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Anti-Mullerian hormone (AMH) is induced by bone morphogenetic protein (BMP) cytokines in human granulosa cells
European Journal of Obstetrics & Gynecology and Reproductive Biology 164 (2012) 44–47
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Anti-Mullerian hormone (AMH) is induced by bone morphogenetic protein (BMP) cytokines in human granulosa cells Sayaka Ogura-Nose, Osamu Yoshino *, Yutaka Osuga *, Jia Shi, Hisahiko Hiroi, Tetsu Yano, Yuji Taketani Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 November 2011 Received in revised form 6 April 2012 Accepted 5 May 2012
Objectives: Serum concentration of anti-Mullerian hormone (AMH) is used as a biomarker in clinical practice. Therefore, it is important to elucidate the mechanism by which AMH is regulated in granulosa cells (GC). An important first step in understanding AMH regulation is to determine which factors upregulate AMH expression. Study design: Human GC, obtained from 28 women undergoing oocyte retrieval for in vitro fertilization, were stimulated with various intraovarian cytokines including bone morphogenetic protein (BMP)-2, -6, -7 -15, activin-A and growth differentiation factor (GDF)-9 (100 ng/ml). The expression of AMH mRNA was evaluated with reverse transcription and quantitative real-time polymerase chain reaction (PCR), and AMH protein in cultured supernatant was measured with EIA kit. Results: BMP-2, -6, -7 and -15, but not activin-A and GDF-9, significantly induced AMH expression in GC at mRNA and protein level, while all stimuli increased FSH receptor mRNA and decreased steroidogenic acute regulatory protein (StAR) mRNA level. Conclusions: Among the transforming growth factor (TGF)-b superfamily, BMP-2, -6, -7 and -15 significantly induced AMH expression in human GC. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: AMH BMP GDF-9 Ovary Granulosa cell
1. Introduction In infertility treatment, the precise assessment of ovarian reserve is necessary to plan a treatment strategy for each patient. Among all available markers of ovarian reserve, much interest has been given to anti-Mullerian hormone (AMH) as a reliable, accurate and reproducible predictor [1,2]. AMH, which belongs to the transforming growth factor (TGF)-b superfamily, is a product of the granulosa cells (GC) in pre-antral and small antral follicles [3]. As serum concentration of AMH is considered an excellent biomarker in clinical practice [1,2], it is important to elucidate the mechanism by which AMH is regulated in GC. Although it has been reported that follicle stimulating hormone (FSH) and estradiol down-regulate AMH expression in GC [4], the factors which upregulate AMH expression are still unknown. Previously, we found that bone morphogenetic protein (BMP)-6, an intra-ovarian factor,
* Corresponding authors at: Department of Obstetrics and Gynecology, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: +81 3 3815 5411x33407; fax: +81 3 3816 2017. E-mail addresses: [email protected] (O. Yoshino), [email protected] (Y. Osuga). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.05.017
increased the expression of AMH in human GC [5]. In addition to BMP-6, other TGF-b superfamily cytokines including BMP-2, -7 and -15, growth differentiation factor (GDF)-9 and activin-A are known to regulate ovarian physiology including gonadogenesis [6,7], folliculogenesis, ovulation and luteinization in various species [8]. In the human ovary, it has been reported that GC express BMP-2, -6 and activin-A, while theca cells express BMP-7 and oocytes express BMP-6, -15 and GDF-9 respectively [5,8–10]. In the present study, we validated the hypothesis that TGF-b superfamily member cytokines might induce AMH expression in GC. 2. Materials and methods 2.1. Reagents and materials Hyaluronidase, fetal bovine serum (FBS), DMEM/F12 and antibiotics (mixture of penicillin, streptomycin, and amphotericin B) were purchased from Sigma (St. Louis, MO). Recombinant human BMP-2, -6, -7, and -15, GDF-9 and activin-A were purchased from R&D Systems (Minneapolis, MN). Recombinant human FSH was kindly provided by Japan Organon (Tokyo, Japan). The concentrations of AMH in supernatant were measured using an enzymatic immunoassay (EIA) kit purchased from Beckman Coulter (Tokyo, Japan).
S. Ogura-Nose et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 164 (2012) 44–47
Written informed consent was obtained from all study participants. Ethical approval was given by Tokyo University Ethics Committee.
