Androgen Excess Down-regulates Connexin43 in a Human Granulosa Cells Line

Elsevier Editorial System(tm) for Fertility and Sterility Manuscript Draft

Manuscript Number: F and S8110R2

Title: Androgen Excess Down-regulates Connexin43 in a Human Granulosa Cells Line Article Type: Correspondence

Section/Category:

Keywords: Connexin43, androgen, human granulosa cells Corresponding Author: Pao-Lin Kuo, MD

Corresponding Author's Institution: National Cheng Kung University, College of Medicine, Tainan, Taiwan.

First Author: Cheng-Hsuan Wu, M.D.

Order of Authors: Cheng-Hsuan Wu, M.D.; Jyuer-Ger Yang, M.S.; Jiann-Jou Yang, Ph.D.; Yueh-Min Lin, M.D.; Hong-Der Tsai, M.D.; Ching-Yuang Lin, M.D. Ph.D.; Pao-Lin Kuo, M.D.

Abstract: Objective: To investigate the effects of androgen excess on connexin43 (Cx43) expression in human granulosa cells in vitro.

Design and Setting: Controlled experimental study in a Medical Center

Intervention(s): Human granulosa cell line (HO-23) was treated by various dosages of

dihydrotestosterone (DHT). The effect of DHT treatment on granulosa cell Cx43 was assessed by using Western blot. Gap junctional intercellular communication (GJIC) between granulosa cells was

investigated by using the scrape-loading and dye transfer. Androgen receptor antagonist, flutamide, was used to test the specificity of the observed androgen responses.

Main Outcome Measure(s): Cx43 protein expression following DHT treatment.

Result(s): Treatment of the HO-23 cells with DHT showed a dose-dependent decrease in Cx43 protein expression. Flutamide significantly blocked the inhibitory effects of DHT on Cx43 expression. DHT-treated cells demonstrated a decreased enhancement of GJIC as assessed by dye transfer experiments. Conclusion(s): High level androgen reduces Cx43 expression and impairs GJIC between human

granulosa cells through androgen receptors. It may impair folliculogenesis, induce follicular atresia and lead to ovulatory dysfunction.

2010/6/16

Alan H. DeCherney, M.D.

Editor-in- Chief

Fertility and Sterility

Department of Obstetrics and Gynecology

UCLA School of Medicine

10833 Le Conte Avenue, 24-153 CHS

Los Angeles, California 90095-1740

Dear Prof. DeCherney:

We would like to resubmit our manuscript entitled “Androgen Excess

Down-regulates Connexin43 in a Human Granulosa Cells Line” for publication in

your cerebrated journal, Fertility and Sterility.

We have answered the reviewers’ and editorial’s comments in the revision as

below:

Editorial’s comment:

1) We had corrected

Reference 4.

2)

Our abstract had been shortened to two sentences.

Reviewer’s comment:

1) We had corrected into “induce” and “lead”. (

Abstract, lines 30, 31)

2)

We change mammalians into mammalian. (Page 10, line 39)

3)

Figure 1-B, bottom: change B+D+ into B+D+F

In addition, we declare that the material contained in this manuscript has not

been published, or is being submitted elsewhere. There is no interest conflict in this

study.

With best wishes,

Looking forward to hearing from you soon, preferably by E-mail or by Fax.

Pao-Lin Kuo, M.D.

Dept. of Obstetrics and Gynecology, National Cheng Kung University Hospital

138 Victory Road, Tainan 70428, Taiwan

E-mail: [email protected] Fax: +886-6-2766185

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F & S 8110 decline and resubmit non-highlighted

Running title: Androgen excess down-regulates Cx43 in granulosa cells

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Androgen Excess Down-regulates Connexin43 in a Human Granulosa Cells Line

Cheng-Hsuan Wu M.D.,

Jyuer-Ger Yang M.S.,

Jiann-Jou Yang Ph.D.,

Yueh-Min Lin M.D.,

Hong-Der Tsai M.D.,

. Ching-Yuang Lin M.D. Ph.D.,

Pao-Lin Kuo M.D.

