Kashino, et al.
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Original paper

Involvement of BMP-15 in glucocorticoid actions on ovariansteroidogenesis by rat granulosa cells.

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Chiaki Kashino, Toru Hasegawa, *Yasuhiro Nakano, *Nahoko Iwata, *Koichiro
Yamamoto, Yasuhiko Kamada, Hisashi Masuyama and *Fumio Otsuka

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Department of Obstetrics and Gynecology and *Department of General Medicine,9

Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, Okayama, Japan

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Running title: Glucocorticoid effects on ovarian steroidogenesis
Key words: Bone morphogenetic protein (BMP), Glucocorticoid, Glucocorticoid

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receptor (GR), Granulosa cells, and Steroidogenesis

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Disclosure Statement: The authors have nothing to disclose.

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Corresponding author: Fumio Otsuka, M.D., Ph.D.
Department of General Medicine, Okayama University Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
Address: 2-5-1 Shikata-cho, Kita-ku, Okayama City, 700-8558, Japan
Phone number: +81-86-235-7342, Fax number: +81-86-235-7345
E-mail address: fumiotsu@md.okayama-u.ac.jp

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Abbreviations:
ActRII, activin type-II receptor; Aldo, aldosterone; ALK, activin receptor-likekinase; 20HSD, 20-hydroxysteroid dehydrogenase; 3βHSD, 3βhydroxysteroid dehydrogenase; BMP, bone morphogenetic protein; BMPRII,BMP type-II receptor; Dex, dexamethasone; FSH, follicle-stimulating hormone;
FSHR, FSH receptor; GR, glucocorticoid receptor; HPO, hypothalamo–pituitary–ovarian; MR, mineralocorticoid receptor; PCOS, polycystic ovary syndrome;
P450arom, P450 aromatase; P450scc, P450 steroid side-chain cleavage
enzyme; RPL19, ribosomal protein L19; and StAR, steroidogenic acuteregulatory protein.

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Word counts: 4431 words,

Figure number: 4 figures
1Kashino, et al.
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Abstract2

To elucidate the impact of glucocorticoids on ovarian steroidogenesis and its

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molecular mechanism by focusing on bone morphogenetic proteins (BMPs), we

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examined the effect of dexamethasone (Dex) on estradiol and progesterone

5synthesis by using primary culture of rat granulosa cells.

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Dex treatment dose-dependently decreased estradiol production but increased

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progesterone production induced by follicle-stimulating hormone (FSH) by

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granulosa cells.

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Dex suppressed P450arom mRNA expression and cAMP synthesis induced by

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FSH.

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mRNAs encoding the enzymes for progesterone synthesis including P450scc

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and 3HSD but not StAR and 20HSD.

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upregulated transcription of the BMP target gene Id-1 and Smad1/5/9

14phosphorylation in the presence of BMP-15 among the key ovarian BMP ligands.

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It was also found that Dex treatment increased the expression level of BMP type-16

I receptor ALK-6 among the type-I and -II receptors for BMP-15.

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Smad6/7 expression was not affected by Dex treatment.

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BMP-15 treatment upregulated glucocorticoid receptor (GR) expression in

It was revealed that

In accordance with the effects of Dex on estradiol synthesis,

Dex treatment in turn enhanced basal as well as FSH-induced levels of2

Of note, Dex treatment significantly

Inhibitory

On the other hand,Kashino, et al.
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granulosa cells.

Collectively, it was revealed that glucocorticoids elicit

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differential effects on ovarian steroidogenesis, in which GR and BMP-15 actions

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are mutually enhanced in granulosa cells.

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Introduction

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The glucocorticoid receptor (GR) is known to be expressed in the ovary

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and it has been shown that glucocorticoids exert direct effects on granulosa cell8functions [1].

9hypermenorrhea can be manifested in patients with glucocorticoid excess.

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the other hand, glucocorticoids can also be used for treatment of polycystic ovary

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syndrome (PCOS) with hyperandrogenism [2].

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glucocorticoids on the reproductive system have not been fully elucidated.

