Gonadotropin’s Gonadotropin’s

BioactivityBioactivity

Dr. Vincenzo VolpicelliDr. Vincenzo Volpicelli

Fertility Center Fertility Center CarditoCardito

Seconda Università degli Studi di Napoli Seconda Università degli Studi di Napoli

Dipartimento di Scienze della VitaDipartimento di Scienze della Vita

SUNfertSUNfert

Seconda Università degli Studi di Napoli Seconda Università degli Studi di Napoli

GonadotropinsGonadotropins

FSH, LH, HCGFSH, LH, HCGglycoproteinsglycoproteinsdimers dimers αα, , ββ (two peptide chain) (two peptide chain)

αα chain aspecific chain aspecificβ β chain specificchain specific ((provides specificity for receptor interaction)provides specificity for receptor interaction)

Glycoproteins are proteins that contain oligosaccharide chains covalently attached to their side-chains.

An oligosaccharide is a saccharide polymer containing a small number (typically three to ten) of

component sugars, also known as simple sugars.

FSHFSH

heterodimeric hormone: heterodimeric hormone:

• 92 amino acids 92 amino acids αα--chain chain

• 111 amino acids 111 amino acids ββ--chainchain

Ben-Rafael Z, Levy T, Schoemaker J. 1995 Pharmacokinetics of follicle-stimulating hormone: clinical significance. Fertil Steril. 63:689–700

Various types of FSH exist according to their sialic acid contentVarious types of FSH exist according to their sialic acid content

The half-life of FSH is 3-4 hours The half-life of FSH is 3-4 hours

LH

• The luteinizing hormone beta subunit gene is localized in the LHB/CGB gene cluster on chromosome 19q13.32

•The gene for the alpha subunit is located on chromosome 6q12.21.

LH/HCG bioactivityLH/HCG bioactivity LH & HCG: the same amino acids in sequence LH & HCG: the same amino acids in sequence LH & HCG both stimulate the same receptorLH & HCG both stimulate the same receptor the hCG the hCG ββ--subunit contains an additional 24 amino acids, subunit contains an additional 24 amino acids, both hormones differ in the composition of their sugar both hormones differ in the composition of their sugar

moieties. moieties. The different composition of these The different composition of these oligosaccharidesoligosaccharides

affects bioactivity and speed of degradation. affects bioactivity and speed of degradation. The biologic The biologic half-lifehalf-life::

LH: LH: 20 minutes20 minutes FSH: FSH: 3-4 hours3-4 hours hCG: hCG: 24 hours24 hours

FSH, LH, HCGFSH, LH, HCG

The protein dimer contains 2 polypeptide units, labeled alpha and beta subunits that are connected by two disulfide bridges

The alpha subunits of LH, FSH, TSH, and hCG are identical, and contain 92 amino acids

The The beta subunitsbeta subunits vary vary

Gn secretionGn secretion

estrogensestrogens

pituitary glandpituitary gland

hypothalamus hypothalamus (arcuate nucleus and preoptic area)(arcuate nucleus and preoptic area)

GnGn

(Gn-RH pulses)(Gn-RH pulses)

ovaryovary

feed-backfeed-back

Estradiol negative feed-backEstradiol negative feed-back

Pituitary gland embryology

Pituitary glandPituitary gland

by diencephalon

by diencephalon

(infundibulum)

(infundibulum)

by Rathke pouch

by Rathke pouch(mouth)(mouth)

Pituitary portal system

Pituitary gland histologyPituitary gland histology

GHGH

HPRLHPRL

FSHFSHLHLHTSHTSHACTHACTH

Gn mode actionGn mode action

• activate a PtdIns(phosphatidylinositol)-calcium second

messenger system

• membrane receptors

•Adenilcyclasi activation

Gn mode of actionGn mode of action

uterine blood uterine blood flow:flow:

increasesincreases the the uterine blood flow uterine blood flow during the early during the early luteal phase, a luteal phase, a periimplantation periimplantation stage stage

50

60

70

80

90

100

110

1° 5° 9° 14° 16° 19° 24°

(Index Resistance)

