Chemerin Suppresses Ovarian Follicular Development and Its Potential Involvement in Follicular Arrest in Rats Treated Chronically With Dihydrotestosterone

Chemerin Suppresses Ovarian Follicular Developmentand Its Potential Involvement in Follicular Arrest inRats Treated Chronically with Dihydrotestosterone

Ji Young Kim, Kai Xue, Mingju Cao, Qi Wang, Jia-yin Liu, Arthur Leader,Jae Yong Han, and Benjamin K. Tsang

Departments of Obstetrics and Gynecology and Cellular and Molecular Medicine (J.Y.K., M.C., Q.W.,A.L., B.K.T.), University of Ottawa, and Chronic Disease Program (J.Y.K., K.X., M.C., Q.W., A.L., B.K.T.),Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1H 8L6; WCU Biomodulation Major(J.Y.K., J.Y.H., B.K.T.), Department of Agricultural Biotechnology, College of Agriculture and LifeSciences, Seoul National University, Seoul 151-742, Republic of Korea; State Key Laboratory ofReproductive Medicine (K.X., J.-y.L.), Clinical Reproductive Medicine Centre, Nanjing Medical University,Nanjing, Peoples’ Republic of China; and Ottawa Fertility Centre (A.L.), Ottawa, Ontario, Canada, K2C3V4

In the present study, we have investigated the cellular mechanisms of androgen-induced antralfollicular growth arrest and the possible involvement of chemerin and its receptor chemokine-likereceptor 1 (CMKLR1) in this process, using a chronically androgenized rat model. We hypothesizethat hyperandrogenism induces antral follicle growth arrest via the action of chemerin and ovarianstructural changes, resulting from granulosa cell and oocyte apoptosis and theca cell survival.Dihydrotestosterone (DHT) treatment resulted in increased expression of chemerin and CMKLR1in antral follicles, absence of corpus luteum, and increased atypical follicles. Addition of chemerinto follicle cultures induced granulosa cell apoptosis and suppressed basal, FSH- and growth dif-ferentiation factor-9-stimulated follicular growth. DHT down-regulated aromatase expression andincreased active caspase-3 content and DNA fragmentation in granulosa cells in vivo. These changeswere accompanied by higher phosphatase and tensin homolog and lower phospho-Akt (Ser473)content in antral follicles and higher calpain expression and down-regulation of cytoskeletal pro-teins in atypical follicles, which were constituted predominantly of theca cells. DHT also activatedgranulosa cell caspase-3, decreased X-linked inhibitor of apoptosis protein, poly(ADP-ribose) poly-merase, and phospho-Akt contents and induced apoptosis in vitro, responses readily attenuatedby forced X-linked inhibitor of apoptosis protein expression. These findings are consistent with ourhypothesis that antral follicular growth arrest in DHT-treated rats results from increased chemerinexpression and action, as well as changes in follicular cell fate and structure, which are a conse-quence of dysregulated interactions of pro-survival and pro-apoptotic modulators in a cell-specificmanner. Our observations suggest that this chronically androgenized rat model may be useful forstudies on the long-term effects of androgens on folliculogenesis and may have implications forthe female reproductive disorders associated with hyperandrogenism.

The mammalian ovary is a complex and highly orga-nized structure, and the follicle is its functional unit,

consisting of an oocyte surrounded by granulosa and thecacells (1). Follicular development is tightly regulated by

gonadotropins, cytokines, and growth factors via crosstalk between granulosa cells, theca cells, and the oocyte.Follicular cell interaction is critical for folliculogenesis andsteroidogenesis (2-4). Thecal androgens stimulate granu-

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2013 by The Endocrine SocietyReceived December 28, 2012. Accepted May 1, 2013.

Abbreviations: CMKLR1, chemokine-like receptor 1; CTL, control; DAPI, 4�,6-diamidino-2-phenylindole; DHT, dihydrotestosterone; GDF9, growth differentiation factor-9; IF, im-munofluoresence; IHC, immunohistochemistry; PARP, poly(ADP-ribose) polymerase;PCOS, polycystic ovarian syndrome; PI3K, phosphoinositide 3-kinase; PTEN, phosphataseand tensin homolog; RT, room temperature; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick end labeling; XIAP, X-linked inhibitor of apoptosis protein.

R E P R O D U C T I O N - D E V E L O P M E N T

doi: 10.1210/en.2013-1001 Endocrinology endo.endojournals.org 1

Endocrinology. First published ahead of print May 21, 2013 as doi:10.1210/en.2013-1001

Copyright (C) 2013 by The Endocrine Society

losa and theca cell proliferation and promote preantral/early antral follicle growth in the primate ovary (5), buthigh concentrations of these steroids can disturb late-stagefollicular development through induction of granulosacell apoptosis and thecal hypertrophy, symptoms oftenassociated with the complex ovarian dysregulation pres-ent in hyperandrogenic anovulation (6, 7). Although hy-perandrogenism is known to be associated with folliculargrowth arrest at the antral stage, the cellular mechanismsinvolved are not completely understood.

Chemerin, a novel adipokine associated with obesityand polycystic ovary syndrome (PCOS) (8, 9), is known toact through its G protein-coupled receptor chemokine-likereceptor 1 (CMKLR1). It is synthesized as an 18-kDaprochemerin protein and undergoes serine protease cleav-age to form an active 16-kDa protein (10, 11). Our recentstudies have shown that ovarian and circulating chemerinlevels are elevated in a chronically androgenized rat modeland that chemerin suppresses FSH-induced steroidogen-esis (12). However, whether and how chemerin is involvedin antral follicular growth arrest has not been reported.

