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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 263-269
Copyright © 1998 by The Endocrine Society

Original Studies

Nongenomic Effects of Androstenedione on Human Granulosa Luteinizing Cells

Véronique Machelon, Françoise Nomé and Jan Tesarik

INSERM, U-355, Institut Paris-Sud sur les Cytokines (V.M., F.N.), 92140 Clamart; Laboratoire d’Eylau (J.T.), 75116 Paris, France

Address all correspondence and requests for reprints to: Dr. Véronique Machelon, INSERM, U-355, Maturation Gamétique et Fécondation, 32 rue des Carnets, 92140 Clamart, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study examines rapid (5–60 s) effects of androgens on the cytosolic free Ca2+ concentration ([Ca2+]i) in human granulosa luteinizing cells. Cells were obtained from human preovulatory follicles, and [Ca2+]i was measured with the use of the Ca2+-responsive fluorescent dye fluo-3. Molar concentrations between 100 pmol/L and 1 µmol/L androstenedione increased [Ca2+]i within 5 s after addition to cells. This [Ca2+]i increase resulted from both Ca2+ influx, as shown by the effects of ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid and the voltage-dependent Ca2+ channel blocker verapamil, and Ca2+ mobilization from the endoplasmic reticulum, as shown by the effects of thapsigargin. Treatment with pertussis toxin and U-73,122, a specific inhibitor of phospholipase C, abolished the effects of androstenedione on [Ca2+]i. Flutamide, a nuclear androgen receptor antagonist, did not block the increase in [Ca2+]i induced by androstenedione. Testosterone (100 pmol/L to 1 µmol/L) had no effect. This is the first report showing that androstenedione increases [Ca2+]i in granulosa cells. These data provide evidence for the presence in granulosa cells of a novel, short term mechanism of androstenedione action involving voltage-dependent Ca2+ channels in the plasma membrane and phospholipase C activation via a pertussis toxin-sensitive G protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROIDS play multiple roles in the physiological regulation of ovarian function, and locally produced steroid hormones may have important autocrine and paracrine effects in the human ovary (reviewed in Ref.1). Androgens are secreted by thecal cells under the control of pituitary gonadotropins and provide a hormonal microenvironment for the preovulatory development of the follicle and the oocyte. In addition to serving as a substrate for estrogen synthesis, androgens may modulate ovarian functions by regulating follicular growth and maturation (2). Androgens may enhance gonadotropin action in granulosa cells (3). On the other hand, they also cause apoptosis in granulosa cells and play an active role in the process of follicular atresia (4). In normal ovulatory cycles, the content of androgens in follicular fluid is related to follicular health and maturity (5). Appropriate concentrations of free, biologically active androgens in follicular fluid are important for successful conception in women undergoing ovarian stimulation for in vitro fertilization; they have been suggested as potential markers for the assessment of oocyte quality, embryo development, and implantation (6). Androgens also play an important role in the etiology of certain disorders of human infertility such as polycystic ovarian disease, characterized by the failure of follicular maturation, the inhibition of ovarian estradiol production, and hyperandrogenization (7).

To better understand the molecular and cellular actions of androgens, it is important to define the signaling mechanisms through which they act. The ovary is a target tissue for androgens. The nuclear androgen receptor, a member of the steroid receptor supergene family of ligand-activated transcription factors that resides predominantly in the nucleus, is expressed in granulosa cells of several species, including human (8, 9, 10). The nuclear androgen receptor was localized immunohistochemically in normal cycling human ovaries, and the staining intensity was strongest in granulosa cells just after ovulation (11). In addition to acting at the nuclear receptors, there is now increasing evidence that ovarian steroids react with the cell surface and thus activate a variety of cell signaling cascades. These effects have been first studied in germ cells. The effect of progesterone on amphibian oocyte maturation is among the earliest discovered nongenomic effects of steroids on cells (12); the action of estradiol on the cell surface implies Ca2+ as a second messenger and contributes to human oocyte capacitation for fertilization (13); the progesterone-initiated human sperm acrosome reaction requires a Ca2+ influx (14, 15), a Cl- efflux (16), and the activation of a protein tyrosine kinase (17). A large body of data now provides evidence for rapid effects of androgens on the cytosolic free calcium concentration ([Ca2+]i) in various cell types (18, 19, 20), including cells of reproductive organs (21). These effects are consistent with the activation of cell surface receptors different from the conventional nuclear receptors.