In a clinical setting, menstrual abnormality, amenorrhea andOn

However, the effects of13

It has been shown that moderate levels of glucocorticoids can support

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female reproductive processes at the anterior pituitary level and may have an15

anti-inflammatory function in the ovary and adjacent tissues [1,3,4]. However,16

stress levels of glucocorticoids adversely affect hypothalamo–pituitary–ovarian17(HPO) function in the adult females.

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secretion of gonadotropins in both genders [5,6], although this effect in males is

Stress and glucocorticoids inhibit the3Kashino, et al.
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modified by inhibitory effects of glucocorticoids on the testes.

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ovarian response to gonadotropins such as glucocorticoid-induced inhibition of

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steroidogenesis by human granulosa-luteum cells [7] and inhibition of

4progesterone synthesis by rat preovulatory follicles [8] has been experimentally

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shown.

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reproductive system seems to be complex and cannot be simply explained by the7interaction of glucocorticoids and GR [6,9-11], in which central factors are also

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involved in the mechanism of glucocorticoid- and/or stress-induced suppression

9of the HPO axis [6,12,13].

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However,

glucocorticoid-induced

suppressionofDiminishedthefemaleNevertheless, the research on the direct effects of glucocorticoids on11

ovarian reproductive functions has been very limited [14-17].

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study, primary granulosa cells obtained from rat ovaries were used to clarify the13

direct effects of glucocorticoids on ovarian steroidogenesis integrated by follicle-

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stimulating hormone (FSH) and various growth factors expressed in the ovary.

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Mutual interactions among gonadotropins and the growth factors including bone16

morphogenetic proteins (BMPs), growth differentiation factors (GDFs) and17

activins/inhibins are critical for normal growth and maturation of ovarian follicles

18[18,19]. Among these factors, the ovarian BMPs play regulatory roles on the

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In the presentKashino, et al.
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activity of FSH receptor (FSHR) expressed on granulosa cells, leading to

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inhibition of progesterone biosynthesis and luteinizing process [20-22].

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present study elucidated specific activity of glucocorticoids in the enhancement4of progesterone production by granulosa cells and the functional interaction with5

the ovarian BMP system.

The

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Materials and Methods

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Reagents and supplies

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Dexamethasone (Dex), diethylstilbestrol (DES), ovine FSH, 4-androstene-3,17-10

dione (androstenedione: a substrate for aromatase) and 3-isobutyl-1-

11methylxanthine (IBMX: a phosphodiesterase inhibitor) were purchased from

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Sigma-Aldrich Co. Ltd. (St. Louis, MO).

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-4, -9 and -15 was purchased from R&D Systems Inc. (Minneapolis, MN).

Recombinant protein of human BMP-2,

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Primary culture of rat granulosa cells

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DES (10 mg) packed in silastic tubes was implanted subcutaneously in the back17

of 22-day-old female Sprague-Dawley rats (Charles River, Wilmington, MA).

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After three days of exposure to DES, the ovaries were resected and punctured

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with a 27-gauge needle.

Granulosa cells were separated from oocytes by

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passing the cell suspension in Medium 199 (Thermo Fisher Scientific, Waltham,3MA) through nylon meshes of different pore sizes (100 and 40 µm) (BD Falcon,

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Bedford, MA), as we previously reported [23-25].

5cultured in McCoy’s 5A (Thermo Fisher Scientific) supplemented with penicillin-

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streptomycin without serum.

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University Institutional Animal Care and Use Committee (OKU-2018704).

The granulosa cells were

The animal protocol was approved by Okayama

89Measurements of ovarian steroids and cAMP

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Granulosa cells were seeded in 96-well plates with McCoy’s 5A (1 × 105 viable

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cells in 200 µl) and androstenedione (100 nM). The cells were cultured for 48 h

12after adding indicated concentrations of Dex to the medium either alone or with

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FSH (10 ng/ml). The levels of ovarian steroids in the medium were measured

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using estradiol and progesterone enzyme-linked immunosorbent assay (ELISA)

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kits (Cayman Chemical Co., Ann Arbor, MI).

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undetectable in the cell-free medium (progesterone < 10 pg/ml and estradiol < 1517

pg/ml). Cellular synthesis of cAMP was evaluated in granulosa cells (1 × 105

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viable cells in 200 µl) cultured with IBMX (0.1 mM) for 48 h in 96-well plates.

