Mechanism of Ovulation By Maryam Hosseinzadeh

1. Ovulation
IN THE NAME OF GOD
By: M, Hosseinzadaeh

2. Ovulation
Ovulation is the occurrence in the
menstrual cycle by which a selected
mature follicle breaks and releases a viable
oocyte from the ovary.
Each month, one egg is released in
humans; but occasionally, two or more can
erupt during the menstrual cycle.
In women with regular menstrual cycles,
the ovulation takes place approximately
two weeks after menstruation.
Ovulation is triggered by the pituitary LH
surge, which initiates a series of ovarian
events, generating a cascade of
paracrine/autocrine factors, enzymes and
transcription factors responsible for the
rupture of the apical follicle wall,
remodeling of extracellular matrix (ECM)
and cell differentiation.

3. Ovulation is the result of a well ordered series of events.
Hypothalamus
Pituitary
Ovary
GnRH
LH
FSH
Estradiol
The main factors responsible for the
function of this axis are:
•Firstly the hypothalamic hormones,
particularly the gonadotrophin
releasing hormone (GnRH),
•Secondly, the pituitary hormones or
gonadotropins (FSH and LH), and
•Finally, the ovarian steroid hormones
(estradiol and progesterone).
These
processes are
controlled by
the
hypothalamic
-pituitary-
ovary axis.
The menstrual cycle is directed
by complex functional
interactions between the ovaries
and the hypothalamus-pituitary
system which control each other
by means of positive or
negative feedback mechanisms.
perogestron

4. Hypothalamus
GnRH is a decapeptide which is synthesized and released by specific neuronal
endings in the nucleus arcuatus of the hypothalamus.
GnRH is transported through the portal vessels towards the anterior pituitary gland.
If GnRH is released in a constant, non-pulsatile manner, gonadotropin release is
suppressed due to an apparent desensitization of the pituitary GnRH receptors.
The hormone is only detectable in the
portal system, being undetectable in the
systemic circulation.
Small quantities of GnRH are sufficient to
effectuate a release of gonadotropins from
the pituitary gland.
The release pattern of the gonadotropins is
dictated by the frequency of GnRH release.
GnRH must be released in a pulsatile
manner, and its effects depend on the
frequency and amplitude of these pulses.

5. Effects of GnRH on Gonadotropins
GnRH acts through its receptor on the pituitary gonadotroph
cells, stimulating production of phospholipase C.
IP3 pathway causes gonadotropin release, while the DAG/PKC
pathway causes gonadotropin synthesis.

6. Pituitary gland
The last step of the hormone cascade is localized in the ovaries, where steroid
hormones are synthesized caused by the gonadotropins action. These hormones
are estradiol and progesterone .
Ovaries
The next step occurs in the pituitary gland. The varying frequency and amplitude of
GnRH release determines the pattern of release of the gonadotropins (FSH and LH)
during the menstrual cycle.

7. FSH release pattern during the cycle
In the last phase of the previous cycle, FSH level increases
due to the drop of estradiol, progesterone and inhibin levels.
This is called positive feedback.
The amount and timing of FSH release by the anterior pituitary changes throughout the ovulatory
cycle.
This rise in FSH concentration stimulates the growth of
antral follicles, resulting in an increase of estradiol and
inhibin B concentrations producing a negative feedback,
a reduction of FSH concentrations.
With the formation of the corpus luteum and the outpouring of both oestradiol and progesterone,
the negative feedback mechanism comes into play and continues its suppression of FSH release
until just before the next menstruation.
At mid-cycle, there is a temporary increase in FSH
secretion. It may be due to the GnRH surge and may
have a function in preparing a cohort of small antral
follicles for the next cycle.

8. Role of FSH
Granulosa cell proliferation and differentiation
Antral follicle development
Estrogen production
Inducing of LH receptor in dominant follicle
Inhibin synthesis
The major role of the FSH surge is to stimulate cumulus
expansion and to facilitate the production of the protease
plasmin.