In the present study the involvement of free cytosolic Ca2+ as a second messenger has been studied in human granulosa luteinizing cells (GLCs). We measured changes in [Ca2+]i in GLCs in response to androgens. After having found that androstenedione causes a rapid rise in cytosolic calcium, we investigated the mechanisms involved in this effect using pharmacological manipulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

Androstenedione, testosterone, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ionomycin, and flutamide, an androgen antagonist used in the treatment of prostate cancer, were purchased from Sigma (St. Quentin Fallavier, France). Acetoxymethylester of fluo-3 (fluo-3/AM) was obtained from Calbiochem (France Biochem, Meudon, France). 1-[6-([17ß-3-metoxyestra-1,3,5(10)-trien-17-yl]-amino)hexyl]-1H-pyrrole-2,5-dione U-73,122 was obtained from Biomol Research Laboratory (Plymouth, MA). RPMI medium, DMEM-Ham’s F-12 medium (vol/vol), trypsin-ethylenediamine tetraacetate solution, and Moloney murine leukemia virus reverse transcriptase (RT) were purchased from Life Technologies (Cergy Pontoise, France). Percoll was obtained from Pharmacia (St. Quentin en Yvelines, France). RNAzol and Taq polymerase were purchased from Bioprobe (Montreuil, France).

Granulosa cell preparations

Cells from follicular fluids obtained from patients treated for in vitro fertilization (22) were isolated and depleted in leukocytes as previously described (23). Granulosa cell preparations depleted in leukocytes were seeded at 104 cells/chamber in Lab-Tek chambered coverglass (Nunc, Naperville, IL), then cultured in Ham’s F-12-DMEM containing 15 mmol/L HEPES, 0.365 g/liter L-glutamine, 50 µg streptomycin, and 50 IU/mL penicillin, supplemented with 10% FCS. After an initial 24-h culture period, medium was removed and replaced by 1% FCS supplemented medium. Cells were then transferred to serum-free medium without phenol red containing 50 µg/mL low density lipoprotein 6 h before their use in experiments for calcium measurement. Ca2+ measurements were performed with leukocyte-depleted cell preparations that were cultured for 72 h. Granulosa luteinizing cells were identified by their 3ß-hydroxysteroid dehydrogenase/{Delta}5,{Delta}4-isomerase activity (24).

Ribonucleic acid (RNA) extraction and RT-PCR

Total RNA was extracted from 106 frozen cells (-80 C) using the RNAzol technique according to the manufacturer’s recommendations, and then extracted with phenol-chloroform. The messenger RNA was then transcribed into complementary DNA using RT primed with random hexamers as previously described (25). We investigated the presence of androgen receptor messenger RNA by RT-PCR using nucleotide sequences in its DNA-binding domain (26). PCR was run for 35 cycles under the conditions of 93 C for 1 min, 59 C for 1 min and 30 s, and 72 C for 1 min 30 s. The 36 cycles were followed by a 10-min extension phase at 72 C. We tested the presence of macrophages by PCR amplification using specific monocyte CD14 oligonucleotide sequences. Sample loading was adjusted to equivalent levels of ß-actin as previously described (23). Specific oligonucleotide sequences used for the detection of androgen receptor, CD14, and ß-actin were designed according to the sequences shown in Table 1Go.