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The levels of steroids wereKashino, et al.
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The concentrations of extracellular cAMP in the medium were measured by a2

cAMP ELISA kit after acetylation, and the assay sensitivity was 0.039 nM (Enzo

3Life Sciences, Inc., Farmingdale, NY).

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Quantitative reverse transcription-PCR of extracted RNA

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Granulosa cells (5 × 105 viable cells in 1 ml) were cultured in 12-well plates with

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serum-free McCoy’s 5A and were treated with FSH (10 ng/ml) and BMP ligands

8(100 ng/ml) either alone or with Dex (100 nM). After the treatments for 24 to 48

9h, total RNA was extracted using TRI Reagent® (Cosmo Bio Co. Ltd., Tokyo) and10

was quantified using a NanoDropTM One spectrophotometer (Thermo Fisher

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Scientific). The primers for genes related to steroidogenesis including Star (also12indicated as StAR), Cyp11a (P450scc), Hsd3b (3βHSD), and Cyp19 (P450arom),

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for genes related to the BMP receptor and the signaling including Acvr1 (ALK-2),

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Bmpr1a (ALK-3), Bmpr1b (ALK-6), Bmpr2 (BMPRII), Acvr2a (ActRIIA), Acvr2b

15(ActRIIB), Smad6, Smad7, and Id1 (inhibitor of DNA binding 1: the BMP target

16gene), for Gr (glucocorticoid receptor), and for a housekeeping gene, Rpl19

17(RPL19) were chosen on the basis of previous reports [23-27].

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were selected from different exons of the corresponding genes to distinguish the7

The primer pairsKashino, et al.
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target PCR products from contaminants in the chromosomal DNA. The primer

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pairs for Hsd20a (20HSD) were selected from 776-795 and 1027-1046 of gene3

accession# AB028066.

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ReverTra Ace® (TOYOBO Co., Ltd. Osaka) with random primers and5deoxynucleotide triphosphate.

6LightCycler® 96 system (Roche Diagnostic Co., Tokyo) under the appropriate

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annealing conditions and amplification efficiency.

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were quantified relatively using the comparative threshold cycle (Ct) method and9were expressed as 2-(ΔΔCt) using Rpl19 as the internal control.

The RNA (1 µg) was reverse-transcribed using

Quantitative PCR was performed using the

The mRNA expression levels

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11Western blotting

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Granulosa cells (2.5 × 105 viable cells in 500 µl) were cultured in 24-well plate in

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serum-free McCoy’s 5A.

14nM), the cells were treated with BMP-15 (100 ng/ml) for 60 min.

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cells were solubilized in 100 µl of RIPA lysis buffer (Upstate Biotechnology, Lake

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Placid, NY) supplemented with 4% β-mercaptoethanol and 2% SDS.

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lysates were subjected to SDS-PAGE/immunoblotting analysis with antibodies

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against phopho-Smad1/5/9 (pSmad1/5/9), total-Smad1 (tSmad1; Cell Signaling

After culturing for 24 h either alone or with Dex (1008

The treated

The cellKashino, et al.
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Technology, Inc., Beverly, MA) and actin (Sigma-Aldrich Co. Ltd.). Each band2

was analyzed using the C-DiGit® Blot Scanner System (LI-COR Biosciences,

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Lincoln, NE). The target signal intensities were expressed as a relative value to4

the corresponding actin intensities.

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Statistics

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The results are presented as means ± SEM of at least three separate

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experiments with triplicated samples.

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ANOVA followed by Fisher's protected least significant difference (PLSD) test and10

the unpaired t-test (StatView 5.0 software, Abacus Concepts, Inc., Berkeley, CA).

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A significant difference was defined as P values < 0.05.

Statistical analysis was performed using

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Results

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We

first examined the effects of Dex

treatment on

ovarian15

steroidogenesis by rat primary granulosa cells. After treatment with Dex (1 to16

300 nM) for 48 h in a serum-free condition, the changes in estradiol and17

progesterone production induced by FSH treatments were examined. As shown18

in Fig. 1A, it was revealed that Dex treatment reduced estradiol production

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induced by FSH (10 ng/ml) but increased progesterone production induced by2

FSH by granulosa cells in a concentration-dependent manner (Fig. 1A).

