Review
Wang and Dey, 2006 Nature Reviews 7:185-199
Lecture X : Comparative Implantation
Wang and Dey, 2006 Nature Reviews 7:185-199
Wang and Dey, 2006 Nature Reviews 7:185-199
Implantation
Rodents (Mouse)
Contact of the blastocyst with the uterus is the first step in the
relationship of a mammal embryo with its environment. This
contact leads to implantation and placentation.
Implantation can be divided into five separate phases:
1. Hatching Phase - Blastocyst loss of Zona pellucida
2. Apposition Phase - Hatched blastocyst aligns and orients itself in preparation for
attachment
3. Adhesion Phase - Attachment between uterine surface epithelium and trophoblast
4. Invasion Phase - After blastocyst becomes firmly anchored to the uterine surface,
the trophoblast cells begin to penetrate the uterine epithelial layer
5. Postimplantation Phase - Trophoblast cells cease invasive activity and the
maternal-fetal relationship is stablized
Mesometrial
Endometrium
Uterine epithelium
Zona pellucida
ICM
Trophectoderm
Antmesometrial
Blastocyst enters uterus
on Day 4
Hatching from the Zona Pellucida
Compartive Placentation (ed. D.H. Steven), 1975
Note the closure
of the lumen
There is extracellular
material between
uterine epithelium
and trophectoderm
Apposition Phase
Blastocyst comes to lie along the
antimesometrial side of uterus. Note
that ICM is oriented toward the
mesometrial side of uterus.
Orientation may be caused by the shape
of embryo which is larger at the embryonic
pole. Treatment with Relaxin can disrupt
the orientation of the blastocysts.
Attachment Phase
Decidualization begins following
adhesion in rodents. Surface
changes in the uterine epithelium
permit adhesion between
trophectoderm and uterine surface
epithelium
Compartive Placentation (ed. D.H. Steven), 1975
Wang and Dey, 2006 Nature Reviews 7:185-199
Receptivity of Mouse Uterus to Blastocyst Attachment
Mucins are large O-linked glycoproteins with ectodomains that extend away
from the surface of the cell apical border.
Mucins may sterically block access to the cell membrane and function as
anti-adhesive molecules.
Muc-1 also referred to as epsialin, may function in the time of blastocyst
attachment in mammals.
Muc-1 is lost from the uterine epithelial apical surface during diestrous
in the mouse. It is expressed only on the apical border during the first
3 days of the estrous cycle.
Muc-1 expression
Muc-1 antibody (green)
Perlecan antibody (red)
Basement membrane
Lumenal Epithelium
Glandular Epithelium
Muc-1 immunocytochemical
staining
A. Proestrous
B. Estrous
C. Metestrous
D. Diestrous
Only in glandular
epithelium, loss of
intensity in surface
Western blot of Muc-1
Surveyor et al. 1995, Endocrinology 136:3639
Northern
Blot
A, B, and C = Days 1, 2 and 3 pregnancy
D = Day 4
Note: Loss of Muc-1 is associated
with time of implantation.
Surveyor et al. 1995, Endocrinology 136:3639
Northern Blot
Estrogen stimulates Muc-1
expression.
Progesterone inhibits Muc-1
expression.
Lane 1 = Day 1 pregnancy
Lane 2 = Day 4 pregnancy
Day 4 of pregnancy mice ovariectomized and
given either P4 (lane 3) or E plus P4 (lane 4).
Surveyor et al. 1995, Endocrinology 136:3639
Model for Muc-1 function as an
antiadhesion moleucle
Surveyor et al. 1995, Endocrinology 136:3639
Role of epithelial surface mucins in steric inhibition of
embryo-endometrial interactions
Aplin, 1997; Rev. Repod. 2:84-93
Lipid Hydrolysis
Prostaglandin Synthesis
Increased
Membrane Permeability
Moulton and Koenig, 1986, In Nidation (ed. Yoshinaga) pp95-109
Heparin Sulfate Proteoglycans
av integrin
through a
bridging
ligand
Heparan sulfate Cadherin
binding to a basic binding
protein. Also possibly
Chondroitin sulfate
Interaction of trophoectoderm and endometrial cells at implantation.
Aplin, 1997; Rev. Repod. 2:84-93
Integrins
a has 17 subunits
b has 8 subunits
Endocannabinoid Signaling
Endocannabinoids (Marijuana) target receptors (CB1 and
CB2) in the mouse embryo.
CB1 localized in trophectoderm and not ICM
CB1 in oviduct and uterus
Knockout of CB1 causes defects in development
and survival
High levels of the endocannabinoid, Anandamide (AEA),
inhibit early embryo development. However, low levels are
needed and stimulate trophoblast development.
Anandamide levels are also important for implantation.
