THE ENDOCRINE ASPECTS OF FOLLICULOGENESIS

1
THE ENDOCRINE ASPECTS OF FOLLICULOGENESIS

• Professor IOANNIS E. MESSINIS MD, PhD (Aberdeen,
  UK)
• Head of Department of Obs/Gynae
• University of Thessalia
• Larissa, GREECE

2
STAGES OF FOLLICULOGENESIS
CLASS 5
CLASS 6
CLASS 4
3-5 mm
6-10 mm
1-2 mm
CLASS 3
CLASS 7
12-16 mm
0.9
HUMAN OVARY
Early antral
0.2
18-20 mm
CLASS 2
0.1
Pre-antral
CLASS 8
CLASS 1
Based on Gougeon, 1986
3
FACTORS INVOLVED IN HUMAN FOLLICULAR GROWTH
INITIATION OF GROWTH (Not FSH)
GONADOTROPHIN DEPENDENT (FSH, LH)
TONIC GROWTH PHASE (FSH possibly important)
70 days
15 days
15 days
CLASS 1
CLASS 5
CLASS 8
Primor- dial
Cyclic recruitment selection
Initial recruitment
Based on Gougeon, 1986 Hum. Reprod. 1, 81-87
4
INITIAL RECRUITMENT

• IGF-1, EGF, TGF-a, TGF-ß, GDNF, KGF, KITL, HGF,
  PDGF, LIF, BMP-4
• Anti-mullerian hormone (AMH or MIS)
• Others (WT1, pRb, myc etc)

5
ANTI-MULLERIAN HORMONE (AMH) AND FOLLICULOGENESIS
IN MICE
FSH
FSH
(-)
(-)
AMH
AMH
()
()
(-)
Primordial Primary Pre-antral
Antral
Visser Themmen, 2005 Mol. Cell. Endocrinol.
234, 81-86
6
FOLLICULOGENESIS (TGF-ß family)
BMP-2,-5,-6 () AMH (-) Inhibin Activin
BMP-4 () BMP-7 () TGF-ß
Oocyte
Granulosa
Theca
GDF-9 () BMP-15 () BMP-6 ()
??? (-) Inhibin Activin BMP-2,-5,-6 ()
Knight Glister, 2006 Reproduction, 132, 191-206
7
FACTORS FROM THE OOCYTE
PREANTRAL FOLLICLES ANTRAL FOLLICLES
GDF-9
()
BMP-15 (GDF-9B) BMP-6 FGF-8 TGF-ß2
(-)
Granulosa cells
Theca cells
Reviews Erickson Shimasaki, 2000 Erickson
Shimasaki, 2001 Moore Shimasaki, 2005
OOCYTE
8
DIFFERENTIATION OF GRANULOSA CELLS
Oocyte morphogens
FSH receptor High mitotic index IGF-1 HAS2 Cox-2
FSH receptor
Cumulus oophorus
Periantral
FSH receptor LH receptor P450arom Kit ligand u-PA
Membrana
Erickson Shimasaki, 2000, TEM, 11, 193-198
9
FACTORS INVOLVED IN HUMAN FOLLICULAR GROWTH
INITIATION OF GROWTH (Not FSH)
GONADOTROPHIN DEPENDENT (FSH, LH)
TONIC GROWTH PHASE (FSH possibly important)
70 days
15 days
15 days
CLASS 1
CLASS 5
CLASS 8
Primor- dial
Cyclic recruitment selection
Initial recruitment
Based on Gougeon, 1986 Hum. Reprod. 1, 81-87
10
INTERCYCLE RISE OF FSH
LUTEAL
FOLLICULAR
6
LH IU/l FSH IU/l
4
2
8
6
4
2
-8 -6 -4 -2 1 3
5 7 DAYS
Messinis et al., 1993 Clin. Endocrinol. 38,
159-163
11
CYCLIC RECRUITMENT SELECTION
Intercycle rise of FSH
FSH
Follicles
10-12 mm
8 mm
16 mm
CLASS 5 CLASS 6 CLASS 7
DOMINANCE
RECRUITMENT SELECTION
12
FSH
FSH
R
R
Induction of FSHR
cAMP
cAMP
aromatase
R
A
E1
Chole- sterole
LH
A
T
T
E2
(-)
5a-reductase
5a-reduced A
Theca cell
Granulosa cell
TWO-CELL TWO-GONADOTROPHIN THEORY (Early
follicular phase)
13
PARACRINE/AUTOCRINE FACTORS

• Inhibin, activin
• Insulin-like growth factors (IGFs) and IGFBPs
• Androgens
• Estrogens
• Others

