REPRODUCTION

Helen MasonSenior 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

HYPOTHALAMIC PITUITARYHYPOTHALAMIC PITUITARYPORTAL CIRCULATIONPORTAL CIRCULATION

increases gene transcription increases gene transcription of gonadotrophin of gonadotrophin andandsub-unitssub-units

gonadotrophgonadotroph

pulsatile release of pulsatile release of LHLH and and FSHFSH

hypothalamus

extrahypothalamicinput

hypothalamic neurotransmitters

GnRH neurons

GnRHGnRH

pituitarypituitary

extrapituitaryextrapituitaryinputinput

remember this GnRHGnRH- decapeptide synthesised and secreted by

specialised neurons within the hypothalamus GnRHGnRH pulse generator- collective group of

neurons that discharge GnRH in orchestrated manner gonadotrophsgonadotrophs- cells in anterior pituitary that

synthesise and secrete LH and FSH in response to GnRH

gonadotrophinsgonadotrophins- LH and FSH stimulate the ovary

GnRH action

GnRHGnRHGnRHGnRH

GnRHGnRH GnRHGnRH

gonadotrophgonadotroph

1 hour

receptor internalisation receptor internalisation

1 min

clinical application

GnRH analogues-agonist or antagonist» structural changes prevent breakdown» pulsatile signal lost» gonadotrophin secretion fallspulsatile continuous pulsatile

Normal follicular phase gonadotrophin pulses

5005004004003003002002001001000000

11

22

33

44

55

66

77

88

LHLHFSHFSH

Time (min)Time (min)

LH/FSH LH/FSH IU/lIU/l

50040030020010000

2

4

6

8

10 LH

FSH

Time Time (min)(min)

LH/FSH LH/FSH IU/lIU/l

gonadotrophin pulses in patient with weight-loss or exercise related

amenorrhoea

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

EE22

PP

LHLHFSHFSH

GnRHGnRH

gonad

testosteronetestosterone

feedbackfeedback

inhibininhibin

hypothalamic/ pituitary/ gonadal axis

HYPOTHALAMUSHYPOTHALAMUS

GnRHGnRH

PituitaryPituitary

LHLHFSHFSH

OVARYOVARY

activinfollistatininhibin

oestrogenprogesterone

positive feedback follicular phase

negative feedback luteal phase

pulsatile release

HYPOTHALAMUSHYPOTHALAMUS

GnRHGnRH

LHLHFSHFSH

PituitaryPituitary

testosteroneDHToestrogen

TESTISTESTIS

activinfollistatininhibin

constantfeedback

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

11 1414 2828

follicular phase luteal phase

ovulationvariable constant14 days

• day 1 is first day of bleedingday 1 is first day of bleeding• regular cycle should have no more than 4 days regular cycle should have no more than 4 days variation from month to monthvariation from month to month• menstruation lasts 3-8 days, written as 7/28 or 5-menstruation lasts 3-8 days, written as 7/28 or 5-6/27-326/27-32

hypothalamic/ pituitary/ ovarian axis

HYPOTHALAMUSHYPOTHALAMUS

GnRHGnRH

PituitaryPituitary

LHLHFSHFSH

OVARYOVARY

activinactivininhibininhibin

oestrogenoestrogenprogesterprogesteroneone

positive feedback positive feedback follicular phasefollicular phase

negative feedback negative feedback luteal phaseluteal phase

+

pulsatile pulsatile releaserelease

the menstrual cycle

late luteallate lutealearly follicularearly follicular

prog declinesprog declinesselectively raises selectively raises

FSH= FSH= intercycle riseintercycle rise

mid follicularmid follicular

E2 increasesE2 increases-ve feedback-ve feedbackFSH fallsFSH falls

2 days of E22 days of E2 >300 pg/ml>300 pg/ml= positive feedback= positive feedback= LH surge= LH surge

high prog=high prog=negative feedbacknegative feedback=low LH/FSH=low LH/FSHP overcomes E2P overcomes E2

mid cyclemid cycle mid lutealmid luteal

PP-ve-ve

LHLH

FSHFSHE2E2-ve-ve

LHLHF S HF S H

E2E2+ve+ve

LHLHF S HF S H

PP-ve-ve

LHLHFSHFSH

menstrual cycle

allows:» cyclical fertility» selection and ovulation of a single

follicle (usually!)» spontaneous ovulation

clinical applications

EE22

PPinhibininhibin

LHLHFSHFSH

GnRH GnRH

analogues

pulsatile treatment

injectionsinjectionsone or bothone or both

disruption ofdisruption ofnegative feedbacknegative feedbackclomidclomid

constant negativeconstant negativefeedback, OCPfeedback, OCP

replacement HRTreplacement HRT

Hypothalamic/pituitary/ovarian axis

E2E2PP

LHLHFSHFSH

inhibin/activininhibin/activinGnSAFGnSAF

follistatinfollistatinE1E1

leptinleptin

IGF-IIGF-I

SHBGSHBG

IGFBP-1IGFBP-1

insulininsulin

AA

GHGHPRL?PRL?

