Endocrine DOI 10.1007/s12020-014-0250-x
ORIGINAL ARTICLE
Dexamethasone altered steroidogenesis and changed redox status of granulosa cells Xiao-Hua Yuan • Bai-Qing Yang • Ying Hu • Yang-Yang Fan • Li-Xia Zhang • Jia-Chen Zhou Ya-Qin Wang • Cai-Ling Lu • Xu Ma
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Received: 20 December 2013 / Accepted: 21 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Glucocorticoids have been widely used in clinical application for anti-inflammatory and immunosuppressive function. Previous study reported that glucocorticoids adversely affect the reproductive system and can directly act on ovary. Here, we found that progesterone production induced by dexamethasone requiring activation of caspase-3 which may mediate differentiation and apoptosis of granulosa cells. Further study displayed that cellular glutathione level was increased and reactive oxygen species was decreased accompanied with unchanged mitochondrial membrane potential which may contribute to the maintenance of steroidogenesis in granulosa cells treated with dexamethasone. Dexamethasone also augmented the level of
Xiao-Hua Yuan, Bai-Qing Yang, and Ying Hu contributed equally to this work. X.-H. Yuan Y. Hu Y.-Y. Fan Y.-Q. Wang (&) Maternity Department, Shaanxi Provincial People’s Hospital, Xi’an 710068, China e-mail: [email protected] X.-H. Yuan B.-Q. Yang C.-L. Lu (&) X. Ma Department of Genetics, National Research Institute for Family Planning, Beijing 100081, China e-mail: [email protected] L.-X. Zhang Clinical Laboratory, Shaanxi Provincial People’s Hospital, Xi’an, China J.-C. Zhou Dalian Medical University, Dalian, China X. Ma (&) Graduate School of Peking Union Medical College; Department of Genetics, National Research Institute for Family Planning, Beijing 100081, China e-mail: [email protected]
anti-Mu¨llerian hormone secreted by preovulatory granulosa cells which indicated that dexamethasone may promote preantral follicles development. Keywords Granulosa cell Follicle Progesterone Dexamethasone Caspase-3 ROS
Introduction Glucocorticoids are hormonal mediator of stress. They have been extensively used in clinical application for antiinflammatory and immunosuppressive function [1]. However, there are many adverse reactions in clinical practice. Mounting evidences suggest that glucocorticoids adversely affect reproduction system. It influences the hypothalamic pituitary adrenal axis at three levels: the hypothalamus; the anterior pituitary gland; the testis/ovary [2–5]. Studies had identified glucocorticoids receptors in ovary cells [5–7], and glucocorticoid can directly act on ovary [8, 9]. The life of follicles goes through four stages: primordial follicles, preantral follicles, antral follicles, preovulatory follicles. In this process, only a small portion of follicles finally develops into ovulatory follicles, and most of the follicles undergo atresia [10]. Coupled with follicles development, granulosa cells proliferate, differentiate, and go through apoptosis around the oocyte and play a critical role in reproductive function by producing steroids. Caspase-3 had been identified the key enzyme mediated apoptosis and differentiation through cleavage of its substrates. A previous study had demonstrated that caspase-3 was activated in response to LH and FSH [11], and our previous work also indicated that caspase-3 played a key role in arsenic-induced progesterone production [12]. The first step of steroidogenesis occurs on mitochondria. The steroidogenic acute regulatory protein (StAR) transfers
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cholesterol from the outer to the inner mitochondria membrane where it is metabolized to pregnenolone by cytochrome P450 cholesterol side chain cleavage enzyme, P450scc [13]. Mounting evidences had identified that the StAR and P450scc proteins are induced by tropic hormone [14–16]. Hales et al. [17] suggested that mitochondria must be energized, polarized, and actively respiring to support steroidogenesis. There are evidences that mitochondrial membrane potential (Dwm) affects steroid synthesis by blocking protein processing into mitochondria [18, 19]. Extra reactive oxygen species (ROS) or redox imbalance damages mitochondria and results steroidogenesis disorder [20–22]. Anti-Mu¨llerian hormone (AMH) is produced by granulosa cells of primary follicles, preantral follicles, and small antral follicles [23]. The level of AMH reaches the maximum in preantral follicles and small antral follicles. However, the production of AMH cannot be detected in follicles in response to FSH and disappears in atresia follicles [24, 25]. AMH is widely used as marker in the assessment of responsiveness of follicles. It is also an indicator of the size of growing follicles pool [25, 26]. It had been reported that dexamethasone induced cystic status of ovary [27]. In the present study, we evaluated the effect of dexamethasone on steroidogenesis of granulosa cells, detected the redox status of granulosa cells treated by dexamethasone ,and revealed the relationship between caspase-3 and progesterone production of granulosa cells. The findings of our study would help evaluate the role of dexamethasone on the reproductive system.
Animals Immature female Sprague–Dawley (SD) rats (21 days old) were issued from the Department of Laboratory Animal Science of Peking University (Beijing, China). The rats were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All the protocols had the approval of the Institutional Committee on Animal Care and Use. Preovulatory granulosa cell culture Female SD rats (21 days old) were injected intraperitoneally with 20 IU/ml PMSG to increase the number of granulosa cells. Two days later, the animals were sacrificed by decapitation, and their ovaries were quickly removed and washed in 0.9 % (w/v) cold normal saline. Granulosa cells were harvested by puncturing the individual ovarian follicles with 25-gauge needles, collected by centrifugation (5009g, 5 min) and cultured in a humidified incubator at 37 °C and 5 % CO2 in DMEM supplemented with 15 % FBS for 24 h before the experimental treatment. Steroid radioimmunoassay (RIA) Conditioned media from granulosa cells (1 9 106/vial) treated with or without dexamethasone (0–20 lL) AcDEVD-CHO (40 lM) were collected; the levels of progesterone in the media were measured by RIA as previously described [28].
Materials and methods
Measurement of caspase-3 activity
Chemicals
Caspases activities were measured using caspase-3 activity kit according to the manufacturer’s instructions. Briefly, GCs, following the treatment of dexamethasone (0–20 lL) for 24 h, were washed with cold phosphate-buffered saline (PBS), resuspended in lysis buffer, and left on ice for 15 min. Activities of caspase-3 were measured with substrate peptides Ac-DEVD-pNA. The release of p-nitroanilide (pNA) was quantified by determining the absorbance with a microplate reader (BioTek Instruments) at 405 nm.
DMSO, b-actin antibody, and dexamethasone were bought from Sigma (Saint Louis, MO, USA). Pregnant mare serum gonadotropin was purchased from HydeVenture Bio Co., Ltd. (Beijing, China). ROS assay kit (DCHF-DA), glutathione (GSH) assay kit, caspase-3 assay kit, Ac-DEVD-CHO, 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolecarbocyanideiodine (JC-1), and Hoechst 33258 were supplied by Beyotime Biotechnology Co. (Haimen, China). AMH Elisa kit was purchased from Huamaike Biotechnology Co. (Beijing, China). Fetal bovine serum (FBS) and DMEM media were purchased from Thermo Scientific HyClone (Logan, UT, USA). Trypsin– EDTA (0.25 %) and penicillin–streptomycin (1009) were obtained from Invitrogen Co., Ltd. (Carlsbad, CA, USA). The StAR antibody and caspase-3 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The P450scc antibody was obtained from Abcam (Abcam, Cambridge, UK).