The objectives of the present study were to better un-derstand the cellular mechanisms of androgen-inducedantral follicular growth arrest and the possible involve-ment of chemerin and its receptor CMKLR1 in this pro-cess, using a chronically androgenized rat model. Ourstudies have demonstrated that chronic androgen admin-istration increases chemerin and CMKLR1 expression,which is associated with suppressed antral follicle devel-opment. The latter response is characterized by dysregu-lated interactions of survival and proapoptotic modula-tors in a cell-specific manner, marked changes in folliclestructure, apoptotic deletion of granulosa cells andoocytes, and the survival and retention of theca cells.These findings support the notion that our rat model is auseful tool, not only for studies on the long-term effects ofandrogens on folliculogenesis, but also has implicationsfor the female reproductive disorders withhyperandrogenism.

Materials and Methods

Reagents and antibodiesDimethyl sulfoxide, PBS tablets, phenylmethyl-sulfonyl flu-

oride, hematoxylin solution, eosin Y solution, paraformalde-hyde, Hoechst-33258, Triton X-100, Tween 20, and phospha-tase inhibitor cocktail were obtained from Sigma-Aldrich(Oakville, Ontario, Canada). Fetal bovine serum, nonessentialamino acids, penicillin, streptomycin, and fungizone were ob-tained from Life Technologies (Gaithersburg, Maryland). Anti-calpain, antivimentin, antiphosphatase and tensin homolog(PTEN), antichemerin, anti-CMKLR1, antigrowth differentia-

tion factor-9 (GDF9), blocking peptides for chemerin, CMKLR1and GDF9, and normal rabbit IgG and normal mouse IgG an-tibodies were from Santa Cruz Biotechnology (Santa Cruz, Cal-ifornia). Anticleaved caspase-3, antifodrin, antipoly (ADP-ri-bose) polymerase (PARP), anti-Akt and anti-phospho-Akt(Ser473) were from Cell Signaling Technology, Inc. (Beverly,Massachusetts). Z-val-ala-asp (OMe)-FMK inhibitor (Z-VAD-FMK) for pan-caspase inhibition was purchased from TocrisBioscience (Ellisville, Missouri). Antirabbit and mouse IgG con-jugated with horseradish peroxidase were purchased from Bio-Rad Laboratories (Mississauga, Ontario, Canada). Fluoresceinisothiocyanate-conjugated secondary antibodies, Alexa Fluor-488 goat antimouse and antirabbit IgG and Alexa Fluor-594goat antimouse and anti-rabbit IgG were from Invitrogen (Bur-lington, Ontario, Canada).

Animals and dihydrotestosterone (DHT)-treated ratmodel

Female Sprague Dawley rats were obtained from CharlesRiver Canada (Montreal, Quebec, Canada) and maintained un-der standard conditions. All animal procedures were carried outin accordance with the guidelines of the Canadian Council onAnimal Care and approved by the Ottawa Hospital ResearchInstitute Animal Care Committee.

Preparation of DHT-treated rats was performed as describedby Mannerås (13) with some modifications. Briefly, rats at 21days of age were randomly divided into 2 experimental groups(control [CTL, n � 10], DHT [n � 12]) and implanted sc with90-day continuous-release SILASTIC capsules (Dow CorningCorp., Midland, Michigan) containing 7.5 mg DHT (daily dose,83 �g; empty SILASTIC capsule as CTL). CTLs received iden-tical pellets lacking the steroid. Rats were monitored twice dailyin the first 3 days and once daily thereafter. Animals wereweighed weekly to monitor weight gain and euthanized at 12weeks after implantation. Ovaries were collected for analysis.

Ovarian morphology and follicle typesOvarian sections from individual animals (10 in CTL group

and 12 in DHT group) were used for the morphologic analysisand follicle-counting experiments. Whole ovary samples werefixed in 10% neutral-buffered formalin, paraffin embedded, andthen serially sectioned at 4 �m, before mounting on positivelycharged glass slides. Technical services for hematoxylin-eosinand periodic acid-Schiff (PAS) staining were provided by theDepartment of Pathology and Laboratory Medicine, Universityof Ottawa, Ontario, Canada. For measurements and photo-graphs, the slides were scanned with a Scan-Scope (Aperio Tech-nologies, Vista, California) and analyzed with ImageScope vir-tual microscopy software (Aperio Technologies). The growingfollicles in every sixth ovary sections (90-135 slides per ovarywere examined per experimental group, depending on ovariansize) were scored according to the following categories: primary,preantral, antral and preovulatory stages, and corpus luteum.Follicles in which the oocyte nucleus was present (the largestcross-section) were scored. Most atypical follicles contained nooocyte, as determined by serial sections.

Ovarian follicle isolation and cultureLarge preantral and early antral follicles (diameter, 150-180

�m) from 14- to 15 day-old rats were isolated in Leibowitz L-15

2 Young Kim et al Chemerin Suppresses Ovarian Follicular Growth Endocrinology

medium with BSA (0.1%, wt/vol), using 28.5-gauge needles(Becton Dickinson and Co., Franklin Lakes, New Jersey) undera microscope. Follicles with intact basement membranes andtheca layers were individually cultured in a 96-well plate(Sarstedt, Newton, North Carolina) in 100 �L of �-MEM sup-plemented with HEPES (10 mM), BSA (0.1%, wt/vol), bovineinsulin (5 �g/mL), transferrin (2 �g/mL), ascorbic acid (25 �g/mL), sodium selenite anhydrous (2 ng/mL), L-glutamine (3 mM),sodium pyruvate (100 �g/mL), streptomycin (100 �g/mL), andpenicillin (100 U/mL) (14). Rats at 14-15 days of age, but notolder, were selected for these experiments because follicles iso-lated exhibit minimal granulosa cell apoptosis and atresia (15).Follicular diameter was measured daily as the average distancebetween the outer edges of the basement membrane in 2 perpen-dicular planes and results were expressed as the change in fol-licular volume. Follicular volume was calculated according to theformula for the volume of a sphere: volume � 4�r3/3, where r isradius. The percent change of follicular volume on day n of cul-ture is defined as the volume difference between day n and day0 (the day of follicle isolation) expressed as a percentage of thevolume at day 0. The culture medium was changed every otherday. The expression of X-linked inhibitor of apoptosis protein(XIAP) and GDF9 in cultured follicles was examined by IF andimmunohistochemistry (IHC), respectively, as previously de-scribed (16, 17).

