1. Performance enhancing drug impact on ovarian steroid hormone biosynthesis
Acknowledgments
References
1) Maravelias C, Dona A, Stefanidou M, Spiliopoulou C 2005 Adverse effects of anabolic
steroids in athletes. A constant threat. Toxicol Lett 158:167-175
2) King SR, LaVoie HA 2012 Gonadal transactivation of STARD1, CYP11A1 and HSD3B. Front
Biosci 17:824-846
Introduction
Steroid hormone structure
P4
Objectives: To determine if the performance enhancing drugs
Superdrol, Dihydrotestosterone (DHT), Mesterolone (a DHT-like analog), Trenbolone (a
progestin with androgen action), and the natural steroid hormone progesterone (given
exogenously) have the ability to directly impact progesterone (P4) production by human
ovarian luteinized granulosa cells (hGC).
Anabolic steroids are widely used as performance enhancing drugs. Women who take
anabolic steroid hormones with androgenic activity will have increased muscle mass, but
can also have side effects such as increased facial hair, acne, water retention, irregular
menstrual cycles, and infertility. Irregular menstrual cycles and infertility can result from
feedback mechanisms that affect pituitary gonadotropin secretion and/or by directly
affecting the ovarian production of natural sex steroid hormone (estrogen and
progesterone). It is unknown if there is a direct effect of many anabolic steroids on human
ovarian steroidogenic cells. Our goal was to determine if the performance enhancing drugs
Superdrol, Dihydrotestosterone (DHT), Mesterolone (a DHT-like analog), Trenbolone (a
progestin with androgen action), and the natural steroid hormone progesterone (given
exogenously) have the ability to directly impact progesterone (P4) production by human
ovarian luteinized granulosa cells (hGC). In steroidogenic cells, the Steroidogenic Acute
Regulatory Protein, encoded by the STARD1 gene, is considered the rate-limiting step in
de novo steroidogenesis including progesterone synthesis. As protein levels of the
STARD1 protein are regulated transcriptionally, we evaluated STARD1 mRNA levels. hGC
were obtained from normal women undergoing oocyte retrieval and cultured for 4 days in
10% fetal calf serum-enriched media prior to incubation in serum-free media. Cells were
treated with water vehicle or 8Br-cAMP (0.25 mM), which is known to stimulate STARD1
mRNA and progesterone synthesis. hGC were also co-cultured with varying concentrations
of Superdrol, DHT, Mesterolone, Trenbolone, and exogenous progesterone for 6-48 h.
STARD1 mRNA levels and P4 secreted in media were quantified by real-time PCR and
ELISA, respectively.
Summary and Conclusions
• Superdrol (0.1-1000 nM) had no effect on mean progesterone concentration in cells treated
with water vehicle or 0.25 mM 8Br-cAMP (n=2, 24 h).
• The fold increase with 8Br-cAMP tended to be reduced with 1000 nM Superdrol compared
to vehicle only.
• Superdrol at 10 nM tended to reduce the 8Br-cAMP stimulated level of STARD1 mRNA, but
did not change the fold response to cAMP.
• At 6 h, 10 nM DHT tended to decrease relative P4 concentration in both water vehicle and
8Br-cAMP treated samples.
• At 24 h, 1 and 10 nM DHT increased basal P4 levels, but did not alter the P4 concentration in
the presence of 8Br-cAMP.
• At 48 h, basal P4 concentration was reduced by 1-10 nM DHT, and the decrease in basal P4
led to an increased fold-response to 8Br- cAMP.
• At 6 and 24 h, but not 48 h, DHT tended to decrease basal STARD1 mRNA. At 24 h and 48
h, 10 nM DHT and 1 nM DHT increased 8Br-cAMP-stimulated STARD1 mRNA levels more
than 2-fold and 3-fold, respectively, when compared to vehicle.
• At 6 h, 100 nM exogenous progesterone increased 8Br-cAMP-stimulated STARD1 mRNA
levels more than 2-fold, and at 24 h a 3-fold reduction in 8Br-cAMP-stimulated STARD1
mRNA levels was observed.
