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They Don’t Just Make a Man

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1 They Don’t Just Make a Man
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

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

5 Hyperandrogenism is Detrimental to
Women’s Health

6 Androgen Priming may work…
The Egg Timer is about to (((Ring)))

7 If Too Many Androgens Cause Unregulated Follicle Overgrowth, Then What Happens When There Are Too Few Androgen?

8 Androgen Receptor Knockout Mice Have Decreased Fertility
Defective folliculogenesis Subfertility Premature ovarian Failure Hu et.al., (2004) PNAS 101 ; Shiina et.al., (2006) PNAS 103 ;

9 Androgen Receptor is Expressed Throughout
the Hypothalamus-Pituitary-Gonadal axis Hypothalamus Granulosa cells Theca cells Pituitary Oocytes Ovary The Follicle Uterus

10 X + Generation of Granulosa cell (GC)-specific
Androgen Receptor knockout (ARKO) Mouse Model X + AR loxp/loxp Amh-RII cre - GC (From Dr. Guido Verhoeven) (From Dr. Martin Matzuk)

11 AR mRNA levels are low in the granulosa cells (GC) of GC-ARKO mice
WT GC AR +/- GC AR -/- Relative expression of AR mRNA * GC Pituitary Hypothalamus Sen et.al., Mol Endo 2010

12 AR Protein levels are low in the granulosa cells (GC) of GC-ARKO mice
+/+ +/- -/- AR Actin GC Sen et.al., Mol Endo 2010

13 AR is Expressed in Theca but not Granulosa cells in GC-ARKO Mice
II III 20X 20X 40X WT * * * GC GC GC * * GC AR -/- * GC 20X 40X Sen et.al., Mol Endo 2010

14 GC-ARKO Mice Have Grossly Normal Reproductive Organs
Ovary Uterus

15 Fertility Test

16 GC-ARKO Mice Have Less Frequent
and Smaller Litters A. 8-9 week old animals Genotype n Litter Pups Pups/litter Litter/female GC AR -/- 8 17 48 2.82 ± 0.2* 2.13 ± 0.22* WT 28 200 7.14 ± 0.29 3.5 ± 0.19 GC AR +/- 195 6.96 ± 0.28 B week old animals Genotype n Litter Pups Pups/litter Litter/female GC AR -/- 6 10 16 1.6 ± 0.23* 1.66 ± 0.21* WT 20 119 5.97 ± 0.25 3.33 ± 0.21 GC AR +/- 19 113 5.65 ± 0.42 3.16 ± 0.16 Sen et.al., Mol Endo 2010

17 Why do GC-ARKO Mice Have Less
Frequent Litters ? Days P D E Stages of Estrus Cycle GC AR +/- GC AR -/- GC-ARKO Mice Cycle Abnormally Sen et.al., Mol Endo 2010

18 GC-ARKO Mice Have Decreased Ovulation Why do GC-ARKO Mice Have
Smaller Litter Size? Number of oocytes * Number of super- ovulated oocytes * GC AR +/- GC AR -/- Sen et.al., Mol Endo 2010

19 Premature Loss of Ovarian Reserve (Premature Ovarian Failure)
GC-ARKO Mice Have Premature Loss of Ovarian Reserve (Premature Ovarian Failure) FSH (ng/ml) *

20 Loss of AR in Granulosa Cells Results in:
Less Frequent Ovulation Decreased number of oocytes ovulated Premature Ovarian Failure

21 Follicle Growth in the Ovary

22 GC-ARKO Mice Have a Normal Follicle Distribution at 4-5 Weeks
% of follicles

23 GC-ARKO Mice Have Decreased Follicle Progression and Increased Follicle Atresia with Age
AF A GC AR +/- GC AR -/- * % of follicles * * *

24 Results in Slow Follicular Growth Rate
Knockout of ARs in GCs Results in Slow Follicular Growth Rate diameter (μm) Follicle * GC AR -/- GC AR +/+ Sen et.al., Mol Endo 2010