In situ localization of apoptosis (terminaldeoxynucleotide transferase-mediated dUTP nickend labeling [TUNEL]) and immunolocalization ofactive-caspase-3

In situ TUNEL was carried out on ovarian sections using aTUNEL kit (Roche, Laval, Ontario, Canada) in accordance withthe manufacturer’s instructions. After TUNEL staining, tissuesections were incubated with anti-cleaved-caspase-3 (1:100)overnight at 4°C. For secondary antibody reactions, the sectionswere incubated with a fluorescence-conjugated secondary anti-body (Alexa Fluor-488 goat antirabbit IgG) at room temperature(RT) for 2 hours, and then incubated with ProLong gold antifadereagent and 4�,6-diamino-2-phenylindole (DAPI, blue, nuclearstain).

IHC and immunofluoresence (IF) microscopyOvarian sections from at least 5 CTL or 5 DHT-treated rats

were used to investigate the expression of target proteins by IHCor IF. Tissues were fixed with 4% paraformaldehyde, washedwith PBS, and incubated with 0.2% Triton X-100 before incu-bation with the appropriate primary antibody in 3% BSA at RT.Sections were incubated with fluorescence-conjugated or horse-radish peroxidase-conjugated secondary antibody (1 h, roomtemperature) and mounted on slides with ProLong gold antifadereagent with DAPI (nuclear stain, blue) and 3,3�-diaminobenzi-dine, respectively. Images were captured with an Olympus BX41fluorescence microscope equipped with an Optronics Magnafiredigital camera and a Prior Proscan motorized driven stage(Olympus, Melville, New York).

Rat granulosa cell isolation and cultureOvaries from immature female Sprague Dawley rats (24-25 d

of age, 10 pups) were collected in M199 medium supplementedwith HEPES (10 mM, pH 7.4), streptomycin-penicillin (100

U/mL), and Fungizone (0.625 �g/mL). Granulosa cells were har-vested by follicle puncture as previously described (12), pooled,washed, and centrifuged (900 � g, 10 min). Cells were plated for48 hours in M199 medium with fetal bovine serum 10% undera humidified atmosphere with 5% CO2. Media were then re-placed with serum-free M199 (Invitrogen, Burlington, Ontario,Canada) supplemented as above, and 1 million granulosa cellswere treated with various concentrations of DHT (0-50 �M) for48 hours or 10 �M DHT for various culture durations (0-96 h).Floating cells that were undergoing apoptosis in this model (15)were removed during medium change before the start of eachexperiment.

Adenoviral X-linked inhibitor of apoptosis protein(XIAP) infection in primary granulosa cell culturesystem

One million granulosa cells were plated in 6-well plates for 48hours and infected with adenoviral sense full-length XIAP cDNAor LacZ (as a CTL adenoviral vector) at 40 MOI, as previouslydescribed (18). DHT was added to culture medium 24 hours afterviral infection, and granulosa cells were cultured for an addi-tional 48 hours. XIAP expression was confirmed by Western blotanalysis.

Western blot analysisWhole-cell lysates were prepared by incubating cell pellets for

30 minutes at 0°C in lysis buffer (30 mM NaCl, 0.5% TritonX-100, 50 mM Tris-HCl [pH 7.4] containing a cocktail of phos-phatase inhibitors [Sigma-Aldrich] and protease inhibitors[Roche, Laval, Canada]). After the insoluble fractions were re-moved by centrifugation (18 000 � g, 4°C, 30 min), supernatantwas collected and protein concentration was determined with aprotein assay kit (Bio-Rad Laboratories, Inc. Mississauga, On-tario, Canada). Equal quantities of protein (30 �g) were sub-jected to SDS-PAGE and electrotransferred onto a nitrocellulosemembrane. The membranes were incubated (1 h, RT) in Tris-buffered saline containing 0.05% Tween 20 (TBS-T [pH 7.4])and nonfat dry milk (5%), and then overnight at 4°C, with di-luted primary antibody (anti-Akt [1:1000], phospho-Akt[Ser473, 1:1000], PTEN [1:2000], �-actin [1:10 000], XIAP [1:2000]) or PARP [1:5000]), followed by horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (1:2000-1:10,000; 1 h, RT). Signal intensity (enhanced chemiluminescencekit [Amersham Pharmacia Biotech, Arlington Heights, Illinois])was assessed densitometrically using Molecular Analyst soft-ware, version 1.4 (Bio-Rad Laboratories). �-Actin was used as aloading CTL.

IF quantification of granulosa cells with activecaspase-3

Isolated granulosa cells were plated on poly-D-lysine (0.05%wt/vol; Sigma) coated 8-well glass culture slides (Becton, Dick-inson and Co.) and cultured in M199 growth media (48 h) beforeDHT treatment. The cells were fixed in paraformaldehyde 4% (1h, RT), washed in PBS. and blocked with 1% BSA and 1% goatserum. Active caspase-3 was detected using an anticleavedcaspase-3 antibody (1:100; Cell Signaling Technology) and Al-exa Fluor 488-conjugated goat antirabbit secondary antibody(1:100; Invitrogen). Actin was detected with red fluorescentTexas Red-X phalloidin (1:100; Invitrogen) as a cytosolic pro-

doi: 10.1210/en.2013-1001 endo.endojournals.org 3

tein. Cells were incubated with ProLong gold antifade reagentcontaining DAPI (blue, nuclear stain). At least 1000 cells pertreatment group were counted in randomly selected fields withthe counter blinded to the sample group to avoid experimentalbias. Cells with active caspase-3 were expressed as a percentageof total cells counted.