VEGFVEGF

TGFTGF

IGF-IIIGF-II IGFBPsIGFBPs

liver

DHEASDHEAS adrenal

pancreas

adipocytesEE

ERER

1 5 10 15 25 2826 20

10

20

0

menses menses

LL LL EE FF MMFF LL FF EE LL MM LL LL LLDAY/PHASE OF CYCLEDAY/PHASE OF CYCLE

30

224466

1100

11 11 22 282822 2211 55 00 55 5566 00

Pnmol

LHsurge>50 IU

FSHIU/l

folliclediametermm

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 million7 million

2 million2 million

400,000400,000

2 months 5 months birth2 months 5 months birth menarchemenarche menopausemenopause

migratory germ migratory germ cellscells

oogoniaoogonia primaryprimaryoocytesoocytes

Primordial follicles

Oocytes into follicles

primaryprimordial intermediate

oocyte

granulosa

theca secondary

Early Follicle Growth

basal lamina

THECA LAYER

GRANULOSA CELL LAYER

oocyte

nucleus

zona pellucida

capillary

stages of follicle growth

0.02 mm0.02 mm

restingresting

INITIATIONINITIATION

.

preantralpreantral antralantral ovulatoryovulatory

RECRUITMENT SELECTIONRECRUITMENT SELECTION

3 cycles3 cycles

basal growth= 65 days+ 5 daysbasal growth= 65 days+ 5 days 10 days 10 days

0.2 mm0.2 mm 2 mm2 mm 20 mm20 mm

parameters of follicle growth

10101010

primordialprimordial preantralpreantral antralantral preovulatorypreovulatory1010 00

1010 22

1010 44

1010 66

1010 88

volumvolumeesurface surface areaarea

radiuradiuss

granulosa granulosa cellscells

absolute absolute increaseincrease

.

0.01 0.1 1 10 mm0.01 0.1 1 10 mmfollicle follicle radius radius

dominant follicle

6 mm6 mm

The Ovarian Follicle

THECA LAYER

GRANULOSA CELLLAYER

CUMULUS CELLS

FOLLICULAR FLUID

o

theca vasculature

Many follicles to one

Thousands of primordial follicles…most die through atresia, a few make it into the menstrual cycle

1 5 10 15 25 2826 20

10

20

0

menses menses

LL LL EE FF MMFF LL FF EE LL MM LL LL LLDAY/PHASE OF CYCLEDAY/PHASE OF CYCLE

30

224466

1100

11 11 22 282822 2211 55 00 55 5566 00

Pnmol

LHsurge>50 IU

FSHIU/l

folliclediametermm

The Inter-cycle rise in FSH

INTER-CYCLERISE IN FSH

140 28

The window of opportunity

FSH level

small follicles

selected follicle

E2 increases

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

follicular phase

LH/FSH

steroidogenesis in the ovary

CholesterolCholesterol PregnenolonePregnenolone ProgesteroneProgesterone

17 17 hydroxypregnenolonehydroxypregnenolone

DehydroepiandrosteroneDehydroepiandrosterone

17 17 hydroxyprogesterone hydroxyprogesterone

AndrostenedioneAndrostenedione

TestosteroneTestosterone

OestradiolOestradiol

AndrostenediolAndrostenediol

SCCSCC

17 17 hydroxylase hydroxylase

17, 20 lyase17, 20 lyase

33HSDHSD

CYP 17CYP 17

aromatasearomatase

17 17 HSDHSD

CYP 19CYP 19

CYP 11aCYP 11a

steroidogenesis

acetateacetate

cholesterol C27cholesterol C27

pregnenelone C21pregnenelone C21

progesterone C21progesterone C21

androgens: testosterone and androstendioneandrogens: testosterone and androstendioneC19 C19 theca onlytheca only

oestrogens C18 oestrogens C18 granulosa onlygranulosa only

side chainside chaincleavage cleavage enzymeenzyme

1717 hydroxylase, hydroxylase,17, 20 lyase17, 20 lyase

aromatasearomatase

theca and granulosa

ditto

ditto

Two-Cell, two gonadotrophin theory of follicular steroidogenesis

A E

THECA

GRANULOSA

basal lamina

Cholesterol

L H FSH

Follicular fluid

capilliaries

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

collagenasecollagenase

Collagen and ECMCollagen and ECMbreakdownbreakdown

Vascular permeabilityVascular permeabilityincreaseincrease

Blood flowBlood flowincreaseincrease

positive intrafollicular pressure=positive intrafollicular pressure=force at oocyte extrusionforce at oocyte extrusion

OVULATIONOVULATION

Follicle wall breakdownFollicle wall breakdown

macrophagmacrophages+ es+ cytokinescytokines

proteolyticenzymes

VEGF, histamine, prostaglandinsVEGF, histamine, prostaglandins

plasminogenplasminogen

ovulation

effects of LH on oocyte

CUMULUS CELLCUMULUS CELL

OOCYTEcAMP

LHLH

CUMULUS CELLCUMULUS CELL SECONDARYOOCYTE

1

2

1st polar body1st polar body23 X23 X

23 Xresumtion of meiosis with resumtion of meiosis with 1st meiotic division1st meiotic division

2nd meiotic division starts then 2nd meiotic division starts then arrested until fertilisationarrested until fertilisation

conservationconservationof cytoplasmof cytoplasm

extra-cellularextra-cellularmatrix secretionmatrix secretion

meiosis arrestedmeiosis arrestedsince before birthsince before birth

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 E2

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

germgerm cellscellsmitosismitosis

lots of lots of oogoniaoogonia

meiosismeiosis

mitosismitosis

more oogoniamore oogonia

oocytesoocytesmeiosis arrested at the dictyate stage.......may last 40 years!meiosis arrested at the dictyate stage.......may last 40 years!46XX46XX

1st meiotic 1st meiotic divisiondivision

sperm penetration of ZPsperm penetration of ZP

head taken head taken into into

cytoplasmcytoplasm

2nd meiotic 2nd meiotic divisiondivision

fusion fusion 46 XY46 XY mitosismitosis 2 cell embryo2 cell embryo

LH surge •only in egg in dominant follicle•cytoplasm is conserved in egg

diploiddiploid

23X23X eggegg polar bodypolar body23X23X

extrusion of extrusion of 2nd Polar 2nd Polar

BodyBodymature mature eggegg

23X23X23X23X

haploidhaploid

•in fetus