REPRODUCTION

Helen Mason
Senior Lecturer in Reproductive Endocrinology
 what has to be acheived?
 sufficient supply of eggs and sperm
 correct number of chromosomes in eggs and
sperm
 egg and sperm have to meet
 creation of new individual with genes from both
parents
 to nurture individual until capable of
‘independent’ life
 the plan !
 hypothalamic/pituitary/gonadal axis
» menstrual cycle
» stimulation of spermatogenesis
 production of gametes
» folliculogenesis
» spematogenesis
 steroidogenesis
 fertilisation
 changes in the female reproductive organs through cycle
 implantation, pregnancy, labour, lactation
 menopause
 what can go wrong, where it goes wrong and how to fix it
hypothalamic/pituitary/gonadal axis

 GnRH secretion and action

 gonadotrophin secretion
 steroid feedback
 endocrinology of the menstrual cycle
 GnRH to gonadotrophins
extrahypothalamic hypothalamic
input neurotransmitters
hypothalamus
GnRH neurons

extrapituitary HYPOTHALAMIC PITUITARY

input GnRH PORTAL CIRCULATION

increases gene transcription pituitary

of gonadotrophin andsub-units
gonadotroph
pulsatile release of LH and FSH
 remember this
 GnRH-
GnRH decapeptide synthesised and secreted by
specialised neurons within the hypothalamus
 GnRH pulse generator- collective group of neurons
that discharge GnRH in orchestrated manner
 gonadotrophs-
gonadotrophs cells in anterior pituitary that
synthesise and secrete LH and FSH in response to GnRH
 gonadotrophins-
gonadotrophins LH and FSH stimulate the ovary
 GnRH action
1 hour
1 min

GnRH GnRH GnRH

GnRH

gonadotroph receptor internalisation

 clinical application
 GnRH analogues-agonist or antagonist
» structural changes prevent breakdown
» pulsatile signal lost
» gonadotrophin secretion falls
pulsatile continuous pulsatile
 Normal follicular phase gonadotrophin pulses

8
LH
7 FSH

5
LH/FSH
4
IU/l
3

0
0 100 200 300 400 500

Time (min)
 gonadotrophin pulses in patient with weight-
loss or exercise related amenorrhoea

10 LH
FSH
8

LH/FSH 6
IU/l
4

0
0 100 200 300 400 500
Time (min)
 LH/FSH/hCG
 peptide hormones with common  sub-unit
and specific  sub-unit
  sub-unit confers specificity of action
 sub-units are glycosylated
 glycosylation confers charge (isoform),

biological activity and half life

 isoform profile changes with menstrual
cycle, age and disorders of feedback
 Gonadotrophins/receptors
 Gonadotrophins act via G-protein linked receptors- 2nd
messenger is cAMP
 FSH low cAMP, LH high cAMP
 Ovary
» FSH receptors only on granulosa cells
» LH receptors always on theca cells and on differentiated
granulosa cells and corpus luteum
 Testis
» FSH receptors on Sertoli cells
» LH receptors on Leydig cells
Hypothalamic/pituitary/gonadal axis

GnRH
feedback

E2
P LH
testosterone FSH

gonad
inhibin
 hypothalamic/ pituitary/ gonadal axis

positive
HYPOTHALAMUS HYPOTHALAMUS
feedback
follicular GnRH pulsatile release GnRH constant
phase feedback
Pituitary Pituitary
negative
feedback
luteal
phase LH LH
FSH FSH
oestrogen activin activin
progesterone follistatin follistatin
testosterone
inhibin inhibin
OVARY DHT TESTIS
oestrogen
 effects of steroid feedback

 steroids
» at hypothalamus inhibit GnRH release
» at pituitary sensitise or densensitise to GnRH
by changing receptor numbers
» when E2 is low exerts negative feedback
» when E2 is high exerts positive feedback
» progesterone exerts negative feedback
 the menstrual cycle

follicular phase luteal phase

variable ovulation constant
14 days

1 14 28
• day 1 is first day of bleeding
• regular cycle should have no more than 4 days variation
from month to month
• menstruation lasts 3-8 days, written as 7/28 or 5-6/27-32
hypothalamic/ pituitary/ ovarian axis