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Western blot analysis GCs (1 9 106/vial) were cultured in six-well plates, incubated with different reagents for the indicated time. The cells were washed with PBS and lysed with a cell lysis buffer containing 0.5 % sodium deoxycholate, 0.1 % SDS, 10 mM NaF, 0.2 mM Na3VO4, 1 mM PMSF, and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Equal amounts of protein were separated on a 12 % SDSPAGE gel, transferred to nitrocellulose membranes, and
Endocrine
probed with b-actin, StAR, P450scc, and caspase-3 antibodies. Detection of signals was performed with an odyssey two-color infrared imaging system (LI-COR, Lincoln, NE). Mitochondrial membrane potential (Dwm) analysis Mitochondrial membrane potential was identified with JC-1 dye. GCs were incubated with dexamethasone (0–20 lL) for 48 h. Then, the cells were incubated at 37 °C and 5 % CO2 for 20 min with medium consisting of 10 mg/ml JC-1, and then the cells were observed under a fluorescence microscope (TE2000, Nikon, Inc., Tokyo, Japan) and detected by the microplate reader with an excitation of 530 nm and an emission of 590 nm (Biotech Instrument).
Measurement of AMH After GCs treated with dexamethasone (0–20 lM) in DMEM-F12 media for 48 h, the conditioned media were collected, and the levels of AMH were measured by Elisa kit. Statistical analysis All data are presented as mean ± SEM. The differences between means were calculated using a one-way analysis of variance followed by a t test with p \ 0.05 considered significant.
Measurement of intracellular GSH
Results
The GSH concentrations in GCs were determined with a dithionitrobenzoic acid–glutathione disulfide reductase recycling assay. The GCs were treated with dexamethasone (0–20 lL) for 48 h. Subsequently, the GSH levels were determined using commercially available kits according to the manufacturer’s instructions.
Dexamethasone stimulates progesterone production in granulosa cells
Measurement of ROS The intracellular ROS of GCs was detected using DCHFDA. At 48 h after dexamethasone (0–20 lL) exposure, the GCs were incubated for 30 min in 5 mM DCHF-DA diluted in DMEM medium. The ROS levels were detected by using a microplate reader with an excitation of 488 nm and an emission of 525 nm (BioTek Instruments, Winooski, VT). Flow cytometry analysis GCs were collected after treatment with dexamethasone (0–20 lM) for 48 h and fixed with 70 % ethanol overnight at 48 °C. The samples were concentrated by removing the ethanol and resuspending the cells in a PBS solution containing PI (50 mg/ml) and RNaseA (25 mg/ml). The apoptotic GCs were detected by flow cytometry (Coulter Epics XL; Beckman-Coulter, Inc., Miami, FL). Hoechst staining After treatment with or without dexamethasone (0–20 lM) for 48 h, GCs growing on the culture plates were fixed with 4 % paraformaldehyde for 30 min at room temperature. Cells were washed with PBS and exposed to 1 mg/ml Hoechst 33258 at room temperature in the dark for 10 min and observed under a fluorescent microscope (TE2000; Nikon, Inc.).
To test the effect of dexamethasone on the production of progesterone in preovulatory granulosa cells, we cultured granulosa cells exposed with various concentrations of dexamethasone. Incubation granulosa cells with dexamethasone (0.2, 2, and 20 lL) for 48 h can significantly increase progesterone production (Fig. 1a). Moreover, dexamethasone (2 lL) treatment induced progesterone production significantly after 6 h (Fig. 1b). Dexamethasone alters the expression of steroidogenic proteins StAR and P450scc In the process of steroidogenesis, the rate-limiting StAR protein transfers cholesterol from the outer to the inner mitochondria membrane where it is metabolized to pregnenolone by the P450scc. To determined whether dexamethasone affects the expression of steroidogenic proteins StAR and P450scc, StAR and P450scc were detected by western blotting. Our result showed that dexamethasone increased StAR and P450scc expression at the concentration range (0.2–20 lL; Fig. 2a). There was no significant difference in the expression of StAR among groups in the concentration range (0.2–20 lL). The P450scc expression was unregulated at 0.2 lL and reached the peak at 2 lL, downregulated slightly at 20 lL (Fig. 2b). Dexamethasone induces caspase-3 activation in preovulatory granulosa cells A previous study reported that LH and FSH enhance caspase-3 and caspase-7 activity of granulosa cells coupled with steroidogenesis. Our result also demonstrated that
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Fig. 1 Dexamethasone stimulated the progesterone production of GCs. Progesterone production of granulosa cells after treatment with various concentrations of dexamethasone for 48 h (a) and at various
time points with a constant concentration of 2 lM dexamethasone (b). The data represent mean ± SEM (n C 4). Columns with asterisks are significantly different from control (0 lL or 0 h). *** p \ 0.0001
Fig. 2 Dexamethasone stimulated steroidogenic protein P450scc and StAR expression. Steroidogenic protein P450scc and StAR were detected by western blot after exposure of GCs to different concentrations of dexamethasone for 24 h. Columns with different superscript letters are significantly different (p \ 0.05)
arsenic induced production in a caspase-3 dependent manner. In light of the previous study, we examined caspase-3 using antibody that can recognize the full length caspase-3 and partially cleaved production 21 KD. We found that dexamethasone (0.2–2 lM) significantly increased the level of 21 KD of caspase-3 at 12 h (Fig. 3a). Caspase-3 fluorometric assay showed that dexamethasone induced caspase-3 activation at 12 h at the concentration of 2 and 20 lL (Fig. 3b). Dexamethasone induces slight apoptosis in preovulatory granulosa cells In most cell types, dexamethasone caused apoptosis, such as rat thymocyte, myeloma, and peripheral blood monocyte. Our result also showed that dexamethasone stimulated activation of caspase-3 which cleaved substrate resulting apoptosis. We further quested that whether dexamethasone also caused apoptosis in preovulatory granulosa cells. By FACS analysis, we determined the subG1 DNA content, a characteristic feature of apoptotic cells. Our result demonstrated that dexamethasone (20 lL) resulted slightly apoptosis in preovulatory granulosa cells. There was no obvious apoptosis of granulosa treated with dexamethasone (0–2 lM; Fig. 4a). Hoechst 33258, a
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Fig. 3 The activation of caspase-3 is involved in dexamethasoneinduced progesterone production. The caspase-3 activity was detected by western blot (a) and caspase-3 activity assay kit (Beyotime China, b) after exposure of GCs to different concentrations of dexamethasone for 12 h
classical way of identifying the morphology of apoptotic cells, also displayed that dexamethasone (20 lL) results in fragment nuclei slightly (Fig. 4b).
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Fig. 4 Dexamethasone induced apoptosis slightly in GCs. After exposure of GCs to dexamethasone (0–20 lL) for 48 h, the sub-G1 content of GCs was analyzed by FACS (a), and Hoechst 33258 was observed under a fluorescence microscope (b, magnification 9100)
Fig. 5 Inhibition caspase-3 activity can inhibit progesterone production of GCs treated with dexamethasone. The progesterone levels were detected after exposure with or without dexamethasone (2 lL) plus caspase-3 inhibitor (AcDEVD-CHO; 40 lL) for 48 h (a). Steroidogenic protein StAR and P450scc were detected after exposure with or without dexamethasone (2 lL) plus caspase-3 inhibitor (Ac-DEVDCHO; 40 lL) for 24 h (b). Columns with different superscript letters are significantly different (p \ 0.05)
Caspase-3 is required in progesterone production induced by dexamethasone in preovulatory GCs To determine whether caspase-3 is required in steroidogenesis regulated by dexamethasone, GCs were incubated
with dexamethasone in the presence or absence of specific caspase-3 inhibitor. As shown in Fig. 5a, specific caspase-3 inhibitor, Ac-DEVD-CHO can cause significantly decrease in progesterone production at 48 h treated with dexamethasone. Further study displayed that specific caspase-3
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Fig. 6 Dexamethasone reduced ROS level and increased GSH level without significant effect on mitochondrial transmembrane potential. After exposure of GCs to various concentrations of dexamethasone for 48 h, the intracellular levels of ROS (a), GSH (b), were
determined. The data represent mean ± SEM (n C 4). Columns with asterisks are significantly different from control (0 lL or 0 h). ** p \ 0.005; *** p \ 0.0001
inhibitor decreased steroidogenic protein P450scc expression but not StAR expression treated by dexamethasone at 24 h (Fig. 5b). These findings indicated that caspase-3 is involved in steroidogenesis in GCs mediated by dexamethasone.