a

Department of Obstetrics and Gynecology ,

Department of Pathology ,

Changhua Christian Hospital, Changhua,

c

Department of Biomedical Science,

Institute of Medicine, Chung Shan

Medical University, Taichung,

Children‟s Medical Center,

Graduate Institute of Clinical Medical Science,

China Medical University, Taichung,

Department of Obstetrics and Gynecology , National Cheng Kung University

Hospital, Tainan, Taiwan, R.O.C

*To whom correspondence should be addressed at:

Ching-Yuang Lin, M.D., Ph.D.,

Children‟s Medical Center, China Medical University Hospital,

2 Yuh-Der Road, Taichung, Taiwan.

TEL: +886-4-22052121 ext 2077 FAX: +886-4-22032798

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Pao-Lin Kuo,M.D.

Department of Obstetrics and Gynecology ,

National Cheng Kung University Hospital,

138 Sheng-Li Road, Tainan, Taiwan 704

Tel: +886-6-2353535 ext 5221

Fax: +886-6-2766185

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Capsule:

Androgen excess reduces Cx43 expression and impairs GJIC between human

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By using a cell model, we found high level androgen reduces connexin 43 expression

and impairs gap junction intercellular communication between human granulosa cells

through androgen receptors. This finding suggests that high level androgen may

impair folliculogenesis which in turn leads to ovulatory dysfunction in polycystic

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Androgen excess is the central defect in polycystic ovarian syndrome (PCOS) patients and androgen excess is believed to be involved in the pathogenesisof follicular development arrest (1,2). Jakimiuk et al. (3) have shown that women with PCOS express elevated levels of 5α reductase mRNA in the granulosa cells. Thus, it is conceivable that the deleterious effects

of androgens in the ovary may be mediated through the conversion to 5α reduced metabolites.

Numerous literatures have shown that normal folliculogenesis relies on the bidirectional talk between granulosa cells and oocytes (4, 5). By facilitating the transfer of ions and small molecules from cell to cell, gap junction intercellular communication (GJIC) between granulosa cells and oocytes plays a critical role in folliculogenesis and oogenesis (6, 7). Connexins (Cx) are membrane-spanning proteins that assemble to form the intercellular channels of gap junctions. At least20 rodent and 21 human connexins have been identified and namedaccording to their molecular weights (8). Gap junctionsbetween ovarian granulosa cells contain predominantly Cx43 which is present at all stages of follicle development(6, 7); while Cx37 is expressed at the oocyte surface in oocyte-somatic cell gapjunctions, with little if any contribution from Cx43 (9; 10). Using chimeric ovaries, Gitten and Kidder (11) demonstrated that Cx37 is required for oocytes but not granulosa cells development; while Cx43 is required for granulosa cells but not oocytes development. The expression level of Cx43 protein is increased as the follicle grows and matures and decreased during follicular atresia (22). Taken together, these data suggested that Cx43 gap junction communication is criticalfor ovarian folliculogenesis.

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hormone, regulate expression of connexins. However, to the best of our knowledge there have

been no reports regarding the effectof androgen on Cx43 expression in human granulosa cells.

We hypothesize that decreased Cx43 expression and GJIC activity are induced by high levels

of androgens in granulosa cells in PCOS ovaries, resulting in impaired folliculogenesis and

anovulation - a hallmark of PCOS. We treated the human granulosa cell line (HO-23) cultured

in vitro with different concentrations of dihydrotestosterone (DHT), a 5α reduced metabolite

of testosterone that has the highest affinity for the androgen receptor (AR) , to investigate the

Cx43 protein levels and to evaluate the effect of androgen on connexins expression in human

granulosa cells.

The HO23 human immortalized luteinized granulosa cell line was provided by Dr. Abraham

Amsterdam, Weizmann Institute of Science, Rehovot 76100, Israel (13). Granulosa cells were

maintained at 37°C in 5% CO2 /air in Dulbecco‟s minimal essential medium (DMEM)/Ham's

F12 (1:1), supplemented with 5% fetal calf serum and antibiotics (100 IU/ml penicillin and

100 μg/ml streptomycin). To test the effect of DHT on Cx43 expression, cells (2 x 106) were

seeded on 100 mm culture dishes and incubated for 24 hours. The media were removed, and

the cells were reincubated in fresh media with different concentrations of DHT. The cells

were harvested for protein assays at 24 hours.