9. CUMULUS CELLS: EXPANSION
the cumulus granulosa cells undergo a series of structural and functional
changes called mucification.
In response to the preovulatory surge of FSH, the cumulus cells secrete
large quantities of a newly synthesized glycoprotein mucous
substance into the extracellular spaces.
This change results in the dispersal of the cumulus cells and causes the
egg-cumulus complex to expand tremendously.
The process of mucification is physiologically critical for the pickup and
transport of the egg in the fallopian tube.

10. Selection of the Dominant Follicle
Progression from primordial, through primary, secondary to pre-antral
follicles does not depend on the gonadotropic stimulation, being a
process controlled and induced by the ovary itself.
Of the millions of primordial follicles that
started life in the ovary, only about 400 will
actually achieve ovulation during the
reproductive life span. That means that
more than 99.9 % of follicles become
atretic.
Nevertheless, the antral follicle already has FSH receptors and grows due
to the FSH action. In the last stage, the preovulatory follicle also becomes
sensitive to the LH action.
In normal cycling women, the dominant
follicle is selected from a cohort of class 5
Follicles (4.7 ± 0.7 mm) at the end of
the luteal phase of the menstrual cycle.
Note

11. At the time of selection, each cohort follicle contains a fully grown oocyte,
about 1 million granulosa cells, a theca interna containing several layers of
TICs, and theca externa composed of smooth muscle cells
A characteristic feature of a dominant follicle is a
high rate of mitosis in the granulosa cells.
As a consequence of increased mitosis, the
dominant follicle continues to grow rapidly
during the follicular phase, reaching 6.9 ± 0.5
mm at days 1 to 5, 13.7 ± 1.2 mm at days 6 to
10, and 18.8 ± 0.5 mm at days 11 to 14.
The key to being chosen as the month’s
ovulatory follicle is sensitivity to FSH.
Decreased estradiol production by the corpus luteum is the principal cause
for the secondary rise in FSH

12. As FSH concentrations fall in response to
rising oestrogen and inhibin B levels and
become less available, only the most
sensitive follicle to FSH, that with the
lowest threshold for a response to FSH,
can survive and continue to thrive and
produce the most oestrogen and LH
receptors.
Selection of the dominant follicle in relation to FSH concentrations
The follicles more sensitive to FSH
rather than those less mature are
selected.
The FSH level declines during the mid
follicular phase.
The follicles most sensitive to FSH will
utilize it to increase aromatase activity
and produce oestrogens and inhibin.
The rest, starved of the possibility of FSH stimulation, become atretic.

13. A. In dominant follicles, FSH in follicular fluid induces P450arom activity that
metabolizes androgen substrate to estradiol (E2 ). In such follicles, E2 and
androstenedione (A4) accumulate in very high concentrations in the follicular
fluid.
B. In nondominant follicles, the low levels of FSH lead to a paucity of granulosa cells
(GC) and low concentrations of estradiol, despite the high levels of A4.
In developing healthy (dominant) follicles (class 5 to 8 follicles), the mean
concentration of follicular fluid FSH increases from about 1.3 mIU/ml
(about 58 ng/ml) to about 3.2 mIU/ml (about 143 ng/ml) through the
follicular phase.In contrast, the levels of FSH are low or undetectable in the
microenvironment of the nondominant cohort follicles
Dominant follicles have a more vascular theca compared with other
antral follicles, and as a result they display an increased uptake of serum
gonadotropins.

14. LH release pattern during the cycle
During the early and mid-follicular phase, the secretion of LH is
relatively quiet with pulses every 60–90 min and a fairly constant low
concentration of circulating LH.
However, this is the calm before the storm.
An enormous climax is reached with the onset of the LH surge in the late
follicular phase, the central event of the ovulatory cycle. Concentrations of
LH rise to 10–20 times their resting level during the rest of the cycle.