• Mean basal P4 tended to be higher with Mesterolone and Trenbolone (1-100 nM, n=2). The
concentration of P4 resulting from 8Br-cAMP stimulation was unaffected by Mesterolone or
Trenbolone, although the fold-response to 8Br-cAMP was less with either steroid.
• Mesterolone and Trenbolone had no effect on basal STARD1 mRNA levels, although basal
levels tended to be lower with 1 nM Mesterolone.
• In the presence of 8Br-cAMP, both Mesterolone and Trenbolone at 10 nM gave a more
robust induction of STARD1 mRNA
• In conclusion, our initial work indicates that several anabolic steroids can have a direct
effect on luteinized granulosa cells of the ovary.
Figure 4. Effects of 6 h cyclic AMP analog or phorbol ester treatment on P3, P4, and total IGF2 mRNA
transcripts in human luteinized granulosa cells. Bars indicate mean plus SEM of IGF2 mRNA levels
relative to the respective vehicle treatment. N = 3 different experiments each performed with a different
patient's cells. Asterisk (*) indicates treatment mean was significantly different from its vehicle treatment,
P < 0.05, as assessed by T-test of ln-transformed data.
Total, P3, and P4 IGF2 transcript levels
Figure 5. Effects of 24 h cyclic AMP analog or phorbol ester treatment on P3, P4, and total
IGF2 mRNA transcripts in human luteinized granulosa cells. Bars are described in Fig. 4 legend.
N = 4 different experiments each performed with a different patient's cells. Asterisks (*) indicate
treatment mean was significantly different from its vehicle treatment, P < 0.05, as assessed by
ANOVA and post-hoc Tukey's test of ln-transformed data.
Figure 6. Effects of 48 h cyclic AMP analog or phorbol ester treatment on P3, P4, and total
IGF2 mRNA transcripts in human luteinized granulosa cells. Bars are described in Fig. 4 legend.
N = 4-7 different experiments each performed with a different patient's cells. Asterisks (*) indicate
treatment mean was significantly different from its vehicle treatment, P < 0.05, as assessed by ANOVA
and post-hoc Tukey's test of ln-transformed data.
0
1
2
3
4
5
6
7
sH20 1 DMSO 20
IGF2mRNAexpressionrelativetovehicle
control
total
P3
P4
8Br-cAMP (mM) PMA (nM)
*
0
1
2
3
4
5
sH20 0.25 1 DMSO 1 20
IGF2mRNAexpressionrelativetovehicle
control
total
P3
P4
8Br-cAMP (mM) PMA (nM)
*
*
8Br-cAMP (mM) PMA (nM)
0
2
4
6
8
10
12
14
16
18
20
sH20 0.25 1 DMSO 1 20
total
P3
P4
*
*
**
*
*
IGF2mRNAexpressionrelativetovehicle
control 6 h treatment
24 h treatment
48 h treatment
Figure 1. Left to right, structures of Superdrol, Proviron,DHT, Trenbolone, and progesterone.
Results
0
5
10
15
20
25
sH20
8Br-cAMP
0 1 10 100 1 10 100
Mesterolone (nM) Trenbolone (nM)
ProgesteroneRelativetoVehicleControl
Figure 3. Summary graph of hGC 1614, 1616, 1617 mRNA & P4 relative to vehicle (n=2 for most, sH20 and cAMP
n=3). A summary of real-time PCR and progesterone assay for patients 1614,1616,1617 is shown.
Treatment conditions are the same, but Y-axis labels differ between mRNA and progesterone. Bars are mean +SME for n=2 for
independent experiments. Mean P4 levels were higher with mesterolone and Trenbolone than other compounds, but was unaffected
by 8Br-cAMP. Neither compound had an effect on mean basal mRNA levels, however, 8Br-cAMP at 10nM had a greater effect on
STARD1 mrNA.
sH20
8Br-cAMP
ProgesteroneRelativetoVehicleControl
0 1 10
DHT (nM)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Figure 2. Steroidogenic cell showing location STARD1 and CYP11A1 protein action.