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  		Table 1. Oligonucleotide sequences of primers for PCR reaction

 
Microscopic fluorescence measurements of single cells

Relative changes in [Ca2+]i were visualized using Bio-Rad MRC 600 confocal laser scanning microscopy unit (Bio-Rad Laboratories, Richmond, CA) and by recording fluorescence emitted by cells loaded with the [Ca2+]i indicator fluo-3. Cells were washed three times with Hanks’ HEPES buffer, pH 7.4 (137 mmol NaCl, 0.441 mmol KH2PO4, 0.442 mmol Na2HPO4, 0.885 mmol MgSO4·7H2O, 27.7 mmol glucose, 1.25 mmol CaCl2, and 20 mmol HEPES), and incubated with 5 µmol fluo-3/AM supplemented with 0.8 µmol pluronic F-127 for 30 min at 37 C in the dark. After loading, cells were washed twice in buffer and allocated to specific treatments, then placed on the heated stage of a Nikon Diaphot inverted microscope (Nikon Corp., Melville, NY), where all [Ca2+]i measurements were performed, as previously described (13). The scanning frequency was one image every 2 s. Tested substances were added in the course of measurement by hand pipetting. The levels of [Ca2+]i were evaluated by analyzing changes in fluorescence intensity. Measurements were performed on single cells. The capacity of cells to increase [Ca2+]i in response to nonspecific stimulation was tested at the end of each experiment by adding to the cells 2 µmol ionomycin. Ionomycin induced a rapid increase in [Ca2+]i in most cells, whereas only a fraction of cells responded to steroid addition. As a consequence, first, the effects of pharmacological treatments were tested on cells that had been previously selected as steroid-responsive cells; second, we controlled that cells could respond to successive additions of steroid. The percentage of responsive cells was also measured in treated preparations vs. untreated preparations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of RT-PCR products

No signal was detected by RT-PCR using CD14 oligonucleotide sequences in leukocyte-depleted GLC preparations. Multimers of a 257 bp of DNA fragment indicate the amplification products of androgen receptor transcripts in GLC preparations obtained after a 72-h culture period (Fig. 1Go).



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  		Figure 1. PCR products from total RNA isolated from granulosa cell preparations. Samples were treated with RT-PCR using oligonucleotide primers corresponding to known androgen receptor, CD14, and ß-actin complementary DNA sequences. Agarose gels were stained with ethidium bromide. The mol wt marker (M) is a DNA ladder. The 237-bp fragment of DNA indicates the amplification products of ß actin (lanes 1 and 3). The 351-bp fragment of DNA indicates the amplification products of CD14 transcripts. It is visualized in original cell preparations (lane 2), but disappears after leukocyte depletion (lane 4). A 257-bp band indicates the amplification products of androgen receptor transcripts in extracts obtained from granulosa cell preparations after a 72-h culture period (lane 5).

 
Steroid effects on [Ca2+]i

The direct effects of androgens, androstenedione, and testosterone (0.1 nmol to 1 µmol) on [Ca2+]i were examined first. The basal [Ca2+]i values were stable, and spontaneous changes in [Ca2+]i were not detected during observation. Steroids were dissolved in ethanol; the final concentration of ethanol never exceeded 0.01%. This ethanol concentration was without effect on the intracellular calcium concentration. Addition of androstenedione increased [Ca2+]i levels within 2–5 s (Fig. 2Go). The Ca2+ surge consisted of a sharp rising phase followed by a relatively slower, but still rapid, return to the resting levels, which was achieved by 30 s after hormone addition. The lowest molar concentration of androstenedione producing a perceivable effect was 100 pmol, and the response was maximal with 10 nmol androstenedione (Fig. 3AGo). Only a fraction of GLCs, ranging from 10–40% according to the preparation, responded to steroid addition. This fraction of reactive cells always rose when doses increased from 100 pmol to 10 nmol. Interestingly, cells were responsive to additional stimuli as soon as intracellular calcium had returned to its basal value and two repetitive exposures of GLCs to 10 nmol androstenedione at an interval of 40 s evoked similar [Ca2+]i responses (Fig. 3BGo). To test whether androgen effects on [Ca2+]i were independent of nuclear action of androgens, cells were preincubated for 6 h with 1 µmol flutamide, an antagonist of androgen genomic action. Calcium measurements were performed after this preincubation period in either the absence or presence of flutamide. In both cases, no significant modification of androstenedione action on Ca2+i was observed. The percentage of cells capable of responding to 10 nmol/L androstenedione by an increase in [Ca2+]i was not significantly modified in flutamide-treated populations vs. untreated populations. Adding 100 pmol to 1 µmol testosterone did not induce any significant increase in intracellular free Ca2+ levels, whereas final treatment with 2 µmol ionomycin induced a strong and rapid response, indicating that intracellular Ca2+ stores were not depleted; a significant percentage of cells responded to androstenedione in parallel experiments (Fig. 4AGo). Adding 10 nmol testosterone had no effect, whereas a calcium surge had been previously induced by exposing cells to 10 nmol androstenedione (Fig. 4BGo). Alternatively, after a previous exposure to 10 nmol testosterone, a subsequent calcium response to 10 nmol androstenedione was observed (Fig. 4CGo).