15. Then, the pituitary gland becomes highly sensitive to GnRH stimulation, due to the
increase of GnRH receptors. Thus, the GnRH surge produces the LH surge.
The causes of the LH surge are:
First, the negative feedback of estradiol at the hypothalamic-pituitary level turns to a
positive feedback when estradiol concentrations reach a critical point.
Following ovulation, increasing concentrations of progesterone slow down the
frequency of LH releasing pulses.
Concentrations of LH once again drop
to baseline levels.

16. The pre-ovulatory LH surge has a number of key
functions:
Triggers of ovulation and follicular rupture about 36 hours
after the surge
Disruption of the cumulus oocyte complex
Luteinization of granulosa cells
The major role of the LH surge is to stimulate meiotic
maturation
formation of the stigma or site of follicle rupture

17. The mid-cycle LH surge induces a dismantling of the gap junctions
between granulosa cells and the oocyte, thus inhibiting the flow of inhibin
factors to the oocyte and allowing the flow of maturation inducing factors
such as calcium, maturation promotor factor (MPF) and another growth
factors.
cAMP is an important mediator in the nuclear maturation of the oocyte.
cAMP activates a protein-kinase that suppress the activation of MPF or
degrades its subunits. The drop of cAMP levels after LH surge favours
oocyte maturation due to the increasing levels of MPF.
Increasing levels of calcium after the LH surge are also necessary to
restore the meiotic arrest.
After oocyte maturation begins and
cAMP levels decline, RIIα-PKA
moves to the mitochondria, and
maturation promoting factor (MPF)
is activated, leading to germinal
vesicle breakdown and maturation
to the MII stage.
LH surge is to stimulate meiotic maturation
Meiosis is arrested here and proceeds no further unless the ovulated egg is
fertilized

18. LH
androgens
estradiol
Theca cells
Granulosa cells
aromatase
FSH
“two cell – two gonadotropin hypothesis”
The mid-cycle surge of LH induces the production of androgens by cells of the
theca follicles. The androgens, androstenedione and testosterone, are then ‘passed
on’ to the neighbouring granulosa cells, where aromatase (CYP19) converts them
into estrogens, mainly estradiol but also estrone.
These events suggest that the function of theca cells and granulosa cells
are controlled by LH and FSH respectively.
Moreover, there is also a fine-tuning achieved by other factors such as
inhibin, insulin-like growth factors (IGFs) I and II and tumor necrosis
factor-α.
Aromatase activity
and therefore,
estrogen production
is controlled by FSH.

19. Oestradiol
Estradiol is the most
important estrogen in the
ovulatory cycle. During
menstruation estradiol
concentrations are low, but
start to increase as FSH
induces follicular
development in the mid-
follicular phase.
When estradiol levels reach a critical point, they activate a positive
feedback mechanism in the hypothalamus and pituitary resulting in LH
and FSH surges.
Following ovulation, estradiol concentrations decrease temporarily but are
revived caused by corpus luteum activity. With the demise of the corpus
luteum, estradiol concentrations drop rapidly to their lowest levels and by
a positive feedback, increase FSH levels immediately preceding
menstruation.

20. The key functions of estradiol in the ovulatory cycle are:
In the mid-late follicular phase
It suppresses the secretion of FSH due to a negative feedback mechanism
leading to the selection of a dominant follicle and preventing multi follicular
development.
In mid-cycle
Triggers the LH surge due to a positive feedback mechanism when its
concentrations rise to a critical level.
In the follicular phase
As a ‘growth hormone’ for the development of the endometrium.
In the ovulatory period
estradiol stimulates the glands of the cervix to secrete a particular type of
mucus which is essential for the sperm to pass through the cervix to reach
the ovum.

21. Progesterone
Progesterone is the main hormone in the luteal phase.
Large quantities are synthesized by luteinized granulosa cells of the corpus
luteum following ovulation.
Progesterone concentrations rise to a peak 7-8 days following ovulation and
fall rapidly with the demise of the corpus luteum.

