Presentation is loading. Please wait.

Presentation is loading. Please wait.

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.

Similar presentations


Presentation on theme: "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."— Presentation transcript:

1 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

2

3

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

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

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

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

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

13 WT GC AR -/- GC 40X GC * * * * * * 40X 20X IIIIII AR is Expressed in Theca but not Granulosa cells in GC-ARKO Mice

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

15 Fertility Test

16 Genotype n LitterPupsPups/litter Litter/female GC AR -/ ± 0.2* 2.13 ± 0.22* WT ± ± 0.19 GC AR +/ ± ± 0.19 Genotype n LitterPups Pups/litter Litter/female GC AR -/ ± 0.23 * 1.66 ± 0.21 * WT ± ± 0.21 GC AR +/ ± ± 0.16 B week old animals A. 8-9 week old animals Sen et.al., Mol Endo 2010 GC-ARKO Mice Have Less Frequent and Smaller Litters

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

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

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

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

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

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

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

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

27

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

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

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

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

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

33 AR EGFR AR MMP Src ERK Ras Raf MEK PXN P-Y PXN P-Y P-S Nucleus PXN P-YP-S Androgens Media DHT Nsp siRNA PXN siRNA 1a1b 2a2b Paxillin is Required for DHT-Induced AR Nuclear Localization Sen et.al., JCI 2012 Androgen Receptor

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

35 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’ GC Primary Culture (WT) Relative expression of miRNA125b *

36

37

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

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

40 Targets of miR-125b Bak1: Bcl-2 homologous killer Bmf: Bcl2 modifying factor Bax: bcl2 associated X protein P 53 : tumor suppressor protein

41 miRNA-125b Suppresses Pro-Apoptotic Protein Expression in Granulosa Cells Bak Bax Bmf GAPDH Nsp anti-miR125b inhibitor DHT P 53 * Relative expression of miRNA125b GC Primary Culture (WT) Nsp Anti-miR125b inhibitor Sen et.al., unpublished

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

43 A In GC-ARKO mice, miRNA-125b Expression Correlates with Apoptosis B Cleaved Caspase 3 ] -/- +/- Granulosa cells Actin Relative expression of miR-125b * WTKO Bak Bax Bmf GAPDH P 53

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? Atresia AR

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

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

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

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

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

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

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

55

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

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

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

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

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

61 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

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

63

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

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

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

67 PABP AAAAAA eIF 5’ Cap PABP Src EGFR AR MMP EGF Raf MEK ERK Paxillin P- Y Paxillin P- S Y Paxillin P- S Y PAB P Paxillin Translation initiation


Download ppt "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."

Similar presentations


Ads by Google