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

26 Ovaries from GC-ARKO Mice Have High
Rate of Apoptosis Cleaved Caspase 3 ] -/- +/- Granulosa cells Actin TUNNEL assay-detects DNA fragmentation that results from apoptosis Sen et.al., Mol Endo 2010 Supplemental Fig 1

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28 Strong enough for a man but
ANDROGENS (Secret) : Strong enough for a man but made for a woman

29 How do Androgens Promote Follicle Growth and Prevent Follicle Atresia?
Androgens Signal?

30 Androgen Signaling Kinases Genomic Cellular Response Androgens AR Non
Gene Expression TF

31 (Matrix Metalloproteinase)
Extra-nuclear Androgen Signaling Membrane bound EGF ligands Androgens EGFR AR MMP PXN P-Y Src Ras (Matrix Metalloproteinase) Raf MEK ERK Sen et.al. JBC 2010; JCI 2012

32 DHT Promotes Paxillin Phophorylation &
Phosphoserine-Paxillin Nuclear Localization Androgens Media DHT P-PXN a b PXN PAXILLIN EGFR AR MMP Src P-Y ERK Ras Raf MEK PXN PXN P-Y P-S PXN P-Y P-S Nucleus Sen et.al., JCI 2012

33 Paxillin is Required for DHT-Induced AR
Nuclear Localization Androgens Androgen Receptor EGFR AR Media DHT Nsp siRNA PXN 1a 1b 2a 2b MMP Src P-Y ERK Ras Raf MEK AR PXN PXN P-Y P-S PXN P-Y P-S Nucleus Sen et.al., JCI 2012

34 How do Androgens Promote Follicle Growth and Prevent Follicle Atresia?
AR Atresia

35 Androgen-Induces miRNA-125b Expression
in Granulosa Cells GC Primary Culture (WT) Relative expression of miRNA125b * miR-125b TATAAA I II III IV ARE: 5’-GGTACAnnnTGTTCT-3’ 5’ UTR region of miR-125b consists of a TATA box and four potential AREs 5’ 3’ Sen et.al., unpublished

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38 Androgen-induced expression of miRNA-125b
Requires the Extranuclear Pathway Media DHT Flutamide U0126 PXN siRNA Relative expression of miRNA125b * GC Primary Culture (WT) EGF EGFR AR MMP Src ERK Ras Raf MEK PXN P-Y P-S Nucleus miRN-125b Sen et.al., unpublished

39 Androgen AR Extranuclear MAPK Paxillin Nuclear (+) miRNA-125b expression Expression of miR-125b in GCs require a synergistic action between the nuclear and extranuclear actions of ARs

40 Targets of miR-125b Bak1: Bcl-2 homologous killer
Bmf: Bcl2 modifying factor Bax: bcl2 associated X protein P53: tumor suppressor protein

41 miRNA-125b Suppresses Pro-Apoptotic
Protein Expression in Granulosa Cells Bak Bax Bmf GAPDH Nsp anti-miR125b inhibitor DHT P53 * Relative expression of miRNA125b GC Primary Culture (WT) Nsp Anti-miR125b inhibitor Sen et.al., unpublished

42 Pro-apoptotic proteins Decreased Follicular atresia
Androgens miRNA-125b expression Pro-apoptotic proteins Decreased Follicular atresia

43 In GC-ARKO mice, miRNA-125b Expression Correlates with Apoptosis
Relative expression of miR-125b * WT KO Bak Bax Bmf GAPDH P53 B Cleaved Caspase 3 ] -/- +/- Granulosa cells Actin

44 Androgen AR Extranuclear MAPK Paxillin Nuclear (+) miRNA-125b expression Pro-apoptotic proteins Decreased Follicular atresia