22. Together with estradiol,
progesterone suppresses
pituitary gonadotropin
release during the luteal
phase.
The initial rise of progesterone concentrations immediately preceding the
LH surge may play a role In the triggering of this surge.
Progesterone
During this phase, FSH is
synthesized and stored
ready for release when
freed from the inhibition
imposed by progesterone
and oestradiol when the
Corpus luteum fails

23. Role of progestrone
The main functions of progesterone secreted by the corpus luteum are To
induce a secretory endometrium, capable to enhance embryo
implantation.
Progesterone also plays a role in the expression of genes needed for
implantation at the level of the endometrium.
stimulates mammary growth
Progesterone is responsible for the stimulation of the theca collagenase,
an enzyme which stimulates the dissociation of cells in the area of the
stigma.

24. Activin is a promoter of many actions of
FSH in that it increases FSH secretion,
promotes ovarian follicular development.
Inhibin
Inhibin is
secreted by
granulosa cells
Inhibin A
Inhibin B
Inhibin A concentrations are low during
most of the follicular phase but start to
rise during its latest stages and peak in
the mid-luteal phase.
inhibin B concentrations start rising early
in the follicular phase, paralleling but
later than the FSH rise. reduces the
synthesis and secretion of FSH and the
number of GnRH receptors in the pituitary
and has an inhibitory effect on the growth
of antral follicles in the ovary.
Activin and follistatin
Follistatin is an activin-binding protein
that neutralizes activin bioactivity.
Estrogens and inhibin B are
both inhibitory factors for the
secretion of FSH.

25. Growth factors
insulin-like growth factors (IGFs) I and II which are very active.
Insulin, as well as binding to IGF receptors, has its own ovarian receptors
and is known to promote androgen production.
The transforming growth factor (TGF) family is also well represented in the
ovary as is epithelial growth factor (EGF). All play a passive role in the
regulation of gonadotropin activity within the follicles.
Anti-Mullerian hormone (AMH)
AMH, a dimeric glycoprotein and member of the transforming growth
factor-beta family, is produced by ovarian follicular granulosa cells in late
pre-antral and small antral follicles. It seems to have a role in the
regulation of folliculogenesis at the two extremes of this process:
(a) by restricting the progression
of development of primordial
follicles
(b) by an inhibition of the sensitivity
of antral follicles to FSH and
inhibition of aromatase activity
during an ovulatory cycle.

26. Mechanism of follicular rupture
On or about the 15th day of an ideal 28-day
cycle, the preovulatory follicle ruptures, and
the egg cumulus complex is released from the
ovary by a process called ovulation.
The preovulatory surges of LH and FSH play a
crucial role in the physiologic mechanism of
ovulation.
A stigma in mammalian reproductive
anatomy refers to the area of the ovarian
surface where the Graafian follicle will burst
through during ovulation and release the
ovum
Stigma formation involves a combination of cell apoptosis, cell migration,
and proteolytic digestion of extracellular matrix layers

27. In response to the LH surge, the preovulatory follicle produces
progesterone and prostaglandin, both of which are obligatory for the
stigma to develop.
PGF2a stimulates the rupture of lysosomes in the apex epithelium
facilitating further stigma formation; it also stimulates ovarian contractions
leading to follicle rupture and; finally stimulates follicle contraction for
actual oocyte expulsion
After the surge in luteinizing hormone prior to
ovulation, vasodilation of capillaries is prominent and
the cross-sectional areas of vasculature lumina
increase. There is evidence of increased vascular
permeability, tissue oedema and ischemia. Capillaries
develop perforations through which blood cells and
platelets escape when ovulation occurs. Shortly
before ovulation, blood flow stops in a small area of
the ovarian surface overlying the bulging follicle.
Prostaglandins production by granulosa cells. Two types of prostaglandins
are known to have some role in the process of ovulation.
PGE2, which stimulates the plasminogen activator, an enzyme which
dissolves connective tissue in the area of the stigma.