Statistical analysisAll data were analyzed using GraphPad Prism 5.0 (GraphPad

Software, Inc., San Diego, California). The results are presented

as mean � SEM of at least 3 independentreplicates as detailed in the figure leg-ends. Data were analyzed by Student’s ttest and 1-way or 2-way ANOVA. A2-way ANOVA was used to assess theeffects and interactions of 2 variables,and multiple comparisons were achievedusing the Bonferroni post hoc test. Sta-tistical significance was defined at P �.05 (*).

Results

DHT-treated rats exhibit markedovarian structural abnormalities

In the present study, we have in-vestigated the molecular and cellularevents associated with antral folliclearrest that also occur in DHT-treated rats. In contrast to normalovarian structure containing folliclesat different stages of development(preantral and large antral) and nor-mal appearance of theca and granu-losa cell layers, and the presence ofcorporea lutea (Figure 1A), DHTtreatment in vivo resulted in amarked reduction in overall ovariansize, the appearance of atypical fol-licles (with the absence of oocytesand reduced granulosa cell numbersbut intact thecal layer), and no cor-pus lutea (Figure 1A). Follicle count-ing revealed that DHT-treated ratovaries harbored a lower percentageof follicles in the preantral to preovu-latory stages but increased numbersof condensed atypical follicles (0.1-0.4 mm diameter) (Figure 1B). In or-der to examine whether the observedDHT-induced changes in follicularmorphology were related to the in-duction of granulosa cell apoptosis,we determined the levels of active

caspase-3 and assessed DNA fragmentation in the DHTgroups. As shown in Figure 1C, DHT induced a varyingdegree of granulosa cell apoptosis in antral follicles (Figure1C, b, c, g, and h), whereas no apoptosis was evident inatypical follicles (Figure 1C, d and i), implying that atyp-ical follicles were depleted of granulosa cells and com-posed mainly of theca cells. Antral follicles exhibit initiallylower levels of granulosa cell apoptosis (Figure 1C, b andg) in DHT-treated rats, which increases with the progres-

Figure 1. Ovarian Features in DHT-Treated Rats A, Hematoxylin and eosin staining of CTL andDHT-treated ovaries. Whereas CTL rats display follicles at different stages and the presence ofcorpora lutea, DHT treatment results in shrinkage of the ovarian structure with minimal largeantral follicles, and the absence of corpora lutea. Selected areas (boxes) indicate enlargedportions in CTL and DHT-treated rats. DHT-treated rat ovaries contain condensed atypical folliclesexhibiting absence of oocyte and reduced granulosa cell numbers. B, Follicle distribution in CTLand DHT-treated rats. Ovaries of DHT-treated rats harbor fewer follicles in preantral topreovulatory stages but a marked increase in number of condensed atypical follicles, and withoutcorpora lutea CTLs. Data were analyzed by an unpaired Student’s t test. *, P � .05; ***, P �.001, compared with CTL groups. C, Ovarian tissues of CTL (a and f) and DHT-treated rats (b–dand g–i) immunostained for active caspase-3 (red, a–e) and TUNEL (green, f–k) on adjacentsections of antral follicle (a and f) in CTL and of antral (b, c, g, and h) and atypical follicles (d andi) in DHT-treated rats. Images were merged with DAPI staining. For TUNEL assay, a positive CTL(k, treated with DNase, 3 �g/mL) and a negative CTL (j, TUNEL buffer without terminaldeoxynucleotidyl transferase) are shown. For caspase-3 detection in IF, a negative control (e,normal rabbit IgG) is shown. PF, primary follicle; PAF, preantral follicle; AF, antral follicle; LAF,large antral follicle; AtyF, atypical follicle; POvF, preovulatory follicle; CL, corpus luteum; GCs,granulosa cells; TCs, theca cells.


sion of the atypical follicles (Figure 1C, c and h), until allgranulosa cells were depleted (Figure 1C, d and i). In thiscontext, DHT-treated rats exhibited distinct reductions inthe antral volume or absence of the antrum, as well asdecreased granulosa cell numbers and retention of thetheca layer in antral follicles and the basement membranein the inner core of the atypical follicles (SupplementalFigure 1 published on The Endocrine Society’s JournalsOnline web site at http://endo.endojournals.org). In ad-dition, NR5A1 (also known as steroidogenic factor-1),expressed predominantly in the theca cells of healthy pre-antral and early antral follicles (19), was localized in atyp-ical follicles of DHT-treated rats (Supplemental Figure 2).

Estrogen is synthesized from androgens by the p450cytochrome aromatase and is responsible for granulosacell proliferation and the ovarian follicular development(20, 21). We then investigated the relationship betweengranulosa cell p450 aromatase expression and the occur-rence of apoptosis by double immunostaining in the ova-ries from CTL and DHT-treated rats. Supplemental Figure3 shows the granulosa cell response to DHT treatment:aromatase expression is down-regulated (SupplementalFigure 3, a and e) while apoptosis increases, as evidencedby higher active caspase-3 level in the follicles of DHT-treated rats (Supplemental Figure 3, b and f). Strong aro-matase expression is clearly visible in the granulosa cells ofantral follicles in CTL rats (Supplemental Figure 3a).H&E staining showed considerable granulosa cell detach-ment and nuclear fragmentation in antral follicles ofDHT-treated rats, indicative of extensive atresia (Supple-mental Figure 3h).