14
ROLE OF INHIBIN/ACTIVIN
INHIBIN
FSH
LH
()
Gr
Th
E
A
()
(-)
ACTIVIN
ACTIVIN
Th Theca cell Gr Granulosa cell
Based on a review by Hillier, 2001
15
FSH ??? IGFs
IGFs
IGFs
IGFBP-4
(-)
IGFBP-4 PROTEASE (PAPP-A)
Granulosa cell
()
E2, P4 and/or LH receptor
R
()
()
R
FSH
Based on reviews by ?rickson Shimasaki 2001
Spicer, 2004
16
ROLE OF IGF-1
HDL/LDL
THECA CELL
R
_

R
C
EGF/TGFa

R
IGF-1
P450ssc

Pr

3ß-HSD
R


?GFß
cAMP
PKA
_
P
P45017a
R
LH
AC
GS
A

ATP

A
cAMP
PKA
P450arom
R
E1
FSH
AC
GS
17ßHSD

E2
ATP
E2
IGF-1

GRANULOSA CELL
17
ROLE OF IGF-2
Androstenedione Testosterone
IGF-2
THECA CELL
R
LH
R
IGF-2
R
FSH
GRANULOSA CELL
E1, E2
R
IGF-2
IGF-2
18
EFFECT OF ANDROGENS (small antral follicles)
GRANULOSA CELL
GRANULOSA CELL
A
A
R
FSH
R
FSH
()
()
T DHT
()
R
E
E
Harlow et al., 1986 Endocrinology 119,
1403-05 Weil et al., 1999 JCEM 84, 2951-56
19
EFFECT OF ANDROGENS VIA OOCYTE FACTORS
14000

DOIGF1
3H-thymidine Incorporation (cpm)

GDF9IGF1
7000



IGF1
0
C
0
5
50
500
IGF1
DHT (nM)
14000

DOIGF1
3H-thymidine Incorporation (cpm)

GDF9IGF1

7000


IGF1

0
Hickey et al., 2005 Biol. Reprod. 73, 825-32
C
0
10
100
1000
IGF1
T (nM)
Porcine GC
20
EFFECT OF ESTROGENS (rat antral follicles)
GRANULOSA CELL
GRANULOSA CELL
R
FSH
arom
R
arom
FSH
()
R
E
Adashi and Hsueh 1982 J. Biol. Chem. 257, 6077-83
21
LEPTIN AND REPRODUCTION
HYPOTHALAMUS
GnRH

FAT TISSUE
PITUITARY

LEPTIN
FSH
LH
?

OVARY
?
Leptin
Based on Messinis
Milingos, 1999 Hum. Reprod. Update 5,
52-63
E2
P4
22
EFFECT OF LEPTIN (human luteinized granulosa
cells)
360000
270000
E2 pmol/l
180000
90000
0
GH
IGF-1
GHL1ng
GHL10ng
IGF-1L1ng
GHL100ng
IGF-1L10ng
IGF-1L100ng
Karamouti et al., 2008 Fertil. Steril. 90, 1444-50
23
GHRELIN AND REPRODUCTION
HYPOTHALAMUS
GnRH
- ?
STOMACH
PITUITARY
-?
GHRELIN
FSH
LH
-
?
Based on
Messini et al., 2009 Hum. Reprod.
Martini et al., 2006
Endocrinology Viani et al., 2008
JCEM
OVARY
E2
P4
24
ATRESIA RESCUE
Androgenic milieu
Estrogenic milieu
LH
FSH
aromatase
5a-reductase
FOLLICLE GROWTH
25
SINGLE FOLLICLE SELECTION
15
FSH IU/L E2 pg/ml
Intercycle rise
10
5
0
400
300
200
van Santbrink et al., 1995, Fertil. Steril.
100
0
M menses D Dominance
M D LH peak
26
FOLLICLE GROWTH IN THE NORMAL CYCLE (Ultrasound)
20
Follicle diameter (mm)
15
10
0
-5 -4 -3 -2 -1
0 1 DAYS FROM LH SURGE ONSET
Templeton, Messinis Baird, 1986 Fertil.
Steril. 46, 1113-1117
Mean values (n11)
27
GROWTH OF THE DOMINANT FOLLICLE
MIDCYCLE
INTERCYCLE
FSH
LH dependence
Intrafollicular regulators
10-12 16 18-20 mm
CLASS 5 CLASS 6 CLASS 7 CLASS 8
Messinis 2008 In Infertility and Assisted
Reproduction Rizk et al., (eds), Cambridge UP
28
INTRAFOLLICULAR REGULATORS

• GRANULOSA
• CELLS
• IGFs
• Inhibin/activin
• Estrogens
• TGF-ß
• FGF
• TNF-a
• IL-1ß

• THECA
• CELLS
• BMP-4
• BMP-7
• TGFa
• HGF
• FGF
• CRH
• KGF (FGF-7)