Gn mode of actionGn mode of action

increase in the number of receptor in preparation for ovulation

After ovulation, the luteinized ovary maintains LH-R-s that allow activation in case there is an implantation

receptors activationreceptors activation

~1% receptor sites activated

•binding LH to the external part of the membrane spanning receptor

•with LH attached, the receptor shifts conformation and thus•mechanically activates the G protein

•and activates the cAMP system

The seven transmembrane α-helix structure of a G protein-coupled receptor such as LHCGR

Gn-R expressionGn-R expression

•Its expression requires appropriate hormonal Its expression requires appropriate hormonal stimulation by FSH and estradiolstimulation by FSH and estradiol

present on:present on:• granulosa cells granulosa cells • theca cellstheca cells• luteal cells luteal cells • interstitial cellsinterstitial cells

Extragonadal Gn-RsExtragonadal Gn-Rs• Gn-Rs have been found in:Gn-Rs have been found in:

the uterus, the uterus, sperm,sperm, seminal vesicles, seminal vesicles, prostate, prostate, skin, skin, breast, breast, adrenals, adrenals, thyroid, thyroid, neural retina, neural retina, neuroendocrine cells, neuroendocrine cells, and (rat) brain.and (rat) brain.

• physiologic role largely unexploredphysiologic role largely unexplored.

Gn action in ovary Gn action in ovary

follicular maturationfollicular maturation

ovulationovulation

luteal function luteal function

Gonadotropin’s avverse effectsGonadotropin’s avverse effects

OHSSOHSS Ovarian volume increasedOvarian volume increased Multiple pregnanciesMultiple pregnancies GynecomastiaGynecomastia

FSH in early follicular phaseFSH in early follicular phaseFSH thresholdFSH threshold: FSH serum concentrations needed to stimulate ovarian follicle : FSH serum concentrations needed to stimulate ovarian follicle growthgrowth (Brown 1978) (Brown 1978)

• At the onset of the menstrual cycle, a cohort of small (2–5 mm) antral follicles is present in each ovary

• This cohort will continue to grow in response to stimulation by FSH

• a process referred to as follicle recruitment • The follicle with the highest sensitivity will benefit most from

increasing FSH levels and will subsequently gain dominance (leader leader)

Brown JB. 1978 Pituitary control of ovarian function: concepts derived from gonadotropin Brown JB. 1978 Pituitary control of ovarian function: concepts derived from gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54 therapy. Aust NZ J Obstet Gynaecol. 18:47–54

Scheele F, Schoemaker J. 1996 The role of follicle-stimulating hormone in the selection of Scheele F, Schoemaker J. 1996 The role of follicle-stimulating hormone in the selection of follicles in human ovaries: a survey of the literature and a proposed model. Gynecol follicles in human ovaries: a survey of the literature and a proposed model. Gynecol Endocrinol. 10:55–66. Endocrinol. 10:55–66.

FSH in early follicular phaseFSH in early follicular phase

• not increase much during a normal not increase much during a normal ovulatory cycle ovulatory cycle

• FSH concentrations only FSH concentrations only 10–30%10–30% above the above the threshold level is sufficient threshold level is sufficient to stimulate to stimulate normal follicle developmentnormal follicle development

**Brown JB. 1978 Pituitary control of ovarian function: concepts derived from Brown JB. 1978 Pituitary control of ovarian function: concepts derived from gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54.gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54.

****Messinis IE, Templeton AA. 1990 The importance of follicle-stimulating hormone Messinis IE, Templeton AA. 1990 The importance of follicle-stimulating hormone increase for folliculogenesis. Hum Reprod. 5:153–156.increase for folliculogenesis. Hum Reprod. 5:153–156.