45 How do Androgens Promote Follicle Growth and Prevent Follicle Atresia?
AR Atresia

46 Follicles Will Not Grow Without FSH
“Androgens augment FSH actions to promote follicular growth” But How ?????

47 Androgens Increase Antral Follicle Number In Whole Ovary Organ Culture
Sen et.al., unpublished

48 Androgens Increase Growth of
Follicles In Vitro DHT (nM) Follicular diameter (μm) * -DHT +DHT Sen et.al., unpublished

49 Androgens are not essential for
FSHR expression Relative expression of FSHR mRNA WT KO FSHR GAPDH Sen et.al., unpublished

50 In Primary Granulosa Cells, Androgens
Increase FSH Protein but not mRNA Expression Relative expression of FSHR mRNA GC Primary Culture (WT) M DHT FSHR GAPDH Sen et.al., unpublished

51 (non-genomic) fashion
While androgens are not essential for FSHR expression, androgens can increase FSHR protein levels in a transcription-independent (non-genomic) fashion

52 AR Signaling in Granulosa Cells Stimulates FSH Receptor Expression,
which Mediates Follicle Growth Extranuclear MAPK Paxillin FSH Receptor Androgen AR Increased Pre-Antral Follicle Growth

53 Androgen Treatment Increases the Sensitivity
Of Follicles Towards FSH Actions FSH (ng/ml) 1 10 100 Percent of Follicles - DHT + DHT Antral Follicle Count

54 AR Signaling in Granulosa Cells Blocks Apoptosis and Promotes Follicle Growth
Androgen AR Increased Pre-Antral Follicle Growth FSHR Extranuclear MAPK Paxillin Nuclear miRNA-125b expression Pro-apoptotic proteins Decreased Follicular atresia

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56 DHEA in DOR Patients DHEA Androstenedione Estrone Estradiol
3bHSD CYP19 Androstenedione Estrone 17bHSD 17bHSD 17bHSD 3bHSD CYP19 Estradiol Androstenediol Testosterone AR

57 DHEA Mimics DHT Effects on Follicular Development
FSH FSH + DHEA FSH + DHEA + Flutamide Percent of Follicles Antral Follicle Count FSH (10ng/ml) DHEA (100nM) Flutamide (100nM)

58 Androgens induce AMH expression Relative expression of AMH mRNA
in granulosa cells * Relative expression of AMH mRNA * GC culture (WT) Sen et.al., unpublished

59 DHEA in DOR Patients- IVF
Testosterone Androstenedione Androstenediol 17bHSD Estradiol CYP19 Estrone 3bHSD AR AMH expression Follicular survival FSH sensitivity # of oocytes retrieved during IVF AFC DHEA

60 Too much androgens: PCOS Too few androgen: Diminished Ovarian Reserve
“Balance is Key” Mr. Miyagi…….Karate kid Too much androgens: PCOS Too few androgen: Diminished Ovarian Reserve

61 Acknowledgements Stephen R Hammes MD PhD CHR Norbert Gleicher MD
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 Catholic University of Leuven, Belgium Guido Verhoeven PhD Baylor College of Medicine Martin Matzuk PhD Funding NIH University of Rochester Foundation of Reproductive Medicine

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

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64 Granulosa cell (GC)- and Oocyte specific
Androgen Receptor knockout female mouse AMHRII / GDF9 promoter AR AR promoter

65 Androgen-Induced Increase in FSHR Protein Levels is Likely Mediated Through
Non-Genomic Actions M DHT ActD CHX FSHR GAPDH Sen et.al., unpublished

66 Androgens Through Extranuclear Actions Induce FSHR Protein levels
Media DHT Flutamide U0126 PXNsiRNA WT PXN Rescue PXNsiRNA + FSHR GAPDH

67 Translation P- P- P- EGF EGFR Y Paxillin Translation initiation eIF
Src AR P- Y MMP Paxillin Translation initiation Raf TSC1 TSC2 MEK AAAAAA eIF 5’ Cap S6K P ERK PABP mTOR-C1 Translation 4EBP1 P Paxillin P- S Y PABP Paxillin Paxillin P- S Y PABP


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