Menstrual cycle II Flashcards
Describe dominant follicle selection and how does the switch to positive feedback occur ?
Where are LHr found on the follicle at this stage?
At the end of follicular phase E2 feedback becomes positive and persistent (300pM, 48H) causing exponential rise in LH that has to exceed a threshold.
This is what causes the release in LH
LH surge relatively precise predictor of timing of ovulation
Where are LHr found on the follicle at this stage?- Theca and granulosa cells in the dominant follicle
Describe the clearance of LH compared to HCG
LH is rapidly cleared from serum, in contrast to hCG which is cleared slowly & binds with great affinity to LHCGR
Describe the changes in the preovulatory follicle prior and after the LH surge
a) ) Preovulatory follicle prior to the LH surge:
- oocyte is surrounded by the zona pelucida and cumulus granulosa cells that connect to the mural granulosa cells that line the interior of the follicle.
-The granulosa cell compartment is separated from the theca cell compartment by a basal lamina. The theca cell compartment is composed of an inner theca interna and an outer theca externa. Unlike the granulosa cell compartment, the theca cell layer is highly vascularized (red). Circulating leukocytes are present in the vessels. The theca externa blends into a layer of connective tissue that is separated from the ovarian surface epithelium by a basal lamina.
The preovulatory follicle after LH stimulation:
-Loss of OSE & breakdown of underlying basal lamina and GC & TC at apex to allow for rupture.
-GC basal lamina is disrupted allowing extension of blood vessels into GC layer and for infiltration of theca cells & leukocytes into GC compartment. COC detaches from surrounding GC to expand.
What factors are responsible for holding the oocyte in meiotic arrest?
-High cAMP → keep maturation promoting factor (MPF) inactive
-cGMP enters oocytes from cumulus cells via gap junctions to inhibit oocyte cAMP phosphodiesterase PDE3A activity (PDE3A normally degrades cAMP)
-H2O2/NO/calcium
other cells/ ovarian environment & integrity of the follicle?
What are the effects of LH in ovulation
Within 3-12h of LH surge we see:
- Detachment of COC from surrounding mural GC, followed by cumulus cell expansion – formation of unique extracellular matrix between cumulus cells (aka “mucification”)
Comprised of long chains of hyaluronan
Visco-elastic properties of CC matrix important for successful ovulation, ovum pick up by oviducts and penetration of sperm - ↓cGMP production and closure of gap junctions
3.Activation of PDE3A → ↓cAMP → activation of pathways leading to breakdown of nuclear membrane in primary oocyte aka germinal vesicle breakdown (GVBD)
4.Resumption of meiosis in oocyte → completion of Meiosis I & release of 1st polar body
5/Arrests again in Metaphase II
How is cAMP produced endogenously
The cAMP is produced endogenously in oocyte through the stimulation of the Gs G-protein by the GPR3, transported into oocyte from adjacent cumulus cells, and/or held by PDE3A inhibitor(s) in the follicular environment.
What are the proposed models for the for maintenance of meiotic arrest at diplotene stage in follicular oocyte.
Refer to diagram form slide 7
The NO produced through nitric oxide synthases in cumulus-granulosa cells stimulate generation of cGMP through GCs pathway. The cGMP from encircling somatic cells is transferred though gap junctions to the oocyte. An increased level of intraoocyte cGMP level may inactivate PDE3A in the oocyte. The NO is also produced by oocyte itself through iNOS-mediated pathway and possibly inhibits PDE3A through cGMP pathway. The inhibition of oocyte PDE3A prevents cAMP hydrolysis and increase intraoocyte cAMP level. The increased cAMP level may activate PKA which in turn inactivate CDC25B phosphatase and thereby MPF. The inactive MPF does not induce meiotic resumption and diplotene arrest is maintained. The reduced production of H2O2 and Ca2+ release from mitochondria may also maintain meiotic arrest at diplotene arrest.
2b: A proposed model of LH/hCG-induced meiotic resumption from diplotene arrest in in preovulatory oocyte. LH/hCG reduces iNOS activity and induces disruption of gap junctions between cumulus-granulosa cells and oocyte. The interruption of communication between cumulus-granulosa cells and oocyte may block the transfer of cGMP produced through NO–GCs pathway. The reduced iNOS activity and thereby decreased intraoocyte NO level further decreases oocyte cGMP level. The net reduction in cGMP level may activate PDE3A that reduces cAMP level generated by oocyte itself through GPR3/AC pathway. The decrease in the cAMP level results in the inactivation of PKA activity, which in turn stimulates CDC25B phosphatase in the oocyte. The activated CDC25B phosphatase induces MPF activity that finally induces resumption of meiosis. Generation of tonic level of ROS and Ca2+ release from mitochondria may also be associated with the induction of meiotic resumption from diplotene arrest
What happens to the oocyte upon the completion of meiosis? & Why is there an unequal division of the cytoplasm ?