PTEN protein content is markedly up-regulated ingranulosa cells of DHT-treated rats, whereasphospho-Akt (Ser473) content is decreased

The phosphoinositide 3-kinase (PI3K)/Akt (protein ki-nase B) pathway plays a pivotal role in regulation of gran-ulosa cell proliferation and survival and is negatively reg-ulated by phosphatase and tensin homolog (PTEN) (22,23). To determine whether the increased levels of granu-losa cell apoptosis after DHT treatment in vivo is associ-ated with dysregulation of the PI3K, follicular phospho-Akt (Ser473) and PTEN contents in CTL and DHT-treated rats were examined by double-label fluorescentIHC. Granulosa cells of DHT-treated rats exhibited con-siderably higher PTEN levels and significantly decreasedAkt phosphorylation, whereas theca phospho-Akt levelswere relatively higher (Figure 2A). Interestingly, PTENexpression was limited to the granulosa cells and was notdetected in the theca layer. Assessment of total Akt levelsrevealed no difference between granulosa and theca cellsof antral follicles in CTL and DHT-treated rats (Figure

2B). In addition, we examined the influence of DHT on thecontent of phospho-Akt and PTEN in granulosa cells invitro. Consistent with our in vivo IF data, DHT increasedPTEN levels and down-regulated phospho-Akt contentbut not total Akt in granulosa cells (Figure 2C). A timecourse experiment show that DHT suppressed p-AKTcontents at 12 and 24 hours without changing total-Aktcontent (Figure 2D).

DHT down-regulates XIAP expression andactivates caspase-3 in the induction of granulosacell apoptosis

We observed that DHT-induced granulosa cell apopto-sis in vivo is associated with caspase-3 activation, and invitro DHT treatment significantly induces the activationof caspase-3 in granulosa cells (Supplemental Figure 4). Toinvestigate the mechanism responsible for granulosa celldeath, we examined the effect of DHT in vitro on thecontent of the antiapoptotic protein XIAP, an importantintracellular protein involved in the regulation of granu-losa cell proliferation and apoptosis (16), and PARP ingranulosa cells. DHT decreased granulosa cell XIAP andPARP contents in a concentration-dependent manner(Figure 3A) and in different time intervals (Figure 3B). Theactivation of caspase-3 and granulosa cell apoptosis wasdirectly related to decreased XIAP expression and in-creased XIAP fragmentation, whereas forced expressionof XIAP attenuated DHT-induced granulosa cellcaspase-3 activation (Figure 3D) and PARP down-regu-lation (Figure 3C). Finally, we found evidence for the roleof caspase-3 activation on DHT-induced apoptosis usingthe pan-caspase inhibitor zVAD-fmk. Pretreatment withzVAD-fmk significantly suppressed DHT-induced down-regulation of XIAP and PARP (Figure 3E) and caspase-3activation (Figure 3F).

Chemerin and its receptor are overexpressed inthe ovarian follicles in DHT-treated rats andsuppress follicular growth by down-regulation ofgranulosa cell XIAP expression and induction ofapoptosis

Recently, we have demonstrated that ovarian mRNAabundance and protein contents of chemerin and its re-ceptor CKMLR1 were higher in DHT-treated rats com-pared with CTLs (12). However, whether and howchemerin and CKMLR1 are involved in the dysregulationof follicular development in this chronically androgenizedrat model is unknown. As shown in Supplemental Figure5A, Chemerin and CKMLR1 were expressed in granulosacells, theca cells, and oocytes at different stages of normalrat follicular development. GDF9 was expressed only inthe oocyte, with expression increasing with follicular mat-


Figure 2. Effect of DHT on PTEN and phospho-Akt (Ser473) Protein Levels in Vivo and in Vitro A, Immunolocalization of PTEN and phospho-Aktin CTL and DHT-treated rats. Ovarian tissue sections were immunostained (magnification, �200) for PTEN (red, a and e) and phospho-Akt (green,b and f) and selected areas of merged images (c and g) with PTEN and phospho-Akt are shown at increased magnification (d and h). PTEN contentwas up-regulated in granulosa cells but undetectable in the theca cells of DHT-treated rats. Phospho-Akt was significantly down-regulated in thegranulosa cells of DHT-treated rats compared with CTLs, whereas its expression in the theca cells was relatively higher. B, There was no differencein total Akt levels in granulosa and theca cells between CTL and DHT-treated rats. C and D, DHT increased PTEN levels but decreased contents ofphospho-Akt, but not total Akt, in vitro. Granulosa cells from antral follicles were cultured with various concentrations of DHT (0-10 �M, 48 h) orwith 10 �M DHT for the indicated time periods (0-24 h). Contents of PTEN, phospho-Akt, and Akt were examined by Western blot. Actin wasused as a loading control. Results are expressed as mean � SEM (n � 3 independent experiments); data were analyzed by 1-way ANOVA followedby Bonferroni post hoc test (*, P � .05; **, P � .01; ***, P � .001 compared with CTL groups). NS, not significant; GCs, granulosa cells; TCs,theca cells.


uration (Supplemental Figure 5B). Whereas the expressionof chemerin and CKMLR1 was higher in granulosa cellsand theca cells in antral follicles of DHT-treated rats com-pared with that in CTL groups (Figure 4), oocyte-derivedgrowth factor GDF9 was down-regulated in DHT-treatedrats (Supplemental Figure 5C).

To examine whether chemerin regulates rat folliculargrowth, ovarian follicles (diameter, 150-180 �m) were

cultured with chemerin (0-1000 ng/mL) for 4 days in vitro. As shown inFigure 5A, chemerin significantlysuppressed basal follicle growth in aconcentration-dependent manner.Whereas 50 ng/mL chemerin signif-icantly suppressed basal folliculargrowth by 50%, maximal suppres-sion was observed at 100 ng/mL ofthe adipokine. Because GDF9 plays acritical role in the regulation of earlyfollicular development and granu-losa cell proliferation (17, 24), wethen examined the influence ofchemerin on GDF9 expression andaction. Chemerin suppressed GDF9expression in the oocyte of follicles ina concentration-dependent manner(Supplemental Figure 5D), suggest-ing that chemerin-induced folliclegrowth arrest may be associated, inpart, with GDF9 down-regulation.In addition, chemerin significantlysuppressed GDF9- and FSH-stimu-lated follicle growth (Figure 5, B andC). These responses were associatedwith down-regulation of XIAP (Fig-ure 5, E and F) and a significant levelof granulosa apoptotic cell death(Figure 5D), suggesting that XIAPdown-regulation by chemerin maybe responsible for the induction ofgranulosa cell apoptosis and follicu-lar growth arrest.

