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ESTROGEN AND ANTI-ANDROGEN INDUCED PENILE MAL-DEVELOPMENT
LILIAN OKUMU
TUSKEGEE UNIVERSITY, TUSKEGEE, AL, USA
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Background
• Endocrine disrupting chemicals interfere
hormonal balance; present in the
environment e.g. in cosmetics, medical
instruments and agriculture
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•Synthetic: diethylstilbestrol (DES), Ethinyl estradiol (EE)
•Phytoestrogens: Plant origin isoflavones (genistein, daidzen)
•Environmental: Bisphenol A (BPA), Methoxychlor (MXC), Pthalates
Background
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Background
•DES daughters have higher incidence of vaginal cancer; sons: testicular cancer and hypospadias
•BPA causes prostate hypertrophy and precancerous growth
•MXC causes lower fertility in females
•EE: More than two million women continue to take contraceptive pills during the first trimester of pregnancy
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Background: effects on wildlife
•Alligators from Lake Apopka (FL) contaminated with industrial estrogenic contaminants have smaller phallus and reduced fertility.
•Turtles from Mody Pond (MA) contaminated with xenobiotics have impaired reproductive functions.
•Fish from some rivers in the USA and Europe contaminated with oral contraceptives are feminized.
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Summary of background
•It is well-established that perinatal exposure to estrogenic compounds (endocrine disruptors) has permanent deleterious effect on the development of reproductive organs in both humans and wildlife.
•However, mechanisms underlying these reproductive disorders are not well-understood.
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•Chemicals: DES (diethylstilbestrol), estradiol valerate (EV),
or ethinyl estradiol (EE). All three compounds caused similar
mal-developments
•Animals: Neonatal rats and mice
•Treatment: 10ng-10µg/day/pup, 1-6 postnatal days, sc
•Tissue collection: Ages at day 7, 12, 18, 28, 41, or adulthood. •Analyses: hormone assays, gene expression (micro array and Q-RT-PCR), WB
Experimental design
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Study-1: Estrogen-induced mal-development of the penis
Goyal et al. (2004) Reproductive Toxicology 18:265-274
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Study-1: Estrogen-induced accumulation of fat cells and loss of smooth muscle cells
Control DES
Goyal et al. (2004) Reproductive Toxicology 18:265-274
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Study-1: Estrogen-induced accumulation of fat cells occurs in the corpora cavernosa, but not in the
corpus spongiosus
Corpus Spongiosus
Corpora Cavernosa
Goyal et al. (2004) Biology of Reproduction 18:265-274
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Study-2: Estrogen-induced penile mal-development is dose-dependent:
Neonatal Exposure to ethinyl estradiol (EE), female
contraceptive, at a dose of 100 ng (0.01 mg/kg) or
higher results in infertility and malformation of penis
in 100% of the treated rats
Mathews et al. (2009) Toxicological Sciences 112:331-343, Okumu et al, in preparation
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Study-2: Radiographs of the penis treated with different doses of EE
Mathews et al. (2009) Toxicological Sciences 112:331-343
Ethinyl Estradiol
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Study-3: Estrogen-induced penile mal-development is dependent upon estrogen exposure during critical
period of development
Goyal et al. (2005) Toxicological Sciences 87:242-254
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Study-4: Estrogen-induced penile mal-development is associated with lower intra-testicular T Surge (typical for rodents from gestation day 17 to postnatal days
7-10)
Goyal et al. (2009) Biology of Reproduction 81:242-254
ng/g
0
500
1000
1500
20004-Day -Old 5-Day -Old 6-Day -Old
Testicular Testosteroneng
/g
0
200
400
600
ng/t
esti
s
0
3
6
7-Day-Old
Fig. 1; Goyal et al.