45
3. Results 3.1. Effect of various intra-ovarian factors and FSH on AMH expression in human GC
2.2. Cell culture of human GC GC were aspirated from 28 patients who had been undergoing ovarian stimulation for in vitro fertilization (IVF). The method for obtaining and culturing human GC was described previously [11]. Briefly, following ovarian suppression by the gonadotropinreleasing hormone analog buserelin acetate (Suprecur, Mochida Pharma, Tokyo, Japan) from the midluteal phase of the preceding cycle, a dose of 150–300 IU human menopausal gonadotropin was given daily until the diameter of the leading follicle reached 17 mm or greater. Then, hCG at a dose of 10,000 IU was administered and transvaginal ultrasound-guided oocyte retrieval was performed 35 h later. The collected human GC were cultured in 500 ml of DMEM/F12 containing 5% FBS and antibiotics in 12-well plates at a density of 2 105 cells/ml and kept at 37 8C in a humidified 5% CO2/95% air environment for 5 days. All of the GC used for the experiments were pre-cultured for 5 days prior to treatments to allow the GC to regain sensitivity to FSH stimulation [12]. The media were changed every 48 h. To evaluate the effects of cytokines, human GC were cultured with or without BMP-2, -6, -7, and -15, GDF-9 or activin-A (100 ng/ml) for 24 h. In the pilot study, we found that this concentration could induce the maximum effect of cytokines on AMH mRNA expression (data not shown). Recombinant cytokines were dissolved in 0.1% bovine serum albumin + 4 mM of HCl as a vehicle. The same amount of vehicle was used for control samples. In some experiments, GC were stimulated with recombinant human FSH (0.5 IU/ml) for 24 h.
Treatment with BMP-2, -6, -7 and -15 (100 ng/ml) stimulated over 9-, 7-, 5-, and 4-fold increases respectively in the AMH mRNA level of human GC (Fig. 1a). Although activin-A (100 ng/ml) induced approximately 2-fold increase of AMH mRNA, there was no statistical difference between control and activin-A stimulation
2.3. Reverse transcription and quantitative real-time polymerase chain reaction analysis Total RNA was extracted from GC, using the RNAeasy minikit (Qiagen, Hilden, Germany). Reverse transcription (RT) was performed using Rever Tra Dash (TOYOBO, Tokyo, Japan). One microgram of total RNA was reverse transcribed in a 20-ml volume. For the quantification of various mRNA levels of AMH, FSH receptor and steroidogenic acute regulatory protein (StAR), real-time polymerase chain reaction (PCR) was performed using LightCycler (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions. The PCR primers were selected from different exons of the corresponding genes to discriminate PCR products that might arise from possible chromosomal DNA contaminants. The primer sequences and PCR conditions have been described elsewhere [5,10]. Expression of each mRNA was normalized by GAPDH mRNA. Experiments were conducted at least three times independently, and each sample assayed in duplicate. 2.4. Measurement of AMH concentration in cultured supernatant AMH concentration in cultured supernatant was measured using the EIA kit according to the instructions. The functional sensitivity of the AMH EIA kit used was above 0.5 pmol/L, and both intra- and inter-assay coefficients of variation of this kit were less than 15%. 2.5. Statistical analysis Data were analyzed by one-way ANOVA with post hoc test for multiple comparisons using Statview software (SAS Institute Inc., Cary, NC). A p-value of less than 0.05 was considered statistically significant.
Fig. 1. mRNA expression induced by stimuli of various intra-ovarian cytokines. Cultured human granulosa cells (GC) were stimulated with BMP-2, -6, -7, and -15, activin-A or GDF-9 (100 ng/ml) for 24 h. Total RNA was extracted from the cells and subjected to real-time PCR to determine various mRNA levels. Data were normalized by GAPDH mRNA level, and shown as the mean standard error of the mean (SEM) relative to an adjusted value of 1.0 for the mean value of the control. *p < 0.05 (vs. control). All experiments were conducted at least three times independently, and each sample was assayed in duplicate. (1-a) Anti-Mullerian hormone (AMH) mRNA; each column represented an average from 7 samples. (1-b) FSH receptor mRNA; representative data from 3 different experiments. (1-c) Steroidogenic acute regulatory protein (StAR) mRNA; representative data from three different experiments.