Implantation competency requires down-regulation of
AEA binding to the blastocyst.
Coordinated down-regulation of CB1 on
blastocyst and decrease in AEA from uterus.
have
Women with elevated peripheral AEA levels
spontaneous pregnancy loss.
Sun and Dey. Mol Cell Endo (2008) 286:S1-S11
Shelesnyak, 1986, In Nidation (ed. Yoshinaga) pp5-22
Dey and Johnson, 1986, In Nidation (ed. Yoshinaga) pp49-62
Role of Growth Factors in Preimplantation Development
IL-6 will
bind to LIFr
as well
Colony Stimulating
Factor (CSF-1)
has been shown to
be involved with
implantation too!
Leukemia Inhibitor Factor has been demonstrated to be involved with
implantation in the mouse.
LIF appears following estrogen
in delayed implantation
LIF stimulates proteinase
expression in the uterus.
Like - MMPs
urokinase type plasminogen
activator.
LIF not present in delayed
implantation
Histological features of the uterus in LIFnull and wild-type mice. Semi-thin resin
sections from days 5 and 6 of pregnancy
were stained with toluidine blue. The insets
show high magnification of differentiated
polygonal stromal cells in the wild-type and
undifferentiated fibroblast-like stroma cells
in the LIF-null mice on day 6 of pregnancy.
E = embryo, scale bars = 50 µm (Reprinted
from Developmental Biology, vol 281,
Fouladi-Nashta et al.
LIF Activation stimulates Heparin Binding Epidermal Growth Factor
Autocrine versus juxtacrine signaling modes. In the simplest model, autocrine signaling is
regulated by the removal of the prepro-extension from the membrane-anchored ligand (step
1) followed by its controlled release from the membrane (step 2). Orientation restrictions are
responsible for the release requirement. In the case of juxtacrine signaling, prepro-extension
release is required (step 1), followed by binding to an auxiliary molecule on a neighboring
cell (step 2). Note that autocrine ligands bind to the cell that produced them, and juxtacrine
ligands bind to a neighboring cell.
Presence of HB-EGF Receptors on Activated Blastocysts
Activation of Implantation by LIF induces HB-EGF in LE of Endometrium
Amphiregulin
HB-EGF
Epiregulin
Immunofluorescence staining for Cox-2 and oncostatin M (OsM) in LIF-null and wild-type
mouse uterus on days 5 and 6 of pregnancy. Cox-2 protein is strongly expressed in the LE and
underlying stromal cells (arrows) at the implantation site on day 5 and expression extended
deeper into the stroma by day 6. In LIF-null mice, expression was limited to the LE cells and
only a few stromal cells expressed Cox-2 in the day-6 uterus. The pattern of expression for
OsM protein in wild-type mice was similar to Cox-2. In LIF-null animals, OsM was
completely absent around the embryo on days 5 and 6 of pregnancy. E = embryo, scale bar =
100µm (Reprinted from Developmental Biology, vol 281, Fouladi-Nashta et al.
Wang and Dey, 2006 Nature Reviews 7:185-199
Kimber (2005) Reproduction 130:131-145
Cross et al. 1994
Science 266:1508
At day 4.5 of mouse
development, the
blastocyst attaches to
the uterine epithelium
and uterine lumenal
closure occurs. A surge
of estrogen from ovary
stimulates implantation.
Estrogen releases the
cytokines such as LIF,
IL-1, HB-EGF and
CSF-1. Adhesion
molecules induce
binding through lectincarborhydrate and
integrin-integrin
interactions
Cross et al. 1994
Science 266:1508
Preimplantation
Implantation
Sherman et al. 1979, In: Matermal Recogniton of Pregnancy, Cibia foundation symposium 64, pp 33-52
Trophoblast cells begin to penetrate epithelium
Decidual cells form
Implantation
Chamber
Invasion Phase
The uterine epithelium degenerates
(apoptosis) and trophoblast cells
come into contact with decidua
Decidualization - uterine stroma undergoes
a transformation, fibroblasts proliferate
and enlarge stromal area
Postimplantation Phase
Trophoblast cells lose their
invasiveness
Compartive Placentation (ed. D.H. Steven), 1975
Mouse blastocyst
implantation Day 7 p.c.
Decidual
Reaction
Note: Position and
orientation of blastocyst.
Surface epithelium is intact
Blastocyst
Mouse blastocyst
implantation Day 8 p.c.
Note: Blastocyst is inside
stroma with loss of
uterine epithelium
Uterine
Lumen
Perry, 1981; J. Reprod. Fert. 62:321
Tachi and Tachi, 1986, In Nidation (ed. Yoshinaga) pp158-182
Day 5 blastocyst implantation
Tung et al. Biol Reprod 35:1045
Tachi and Tachi, 1986, In Nidation (ed. Yoshinaga) pp158-182
Rat Implantation site on Day 7 p.c.