A
B
C
**** *
7-Day-Old
* * *
* * * * *
(5)
(14)
(8)
(14)
(11)
(20)
(14)
(8)
(14)
(15) (7)
(8)
(8) (8)
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Control DES
Study-5: Estrogen-induced penile mal-development is associated with ERα up-
regulation
Goyal et al (2004) Biology of Reproduction 70:284-297Goyal et al (2007) Reproduction 134:199-208
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Study-6: ERα presence is essential for estrogen-induced penile mal-development
Goyal et al (2007) Reproduction 133:1057-1067
ERKO Data
Pen
is W
eigh
t (g)
0.00
0.01
0.02
0.03
0.04
0.05
Pen
is L
engt
h (m
m)
4
8
12
16
WC WD KC KD
*
*
WC WD KC KD
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Study-7: Estrogen-induced penile mal-development is mitigated by ER antagonist ICI 182780 and AR agonist DHT or testosterone
(T)
Goyal et al. (2009) Biology Reproduction 81:242-254
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Studies-9&10: Estrogen induced penile mal-development is characterized by down-regulation of smooth muscle cell
markers in the penis and steroidogenic enzymes in the testis
Okumu et al., 2012
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Study-9: similar to estrogen, GnRH antagonist antide (GnRH-A) supressed intratesticular testosterone surge and mRNA for
steroidogenic enzymes
Okumu et al., 2014
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Fo
ld c
han
ge
in g
ene
exp
ress
ion
rel
ativ
e to
Co
ntr
ol
0
1
2
3
4
521-day-old
a aa
a a
b
a,b a,b
b b
a
a a
c
a
b
a
c6 6 5 5 6 4
PPARMyh11Esr1
Estrogen up-regulates Esr1 and Pparγ mRNA and down-regulates Myh11 mRNA and these alterations are Mitigated
by Estrogen Receptor Antagonist ICI and DHT
Okumu et al., 2012
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Both estrogen and GnRH-A treatment induced similar effects in the penis
Ctrl DES
DES+DHT
DES+ICI DHT IC
I0
1
2
3
4
5
Rela
tive
fold
cha
nge
b
a,c
aa,c
a,c
a,c
Control Antide Ant + DHT Ant + ICI0
2
4
6
8
Rela
tive
fold
cha
nge
a
b
c
b,d
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MYH11 protein expression in the penis
Control DES0
0.4
0.8
1.2
Ban
d d
en
sity
***
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ACTA immunolocalization
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Results: ACTA IHC
Control DES-treated
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Down-regulation of the smooth muscle cell marker Myh11 was specific to the corpus cavernosum penis
10
2
4
6
8
10
12
14
16
18Q-RT-PCR: Myh11 expression
Control GnRH-A EE
RFC
in M
yh11 e
xpre
ssio
n
Corpus cavernosum Corpus spongiosum Prostate
*
***
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Similarly, estrogen and anti-androgen induced effects on Pde5a were limited to the corpus cavernosum penis
10
1
2
3
4
5
6 Q-RT-PCR: Pde5a expression
Control GnRH-A EE
RFC
in P
de5a e
xpre
ssio
n
Corpus cavernosum Corpus spongiosum Prostate
** **
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PDE5A protein is downregulated in GnRH-A treated penile tissues
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Summary of results
Neonatal treatment with DES and antide…
Suppressed testicular testosterone surge at
day 7
Decreased MYH11, ACTA2 and PDE5A
expression
Increased PPARG & ESR1 expression
Both DHT and ICI mitigated DES-induced
effects in the penis
However, ICI unable to mitigate antide effects
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Conclusions
Anti-androgens cause permanent mal-development
of the penis, similar to estrogenic exposure
Low androgen levels/action due to DES impedes
differentiation of stromal cells into smooth muscle
cells in the corpus cavernosum while enhancing
adipogenesis.
ESR1 mediated pathway and low T involved in DES;
low T in GnRH-A
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E
Leydig Cell Stromal Cell Stromal Cell
Hypothesis for mechanism of estrogen-induced mal-development of the penis
Testosterone ERaPPARg,
MYH11, ACTA2
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Acknowledgements
Dr. Hari Goyal Dr. Liz SimonDr. Tim BradenMs. Carol S. WilliamsMr. John W. WilliamsDr. Datiri & CMRC staff
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THANK YOU