The role of growth factors in regulating cellular events during ovarian follicular development Leon J. Spicer

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1 The role of growth factors in regulating cellular events during ovarian follicular development Leon J. Spicer Department of Animal Science, Oklahoma State University, Stillwater, OK USA SESSION #54 EAAP 2015

2 Outline: General/Background on: IGF, TGFB, Hh, FGFs Systems Data on: IGFs; GDFs, BMPs; Hhs; FGFs & Ovarian Follicle Development Future Directions

3 Background

4 Background: Possible Metabolic Mediators of Reproduction adipose muscle liver leptin? TNFa? glycerol / FA insulin glucagon pancreas T3, T4 IGF-I thyroid lactate / aa cortisol epi/norepi IGF-I & -II adrenal IGFBP-3 & -2 glucose / ketones GH LDL / HDL Blood Plasma pituit ary From: Spicer, 2001 (Dom Anim Endocr 21: )

5 Endocrine- Autocrine- Paracrine Action of Factors in Ovarian Follicles Blood Vessels OOCYE GRANULOSA CELLS THECA CELLS Oocyte Factors Granulosa Factors Theca factors Modified from: Shimasaki et al., 2004 (Endocr. Rev. 25:72-101)

6 PARACRINE EFFECTS AUTOCRINE EFFECTS ENDOCRINE EFFECTS

7 Normal Ovarian Follicle Growth

8 Follicle Size Ovulation IGFs, TGFB, Hh, FGFs Ovulation Dominance FSH Deviation LH Subordinate Recruitment Atresia Estrus Day of estrous cycle Estrus

9 The complexity of the IGF system IGFBP-1 IGFBP-2 IGFBP-3 X X IGFBP-4 IGFBP-5 IGFBP-6 IGFBP Proteases (e.g., PAPP-A) IGFBP fragment s Insulin IGF-I IGF-2 Low-affinity IGFBP binders: IGFBP-7 IGFBP-8 IR IGF-IR IGF-IR/IR IGF-2R hybrid

10 During Follicular Growth: Estradiol and Androstenedione levels in follicular fluid (FFL) are greater in Dominant vs. Subordinate follicles. IGF-I Levels (total) in FFL do not differ between Dominant and Subordinate follicles FREE concentrations are greater. Primarily endocrine in origin. stimulates steroidogenesis and mitogenesis of ovarian cells.

11 Follicular Fluid IG F-I, ng/ml Free IGF-I: DF > SF Total IGF-I Free IGF-I DF-24 SF-24 DF-48 SF-48 Follicle Type and Collection Time (Santiago et al., 2005; Domest Anim Endocr 28:48-63)

12 log Free IGF-I Correlation log Estradiol of Estradiol vs log Free & IGF-I Free IGF-I Bovine Follicles Log Estradiol log Free IGF-I positively correlated (P<0.001) with E 2 (r=0.73) (Santiago et al., 2005; Domest Anim Endocr 28:48-63)

13 48 h 10% FCS then 48 h Serum-free Cultured Granulosa Cells From bovine follicles Cultured Thecal Cells

14 IGF-I Stimulates Aromatase Activity in Bovine Granulosa Cells Small Follicles ng/ml ED 50 = 5 vs 30 ng/ml ng/ml IGF-I, ng/ml Spicer et al., 2002 (Domest Anim Endocr 22:237-54)

15 IGF-2 Stimulates Aromatase Activity in Bovine Granulosa Cells Large Follicles ED 50 = 12 ng/ml (Spicer & Aad, 2007; Biol. Reprod. 77:18-27)

16 IGFs Stimulates CYP19A1 in Granulosa Cells (Spicer & Aad, 2007; Biol. Reprod. 77:18-27) (100 ng/ml)

17 Androstenedione (pg/10 5 cells/24 h) IGFs Stimulates Steroidogenesis in Thecal Cells 500 Control + LH CON IGF-1 IGF-2 Treatment (100 ng/ml) Spicer et al., 2004 (Mol. Cell. Endocr. 227:1-7)