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S. Ogura-Nose et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 164 (2012) 44–47
1.2
AMH mRNA
fold increase
1 0.8
*
0.6 0.4 0.2
* P<0.05
0
Control
FSH
Fig. 2. Anti-Mullerian hormone (AMH) mRNA expression stimulated with FSH. Cultured human granulosa cells (GC) were stimulated with recombinant FSH (0.5 IU/ml) for 24 h. Total RNA was extracted from the cells and subjected to realtime PCR to determine AMH mRNA levels. Data were normalized by GAPDH mRNA level, and shown as the mean standard error of the mean (SEM) relative to an adjusted value of 1.0 for the mean value of the control. *p < 0.05 (vs. control). Experiments were conducted three times independently and each sample was assayed in duplicate. Combined data from 3 different experiments were shown.
with post hoc analysis. Also, GDF-9 (100 ng/ml) did not change AMH mRNA expression in human GC. With these stimuli of BMP-2, -6, -7 and -15, activin-A and GDF-9, GC exhibited an increased level of FSH receptor mRNA and decreased level of StAR mRNA compared to controls (Fig. 1b and c). With the stimulation of recombinant FSH, the expression of AMH mRNA in GC was decreased significantly compared to control (Fig. 2). 3.2. Concentration of AMH in cultured supernatant The concentration of AMH in the cultured supernatant is shown in Fig. 3. AMH protein was undetectable in the supernatant of GC without any stimuli (control). AMH was detected with BMP cytokines stimuli, but with neither activin-A nor GDF-9 stimulation. 4. Comment In the present study, we have shown that BMP-2, -6, -7 and -15 induced AMH expression in human GC. Recently, AMH has been demonstrated to play an important role in ovarian function with its inhibitory effect on follicle recruitment, the process by which 3
AMH concentraon
2.5
pm mol/L
2 1.5 1 0.5 0
N.D. cont
N.D. BMP2
BMP6
BMP7
BMP15
acvinA
N.D. GDF9
Fig. 3. Concentration of AMH in cultured supernatant. Cultured human granulosa cells (GC) were stimulated with BMP-2, -6, -7, and -15, activin-A or GDF-9 (100 ng/ ml) for 24 h. AMH concentration in cultured supernatant was measured using EIA kit. Experiments were conducted three times independently and each sample was assayed in duplicate. Combined data from three different experiments were shown as mean standard error of the mean (SEM). N.D., not detected.
primordial follicles enter the growing pool of primary follicles [3]. Moreover, serum concentration of AMH is considered as an excellent biomarker in clinical practice to estimate the ovarian reserve of individuals [1,2]. In addition to AMH, activin-A and BMP cytokines are known to be important intra-ovarian factors in the regulation of ovarian function. Activin-A is known to be expressed strongly in GC of healthy follicles [13]. Among BMP cytokines, BMP-2, -6, -7, -15 and BMP-15’s closest homologue, GDF-9, are expressed in the human ovary [5,10,14]. BMP-15 and GDF-9, which are expressed exclusively in the oocytes, have attracted much attention, because mutations of these genes have been observed in patients with premature ovarian failure (POF) [15–17]. As shown in Fig. 1, treatment with BMP-2, -6, -7 and -15 (100 ng/ml) significantly increased AMH mRNA expression, while activin-A induced only a minimal increase in AMH expression, and GDF-9 exhibited no effect on AMH expression. These differences might arise from the different cell signaling pathways, in which BMP-2, -6, -7, and-15 activate mothers against decapentaplegic homolog (Smad)-1, 5 and 8, while activin-A and GDF-9 activate Smad-2 and 3 [8]. Activin-A and GDF-9 are known to induce mRNA expression of FSH receptor [18,19], and decrease StAR mRNA [20], which is one of the rate-limiting factors in progesterone production by mediating translocation of cholesterol from the outer to the inner mitochondrial membrane [21]. In Fig. 1b and c, both activin-A and GDF-9 induced FSH receptor mRNA and decreased StAR mRNA, confirming the biological activity of recombinant activin-A and GDF-9 used in our experiments. Further study is needed regarding the mechanism of activin-A and GDF-9 on FSH receptor and StAR, but not AMH. We also confirmed that BMP-2, -6, -7 and -15 induced FSH receptor mRNA expression and decreased StAR mRNA expression, as