AMH secretion significantly at the concentration of 0.2 and 2 lM. Although the level AMH of group treated by dexamethasone (20 lM) compared with control group also increased, there was not statistically significant which may due to the apoptosis of granulosa cells (Fig. 8).
Effect of dexamethasone on ROS, GSH, and mitochondrial membrane potential (DWm)
Discussion
The intact mitochondrial membrane and redox balance are the requirement of steroidogenesis. The increase in ROS or depletion in reduced GSH would damage mitochondrial membrane and result in inhibition of steroidogenesis. Redox imbalance and the damage of mitochondrial membrane can also result caspase-3 activation and apoptosis. Our result displayed that steroidogenesis, caspase-3 activation, and apoptosis appeared in a process simultaneously. To further study the process of steroidogenesis stimulated by dexamethasone in preovulatory granulosa cells, we examined the levels of ROS, GSH, and mitochondrial membrane potential (DWm). Our result showed that dexamethasone enhanced the levels of GSH in GCs (Fig. 6b). The ROS levels treated with various doses of dexamethasone were decreased (Fig. 6a). JC-1 is widely used to detect cellular mitochondrial membrane potential. The aggregated JC-1 which indicated the intact mitochondrial membrane was detected by fluorescence microplate reader and fluorescence microscope. We found that there was no significant difference between treated groups and control group (Fig. 7a, b). Effect of dexamethasone on AMH AMH is produced by granulosa of preantral follicles and small antral follicles. It reflects the responsiveness of follicles. Our result showed that dexamethasone induced
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Glucocorticoids have served as anti-inflammatory and immunosuppressive agent for a long time in clinical application. Dexamethasone is typical representative of glucocorticoid. High dose of dexamethasone can induce diabetes, peptic ulcer, a series of symptoms similar with Cushing syndrome [29–31]. Dexamethasone also acts on reproduction system. Previous study reported that glucocorticoids enhanced progesterone production in luteinized granulosa cells from mature follicles and increased progesterone production in preovulatory granulosa cells stimulated by FSH or 8-Br-cAMP [32, 33], whereas dexamethasone inhibited progesterone synthesis of preovulatory granulosa in response to LH [34]. Here, we found that dexamethasone stimulated progesterone production in granulosa cells from preovulatory follicles of unmatured SD rat. The discrepancy may due to different dose, different stimulating factor combined with glucocorticoids, and the different status of follicle development. The responses of GCs to LH and FSH are up-regulation steroidogenic protein StAR and P450scc following with steroidogenesis. Our finding also showed that dexamethasoneinduced progesterone production similar to LH and FSH acts through StAR and P450scc. As apoptosis-inducing agent, dexamethasone induces activation caspase-3 and apoptosis in a variety of tissues and cells [4, 35, 36]. In our experiment, dexamethasone increased caspase-3 activation (0.2, 2, and 20 lM) and slight
Endocrine Fig. 7 Mitochondrial membrane potentials of preovulatory granulosa cells treated with various concentrations of dexamethasone remained unchanged. After exposure of GCs to various concentrations of dexamethasone for 48 h, the aggregated JC-1 was examined by microplate reader and detected under fluorescent microscope
Fig. 8 Dexamethasone stimulated the AMH production of GCs. After exposure of GCs to various concentrations of dexamethasone for 48 h, the AMH levels of GCs were analyzed by AMH Elisa. Columns with asterisks are significantly different from control (0 lL). ** p \ 0.005; *** p \ 0.0001
apoptosis (20 lM) coupled with progesterone production. Interestingly, Yacobi et al. [11] reported that LH and FSH induced increase in caspase-3 and caspase-7 in preovulatory follicles. Subsequently, they found that LH-induced caspase activation in preovulatory follicles was coupled with mitochondrial steroidogenesis [37]. Caspase-3 plays a key role both in differentiation and apoptosis [38, 39]. The destination of developing follicles (ovulation or atresia) is dependent on the cells within them (proliferation, differentiation, or apoptosis) regulated by endocrine, autocrine, and paracrine. Coupled with granulosa differentiation dominant follicles develop into ovulatory follicles following with luteinization, meanwhile, a large number of follicles
undergo atresia accompanied by granulosa cells apoptosis [10]. The ability of progesterone production of granulosa cells gradually increases with strengthening degree of differentiation. There were also evidences that the progesterone production was increased in atresia follicles in which apoptotic granulosa cell reached 20–30 % [40]. Afterward, Hummitzsch et al. [41] found that steroidogenesis was coupled with programmed cell death through establishing programmed cell death model with spheroids of granulosa cells in vitro. In our studies, inhibition of caspase-3 activity results to a decrease in progesterone production of granulosa cells stimulated by dexamethasone (Fig. 5). Our previous study also identified that inhibition caspase-3 activity resulted in a decrease in progesterone synthesis of granulosa cells stimulated by arsenic and FSH [12]. Current evidences indicated that progesterone production required caspase-3 activation through mediating differentiation or apoptosis of granulosa cells. For steroidogenesis, the initial step occurs within the mitochondrion. Hales et al. [17] suggested that mitochondria must be intact, energized, polarized to maintain steroidogenesis. The depletion of antioxidant GSH and extra ROS would damage the mitochondrial membrane resulting decrease in steroidogenesis [22, 42]. Dexamethasone, as apoptosis-inducing agent, always leads to ROS generation in a variety of cells [36, 43]. However, our study demonstrated that the GSH was increased coupled with ROS decline in granulosa cells treated with dexamethasone (Fig. 6) and the mitochondrial membrane potential remained unchanged, a state that is necessary for steroidogenesis (Fig. 7). Amsterdam [44] reported when immortalized granulosa cells were stimulated to undergo
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apoptosis, there was a dramatic increase in progesterone production, and the mitochondria were preserved intact. To some extent, Abraham’s experiment was confirmed by our results. How mitochondria were kept intact to support progesterone synthesis when apoptosis-inducing agent was added in preovulatory granulosa cells? Our previous study displayed that arsenic which induces oxidative damage and apoptosis in a variety of cells induced progesterone production and stimulated cellular antioxidant activity coupled with mitochondrial membrane potential increased. Behera et al. [45] reported that progesterone stimulated mitochondrial membrane potential and cellular ATP production in MCF-10A benign breast epithelial cells. Progesterone also relives myocardial ischemia/reperfusion injury in rats, enhances superoxide dismutase activity, and increases reduced GSH levels of serum. Thereafter, we concluded that progesterone enhanced by dexamethasone may modulate cellular redox status to protect against mitochondrial damage. We would explore the mechanism of antioxidant function mediated by progesterone in our future study. AMH has been identified and proposed for evaluation of response of follicles. It is predominantly produced by granulosa cells of preantral follicles and small antral follicles. The level of AMH was related to polycystic ovaries. Previous study had reported dexamethasone induced cystic status of ovary [27]. Our result showed that dexamethasone induced AMH production of preovulatory granulosa cells which indicated that dexamethasone may promote preantral follicles development. In conclusion, our experiment demonstrated that dexamethasone-induced progesterone production was requiring for activation of caspase-3 which may mediate differentiation and apoptosis of granulosa cells. The cellular ROS was decreased coupled with GSH increased, and the mitochondrial membrane potential remained unchanged which may contribute to steroidogenesis. Although the results obtained from vitro experiments may not be completely on behalf of the in vivo situation, we hope our data would help provide valuable information to clarify the impact of dexamethasone on reproductive system. Further study is required to explore the impact of dexamethasone on follicle development and steroidogenesis in vivo and the relative mechanism. Acknowledgments This work was supported by National Natural Science Foundation of China (81200418). Core facilities used in this research were provided by the Department of Genetics, National Research Institute for Family Planning and Central Laboratory, Shaanxi Provincial People’s Hospital. Conflict of interest We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for
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this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing, we confirm that we have followed the regulations of our institutions concerning intellectual property.
