIden-tification of hepsin and protein disulfide isomerase A3 as targetsof gelatinolytic action in rat ovarian granulosa cells during theperiovulatory periodrdquo Biology of Reproduction vol 85 no 4 pp858ndash866 2011

[38] K C Lewandowski J Komorowski C J OrsquoCallaghan et alldquoIncreased circulating levels of matrix metalloproteinase-2 and-9 in women with the polycystic ovary syndromerdquo Journal ofClinical Endocrinology and Metabolism vol 91 no 3 pp 1173ndash1177 2006

[39] V A Gomes C S Vieira A L Jacob-Ferreira et al ldquoImbal-anced circulating matrix metalloproteinases in polycystic ovarysyndromerdquoMolecular and Cellular Biochemistry vol 353 no 1-2 pp 251ndash257 2011

[40] X Liao J BThrasher J Pelling J Holzbeierlein Q-X A Sangand B Li ldquoAndrogen stimulates matrix metalloproteinase-2expression in human prostate cancerrdquo Endocrinology vol 144no 5 pp 1656ndash1663 2003

[41] S Goldman and E Shalev ldquoMMPS and TIMPS in ovarian phys-iology and pathophysiologyrdquo Frontiers in Bioscience vol 9 pp2474ndash2483 2004

[42] V J Cristofalo C Volker M K Francis and M Tresini ldquoAge-dependent modifications of gene expression in human fibrob-lastsrdquo Critical Reviews in Eukaryotic Gene Expression vol 8 no1 pp 43ndash80 1998

[43] E Acuna R Fornes D Fernandois et al ldquoIncreases in nore-pinephrine release and ovarian cyst formation during ageing inthe ratrdquo Reproductive Biology and Endocrinology vol 7 article64 2009

[44] R K Gupta K P Miller J K Babus and J A Flaws ldquoMethoxy-chlor inhibits growth and induces atresia of antral folliclesthrough an oxidative stress pathwayrdquo Toxicological Sciences vol93 no 2 pp 382ndash389 2006


[45] A Bukovskya M E Ayala R Dominguez et al ldquoPostnatalandrogenization induces premature aging of rat ovariesrdquo Ster-oids vol 65 no 4 pp 190ndash205 2000

[46] A R Genazzani N Pluchino S Luisi and M Luisi ldquoEstrogencognition and female ageingrdquoHumanReproductionUpdate vol13 no 2 pp 175ndash187 2007

[47] N I Parikh S Cnattingius M A Mittleman J F Ludvigssonand E Ingelsson ldquoSubfertility and risk of later life maternalcardiovascular diseaserdquoHuman Reproduction vol 27 no 2 pp568ndash575 2012

[48] R D Semba E J Nicklett and L Ferrucci ldquoDoes accumulationof advanced glycation end products contribute to the agingphenotyperdquo The Journals of Gerontology Series A vol 65 no9 pp 963ndash975 2010

[49] M Szafarowska and M Jerzak ldquoOvarian aging and infertilityrdquoGinekologia Polska vol 84 no 4 pp 298ndash304 2013

[50] E Diamanti-Kandarakis C Piperi E Patsouris et al ldquoImmun-ohistochemical localization of advanced glycation end-pro-ducts (AGEs) and their receptor (RAGE) in polycystic and nor-mal ovariesrdquoHistochemistry and Cell Biology vol 127 no 6 pp581ndash589 2007

[51] E Diamanti-Kandarakis I Katsikis C Piperi et al ldquoIncreasedserum advanced glycation end-products is a distinct finding inlean women with polycystic ovary syndrome (PCOS)rdquo ClinicalEndocrinology vol 69 no 4 pp 634ndash641 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Several mechanisms seem involved in aging which oxida-tive stress (OS) is considered as one of the most importantones [4] OS occurs due to excessive formation of oxygen-derived andor nitrogen-derived toxic products in the pres-ence of minimal antioxidant activity [5]

At moderate concentrations reactive oxygen species(ROS) regulate physiologic functions in female reproductionsuch as folliculogenesis oocyte maturation steroidogenesiscorpus luteal function and luteolysis [5] However increasedOS during ovarian aging may contribute to follicular atresiaand diminishing quantity and quality of oocytes [6] Substan-tial theories explain that a long lasting status of overproducedtoxic free radicals insufficient antioxidants and exposure toendocrine disrupting chemicalsmay be involved in reproduc-tive disorders which eventually confer acceleration of gonadssenescence and premature reproductive aging

Polycystic ovarian syndrome (PCOS) as an inflamma-tory condition is the leading cause of anovulatory infertili-ty in reproductive-aged women It is characterized byincreasing androgen secretion menstrual irregularity oligo-ovulationanovulation polycystic ovaries (PCO) infertilityand pregnancy complications [7] An ovarian cyst is devel-oped as a result of hormonal imbalance and has an atreticfluid-filled follicular structure with thin granulosa cell walls[8] Not only PCOSpatients display reproductive features butalso they develop several metabolic risks of type 2 diabetesand cardiovascular diseases including abdominal obesityinsulin resistance (IR) hyperinsulinemia glucose intoler-ance hypertension and metabolic syndrome [9] Thereare several different diagnostic criteria for PCOS Henceregardless of ethnicity PCOS has different prevalence inwomen at their functional reproductive period According toRotterdam criteria it is estimated that the average prevalenceof PCOS is approximately 18 that is too high [10]

