REPRODUCTIVE ENDOCRINOLOGY Neuroendocrinology, Gonadotropins, Sex steroids, Prostaglandins, Ovulation, Menstruation, Hormone Assay REPRODUCTIVE ENDOCRINOLOGY

REPRODUCTIVE ENDOCRINOLOGY

Neuroendocrinology, Gonadotropins, Sex steroids, Prostaglandins, Ovulation,

Menstruation, Hormone AssayLentz GM, Lobo RA, Gershenson DM, and Katz VL. Comprehensive Gynecology 2012. 6th

Edition.

• To review the basic anatomic, histologic, and biochemical concepts governing the hypothalamic-pituitary-ovarian (HPO)axis.

• To discuss the cyclic changes in hypothalamic, pituitary, and

ovarian hormones and other growth factors in relation to the menstrual cycle.

OBJECTIVES

Lentz GM, Lobo RA, Gershenson DM, and Katz VL. Comprehensive Gynecology 2012. 6th Edition.

HPO Axis: An overview

HYPOTHALAMUS release of GnRH which stimulates release of LH and FSH from the adenohypophysis (ANTERIOR PITUITARY)

Reproductive Endocrinology

GnRH neurons originate from progenitor cells in the embryonic olfactory placode where they develop and migrate during ealry fetal life toward the brain.

16 weeks fetal life: functional connections between GnRH neurons and the hypophyseal portal system is established.

Failure of GnRH neuronal migration

and subsequent establishment of

functional connection between

brain and hypophyseal portal system results in

KALLMANN SYNDROME.


· Hypothalamic-Pituitary Transport of GnRHVia Portal Vessels of the Superior Hypophyseal Artery


ARCUATE NUCLEUS - greatest number of GnRH neurons


GnRH: a Decapeptide (10 a.a.)


GnRH gene is situated on Chr 8p encoding for a 92 a.a. precursor molecule for GNRH:1) signal peptide sequence (13 a.a. long)2) GnRH sequence itself (10 a.a. long)3) posttranscriptional processing signal (3 a.a. long)4) GAP/ GnRH associated peptide (56 amino acid peptide)

GnRH (short half-life): t 1/2 of 2-4 mins.


HYPOTHALAMUS: Pulsatile Release of GnRH


GnRH pulses:occurs at an hourly interval

Rising edge of each GnRH pulse is abrupt, increasing by a factor of 50 within 1 minute.

GnRH Pulse Generator


• Mechanisms Responsible for GnRH Pulsatility:The cellular basis and the mechanisms that determine the timing of the increase in

multiunit activity resulting in pulsatile GnRH activity are still under study.

1) There is a growing consensus that pulsatile activity originates from an inherent pace-making activity of the GnRH neuron itself: synchronized action occur through gap junctions between GnRH neuron

2) Role of Kisspeptin (KISS1), product of KISS1 gene and its receptor (GPR54 or KISS1R) in GnRH regulation - KISS1 neurons directly innervate and stimulate GnRH neurons and is now implicated in mediating sex steroids feedback loops especially during preovulatory GnRH/LH surge. The presence of the estrogen and the androgen receptors in KISS1 neurons within the arcuate nucleus modulates the feedback loops.

KISS1 also has been shown to play a role in initiation of puberty.Mutation or targeted deletions in KISS1 or its receptor = HYPOGONADOTROPIC

HYPOGONADISM



• Modulatory influences on GnRH release:

Foremost modulatory influence on frequency and amplitude of GnRH pulses is the ovarian steroid hormones.

Estradiol decrease GnRH pulse amplitude.Progesterone decreases

GnRH pulse frequency.



• Modulatory influences on GnRH release:



3 Stimulatory inputs

4 Inhibitory inputs

Biogenic amine Norepinephrine (NE) Dopamine (DA)Amino acid Glutamate Gamma-

aminobutyric acid (GABA)

Peptide Neuropeptide Y (NPY)

Corticotropin-releasing hormone (CRH)

Endogenous opioid

B-endorphin

These systems may affect the GnRH pulse generator either tonally or conditionally.

• Metabolic influences on GnRH release:



LEPTIN NEUROPEPTIDE Y (NPY)

Anorexigenic protein Orexigenic protein

Ob gene product

Produced by adipocytes Arcuate nucleus

Decreased by fasting (d/t body fat stores)

Increased by fasting

Stimulates GnRH release

Has 2 contradictory actions on GnRH release:Pulsatile stimulation – GnRH releaseContinuous stimulation – GnRH release

Growing evidence indicates that complex and extensively integrated physiologic

mechanisms connect an active reproductive axis to the metabolic state (nutritional

status).

