Methods
We searched for articles published in English in the PubMed and Google Scholar databases.
The keywords were as follows: menstrual cycle, menstrual cycle physiology, folliculogenesis, theca cells, granulosa cells, oocyte, oocyte-cumulus complex, follicular wall digestion, cumulus-oocyte-complex expansion, oocyte maturation, gene expression, FSH, LH, and progesterone receptor. We expanded the search to the references of the retrieved articles.
Follicular Dynamics and Folliculogenesis
The more advanced stages of follicle development are characterized by the appearance
of intercellular space filled by antral fluid. At this stage, the granulosa cells
are differentiated into two distinct populations: cumulus cells, which are those closely
linked to the oocyte, and wall or mural granulosa cells, which internally line the
follicular wall. Although these two cell types share a common origin, there are differences
in the production of transcribers and proteins.[3] At the end of follicular development, the FSH and estradiol promote the expression
of the LH receptor (LHR) in granulosa cells. Most LH molecules bind to mural granulosa
cells rather than to cumulus cells.[4] Cumulus cells provide energy input to the oocyte, controlling its growth and metabolism.[5] On the other hand, mural granulosa cells are responsible for steroid synthesis and
differentiation in luteum cells after ovulation.[6] Cumulus granulosa cells play a smaller role regarding the function of the corpus
luteum. Follicular architecture is provided by the inner and outer theca-cell layers
([Fig. 1]). The theca cells, provided with LHR, are responsible for the capture of the substrate
cholesterol and its enzymatic conversion into androgens, mainly testosterone (T) and
androstenedione (A4). In turn, granulosa cells, which are adjacent to the theca cells,
capture A4 and T and, by the action of the aromatase enzyme, convert them into estrone
and estradiol respectively ([Fig. 2]).[7]
Fig. 1 Structure of the periovulatory follicle showing internal and external theca cell
layers, granulosa cells, and the oocyte.
Fig. 2 Scheme showing the steroidogenesis of theca and granulosa ovarian cells. Abbreviations:
P450ssc, cytochrome P450 for cleavage of cholesterol side chains; 3-βSHD, 3 β hydroxysteroid
dehydrogenase; 17-HSD3, 17-hydroxysteroid dehydrogenase. Source: Medeiros et al.[7]
Folliculogenesis begins with the formation of the primordial follicle, and ends with
the preovulatory follicle.[8] The FSH, released by the anterior pituitary gland, promotes the recruitment of follicular
waves that, in response, secrete estradiol and inhibin. When synthesized, these hormones
modulate the release of pituitary FSH and LH in a pulsatile way. At the end of folliculogenesis,
the preovulatory peaks of FSH and LH induce a complex sequence (or even a concurrence)
of events: oocyte maturation, cumulus cell expansion, follicular wall digestion, and
release of the cumulus-oocyte complex.[9]
Ovulation Process
Genetic Aspects Determining Ovulation
The ovulation process occurs in a coordinated and interrelated way in five complex
steps: interruption of granulosa cell proliferation, resumption of meiosis, expansion
of the cumulus with oocyte release inside the antrum, lysis of the follicular wall,
and oocyte extrusion at the metaphase II (MII) stage. In mammals, oocytes are stationed
in meiosis I at prophase I. The resumption of meiosis I occurs during puberty as a
result of the gonadotropic stimulus in follicles in the preovulatory stage, culminating
in the rupture of the germ vesicle.[10] The increase in the concentrations of LH and FSH in the mid-cycle in the presence
of the preovulatory follicle, now provided with LHR in granulosa cells, promotes the
activation of several genes that encode the synthesis of various proteins. This process
is similar to inflammatory processes.[11] The LH activates cyclase, resulting in intracellular increases in cyclic adenosine
monophosphate (cAMP) that activate cAMP-dependent kinases and the expression of the
hyaluronic synthase 2 (HAS-2) and cyclooxygenase 2 (COX-2) enzymes, the tumor necrosis
factor-inducible gene 6 protein (TSG-6), pentraxin 3 (PTX-3), and genes of the epidermal
growth factor (EGF)-like family, such as amphiregulin (AREG), epiregulin (EREG), and
betacellulin (BTC).[12]
[13]
[14] Tissue rearrangement occurs as a result of the activation of these genes participating
in the cascade of ovulation events.
The Role of the Follicle-stimulating Hormone
Periovulatory gene expression induced by the FSH in cumulus cells plays a minor but
necessary role in the mediation of ovulation ([Fig. 3]). The occurrence of the FSH peak activates its own receptor (FSHR), stimulates the
expression of steroidogenic factors, and induces LHR synthesis in granulosa cells.
