Menstrual Cycle II Flashcards
How is the dominant follicle ovulated (referring to the LH surge)?
- Selection and exponential growth of the dominant follicle
- Switch to positive feedback occurs; at the end of the follicular phase, E2 feedback becomes positive and persistent (300pM, 48h) causing exponential rise in LH that has to exceed a threshold.
- LH surge lasts for 36-48 hours & triggers ovulation (timing varies from species-species)
- LH surge relatively precise predictor of timing of ovulation
- LH is rapidly cleared from serum, in contrast to hCG which is cleared slowly & binds with great affinity to LHCGR
- At this stage, LHr are found on both theca and granulosa cells of the dominant follicle.
- Must have selection and growth of a dominant follicle = at least >15mm diameter on ultrasound
- Onset of LH surge precedes ovulation by 36h but the peak precedes ovulation by 10-12 hours.
- Point of the menstrual cycle leads up to selection and exponential growth of the dominant follicle for menstruation
- High, sustained oestrogen over 2 days causes an exponential rise in LH that has to exceed a threshold (women with PCOS can get little surges in LH but ovulation won’t occur without surpassing this threshold of 25 mIU (can be expressed in different units)). When this threshold is exceeded, that generates enough of an LH surge to trigger ovulation. It lasts for about 36 to 48 hours, but this really varies between species. Ovulation is triggered in that time frame. It is usually a really precise indicator of ovulation. If you can measure the LH surge and it has exceeded that threshold, you generally know ovulation is going to occur.
- hCG is released from the embryo itself. CL is maintained if the egg is fertilised as it produces hCG which binds to the LH receptors with great affinity.
- The dominant follicle moves outwards again to be released for ovulation.
When the LH surge occurs, it leads to breakdown of the surface epithelium of the basement membrane and digestion of all the theca and granulosa cells as well as the basement membrane above the mural granulosa cells (below the theca). This allows invasion of blood vessels into the follicle. Remember, the granulosa cells do not have any vasculature, all the vasculature is in the theca externa. Those invade through into the follicle, and they bring loads of inflammatory cytokines and other leukocytes which will continue this process of ovulation. The cumulus-oocyte complex will detach off from those mural granulosa cells and undergo certain changes. These complex things occur in the preovulatory follicle upon the LH surge. The LH surge is really important to allow the whole follicle to rupture and release the egg.
Compare the structure of the preovulatory follicle prior to the LH surge and following LH stimulation immediately prior to ovulation.
1) The preovulatory follicle prior to the LH surge
- The 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.
2) Preovulatory follicle following LH stimulation immediately prior to ovulation. Disruption of the granulosa cell basal lamina allows extension of vessels into the granulosa cell compartment. Theca cells and leukocytes also enter into the granulosa cell compartment. The cumulus oocyte complex detaches from the surrounding granulosa cells and undergoes cumulus expansion. At the follicular apex (top of image), there is a loss of ovarian surface epithelium and breakdown of the underlying basal lamina, and theca cells and granulosa cells to allow for rupture. Rupture will occur at the follicle apex.
- The schematic shows the preovulatory follicle prior to the LH surge. The outer cortex is filled with primordial follicles. As they activate and grow, follicles will activate and grow into the medulla of the ovary where there is a rich blood supply and the dominant follicle, as it grows, will move back up towards the surface of the ovary ready for ovulation. It is bulging underneath the ovarian surface epithelium and that is resting on the basal lamina (basement membrane). Then there are the theca cells, both the theca interna and theca externa, which also rest on a basement membrane, separating it from the mural granulosa cells. Can see the egg with cumulus cells.
The dominant follicle moves outwards again to be released for ovulation.
When the LH surge occurs, it leads to breakdown of the surface epithelium of the basement membrane and digestion of all the theca and granulosa cells as well as the basement membrane above the mural granulosa cells (below the theca). This allows invasion of blood vessels into the follicle. Remember, the granulosa cells do not have any vasculature, all the vasculature is in the theca externa. Those invade through into the follicle, and they bring loads of inflammatory cytokines and other leukocytes which will continue this process of ovulation. The cumulus-oocyte complex will detach off from those mural granulosa cells and undergo certain changes. These complex things occur in the preovulatory follicle upon the LH surge. The LH surge is really important to allow the whole follicle to rupture and release the egg.