And overall, in regard to the reproductive system, the GnRH pulse generator actually acts as the link between the environment, the

internal milieu, and the reproductive axis.



· Anterior Pituitary: also called Adenohypophysis

Is derived from Rathke’s pouch ~ 3rd week of life, in contrast to the posterior pituitary (neurohypophysis) which is a direct extension of the the brain.

Contain gonadotropes – specialized cells that produce gonadotropins.

Gonadotropes contain high affinity GnRH receptors (GnRH-R) found along their cell membranes.



GnRH-R • Encoded by Chr 4q13.2-13.3

and spans 18.9kb.• Belongs to the family of G protein-coupled receptors

• It contains 7 TM helices connected by 6 alternating IC and EC loops (~NH2

terminus is located on EC side) and lacks a carboxy terminus on IC site in contrast to other protein receptors (i.e LH/ FSH/TSH/ hCG).


GnRH-R • Activation of GnRH-R stimulates

cellular production of specific membrane-associated DAG* (diacylglycerols) that act as 2nd messenger to activate cellular proteins:

Protein kinase C (PKC) and Mitogen-activated protein kinase (ERK)

Phosphorylated ERK activates transcription factors (TF)

Gene transcription of gonadotropin subunits and synthesis of both gonadotropins

transient rapid influx of IC Ca++ of EC Ca++

burst of exocytosis activates calmodulin

a Calcium-binding protein

rapidly release maintaining LH and FSH gonadotropin release

*DAG amplify the action of Ca++-calmodulin, synergistically enhancing the release of gonadotropins.

Calmodulin antagonist = GnRH-stimulated gonadotropin release.




GnRH-R and Estrogen• Estradiol decreases GnRH pulse

amplitude but its effect on GnRH-R is to increase it.

• Increase GnRH-R leads to increase Ca++ response with amplified gonadotropin release per GnRH pulse

• Thus GnRH pulses of similar amplitude elicit greater gonadotropin responses during the late follicular phase and luteal phase (when estradiol levels are highest), but the responses are lower during the early follicular phase (when estradiol levels are lowest)



• GnRH pulse frequency besides pulse amplitude regulates gonadotropin subunit gene transcription:

Low GnRH pulse frequency = FSH synthesis (progesterone effect)

High GnRH pulse frequency = LH synthesis

• Changing pulsatile infusion from a high to a low pulse frequency results in an FSH: LH ratio.

• This phenomenon may play a role in the changing FSH:LH ratio that occurs during the passage from one menstrual cycle to another

GnRH Pulse frequency and Gonadotropin release

Clinical example: PCOS = high GnRH pulse frequency, resulting in the pathologic elevation in LH:FSH ratio

LH:FSH = PCOS

FSH:LH



GnRH-R desensitization

• GnRH pulse (pulsatile release) causes rapid release of both FSH and LH (within minutes).

• GnRH pulse (continuous release) results in sustained GnRH-R exposure causing reduce response to GnRH stimulation = homologous desensitization/ down-regulation of the receptor. This takes a few days reflecting loss of active cell surface receptors and maintained by a loss of functional Ca++ channels.

GnRH Agonist GnRH Antagonist

Chemical structure(Normal peptidase action: cleaving the

decapeptide molecule at the Gly6 to Leu7 and at the

Pro9 to Gly10 bonds)

A.A. 6 substituted with d-aminoA.A. 10 subs w/ N-ethylamide or Aza-Gly moiety

A.A. 2(His) or A.A.3(Trp)

substitution

Structural effect Greater resistance to enzymatic proteolysis resulting in longer half-life (hours vs 2-4 mins.)

Action on GnRH-R Initial stimulation of gonadotropin release

(flare effect) followed by desensitization

Competes with GnRH for receptor sites

Common clinical application

In women: Endometriosis, Uterine leiomyoma, DUB, Contraception, Breast cancer, Ovulation inhibitionIn men: Prostatic cancer, Cryptorchidism

GnRH agonist versus GnRH antagonist: Action on the GnRH-R



1 2 3 4 5 6 7 8 9 10

Anterior Pituitary: Gonadotropins



FSH LHChemical structure similarity

HMW glycoproteins: heterodimers (two monomeric units = α and β subunits)Same α subunit = 92 a.a. also shared with hCG and TSH

Chemical structure difference

1. β subunit – confers specific biologic activity2. Sugar moieties - affects bioactivity and speed of

degradation

Biologic half-life 3-4 hours 20 minutesActs on Granulosa cells Theca cells and Luteal

cellsPrimary action Stimulate follicular

growth (folliculogenesis)Stimulate ovarian steroid hormone

production (steroidogenesis)