partially reported previously [5,10,18]. We also confirmed that BMP cytokines induced AMH protein in the cultured supernatant (Fig. 3), but among BMP cytokines, the level of AMH mRNA did not correlate directly with AMH protein level (Figs. 1a and 3). As activin-A and GDF-9 could not induce AMH expression at mRNA and protein level, a certain level of AMH mRNA might be needed to produce AMH protein. Because production of TGF-b superfamily cytokines is regulated not only by mRNA level, but also by post-transcriptional level [22], other factor(s) including protein convertases might be involved in producing AMH protein. BMP cytokines are expressed mainly in growing follicles [8]; thus, GC of growing follicles are the likely target of BMP action. AMH is known to suppress the recruitment of primordial follicles [23]. Therefore, one can speculate that AMH, derived from growing follicles with the stimulation of BMP cytokines, might suppress the growth of the surrounding primordial follicles, preventing their progression to the primary follicle stage, thereby preserving the ovarian reserve. On the other hand, in the growing follicle, BMP cytokines can also induce FSH receptor, which enables follicles to respond to serum FSH [5,10,18]. Activated FSH receptors can increase estradiol concentration in the follicle via induction of aromatase expression [24]. Consequently, elevated FSH and/or estradiol in the follicle are known to decrease the expression of AMH [4]. In the present study, we found that FSH could decrease the expression of AMH mRNA in GC (Fig. 2), confirming the regulation of AMH expression by FSH stimulation. In summary, among TGF-b superfamily cytokines which are known to be important factors for folliculogenesis, BMP-2, -6, -7 and -15, but not activin-A and GDF-9, induced AMH expression in GC. Cultured GC used in this study may not represent the stages of growing follicles in which AMH expression is at its highest level [3]. Our findings that AMH mRNA levels are clearly up-regulated by BMP cytokines in human GC are novel, however, and open new insights into our understanding of AMH regulation in the human ovary.
S. Ogura-Nose et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 164 (2012) 44–47
Financial support This work was supported in part by Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.
[11]
[12]
[13]
Acknowledgment We thank Dr. Heather M. Martinez for her helpful discussion and critical reading of the manuscript. References [1] Fanchin R, Taieb J, Lozano DH, Ducot B, Frydman R, Bouyer J. High reproducibility of serum anti-Mullerian hormone measurements suggests a multistaged follicular secretion and strengthens its role in the assessment of ovarian follicular status. Human Reproduction 2005;20:923–7. [2] van Rooij IA, Broekmans FJ, te Velde ER, et al. Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve. Human Reproduction 2002;17: 3065–71. [3] Gruijters MJ, Visser JA, Durlinger AL, Themmen AP. Anti-Mullerian hormone and its role in ovarian function. Molecular and Cellular Endocrinology 2003;211:85–90. [4] Baarends WM, Uilenbroek JT, Kramer P, et al. Anti-mullerian hormone and anti-mullerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth. Endocrinology 1995;136:4951–62. [5] Shi J, Yoshino O, Osuga Y, et al. Bone morphogenetic protein-6 stimulates gene expression of follicle-stimulating hormone receptor, inhibin/activin beta subunits, and anti-Mullerian hormone in human granulosa cells. Fertility and Sterility 2009;92:1794–8. [6] Ross A, Munger S, Capel B. Bmp7 regulates germ cell proliferation in mouse fetal gonads. Sex Deviations 2007;1:127–37. [7] Lawson KA, Dunn NR, Roelen BA, et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes and Development 1999;13:424–36. [8] Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in mammalian reproduction. Endocrine Reviews 2004;25: 72–101. [9] Knight PG, Glister C. TGF-beta superfamily members and ovarian follicle development. Reproduction 2006;132:191–206. [10] Shi J, Yoshino O, Osuga Y, et al. Bone morphogenetic protein-2 (BMP-2) increases gene expression of FSH receptor and aromatase and decreases gene
