androgens: they don’t just make a man out of you ! aritro sen ph.d. university of rochester school...

Androgens:

They Don’t Just Make a Man

Out of You !

Aritro Sen Ph.D. University of Rochester School of Medicine & Dentistry

Department of Medicine

Division of Endocrinology & Metabolism

Androgens: They Don’t Just Make a Man Out of You !

Hyperandrogenism is Detrimental to Women’s Health

Androgen Priming may work…

The Egg Timer is about to (((Ring)))

If Too Many Androgens Cause

Unregulated Follicle Overgrowth,

Then What Happens When There Are

Too Few Androgen?

Shiina et.al., (2006) PNAS 103 ; 1 224-229

• Defective folliculogenesis

• Subfertility

• Premature ovarian Failure

Hu et.al., (2004) PNAS 101 ; 11209-11214

Androgen Receptor Knockout Mice

Have Decreased Fertility

Pituitary

Uterus

Ovary

Theca cells

Granulosa cells

Oocytes

The Follicle

Androgen Receptor is Expressed Throughout the Hypothalamus-Pituitary-Gonadal axis

Hypothalamus

AR loxp/loxp

+ XAmh-RII cre - GC

Generation of Granulosa cell (GC)-specific

Androgen Receptor knockout (ARKO)

Mouse Model

(From Dr. Guido Verhoeven) (From Dr. Martin Matzuk)

AR mRNA levels are low in the

granulosa cells (GC) of GC-ARKO mice

GC Pituitary Hypothalamus

WT GC AR +/- GC AR -/-

Rela

tive

expr

essi

on

of A

R m

RNA

*0

1

2

Sen et.al., Mol Endo 2010

+/+ +/- -/-AR

ActinGC

AR Protein levels are low in the

granulosa cells (GC) of GC-ARKO mice



WT

GC

AR -/

-

GCGC

40XGC

GC

**

*

*

* *

40X

20X

20X20XI II III

AR is Expressed in Theca but not Granulosa cells in GC-ARKO Mice

GC AR +/- GC AR -/-

Ovary

Uterus

GC-ARKO Mice Have Grossly Normal Reproductive Organs

Fertility Test

Genotype n Litter Pups Pups/litter Litter/female

GC AR -/- 8 17 48 2.82 ± 0.2* 2.13 ± 0.22*

WT 8 28 200 7.14 ± 0.29 3.5 ± 0.19

GC AR +/- 8 28 195 6.96 ± 0.28 3.5 ± 0.19

Genotype n Litter Pups Pups/litter Litter/female

GC AR -/- 6 10 16 1.6 ± 0.23* 1.66 ± 0.21*

WT 6 20 119 5.97 ± 0.25 3.33 ± 0.21

GC AR +/- 6 19 113 5.65 ± 0.42 3.16 ± 0.16

B. 24-25 week old animals

A. 8-9 week old animals


GC-ARKO Mice Have Less Frequent and Smaller Litters

Why do GC-ARKO Mice Have Less Frequent Litters ?


0 10 20 30Days

PDE

Stag

es o

f Es

trus

Cyc

le GC AR +/-

GC AR -/-PDE

GC-ARKO Mice Cycle Abnormally

GC AR +/-GC AR -/-

8-9 weeks 24-25 weeks0

5

10

Num

ber o

f oo

cyte

s

**

8-9 weeks 24-25 weeks0

510152025

Num

ber o

f sup

er-

ovul

ated

ooc

ytes

*

Why do GC-ARKO Mice Have Smaller Litter Size?


GC-ARKO Mice Have Decreased Ovulation

GC AR +/- GC AR -/-0

20

40

FSH

(ng/

ml)

GC-ARKO Mice HavePremature Loss of Ovarian Reserve

(Premature Ovarian Failure)

*

Loss of AR in Granulosa Cells Results in:

• Less Frequent Ovulation

• Decreased number of oocytes ovulated

• Premature Ovarian Failure

Follicle Growth in the Ovary

% o

f fol

licle

s

Pr P PA A CL AF0

20

40

60

GC AR +/- GC AR -/-

GC-ARKO Mice Have a Normal Follicle Distribution at 4-5 Weeks

Pr P PA A CL AF0

20

40

60

**

*

*

CL

AFAFAF

AF AF

AF

AF

AF

A

A

A

ACL

CL

A

GC AR +/- GC AR -/-

GC-ARKO Mice Have Decreased Follicle Progression and Increased Follicle Atresia with Age

% o

f fol

licle

s

Folli

cle

diam

eter

(μm

)

D0 D40

150

300

*GC AR -/-GC AR +/+

Knockout of ARs in GCs Results in Slow Follicular Growth Rate


The AR is Required for Follicle Progression from Pre-Antral to Antral Stage

Supplemental Fig 1

AOvaries from GC-ARKO Mice Have High

Rate of Apoptosis


TUNNEL assay-detects DNA fragmentation that results from apoptosis

CleavedCaspase 3

Caspase 3]

-/-+/-

Gra

nulo

sa c

ells

Actin

ANDROGENS (Secret) :

Strong enough for a man but made for a woman

How do Androgens Promote Follicle Growth and Prevent Follicle Atresia?