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  		Figure 2. Transients of [Ca2+]i of single human granulosa luteinizing cells after adding 10 nmol androstenedione (A). Relative changes in [Ca2+]i are expressed as changes in fluo-3 fluorescence intensity. Within a few seconds after adding 10 nmol A, a rapid increase in free cellular Ca2+ was visualized by an increase in fluorescence intensity inside the responsive cells. Theses pictures are representative of responsive cells.

 


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  		Figure 3. Direct effects of androstenedione (A) on [Ca2+]i in single human luteinizing granulosa cells. A, Increase in [Ca2+]i after addition of A concentrations increasing from 100 pmol to 10 nmol. B, Increase in [Ca2+]i after two successive additions of 10 nmol A. The final addition of 2 µmol ionomycin (Io) induced a rapid increase in [Ca2+]i. These traces are representative of responsive cells, and experiments were successfully repeated in three preparations.

 


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  		Figure 4. Effect of testosterone (T) on [Ca2+]i in single human luteinizing granulosa cells. A, Absence of significant [Ca2+]i changes after adding T concentrations increasing from 0.1 nmol to 1 µmol. B, First addition of 10 nmol androstenedione (A) followed by addition of 10 nmol T: cells that were responsive to A did not respond to T. C, First addition of 10 nmol T followed by addition of 10 nmol A; cells responsive to A were not previously responsive to T. A final addition of 2 µmol ionomycin (Io) induced a rapid increase in [Ca2+]i in each set of experiments, showing that cells were not depleted of Ca2+. These traces are representative of responsive cells, and experiments were successfully repeated in three preparations.

 
Transmembrane calcium flux

To investigate whether the action of androstenedione on [Ca2+]i was due to an influx of Ca2+ from the extracellular milieu, two types of blocking experiments were performed. In the first one, a small excess of EGTA (2 mmol) was added to chelate extracellular free calcium. Androstenedione (10 nmol) was added 30 s after adding EGTA, when a new steady state level of [Ca2+]i had been reached (27). Cells that responded to the first addition of androstenedione did not respond to any further addition of androstenedione after EGTA treatment (Fig. 5AGo). In the second one, verapamil, a selective blocker of Ca2+ entry via L-type voltage-dependent Ca2+ channels, was added to a final concentration of 10 µmol. Androstenedione at 10 nmol was added 1 min later. Treating cells with verapamil abolished the increase in [Ca2+]i induced by androstenedione (Fig. 5BGo). Cells responded to successive additions of androstenedione in control experiments.



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  		Figure 5. Effects of EGTA and verapamil on androstenedione-triggered Ca2+ transients. A, Extracellular calcium was removed by treating cells with 2 µmol EGTA, then 10 nmol androstenedione (A) were added after 30 s. EGTA treatment abolished the ability of cells to respond to A; the response to 2 µmol ionomycin was not abolished. B, One minute after adding 10 µmol verapamil, cells were exposed to 10 nmol A; verapamil treatment abolished the response to A; the response to 2 µmol ionomycin was not abolished. These traces are representative of responsive cells, and experiments were successfully repeated in three preparations.