· Hypothalamic pulsatile release of GnRH

· Anterior pituitary pulsatile release of FSH and LH

· FSH and LH reaches the Ovary to stimulate cyclic estrogen and progesterone production

· Estrogen and progesterone stimulates ovarian folliculogenesis and endometrial menstrual changes



THE MENSTRUAL CYCLE

OVARIES: OOGENESIS/ GAMETOGENESIS

600,000 at 2nd month of fetal life

7 million by 6th to 7th month ~ UNDERGO MEIOSIS I forming Primary Oocyte2-4 Million at birth300,000 at puberty (90% depleted)25,000 at age 371000 at age 50

Primary oocytes are arrested at Meiosis I – Diplotene stage

(germinal vesicular stage) of Prophase at birth

Meiosis II is only completed if the oocyte is fertilized


OOGONIA or PRIMORDIAL GERM CELLS develop from endoderm then migrate to the genital ridge at 5-6 weeks

Arrested in metaphase II

Arrested in prophase I

THE MENSTRUAL CYCLE


· OVARIES: FOLLICULOGENESIS

1) PRIMORDIAL FOLLICLE – primary oocyte surrounded by a single layer of granulosa cells (GC).

2) PRIMARY/ PREANTRAL FOLLICLE– primary oocyte surrounded by a multiple layers of granulosa cells (GC); development is not gonadotropin-dependent but influenced by intra-ovarian, nonsteroidal process (occurs during non-ovulatory stages of childhood, pregnancy, oral contraceptive use, and ovulatory cycles).

3) SECONDARY/ ANTRAL FOLLICLE – under hypothalamic-pituitary control and requires FSH.

4) MATURE/ PRE-OVULATORY FOLLICLE

THE MENSTRUAL CYCLE


· OVARIES: STEROIDOGENESIS

1) ESTRADIOL2) PROGESTERONE3) ANDROSTENEDIONE

Also secrete in varying amounts:• Estrone• Pregnenolone• 17-OH progesterone• Testosterone• DHEA

Does not synthesize (d/t lack of appropriate enzymes):• Mineralocorticoid• Glucocorticoid

2- carbon compound

27-carbon steroid

21 carbon atoms

19 carbon atoms

18 carbon atoms


STEROIDOGENESISSteroidogenic acute regular protein

STEROIDOGENESIS


Aromatase enzyme• Converts androgens to estrogens

(estrone/estradiol)

• Complex enzyme comprised of 2 proteins: •

1) P450 arom –member of the cytochrome P450 superfamily of genes, catalyzes reactions for formation of phenolic A ring.

2) NADPH-cytochrome P450 reductase – an ubiquitous protein required for transferring reducing equivalents from NADPH to any microsmal form of cytochrome P450 which it comes in contact.

• Found in: ovaries, endometrium, brain, placenta, bone, skin, adipose tissue.

In the ovary, the main androgen source is ovarian testosterone and thus the main estrogen product from the ovary is estradiol, whereas In adipose tissue, the main androgen source is circulating androstenedione (produced by the adrenals) and hence the principal estrogen produced is estrone.

Overall, the C18 estrogen produced in different tissue sites is specific and dependent on the nature of the C19 steroid presented to the aromatase enzyme:

Clinical application:

CYP 19 aromatase gene Mutation = an autosomal recessive disorder leading to aromatase deficiency syndrome. Manifested by accumulation of androgens during pregnancy leading to virilization at birth and hypoestrogenism.

AROMATASE INHIBITORS = useful in management of patients with ER + tumors, i.e. Breast CA.



Ovarian Steroid: Blood Transport and Metabolism• Transport: 1) SHBG – a β-globulin synthesized by the liver; DHT>Testosterone>E2 affinity 2) albumin – most biologically important fraction bec. free to diffuse or actively transported through capillary wall and bind to its receptor 3) corticosteroid binding protein (CBG or transcortin) – primarily binds adrenal steroids and progesterone to a lesser degree

Both SHBG & CBG – high affinity but low capacity for steroids vs. Albumin – low affinity but high capacity hence steroids can readily dissociate and enter target cells.

• Metabolism: Liver via oxidation (bile acids) and Kidneys via conjugation to form water soluble products (estradiol-17 glucuronide, estrone sulfate, and pregnanediol-3-glucuronide, the major urinary metabolite of progesterone)

Testosterone Estradiol

SHBG 65% 60%

Albumin 30% 38%

Free 5% 2-3%



Ovaries: Prostaglandin action• A subclass of eicosanoids and prostanoids and are general mediators of inflammatory and

anaphylactic reactions.