18 IGFs Stimulates Proliferation of Granulosa Cells (Spicer & Aad, 2007; Biol. Reprod. 77:18-27) (100 ng/ml)

19 IGFBPs in Dominant vs. Subordinate Bovine Follicles BP-3 BP-2 BP-5 BP kDa 34-kDa kDa 22-kDa DF DF DF From Spicer et al., 2001 (Domest. Anim. Endocr. 21:1-15)

20 IGFBP-2 Activity is Lower in DF From Stewart et al., 1996 (Endocrinology 137:2842)

21 IGFBP-3 Inhibits IGF-I-induced Aromatase Activity by Granulosa Cells Spicer & Chamberlain, 1999 (Dom. Anim. Endocr. 16:19-29)

22 IGF2R decreases with increased follicle size (Spicer & Aad, 2007; Biol. Reprod. 77:18-27)

23 IGF2Rc mrna is decreased in Granulosa cells of Twinner cattle (Modified from: Aad et al., 2013; Domest. Anim. Endocr. 45:187)

24 Summary: IGF-I & -2 stimulate steroid production and cell proliferation. IGFBPs block IGF binding and cell responses. IGF2Rc is reduced in co-dominant follicles

25 During Follicular Growth: IGFBPs IGFBP-2, -4 and -5 levels decrease. (which bind IGF-I and 2 and block IGF action) thus, decreased IGFBPs cause increased free IGF which further stimulate follicle development.

26 Hormones inhibit IGFBP & IGF2R production & stimulate proteases FSH Day 5 Low IGFBPs E2 & Free IGF-I & -2 IGF-I inhibits IGF2R production Decreased IGF2R & IGFBPs Day 10 Ovulation LH Atresia Hormones stimulate IGFBP & IGF2R production & inhibit proteases Development of the Dominant Follicle

27 Impact of TGFB family on Granulosa and Thecal Cell Steroidogenesis

28 TGFB Family of Proteins From: Mazerbourg & Hsueh, 2006 (Hum. Reprod. Update 12:373-83)

29 Smad7 Smad6 Intracellular signaling of GDF-9 vs. BMP-2/4

30 Redrawn from: Spicer et al., 2008 (Biol. Reprod. 78:243)

31 2.6- to 3.2-fold increases Redrawn from: Spicer et al., 2008 (Biol. Reprod. 78:243)

32 From: Spicer et al., 2006 (J. Endocrinol. 189:329) Spicer et al., 2015 (unpublished)

33 Summary: GDF9 stimulates cell proliferation and inhibits gonadotropin-induced steroid production in granulosa & theca cells (Spicer et al., 2006; 2008). BMP4 is a potent inhibitor of steroidogenesis in granulosa and theca cells & stimulates granulosa cell proliferation.

34 Microarray gene expression comparisons may aid in understanding additional causes of anovulation and ovarian follicular cyst formation. 23,000 gene inquiries VS. Hand full of candidate genes

35 Microarray Analysis: Affymetrix Bovine GeneChips 24,072 probe sets for Interrogation of approximately 23,000 bovine transcripts. N = 163 genes P < 0.01

36 Target genes: Comparison of fold changes estimated through MA and RT-PCR (after log transformation): N=3 vs. 3 N=24 vs. 23 P<0.05 Of 19 genes Of 144 genes BRB Modified from: Grado-Ahuir et al., 2011 (J. Anim. Sci. 89: )

37 Impact of hedgehog proteins on Thecal Cell Steroidogenesis

38

39 Hh-interacting-protein=Hip-1- bound hh / SAG SANT1-4, Cur61414 KAAD- Smo Ptch1 Ptch1= Patched Smo= Smoothened 7-trans-m-p Modified from: Wijgerde et al., 2005 (Endocrinology 146: )

40 Cellular localization in postnatal Amh mouse ovary Indian hh Desert hh Ihh and Dhh mrnas located in GC of preantral and antral follicles (healthy). Ptch1 mrna mainly in adjacent theca cell compartment. Ptch 1 None in CL. Large follicles CL Atretic follicles From: Wijgerde et al., 2005

41 Ihh mrna in large vs. small bovine follicles 41-fold 19-fold Redrawn from: Spicer, Sudo et al., 2009 (Reproduction 138:329)

42 Ptch1 mrna is greater in large vs. small bovine follicles 2.7-fold Redrawn from: Spicer, Sudo et al., 2009 (Reproduction 138:329)