Protein content in cell lysate was determined by the bicinchoninic acid (BCA) protein assay

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polyvinylidene difluoride membrane (Bio-Rad Laboratories, Ca, USA). The membranes were

blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 1 hours and incubated with

mouse anti-human Cx43 antibody (Sigma-Aldrich Co. , USA) (1:3000) in 0.1% nonfat dry

milk overnight at 4°C. After three washes in TBS containing 0.1% Tween-20, the membranes

were incubated with goat anti-mouse IgG (Chemicon, USA) (1:5000) conjugated with

alkaline phosphatase (Sigma-Aldrich) for 1 hour. The membranes were washed again as

above, applied to the enhanced chemilunimescence (ECL) detection kit (Amersham, UK), and

exposed to X-ray film (BioMAx films, Eastman Kodak Company, Rochester, NY, USA. To

assess the amount of Cx43 protein loaded, filterswere treated with 2% SDS and 100 mM β-mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8) for 30 min at 60°C and reprobed for

β-actin. Films were scanned, and the optical density of the bandswas measured with Scion Image. Data shown are representativeof at least three independent experiments with similar

results.

Assessment of GJIC was determinedusing the scrape-loading and dye transfer (SLDT)

technique with a fluorescent dye, LuciferYellow (LY, Sigma, USA) (14). Briefly, human

granulosa cells, cultured as described above, were washed thoroughlywith PBS. SL was

performed applying three cuts on cell monolayerwith a surgical scalpel, and then a

mixture of 1% LY and 1% of rhodamine dextran in PBS was added to the cells.The cells

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fluorescence. The cells were then fixed with 4% paraformaldehyde and photographed with a

fluorescent microscope equipped with a camera. Cells that receivedthe LY from the

scrape-loaded cells were considered as communicating.The dye-coupled cell layers on either

sides of the scrape were counted to evaluate the GJIC.

Data are expressed as mean ± SEM of at least three independent experiments performed at

different time points. All data were analyzed using one-way analysis of variance (ANOVA).

If the differences were significant, a Dunnett post hoc test was used for post-ANOVA

multiple comparisons. Statistical significance was determined asP < 0.05.

The effect of DHT on Cx43 protein expression in response to 8-Br-cAMP was examined by

Western blot analysis. 8-Br-cAMP, an analogue of cyclic AMP, is known to activate

adenylate cyclase and increase the adenosine cAMP pool in the granulosa cells and

up-regulates the Cx43 expression (15). A previous study showed the range of DHT in

follicularfluid of normal population is 0.77 ± 0.11 ng/ml (16). To demonstrate the effect of

androgen excess on Cx43 expression, cultured human granulosa cells were treated with

increasing doses of DHT at 0, 1, 10, or 100 ng/ml. Western blot analysis of DHT-treated

granulosa cell protein samples demonstrated decreasing amount of Cx43 protein in a

dose-dependent manner. In addition, a significant decrease in the expression could be

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To assess whether the suppressive effect of DHT on Cx43 is mediated through the AR, cells

were incubated with DHT (10 ng/ml) and flutamide (100 ng/ml, a 10-fold excessabove that of

DHT). Flutamide, an AR antagonist, significantly blocked the inhibitoryeffects of DHT on

Cx43 expression as shown in Figure 1-A.

To understand the association between reduced Cx43 expression and GJIC activity, we

assessed the ability of HO-23 granulosa cells to transfer Lucifer yellow through gap junctions

using the scrape-loading dye transfer (SLDT) technique in corresponding cultures of cells

with reduced Cx43 expression. As shown in Figure 1-B, the number of dye-coupled cell

layers was significantly reduced on either side of the scrape in HO-23 granulosa cells grown

in 8-Br-cAMP with DHT medium compared with cells grown in 8-Br-cAMP medium. The

phenomenon wasprevented upon replenishing the medium with flutamide. This gap

junctional defect could be induced with DHT and the effect was recovered with addition of

flutamide to the culture medium.