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To try to determine the mechanism by which Dex regulates ovarian4

steroidogenesis, the mRNA expression levels of steroidogenetic protein and5

enzymes were evaluated.

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ng/ml) or Dex (100 nM) for 48 h were extracted and mRNA levels of

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steroidogenetic factors and enzymes were quantified by real-time RT-PCR.

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accordance with the effects of Dex on estradiol synthesis, Dex (100 nM)

9treatment suppressed P450arom mRNA expression (Fig. 2A).

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synthesis stimulated by FSH (10 ng/ml) was also suppressed by Dex (100 nM)

11treatment for 48-h culture of granulosa cells (Fig. 2B).

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Dex treatment significantly enhanced the expression of mRNAs encoding the13

enzymes for progesterone synthesis including P450scc and 3HSD, but not StAR,

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induced by FSH (10 ng/ml).

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were also increased by treatment with Dex (100 nM) for 48 h (Fig. 2B), although16

the mRNA levels of StAR and 20HSD, which alters progesterone to an inactive

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form, were not affected by the Dex treatment.

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predominantly converts progesterone to its biologically inactive form, 20-Total RNAs of granulosa cells treated with FSH (10In

The cAMPAs shown in Fig. 2C,

Basal levels of mRNAs of P450scc and 3HSD

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The enzyme 20HSDKashino, et al.
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1hydroxyprogesterone, and has an important role in the termination of pregnancy

2and the initiation of parturition [28,29].

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Next, the effects of Dex on BMP receptor signaling by granulosa cells

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were evaluated.

Total RNAs of granulosa cells treated with BMPs (100 ng/ml)

5in combination with Dex (100 nM) for 48 h were extracted and mRNA levels of

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steroidogenetic factors and enzymes were quantified by quantitative PCR.

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note, Dex treatment significantly upregulated transcription of the BMP-target

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gene Id-1 induced by BMP-15 (100 ng/ml) compared with that induced by other

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BMP ligands including BMP-2, -4 and -9 (Fig. 3A).

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analysis using cell lysates showed that treatments with both BMP-15 and Dex

11effectively upregulated Smad1/5/9 phosphorylation by granulosa cells (Fig. 3B).

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It was also found that Dex treatment increased the expression level of the BMP

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type-I receptor ALK-6 among the type-I and -II receptors for BMP-15 (Fig. 3C).

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Inhibitory Smad6/7 expression was not affected by Dex treatment. On the other15

hand, BMP-15 treatment upregulated GR expression in granulosa cells (Fig. 3D).

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Thus, it was revealed that glucocorticoids elicit differential effects on ovarian

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steroidogenesis, in which GR and BMP-15 actions are mutually enhanced in

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granulosa cells.

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Of

Furthermore, Western blot

Kashino, et al.
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Discussion

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In the present study, it was revealed that Dex treatment decreased

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estradiol production but increased progesterone production induced by FSH by

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granulosa cells in vitro (Fig. 4).

6mRNA expression and cAMP synthesis induced by FSH, whereas Dex enhanced

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basal as well as FSH-induced levels of mRNAs encoding the enzymes for8

progesterone synthesis including P450scc and 3HSD.

9treatment was found to upregulate BMP-Smad1/5/9 signaling in the presence of10

BMP-15 among the key ovarian BMP ligands via upregulating ALK-6, which is the11

main type-I receptor for BMP-15, while BMP-15 upregulated GR expression in

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granulosa cells.

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ovarian steroidogenesis, in which GR and BMP-15 activities seem to be mutually

14enhanced in granulosa cells (Fig. 4).

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Dex treatment also suppressed P450arom

Importantly, Dex

Namely, glucocorticoids can elicit differential effects on

In the ovarian follicles, the BMP system including BMP ligands and the16

receptors has been known to be expressed in a cell-specific manner [20].

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mammals, the ovarian BMP system has been shown to play crucial roles in the18

maintenance of female fertility [19-22].

12In

In addition to the ligand-dependentKashino, et al.
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actions of BMPs in the process of normal folliculogenesis, a common key function

2of BMPs has been recognized to regulate the FSHR sensitivity expressed on

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granulosa cells, [30-32].