43 Estradiol stimulates 3-fold & IGF1 inhibits IHH mrna 2-fold From: Aad et al., 2012 (Biol Reprod 87:79) 300 ng/ml FSH had no effect on IHH mrna

44 IGF1 inhibits theca cell PTCH1 30 ng/ml 50% 30 ng/ml From: Aad et al., 2012 (Biol Reprod 87:79) E2 increased Ptch1 mrna

45 Hedgehog stimulates bovine theca cell proliferation C + IGF1 From: Spicer, Sudo et al., 2009 (Reproduction 138:329)

46 Hedgehog stimulates bovine theca cell androstenedione production From: Spicer, Sudo et al., 2009 (Reproduction 138:329)

47

48 Summary: Hh proteins stimulate cell proliferation and stimulates gonadotropin-induced steroid production in bovine theca cells (Spicer et al., 2009). IGF1 inhibits Hh system in bovine granulosa & theca cells. GDF9 stimulates Hh system in bovine granulosa cells.

49 Impact of FGFs on Granulosa and Thecal Cell Steroidogenesis

50 FGF Family of Proteins 21 members of the FGF family signal through high affinity transmembrane tyrosine kinase receptors. There are 4 main FGF receptors (FGFR): FGFR1 through FGFR4. All receptors except FGFR4 have 2 to 3 isoforms (IIIa, IIIb, IIIc). FGF9 only binds to FGFR1IIIc, FGFR2IIIc, FGFR3IIIb, FGFR3IIIc, and FGFR4. Potential interactions with Heparin-related molecules.

51 Steroid production is inhibited & proliferation is stimulated by FGF9 in bovine theca cells Schreiber et al., 2012 (J. Endocrinol. 215:167-75)

52 Steroid production is inhibited & proliferation is stimulated by FGF9 in bovine granulosa cells Schreiber & Spicer, 2012 (Endocrinology 153: )

53 FGF9 mrna is inhibited by IGF1 & TNFα in bovine granulosa cells Schreiber & Spicer, 2012 (Endocrinology 153: )

54 Summary: FGF9 stimulates cell proliferation and inhibits gonadotropin-induced steroid production in bovine granulosa & theca cells (Schreiber & Spicer, 2012; Schreiber et al., 2012). IGF1 & TNFα inhibits FGF9 mrna in bovine granulosa cells.

55 Hormones inhibit IGFBP & IGF2R production & stimulate proteases IGF-I inhibits IHH & stimulates BRB production Day 5 increased vascularity Day 10 Ovulation X CYST Low IGFBPs E2 & Free IGF-I & -2 FGF9 slows theca proliferation & allows for increased steroidogenesis Development of Cystic Follicles

56 Conclusions: Can microarray studies help identify new factors? YES GENES identified point toward IGF-1 & growth factor regulation of mitogenic & angiogenic factors.

57 In Bovine Granulosa Cells Angiogenin stimulates proliferation Bovine Brain Ribonuclease is induced by IGF1 Spicer, unpublished

58 Vascular Cast of 5 mm Bovine Follicle Yamada et al., 1995 (Arch. Histol. Jap. 58:567)

59 Vascular Cast of Bovine Antral Follicle Yamada et al., 1995 (Arch. Histol. Jap. 58:567)

60 Conclusions: IGFs & IHH may regulate mitogenic & angiogenic factors produced by the follicle, including cell proliferation while stimulating FSH/LH-induced differentiation. GDF9, BMP4 & FGF9 may induce mitosis & ihibit steroidogenesis of granulosa & theca cells. Ribonuclease A proteins (i.e., BRB & ANG) may be physiologically relevant and hormonally regulated in granulosa cells.

61 Future Directions: Will alteration in these systems: 1. Reduce the number of follicles that undergo atresia? 2. Improve a female s response to superovulation/ synchronization? 3. Prevent development of cystic ovaries?

62 FGF9-BRB team Dziekuje GDF9-IHH team

63 Proliferation Angiogenesis Cyst Hypothesis - Cow: Theca cell A4 - IHH FGF9 IGF1 & 2 ( Free) Angiogenin IGF1Rc? TNF Granulosa cell