Wang and Dey, 2006 Nature Reviews 7:185-199

Wang and Dey, 2006 Nature Reviews 7:185-199

Review


Lecture X : Comparative Implantation

Implantation

Rodents (Mouse)Contact of the blastocyst with the uterus is the first step in therelationship of a mammal embryo with its environment. Thiscontact leads to implantation and placentation.

Implantation can be divided into five separate phases:

1. Hatching Phase -

2. Apposition Phase -

3. Adhesion Phase -

4. Invasion Phase -

5. Postimplantation Phase -

Blastocyst loss of Zona pellucida

Hatched blastocyst aligns and orients itself in preparation forattachment

Attachment between uterine surface epithelium and trophoblast

After blastocyst becomes firmly anchored to the uterine surface, the trophoblast cells begin to penetrate the uterine epithelial layer

Trophoblast cells cease invasive activity and thematernal-fetal relationship is stablized

Mesometrial

Endometrium

Uterine epithelium

Zona pellucidaICMTrophectoderm

Antmesometrial

Blastocyst enters uteruson Day 4

Hatching from the Zona Pellucida

Compartive Placentation (ed. D.H. Steven), 1975

Apposition PhaseAttachment Phase

Blastocyst comes to lie along theantimesometrial 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 embryonicpole. Treatment with Relaxin can disruptthe orientation of the blastocysts.

Note the closureof the lumen

Decidualization begins followingadhesion in rodents. Surface changes in the uterine epitheliumpermit adhesion betweentrophectoderm and uterine surfaceepithelium

There is extracellularmaterial between

uterine epithelium and trophectoderm


Receptivity of Mouse Uterus to Blastocyst Attachment

Mucins are large O-linked glycoproteins with ectodomains that extend awayfrom the surface of the cell apical border.

Mucins may sterically block access to the cell membrane and function asanti-adhesive molecules.

Muc-1 also referred to as epsialin, may function in the time of blastocystattachment in mammals.

Muc-1 is lost from the uterine epithelial apical surface during diestrousin the mouse. It is expressed only on the apical border during the first3 days of the estrous cycle.

Surveyor et al. 1995, Endocrinology 136:3639

Western blot of Muc-1

Muc-1 expressionMuc-1 antibody (green)Perlecan antibody (red)

Basement membrane

Lumenal Epithelium

Glandular Epithelium

Muc-1 immunocytochemicalstaining

A. ProestrousB. EstrousC. MetestrousD. Diestrous

Only in glandularepithelium, loss ofintensity in surface


A, B, and C = Days 1, 2 and 3 pregnancyD = Day 4

NorthernBlot

Note: Loss of Muc-1 is associatedwith time of implantation.


Northern Blot

Lane 1 = Day 1 pregnancyLane 2 = Day 4 pregnancyDay 4 of pregnancy mice ovariectomized and given either P4 (lane 3) or E plus P4 (lane 4).

Estrogen stimulates Muc-1expression.Progesterone inhibits Muc-1expression.


Model for Muc-1 function as anantiadhesion moleucle

Role of epithelial surface mucins in steric inhibition ofembryo-endometrial interactions

Aplin, 1997; Rev. Repod. 2:84-93

Lipid HydrolysisProstaglandin Synthesis

Increased Membrane Permeability

Moulton and Koenig, 1986, In Nidation (ed. Yoshinaga) pp95-109

Interaction of trophoectoderm and endometrial cells at implantation.

v integrinthrough a bridging

ligand

Heparan sulfatebinding to a basic

protein. Also possiblyChondroitin sulfate

Cadherinbinding

Aplin, 1997; Rev. Repod. 2:84-93

Heparin Sulfate Proteoglycans

Integrins has 17 subunits has 8 subunits

Endocannabinoid Signaling

Endocannabinoids (Marijuana) target receptors (CB1 and CB2) in the mouse embryo.

CB1 localized in trophectoderm and not ICMCB1 in oviduct and uterusKnockout 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.

Women with elevated peripheral AEA levels have 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

Leukemia Inhibitor Factor has been demonstrated to be involved withimplantation in the mouse.

IL-6 willbind to LIFras well

Colony StimulatingFactor (CSF-1)has been shown to be involved with implantation too!