Although anovulation is frequently observed in PCOS patients with androgen excess, the

underlying mechanism has not been fully elucidated. Several possibilities have been raised to

explain how androgens might mediate anovulation. In this study, we propose a new pathway

through which high androgen level down-regulates Cx43 expression, reduce GJIC in human

granulosa cells, and finally lead to impaired folliculogenesis.

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ovarian physiology in animals (17, 18). One of the targets of this disruptive action is the

granulosa cell within the ovarian follicle. While both androgen and gap junctions are reported

to have important roles in follicular development, our study provides a novel mechanism

which links androgen excess and Cx43 expression in the granulosa cells. We showed reduced

GJIC activity in the milieu of high androgen and reduced Cx43 expression which occurs at

the translation level mediated through androgen receptor.

A link between reduced GJIC activity and reduced connexin gene expression has been

previously reported in rat ovarian cells exposed to luteinizing hormone (LH). LH inhibited

GJIC activity by down-regulating Cx43 synthesis (19). Recently, Wang et al. (20) provided

the evidence that the strength of gap junctional conductance was positively correlated with

Cx43 level in human cumulus cells. Although it is possible that the effect of high androgen on

GJIC activity may be mediated via other junction protein components, our findings indicate

that reduced GJIC activity in human granulosa cells exposed to high androgen is at least in

part due to inhibition of Cx43 gene expression.

The expression and function of Cx43 protein in the ovary of mammalian species have

been discussed previously (21). In the mouse ovary, Cx43 is an important gap junction

protein required for granulosa cell to granulosa cell communication, which in turn is required

for follicle and oocyte maturation (7). Decreased Cx43 protein level is associated with

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a coordinating role in the process of atresia (12). Disruption of granulosa cell couplingnot

only impairs follicle growth, but also impacts negativelyon the quality of the enclosed

oocytes. The knock-out mice of Cx43 showed that GJIC activity was reduced and oocyte

growth in mutant follicles was retarded (22). However, we know little about Cx43 in human

ovarian follicles and their roles in human folliculogenesis and fertility. A recent study shows

that Cx43 was primarily localized in the membrane where it forms gap junction-like plaques

between human cumulus cells, and its expression level may influence pregnancy outcome

from in vitro fertilization (20). Furthermore, high Cx43 levels in granulosa cells are linked to

good prognosis of human oocytes (9).

The roles of androgen and androgen receptor (AR) are well-established in the

development of male reproductive organs and spermatogenesis. However, the functions of

this steroid and its receptor in the ovary remain elusive. AR expression in the ovary has been

described at various stages of folliculogenesis in several species. In human ovaries, AR

protein expression is observed at different stages in granulosa and theca cells (23). Using a

Cre–loxP system to generate AR-knockout (ARKO) mice, investigators showed that activated

ARs are indispensable for normal folliculogenesis (24, 25). In this study, we demonstrate that

androgen regulates Cx43 expression in cultured human granulosa cells by an AR-dependent

process considering suppressive effectof DHT on Cx43 was blocked by an AR antagonist,

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treatment ofpatients with PCOS and anovulation. In fact, flutamide is able to restore

ovulation in some women with PCOS (26, 27). However, there is still concern about the use

of flutamide in women with infertility considering its possible teratogenic effect (28).

The root cause of anovulation in PCOS is now thought to be associated with the

significant abnormalities in the very earliest stages offolliculogenesis (29). The normal

„dialogue‟ between oocyte and granulosa cells in these early growing follicles is altered. Recently, Li et al. (30) raised a hypothesis that if dysfunctional communications exist

between granulosa cells or granulosa cells and the oocyte, unnecessary androgen will enter

the oocyte and bind to the AR, provoking some follicles to undergo atresia. We therefore

propose a working mode for the effect of androgen excess on folliculogenesis and oogenesis.

High androgen level down-regulates Cx43 translation and impairs the communication

between granulosa cells and possibly communications between granulosa cells and oocyte.

Impaired communications negatively impacts folliculogenesis. The adverse effect of

androgen excess is further strengthened by “shunting” of excessive androgens to the oocytes secondary to impaired communications between granulosa cells.

In conclusion, the presentstudy provides the first evidence that increased levels of

androgen down-regulate Cx43 expression and impair communications between granulosa

cells. The effect may compromise folliculogenesis and oogenesis in PCOS patients. Caution

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clinical setting considering few clinical data in this study. In addition, how DHT affects Cx43

translation still awaits further investigation.