• Formed from arachidonic acid which is derived from linoleic acid supplied in the diet.

• Formation is inhibitied by NSAIDS type 1 (aspirin and indomethacin) and type 2 (phenylbutazone) and corticosteroids.

Ovary Endometrium

Prostaglandin:modulate the responses of endogenous stimulators and inhibitors

1) Prostaglandin F2a (PGF2a) regulates ovarian receptor availability during ovarian stimulation by LH2) Help control early follicular growth by increasing blood supply to certain follicles and inducing FSH receptors in granulosa cells of preovulatory follicles.3) Both PGF2a and PGE2 are concentrated in follicular fluid of preovulatory follicles and may assist in the process of follicular rupture by facilitating proteolytic enzyme activity in the follicular walls.

Concentrations of PGE2 and PGF2a increase progressively from the proliferative to the secretory phase of the cycle, with highest levels at menstruation.

~ may help regulate myometrial contractility and may also play a role in regulating the process of menstruation.

· Communication within the HPO endocrine axis:

1) Steroid receptors – found in cytoplasm or nucleus and may undergo shuttling in the absence of hormone(vs cell membrane for peptide hormones); inactive state via multiprotein inhibitory complex (i.e. heat shock proteins, hsp) and activated state that allows binding to hormone responsive element HRE in DNA-binding site.

2) The Ovarian-hypothalamic-pituitary Feedback Loops:

The Negative Steroid Feedback Loop The Positive Estradiol Feedback Loop Ovarian Peptides Feedback Loops




The Negative Steroid Feedback Loop

Estradiol-17b • Mainly by controlling the amplitude of

each LH pulse, relayed by estrogen-receptive kisspeptin and possibly GABA neurons (hypothalamus).

• Inhibitory effect on the pituitary gonadotrope: decrease gonadotropin response to GnRH (involving GnRH-R)

Progesterone• affects mainly the GnRH pulse

generator by slowing the frequency of pulses (becomes more pronounced during luteal phase)




The Positive Estradiol Feedback Loop

• dependent on rapidly rising estradiol levels, in combination with a small but significant progesterone rise, both produced by the mature dominant follicle and responsible for the generation of the preovulatory LH and FSH surge.

• serves as the critical signal to the hypothalamic-pituitary axis that the dominant follicle is ready to ovulate.




Ovarian Peptides Feedback Loops

• nonsteroid ovarian factors exert negative feedback effects on the anterior pituitary

• Inhibin A (pk: midluteal) and B (pk: following LH surge)

Preferential inhibition of FSH over LH through their own negative feedback loop ~functions at a significantly slower rate (hours) vs steroid negative feedback loop (minutes)

• Activin A and B Stimulate FSH release from the pituitary.



THE MENSTRUAL CYCLE


Ovarian cycle Follicular

phase Luteal phase

Uterine cycle Proliferative

phase Secretory

phase

THE MENSTRUAL CYCLE



phase Luteal phase

FOLLICULAR LUTEAL

THE MENSTRUAL CYCLE



phase Luteal phase

FOLLICULAR LUTEAL

1.At the beginning each menstrual cycle → low levels of gonadal steroids

2. With the demise of the corpus luteum, FSH levels begin to rise

3. Rising estrogen levels provide negative feedback on pituitary FSH secretion

4. At the end of the follicular phase (just before ovulation), FSH-induced LH receptors are present on granulosa cells and, with LH stimulation, modulate the secretion of progesterone

THE MENSTRUAL CYCLE



phase Luteal phase

FOLLICULAR LUTEALAfter a sufficient degree of estrogenic stimulation, the pituitary LH surge is triggered, which is the proximate cause of ovulation that occurs 24 to 36 hours later

Both estrogen and progesterone remain elevated throughout the lifespan of the corpus luteum and then wane with its demise, setting the stage for the next cycle

Progesterone levels rise precipitously after ovulation and can be used as a presumptive sign that ovulation has occurred

1) Recruitment of a Cohort of Antral Follicles – by FSH

2) Selection of a Dominant Follicle – completed by Day 5 and most probably reflects the competitive advantage of the dominant follicle, characterized by a well-vascularized theca layer better gonadotropin supply to their target receptors, greater local estradiol secretion, which increases the density of gonadotropin receptors and promotes cell multiplication.