28. FSH, at very low levels,
stimulates the production of
plasminogen activator (PA) by
the granulosa cells.
1
PA catalyzes the conversion of
plasminogen from the
granulosa cells to plasmin
3
that targets the cells of the
follicular wall, weakening their
intercellular adhesion.
5
Plasmin is a serine protease derived from plasminogen by enzymatic
activation. Two forms of plasminogen activators have been characterized,
urokinase (uPA) and tissue (tPA) types. Both uPA and tPA appear to
contribute to ovary plasmin biosynthesis and ovulation. The follicular fluid
contains relatively high levels of the plasmin precursor, plasminogen. The
preovulatory surge of FSH appears to stimulate granulosa cells to secrete
plasminogen activator, which converts plasminogen to the active protease
plasmin. Plasmin appears to play a role in the degradation of the
granulosa cells and basal lamina in the presumptive stigma.

29. FSH also induces the huge
expansion of the cumulus
oophorus (by mucification)
which also includes up regulation
of LH receptors on the granulosa
cells’ surfaces prior to the LH
surge.
2
The only targets of FSH in the
ovary are the granulosa cells
Luteinizing hormone (LH)
induces the resumption of
meiosis (by the mid-cycle
surge)
LH also induces the follicle
corona cells to make and
release progesterone that
induces prostaglandins
(PG’s) E and F production.
6
7
These PG’s trigger the presumptive
stigma cells of the ovarian wall to
make and release a number of
proteases that break down the
ovarian matrix and the follicular
tunica albuginea
8

30. this ultimately allows for follicular expansion and
exit of the ovulated oocyte at the stigma
9 10
LH luteinization of the granulosa and theca lutein cells (right
image) already initiating the formation of the corpus luteum.
Just before ovulation, the apical region becomes avascular, and in the periapical
vessels, leakage of intravascular contents into intercapillary spaces reflects
increased permeability.

31. Scanning electron micrograph of the protruding surface of an ovulatory
follicle.

32. NGF/TrkA interactions lead to a loss
of intercellular communication by
disrupting gap junctions between
theca cells and result in increased
migratory behavior.
Nerve growth factor (NGF) and one of its receptors, the tyrosine
kinase receptor TrkA
NGF and TrkA expression
are induced in theca cells in
response to the LH surge.
Vascular endothelial growth factor (VEGF) and its downstream signaling
pathways are required for follicle angiogenesis during antral follicle
growth, and there is evidence in the primate that VEGF is important for
follicle rupture.
VEGF
VEGF promotes vascular permeability, allowing more efficient
delivery of bloodborne Factors including LH and FSH, and immune
cells to the follicle.
α-Adrenergic agents, such as
norepinephrine, exert a
stimulatory effect on ovarian
contractions, whereas β-
adrenergic agents are inhibitory.

33. Enzymes such as matrix metalloproteinases (MMPs) and ADAMTS
proteases are involved (although not all are essential):
Tissue inhibitors of MMPs (TIMPs) are also involved in the ovulatory
process and the pattern of in vivo expression suggests that decreased
TIMPs levels and increased MMPs are involved in follicle rupture.
The extracellular matrix remodeling is completed by a process similar to
the inflammatory process, with the participation of macrophages,
neutrophils, cytokines produced by leukocytes, platelet activating factor
and free radicals.
Degrading of the
basement membrane
Remodeling of the extracellular
matrix
Rupturing of the follicular apex.
ovulatory induces with mediators including endothelin-2, interleukin-6
and cGMP-dependent protein kinase II.

34. Relaxin
Theca interna constitutes the primary source of follicular
relaxin
Oxytocin synthesized by the ovary
Arachidonic acid
play an essential role in gonadotrophin activation of
ovarian collagenolysis, needed for follicle rupture at
ovulation.
This is supported by the fact that inhibitors of cyclooxygenase and lipoxygenase
pathways of arachidonic acid metabolism inhibit follicle rupture at ovulation.
large amounts of sex steroids present in the follicle
during the early preovulatory phase can stimulate
growth of lysosomes
sex steroids
Other substances that appear to influence the process of ovulation at a
local ovarian level include various cytokines, oxygen free-radicals, nitric
oxide and angiotensin II.