FSH concentrations reach a maximum in the early follicular phase of the FSH concentrations reach a maximum in the early follicular phase of the normal menstrual cycle and decrease thereafternormal menstrual cycle and decrease thereafter

FSH in follicular phaseFSH in follicular phase

Stimulates:1. follicular growth,

2. granulosa cell aromatase activity,

3. induction of LH receptors on the granulosa cell membrane,

4. estradiol secretion

•enzyme of the cytochrome P450 group

•mediate androgens aromatization:

producing estrogens producing estrogens sexual developmentsexual development

Aromatase

FSH in late follicular phaseFSH in late follicular phasedecreasedecrease due to increased ovarian secretion of:due to increased ovarian secretion of:

EE22

ββ--inhibin inhibin

Hotchkiss J, Knobil E. 1994 The menstrual cycle and its neuroendocrine control. In: Knobil Hotchkiss J, Knobil E. 1994 The menstrual cycle and its neuroendocrine control. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 711–750. E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 711–750.

Groome NP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimeric inhibin B Groome NP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab. 81:1401–1405.throughout the human menstrual cycle. J Clin Endocrinol Metab. 81:1401–1405.

negative feedback at the hypothalamic-pituitary levelnegative feedback at the hypothalamic-pituitary level

Thecal Cell Granulosa CellLHLH

cAMcAMPP

Protein Protein Kinase Kinase AA

cholesterol

pregnenolone

17-OH-P

DHEA

A

CYP11

CYP17

CYP17

3βHSD

A

E1

E2

FSHFSH

RR

RR

blood

Protein kinase

cAMP

P450

17βHSD

BBaasseemmeenntt

MMeemmbbrraannee

P4P4

AldostAldostCortisolCortisol

Steroidogenesis

FSH follicular decreasingFSH follicular decreasing

• strict relationship with dominant follicle strict relationship with dominant follicle developmentdevelopment

Schipper I, Hop J and Fauser B:Schipper I, Hop J and Fauser B: “ “The Follicle-Stimulating Hormone (FSH) Threshold/Window Concept Examined by The Follicle-Stimulating Hormone (FSH) Threshold/Window Concept Examined by

DifferentDifferent Interventions with Exogenous FSH during the Follicular Phase of the Normal Menstrual Cycle: Duration, Interventions with Exogenous FSH during the Follicular Phase of the Normal Menstrual Cycle: Duration, Rather Than Magnitude, of FSH Increase Affects Follicle Development”. The Journal of Clinical Endocrinology & Rather Than Magnitude, of FSH Increase Affects Follicle Development”. The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 4 1292-1298 Metabolism Vol. 83, No. 4 1292-1298

• As a consequence, other recruited As a consequence, other recruited follicles lackfollicles lack sufficient stimulation by sufficient stimulation by FSH and enter atresiaFSH and enter atresia

Zeleznik AJ, Hutchison JS, Schuler HM. 1985 Interference with the gonadotropin-Zeleznik AJ, Hutchison JS, Schuler HM. 1985 Interference with the gonadotropin-suppressing actions of estradiol in macaques overrides the selection of a single suppressing actions of estradiol in macaques overrides the selection of a single preovulatory follicle. Endocrinology. 117:991–999.preovulatory follicle. Endocrinology. 117:991–999.

FSH follicular decreasingFSH follicular decreasing

• Apparently, the maturing dominant follicle requires Apparently, the maturing dominant follicle requires less FSH to continue its growth. less FSH to continue its growth.

• It’s due to up-regulated FSH-sensitivity of leading It’s due to up-regulated FSH-sensitivity of leading follicle for: follicle for:

1.1. induction of locally various growth factors (IGF-I, induction of locally various growth factors (IGF-I, AMH, AMH, inibina B, leptina, ICAM-1, VCAM-1, VEGFinibina B, leptina, ICAM-1, VCAM-1, VEGF))

2.2. induction of LH receptors that enhance FSH induction of LH receptors that enhance FSH sensitivitysensitivity

•Erickson GF. 1996 The ovarian connection. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive endocrinology, surgery, and technology. Philadephia: Lippincott-Raven; 1141–1160.