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predominantly enhanced in granulosa cells derived from ovarian tissues of PCOS

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patients compared with normal patients [33-35], implying that upregulation of

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BMPs are involved in the disruption of follicular development in the ovaries of

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PCOS.

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ALK-6 was revealed to be upregulated by glucocorticoid treatment, implying a9functional interaction between GR and the BMP system in granulosa cells.

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For instance, the expression of BMP-6 was

In the present study, the expression level of the BMP type-I receptor

Heush et al. [14] reported the effects of glucocorticoids on

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steroidogenesis of ovarian granulosa cells.

Treatments with cortisol and12dexamethasone suppressed the increase in P450arom activity induced by FSH

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in rat granulosa cell culture.

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production was stimulated to a maximum of ~1.7 fold of the control level. These

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differential effects of glucocorticoids on estrogen production and progesterone

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production indicate that glucocorticoids elicit selective inhibition of P450arom

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activity induced by FSH via a mechanism other than interfering with the18

aromatization reaction in granulosa cells.

In the same culture condition, progesterone

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In this regard, Adashi et al. [15] also reported direct effects of2

glucocorticoids on basal and FSH-stimulated production of progesterone by3

ovarian granulosa cells from immature, hypophysectomized, estrogen-treated

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rats.

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with glucocorticoid rather than mineralocorticoid potencies.

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underlying mechanism, treatment with glucocorticoids led to a significant

7decrease in the activity of 20HSD detected by the conversion rate of

8progesterone, leading to an increase in progesterone production [15]. However,9

the results were not reproduced by our experiments, which showed that 20HSD

10mRNA expression was not altered by Dex treatment in granulosa cells.

In that study, the stimulatory effects of the corticoids tested were correlated
As for the11

In addition to glucocorticoid activity in granulosa cells, we found in our

12earlier study that aldosterone (Aldo) treatment enhanced FSH-induced

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progesterone, but not estradiol, production [36].

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Aldo treatment suppressed BMP-6-induced Smad1/5/9 signaling with increased

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expression of inhibitory Smad6.

16(MR) expression in granulosa cells was downregulated by BMP-6, showing the

17functional interaction as a negative feedback between BMP and MR signaling.

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Given that the ovarian BMP system acts as suppressor for progesterone

Of interest, it was found thatOn the other hand, mineralocorticoid receptor

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biosynthesis as a luteinizing inhibitor [20-22], Aldo plays an inhibitory role in the2endogenous BMPs, leading to the progesterone accumulation in the growing

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follicles.

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Increased androgen action and its receptor activity in various stages of5developing follicles have been shown to be involved in the pathogenesis of PCOS

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[37].

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growth factor-I enhanced FSH-induced progesterone synthesis by rat granulosa

8cells [23].

9production was similar to the effects of Aldo, both of which are functionally linked

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to the counteraction of endogenous BMPs in granulosa cells, leading to the11enhancement of progesterone biosynthesis.

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BMP activity, which was different from that of androgen or Aldo, resulted in the13

upregulation of BMP-15 signaling.

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BMP-receptor signaling may reflect a negative feedback function to suppress

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progesterone production directly enhanced by Dex treatment by granulosa cells.

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In a clinical setting, glucocorticoids such as prednisone andWe previously reported that androgen in combination with insulin-like

The impact of androgen action that enhanced progesterone

However, the effects of Dex on

This finding implies that the upregulation of17

dexamethasone have been used to induce ovulation [2].

18a high adrenal androgen level, low-dose dexamethasone can be used to attain a

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In PCOS patients with

Kashino, et al.
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higher ovulation rate and higher cumulative pregnancy rate.

Such effects of2

glucocorticoids on successful ovulation and/or pregnancy may be involved in the3

activity of glucocorticoids on the enhancement of progesterone as well as BMP

4signaling in granulosa cells as shown in the present study.

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Collectively, the results indicated that glucocorticoids elicit differential

6effects on ovarian steroidogenesis of estradiol and progesterone, in which GR

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and BMP-15 actions are mutually enhanced in granulosa cells.