Follicles in DHT-treated ratsexhibit high calpain expressionand down-regulatedcytoskeletal proteins

DHT-treated rats exhibitedmarkedly increased numbers of con-densed atypical follicles and dra-matic changes in the structure of theovary (Figure 1). To determinewhether the structural changes ob-

served in the condensed atypical follicles of DHT-treatedrats are associated with changes in cytoskeleton-remod-eling enzymes, the expression of calpain and its substratesvimentin, fodrin, and �-tubulin in DHT-treated rats wasanalyzedby fluorescence IHC.Antral andatypical folliclesexhibited higher calpain expression in DHT-treated ratscompared with nontreated CTLs (Figure 6A). The inter-

Figure 3. DHT Down-Regulates XIAP and Activates Caspase-3 in Vitro A and B, DHT suppressedXIAP and PARP contents in a concentration-dependent manner (A) and in different time intervals(B) in vitro. Cleaved XIAP content was increased by DHT. Granulosa cells were cultured withvarious concentrations of DHT (A, 0-10 �M, 48 h) or with 10 �M DHT for the indicated timeperiods (B, 0-96 h). XIAP and PARP content was examined by Western blot. Actin was used as aloading control. Results are expressed as mean � SEM (n � 3 independent experiments); datawere analyzed by 1-way ANOVA (A) and 2-way ANOVA (B) followed by Bonferroni post hoc test(*, P � .05; **, P � .01; ***, P � .001, compared with CTL groups). NS, not significant. C andD, XIAP overexpression attenuated DHT-induced down-regulation of PARP and caspase-3activation. Granulosa cells were infected with adenoviral XIAP sense cDNA or control LacZ(MOI � 40; 24 h) and cultured with DHT (10 �M, 48 h). Granulosa cells with active caspase-3were quantified by IF. A minimum of 1000 cells per treatment group were assessed. Results areexpressed as mean � SEM (n � 4 independent experiments); data were analyzed by 2-wayANOVA followed by Bonferroni post hoc test (***. P � .001). E and F, Pan-caspase inhibitor(zVAD-fmk) suppressed DHT-induced XIAP cleavage, PARP down-regulation, and caspase-3activation. Granulosa cells were precultured with zVAD-fmk (10 �M, 2 h) before DHT treatment(10 �M, 48 h). XIAP and PARP content was examined by Western blot. Results are expressed asmean � SEM (n � 3 independent experiments); data were analyzed by 2-way ANOVA followedby Bonferroni post hoc test (***, P � .001).


mediate filaments, vimentin and fodrin, as well as the mi-crotubule �-tubulin were significantly down-regulated inatypical follicles in DHT-treated rats (Figure 6B, a–c). Incontrast, actin, which is not a substrate of calpain, re-mained unaffected (Figure 6B, d).

Discussion

In the present study, we have investigated the role ofchemerin and the cellular mechanisms involved in andro-gen-induced antral follicular growth arrest, using a chron-ically androgenized rat model. Our studies demonstratethat chronic androgen administration increases chemerinand CKMLR1 expression that is involved in the inductionof antral follicle growth arrest. The latter response is char-acterized by dysregulated interactions of survival (p-Akt,XIAP, PARP) and proapoptotic (PTEN, caspase-3) factorsin a cell-specific manner. This hyperandrogenic state isalso accompanied by marked changes in follicle structure,including up-regulation of calpain expression and de-creased cytoskeletal proteins, apoptotic deletion of gran-ulosa cells and oocytes, and the survival and retention oftheca cells.

Aromatase catalyzes the conversion of androgens toestrogens, and estrogens are known to promote granulosacell proliferation and follicular growth in vivo (20, 21). Inthe present study, active caspase-3 level and nuclear frag-mentation in granulosa cells were markedly increased inDHT-treated rats, a response accompanied by down-reg-ulation of aromatase expression. In addition, it is alsoknown that PI3K/Akt signaling is important for aromatase

expression during granulosa cell dif-ferentiation (22). Our results revealthat DHT-treated rats exhibit de-creased granulosa cell Akt phos-phorylation that was accompaniedby higher PTEN level, raising thepossibility that they may be involvedin the regulation of granulosa cell de-mise. PTEN, as a phosphatase, isknown to dephosphorylate phos-phatidylinositol (3,4,5)-trisphos-phate, thereby negatively regulatingPI3K/Akt signaling (23). A recentstudy reports that PTEN mutantmice exhibit enhanced ovulation, de-creased apoptosis, and increasedproliferation in granulosa cells (25).

Moreover, our current study alsosuggests that the dysregulated follic-ular growth in DHT-treated rats iscontrolled by complex interactions

and dysregulation of survival (down-regulation of p-Akt,XIAP, and PARP) and proapoptotic (up-regulation ofPTEN and caspase-3) modulators in a cell-specific man-ner. The mechanism by which phospho-Akt down-regu-lation influences granulosa cell apoptosis in the DHT-treated rat model remains unknown. It is possible thatphospho-Akt down-regulation reduces the expression andstability of XIAP. We have previously demonstrated theantiapoptotic role of XIAP in granulosa cells (16, 18, 26),and here we show that physiologically relevant concen-trations of DHT decrease XIAP and phospho-Akt con-tents and enhance active caspase-3 level in granulosa cellsin vitro. Because Akt phosphorylates XIAP and preventsits auto-ubiquitination and proteasomal degradation(27), it is conceivable that the decreased granulosa cellphospho-Akt content may lead to XIAP destabilizationand caspase-3 activation. This was further supported bythe observation that forced expression of XIAP signifi-cantly attenuated DHT-induced caspase-3 activation andPARP down-regulation. These findings suggest that theinteraction of survival and antiapoptotic factors plays acritical role in regulating DHT-induced granulosa cellapoptosis.