THE MENSTRUAL CYCLE


Ovarian cycle Follicular phase

FOLLICULAR LUTEAL

THE MENSTRUAL CYCLE


Ovarian cycle Follicular phase

FOLLICULAR LUTEAL

3) Growth of the Dominant Follicle: The Maturing Secondary or Antral Follicle

(Max mean D = 19.5 mm, 18-25 mm; Mean max follicular vol = 3.8mL 3.1-8.2 mL)

THE MENSTRUAL CYCLEFOLLICULAR LUTEAL

During the ovulatory surge: LH levels increase 10-fold over 2 to 3 days FSH levels increase 4-fold.

This gonadotropin surge is an absolute requirement for the final maturation of the oocyte and the initiation of the follicular rupture.

LH surge initiates germinal vesicle (or nucleus) disruption, and the fully grown oocyte resumes meiosis (meiotic maturation) from the diplotene stage of the first to metaphase II of the second meiotic division and completed only during fertilization.

Ovulation: 24 hrs after E2 peak 32 hrs after initial LH rise 12-16 hrs after LH pk in serum

Ovarian cycle Ovulation


THE MENSTRUAL CYCLE


Ovarian cycle Luteal phase

FOLLICULAR LUTEAL

Normal function of the corpus luteum depends primarily on LH stimulation throughout the luteal phase.

Progesterone dominance during the luteal phase affects the hypothalamic thermoregulatory center = small increase in basal body temperature (BBT)

Progesterone levels: <1 ng/mL before ovulation vs10-20 ng/mL at midluteal

THE MENSTRUAL CYCLE


Ovarian cycle Luteal-Follicular

transition phase

FOLLICULAR LUTEAL

The end of the luteal phase is characterized by a dramatic decrease in progesterone, estradiol, and inhibin A.

This is accompanied by a characteristic divergence in the FSH:LH ratio, now favoring a specific rise in FSH

THE MENSTRUAL CYCLE

Lentz GM, Lobo RA, Gershenson DM, and Katz VL. Comprehensive Gynecology 2012. 6th Edition. PROLIFERATIVE SECRETORY

Uterine cycle Proliferative

phase

The estrogen from the developing follicles acts on the endometrium in the ‘Follicular’ phase, stimulating it to grow or ‘proliferate’.

Ovarian Follicular Phase = Endometrial Proliferative Phase

FOLLICULAR LUTEAL

THE MENSTRUAL CYCLE


Uterine cycle Secretory

phase

The progesterone from the corpus luteum acts on the endometrium in the ‘Luteal’ phase, stimulating the glands to ‘secrete’ and prepare for the implantation of a fertilised egg.

The spiral arterioles begin to coil and later become kinked.

Ovarian Luteal Phase = Endometrial Secretory Phase

FOLLICULAR LUTEAL

THE MENSTRUAL CYCLE


Uterine cycle Menstruation

phase

FOLLICULAR LUTEAL

The life span of the corpus luteum is about 12-14 days

If pregnancy does not occur, it self destructs (‘luteolysis’)

As progesterone production declines, the endometrium loses it support and with the resorption of stromal oedema, the spiral arterioles become more kinked and constricted.

The endometrial tissue becomes necrotic and sheds as menstrual effluent

THE MENSTRUAL CYCLE


Endometrial Cyclic changes

FOLLICULAR LUTEAL

Decidua functionalis - superficial 2/3 & cycling portion of the endometrium; proliferates and is ultimately shed with each cycle if pregnancy does not occur. stratum spongiosum - deeply

situated intermediate zone stratum compactum -

superficial compact zone the endometrium.

Decidua basalis - deepest region of endometrium. It does not undergo significant monthly proliferation & is the source of endometrial regeneration after each menses

CONSTANT VARIABLE

THE MENSTRUAL CYCLE


HORMONAL REGULATION OF OOGENSIS AND OVULATION

FOLLICULAR PHASE LUTEAL PHASEOVULATION

10-20 primordial follicles begin to develop in response to FSH and LH levels

FSH and LH stimulate theca and granulosa production of estrogen and progesterone

surge of LH induces ovulation

theca and granulosa cells transform into the corpus luteum and secrete large amounts of progesterone

if fertilization does not occur, corpus luteum degenerates ... if fertilization does occur, HCG released from the embryo maintains corpus luteum


In Summary: HPO Axis and Menstrual Cycle

THE MENSTRUAL CYCLE


Documents

REPRODUCTIVE ENDOCRINOLOGY Neuroendocrinology, Gonadotropins, Sex steroids, Prostaglandins, Ovulation, Menstruation, Hormone Assay REPRODUCTIVE ENDOCRINOLOGY