References 1. P.J. Barnes, I. Adcock, Anti-inflammatory actions of steroids: molecular mechanisms. Trends Pharmacol. Sci. 14(12), 436–441 (1993) 2. S. Jeyamohan et al., 158 Dexamethasone’s effect in multiplelevel anterior cervical discectomy and fusion. Neurosurgery. 60(Suppl 1), 172–173 (2013) 3. J. Lindholm, Cushing’s disease, pseudo-Cushing states and the dexamethasone test: a historical and critical review. Pituitary. doi:10.1007/s11102-013-0509-x 4. M. Poulain et al., Dexamethasone induces germ cell apoptosis in the human fetal ovary. J. Clin. Endocrinol. Metab. 97(10), E1890–E1897 (2012) 5. D.V. Milutinovic et al., Hypothalamic–pituitary–adrenocortical axis hypersensitivity and glucocorticoid receptor expression and function in women with polycystic ovary syndrome. Exp. Clin. Endocrinol. Diabetes 119(10), 636–643 (2011) 6. R. Sakumoto, S. Ito, K. Okuda, Changes in expression of 11betahydroxysteroid dehydrogenase type-1, type-2 and glucocorticoid receptor mRNAs in porcine corpus luteum during the estrous cycle. Mol. Reprod. Dev. 75(5), 925–930 (2008) 7. M. Tetsuka et al., Expression of 11beta-hydroxysteroid dehydrogenase, glucocorticoid receptor, and mineralocorticoid receptor genes in rat ovary. Biol. Reprod. 60(2), 330–335 (1999) 8. M. Irahara et al., Glucocorticoid receptor-mediated post-ceramide inhibition of the interleukin-1beta-dependent induction of ovarian prostaglandin endoperoxide synthase-2 in rats. Biol. Reprod. 60(4), 946–953 (1999) 9. S. Kol et al., Glucocorticoids suppress basal (but not interleukin1-supported) ovarian phospholipase A2 activity: evidence for glucocorticoid receptor-mediated regulation. Mol. Cell. Endocrinol. 137(2), 117–125 (1998) 10. E.A. McGee, A.J. Hsueh, Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21(2), 200–214 (2000) 11. K. Yacobi et al., Gonadotropins enhance caspase-3 and -7 activity and apoptosis in the theca-interstitial cells of rat preovulatory follicles in culture. Endocrinology 145(4), 1943–1951 (2004) 12. X.H. Yuan et al., Arsenic induced progesterone production in a caspase-3-dependent manner and changed redox status in preovulatory granulosa cells. J. Cell Physiol. 227(1), 194–203 (2012) 13. G.D. Niswender, Molecular control of luteal secretion of progesterone. Reproduction 123(3), 333–339 (2002) 14. N. Pescador et al., Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. Biol. Reprod. 57(3), 660–668 (1997) 15. K. Tajima et al., Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val135) and Ha-ras genes. Mol. Hum. Reprod. 8(1), 48–57 (2002) 16. Y.J. Chen et al., Interplay of PI3K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFbeta1 enhancement of FSH-
Endocrine
17. 18.
19.
20.
21.
22.
23.
24. 25.
26.
27.
28.
29.
30.
31.
stimulated steroidogenesis in rat ovarian granulosa cells. J. Endocrinol. 192(2), 405–419 (2007) D.B. Hales et al., Mitochondrial function in Leydig cell steroidogenesis. Ann. NY Acad. Sci. 1061, 120–134 (2005) J.A. Allen et al., Energized, polarized, and actively respiring mitochondria are required for acute Leydig cell steroidogenesis. Endocrinology 147(8), 3924–3935 (2006) S.R. King et al., Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) protein. J. Steroid Biochem. Mol. Biol. 69(1–6), 143–154 (1999) X.M. Qi et al., ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes. Acta Pharmacol. Sin. 34(9), 1229–1236 (2013) L. Zhou et al., Oxidative stress and phthalate-induced downregulation of steroidogenesis in MA-10 Leydig cells. Reprod. Toxicol. 42, 95–101 (2013) H. Chen et al., Effect of glutathione depletion on Leydig cell steroidogenesis in young and old brown Norway rats. Endocrinology 149(5), 2612–2619 (2008) W.M. Baarends 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 136(11), 4951–4962 (1995) J.A. Visser et al., Anti-Mullerian hormone: a new marker for ovarian function. Reproduction 131(1), 1–9 (2006) D.B. Seifer et al., Early follicular serum Mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil. Steril. 77(3), 468–471 (2002) I.A. van Rooij et al., Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve. Hum. Reprod. 17(12), 3065–3071 (2002) B. Jana et al., Dexamethasone-induced changes in sympathetic innervation of porcine ovaries and in their steroidogenic activity. J. Reprod. Dev. 51(6), 715–725 (2005) P.F. Terranova, F. Garza, Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH. Biol. Reprod. 29(3), 630–636 (1983) L.P. Roma et al., Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways. Gen. Physiol. Biophys. 31(1), 65–76 (2012) J.M. De Corral Saleta, J.C. Penhos, A.F. Cardeza, Diabetogenic and antidiabetogenic action of triamcinolone and dexamethasone. C. R. Seances Soc. Biol. Fil. 154, 2371–2372 (1960) U. Bandyopadhyay et al., Dexamethasone makes the gastric mucosa susceptible to ulceration by inhibiting prostaglandin
32.
33.
34.
35.
36.
37.
38. 39. 40.
41.
42.
43.
44. 45.
synthetase and peroxidase—two important gastroprotective enzymes. Mol. Cell. Biochem. 202(1–2), 31–36 (1999) Z. Ben-Rafael et al., Cortisol stimulation of estradiol and progesterone secretion by human granulosa cells is independent of follicle-stimulating hormone effects. Fertil. Steril. 49(5), 813–816 (1988) J.G. Yang, C.C. Yu, P.S. Li, Dexamethasone enhances follicle stimulating hormone-induced P450scc mRNA expression and progesterone production in pig granulosa cells. Chin. J. Physiol. 44(3), 111–119 (2001) T.J. Huang, P.S. Li, Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles. Biol. Reprod. 64(1), 163–170 (2001) A.J. Bhatt et al., Dexamethasone induces apoptosis of progenitor cells in the subventricular zone and dentate gyrus of developing rat brain. J. Neurosci. Res. 91(9), 1191–1202 (2013) G.B. Park et al., ROS and ERK1/2-mediated caspase-9 activation increases XAF1 expression in dexamethasone-induced apoptosis of EBV-transformed B cells. Int. J. Oncol. 43(1), 29–38 (2013) K. Yacobi, A. Tsafriri, A. Gross, Luteinizing hormone-induced caspase activation in rat preovulatory follicles is coupled to mitochondrial steroidogenesis. Endocrinology 148(4), 1717–1726 (2007) L. Oliver, F.M. Vallette, The role of caspases in cell death and differentiation. Drug Resist. Updates 8(3), 163–170 (2005) M. Lamkanfi et al., Caspases in cell survival, proliferation and differentiation. Cell Death Differ. 14(1), 44–55 (2007) R.H. Braw, S. Bar-Ami, A. Tsafriri, Effect of hypophysectomy on atresia of rat preovulatory follicles. Biol. Reprod. 25(5), 989–996 (1981) K. Hummitzsch et al., Spheroids of granulosa cells provide an in vitro model for programmed cell death coupled to steroidogenesis. Differentiation 77(1), 60–69 (2009) T. Diemer et al., Reactive oxygen disrupts mitochondria in MA10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology 144(7), 2882–2891 (2003) C.L. Kao et al., Resveratrol promotes osteogenic differentiation and protects against dexamethasone damage in murine induced pluripotent stem cells. Stem Cells Dev. 19(2), 247–258 (2010) A. Amsterdam et al., Steroidogenesis and apoptosis in the mammalian ovary. Steroids 68(10–13), 861–867 (2003) M.A. Behera et al., Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells. Am. J. Physiol. Endocrinol. Metab. 297(5), E1089–E1096 (2009)
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ORIGINAL ARTICLE
Dexamethasone altered steroidogenesis and changed redox status of granulosa cells Xiao-Hua Yuan • Bai-Qing Yang • Ying Hu • Yang-Yang Fan • Li-Xia Zhang • Jia-Chen Zhou Ya-Qin Wang • Cai-Ling Lu • Xu Ma
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Received: 20 December 2013 / Accepted: 21 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Glucocorticoids have been widely used in clinical application for anti-inflammatory and immunosuppressive function. Previous study reported that glucocorticoids adversely affect the reproductive system and can directly act on ovary. Here, we found that progesterone production induced by dexamethasone requiring activation of caspase-3 which may mediate differentiation and apoptosis of granulosa cells. Further study displayed that cellular glutathione level was increased and reactive oxygen species was decreased accompanied with unchanged mitochondrial membrane potential which may contribute to the maintenance of steroidogenesis in granulosa cells treated with dexamethasone. Dexamethasone also augmented the level of