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15 acts as an inhibitor for progesterone production by suppressing FSH-receptor

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actions [38,39], the results suggested that glucocorticoids are functionally linked

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to the enhancement of endogenous BMP-15, leading to negative feedback

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toward the progesterone overproduction induced by FSH and Dex in granulosa

12cells (Fig. 4).

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effects on ovarian steroidogenesis, in which GR and BMP-15 actions are mutually

14enhanced in granulosa cells.

Given that BMP-

Hence, it was revealed that glucocorticoids elicit differential

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Acknowledgements

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This work was supported in part by Grants-in-Aid for Scientific Research(18K08479 and 21K08556).

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Kashino, et al.
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1

Figure Legends:

2

Fig. 1. Effects of glucocorticoid on FSH-induced steroidogenesis by rat

3

granulosa cells.

4of dexamethasone (Dex) either alone or in combination with FSH in a serum-free5

condition for 48 h.

6

the medium were evaluated by ELISA and expressed as fold changes.

7

in all panels are shown as means ± SEM of data from at least three individual

8experiments with triplicated samples.

9

Fisher's PLSD test.

Granulosa cells were cultured with indicated concentrations

The concentrations of A) estradiol and B) progesterone in
Results

The results were analyzed by ANOVA with

10

11

Fig. 2. Effects of glucocorticoid on steroidogenetic enzyme expression by

12

rat granulosa cells.

13

treated with FSH and Dex for 48 h.

14

levels were determined by real-time PCR. The target gene mRNA levels were15

standardized by Rpl19 levels and expressed as fold changes.

16

cultured with Dex either alone or in combination with FSH in a serum-free medium

17

containing IBMX for 48 h.

18

ELISA.

A, C) Cellular RNA was extracted from granulosa cells
Cyp19, Star, Cyp11a, and Hsd3b mRNAB) Cells were

The cAMP levels in the medium were examined by

Results in all panels are shown as means ± SEM of data from at least

21

Kashino, et al.
BBRC

1

three individual experiments with triplicated samples.

The results were2

analyzed by ANOVA with Fisher's PLSD test and the unpaired t-test. Values with3

different superscript letters show significant difference at P < 0.05; and *P < 0.05

4

vs. control group.

5

6

Fig. 3. Effects of glucocorticoid activity on BMP signaling in rat granulosa

7cells.

82, - 4, -9 and -15 in the presence or absence of Dex for 24 h, and Id1 mRNA

9levels were determined by real-time PCR. The target gene mRNA levels were10

standardized by Rpl19 levels and expressed as fold changes.

11

cultured in a serum-free condition in the presence or absence of Dex for 48 h.

12

The cells were lysed and subjected to Western blot analysis using anti-Smad1/5/9,

13

tSmad1 and anti-actin antibodies.

14

Smad6 were standardized by actin signal intensities in each sample and15

expressed as fold changes.

16

treated with Dex or BMP-15 for 48 h and mRNA levels of BMP receptors,

17

inhibitory Smads and Gr were determined by real-time PCR.

18

panels are shown as means ± SEM of data from at least three individual

A, C) Cellular RNA was extracted from granulosa cells treated with BMP-

B) Cells were

The signal intensities of pSmad1/5/9 andC, D) RNA was extracted from granulosa cells

22

Results in allKashino, et al.
BBRC

1experiments with triplicated samples.

The results were analyzed by ANOVA with2

Fisher's PLSD test and the unpaired t-test.

*P < 0.05 vs. control group.

3

4

Fig. 4. Mechanism by which glucocorticoid regulates steroidogenesis via

5

BMP-Smad signaling in granulosa cells. Dex decreased estradiol production

6

but increased progesterone production induced by FSH by granulosa cells.

7

also suppressed P450arom mRNA expression and cAMP synthesis induced by

8

FSH, whereas Dex enhanced basal as well as FSH-induced levels of mRNAs

9

encoding the enzymes for progesterone synthesis including P450scc and 3HSD.

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Dex upregulated BMP-15/Smad1/5/9 signaling via upregulating the BMP type-I

11

receptor, while BMP-15 upregulated GR expression.

12

were mutually enhanced in granulosa cells.

13

BMP receptors; and FSHR, FSH receptor.

14

23

Dex

GR and BMP-15 actions

AC, adenylate cyclase; BMPRs,