What factors are responsible for holding the oocyte in meiotic arrest (Regulation of Meiotic Arrest)?
- The oocyte in the cumulus cells is in meiotic arrest and has been from when it was laid down and formed in the foetus.
- Following the LH surge, it is ready to complete meiosis I now.
1) High cAMP → keep maturation promoting factor (MPF) inactive
2) cGMP enters oocytes from cumulus cells via gap junctions to inhibit oocyte cAMP phosphodiesterase PDE3A activity (PDE3A normally degrades cAMP). cGMP keeps cAMP high. It is formed in the surrounding cumulus cells. They have all of these projections and gap junctions that go into the egg, allowing it to communicate with the egg. Cyclic GMP will enter the oocyte and inhibit this enzyme which normally degrades cAMP. The enzyme is kept inactive by cyclic GMP, so cyclic AMP remains high within the oocyte. It keeps the nucleus in meiotic arrest.
3) H2O2/NO/calcium
4) other cells/ ovarian environment & integrity of the follicle?
What are the effects of the LH surge?
- Within 3 to 12 hours of the LH surge, various events are seen
1) Detachment of COC from surrounding mural GC, followed by cumulus cell expansion – formation of unique extracellular matrix between cumulus cells (aka “mucification”). This sticky matrix is comprised of long chains of hyaluronan. The sperm have to fight their way through all of this sticky mass to reach the egg. It is another ‘selection of the fittest’ type hurdle that the sperm have to compete. This is why there are millions of sperm that are released. Visco-elastic properties of CC matrix important for successful ovulation, ovum pick up by oviducts and penetration of sperm. The breakup of the ovarian surface epithelium, infiltration of the blood vessels, degradation of the basement membranes and the egg with the cumulus cells detach away from the stalk which connects it to the mural granulosa cells. They will become expanded, sticky and mucified (mucificatiton). Hyaluronan makes up an extracellular matrix that is secreted, making the eggs very sticky, which aids the pick up. The tube is not attached to the egg; it has to move and pick up the egg. That pickup is aided by the fact that it is sticky and mucified. This is known because pictures are seen from IVF clinics and IVF when they retrieve the eggs and fertilise them in vitro.
2) ↓cGMP production and closure of gap junctions; The LH surge also results in a decrease in cyclic GMP. The gap junctions that connect the egg and the cumulus cells close up, so cGMP can’t enter anymore. Cyclic AMP is reduced and it leads to the maturation promotion factor. Results in breakdown of the nuclear membrane (germinal vesicle breakdown) and completion of meiosis I. Meiosis I will start and complete. It will release the chromosomes into a daughter cell, known as a polar body. Reforms the spindle for meiosis II and arrests again in metaphase II.
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
- Ovulation occurs ~12-18 hours after the peak of LH surge
- Follicle moves back toward surface ready for ovulation. There are the groups of surface epithelial cells in the presumptive stigma (a flower-like bouquet), which are in the process of being sloughed off.
How is meiotic arrest maintained?
- 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.
- A proposed model for maintenance of meiotic arrest at diplotene stage in follicular oocyte. 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. - Shows current understanding of the model
- Cyclic GMP is produced in the cumulus cells which enters via the gap junctions. Keeps that enzyme, PDE3A, inactive and keeps cAMP levels high. As cAMP is high, it doesn’t allow for this step. This maturation promoting factor is inactive and all these other things also play a role. When there is an LH surge, LH binds to its receptors, triggers this pathway which will degrade cyclic GMP or reduce favourable formation of cGMP, closes gap junctions. This will increase this activity, degrade cyclic AMP which will allow for that to be activated, germinal vesicle breakdown. Allows meiosis I to resume and complete.
What is the result of meiosis I?
- 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)
- Need to conserve for the inside or the material synthesise earlier so there is unequal division of cytoplasm → takes fertilised zygote through growth and implantation
- Early oocytes are also classified as immature (germinal vesicle (GV) or metaphase I (MI) stage). The breakdown of the germinal vesicle indicates a resumption of meiosis and the extrusion of the first polar body (1 PB) indicates completion of the first meiotic division in human oocytes.