Human luteinized granulosa
cells from IVF program
Primary culture
DMEM/F12 + 10% FBS
Purify by
Percoll gradient
Collect media
Isolate RNA
Synthesize cDNA
Real-time PCR quantification
of individuals with specific
treatments.
4 days
Treatment 6, 24, 48 h
Serum-free media
sH20 Control
8Br-cAMP 0.25 mM
Superdrol
DHT
Mesterolone
Trenbolone
Progesterone
ELISA of diluted media
for progesterone quantity
Methods
sH20
8Br-cAMP
0 1 10 100 1 10 100
Mesterolone (nM) Trenbolone (nM)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
STARD1mRNA/TBPmRNARelativetoVehicleControl
Figure 4. Summary graph of hGC 1513 mRNA & P4 treated with DHT and exogenous progesterone.(n=1). A
summary of real-time PCR and progesterone assay for patient 1513 treated at 6h with n=1 per time point. At 6 h, 10 nM
DHT tended to decrease relative P4 concentration in both water vehicle and 8Br-cAMP treated samples. DHT also tended
t o decrease basal STARD1 mRNA. P4 treated cells at 100 nM concentrations also had a 2 fold increase in 8Br-cAMP
stimulated STARD1 mRNA.
sH20
8Br-cAMP
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
STARD1mRNA/TBPmRNARelativetoVehicleControl
0 1 10 1 10 100
DHT (nM) P4 (µM)
sH20
8Br-cAMP
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
STARD1mRNA/TBPmRNARelativetoVehicleControl
0 1 10 1 10 100
DHT (nM) P4 (µM)
ProgesteroneRelativetoVehicleControl
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 1 10
DHT (nM)
sH20
8Br-cAMP
Figure 5. Summary graph of hGC 1514 mRNA & treated with DHT and exogenous progesterone.(n=1). A summary of
real-time PCR and progesterone assay for patients1514 treated at 24h with n=1 per time point. 1 and 10 nM DHT
increased basal P4 levels, but did not alter the P4 concentration in the presence of 8Br-cAMP. The increase in basal P4
with DHT did reduce the fold-response to 8Br-cAMP. Similar to 6 h treatment, DHT tended to decrease basal STARD1
mRNA. At 24 h 10 nM DHT and 1 nM DHT increased 8Br-cAMP-stimulated STARD1 mRNA levels more than 2-fold and 3-
fold, respectively, when compared to vehicle.
sH20
8Br-cAMP
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
0 1 10 1 10 100
DHT (nM) P4 (µM)
STARD1mRNA/TBPmRNARelative
toVehicleControl
ProgesteroneRelativetoVehicleControl
0 1 10
DHT (nM)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
sH20
8Br-cAMP
Figure 6. Summary graph of hGC 1516 mRNA & P4 relative to vehicle (n=1). A summary of
real-time PCR and progesterone assay for patient1516 treated at 48h with n=1 per time
point. basal P4 concentration was reduced by 1-10 nM DHT, and the decrease in basal P4 led to
an increased fold-response to 8Br- cAMP. 48 h treated cells had similar effects with 24h cells with
10 nM DHT and 1 nM DHT increased 8Br-cAMP-stimulated STARD1 mRNA levels .
sH20
8Br-cAMP
Superdrol (nM)
STARD1mRNA/TBPmRNARelative
toVehicleControl
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
0 0.1 1 10 100 1000
sH20
8Br-cAMP
Superdrol (nM)
ProgesteroneRelativetoVehicleControl
0.0
0.5
1.0
1.5
2.0
2.5
0 0.1 1 10 100 1000
Figure 7. Summary graph of hGC 1434 &1472 mRNA & P4 treated with Superdrol(n=2). A
summary of real-time PCR and progesterone assay for patient 1434 and 1472. treated at 24h
with n=2 per time point. Superdrol (0.1-1000 nM) had no effect on mean progesterone
concentration in cells treated with water vehicle or 0.25 mM 8Br-cAMP .Superdrol at 10nM
tended to reduce 8Br-cAMP STARD1 mRNA levels, but had no effect on fold response of cAMP.