Xiao-Hua Yuan, Bai-Qing Yang, and Ying Hu contributed equally to this work. X.-H. Yuan Y. Hu Y.-Y. Fan Y.-Q. Wang (&) Maternity Department, Shaanxi Provincial People’s Hospital, Xi’an 710068, China e-mail: [email protected] X.-H. Yuan B.-Q. Yang C.-L. Lu (&) X. Ma Department of Genetics, National Research Institute for Family Planning, Beijing 100081, China e-mail: [email protected] L.-X. Zhang Clinical Laboratory, Shaanxi Provincial People’s Hospital, Xi’an, China J.-C. Zhou Dalian Medical University, Dalian, China X. Ma (&) Graduate School of Peking Union Medical College; Department of Genetics, National Research Institute for Family Planning, Beijing 100081, China e-mail: [email protected]
anti-Mu¨llerian hormone secreted by preovulatory granulosa cells which indicated that dexamethasone may promote preantral follicles development. Keywords Granulosa cell Follicle Progesterone Dexamethasone Caspase-3 ROS
Introduction Glucocorticoids are hormonal mediator of stress. They have been extensively used in clinical application for antiinflammatory and immunosuppressive function [1]. However, there are many adverse reactions in clinical practice. Mounting evidences suggest that glucocorticoids adversely affect reproduction system. It influences the hypothalamic pituitary adrenal axis at three levels: the hypothalamus; the anterior pituitary gland; the testis/ovary [2–5]. Studies had identified glucocorticoids receptors in ovary cells [5–7], and glucocorticoid can directly act on ovary [8, 9]. The life of follicles goes through four stages: primordial follicles, preantral follicles, antral follicles, preovulatory follicles. In this process, only a small portion of follicles finally develops into ovulatory follicles, and most of the follicles undergo atresia [10]. Coupled with follicles development, granulosa cells proliferate, differentiate, and go through apoptosis around the oocyte and play a critical role in reproductive function by producing steroids. Caspase-3 had been identified the key enzyme mediated apoptosis and differentiation through cleavage of its substrates. A previous study had demonstrated that caspase-3 was activated in response to LH and FSH [11], and our previous work also indicated that caspase-3 played a key role in arsenic-induced progesterone production [12]. The first step of steroidogenesis occurs on mitochondria. The steroidogenic acute regulatory protein (StAR) transfers
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cholesterol from the outer to the inner mitochondria membrane where it is metabolized to pregnenolone by cytochrome P450 cholesterol side chain cleavage enzyme, P450scc [13]. Mounting evidences had identified that the StAR and P450scc proteins are induced by tropic hormone [14–16]. Hales et al. [17] suggested that mitochondria must be energized, polarized, and actively respiring to support steroidogenesis. There are evidences that mitochondrial membrane potential (Dwm) affects steroid synthesis by blocking protein processing into mitochondria [18, 19]. Extra reactive oxygen species (ROS) or redox imbalance damages mitochondria and results steroidogenesis disorder [20–22]. Anti-Mu¨llerian hormone (AMH) is produced by granulosa cells of primary follicles, preantral follicles, and small antral follicles [23]. The level of AMH reaches the maximum in preantral follicles and small antral follicles. However, the production of AMH cannot be detected in follicles in response to FSH and disappears in atresia follicles [24, 25]. AMH is widely used as marker in the assessment of responsiveness of follicles. It is also an indicator of the size of growing follicles pool [25, 26]. It had been reported that dexamethasone induced cystic status of ovary [27]. In the present study, we evaluated the effect of dexamethasone on steroidogenesis of granulosa cells, detected the redox status of granulosa cells treated by dexamethasone ,and revealed the relationship between caspase-3 and progesterone production of granulosa cells. The findings of our study would help evaluate the role of dexamethasone on the reproductive system.
Animals Immature female Sprague–Dawley (SD) rats (21 days old) were issued from the Department of Laboratory Animal Science of Peking University (Beijing, China). The rats were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All the protocols had the approval of the Institutional Committee on Animal Care and Use. Preovulatory granulosa cell culture Female SD rats (21 days old) were injected intraperitoneally with 20 IU/ml PMSG to increase the number of granulosa cells. Two days later, the animals were sacrificed by decapitation, and their ovaries were quickly removed and washed in 0.9 % (w/v) cold normal saline. Granulosa cells were harvested by puncturing the individual ovarian follicles with 25-gauge needles, collected by centrifugation (5009g, 5 min) and cultured in a humidified incubator at 37 °C and 5 % CO2 in DMEM supplemented with 15 % FBS for 24 h before the experimental treatment. Steroid radioimmunoassay (RIA) Conditioned media from granulosa cells (1 9 106/vial) treated with or without dexamethasone (0–20 lL) AcDEVD-CHO (40 lM) were collected; the levels of progesterone in the media were measured by RIA as previously described [28].
Materials and methods
Measurement of caspase-3 activity
Chemicals
Caspases activities were measured using caspase-3 activity kit according to the manufacturer’s instructions. Briefly, GCs, following the treatment of dexamethasone (0–20 lL) for 24 h, were washed with cold phosphate-buffered saline (PBS), resuspended in lysis buffer, and left on ice for 15 min. Activities of caspase-3 were measured with substrate peptides Ac-DEVD-pNA. The release of p-nitroanilide (pNA) was quantified by determining the absorbance with a microplate reader (BioTek Instruments) at 405 nm.
DMSO, b-actin antibody, and dexamethasone were bought from Sigma (Saint Louis, MO, USA). Pregnant mare serum gonadotropin was purchased from HydeVenture Bio Co., Ltd. (Beijing, China). ROS assay kit (DCHF-DA), glutathione (GSH) assay kit, caspase-3 assay kit, Ac-DEVD-CHO, 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolecarbocyanideiodine (JC-1), and Hoechst 33258 were supplied by Beyotime Biotechnology Co. (Haimen, China). AMH Elisa kit was purchased from Huamaike Biotechnology Co. (Beijing, China). Fetal bovine serum (FBS) and DMEM media were purchased from Thermo Scientific HyClone (Logan, UT, USA). Trypsin– EDTA (0.25 %) and penicillin–streptomycin (1009) were obtained from Invitrogen Co., Ltd. (Carlsbad, CA, USA). The StAR antibody and caspase-3 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The P450scc antibody was obtained from Abcam (Abcam, Cambridge, UK).
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Western blot analysis GCs (1 9 106/vial) were cultured in six-well plates, incubated with different reagents for the indicated time. The cells were washed with PBS and lysed with a cell lysis buffer containing 0.5 % sodium deoxycholate, 0.1 % SDS, 10 mM NaF, 0.2 mM Na3VO4, 1 mM PMSF, and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Equal amounts of protein were separated on a 12 % SDSPAGE gel, transferred to nitrocellulose membranes, and
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probed with b-actin, StAR, P450scc, and caspase-3 antibodies. Detection of signals was performed with an odyssey two-color infrared imaging system (LI-COR, Lincoln, NE). Mitochondrial membrane potential (Dwm) analysis Mitochondrial membrane potential was identified with JC-1 dye. GCs were incubated with dexamethasone (0–20 lL) for 48 h. Then, the cells were incubated at 37 °C and 5 % CO2 for 20 min with medium consisting of 10 mg/ml JC-1, and then the cells were observed under a fluorescence microscope (TE2000, Nikon, Inc., Tokyo, Japan) and detected by the microplate reader with an excitation of 530 nm and an emission of 590 nm (Biotech Instrument).