• GDF-9
• BMP-15
• BMP-6
• ??F-a

OOCYTE
29
ROLE OF THECA-DERIVED BMPs
FSH
LH
18-20 mm
Follicles
FSH
LH
P4
-
StAR
BMP-4, -7
Th
Gr
Based on Tajima, 2007 RBMOnline 15, 591-609
P450

arom
A
E2
A
30
LH DEPENDENCE
LH
FSH
R
R
Induction of LHR
cAMP
cAMP
aromatase
R
A
E1
Chole- sterole
LH
A
T
T
(-)
E2
5a-reductase
5a-reduced A
Theca cell
Granulosa cell
MID- TO LATE FOLLICULAR PHASE (Dominant follicle)
31
EXPRESSION LEVEL OF FSHR AND LHR IN IVM-GCs
6
?
5
FSHR
?
4
LHR
Relative expression
3
2
1
0
C
A
AHCG
AFSH
AHCG

FSH
Human unluteinized granulosa cells
Lindeberg et al., 2007 Hum. Reprod. 22, 401-6
32
DOMINANT FOLLICLE Gonadotrophin dependence
2
FSHR
R²69.5 Plt0.01
DF gene expression relative to reference (log2
transformed)
ESR2
1
R²57.5 Plt0.01
0
-1
R²63.7, Plt0.01
-2
LHCGR
-3
1
2
3
4
5
6
Days after wave emergence
Mihm et al., 2006 Biol. Reprod. 74,
1051-9 (modified)
In cattle
33
NORMAL CYCLE
LH SURGE
FOLLICULAR PHASE Early Mid- Late
LUTEAL PHASE
LUTEAL PHASE
FSH

• FSH
• Aromatase ?
• E2 ?
• FSHR ?
• LHR ?
• GC prolif. ?
• LH
• A ?

• FSH
• Aromatase?
• E2?
• FSHR?
• LHR?
• GC prolif. ?
• LH
• A ?
• E2 ?
• GC prolif. ?

34
FSH OR LH?
Cetrorelix 3 mg sc
E2
LH
FSH
Follicle diameter
0 24 hours
17 mm
Based on Leroy et al., 1994 Fertil. Steril. 62,
461-7
35
FSH OR LH? (Follicle growth rate)
20
FD mm
18
16
14
12
10
Spontaneous cycles
8
GnRH antagonist FSH (from FD 14 mm)
6
4
Spontaneous cycles
2
0
-6 -5 -4 -3
-2 -1 0
DAYS RELATIVE TO HCG
36
HYPOGONADOTROPHIC HYPOGONADISM
FSH (IU/L) Follicle diameter (mm)
E2 (pmol/L)
20
Follicle
15
FSH
E2
10
5
200
100
0 6 10 15 20 25 30
35 Days
recFSH
75 IU
Shoham et al., 1993 Fertil. Steril. 59, 738-42
7 14 21
37
HYPOGONADOTROPHIC HYPOGONADISM (Use of rFSH with
rLH)
FSH LH E2
IU IU (pmol/l) Couzinet et al., 1988
225 225 2753
(HMG) (HMG) Kousta et al.,1996 150
225 780 El-Shawarby et al., 2004 50-75
75 3155
Based on a review by Messinis, 2005 Hum.
Reprod. 20, 2688-97
38
LH IN FOLLICULAR MATURATION
15
FSH IU/L
DAY 0 (FD 14 mm)
10
5
DAY 1
0
DAY 2 (day of HCG)

4
LH IU/L

2
0
2000
E2 pg/ml
1000
Sullivan et al., 1999 JCEM 84, 228-232
FSH up to 14 mm GnRH agonist
0
FSH
LH-H
LH-L
S
39
FSH CYCLE
FOLLICULAR PHASE
LUTEAL PHASE
LUTEAL PHASE
FSH
LH

• FSH leads the follicles to the pre-ovulatory stage

• LH is required for the production of steroids

40
LH RECEPTORS

• FSH is unable to stimulate development of mice
  early antral follicles to the preovulatory stage
  in the absence of LHR

Pakarainen et al., 2005 Mol. Endocrinol. 19,
2591-2602
41
DUAL ACTION OF LH ON THECA CELLS AND LH
ceiling concept
LH
FSH
Luteinization of theca and
granulosa cells
Steroidogenesis (androstendione)
Based on a review by Tajima, 2007 RBMOnline 15,
591-07
42
CONCLUSIONS - 1

• Both FSH and LH are important for the final
  stages of follicle maturation
• The action of FSH predominates in the first half
  and that of LH in the second half of the
  follicular phase

43
CONCLUSIONS - 2

• Different intrafollicular regulators are
  important for the selection and the growth of the
  dominant follicle
• Under pharmacological interventions, FSH is
  capable to initiate and sustain the growth of the
  dominant follicle, while LH is required for
  steroids production