It has been demonstrated that chemerin levels in thecirculation are elevated in obese women (8) and PCOSpatients (9). In the current studies, we observed a higherovarian chemerin and CMKLR1 expression in the DHT-treated rats when compared with CTLs. Our unpublisheddata indicated that the levels of chemerin and CMKLR1are elevated in ovarian follicles of human PCOS subjectscompared with the CTLs. In addition, chemerin levels in

Figure 4. Expression of Chemerin and CMKLR1 in Vivo Chemerin and CMKLR1 levels arehigher in antral follicles of DHT-treated rats compared with CTL. Ovarian tissue sections wereimmunostained for chemerin (a and b) and CMKLR1 (d and e). Negative CTLs (blocking peptidesfor chemerin [c] and CMKLR1 [(f)]) are shown. OO, oocyte; GC, granulosa cells; TCs, theca cells;CLs, corpus luteum.


Figure 5. Chemerin Induces Follicular Growth Arrest Which Is Associated with the Induction of Granulosa Cell Apoptosis and Down-Regulation ofXIAP A–C, Chemerin suppressed basal and GDF9- and FSH-stimulated rat follicle growth in vitro. Follicles (diameter, 150-180 �m) isolated from14-day-old rats were cultured with different concentrations of chemerin (A; chem, 0, 10, 50, 100, and 1000 ng/mL), chemerin (B; chem, 0, 100,and 1000 ng/ml) � GDF9 (100 ng/ml) or (C; chem, 0, 100, and 1000 ng/mL) � FSH (10 ng/mL) for 4 days. Follicular diameter was determined andchanges in follicular volume were calculated as we reported earlier (17). n � 5 replicate experiments each with 10 follicles per group. (Data wereanalyzed by 2-way ANOVA followed by Bonferroni post hoc test. In panel A: *, P � .05; **, P � .01; ***, P � .001 compared with CTL group. Inpanels B and C: *, P � .05; **, P � .01; ***, P � .001 compared with GDF9 or FSH only group). D, Follicles were cultured with chemerin (0-1000ng/mL) for 4 days and in situ detection of apoptotic cells (TUNEL staining) was performed. A positive CTL (DNase, 3 �g/mL) and a negative CTL(TUNEL buffer without terminal deoxynucleotidyl transferase) are shown. Images were merged with DAPI staining. Chemerin induced significantlygranulosa cells apoptosis. (n � 10 follicles per group); data were analyzed by 1-way ANOVA followed by Bonferroni post hoc test (*, P � .05; **,P � .01 compared with CTL group). E, Follicles were cultured with and without chemerin (100 ng/mL) 4 days. Follicles tissue sections wereimmunostained for XIAP and normal rabbit IgG (as a negative CTL). Images were merged with DAPI staining. XIAP fluorescence intensity wasanalyzed by Image J. Chemerin suppressed XIAP expression in granulosa cells of follicles. (n � 10 follicles per group); data were analyzed by anunpaired Student t test (*,P � .05 compared with CTL group) F, Granulosa cells were cultured with chemerin (0-1000 ng/mL) for 4 days. XIAPcontent was examined by Western blot and actin was used as a loading control. Chemerin down-regulated XIAP protein levels. Results areexpressed as mean � SEM (n � 3 independent replicates); data were analyzed one-way ANOVA followed by Bonferroni post hoc test (*P � .05compared with CTL group). Chem, chemerin.


Figure 6. A, Fluorescent immunolocalization of calpain and cytoskeletal proteins (vimentin, fodrin, �-tubulin, and actin) in CTL and DHT-treatedrats (magnification, �200). Ovarian tissue sections were immunostained for calpain (a–c) or normal rabbit IgG (negative CTL, d). Panels e–h showDAPI staining. The expression of calpain in granulosa and theca cells was considerably higher in the antral and atypical follicles of DHT-treated ratswhen compared with CTL. B, Immunoreactivities of the intermediate filament proteins fodrin (a) and vimentin (b), microtubular protein �-tubulin(c), and microfilamental protein actin (d) in DHT-treated rats. Merged images include DAPI staining. Vimentin, fodrin, and �-tubulin weresignificantly down-regulated in atypical follicles of DHT-treated rats. In contrast, actin (which is not a substrate of calpain), remained unaffected byDHT treatment. C, A hypothetical model illustrating the cellular events involved in dysregulated ovarian follicular growth under chronichyperandrogenization in the rat. Excess androgen increases the levels of chemerin and CMKLR1 and decreases granulosa cell aromataseexpression, a response accompanied by increased active caspase-3 content and DNA fragmentation. These events result in apoptotic clearance ofthe oocyte and granulosa cells but survival and retention of the theca cell layer. The latter collapses with increased calpain expression and activitydown-regulates the cytoskeletal substrates vimentin, fodrin, and �-tubulin in the atypical follicles. D, Proposed signaling pathways involved inchemerin-induced follicular growth arrest in a chronically androgenized rat model showing up-regulation of intracellular proapoptotic mediatorsPTEN and caspases-3 and down-regulation of antiapoptotic factors XIAP, PARP, and phospho-Akt in granulosa cells and oocyte-expressing GDF9,with phospho-Akt being up-regulated for theca cell survival. F, follicle; CL, corpus luteum; GCs, granulosa cells; TCs, theca cells; OO, oocyte; An,antrum.