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expression of LH receptor and StAR in human granulosa cells. American Journal of Reproductive Immunology 2011;65:421–7. Yoshino O, Osuga Y, Koga K, et al. Upregulation of interleukin-8 by hypoxia in human ovaries. American Journal of Reproductive Immunology 2003;50: 286–90. Erickson GF, Garzo VG, Magoffin DA. Insulin-like growth factor-I regulates aromatase activity in human granulosa and granulosa luteal cells. The Journal of Clinical Endocrinology and Metabolism 1989;69:716–24. Yamoto M, Minami S, Nakano R, Kobayashi M. Immunohistochemical localization of inhibin/activin subunits in human ovarian follicles during the menstrual cycle. The Journal of Clinical Endocrinology and Metabolism 1992;74:989–93. Aaltonen J, Laitinen MP, Vuojolainen K, et al. Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis. The Journal of Clinical Endocrinology and Metabolism 1999;84:2744–50. Dixit H, Rao LK, Padmalatha VV, et al. Missense mutations in the BMP15 gene are associated with ovarian failure. Human Genetics 2006;119:408–15. Di Pasquale E, Rossetti R, Marozzi A, et al. Identification of new variants of human BMP15 gene in a large cohort of women with premature ovarian failure. The Journal of Clinical Endocrinology and Metabolism 2006;91: 1976–9. Zhao H, Qin Y, Kovanci E, Simpson JL, Chen ZJ, Rajkovic A. Analyses of GDF9 mutation in 100 Chinese women with premature ovarian failure. Fertility and Sterility 2007;88:1474–6. Shi J, Yoshino O, Osuga Y, Nishii O, Yano T, Taketani Y. Bone morphogenetic protein 7 (BMP-7) increases the expression of follicle-stimulating hormone (FSH) receptor in human granulosa cells. Fertility and Sterility 2010;93: 1273–9. Kobayashi N, Orisaka M, Cao M, et al. Growth differentiation factor-9 mediates follicle-stimulating hormone-thyroid hormone interaction in the regulation of rat pre-antral follicular development. Endocrinology 2009;150: 5566–74. Shi FT, Cheung AP, Klausen C, Huang HF, Leung PC. Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells. The Journal of Clinical Endocrinology and Metabolism 2010;95: E172–80. Hiroi H, Christenson LK, Strauss 3rd JF. Regulation of transcription of the steroidogenic acute regulatory protein (StAR) gene: temporal and spatial changes in transcription factor binding and histone modification. Molecular and Cellular Endocrinology 2004;215:119–26. Steiner DF. The proprotein convertases. Current Opinion in Chemical Biology 1998;2:31–9. Durlinger AL, Visser JA, Themmen AP. Regulation of ovarian function: the role of anti-Mullerian hormone. Reproduction 2002;124:601–9. Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocrine Reviews 1996;17:121–55.
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Anti-Mullerian hormone (AMH) is induced by bone morphogenetic protein (BMP) cytokines in human granulosa cells Sayaka Ogura-Nose, Osamu Yoshino *, Yutaka Osuga *, Jia Shi, Hisahiko Hiroi, Tetsu Yano, Yuji Taketani Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 November 2011 Received in revised form 6 April 2012 Accepted 5 May 2012
Objectives: Serum concentration of anti-Mullerian hormone (AMH) is used as a biomarker in clinical practice. Therefore, it is important to elucidate the mechanism by which AMH is regulated in granulosa cells (GC). An important first step in understanding AMH regulation is to determine which factors upregulate AMH expression. Study design: Human GC, obtained from 28 women undergoing oocyte retrieval for in vitro fertilization, were stimulated with various intraovarian cytokines including bone morphogenetic protein (BMP)-2, -6, -7 -15, activin-A and growth differentiation factor (GDF)-9 (100 ng/ml). The expression of AMH mRNA was evaluated with reverse transcription and quantitative real-time polymerase chain reaction (PCR), and AMH protein in cultured supernatant was measured with EIA kit. Results: BMP-2, -6, -7 and -15, but not activin-A and GDF-9, significantly induced AMH expression in GC at mRNA and protein level, while all stimuli increased FSH receptor mRNA and decreased steroidogenic acute regulatory protein (StAR) mRNA level. Conclusions: Among the transforming growth factor (TGF)-b superfamily, BMP-2, -6, -7 and -15 significantly induced AMH expression in human GC. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: AMH BMP GDF-9 Ovary Granulosa cell