HYPOTHALAMUS
pulsatile
positive feedback GnRH release
follicular phase
Pituitary

negative feedback
luteal phase LH
FSH activin
oestrogen
progesterone inhibin

+ OVARY
 the menstrual cycle
late luteal mid follicular mid cycle mid luteal
early follicular

LH E2 LH E2 LH P LH
P FSH
-ve FSH -ve FSH +ve FSH -ve

prog declines E2 increases 2 days of E2 high prog=

selectively raises -ve feedback >300 pg/ml negative feedback
FSH= FSH falls = positive feedback =low LH/FSH
intercycle rise = LH surge P overcomes E2
 menstrual cycle

 allows:
» cyclical fertility
» selection and ovulation of a single follicle
(usually!)
» spontaneous ovulation
 clinical applications

analogues
disruption of GnRH
pulsatile treatment
negative feedback
clomid

E2 LH injections
constant negative P FSH one or both
feedback, OCP inhibin

replacement HRT
 Hypothalamic/pituitary/ovarian axis

liver
IGF-I

IGFBP-1

E2 GH LH SHBG
P PRL? FSH
GnSAF inhibin/activin
E1 pancreas
leptin follistatin VEGF
insulin

E IGF-II IGFBPs
adipocytes ER A
TGF
DHEAS adrenal
 26 1 5 10 15 20 25 28

follicle 20
diameter menses menses
mm

10

LH 30

surge P
>50 IU nmol
10

FSH 6
4
IU/l 2

26 1 5 10 15 20 25 28
DAY/PHASE OF CYCLE
LL EF MF LF EL ML LL
 main points to remember
 intercycle rise in FSH followed by slow
decline
 slow rise in LH in follicular phase to
exponential mid-cycle rise
 2 peaks in oestradiol- different shapes
 single luteal phase rise in progesterone
How does a patient work out when they
are going to ovulate?
ovulation?
Folliculogenesis and Steroidogenesis
 oocytes
embryo-menopause

7 million

2 million
400,000

2 months 5 months birth menarche menopause

migratory germ oogonia primary

cells oocytes
Primordial follicles
 Oocytes into follicles

primordial intermediate primary

oocyte

theca secondary
granulosa
 Early Follicle Growth

nucleus
oocyte

zona pellucida
GRANULOSA CELL LAYER

basal lamina

capillary THECA LAYER

 stages of follicle growth

3 cycles

basal growth= 65 days+ 5 days 10 days

INITIATION RECRUITMENT SELECTION

0.02 mm 0.2 mm 2 mm 20 mm
.

resting preantral antral ovulatory

 parameters of follicle growth
10 10 volume
surface area
10 8 granulosa cells
absolute radius
10 6
increase

10 4

10 2

10 0
primordial preantral antral preovulatory

follicle radius 0.01 0.1 1 10 mm

dominant follicle
6 mm
 The Ovarian Follicle

THECA LAYER

CUMULUS
CELLS

o
GRANULOSA CELL
LAYER

FOLLICULAR FLUID
theca vasculature
 Many follicles to one

Thousands
of
primordial
follicles…
most die
through
atresia, a
few make it
into the
menstrual
cycle
 26 1 5 10 15 20 25 28

follicle 20
diameter menses menses
mm

10

LH 30

surge P
>50 IU nmol
10

FSH 6
4
IU/l 2

26 1 5 10 15 20 25 28
DAY/PHASE OF CYCLE
LL EF MF LF EL ML LL
The Inter-cycle rise in FSH