Acknowledgment:

This study was supported by grants from the Changhua Christian Hospital

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Figure legends

Figure 1-A. Antiandrogen treatment blocked the inhibitoryeffects of DHT on Cx 43

expression. HO-23 cells were cultured for 24 h under serum-free conditions in the presence of

8-Br-cAMP (0.1 mM), DHT (10ng/ml), and the antiandrogen flutamide (100 ng/ml). Data are

expressed as the mean ± SD of three different experiments. (＊: p < 0.05 vs. 8-Br-cAMP)

B: 8-Br-cAMP D: DHT F: flutamide

Figure 1-B: Analysis of GJIC activity in HO-23 cells using SLDT technique. RD remains in

the cells immediately bordering the scrape, while the LY diffuses to neighboring cells in gap

junction-enabled cells. GJIC was evaluated as the counts from dye-coupled cell layers and is

represented as a histogram. GJIC is blocked in HO-23 cells treated with 8-Br-cAMP (0.1 mM)

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blockage of GJIC induced by DHT to a comparable level to 8-Br-cAMP (ns, not significant

vs 8-Br-cAMP). Data are expressed as the mean ± SD of three different experiments.

B: 8-Br-cAMP D: DHT F: flutamide

GJIC: gap junction intercellular communication

SLDT: scrape-loading and dye transfer

Figure 1-A.

Cx 43

β- Actin

Control B B+D B+F B+D+F

0 20 40 60 80 100 120

*

Opt

ic

D

en

si

ty

Figure 1-B.

LY

RD

Control

B

B+D

B+D+F

*

N

o of

Dye

Cou

pling

C

ell

Layer

s

F & S 8110 decline and resubmit highlighted

Running title: Androgen excess down-regulates Cx43 in granulosa cells

Androgen Excess Down-regulates Connexin43 in a Human Granulosa Cells Line

Cheng-Hsuan Wu M.D.,

Jyuer-Ger Yang M.S.,

Jiann-Jou Yang Ph.D.,

Yueh-Min Lin M.D.,

Hong-Der Tsai M.D.,

. Ching-Yuang Lin M.D. Ph.D.,

Pao-Lin Kuo M.D.

a

Department of Obstetrics and Gynecology ,

Department of Pathology ,

Changhua Christian Hospital, Changhua,

c

Department of Biomedical Science,

Institute of Medicine, Chung Shan

Medical University, Taichung,

Children‟s Medical Center,

Graduate Institute of Clinical Medical Science,

China Medical University, Taichung,

Department of Obstetrics and Gynecology , National Cheng Kung University

Hospital, Tainan, Taiwan, R.O.C

*To whom correspondence should be addressed at:

Ching-Yuang Lin, M.D., Ph.D.,

Children‟s Medical Center, China Medical University Hospital,

2 Yuh-Der Road, Taichung, Taiwan.

TEL: +886-4-22052121 ext 2077 FAX: +886-4-22032798

Pao-Lin Kuo,M.D.

Department of Obstetrics and Gynecology ,

National Cheng Kung University Hospital,

138 Sheng-Li Road, Tainan, Taiwan 704

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Androgen excess reduces Cx43 expression and impairs GJIC between human

By using a cell model, we found high level androgen reduces connexin 43 expression

and impairs gap junction intercellular communication between human granulosa cells

through androgen receptors. This finding suggests that high level androgen may

impair folliculogenesis which in turn leads to ovulatory dysfunction in polycystic

Androgen excess is the central defect in polycystic ovarian syndrome (PCOS) patients and androgen excess is believed to be involved in the pathogenesisof follicular development arrest (1,2). Jakimiuk et al. (3) have shown that women with PCOS express elevated levels of 5α reductase mRNA in the granulosa cells. Thus, it is conceivable that the deleterious effects

of androgens in the ovary may be mediated through the conversion to 5α reduced metabolites.