36. 1-5 Minutes before
Follicular Rupture
½-1 Hour before
Follicular Rupture
10 Hours before Follicular
RuptureAnatomy of Ovulation

37. Ovulation is initiated by the surge of LH (Step 1) that induces the expression of
specific inflammatory-related genes in granulosa cells, which then leads to an
intrafollicular signaling cascade and the expression of similar genes in cumulus cells:
Areg, Ptgs2, Has2 and Tnfaip6. Once activated in COCs, these genes establish an
autocrine regulatory loop whereby PGE regulates Areg expression and AREG
regulates Ptgs2 mRNA; together they induce genes that control the production and
stabilization of HA-rich matrix (Step 2). As expansion proceeds, genes associated
with innate immune cell responses, such as Il6, are induced (Step 3) and continue to
be expressed in ovulated COCs.
hypothesize that
ovulation
involves
coordinated and
sequential
inflammatory
and innate
immune
responses

38. FSH binds FSH receptors on cumulus cells to activate AC, cAMP production
and PKA.
This pathway activates Erk as do Egf receptors in response to Egf-L
amphiregulin (AR), betacellulin (BC) and epiregulin (ER).
Transcription factors regulated by these
kinase cascades include AP-1 factors,
Elk-1 and cAMP response element-
binding protein (CREB).
In growing follicles, cAMP is
translocated to the oocyte via gap
junctions until these are inactivated
through phosphorylation by Erk and
cells separate from the oocyte through
cumulus expansion.
Signal transduction in cumulus cells
in the mouse periovulatory phase.
Consequently, cAMP levels in the oocyte
fall.

39. In a separate pathway, oocyte-secreted growth and differentiation factor-
9 (Gdf-9) and bone morphogenetic protein-15 (BMP-15) activate integral
receptor kinase activity on cumulus cells, which results in phosphorylation
of SMAD2/3, which translocate to the nucleus in dimers with SMAD4.
These transcription factors
promote expression of key
cumulus genes required for
specification of the cumulus-
specific response to the
ovulatory surge.
PGE2 activates the EP2 receptor to mediate a signal transduction pathway
similar to FSH, and RGS-2 may control the activation of G-proteins.
Genes induced via these two
signal transduction
mechanisms include HAS-2,
TSG-6, PTX-3, COX-2, PGE2
receptor (EP2) and regulator
of GPCR-2 (RGS-2).

40. Transcriptional complexes modify chromatin structure and stabilize
transcriptional machinery to transiently activate high gene expression.
Genes expressed in this rapid transient fashion via this signalling cascade
include PR, a disintegrin and metalloproteinase with thrombospondin
repeats (ADAMTS-1), Egr-1 cathepsin L and versican.
Signal transduction in mural granulosa cells in the
mouse periovulatory phase.
LH interacts with its G-protein (Gas)-
coupled receptor activating AC and
hence intracellular cAMP production
as well as iP3/Ca Þþ mobilization.
Consequently, PKA and Erk activation
result in phosphorylation (line
arrows) of transcription factors
including cAMP response element-
binding protein (CREB) and
stimulatory proteins 1 and 3 (Sp1/3).
Transcriptional complexes assembled on the promoters of periovulatory
genes expressed in mural granulosa cells include Sp1/3 and additional
factors including CREB, reproductive homeobox 5 (Rhox5) or other induced
transcription factors.

41. Clinical Signs and Symptoms of Ovulation
Many women refer heightened sense of smell and sexual desire in
the several days immediately before ovulation.
Towards the time of ovulation the pH in the vagina becomes less acidic
The cervical mucus becomes more copious and less viscous, all of
which favour the progress of motile sperm towards the released oocyte.
Moreover, the action of progesterone increases basal body
temperature by one-quarter to one-half degree Celsius (one-half to one
degree Fahrenheit).
Many women experience secondary fertility signs including Mittelschmerz
(pain associated with ovulation).

42. Thanks for your attention