Measurement of AMH After GCs treated with dexamethasone (0–20 lM) in DMEM-F12 media for 48 h, the conditioned media were collected, and the levels of AMH were measured by Elisa kit. Statistical analysis All data are presented as mean ± SEM. The differences between means were calculated using a one-way analysis of variance followed by a t test with p \ 0.05 considered significant.
Measurement of intracellular GSH
Results
The GSH concentrations in GCs were determined with a dithionitrobenzoic acid–glutathione disulfide reductase recycling assay. The GCs were treated with dexamethasone (0–20 lL) for 48 h. Subsequently, the GSH levels were determined using commercially available kits according to the manufacturer’s instructions.
Dexamethasone stimulates progesterone production in granulosa cells
Measurement of ROS The intracellular ROS of GCs was detected using DCHFDA. At 48 h after dexamethasone (0–20 lL) exposure, the GCs were incubated for 30 min in 5 mM DCHF-DA diluted in DMEM medium. The ROS levels were detected by using a microplate reader with an excitation of 488 nm and an emission of 525 nm (BioTek Instruments, Winooski, VT). Flow cytometry analysis GCs were collected after treatment with dexamethasone (0–20 lM) for 48 h and fixed with 70 % ethanol overnight at 48 °C. The samples were concentrated by removing the ethanol and resuspending the cells in a PBS solution containing PI (50 mg/ml) and RNaseA (25 mg/ml). The apoptotic GCs were detected by flow cytometry (Coulter Epics XL; Beckman-Coulter, Inc., Miami, FL). Hoechst staining After treatment with or without dexamethasone (0–20 lM) for 48 h, GCs growing on the culture plates were fixed with 4 % paraformaldehyde for 30 min at room temperature. Cells were washed with PBS and exposed to 1 mg/ml Hoechst 33258 at room temperature in the dark for 10 min and observed under a fluorescent microscope (TE2000; Nikon, Inc.).
To test the effect of dexamethasone on the production of progesterone in preovulatory granulosa cells, we cultured granulosa cells exposed with various concentrations of dexamethasone. Incubation granulosa cells with dexamethasone (0.2, 2, and 20 lL) for 48 h can significantly increase progesterone production (Fig. 1a). Moreover, dexamethasone (2 lL) treatment induced progesterone production significantly after 6 h (Fig. 1b). Dexamethasone alters the expression of steroidogenic proteins StAR and P450scc In the process of steroidogenesis, the rate-limiting StAR protein transfers cholesterol from the outer to the inner mitochondria membrane where it is metabolized to pregnenolone by the P450scc. To determined whether dexamethasone affects the expression of steroidogenic proteins StAR and P450scc, StAR and P450scc were detected by western blotting. Our result showed that dexamethasone increased StAR and P450scc expression at the concentration range (0.2–20 lL; Fig. 2a). There was no significant difference in the expression of StAR among groups in the concentration range (0.2–20 lL). The P450scc expression was unregulated at 0.2 lL and reached the peak at 2 lL, downregulated slightly at 20 lL (Fig. 2b). Dexamethasone induces caspase-3 activation in preovulatory granulosa cells A previous study reported that LH and FSH enhance caspase-3 and caspase-7 activity of granulosa cells coupled with steroidogenesis. Our result also demonstrated that
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Fig. 1 Dexamethasone stimulated the progesterone production of GCs. Progesterone production of granulosa cells after treatment with various concentrations of dexamethasone for 48 h (a) and at various
time points with a constant concentration of 2 lM dexamethasone (b). The data represent mean ± SEM (n C 4). Columns with asterisks are significantly different from control (0 lL or 0 h). *** p \ 0.0001
Fig. 2 Dexamethasone stimulated steroidogenic protein P450scc and StAR expression. Steroidogenic protein P450scc and StAR were detected by western blot after exposure of GCs to different concentrations of dexamethasone for 24 h. Columns with different superscript letters are significantly different (p \ 0.05)
arsenic induced production in a caspase-3 dependent manner. In light of the previous study, we examined caspase-3 using antibody that can recognize the full length caspase-3 and partially cleaved production 21 KD. We found that dexamethasone (0.2–2 lM) significantly increased the level of 21 KD of caspase-3 at 12 h (Fig. 3a). Caspase-3 fluorometric assay showed that dexamethasone induced caspase-3 activation at 12 h at the concentration of 2 and 20 lL (Fig. 3b). Dexamethasone induces slight apoptosis in preovulatory granulosa cells In most cell types, dexamethasone caused apoptosis, such as rat thymocyte, myeloma, and peripheral blood monocyte. Our result also showed that dexamethasone stimulated activation of caspase-3 which cleaved substrate resulting apoptosis. We further quested that whether dexamethasone also caused apoptosis in preovulatory granulosa cells. By FACS analysis, we determined the subG1 DNA content, a characteristic feature of apoptotic cells. Our result demonstrated that dexamethasone (20 lL) resulted slightly apoptosis in preovulatory granulosa cells. There was no obvious apoptosis of granulosa treated with dexamethasone (0–2 lM; Fig. 4a). Hoechst 33258, a
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Fig. 3 The activation of caspase-3 is involved in dexamethasoneinduced progesterone production. The caspase-3 activity was detected by western blot (a) and caspase-3 activity assay kit (Beyotime China, b) after exposure of GCs to different concentrations of dexamethasone for 12 h
classical way of identifying the morphology of apoptotic cells, also displayed that dexamethasone (20 lL) results in fragment nuclei slightly (Fig. 4b).
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Fig. 4 Dexamethasone induced apoptosis slightly in GCs. After exposure of GCs to dexamethasone (0–20 lL) for 48 h, the sub-G1 content of GCs was analyzed by FACS (a), and Hoechst 33258 was observed under a fluorescence microscope (b, magnification 9100)
Fig. 5 Inhibition caspase-3 activity can inhibit progesterone production of GCs treated with dexamethasone. The progesterone levels were detected after exposure with or without dexamethasone (2 lL) plus caspase-3 inhibitor (AcDEVD-CHO; 40 lL) for 48 h (a). Steroidogenic protein StAR and P450scc were detected after exposure with or without dexamethasone (2 lL) plus caspase-3 inhibitor (Ac-DEVDCHO; 40 lL) for 24 h (b). Columns with different superscript letters are significantly different (p \ 0.05)
Caspase-3 is required in progesterone production induced by dexamethasone in preovulatory GCs To determine whether caspase-3 is required in steroidogenesis regulated by dexamethasone, GCs were incubated
with dexamethasone in the presence or absence of specific caspase-3 inhibitor. As shown in Fig. 5a, specific caspase-3 inhibitor, Ac-DEVD-CHO can cause significantly decrease in progesterone production at 48 h treated with dexamethasone. Further study displayed that specific caspase-3
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Fig. 6 Dexamethasone reduced ROS level and increased GSH level without significant effect on mitochondrial transmembrane potential. After exposure of GCs to various concentrations of dexamethasone for 48 h, the intracellular levels of ROS (a), GSH (b), were
determined. The data represent mean ± SEM (n C 4). Columns with asterisks are significantly different from control (0 lL or 0 h). ** p \ 0.005; *** p \ 0.0001
inhibitor decreased steroidogenic protein P450scc expression but not StAR expression treated by dexamethasone at 24 h (Fig. 5b). These findings indicated that caspase-3 is involved in steroidogenesis in GCs mediated by dexamethasone.