Because of its complex pathogenesis and unrecognizedetiology no preventive measure has been implemented forPCOS to dateThe roles of OS and chronic low-grade inflam-mation in the pathogenesis of PCOS and the potential ben-efits of antioxidants have been the subjects of recent studies[11 12] High levels of androgen have been proposed as aninitial step in themajority of PCOS cases Hyperandrogenismimpairs maturation of developing follicles in ovaries andconsequently leads to developing cystogenesis [13] Extensiveanimal experimentations have indicated that prepubertal orpubertal exposure of low doses of androgens via aromatiza-tion to estrogen results in long-term reproductive consequen-ces including constant estrous cycles hyposteroidogenesisanovulation and development of cystic follicles at adulthood[14] Moreover exposure to exogenous estrogen in adulthoodhas been determined by having deleterious effects on theovarian physiology and endocrinology which may ultimatelylead to cystogenesis loss of follicle pool and early senescence[15] There are some evidences implying that there might bean association between aging and PCO For instance it hasbeen recently shown that accelerated aging inmouse inducedby D-galactose was concurrent with ovarian cystogenesis[16] However the causal link between hyperandrogenismand accelerated ovarian aging is not definitely recognizedIt is therefore essential to perceive what hormonal cellular

molecular metabolic environmental andor genetic factorscontribute to this condition and how they affect ovarian folli-cle development We have recently documented the poten-tial role of oxidativenitrosative stress and inflammatoryresponses in the pathogenesis of hyperandrogenism-inducedPCO in rat [17 18] However negligible information exists onthe hyperandrogenization effects on ovarian aging and themolecular basis of disturbed folliculogenesis and cystogen-esis

Overall it should be of considerable interest to investigatepossible development of ovarian aging in hyperandrogenicPCO rats Therefore the main objective of this study wasto investigate the association of ovarian aging with hyperan-drogenism-induced PCO through cellular molecular histo-pathological biochemical and endocrinal evaluations

2 Materials and Methods

21 Reagents Unless otherwise stated all chemicals werepurchased from Sigma-Aldrich (USA) Krebs-ringer-bicar-bonate (KRB) and ethyl acetate from Fluka (USA) rat TNF-120572 ELISA kit from Bender Med System (Austria) rat MMP-2 ELISA kitfrom Cusabio Biotech Co Ltd (China) steroidhormone radioimmunoassay kits from Neogen (USA) andletrozole from SOHA Pharmaceutical Co (Iran) were usedin this study

22 Rats Adult female albino Wistar rats (180ndash200 g) withnormal estrous cycle were used in the study Animals wererandomly divided into two groups containing 10 rats eachAll rats had ad libitum access to pelleted food and tap waterand housed under controlled temperature (22ndash25∘C) with arelative humidity of 40ndash55 and 12 h lights and dark cycleOnly females with at least three consecutive 4-5 d regularestrous cycles were used in the experiment Throughout theentire treatment animals were weighed and a vaginal smearwas done daily (to determine the stage of the reproductivecycle) up to the day of autopsy All rats received care inaccordance with the national health guidelines and the studyprotocol was approved by Tehran University of MedicalSciences (TUMS) review board

23 Sexual Cycle To study whether treatments altered theestrous cycle smears were obtained daily by vaginal washingand evaluated microscopically during the treatment periodAs determined in previous studies [17 18] the observationof cornified cells in the smears during a minimum of 10consecutive days was defined as persistent estrous indicatinganovulation and development of follicular cysts At thebeginning of the experiments all rats had regular cycles

24 Treatments The Control group of ten rats received onlyvehicle (09NaCl solution) orally once dailyThe treatmentgroup of ten rats was gavaged with letrozole once daily at aconcentration of 1mgkg orally dissolved in 09NaCl Effec-tive dose of letrozole (SOHA Pharmaceutical Co TehranIran) were selected upon previous experiments The treat-ment period was 21 days [17 18]







2 20mM


3 17mM ADP










3 Results








4 Discussion






lowastlowastlowast





lowastlowastlowast




lowastlowastlowast






lowastlowastlowast












lowastlowastlowast





(a)

(b)

(c)

(d)

(e)

(F)

Control PCO

CL CL

CL

CL

CC

CC

C

C

C CCCCC

CC

C

C

PAFAPF

HTC

AAF

TZP

OS

ZPBPGC

LGC

LGC Malformedoocyte


















5 Conclusion






Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2 20mM


3 17mM ADP










3 Results








4 Discussion






lowastlowastlowast





lowastlowastlowast




lowastlowastlowast






lowastlowastlowast












lowastlowastlowast





(a)

(b)

(c)

(d)

(e)

(F)

Control PCO

CL CL

CL

CL

CC

CC

C

C

C CCCCC

CC

C

C

PAFAPF

HTC

AAF

TZP

OS

ZPBPGC

LGC

LGC Malformedoocyte


















5 Conclusion






Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results








4 Discussion






lowastlowastlowast





lowastlowastlowast




lowastlowastlowast






lowastlowastlowast












lowastlowastlowast





(a)

(b)

(c)

(d)

(e)

(F)

Control PCO

CL CL

CL

CL

CC

CC

C

C

C CCCCC

CC

C

C

PAFAPF

HTC

AAF

TZP

OS

ZPBPGC

LGC

LGC Malformedoocyte


















5 Conclusion






Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















lowastlowastlowast





lowastlowastlowast




lowastlowastlowast






lowastlowastlowast












lowastlowastlowast





(a)

(b)

(c)

(d)

(e)

(F)

Control PCO

CL CL

CL

CL

CC

CC

C

C

C CCCCC

CC

C

C

PAFAPF

HTC

AAF

TZP

OS

ZPBPGC

LGC

LGC Malformedoocyte


















5 Conclusion






Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(c)

(d)

(e)

(F)

Control PCO

CL CL

CL

CL

CC

CC

C

C

C CCCCC

CC

C

C

PAFAPF

HTC

AAF

TZP

OS

ZPBPGC

LGC

LGC Malformedoocyte


















5 Conclusion






Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























5 Conclusion






Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article ovarian aging-like phenotype in the...

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