How do Androgens Signal?

TF

Cellular Response

Gene ExpressionGenomic

Kinases

Androgens

AR

ARNonGenomic

Androgen Signaling

Extra-nuclear Androgen Signaling

Src

EGFR

ARMMP

ERK

Ras

Raf

MEK

Membrane bound

EGF ligands

Androgens

Sen et.al. JBC 2010; JCI 2012

PXNP-Y(Matrix Metalloproteinase)

EGFR

ARMMP Src

ERK

Ras

Raf

MEK

PXNP-Y

PXNP-Y P-S

Nucleus

PXNP-Y P-S

Androgens

DHT Promotes Paxillin Phophorylation & Phosphoserine-Paxillin Nuclear Localization

Sen et.al., JCI 2012

Media DHT

P-PXN

a b

PXN

PAXILLIN

AR

EGFR

ARMMP Src

ERK

Ras

Raf

MEK

PXNP-Y

PXNP-Y P-S

Nucleus

PXNP-Y P-S

Androgens

Media DHT

Nsp

si

RNA

PXN

si

RNA

1a 1b

2a 2b

Paxillin is Required for DHT-Induced ARNuclear Localization

Sen et.al., JCI 2012

Androgen Receptor


Atresia

AR

Androgen-Induces miRNA-125b Expression

in Granulosa Cells

Sen et.al., unpublished

miR-125b

TATAAA

IIIIIIIV

ARE: 5’-GGTACAnnnTGTTCT-3’

5’ UTR region of miR-125b consists of

a TATA box and four potential AREs

5’ 3’

Media DHT0

5

10

15

GC Primary Culture (WT)

Rela

tive

expr

essi

on

of m

iRN

A125

b *


EGFR

ARMMP Src

ERK

Ras

Raf

MEK

PXNP-Y

PXNP-Y P-S

Nucleus

AR

PXNP-Y P-S

miRN-125b

Androgen-induced expression of miRNA-125b Requires the Extranuclear Pathway

Med

ia

DH

T

Flut

amid

e

U01

26

PXN

siRN

A

DHT

Rela

tive

expr

essi

on

of m

iRN

A125

b *


0

4

8

EGF

Expression of miR-125b in GCs require a synergistic action between the nuclear

and extranuclear actions of ARs

Androgen ARExtranuclear

• MAPK• Paxillin

Nuclear

miRNA-125b expression(+)

Targets of miR-125b

• Bak1: Bcl-2 homologous killer • Bmf: Bcl2 modifying factor• Bax: bcl2 associated X protein• P53: tumor suppressor protein

miRNA-125b Suppresses Pro-Apoptotic Protein Expression in Granulosa Cells

BakBaxBmf

GAPDH

Nsp anti-

miR

125b

inhi

bito

r

DHT

P53

0

0.4

0.8

1.2

*

Rela

tive

expr

essi

on

of m

iRN

A125

b


Nsp Anti-miR125b inhibitor


miRNA-125b expression

Pro-apoptotic proteins

Decreased Follicular atresia

Androgens

AIn GC-ARKO mice, miRNA-125b Expression Correlates with Apoptosis

B

CleavedCaspase 3

Caspase 3

]

-/-+/-

Gra

nulo

sa c

ells

Actin

WT KO0

0.5

1

1.5

Rela

tive

expr

essi

on

of m

iR-1

25b

*

WT KOBakBaxBmf

GAPDHP53

Androgen ARExtranuclear


Nuclear


Pro-apoptotic proteins

Decreased Follicular atresia

(+)


Atresia

AR

Follicles Will Not Grow Without FSH

“Androgens augment FSH actions to promote follicular growth”

But How ?????