 
Release of calcium from intracellular stores

To determine the part of the [Ca2+]i transient that was due to release from intracellular stores, we used the naturally occurring sequiterpene lactone, thapsigargin, at 1 µmol, a concentration at which it had the greatest effect on [Ca2+]i (28, 29). Thapsigargin inhibits the endoplasmic reticulum ATP-dependent Ca2+ pump, and Ca2+ from the associated store is released with little or no effect on plasma membrane and sarcoplasmic reticulum. The rise in [Ca2+]i induced by 1 µmol thapsigargin reached a peak within 90 s and then slowly decreased. Androstenedione (10 nmol) was added 10 min after thapsigargin. Treatment with thapsigargin abolished the androstenedione-induced Ca2+ spike in most responsive cells, although final treatment with 2 µmol ionomycin induced a strong and rapid response (Fig. 6AGo). To test the involvement of phospholipase C (PLC) in the rapid response to androstenedione, U-73,122, a specific inhibitor of PLC, was used (30, 31). GLCs were treated with 5 µmol U-73,122 for 2 min before the addition of 10 nmol androstenedione. Cells that responded to the first addition of androstenedione by a transient increase in [Ca2+]i did not respond to any further addition of androstenedione after treatment, whereas the peak induced by 2 µmol ionomycin was not abolished (Fig. 6BGo). In control experiments, cells responded to successive additions of androstenedione. To test the involvement of G proteins, we used pertussis toxin (PTX), which inactivates sensitive G proteins by ADP ribosylation of the {alpha}-subunit. GLCs were preincubated for 24 h with 100 ng/mL PTX before fluo-3 loading and [Ca2+]i measurements. No cell responded to 10 nmol androstenedione, whereas cells responded to 100 pmol progesterone, and most cells responded to the final addition of 2 µmol ionomycin (Fig. 6CGo). In control experiments performed with untreated populations, a significant fraction of cells (>10%) responded to the addition of androstenedione.



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  		Figure 6. Effects of thapsigargin, U-73,122, and PTX on androstenedione-triggered Ca2+ transients. A, Cells were incubated for 10 min with 1 µmol thapsigargin before adding 10 nmol androstenedione (A). Thapsigargin (1 µmol) caused a monotonic sustained increase in [Ca2+]i; after basal calcium levels were restored, 10 nmol A was added; thapsigargin treatment inhibited the increase in [Ca2+]i induced by A; the response to 2 µmol ionomycin was not abolished. B, Cells were incubated for 2 min with 5 µmol U-73,122, a specific PLC inhibitor, before adding 10 nmol A; U-73122 treatment abolished the increase in [Ca2+]i triggered by A. C, Cells were pretreated for 24 h with 100 ng/mL PTX before adding 10 nmol A. PTX treatment abolished the androstenedione-triggered Ca2+ transients, whereas it did not alter the response to 100 pmol/L progesterone (P) or 2 µmol ionomycin (Io). These traces are representative of responsive cells. Experiments were successfully repeated in three preparations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As previously shown for estradiol and progesterone in chicken and pig granulosa cells (32, 33), the present data demonstrate by using confocal laser scanning microscopy that androstenedione, but not testosterone, may induce a transient [Ca2+]i increase in human GLCs through a nongenomic action. This action involved PLC- and PTX-sensitive G protein activation as well as direct activation of transmembrane influx of extracellular calcium via activation of voltage-dependent calcium channels. Although GLCs might contain high levels of androstenedione when harvested, androstenedione was unlikely to be released by cultured GLCs. Firstly, granulosa cells do not secrete androstenedione, whose production is limited to thecal cells according to the two-cell theory (34, 35); secondly, human GLCs display high aromatase activity in culture (36), leading to metabolization of androstenedione into estradiol. The minimal effective concentration of androstenedione to increase [Ca2+]i in human GLC was 100 pmol, and the effect was maximal at 10 nmol. The androstenedione effect was rapid, taking less than 5 s from steroid addition to the beginning of [Ca2+]i increase. Accordingly, the effect of androstenedione addition could not be dependent on its transformation into estradiol. The effect of androstenedione was not inhibited by preincubation with flutamide, a pure nonsteroidal antiandrogen. Thus, androstenedione action on intracellular calcium was not transmitted via nuclear receptors. Testosterone, a steroid closely related to androstenedione, was shown to induce no transient [Ca2+]i regardless of whether GLC were exposed to testosterone before or after the addition of androstenedione. These data reveal the high specificity of the membrane response of granulosa cells to androgens. Moreover, testosterone, the major testicular steroid, rapidly and specifically increases intracellular calcium in Sertoli cells (21), suggesting that the membrane response of gonadal cells to steroids may be sex specific. To reinforce this suggestion, estradiol and progesterone, two major ovarian steroids, have minor or no membrane effects in Sertoli cells (21), whereas both have been shown to induce an increase in intracellular calcium in granulosa cells (32, 33).