Such functions of the FSHR are related to the FSH activation of cAMP synthesis, and
are triggered mainly through the expression of protein kinases A (PKA) and C (PKC)
enzymes in granulosa cells.[15] The FSH activates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt)
pathway to mediate cell survival and granulosa proliferation, including the expression
of the vascular endothelial growth factor (VEGF) gene, and it activates extracellular-regulated
kinase (ERK) signaling in mural granulosa and cumulus cells, facilitating cumulus
expansion.[16] The FSH may also induce COX-2 and other prostaglandin synthases through cAMP/PKA
activation.[17] Activation of the COX-2 gene results mainly in prostaglandin F-α (PGF2α) that induces
changes in the gene expression of the cumulus-oocyte-complex, which is critical for
cumulus-oocyte-complex expansion.[18] Additionally, the FSH induces the expression of genes belonging to the family of
disintegrin and metalloproteinases (A disintegrin and metalloproteinase with thrombospondin
motifs, ADAMTS), molecules relevant in the process of cleavage of the extracellular
matrix ([Fig. 3]). It seems that these proteins are the main regulators of the release of EGF-like
proteolytic factors in a soluble form (AREG, EREG, and BTC),[19] which activate the EGF receptor tyrosine kinase and the extracellular signal-regulated
kinase (ERK) involved in cumulus expansion.[13] Metalloproteinases ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMS-16, genes expressed in
granulosa cells, are involved in the dissociation of the cumulus-oocyte complex and
in the formation of the corpus luteum.[20]
[21] Then, the FSH, in the same way as in the mucification of the cumulus, plays a role
with the LH in the synthesis of enzymes responsible for the digestion of the follicle
wall.
Fig. 3 Biochemical events initiated by the follicle stimulating hormone (FSH) in the preovulatory
follicle. Abbreviations: VEGF, vascular endothelial growth factor; COX-2, cyclaoxygenase-2;
PKA, protein kinase A; PKC, protein kinase C.
The Role of the Luteinizing Hormone
The role the LH in the ovulation process is complex and fundamental for the resumption
of meiosis, loosening of the cumulus cells, and rupture of the follicle.[22] With the peak of the LH, the messenger ribonucleic acid (mRNA) for the progesterone
receptor (PR) as well as other genes is now transcribed into the granulosa cells of
preovulatory follicles ([Fig. 4]).[23] The PR has an indirect influence on the synthesis of proteolytic enzymes cathepsin
L and ADAMTS-1, which together play a role in tissue degradation and the remodeling
of the extracellular matrix at the apex of the preovulatory follicle until ovulation
occurs.[24] The LH peak, modulated by AMP, participates in the process of suppression of the
proliferation of granulosa cells, and restarts meiosis, dissociation of the granulosa,
digestion of the follicle wall, and luteinization.
Fig. 4 Expression of several genes induced by the luteinizing hormone (LH) peak. Source: Richards et al.[23] Abbreviations: PO, preovulatory; PR, progesterone receptor; PGS, prostaglandins;
C/EBPβ, CAAT enhancer-binding protein β; p27KIP1, cyclin-dependent kinase inhibitor 1B; Start
5, steroidogenic enzymes; IGF, insulin growth factor; COX, cyclooxygenase.
Biochemical Aspects Determining Ovulation
Mucification and Cumulus Expansion
The genetic and biochemical events responsible for cumulus mucification are summarized
in [Fig. 5].[23] The matrix on which the cumulus cells move has three major components: hyaluronic
acid (HA) and two HA binding proteins, TSG-6, and inter-α-trypsin inhibitor (ITI).[9]
[25] Induced by the peaks of FSH and LH, HAS-2 is the main enzyme responsible for the
synthesis of arachidonic acids and HAs in the cumulus-oocyte complex, and, in synergy
with COX-2, causes the synthesis of prostaglandins (PGs) from arachidonic acid in
the granulosa cells of the cumulus. Thus, the expression of COX-2 in the cumulus cells
promotes the synthesis of PGs, mainly prostaglandin E (PGE), and ensures the expansion
of the cumulus.[18]
[25]
[26] However, cumulus expansion occurs only when the ITI enters the follicle. The TSG-6
and the proteoglycans brevican and versican, induced by high concentrations of LH
and HA stabilization, are rapidly expressed in the cumulus granulosa cells of preovulatory
follicles.[16] In the context of deficiency of the TSG-6 enzyme, the extracellular matrix is not
structured, compromising cumulus expansion.[27] The PTX-3 protein, with an affinity for TSG6, is also responsible for the stability
of the cumulus matrix. The interaction between these enzymes appears to be crucial
for the structuring and expansion of the cumulus matrix, enabling the dispersion of
the cumulus cells away from the oocyte.[20] Collectively, these observations indicate that HA, ITI, and COX-2, induced by the
TSG-6 gene, are critical for cumulus matrix formation, cumulus cell differentiation,
and, ultimately, cumulus expansion.