AMH secretion significantly at the concentration of 0.2 and 2 lM. Although the level AMH of group treated by dexamethasone (20 lM) compared with control group also increased, there was not statistically significant which may due to the apoptosis of granulosa cells (Fig. 8).
Effect of dexamethasone on ROS, GSH, and mitochondrial membrane potential (DWm)
Discussion
The intact mitochondrial membrane and redox balance are the requirement of steroidogenesis. The increase in ROS or depletion in reduced GSH would damage mitochondrial membrane and result in inhibition of steroidogenesis. Redox imbalance and the damage of mitochondrial membrane can also result caspase-3 activation and apoptosis. Our result displayed that steroidogenesis, caspase-3 activation, and apoptosis appeared in a process simultaneously. To further study the process of steroidogenesis stimulated by dexamethasone in preovulatory granulosa cells, we examined the levels of ROS, GSH, and mitochondrial membrane potential (DWm). Our result showed that dexamethasone enhanced the levels of GSH in GCs (Fig. 6b). The ROS levels treated with various doses of dexamethasone were decreased (Fig. 6a). JC-1 is widely used to detect cellular mitochondrial membrane potential. The aggregated JC-1 which indicated the intact mitochondrial membrane was detected by fluorescence microplate reader and fluorescence microscope. We found that there was no significant difference between treated groups and control group (Fig. 7a, b). Effect of dexamethasone on AMH AMH is produced by granulosa of preantral follicles and small antral follicles. It reflects the responsiveness of follicles. Our result showed that dexamethasone induced
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Glucocorticoids have served as anti-inflammatory and immunosuppressive agent for a long time in clinical application. Dexamethasone is typical representative of glucocorticoid. High dose of dexamethasone can induce diabetes, peptic ulcer, a series of symptoms similar with Cushing syndrome [29–31]. Dexamethasone also acts on reproduction system. Previous study reported that glucocorticoids enhanced progesterone production in luteinized granulosa cells from mature follicles and increased progesterone production in preovulatory granulosa cells stimulated by FSH or 8-Br-cAMP [32, 33], whereas dexamethasone inhibited progesterone synthesis of preovulatory granulosa in response to LH [34]. Here, we found that dexamethasone stimulated progesterone production in granulosa cells from preovulatory follicles of unmatured SD rat. The discrepancy may due to different dose, different stimulating factor combined with glucocorticoids, and the different status of follicle development. The responses of GCs to LH and FSH are up-regulation steroidogenic protein StAR and P450scc following with steroidogenesis. Our finding also showed that dexamethasoneinduced progesterone production similar to LH and FSH acts through StAR and P450scc. As apoptosis-inducing agent, dexamethasone induces activation caspase-3 and apoptosis in a variety of tissues and cells [4, 35, 36]. In our experiment, dexamethasone increased caspase-3 activation (0.2, 2, and 20 lM) and slight
Endocrine Fig. 7 Mitochondrial membrane potentials of preovulatory granulosa cells treated with various concentrations of dexamethasone remained unchanged. After exposure of GCs to various concentrations of dexamethasone for 48 h, the aggregated JC-1 was examined by microplate reader and detected under fluorescent microscope
Fig. 8 Dexamethasone stimulated the AMH production of GCs. After exposure of GCs to various concentrations of dexamethasone for 48 h, the AMH levels of GCs were analyzed by AMH Elisa. Columns with asterisks are significantly different from control (0 lL). ** p \ 0.005; *** p \ 0.0001
apoptosis (20 lM) coupled with progesterone production. Interestingly, Yacobi et al. [11] reported that LH and FSH induced increase in caspase-3 and caspase-7 in preovulatory follicles. Subsequently, they found that LH-induced caspase activation in preovulatory follicles was coupled with mitochondrial steroidogenesis [37]. Caspase-3 plays a key role both in differentiation and apoptosis [38, 39]. The destination of developing follicles (ovulation or atresia) is dependent on the cells within them (proliferation, differentiation, or apoptosis) regulated by endocrine, autocrine, and paracrine. Coupled with granulosa differentiation dominant follicles develop into ovulatory follicles following with luteinization, meanwhile, a large number of follicles
undergo atresia accompanied by granulosa cells apoptosis [10]. The ability of progesterone production of granulosa cells gradually increases with strengthening degree of differentiation. There were also evidences that the progesterone production was increased in atresia follicles in which apoptotic granulosa cell reached 20–30 % [40]. Afterward, Hummitzsch et al. [41] found that steroidogenesis was coupled with programmed cell death through establishing programmed cell death model with spheroids of granulosa cells in vitro. In our studies, inhibition of caspase-3 activity results to a decrease in progesterone production of granulosa cells stimulated by dexamethasone (Fig. 5). Our previous study also identified that inhibition caspase-3 activity resulted in a decrease in progesterone synthesis of granulosa cells stimulated by arsenic and FSH [12]. Current evidences indicated that progesterone production required caspase-3 activation through mediating differentiation or apoptosis of granulosa cells. For steroidogenesis, the initial step occurs within the mitochondrion. Hales et al. [17] suggested that mitochondria must be intact, energized, polarized to maintain steroidogenesis. The depletion of antioxidant GSH and extra ROS would damage the mitochondrial membrane resulting decrease in steroidogenesis [22, 42]. Dexamethasone, as apoptosis-inducing agent, always leads to ROS generation in a variety of cells [36, 43]. However, our study demonstrated that the GSH was increased coupled with ROS decline in granulosa cells treated with dexamethasone (Fig. 6) and the mitochondrial membrane potential remained unchanged, a state that is necessary for steroidogenesis (Fig. 7). Amsterdam [44] reported when immortalized granulosa cells were stimulated to undergo
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apoptosis, there was a dramatic increase in progesterone production, and the mitochondria were preserved intact. To some extent, Abraham’s experiment was confirmed by our results. How mitochondria were kept intact to support progesterone synthesis when apoptosis-inducing agent was added in preovulatory granulosa cells? Our previous study displayed that arsenic which induces oxidative damage and apoptosis in a variety of cells induced progesterone production and stimulated cellular antioxidant activity coupled with mitochondrial membrane potential increased. Behera et al. [45] reported that progesterone stimulated mitochondrial membrane potential and cellular ATP production in MCF-10A benign breast epithelial cells. Progesterone also relives myocardial ischemia/reperfusion injury in rats, enhances superoxide dismutase activity, and increases reduced GSH levels of serum. Thereafter, we concluded that progesterone enhanced by dexamethasone may modulate cellular redox status to protect against mitochondrial damage. We would explore the mechanism of antioxidant function mediated by progesterone in our future study. AMH has been identified and proposed for evaluation of response of follicles. It is predominantly produced by granulosa cells of preantral follicles and small antral follicles. The level of AMH was related to polycystic ovaries. Previous study had reported dexamethasone induced cystic status of ovary [27]. Our result showed that dexamethasone induced AMH production of preovulatory granulosa cells which indicated that dexamethasone may promote preantral follicles development. In conclusion, our experiment demonstrated that dexamethasone-induced progesterone production was requiring for activation of caspase-3 which may mediate differentiation and apoptosis of granulosa cells. The cellular ROS was decreased coupled with GSH increased, and the mitochondrial membrane potential remained unchanged which may contribute to steroidogenesis. Although the results obtained from vitro experiments may not be completely on behalf of the in vivo situation, we hope our data would help provide valuable information to clarify the impact of dexamethasone on reproductive system. Further study is required to explore the impact of dexamethasone on follicle development and steroidogenesis in vivo and the relative mechanism. Acknowledgments This work was supported by National Natural Science Foundation of China (81200418). Core facilities used in this research were provided by the Department of Genetics, National Research Institute for Family Planning and Central Laboratory, Shaanxi Provincial People’s Hospital. Conflict of interest We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for
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this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing, we confirm that we have followed the regulations of our institutions concerning intellectual property.