Numerous literatures have shown that normal folliculogenesis relies on the bidirectional talk between granulosa cells and oocytes (4, 5). By facilitating the transfer of ions and small molecules from cell to cell, gap junction intercellular communication (GJIC) between granulosa cells and oocytes plays a critical role in folliculogenesis and oogenesis (6, 7). Connexins (Cx) are membrane-spanning proteins that assemble to form the intercellular channels of gap junctions. At least20 rodent and 21 human connexins have been identified and namedaccording to their molecular weights (8). Gap junctionsbetween ovarian granulosa cells contain predominantly Cx43 which is present at all stages of follicle development(6, 7); while Cx37 is expressed at the oocyte surface in oocyte-somatic cell gapjunctions, with little if any contribution from Cx43 (9; 10). Using chimeric ovaries, Gitten and Kidder (11) demonstrated that Cx37 is required for oocytes but not granulosa cells development; while Cx43 is required for granulosa cells but not oocytes development. The expression level of Cx43 protein is increased as the follicle grows and matures and decreased during follicular atresia (22). Taken together, these data suggested that Cx43 gap junction communication is criticalfor ovarian folliculogenesis.

hormone, regulate expression of connexins. However, to the best of our knowledge there have

been no reports regarding the effectof androgen on Cx43 expression in human granulosa cells.

We hypothesize that decreased Cx43 expression and GJIC activity are induced by high levels

of androgens in granulosa cells in PCOS ovaries, resulting in impaired folliculogenesis and

anovulation - a hallmark of PCOS. We treated the human granulosa cell line (HO-23) cultured

in vitro with different concentrations of dihydrotestosterone (DHT), a 5α reduced metabolite

of testosterone that has the highest affinity for the androgen receptor (AR) , to investigate the

Cx43 protein levels and to evaluate the effect of androgen on connexins expression in human

granulosa cells.

The HO23 human immortalized luteinized granulosa cell line was provided by Dr. Abraham

Amsterdam, Weizmann Institute of Science, Rehovot 76100, Israel (13). Granulosa cells were

maintained at 37°C in 5% CO2 /air in Dulbecco‟s minimal essential medium (DMEM)/Ham's

F12 (1:1), supplemented with 5% fetal calf serum and antibiotics (100 IU/ml penicillin and

100 μg/ml streptomycin). To test the effect of DHT on Cx43 expression, cells (2 x 106) were

seeded on 100 mm culture dishes and incubated for 24 hours. The media were removed, and

the cells were reincubated in fresh media with different concentrations of DHT. The cells

were harvested for protein assays at 24 hours.

Protein content in cell lysate was determined by the bicinchoninic acid (BCA) protein assay

polyvinylidene difluoride membrane (Bio-Rad Laboratories, Ca, USA). The membranes were

blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 1 hours and incubated with

mouse anti-human Cx43 antibody (Sigma-Aldrich Co. , USA) (1:3000) in 0.1% nonfat dry

milk overnight at 4°C. After three washes in TBS containing 0.1% Tween-20, the membranes

were incubated with goat anti-mouse IgG (Chemicon, USA) (1:5000) conjugated with

alkaline phosphatase (Sigma-Aldrich) for 1 hour. The membranes were washed again as

above, applied to the enhanced chemilunimescence (ECL) detection kit (Amersham, UK), and

exposed to X-ray film (BioMAx films, Eastman Kodak Company, Rochester, NY, USA. To

assess the amount of Cx43 protein loaded, filterswere treated with 2% SDS and 100 mM β-mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8) for 30 min at 60°C and reprobed for

β-actin. Films were scanned, and the optical density of the bandswas measured with Scion Image. Data shown are representativeof at least three independent experiments with similar

results.

Assessment of GJIC was determinedusing the scrape-loading and dye transfer (SLDT)

technique with a fluorescent dye, LuciferYellow (LY, Sigma, USA) (14). Briefly, human

granulosa cells, cultured as described above, were washed thoroughlywith PBS. SL was

performed applying three cuts on cell monolayerwith a surgical scalpel, and then a

mixture of 1% LY and 1% of rhodamine dextran in PBS was added to the cells.The cells

fluorescence. The cells were then fixed with 4% paraformaldehyde and photographed with a

fluorescent microscope equipped with a camera. Cells that receivedthe LY from the

scrape-loaded cells were considered as communicating.The dye-coupled cell layers on either

sides of the scrape were counted to evaluate the GJIC.