The sources of Ca2+ mediating the nongenomic response of human GLCs to androstenedione were also investigated. The ability of GLCs to respond to androstenedione was diminished by removal of extracellular calcium with EGTA and by pharmacological blockage of voltage-activated membrane calcium channels with the use of verapamil, a blocker of voltage-dependent Ca2+ channels. Furthermore, thapsigargin, which alters calcium sequestration by the endoplasmic reticulum, blocked the androstenedione-induced Ca2+ transient. These results suggest that the calcium spike induced by androstenedione was due to both transmembrane influx through activation of voltage-dependent Ca2+ channel and mobilization of Ca2+ from endoplasmic reticulum. The response induced by androstenedione was probably a combination of Ca2+ mobilization from endoplasmic reticulum and Ca2+ entry from the extracellular medium. A cross-amplification requiring both the Ca2+ mobilization and the Ca2+ influx pathways appears to be needed for a measurable Ca2+ response, as the pharmacological blockage of either pathway led to a disappearance of a perceptible androstenedione effect. Further study is needed to determine whether the primary action of androstenedione is at the GLC membrane inducing a Ca2+ influx, which is subsequently amplified by Ca2+-induced Ca2+ release, or whether the primary action is at intracellular Ca2+ stores with subsequent amplification through capacitative Ca2+ entry (37).

The direct PLC inhibitor, U-73,122 inhibited the response to androstenedione in terms of [Ca2+]i. Furthermore, treating the cells with PTX resulted in inhibiting the response to androstenedione. In contrast, PTX treatment did not inhibit the response to progesterone as previously demonstrated (33). These results suggest that the mobilization of Ca2+ from endoplasmic reticulum implicates PLC activation involving G proteins sensitive to PTX, possibly Gi{alpha} subtypes that are expressed in human GLCs (38).

Cytosolic calcium may represent an element of a new biochemical pathway for androgen action on GLCs, and the ability of androstenedione to trigger the influx and release of Ca2+ may be a new feature of its action. In pregnancy-associated follicles after in vitro fertilization, concentrations of free androstenedione range from 1.9–8.2 nmol (39). Similar concentrations were found in unstimulated cycles (6). These concentrations of androstenedione were also those that induced a rapid increase in [Ca2+]i in cultured GLCs, arguing that membrane steroid action is a physiological relevant effect. The resulting surge of cytosolic calcium and the PLC-activated phosphoinositide turnover may induce a cascade of intracellular phosphorylations. These phosphorylations may interact with numerous cellular metabolic pathways, more particularly by modulating the activation of androgen nuclear receptor (40). Androgen receptor is expressed in granulosa cells in primate ovaries (9), mostly in preantral/early antral follicles and more faintly in preovulatory follicles (41). Androgen has been implicated in the local control of human ovarian function. The present data suggest that androgen action may be related to its membrane effect and raise the following question: what are the physiological consequences of membrane action of androst-enedione?

In conclusion, the present study demonstrates, for the first time, a calcium-mobilizing effect of androstenedione in human GLCs, which may be independent of nuclear receptor expression. Androstenedione acts at two distinct levels: opening of plasma membrane voltage-dependent Ca2+ channel and activation of a complex signal transduction cascade including a pertussis-sensitive G protein and PLC activation. These findings provide evidence for a novel, short term mechanism of androstenedione action different from the classical long term genomic effect. The data presented also demonstrate the specificity of androstenedione action on human luteinizing granulosa cells.


   Acknowledgments
 
We thank the Center for Reproductive Medicine (American Hospital of Paris, Neuilly, France) for providing us with follicular aspirates and the technicians of the Center for their helpful assistance. We are grateful to Dr. M. Lieberherr for her fruitful discussions and helpful critical advice.

Received February 10, 1997.

Revised July 9, 1997.

Revised September 3, 1997.

Accepted September 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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