INTER-CYCLE
RISE IN FSH

0 14 28
The window of opportunity
selected follicle

E2 increases

FSH level

small follicles
 Follicle selection
 raised FSH presents a “window” of
opportunity
 most sensitive follicle responds first
 known as FSH threshold hypothesis
 falling levels prevent further follicle
growth
 follicular phase gonadotrophins

inter-cycle
rise in FSH surge
LH/
FSH LH

follicular phase
 steroidogenesis in the ovary
SCC 3  HSD
Cholesterol Pregnenolone Progesterone
CYP 11a
17 hydroxylase
17hydroxypregnenolone
hydroxypregnenolone 17 hydroxyprogesterone
17, 20 lyase CYP 17
Dehydroepiandrosterone Androstenedione
17 HSD
Androstenediol
Androstenediol Testosterone
aromatase CYP 19
Oestradiol
 steroidogenesis
acetate

cholesterol C27 theca and granulosa

side chain
cleavage pregnenelone C21 ditto
enzyme
progesterone C21 ditto
17 hydroxylase,
17, 20 lyase
androgens: testosterone and androstendione
aromatase C19 theca only

oestrogens C18 granulosa only

Two-Cell, two gonadotrophin theory
of follicular steroidogenesis
THECA
capilliaries b asal lamina

GRANULOSA

Chol esterol
Follicular fluid

A E
LH FSH
The LH surge and ovulation
 LH surge
 at end of follicular phase E2 feedback becomes positive
causing exponential rise in LH
 E2 production falls and P is stimulated
 granulosa cells stop dividing
 above result in luteinisation of follicle cells
 blood flow to the follicle increases
» increase in vascular permeability
 appearance of apex or stigma on ovary wall
 cascade of events
 ovulation
macrophages+ proteolytic VEGF, histamine, prostaglandins
cytokines enzymes

collagenase
plasminogen
Vascular permeability Blood flow
increase increase
Collagen and ECM
breakdown
positive intrafollicular pressure=
Follicle wall breakdown force at oocyte extrusion

OVULATION
ovulation
 effects of LH on oocyte
1
CUMULUS CELL
cAMP
meiosis arrested OOCYTE
since before birth

LH
conservation
of cytoplasm
CUMULUS CELL SECONDARY
2
OOCYTE extra-cellular
resumtion of meiosis with
1st meiotic division 23 X matrix secretion

2nd meiotic division starts then 1st polar body

arrested until fertilisation 23 X
Fractured follicle
 ovulation
 18 hrs after peak of LH, ovulation occurs
 secondary oocyte with cumulus cells is
extruded from the ovary
 follicular fluid may pour into Pouch of Douglas
 egg ‘collected’ by fimbria of Fallopian tube
 egg progresses down tube by peristalsis and
action of cilia
The journey of the oocyte

LH and ovulation
converts the
primary oocyte to
secondary oocyte
plus 1st polar body.
 corpus luteum formation
 basal lamina of follicle breaks down
 blood vessels and blood invade granulosa cells
 blood and fibrin clot forms in centre
 follicle collapses
 corpus luteum is formed, ‘yellow body’
 progesterone production increases, also E 2
 CL contains large nos. LH receptors, CL supported by
LH and hCG: luteotrophic factors
Corpus luteum
Section of CL
 secretions of CL
 progesterone:
» supports oocyte in its journey
» prepares the endometrium
» controls cells in Fallopian tubes
» alters secretions of cervix
» acts in paracrine manner to stimulate its own
release
 oestradiol:
» for endometrium
 role of LH in CL formation
and support
 luteinisation of follicular cells
 production of proteolytic enzymes
 stimulation of angiogenic factors
maintainance of the CL---luteotrophin
 stimulation of cholesterol availablity
 stimulation of side-chain cleavage enzyme
 stimulation of aromatase
 CL- demise
 if fertilisation does not occur, CL has finite lifespan of 14
days.
 removal of CL essential to initiate new cycle
 LH receptor numbers fall, vascularity falls resulting in
regression or luteolysis
 Demise thought to be due to prostaglandin and immune
cells (cytokines)
 Cell death occurs, vasculature breakdown, CL shrinks
 Process is not well understood
 oogonia--oocytes--eggs--fertilisation
lots of oogonia germ cells
•in fetus
mitosis mitosis

diploid meiosis more oogonia

46XX oocytes meiosis arrested at the dictyate stage.......may last 40 years!

haploid LH surge 1st meiotic division •only in egg in

polar body 23X dominant follicle
23X egg •cytoplasm is
2nd meiotic division conserved in egg
extrusion of 23X
23X mature 2nd Polar
egg Body sperm penetration of ZP
head taken
into
cytoplasm
fusion 46 XY mitosis 2 cell embryo