1. Introduction In infertility treatment, the precise assessment of ovarian reserve is necessary to plan a treatment strategy for each patient. Among all available markers of ovarian reserve, much interest has been given to anti-Mullerian hormone (AMH) as a reliable, accurate and reproducible predictor [1,2]. AMH, which belongs to the transforming growth factor (TGF)-b superfamily, is a product of the granulosa cells (GC) in pre-antral and small antral follicles [3]. As serum concentration of AMH is considered an excellent biomarker in clinical practice [1,2], it is important to elucidate the mechanism by which AMH is regulated in GC. Although it has been reported that follicle stimulating hormone (FSH) and estradiol down-regulate AMH expression in GC [4], the factors which upregulate AMH expression are still unknown. Previously, we found that bone morphogenetic protein (BMP)-6, an intra-ovarian factor,
* Corresponding authors at: Department of Obstetrics and Gynecology, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: +81 3 3815 5411x33407; fax: +81 3 3816 2017. E-mail addresses: [email protected] (O. Yoshino), [email protected] (Y. Osuga). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.05.017
increased the expression of AMH in human GC [5]. In addition to BMP-6, other TGF-b superfamily cytokines including BMP-2, -7 and -15, growth differentiation factor (GDF)-9 and activin-A are known to regulate ovarian physiology including gonadogenesis [6,7], folliculogenesis, ovulation and luteinization in various species [8]. In the human ovary, it has been reported that GC express BMP-2, -6 and activin-A, while theca cells express BMP-7 and oocytes express BMP-6, -15 and GDF-9 respectively [5,8–10]. In the present study, we validated the hypothesis that TGF-b superfamily member cytokines might induce AMH expression in GC. 2. Materials and methods 2.1. Reagents and materials Hyaluronidase, fetal bovine serum (FBS), DMEM/F12 and antibiotics (mixture of penicillin, streptomycin, and amphotericin B) were purchased from Sigma (St. Louis, MO). Recombinant human BMP-2, -6, -7, and -15, GDF-9 and activin-A were purchased from R&D Systems (Minneapolis, MN). Recombinant human FSH was kindly provided by Japan Organon (Tokyo, Japan). The concentrations of AMH in supernatant were measured using an enzymatic immunoassay (EIA) kit purchased from Beckman Coulter (Tokyo, Japan).
S. Ogura-Nose et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 164 (2012) 44–47
Written informed consent was obtained from all study participants. Ethical approval was given by Tokyo University Ethics Committee.
45
3. Results 3.1. Effect of various intra-ovarian factors and FSH on AMH expression in human GC
2.2. Cell culture of human GC GC were aspirated from 28 patients who had been undergoing ovarian stimulation for in vitro fertilization (IVF). The method for obtaining and culturing human GC was described previously [11]. Briefly, following ovarian suppression by the gonadotropinreleasing hormone analog buserelin acetate (Suprecur, Mochida Pharma, Tokyo, Japan) from the midluteal phase of the preceding cycle, a dose of 150–300 IU human menopausal gonadotropin was given daily until the diameter of the leading follicle reached 17 mm or greater. Then, hCG at a dose of 10,000 IU was administered and transvaginal ultrasound-guided oocyte retrieval was performed 35 h later. The collected human GC were cultured in 500 ml of DMEM/F12 containing 5% FBS and antibiotics in 12-well plates at a density of 2 105 cells/ml and kept at 37 8C in a humidified 5% CO2/95% air environment for 5 days. All of the GC used for the experiments were pre-cultured for 5 days prior to treatments to allow the GC to regain sensitivity to FSH stimulation [12]. The media were changed every 48 h. To evaluate the effects of cytokines, human GC were cultured with or without BMP-2, -6, -7, and -15, GDF-9 or activin-A (100 ng/ml) for 24 h. In the pilot study, we found that this concentration could induce the maximum effect of cytokines on AMH mRNA expression (data not shown). Recombinant cytokines were dissolved in 0.1% bovine serum albumin + 4 mM of HCl as a vehicle. The same amount of vehicle was used for control samples. In some experiments, GC were stimulated with recombinant human FSH (0.5 IU/ml) for 24 h.