References 1. P.J. Barnes, I. Adcock, Anti-inflammatory actions of steroids: molecular mechanisms. Trends Pharmacol. Sci. 14(12), 436–441 (1993) 2. S. Jeyamohan et al., 158 Dexamethasone’s effect in multiplelevel anterior cervical discectomy and fusion. Neurosurgery. 60(Suppl 1), 172–173 (2013) 3. J. Lindholm, Cushing’s disease, pseudo-Cushing states and the dexamethasone test: a historical and critical review. Pituitary. doi:10.1007/s11102-013-0509-x 4. M. Poulain et al., Dexamethasone induces germ cell apoptosis in the human fetal ovary. J. Clin. Endocrinol. Metab. 97(10), E1890–E1897 (2012) 5. D.V. Milutinovic et al., Hypothalamic–pituitary–adrenocortical axis hypersensitivity and glucocorticoid receptor expression and function in women with polycystic ovary syndrome. Exp. Clin. Endocrinol. Diabetes 119(10), 636–643 (2011) 6. R. Sakumoto, S. Ito, K. Okuda, Changes in expression of 11betahydroxysteroid dehydrogenase type-1, type-2 and glucocorticoid receptor mRNAs in porcine corpus luteum during the estrous cycle. Mol. Reprod. Dev. 75(5), 925–930 (2008) 7. M. Tetsuka et al., Expression of 11beta-hydroxysteroid dehydrogenase, glucocorticoid receptor, and mineralocorticoid receptor genes in rat ovary. Biol. Reprod. 60(2), 330–335 (1999) 8. M. Irahara et al., Glucocorticoid receptor-mediated post-ceramide inhibition of the interleukin-1beta-dependent induction of ovarian prostaglandin endoperoxide synthase-2 in rats. Biol. Reprod. 60(4), 946–953 (1999) 9. S. Kol et al., Glucocorticoids suppress basal (but not interleukin1-supported) ovarian phospholipase A2 activity: evidence for glucocorticoid receptor-mediated regulation. Mol. Cell. Endocrinol. 137(2), 117–125 (1998) 10. E.A. McGee, A.J. Hsueh, Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21(2), 200–214 (2000) 11. K. Yacobi et al., Gonadotropins enhance caspase-3 and -7 activity and apoptosis in the theca-interstitial cells of rat preovulatory follicles in culture. Endocrinology 145(4), 1943–1951 (2004) 12. X.H. Yuan et al., Arsenic induced progesterone production in a caspase-3-dependent manner and changed redox status in preovulatory granulosa cells. J. Cell Physiol. 227(1), 194–203 (2012) 13. G.D. Niswender, Molecular control of luteal secretion of progesterone. Reproduction 123(3), 333–339 (2002) 14. N. Pescador et al., Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. Biol. Reprod. 57(3), 660–668 (1997) 15. K. Tajima et al., Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val135) and Ha-ras genes. Mol. Hum. Reprod. 8(1), 48–57 (2002) 16. Y.J. Chen et al., Interplay of PI3K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFbeta1 enhancement of FSH-
Endocrine
17. 18.
19.
20.
21.
22.
23.
24. 25.
26.
27.
28.
29.
30.
31.
stimulated steroidogenesis in rat ovarian granulosa cells. J. Endocrinol. 192(2), 405–419 (2007) D.B. Hales et al., Mitochondrial function in Leydig cell steroidogenesis. Ann. NY Acad. Sci. 1061, 120–134 (2005) J.A. Allen et al., Energized, polarized, and actively respiring mitochondria are required for acute Leydig cell steroidogenesis. Endocrinology 147(8), 3924–3935 (2006) S.R. King et al., Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) protein. J. Steroid Biochem. Mol. Biol. 69(1–6), 143–154 (1999) X.M. Qi et al., ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes. Acta Pharmacol. Sin. 34(9), 1229–1236 (2013) L. Zhou et al., Oxidative stress and phthalate-induced downregulation of steroidogenesis in MA-10 Leydig cells. Reprod. Toxicol. 42, 95–101 (2013) H. Chen et al., Effect of glutathione depletion on Leydig cell steroidogenesis in young and old brown Norway rats. Endocrinology 149(5), 2612–2619 (2008) W.M. Baarends 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 136(11), 4951–4962 (1995) J.A. Visser et al., Anti-Mullerian hormone: a new marker for ovarian function. Reproduction 131(1), 1–9 (2006) D.B. Seifer et al., Early follicular serum Mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil. Steril. 77(3), 468–471 (2002) I.A. van Rooij et al., Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve. Hum. Reprod. 17(12), 3065–3071 (2002) B. Jana et al., Dexamethasone-induced changes in sympathetic innervation of porcine ovaries and in their steroidogenic activity. J. Reprod. Dev. 51(6), 715–725 (2005) P.F. Terranova, F. Garza, Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH. Biol. Reprod. 29(3), 630–636 (1983) L.P. Roma et al., Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways. Gen. Physiol. Biophys. 31(1), 65–76 (2012) J.M. De Corral Saleta, J.C. Penhos, A.F. Cardeza, Diabetogenic and antidiabetogenic action of triamcinolone and dexamethasone. C. R. Seances Soc. Biol. Fil. 154, 2371–2372 (1960) U. Bandyopadhyay et al., Dexamethasone makes the gastric mucosa susceptible to ulceration by inhibiting prostaglandin
32.
33.
34.
35.
36.
37.
38. 39. 40.
41.
42.
43.
44. 45.
synthetase and peroxidase—two important gastroprotective enzymes. Mol. Cell. Biochem. 202(1–2), 31–36 (1999) Z. Ben-Rafael et al., Cortisol stimulation of estradiol and progesterone secretion by human granulosa cells is independent of follicle-stimulating hormone effects. Fertil. Steril. 49(5), 813–816 (1988) J.G. Yang, C.C. Yu, P.S. Li, Dexamethasone enhances follicle stimulating hormone-induced P450scc mRNA expression and progesterone production in pig granulosa cells. Chin. J. Physiol. 44(3), 111–119 (2001) T.J. Huang, P.S. Li, Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles. Biol. Reprod. 64(1), 163–170 (2001) A.J. Bhatt et al., Dexamethasone induces apoptosis of progenitor cells in the subventricular zone and dentate gyrus of developing rat brain. J. Neurosci. Res. 91(9), 1191–1202 (2013) G.B. Park et al., ROS and ERK1/2-mediated caspase-9 activation increases XAF1 expression in dexamethasone-induced apoptosis of EBV-transformed B cells. Int. J. Oncol. 43(1), 29–38 (2013) K. Yacobi, A. Tsafriri, A. Gross, Luteinizing hormone-induced caspase activation in rat preovulatory follicles is coupled to mitochondrial steroidogenesis. Endocrinology 148(4), 1717–1726 (2007) L. Oliver, F.M. Vallette, The role of caspases in cell death and differentiation. Drug Resist. Updates 8(3), 163–170 (2005) M. Lamkanfi et al., Caspases in cell survival, proliferation and differentiation. Cell Death Differ. 14(1), 44–55 (2007) R.H. Braw, S. Bar-Ami, A. Tsafriri, Effect of hypophysectomy on atresia of rat preovulatory follicles. Biol. Reprod. 25(5), 989–996 (1981) K. Hummitzsch et al., Spheroids of granulosa cells provide an in vitro model for programmed cell death coupled to steroidogenesis. Differentiation 77(1), 60–69 (2009) T. Diemer et al., Reactive oxygen disrupts mitochondria in MA10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology 144(7), 2882–2891 (2003) C.L. Kao et al., Resveratrol promotes osteogenic differentiation and protects against dexamethasone damage in murine induced pluripotent stem cells. Stem Cells Dev. 19(2), 247–258 (2010) A. Amsterdam et al., Steroidogenesis and apoptosis in the mammalian ovary. Steroids 68(10–13), 861–867 (2003) M.A. Behera et al., Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells. Am. J. Physiol. Endocrinol. Metab. 297(5), E1089–E1096 (2009)
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