FSH in late luteal phaseFSH in late luteal phase

• At the end of the luteal phase, there is a At the end of the luteal phase, there is a slight rise in FSH that seems to be of slight rise in FSH that seems to be of importance to start the next ovulatory cycleimportance to start the next ovulatory cycle

• a cohort of small antral follicles is prevented a cohort of small antral follicles is prevented from undergoing atresia and is stimulated from undergoing atresia and is stimulated for further developmentfor further development

Hodgen GD. 1982 The dominant ovarian follicle. Fertil Steril. 38:281–300 Hodgen GD. 1982 The dominant ovarian follicle. Fertil Steril. 38:281–300

LH mode actionLH mode action With the rise in estrogens, LH receptors are also With the rise in estrogens, LH receptors are also

expressed on the maturing follicleexpressed on the maturing follicle estrogen rise leads via the hypothalamic interface to the estrogen rise leads via the hypothalamic interface to the

“positive LH feed-back” effect, a release of LH over a “positive LH feed-back” effect, a release of LH over a 24-48 hour period24-48 hour period

This 'LH surge' triggers ovulationThis 'LH surge' triggers ovulation LH is necessary to maintain luteal function (P4) for the LH is necessary to maintain luteal function (P4) for the

first two weeksfirst two weeks LH supports thecal cells in the ovary that provide LH supports thecal cells in the ovary that provide

androgens and hormonal precursors for estradiol androgens and hormonal precursors for estradiol productionproduction

In case of a pregnancy luteal function will be further In case of a pregnancy luteal function will be further maintained by the action of hCG (a hormone very maintained by the action of hCG (a hormone very similar to LH) from the newly established pregnancysimilar to LH) from the newly established pregnancy

FSH geneFSH gene

αα-chain gene-chain gene locate in arme 6p21.1-23 locate in arme 6p21.1-23

ββ-chain -chain gene: gene: locate in 11p13 only in gonadotrope cells of locate in 11p13 only in gonadotrope cells of

pituitary gland pituitary gland increased by Gn-RH and activine increased by Gn-RH and activine decreased by inhibinedecreased by inhibine

Deficient gonadotropin’s levelDeficient gonadotropin’s level

hypogonadism and amenorrhoeahypogonadism and amenorrhoea: Kallmann syndrome Kallmann syndrome Hypothalamic suppressionHypothalamic suppressionHypopituitarismHypopituitarismEating disorder (leptine)Eating disorder (leptine)HyperprolactinemiaHyperprolactinemiaGonadotropin deficiencyGonadotropin deficiencyGonadal suppression therapy Gonadal suppression therapy

• GnRH antagonistGnRH antagonist• GnRH agonist (downregulation)GnRH agonist (downregulation)

LH-R abnormalitiesLH-R abnormalities

• in females can lead to infertilityin females can lead to infertility

• masculinization masculinization

• In 46, XY pseudohermaphroditism, In 46, XY pseudohermaphroditism,

• hypospadiashypospadias

• micropenismicropenis

Antibodies to LH-R can interfere with LH-R activityAntibodies to LH-R can interfere with LH-R activity

High Gonadotropin levelsHigh Gonadotropin levels

Premature menopause Premature menopause Gonadal dysgenesis, Turner syndrome Gonadal dysgenesis, Turner syndrome CastrationCastration Swyer syndrome Swyer syndrome Polycystic Ovary Syndrome Polycystic Ovary Syndrome Certain forms of CAHCertain forms of CAH Testicular failureTesticular failure

Persistently high LH levels are indicative of situations where the normal restricting Persistently high LH levels are indicative of situations where the normal restricting feedback from the gonad is absent, leading to a pituitary production of both LH and FSH.feedback from the gonad is absent, leading to a pituitary production of both LH and FSH.

typical in the menopausetypical in the menopause

FSH in COHFSH in COH• multiple follicle development is induced by elevating

FSH concentrations far above the threshold • By starting with a lower dose of gonadotropins and

stepwise small increments, chances of inducing monofollicular growth should increase with a concomitant reduction of complications (step-up protocol)

• However, these stimulation protocols are characterized by FSH concentrations remaining above the threshold

Polson DW, Mason HD, Saldahna MBY, Franks S. 1987 Ovulation of a single dominant follicle during treatment with low-dose pusatile follicle stimulating hormone in women with polcystic ovary syndrome. Clin Endocrinol

(Oxf). 26:205–212. White DM, Polson DW, Kiddy D, et al. 1996 Induction of ovulation with low-dose gonadotropins in polycystic

ovary syndrome: an analysis of 109 pregnancies in 225 women. J Clin Endocrinol Metab. 81:3821–3824.