Data are expressed as mean ± SEM of at least three independent experiments performed at

different time points. All data were analyzed using one-way analysis of variance (ANOVA).

If the differences were significant, a Dunnett post hoc test was used for post-ANOVA

multiple comparisons. Statistical significance was determined asP < 0.05.

The effect of DHT on Cx43 protein expression in response to 8-Br-cAMP was examined by

Western blot analysis. 8-Br-cAMP, an analogue of cyclic AMP, is known to activate

adenylate cyclase and increase the adenosine cAMP pool in the granulosa cells and

up-regulates the Cx43 expression (15). A previous study showed the range of DHT in

follicularfluid of normal population is 0.77 ± 0.11 ng/ml (16). To demonstrate the effect of

androgen excess on Cx43 expression, cultured human granulosa cells were treated with

increasing doses of DHT at 0, 1, 10, or 100 ng/ml. Western blot analysis of DHT-treated

granulosa cell protein samples demonstrated decreasing amount of Cx43 protein in a

dose-dependent manner. In addition, a significant decrease in the expression could be

To assess whether the suppressive effect of DHT on Cx43 is mediated through the AR, cells

were incubated with DHT (10 ng/ml) and flutamide (100 ng/ml, a 10-fold excessabove that of

DHT). Flutamide, an AR antagonist, significantly blocked the inhibitoryeffects of DHT on

Cx43 expression as shown in Figure 1-A.

To understand the association between reduced Cx43 expression and GJIC activity, we

assessed the ability of HO-23 granulosa cells to transfer Lucifer yellow through gap junctions

using the scrape-loading dye transfer (SLDT) technique in corresponding cultures of cells

with reduced Cx43 expression. As shown in Figure 1-B, the number of dye-coupled cell

layers was significantly reduced on either side of the scrape in HO-23 granulosa cells grown

in 8-Br-cAMP with DHT medium compared with cells grown in 8-Br-cAMP medium. The

phenomenon wasprevented upon replenishing the medium with flutamide. This gap

junctional defect could be induced with DHT and the effect was recovered with addition of

flutamide to the culture medium.

Although anovulation is frequently observed in PCOS patients with androgen excess, the

underlying mechanism has not been fully elucidated. Several possibilities have been raised to

explain how androgens might mediate anovulation. In this study, we propose a new pathway

through which high androgen level down-regulates Cx43 expression, reduce GJIC in human

granulosa cells, and finally lead to impaired folliculogenesis.

ovarian physiology in animals (17, 18). One of the targets of this disruptive action is the

granulosa cell within the ovarian follicle. While both androgen and gap junctions are reported

to have important roles in follicular development, our study provides a novel mechanism

which links androgen excess and Cx43 expression in the granulosa cells. We showed reduced

GJIC activity in the milieu of high androgen and reduced Cx43 expression which occurs at

the translation level mediated through androgen receptor.

A link between reduced GJIC activity and reduced connexin gene expression has been

previously reported in rat ovarian cells exposed to luteinizing hormone (LH). LH inhibited

GJIC activity by down-regulating Cx43 synthesis (19). Recently, Wang et al. (20) provided

the evidence that the strength of gap junctional conductance was positively correlated with

Cx43 level in human cumulus cells. Although it is possible that the effect of high androgen on

GJIC activity may be mediated via other junction protein components, our findings indicate

that reduced GJIC activity in human granulosa cells exposed to high androgen is at least in

part due to inhibition of Cx43 gene expression.

The expression and function of Cx43 protein in the ovary of mammalian species have

been discussed previously (21). In the mouse ovary, Cx43 is an important gap junction

protein required for granulosa cell to granulosa cell communication, which in turn is required

for follicle and oocyte maturation (7). Decreased Cx43 protein level is associated with

a coordinating role in the process of atresia (12). Disruption of granulosa cell couplingnot

only impairs follicle growth, but also impacts negativelyon the quality of the enclosed

oocytes. The knock-out mice of Cx43 showed that GJIC activity was reduced and oocyte

growth in mutant follicles was retarded (22). However, we know little about Cx43 in human

ovarian follicles and their roles in human folliculogenesis and fertility. A recent study shows

that Cx43 was primarily localized in the membrane where it forms gap junction-like plaques

between human cumulus cells, and its expression level may influence pregnancy outcome

from in vitro fertilization (20). Furthermore, high Cx43 levels in granulosa cells are linked to

good prognosis of human oocytes (9).