Treatment with BMP-2, -6, -7 and -15 (100 ng/ml) stimulated over 9-, 7-, 5-, and 4-fold increases respectively in the AMH mRNA level of human GC (Fig. 1a). Although activin-A (100 ng/ml) induced approximately 2-fold increase of AMH mRNA, there was no statistical difference between control and activin-A stimulation
2.3. Reverse transcription and quantitative real-time polymerase chain reaction analysis Total RNA was extracted from GC, using the RNAeasy minikit (Qiagen, Hilden, Germany). Reverse transcription (RT) was performed using Rever Tra Dash (TOYOBO, Tokyo, Japan). One microgram of total RNA was reverse transcribed in a 20-ml volume. For the quantification of various mRNA levels of AMH, FSH receptor and steroidogenic acute regulatory protein (StAR), real-time polymerase chain reaction (PCR) was performed using LightCycler (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions. The PCR primers were selected from different exons of the corresponding genes to discriminate PCR products that might arise from possible chromosomal DNA contaminants. The primer sequences and PCR conditions have been described elsewhere [5,10]. Expression of each mRNA was normalized by GAPDH mRNA. Experiments were conducted at least three times independently, and each sample assayed in duplicate. 2.4. Measurement of AMH concentration in cultured supernatant AMH concentration in cultured supernatant was measured using the EIA kit according to the instructions. The functional sensitivity of the AMH EIA kit used was above 0.5 pmol/L, and both intra- and inter-assay coefficients of variation of this kit were less than 15%. 2.5. Statistical analysis Data were analyzed by one-way ANOVA with post hoc test for multiple comparisons using Statview software (SAS Institute Inc., Cary, NC). A p-value of less than 0.05 was considered statistically significant.
Fig. 1. mRNA expression induced by stimuli of various intra-ovarian cytokines. Cultured human granulosa cells (GC) were stimulated with BMP-2, -6, -7, and -15, activin-A or GDF-9 (100 ng/ml) for 24 h. Total RNA was extracted from the cells and subjected to real-time PCR to determine various mRNA levels. Data were normalized by GAPDH mRNA level, and shown as the mean standard error of the mean (SEM) relative to an adjusted value of 1.0 for the mean value of the control. *p < 0.05 (vs. control). All experiments were conducted at least three times independently, and each sample was assayed in duplicate. (1-a) Anti-Mullerian hormone (AMH) mRNA; each column represented an average from 7 samples. (1-b) FSH receptor mRNA; representative data from 3 different experiments. (1-c) Steroidogenic acute regulatory protein (StAR) mRNA; representative data from three different experiments.
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S. Ogura-Nose et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 164 (2012) 44–47
1.2
AMH mRNA
fold increase
1 0.8
*
0.6 0.4 0.2
* P<0.05
0
Control
FSH
Fig. 2. Anti-Mullerian hormone (AMH) mRNA expression stimulated with FSH. Cultured human granulosa cells (GC) were stimulated with recombinant FSH (0.5 IU/ml) for 24 h. Total RNA was extracted from the cells and subjected to realtime PCR to determine AMH mRNA levels. Data were normalized by GAPDH mRNA level, and shown as the mean standard error of the mean (SEM) relative to an adjusted value of 1.0 for the mean value of the control. *p < 0.05 (vs. control). Experiments were conducted three times independently and each sample was assayed in duplicate. Combined data from 3 different experiments were shown.
with post hoc analysis. Also, GDF-9 (100 ng/ml) did not change AMH mRNA expression in human GC. With these stimuli of BMP-2, -6, -7 and -15, activin-A and GDF-9, GC exhibited an increased level of FSH receptor mRNA and decreased level of StAR mRNA compared to controls (Fig. 1b and c). With the stimulation of recombinant FSH, the expression of AMH mRNA in GC was decreased significantly compared to control (Fig. 2). 3.2. Concentration of AMH in cultured supernatant The concentration of AMH in the cultured supernatant is shown in Fig. 3. AMH protein was undetectable in the supernatant of GC without any stimuli (control). AMH was detected with BMP cytokines stimuli, but with neither activin-A nor GDF-9 stimulation. 4. Comment In the present study, we have shown that BMP-2, -6, -7 and -15 induced AMH expression in human GC. Recently, AMH has been demonstrated to play an important role in ovarian function with its inhibitory effect on follicle recruitment, the process by which 3
AMH concentraon
2.5
pm mol/L
2 1.5 1 0.5 0
N.D. cont
N.D. BMP2
BMP6
BMP7
BMP15
acvinA
N.D. GDF9
Fig. 3. Concentration of AMH in cultured supernatant. Cultured human granulosa cells (GC) were stimulated with BMP-2, -6, -7, and -15, activin-A or GDF-9 (100 ng/ ml) for 24 h. AMH concentration in cultured supernatant was measured using EIA kit. Experiments were conducted three times independently and each sample was assayed in duplicate. Combined data from three different experiments were shown as mean standard error of the mean (SEM). N.D., not detected.