FSH gateFSH gate• the "FSH-gate" or "FSH-window" concept has been the "FSH-gate" or "FSH-window" concept has been

proposed, which adds the element of time to the FSH proposed, which adds the element of time to the FSH threshold theory and emphasizes the significance of a threshold theory and emphasizes the significance of a transient increase in FSH above the threshold level for transient increase in FSH above the threshold level for single dominant follicle development single dominant follicle development **

• Moreover, step-down dose regimen COH, has proven Moreover, step-down dose regimen COH, has proven successful in reducing the incidence of multiple follicle successful in reducing the incidence of multiple follicle developmentdevelopment ****

** ** van Santbrink EJP, Donderwinkel PFJ, van Dessel HJHM, Fauser BCJM. 1995 van Santbrink EJP, Donderwinkel PFJ, van Dessel HJHM, Fauser BCJM. 1995

Gonadotrophin induction of ovulation using a step-down dose regimen: single-centre clinical Gonadotrophin induction of ovulation using a step-down dose regimen: single-centre clinical experience in 82 patients. Hum Reprod. 10:1048–1053experience in 82 patients. Hum Reprod. 10:1048–1053

**Baird DT. 1987 A model for follicular selection and ovulation: lessons from superovulation. Baird DT. 1987 A model for follicular selection and ovulation: lessons from superovulation.

J Steroid Biochem. 27:15–23 J Steroid Biochem. 27:15–23

FSH windowFSH window

• the FSH window concept has been the FSH window concept has been proposed, proposed, stressingstressing the significance of the significance of the (limited) the (limited) duration of FSH elevationduration of FSH elevation above the threshold level above the threshold level

• rather than the height of the elevationrather than the height of the elevation of of FSH for single dominant follicle selectionFSH for single dominant follicle selection

Fauser BCJM, van Heusden AM. 1997 Manipulation of human ovarian function: physiological Fauser BCJM, van Heusden AM. 1997 Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev. 18:71–106.concepts and clinical consequences. Endocr Rev. 18:71–106.

Gn dosage

• For assisted reproductive technology procedures, the usual initial dose is 150 IU to 225 IU daily for 5 days.

• The dose is then adjusted according to response and is usually continued for 6 to 12 days.

• When an adequate response is achieved, this medication is stopped and another medication, hCG, is given to induce ovulation.

FSH initial dosesFSH initial doses

patient’s agepatient’s age basal FSH basal FSH PCOSPCOS

HCG HCG

Gonasi fl i.m. 1000, 2000, 5.000 UIGonasi fl i.m. 1000, 2000, 5.000 UI

• HCGHCG• pregnant women urinepregnant women urine• made by the placenta made by the placenta • LH-activity likeLH-activity like• > half-life LH (4 h vs. 15 min)> half-life LH (4 h vs. 15 min)

hCG in normal pregnancy hCG in normal pregnancy

0

20000

40000

60000

80000

100000

120000

140000

0 7 8 9 10 11 12 13 14 16 19 20 39

HCG HCG

It is heterodimeric glycoprotein:It is heterodimeric glycoprotein:–α subunit identical to LH, FSH, TSH α subunit identical to LH, FSH, TSH –β subunit unique to hCGβ subunit unique to hCG–92 + 152 amino acids92 + 152 amino acids

HCG mode actionHCG mode action

interacts with the LHCG receptor interacts with the LHCG receptor

Follicle rupture inductionFollicle rupture induction

maintenance of the corpus luteum maintenance of the corpus luteum during the beginning of pregnancy, during the beginning of pregnancy,

causing it to secrete P4causing it to secrete P4

meiosis restarting meiosis restarting