Luminal epith
Collagen Fibers
Decidual cells
Tung et al. Biol Reprod 35:1045
Capillary
Blastocyst
Implantation
Chamber
Enders and Schlafke, 1979, In: Matermal Recogniton of Pregnancy, Cibia foundation symposium 64, pp 3-32
Tachi and Tachi, 1986, In Nidation (ed. Yoshinaga) pp158-182
Tung et al. Biol Reprod 35:1045
Restriction of dye from the primary decidual zone
Tung et al. Biol Reprod 35:1045
Salamonsen, 1999;Rev. Reprod. 4:11-22
Role of Metalloproteinases and Tissue Inhibitor of Metalloproteinases
Urokinase-type
plasminogen activator
Plasminogen
MMPs can be regulated
by steroids- P4 negative role
Cytokines - IL-1, TNF-a
MMP secreted in their
latent form and most
be cleaved to be activated
Tissue inhibitor of metalloproteinases
a-macroglobulin
Salamonsen, 1999;Rev. Reprod. 4:11-22
Development of Fetal Membranes in the Rat
Development of the embryo and fetal membranes in the rat is one of space
conservation. The conservation of space is achieved through inversion of the
germ layers which results in the yolk sac surrounding the embryo.
Polar
Trophoblast
Mural
Trophoblast
After loss of the zona (D 4-5) the rat blastocyst attaches eccentrically in a
groove on the antimesometrial aspect of the uterine wall. The polar trophoblast
cells continue to divide to become the ectoplacental cone; these cells engulf and
digest maternal erythrocytes. Mural trophoblast cells do not divide, but their
DNA content rises as they become transformed into primary giant cells
Compartive Placentation (ed. D.H. Steven), 1975
Parietal endoderm
Capillary channels
in the decidual tissue
enter a sinus-like
space between the
trophoblast and
Reichert’s membrane.
Because of inversion
of germ layers, the
Chorion does not
surround the embryo.
Viseral endoderm
The ICM cells continue to increase in number
and are pushed down into the blastocoele so that
the endoderm surrounds the ectoderm cells
(layer inversion) which forms the primary
embryonic cavity. A extra-embryonic membrane
called Reichert’s membrane appears on Day 6.
It first appears as a amorphous secretion along
the inner wall of the trophoblast. It lies between
the Trophoblastic giant cells and parietal endoderm.
Compartive Placentation (ed. D.H. Steven), 1975
Compartive Placentation (ed. D.H. Steven), 1975
Compartive Placentation (ed. D.H. Steven), 1975
Perry, 1981; J. Reprod. Fert. 62:321
Day 7.5 Mouse Embryo
Decidual response has
occurred.
Uterine epithelium have
undergone apoptosis.
Transformation and
growth of stroma cells
into decidual cells.
Trophoblast giant cells
invade the decidua.
Invasion regulated by
MMP’s and TIMP’s as
well as integrins
Cross et al. 1994
Science 266:1508
Compartive Placentation (ed. D.H. Steven), 1975
The hemochorial placenta is formed from the ectoplacental cone with its
lacunae of maternal blood, in relation to the chorionic ectoderm.
Compartive Placentation (ed. D.H. Steven), 1975
Day 9.5 Mouse Embryo
Chorioallantoic Placenta
Allantois fuses with the
chorion.
Labyrinthine layer
develops in which there
is extensive
intermingling of
maternal blood and fetal
blood vessels.
Decidua has largely
regressed and placental
development complete.
Cross et al. 1994
Science 266:1508
Stimulus of Implantation
1. The luminal epithelial cells initially differentiate so that their free surface
becomes organized is such a way to allow attachment of the trophoblast.
2. During attachment phase, the apposing luminal surfaces of the endometrium
come in close contact in all parts of the uterus. Stimulated by estrogen.
3. The blastocyst attachment is antimesometrial with the inner cell mass oriented
mesometrially.
4. The blastocyst invades through the uterine epithelium by phagocytosis of the
uterine epithelium which has undergone apoptosis. Caused by estrogen not
embryo.
5. During invasion process the stroma surrounding the blastocyst has undergone
decidualization. Blood vessels in the stroma become more permeable leading to
edema with fibroblast cells undergoing waves of cell division with formation
of gap junctions between cells.
Function of Decidual Cells
1. Barrier against possible immunological rejection of fetus.
2. Protection of the uterus from ravages of the invading trophoblast.
3. Nutritive role until formation of allantois.
4. Endocrine function - estrogen, prolactin, placental lactogen
5. Maternal recognition of pregnancy