The roles of androgen and androgen receptor (AR) are well-established in the

development of male reproductive organs and spermatogenesis. However, the functions of

this steroid and its receptor in the ovary remain elusive. AR expression in the ovary has been

described at various stages of folliculogenesis in several species. In human ovaries, AR

protein expression is observed at different stages in granulosa and theca cells (23). Using a

Cre–loxP system to generate AR-knockout (ARKO) mice, investigators showed that activated

ARs are indispensable for normal folliculogenesis (24, 25). In this study, we demonstrate that

androgen regulates Cx43 expression in cultured human granulosa cells by an AR-dependent

process considering suppressive effectof DHT on Cx43 was blocked by an AR antagonist,

treatment ofpatients with PCOS and anovulation. In fact, flutamide is able to restore

ovulation in some women with PCOS (26, 27). However, there is still concern about the use

of flutamide in women with infertility considering its possible teratogenic effect (28).

The root cause of anovulation in PCOS is now thought to be associated with the

significant abnormalities in the very earliest stages offolliculogenesis (29). The normal

„dialogue‟ between oocyte and granulosa cells in these early growing follicles is altered. Recently, Li et al. (30) raised a hypothesis that if dysfunctional communications exist

between granulosa cells or granulosa cells and the oocyte, unnecessary androgen will enter

the oocyte and bind to the AR, provoking some follicles to undergo atresia. We therefore

propose a working mode for the effect of androgen excess on folliculogenesis and oogenesis.

High androgen level down-regulates Cx43 translation and impairs the communication

between granulosa cells and possibly communications between granulosa cells and oocyte.

Impaired communications negatively impacts folliculogenesis. The adverse effect of

androgen excess is further strengthened by “shunting” of excessive androgens to the oocytes secondary to impaired communications between granulosa cells.

In conclusion, the presentstudy provides the first evidence that increased levels of

androgen down-regulate Cx43 expression and impair communications between granulosa

cells. The effect may compromise folliculogenesis and oogenesis in PCOS patients. Caution

clinical setting considering few clinical data in this study. In addition, how DHT affects Cx43

translation still awaits further investigation.

Acknowledgment:

This study was supported by grants from the Changhua Christian Hospital

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Figure legends

Figure 1-A. Antiandrogen treatment blocked the inhibitoryeffects of DHT on Cx 43

expression. HO-23 cells were cultured for 24 h under serum-free conditions in the presence of

8-Br-cAMP (0.1 mM), DHT (10ng/ml), and the antiandrogen flutamide (100 ng/ml). Data are

expressed as the mean ± SD of three different experiments. (＊: p < 0.05 vs. 8-Br-cAMP)

B: 8-Br-cAMP D: DHT F: flutamide

Figure 1-B: Analysis of GJIC activity in HO-23 cells using SLDT technique. RD remains in

the cells immediately bordering the scrape, while the LY diffuses to neighboring cells in gap

junction-enabled cells. GJIC was evaluated as the counts from dye-coupled cell layers and is

represented as a histogram. GJIC is blocked in HO-23 cells treated with 8-Br-cAMP (0.1 mM)

blockage of GJIC induced by DHT to a comparable level to 8-Br-cAMP (ns, not significant

vs 8-Br-cAMP). Data are expressed as the mean ± SD of three different experiments.

B: 8-Br-cAMP D: DHT F: flutamide

GJIC: gap junction intercellular communication

SLDT: scrape-loading and dye transfer

We have answered the editorial’s and reviewers’ comments in the revision as

below:

Editorial’s comment:

1) We had corrected

Reference 4.

2)

Our abstract had been shortened to two sentences.

Reviewer’s comment:

1) We had corrected into “induce” and “lead”. (

Abstract, lines 30, 31)

2)

We change mammalians into mammalian. (Page 10, line 39)

3)

Figure 1-B, bottom: change B+D+ into B+D+F

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Cheng-Hsuan Wu