primordial follicles enter the growing pool of primary follicles [3]. Moreover, serum concentration of AMH is considered as an excellent biomarker in clinical practice to estimate the ovarian reserve of individuals [1,2]. In addition to AMH, activin-A and BMP cytokines are known to be important intra-ovarian factors in the regulation of ovarian function. Activin-A is known to be expressed strongly in GC of healthy follicles [13]. Among BMP cytokines, BMP-2, -6, -7, -15 and BMP-15’s closest homologue, GDF-9, are expressed in the human ovary [5,10,14]. BMP-15 and GDF-9, which are expressed exclusively in the oocytes, have attracted much attention, because mutations of these genes have been observed in patients with premature ovarian failure (POF) [15–17]. As shown in Fig. 1, treatment with BMP-2, -6, -7 and -15 (100 ng/ml) significantly increased AMH mRNA expression, while activin-A induced only a minimal increase in AMH expression, and GDF-9 exhibited no effect on AMH expression. These differences might arise from the different cell signaling pathways, in which BMP-2, -6, -7, and-15 activate mothers against decapentaplegic homolog (Smad)-1, 5 and 8, while activin-A and GDF-9 activate Smad-2 and 3 [8]. Activin-A and GDF-9 are known to induce mRNA expression of FSH receptor [18,19], and decrease StAR mRNA [20], which is one of the rate-limiting factors in progesterone production by mediating translocation of cholesterol from the outer to the inner mitochondrial membrane [21]. In Fig. 1b and c, both activin-A and GDF-9 induced FSH receptor mRNA and decreased StAR mRNA, confirming the biological activity of recombinant activin-A and GDF-9 used in our experiments. Further study is needed regarding the mechanism of activin-A and GDF-9 on FSH receptor and StAR, but not AMH. We also confirmed that BMP-2, -6, -7 and -15 induced FSH receptor mRNA expression and decreased StAR mRNA expression, as partially reported previously [5,10,18]. We also confirmed that BMP cytokines induced AMH protein in the cultured supernatant (Fig. 3), but among BMP cytokines, the level of AMH mRNA did not correlate directly with AMH protein level (Figs. 1a and 3). As activin-A and GDF-9 could not induce AMH expression at mRNA and protein level, a certain level of AMH mRNA might be needed to produce AMH protein. Because production of TGF-b superfamily cytokines is regulated not only by mRNA level, but also by post-transcriptional level [22], other factor(s) including protein convertases might be involved in producing AMH protein. BMP cytokines are expressed mainly in growing follicles [8]; thus, GC of growing follicles are the likely target of BMP action. AMH is known to suppress the recruitment of primordial follicles [23]. Therefore, one can speculate that AMH, derived from growing follicles with the stimulation of BMP cytokines, might suppress the growth of the surrounding primordial follicles, preventing their progression to the primary follicle stage, thereby preserving the ovarian reserve. On the other hand, in the growing follicle, BMP cytokines can also induce FSH receptor, which enables follicles to respond to serum FSH [5,10,18]. Activated FSH receptors can increase estradiol concentration in the follicle via induction of aromatase expression [24]. Consequently, elevated FSH and/or estradiol in the follicle are known to decrease the expression of AMH [4]. In the present study, we found that FSH could decrease the expression of AMH mRNA in GC (Fig. 2), confirming the regulation of AMH expression by FSH stimulation. In summary, among TGF-b superfamily cytokines which are known to be important factors for folliculogenesis, BMP-2, -6, -7 and -15, but not activin-A and GDF-9, induced AMH expression in GC. Cultured GC used in this study may not represent the stages of growing follicles in which AMH expression is at its highest level [3]. Our findings that AMH mRNA levels are clearly up-regulated by BMP cytokines in human GC are novel, however, and open new insights into our understanding of AMH regulation in the human ovary.
S. Ogura-Nose et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 164 (2012) 44–47
Financial support This work was supported in part by Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.
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