Androgens Increase Antral Follicle NumberIn Whole Ovary Organ Culture


0 10 25 1000

100

200

300

400 D0 D4

DHT (nM)

Fo

llic

ula

r di

amet

er (

μm

)

* *

Androgens Increase Growth of Follicles In Vitro


-DHT

+DHT

WT GC-ARKO0

0.6

1.2

Rela

tive

expr

essi

on

of F

SHR

mRN

A

WT KOFSHR

GAPDH

Androgens are not essential for FSHR expression


In Primary Granulosa Cells, Androgens Increase FSH Protein but not mRNA Expression

M DHTFSHR

GAPDH

M DHT0

0.6

1.2

Rela

tive

expr

essi

on

of F

SHR

mRN

A



While androgens are not essential for FSHR expression, androgens can increase FSHR protein levels in a

transcription-independent (non-genomic) fashion

Androgen AR

AR Signaling in Granulosa Cells Stimulates FSH Receptor Expression,

which Mediates Follicle Growth

FSH Receptor

Increased Pre-AntralFollicle Growth

Extranuclear


Androgen Treatment Increases the Sensitivity Of Follicles Towards FSH Actions

0

25

50

FSH (ng/ml)0 1 10 100

Perc

ent o

f Fol

licle

s

- DHT

+ DHT

Antral Follicle Count

AR Signaling in Granulosa Cells Blocks Apoptosis and Promotes Follicle Growth

Androgen AR FSHR Increased Pre-Antral

Follicle GrowthExtranuclear


Nuclear


Pro-apoptotic proteins Decreased Follicular atresia

DHEA in DOR Patients

DHEA

Testosterone

Androstenedione

Androstenediol

17b

HSD

Estradiol

CYP19Estrone

3bHSD

CYP193bHSD 17b

HSD

17b

HSD

AR

FSH (10ng/ml)DHEA (100nM) Flutamide (100nM)

0

10

20

FSH FSH +DHEA

FSH + DHEA+ Flutamide

Perc

ent o

f Fol

licle

s

Antral Follicle Count

DHEA Mimics DHT Effects on Follicular Development

M DHT0

4

8

12

Rela

tive

expr

essi

on o

f AM

H m

RNA

GC culture (WT)*

WT GC-ARKO0

0.7

1.4

*

Androgens induce AMH expression in granulosa cells


DHEA in DOR Patients- IVF

DHEA

DHEA

Testosterone

Androstenedione

Androstenediol

17b

HSD

Estradiol

CYP19Estrone

3bHSD

AR

AMH expression

Follicular survival

FSH sensitivity

# of oocytes retrieved during IVF

AFC

3bHSD CYP19

17b

HSD

17b

HSD

“Balance is Key”Mr. Miyagi…….Karate kid

Too much androgens: PCOSToo few androgen: Diminished Ovarian Reserve

Acknowledgements

Funding

NIH

University of Rochester

Foundation of Reproductive Medicine

Stephen R Hammes MD PhD

Melissa Rasar MD PhD

Amber West

Liliana Carbajal PhD

Kristen Evaul PhD

James Deng PhD

Tamara Strauss PhD

Francesco Caiazza PhD

Anindita Biswas MS

Hen Prizant

Ismary DeCastro MD

Allison Light

CHR

Norbert Gleicher MD

David Barad MD

Ho-Joon Lee PhD

Tamar Michaeli PhD

Aya Shohat-Tal PhD

Baylor College of Medicine

Martin Matzuk PhD

Catholic University of Leuven, Belgium

Guido Verhoeven PhD

Now….. who says I don’t have ANDROGENS!!!!!!

THANK YOU

Granulosa cell (GC)- and Oocyte specific Androgen Receptor knockout female mouse

ARAMHRII / GDF9 promoter

AMHRII / GDF9 promoter

AR

promoter

AR

AR

Androgen-Induced Increase in FSHR Protein Levels is Likely Mediated Through

Non-Genomic Actions

M DHT ActD CHX

DHT

FSHR

GAPDH


Med

iaDH

TFlu

tam

ide

U0126

PXNsiR

NA

DHT

PXNsiR

NA +

WT

PXN R

escu

e

Androgens Through Extranuclear ActionsInduce FSHR Protein levels

FSHR

GAPDH

PABPAAAAAA

eIF

5’ Cap

PABP

Src

EG

FR

ARMMP

EGF

Raf

MEK

ERK

PaxillinP-Y

PaxillinP-SP-Y

PaxillinP-SP-Y

PABPPaxillin

TSC1TSC2

mTOR-C1

S6KP

4EBP1P

Tran

slat

ion

Translationinitiation

androgens: they don’t just make a man out of you ! aritro sen ph.d. university of rochester school...

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