- Need to conserve for the oocyte all the materials synthesised earlier, because it will need this to take fertilized zygote forward in growth and implantation
- Unequal division occurs predominantly because the sperm only brings chromosomes and mitochondria (designed for speed/travel to egg)
- After the LH surge, the metaphase II spindle has formed. Half the chromosomes from meiosis I are in the polar body with a tiny amount of cytoplasm. The oocyte retains all of the organelles and proteins because the sperm is designed for travel and only brings the chromosomes. It brings its chromosomes (two haploid gametes come together to create an individual) and the other parts, e.g. the tail, are packed with mitochondria for energy. Everything required for early preimplantation embryo development will come from the egg. This is why all the cytoplasm is retained in the egg; it provides everything that the early embryo needs for early divisions and survival.
When is meiosis II completed?
- Meiosis II is only completed on fertilisation. If fertilisation doesn’t occur, if sperm doesn’t penetrate through, it doesn’t finish and it just degrades.
- Egg is ovulated in this arrested state. Picked up by the fimbriae, travels down the fallopian tube and, if it is fertilised, it will complete meiosis II.
- 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 is luteinisation?
- LH surge induces expression of progesterone receptor (PR) in GC in all species and results in luteinisation of DF cells (both granulosa and theca – now known as granulosa luteal and theca luteal cells)
- E2 production falls and P is stimulated (both P & 17α-OHP – can be measured in blood)
- Blood flow to the follicle increases & new vessels appear in avascular GC (invade into the avascular GC)
- Prostaglandins and proteolytic enzymes, e.g. collagenase and plasmin, are increased in response to LH and progesterone. They digest collagen in follicle wall. Digesting the stroma and other cells to allow for the follicle to be ovulated.
- Appearance of apex or stigma on ovary wall
- The appearance of mammalian follicles at the time of ovulation has been described by numerous observers. Importance features to which attention has been directed are: changes in the vascularization of the follicle, the appearance of an avascular area known as the macula pellucida or stigma, the formation of small blood clots near the stigma and some extravasation of blood, and the rupture of the follicle at the stigma and exist of the oocyte and follicular fluid.
Describe the cascade of events that lead to 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) to digest the follicle and ovary wall for release of the egg
- HETEs (metabolites)may stimulate angiogenesis and hyperemia (↑blood flow). Want very rich vasculature to occur; the corpus luteum has a very good blood supply.
- Ovulation is considered to be an inflammatory response.
- Not understood why progesterone is important. Although the majority of the progesterone comes from the corpus luteum, if progesterone inhibitor is given, it will suppress ovulation.
- Angiogenesis = new blood vessels
- 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
How does the follicle rupture for 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.
- 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.
- Ovarian surface epithelium rests on the basement membrane. That TA is a very fibrous, collagen sheet which encapsulates the ovary. In PCOS, it can be quite thick. The cumulus cells are around oocyte with mural granulosa cells.
- It is about digesting of these walls. What restrains it is being degraded/digested off so that it weakens and thins out for release.
- 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
- LH surge binds to receptors on the ovarian surface epithelium. Causes release or secretion of plasminogen activator, which converts plasminogen to plasmin. Plasmin will then activate these enzymes, collagenase, via another intermediary (matrix metalloproteinase-1). These enzymes will break apart and digest both basement membranes and this whole network of fibrils, fibrous layers and tubules etc. in the theca and the tunica. All of those are digested to allow release. This is all reinforced by release of TNF-alpha, a cytokine which again reinforces this whole pathway. Eventually, the follicle rupture and release the egg.
- 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.
- Plasminogen activates the matrix metalloproteinases.
What is 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?
- Residual part of follicle collapses into space left by fluid – a clot forms and whole structure become corpus luteum
- Oestrogen – also important is oestrogen:progesterone ratio
- Called secondary oocyte because it is in metaphase II
- It is important to remember that the corpus luteum will contain both luteinised granulosa and theca cells.
What inflammation is 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!