human follicular fluid are 8-fold higher than those in se-rum (Wang, Q., and K. Xue, unpublished data). Theseobservations suggest that chemerin may be a potential in-traovarian regulator of ovarian follicular development.For the first time, we demonstrate that chemerin down-regulates XIAP expression and induces apoptosis in gran-ulosa cells, thereby suppressing follicle growth. It isknown that the oocyte-derived factor GDF9 promotesgranulosa cell proliferation and preantral/early antral fol-licle growth (17, 24). In this study, we also observed thatchemerin suppresses GDF9 expression and GDF9-in-duced follicular growth in vitro. Whereas GDF9 levels inoocytes were reduced in DHT-treated rats, an aberrantGDF9 expression (28) and 5 novel missense GDF9 muta-tions have been reported in PCOS patients (29). Therefore,defining the interplay between chemerin and GDF9 ex-pression and action could provide important insights intothe dysregulated follicular development in this complexsyndrome.

In addition to its role in the control of ovarian folliculargrowth, chemerin is important in the regulation of follic-ular steroidogenesis (12). We have previously reportedthat chemerin inhibits FSH-induced aromatase expressionand estrogen secretion in granulosa cells and that this in-fluence is mediated through increased expression and ac-tion of the mitochondrial protein (12, 30). This observa-tion, together with our present finding that elevatedchemerin levels and down-regulated aromatase expres-sion are positively related to increased granulosa cell ap-optosis in DHT-treated rats, supports the hypothesis thatchemerin plays a paracrine and/or autocrine-regulatoryrole in the ovary and contributes to the dysfunction of theovarian function. Further studies on the downstream sig-naling pathway triggered by chemerin in the dysregulationof follicular development may provide important clues inthe understanding of the precise role of chemerin in folliclegrowth arrest.

In the present study, we have shown that DHT-treatedrats display dysregulated antral follicular growth and havebegun to define the cellular mechanisms involved in itsstructural changes. Our observation that the ovarianweight in DHT-treated rats was decreased compared withthat in CTLs is in good agreement with other PCOS rodentmodels induced by androgen or estrogen treatment (13,31-33) but is in contrast to the human phenotype. Themechanisms responsible for the reduced ovarian weight inthese rodent models remain unknown. We have demon-strated here that DHT administration induces shrinkageof the follicular structure, the appearance of atypical fol-licles with oocytes absent, a marked reduction in granu-losa cell number and follicular fluid volume, and retentionof the theca layer. It is possible that the observed changes

in cytoskeletal proteins and structural perturbations inatypical follicles could decrease the overall permeability ofcritical areas required for fluid influx. The calcium-depen-dent protease calpain degrades cytoskeletal componentssuch as microtubule and intermediate filament proteinsand is a critical regulator of cytoskeletal reorganization,cell morphology, and apoptosis (34-36). In the presentstudies, antral and atypical follicles of DHT-treated ratsexhibited markedly higher calpain expression comparedwith CTLs, an observation associated with decreased ex-pression of the cytoskeletal substrates, vimentin, fodrin,and �-tubulin, but not of actin (not a calpain substrate).Taken together, these findings demonstrate, for the firsttime, that increased calpain expression, with concomitantdown-regulation of cytoskeletal substrates, is closely as-sociated with the follicle collapse and development ofatypical follicles, the final stage of follicular dysregulationin DHT-treated rats.

In conclusion, using a chronically androgenized rodentmodel, we have defined the cellular and molecular eventsleading to the dysregulation of ovarian follicular growthand offered a new insight into the mechanism of ovarianfollicular growth arrest in the pathology of hyperandro-genism. Notably, we have provided novel evidence thatchemerin is an important intraovarian regulator and maycontribute to the dysregulation of follicular development.To facilitate future investigations, we hypothesize that an-tral follicle growth promoted by gonadotropin and GDF9is arrested in DHT-treated rats as a result of enhancedchemerin expression and action, as well as of ovarianstructural changes following apoptotic clearance of theoocyte and granulosa cells and the survival and retentionof the theca cell layer. The follicle collapses with increasedcalpain expression and down-regulation of its cytoskeletalsubstrates (Figure 6C). We also propose that the dysregu-lated ovarian follicular development in DHT-treated ratsis a consequent of chemerin-induced up-regulation of in-tracellular proapoptotic mediators (PTEN andcaspases-3) and down-regulation of antiapoptotic factors(XIAP and phospho-Akt) and their complex interactions(Figure 6D), many of which remain to be elucidated. Al-though inherent limitations exist in all animal models de-veloped for human diseases (37), the present studies sig-nificantly contribute to the current understanding of thechronic influence of excess androgen on ovarian folliculargrowth as well as the pathology of female reproduction.

Acknowledgments

We thank Lee Farrand for editing the manuscript.


Address all correspondence and requests for reprints to: Ben-jamin K. Tsang, Ph.D., Ottawa Hospital Research Institute, TheOttawa Hospital, General Campus, 501 Smyth Road, Mail Box511, Ottawa, Ontario K1H 8L6 Canada. E-mail:[email protected].

This work was supported by grant MOP-119381 from theCanadian Institutes of Health Research (CHIR); grant R31-10056 from the World Class University (WCU) programthrough the Ministry of Education, Science and Technologyfunded by the National Research Foundation of Korea (R31-10056); grants from the Human Reproduction Research Fund ofthe Ottawa Fertility Centre; CIHR-QTNPR postdoctoral fel-lowship and graduate scholarship (to J.Y.K. and Q.W., respec-tively); and CIHR-REDIH graduate scholarship (to K.X.) andCIHR-STIRRHS postdoctoral fellowship (to M.J.C.).

Disclosure Summary: The authors declare no conflict ofinterest.

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