Fig. 5 Combined actions of the FSH and LH in the expansion of oocyte-cumulus cells. Source: Richards et al.[23] Abbreviations: HA, hyaluronic acid; GDF, growth defferentation factor; BMP, bone
morphogenetic protein; PE, prostaglandin E receptor.
Oocyte Maturation
The oocyte maturation process aims to empower the female gamete and ensure its subsequent
development until the activation of the embryonic genome occurs. Therefore, chromatin
condensation is relevant in the continuity of meiosis, redistribution of organelles
in the cytoplasm, and alterations in the cytoskeleton; all of these modifications
are precisely regulated and coordinated ([Fig. 6]).[28] For this to happen, there is paracrine cross-talk between the oocyte and cumulus
cells. Cumulus cells penetrate the zona pellucida and limit the ooelema gap junction
between the cumulus and the oocyte transfer of small molecules.[28] Biochemically, the oocyte regulates the metabolism of cumulus cells, which in turn
provide ions, metabolites, amino acids, and small oocyte regulatory molecules ([Fig. 7]).[29] Paracrine oocyte factors are soluble, and are generically referred to as oocyte-secreted
factors (OSFs).[30] The growth differentiation factor 9 (GDF-9), the bone morphogenetic protein 15 (BMP15),
and, to a lesser extent, the BMP6 are considered OSFs; all belong to the family of
transforming growth factors β (TGFβ).[30]
[31] These factors coordinate the differentiation lineage and function of granulosa cells.
Fig. 6 The role of the LH in meiosis resumption. Source: Coticchio et al.[28] Abbreviations: TGFβ, transforming growth factor β; EGF, epidermal growth factor.
Fig. 7 Cell-cell signaling between the oocyte and granulosa cells in the final stage of
follicle development. Source: Adapted from: Sutton et al.[29] Abbreviations: GDF-9, growth differentiation factor 9; BMP-15, bone morphogenetic
protein 15; FGF-8B, fibroblast growth factor 8B.
The functions of the OSFs include growth stimulation, prevention of apoptosis, inhibition
of luteinization, regulation of energy metabolism, cholesterol biosynthesis, and regulation
of cumulus expansion.[32]
[33]
[34] The factors that regulate the relationship between cumulus granulosa cells and the
oocyte include ions, metabolites, amino acids, and small intracellular signaling molecules
such as cAMP, cyclic guanosine monophosphate (cGMP), and inositol triphosphate-3 (IP3).[6]
[32] In the regulation of meiosis, cAMP synthesized by the oocyte itself and by cells
of the mural granulosa and cumulus reaches the oocyte through the junctions of the
hexameric lacunar canal composed of connectin proteins.[35]
[36]
In general, the properties of lacunar junctions enable the direct and bidirectional
transport of small molecules between the oocyte and the granulosa cells. High intraoocyte
levels of cAMP maintain the oocyte in the stage of germ vesicle, through suppression
of the activity of the maturation-proimoting factor (MPF).[37]
[38]
[39] Follicle somatic cells also provide cGMP to the oocyte, inhibiting the phosphodiesterase
enzyme type 3A (PDE3A), thereby preventing the degradation of cAMP with the accumulation
of this factor and inhibition of the resumption of meiosis.[38]
[40] With the LH stimulus at high concentrations, the connectins close, decreasing the
contribution of cAMP and cGMP from the cumulus cells to the oocyte. Therefore, the
decrease in cAMP levels leads to the phosphorylation of PDE3A that degrades the cAMP.
The degradation of cAMP enables the synthesis of the MPF, which promotes the resumption
of meiosis I.[41]
In a recent study[42] in mice, the expression of natriuretic peptide type C (NPPC) was found in the mural
granulosa cells, and natriuretic peptide receptor 2 (NPR2) was found in cumulus cells.
With the communication between these two cell types the NPPC ligand and NPR2 stimulate
the secretion of cGMP and cAMP. By adding NPPC to the culture media, an increase in
the rates of oocytes that did not resume meiosis was observed, favoring the synchrony
between nuclear maturation and cytoplasmic maturation.[42] During cytoplasmic maturation, there is a physical rearrangement of mitochondrial
groups and endoplasmic reticulum, following the maturation time and energy dependence
of the meiotic spindles so that chromatin is divided.
The meiotic spindles are responsible for the continuity of the meiotic division and
extrusion of the two polar corpuscles. Initially, the mitochondrial groups are in
a central position in the oocyte. As the maturation progresses, they migrate to the
edges of the oocyte, close to the extruding regions of the polar body.[43]
[44] The MPF is the factor directly involved in cytoplasmic maturation, because, in addition
to inducing the breakdown of the germ vesicle, it promotes the condensation of chromosomes,
moving them from prophase I to metaphase I (MI), in which there is the formation of
the meiotic spindle and the alignment of chromosomes in the center of the spindle.