Early oocytes classified as immature i.e. at germinal vesicle (GV) or metaphase 1 stage
Meiosis I is completed with half chromosomes but nearly all cytoplasm remaining in the secondary oocyte
Remaining chromosomes move with small bag of cytoplasm to form discarded polar body (PB)
Why unequal division of cytoplasm?
secondary oocyte contains all the nutrients to nurture the oocyte. Need to conserve for the oocyte all the materials synthesised earlier - takes the fertilised zygote through growth and implantation
describe meiosis in the oocyte
Chromosomes of secondary oocyte immediately enter 2nd meiotic division, form the 2nd metaphase spindle and arrest
This arrest is maintained by cytostatic factor (protein complex)
Egg is ovulated in this arrested state
What are the other effects of the LH surge
LH surge induces expression of progesterone receptor (PR) in GC in all species and results in luteinisation of DF cells (both granulosa and theca)
E2 production falls and P is stimulated (P & 17α-OHP)
Blood flow to the follicle increases & new vessels appear in avascular GC
Prostaglandins and proteolytic enzymes eg collagenase and plasmin, are increased in response to LH and progesterone. What do they do?
Digest collagen in follicle wall
appearance of apex or stigma on ovary wall
What stimulates ovulation?
Cascade of events → release of COC → Ovulation
Increased secretion of chemokine/cytokines from GC & TC triggers massive infiltration of leukocytes from circulation → acute inflammatory response
In humans – ovulation occurs randomly from either ovary during a given cycles, some indication more common from right ovary
Progesterone essential for ovulation
Progesterone inhibitor (RU486) suppress ovulation
Prostaglandins-E and -F and hydroxyeicosatetraenoic acid (HETE metabolite of arachidonic acid) reach a peak level in follicular fluid just prior to ovulation
Prostaglandins stimulate proteolytic enzymes (proteases)
HETEs may stimulate angiogenesis and hyperemia (↑blood flow)
Of interest, some studies have suggested that ovulation occurs more commonly from the right ovary and right sided ovulation carries a higher potential for pregnancy [34].
The mechanism causing the postovulatory fall in LH is unknown. The decline in LH may be due to the loss of the positive feedback effect of estrogen, due to the increasing inhibitory feedback effect of progesterone, or due to a depletion of LH content of the pituitary from downregulation of GnRH receptors
Hydroxyeicosatetraenoic acid methyl esters (HETEs) are metabolites of arachidonic acid that are generated along with prostaglandins (PGs) during acute inflammatory reactions.
Hyperemia = increase of blood flow
What is the fundamental aspect of follicle rupture in ovulation
No increase in intra-follicular pressure
Progressive weakening of stigma region and OSE overlying follicle prior to rupture – fundamental aspect
The first major advance beyond this descriptive stage came with the demonstration that antral pressure does not increase prior to follicle rupture. This observation eliminated a number of hypotheses concerning ovulation and focused attention on the deterioration and weakening of the follicle wall at the stigma. That the follicle wall weakness prior to rupture is established beyond doubt by gross observations of its increased fragility during various manipulative procedures, by stress-strain measurements in vivo and in vitro, and by histological observations. Progressive weakening of the stigma region by thinning and degeneration is a fundamental aspect of the preovulatory maturation of follicles, and it would appear to be a necessary prerequisite to rupture.
What is involved in the rupture of the ovarian wall?
check diagram
OSE=simple layer of epithelieal cells (squamous/cuboidal/columnar depending on location), which is supported by a basement membrane that lies over the TA (held together by desmosomes and gap/tight junctions). Preferential growth of the DF brings it in close apposition with the OSE.
LH stimulates secretion of Plasminogen Activator (PA)
Collagenase disrupts fibril network of theca & tunica albuginea & promotes digestion of basement membrane of follicle and OSE
TNF induces cell death, proteolysis, stigma formation and eventual follicular rupture
detailed exp: LH from circulation (acts on follicle of course to resume meiosis etc) but also binds to LHRs on OSE, to cause release of plasminogen activator, which converts Plasminogen to Plasmin. This then activated collagenase via MMP-1 formation to disrupt the fibril network in tunica albuginea and also digest basement membranes of follicle and OSE. Also get cytokine production, primarily TNFalpha, which also induces proteolysis via MMP-2 production and promotes apoptosis, stigma formation and eventual rupture.
What happens upon ovulation
Secondary oocyte (arrested in metaphase II) with cumulus cells is extruded from the ovary
follicular fluid may pour into Pouch of Douglas
egg ‘collected’ by fimbria of uterine tube
egg progresses down tube by peristalsis and action of cilia
Ciliated cells are controlled by which hormones?- oestrogen
Residual part of follicle collapses into space left by fluid – a clot forms and whole structure become corpus luteum
How is inflammation associated with ovulation?
The follicular fluid is “inflammatory”
Inflammation definitely present, but too much is detrimental…
Higher “inflammation markers” in FF associated with decreased pregnancy rate (specifically C Reactive Protein, CRP)
Gingivitis associated with poorer IVF outcomes!