LIF stimulates proteinaseexpression in the uterus.Like - MMPs urokinase type plasminogen activator. LIF not present in delayed implantation

LIF appears following estrogenin delayed implantation

Histological features of the uterus in LIF-null 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.

Kimber (2005) Reproduction 130:131-145

Cross et al. 1994Science 266:1508

At day 4.5 of mousedevelopment, the blastocyst attaches to the uterine epithelium and uterine lumenalclosure occurs. A surgeof estrogen from ovarystimulates implantation.Estrogen releases thecytokines such as LIF,IL-1, HB-EGF andCSF-1. Adhesion molecules inducebinding through lectin-carborhydrate and integrin-integrininteractions


PreimplantationImplantation

Sherman et al. 1979, In: Matermal Recogniton of Pregnancy, Cibia foundation symposium 64, pp 33-52

Invasion Phase

Postimplantation PhaseThe uterine epithelium degenerates(apoptosis) and trophoblast cellscome into contact with decidua Trophoblast cells lose their

invasiveness

ImplantationChamberDecidual cells form

Trophoblast cells begin to penetrate epithelium

Decidualization - uterine stroma undergoesa transformation, fibroblasts proliferateand enlarge stromal area


Perry, 1981; J. Reprod. Fert. 62:321

Mouse blastocystimplantation Day 7 p.c.

DecidualReaction

Note: Position andorientation of blastocyst.Surface epithelium is intact

Mouse blastocyst implantation Day 8 p.c.

Note: Blastocyst is inside stroma with loss of uterine epithelium

Blastocyst

UterineLumen

Tachi and Tachi, 1986, In Nidation (ed. Yoshinaga) pp158-182

Tung et al. Biol Reprod 35:1045

Day 5 blastocyst implantation


Rat Implantation site on Day 7 p.c.

Capillary

Luminal epithCollagen Fibers

Decidual cells

Blastocyst

ImplantationChamber

Enders and Schlafke, 1979, In: Matermal Recogniton of Pregnancy, Cibia foundation symposium 64, pp 3-32


Restriction of dye from the primary decidual zone

Salamonsen, 1999;Rev. Reprod. 4:11-22

Role of Metalloproteinases and Tissue Inhibitor of Metalloproteinases


MMPs can be regulatedby steroids- P4 negative roleCytokines - IL-1, TNF-MMP secreted in theirlatent form and mostbe cleaved to be activated

Tissue inhibitor of metalloproteinases-macroglobulin

Urokinase-typeplasminogen activatorPlasminogen

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 thegerm layers which results in the yolk sac surrounding the embryo.

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 trophoblastcells continue to divide to become the ectoplacental cone; these cells engulf and digest maternal erythrocytes. Mural trophoblast cells do not divide, but theirDNA content rises as they become transformed into primary giant cells

Mural Trophoblast

Polar Trophoblast


The ICM cells continue to increase in numberand are pushed down into the blastocoele so thatthe endoderm surrounds the ectoderm cells(layer inversion) which forms the primary embryonic cavity. A extra-embryonic membranecalled Reichert’s membrane appears on Day 6.It first appears as a amorphous secretion alongthe inner wall of the trophoblast. It lies betweenthe Trophoblastic giant cells and parietal endoderm.

Viseral endoderm

Parietal endoderm

Capillary channelsin the decidual tissueenter a sinus-like space between the trophoblast and Reichert’s membrane.Because of inversion of germ layers, the Chorion does not surround the embryo.


Perry, 1981; J. Reprod. Fert. 62:321

Day 7.5 Mouse Embryo

Decidual response hasoccurred.Uterine epithelium haveundergone apoptosis.Transformation and growth of stroma cells into decidual cells.Trophoblast giant cellsinvade the decidua.Invasion regulated byMMP’s and TIMP’s aswell as integrins


The hemochorial placenta is formed from the ectoplacental cone with itslacunae of maternal blood, in relation to the chorionic ectoderm.


Day 9.5 Mouse Embryo

Chorioallantoic PlacentaAllantois fuses with the chorion.

Labyrinthine layer develops in which thereis extensiveintermingling of maternal blood and fetal blood vessels.

Decidua has largely regressed and placentaldevelopment complete.


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

Wang and Dey, 2006 Nature Reviews 7:185-199

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