Then, anaphase I occurs, which consists of the separation of homologous chromosomes.
Sequentially, telophase I begins with the extrusion of the first polar body, and the
oocyte is in the metastasis II stage. At this stage, there is the formation of the
second meiotic spindle and alignment of chromosomes, following anaphase II and telophase
II and, finally, the extrusion of the second polar body.[45]
[46] The oocyte remains in this stage until ovulation occurs and there is the penetration
of the sperm.
Follicular Wall Digestion
Morphological and biochemical changes that result in rupture of the follicular wall
and oocyte extrusion occur basically by the action of the LH, because it induces the
synthesis and secretion of various enzymes ([Fig. 8]). The role of the FSH is smaller in this process, when the oocyte and cumulus cells
are still fixed in the extracellular matrix (ECM). With the LH peak, LHR on the surface
of the granulosa cells activates the digestion of the ECM within the theca layers
and tunica albuginea at the ovarian surface via adenyl cyclase. The effectiveness
of ECM digestion occurs through the balance between matrix components and proteases
in the cumulus, oocyte, and endothelium cells that form the corpus luteum.[21] Theca cells express a variety of matrix metalloproteinases (MMPs), including MMP2
(gelatinase A), MMP9 (gelatinase B), MMP13 (collagenase), MMP14, MMP16, MMP19, and
tissue inhibitor of MMPs-1 (TIMP-1).[9]
Fig. 8 Role of LH-induced genes in the digestion of the follicular wall.
The ADAMTS 16, present in luteinized granulosa cells, responds to FSH stimulation
and actively participates in the process of structural follicle remodeling at the
time of ovulation. The role of the LH on PR is mimicked by cAMP-inducing agonists
(FSH, forskolin). Targets of PR appear to control the rupture of the follicle, mainly
ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin) and cathepsin L.
Among the proteases involved, thrombospondins 1 and 4 (ADAMTS1/4) promote the breakdown
of the proteoglycan family structures, such as versican, through granulosa activation
by PRs,[47] thereby contributing to the follicular rupture. Through its receptor in granulosa
cells, the LH induces the transcription of early growth regulatory factor-1 (EGR-1),
CAAT enhancer-binding protein β (C/EBPβ), PR, and other activator protein-1 family members
(proto-oncogenes, c-Fos, c-Jun, Fra2, JunD), all involved in the functional activity
of the granulosa cells of the ovulating follicle.
The proteoglycan (versican, brevican) components of the ECM induced by the LH peak,
on either granulosa or theca cells, serve as substrates preferably for ADAMTS 1, culminating
in follicular rupture.[47] Metalloproteinases such as plasminogen and collagenase are part of the follicular
digestion process, and their control is mediated by metalloproteinase inhibitors,
ensuring local homeostasis and completion of the ovulation process.[48] To illustrate, the model proposed by Ogiwara et al.[49] in the Japanese rice fish, also known as medaka, shows the involvement of proteinases
in the lysis of the follicular wall ([Fig. 9]).
Fig. 9 A model of follicle rupture during ovulation in the Japanese rice fish, also known
as medaka. (A) In the follicle, a few hours before ovulation, progelatinase A is activated by membrane
type 1-matrix metalloproteinase (MT1-MMP) on the surface of the oocyte, whereas gelatinase
A is immediately inactivated by the tissue inhibitor of matrix metalloproteinases-2b
(TIMP-2b). (B) At the time of ovulation, the hydrolysis of basement membrane type-IV collagen is
initiated by active gelatinase A at the follicle–ovarian surface contact site. (C) membrane type 2-matrix metalloproteinase (MT2-MMP), which is now expressed on the
surface of the granulosa cells, can degrade the type-I collagen that is present in
the theca cell layer. (D) As a result, the oocyte is exposed at the contact site, leading to ovulation. Abbreviations:
BM, basement membrane; EC, epithelial cell; GC, granulosa cell; OC, oocyte; TC, theca
cell. Source: Ogiwara et al.[49] We would like to thank the National Academy of Sciences of the United States by
permission
After the rupture of the follicular wall, there is tissue reorganization by the activation
of promatrix factors, which, in an organized and vascularized way, causes granulosa
cell differentiation into luteal cells, thereby originating the corpus luteum. The
corpus luteum is composed of functional cells for the synthesis of progesterone, the
main regulator of the pituitary secretion of gonadotropins, the principal factors
involved in the maintenance of the corpus luteum until initial gestation.[50] In the absence of maternal recognition of pregnancy, the corpus luteum regresses
rapidly, and the ovarian cycle is resumed.[51]