- It is known the follicular fluid is inflammatory because various inflammatory markers and macrophages can be seen. They are very much involved in ovulation and repair. It is known to be an inflammatory process, but if it is too strong an inflammatory process, it can be associated with negative outcomes (don’t want it to go too much that way).
How does the ovulatory wound heal?
- Ovary faces monumental task of repairing damage caused by follicle rupture after each ovulation
- Basic steps are known but the underlying mechanisms are still unknown
- Interestingly the ovulation wounds scar, but not for long – quick resolution
- Maybe steroidogenic environment helps – mitogenic (oestrogen)
- Recently identified stem cell/progenitor population that may contribute to maintenance of OSE (Ng et al (2014) Nature Cell Biology 16:745-757)
- Ovary repairs quickly and effectively (one of the fastest recovering organs in the body; mechanism could be applied to healing other wounds quicker if figured out)
- It is thought that maybe the oestrogenic environment helps, since oestrogen is a known mitogen (promotes cell growth etc.). It is also thought that maybe a stem cell population contributes to it as well.
- Can see the fibrin clot (this is after ovulation).
- Ovulated egg is picked up, travels down, meets the sperm within the fallopian tube and fertilisation will occur. If no sperm is present/no fertilisation occurs, it just travels down and is degraded, can shed out with the endometrium.
What are the signs of ovulation?
1) A slight rise in basal body temperature, typically 0.5 to 1 degree, measured by a thermometer. Need to keep a chart of basal body temp from day 1 of LMP. You won’t feel the change, but a few days after you ovulate, your basal body temperature rises. (Your basal body temperature, or BBT, is your lowest body temperature in a 24-hour period.) This tiny uptick is only 0.4 to 1.0 degree Fahrenheit. You can detect it by taking your BBT every morning with a special thermometer. Another important point to note when monitoring temperature patterns, is that there will not always be a drop in temperature before or when ovulation occurs. The rise in temperature does almost always indicate that ovulation has occurred.
It is for this reason that a number of cycles will have to be monitored and recorded, if using temperature as an ovulation predictor. Temperature fluctuates on a daily basis so needs to be monitored for the entire cycle. Jumps up a little bit higher just up to ovulation (wouldn’t know this without keeping a daily record)
2) Tender breasts. There are other signs, again driven by hormones, e.g. tender breasts, that are very characteristic of ovulation.
3) Abdominal bloating
4) Light spotting
5) Changes in cervical mucus
6) Slight pain or ache on one side of the abdomen. Some women can actually feel that they have ovulated on one side or another (a preovulatory follicle can reach up to 20mm in size; almost the size of the ovary and the ovary is connected to peduncle anatomically). The ovary can torque or twist to allow the fimbriae to reach the egg. This is why women who have had a lot of pelvic or abdominal surgery can develop adhesions or lesions which can restrict their fertility. This is why they put dyes down the tube to see how patent (open) tubes are and how things are moving.
How does cervical mucus act as a sign of ovulation?
- The cervical mucus or cervical fluid changes throughout MC
- Immediately after menstruation, the cervical mucous is scant and viscous.
- In late follicular phase, ↑ E2 levels, the cervical mucous becomes clear, copious and elastic.
- Quantity ↑ 30 fold compared to EFP
- The stretchability/elasticity of cervical mucous evaluated between two glass slides and recorded as the spinnbarkeit
- After ovulation, ↑progesterone levels, the cervical mucous again becomes thick, viscous and opaque and ↓ quantity produced
- The cervical mucus changes because it is responding to oestrogens and progesterone.
- Cervical mucus is often evaluated as part of a fertility workup
- Just because these changes in the mucus are seen does not always mean ovulation has occurred. Can sometimes get fluctuations in hormones without ovulation occurring. This is why it should be looked at with other signs (temperature).
- If you look closely at the cervical mucus, you will notice that there can be discharges of watery or egg white mucus more than once during your cycle. You might have egg white cervical mucus and still not ovulate. That is why it is necessary to use more than one method of tracking your fertility days. Combining cervical mucus ovulation monitoring with charting your basal body temperature is a very good combination. They are complementary, as watching the cervical mucus you can see when you are about to ovulate and the change in basal body temperatures shows when you have already ovulated.
