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Molecular Mechanism of Implantation

Michiko N. Fukuda

ABSTRACT

Implantation following placentation is a unique system for mammals to reproduce. The initial
attachment of the embryo to the uterus occurs via the apical cell membranes of two epithelial cells,
trophoblast of the blastocyst and surface epithelial cells of the endometrium. Recently, a novel cell
adhesion molecule complex, trophinin and tastin, potentially mediating the initial attachment of
trophoblast to the endometrial epithelium was discovered (Fukuda, M. N., Sato, T., Nakayama, J.,
Klier, G., Mikami, M., Aoki, D., Nozawa, S., 1995, Genes & Development, 9, 1199-1210). This review
provides a brief overview of cell adhesion molecules involved in implantation and introduces
identification and characterization of trophinin and tastin.

KEY WORDS
Ovum implantation, Cytoskeleton, Molecular cloning, Cell adhesion molecule, Embryo

INTRODUCTION

Implantation is a unique system for mammals to reproduce. Studies on implantation at subcellular

and molecular levels have been difficult due to the limitation of the materials to be analyzed.
Recently a major break through was made in this area that is a discovery of trophinin and tastin.
Availability of genetic probe of these new molecules in-volved in implantation enable us to analyze
further the molecular mechanism underlying implantation, an investigation that may also shed light
on the process of metastasis and invasion of tumor cells.

EMBRYO IMPLANTATION: A CELL BIOLOGICAL PARADOX

A fertilized egg undergoes development from single cell stage to blastocyst. The blastocyst is
composed of outer cellular layer and inner cell mass. Inner cell mass is made of the embryonic stem
cells. Embryonic stem cells are pluripotent, are able to differentiate into a variety of cell types, and
becomes a baby. Outer cellular layer of the blastocyst is composed of trophoblasts, adheres to the
surface of endometrium, and becomes placenta.

The early events of implantation are an initial apposition of the trophoblast to the uterus and
subsequent adhesion and penetration of trophoblast into uterine epi-thelium1). The initial attachment
of the trophoblast to the endometrial epithelium is described as "a cell biological paradox"2) since
this cell-to-cell contact occurs via the apical cell membranes of two epithelial cells, trophoblast and
endometrial epithelial cells. In general, the apical surface of the epithelium is not adhesive, whereas
the basolateral surfaces support adhesion. One hypothesis to explain such cell adhesion is that the
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apical surfaces of the trophectoderm and the uterine epithelium express a "basolateral" character
that promotes adhesion between these two cell types 3,4,5). Alternatively, the apical plasma
membranes of the trophoblast and endometrial epithelial might express unique cell adhesion
molecule(s) that mediate an initial attachment between these cells 6).

SEARCH FOR CELL ADHESION MOLECULES INVOLVED IN IMPLANTATION

Attempts to identify molecules involved in embryo implantation have been conducted both in vivo
and in vitro3,4,5,7,8,9,10,11,12). By using specific antibodies to the cell adhesion molecules such as
cadherins and integrins, trophoblasts and endometrial epithelial cells at the time of implantation
were investigated4,5,7,8,9,10). For example, when the blastocyst attached to the uterine epithelium, E-
and P-cadherins were detected at the cell-cell boundaries but not at the apical surface of either
epithelia, suggesting that the initial adhesion of trophoblast cells to the uterine epithelium is
mediated by molecules other than E- and P-cad-herins 7,8,9). These studies also suggested that
disappearance of cad herins is linked to the acquisition of trophoblast invasiveness 13). An integrin
such as 1-integrin is another molecule thought to be involved in implantation. However, recent
report of 1-integrin gene knock-out mouse revealed that 1 -integrin is not necessary for
implantation itself, but both inner cell mass and trophoblasts of 1-integrin null blastocysts
degenerated about one day after implantation14).

Several lines of evidence indicate that changes in the state of uterine receptivity correlate with
changes in cell surface carbohydrate structures. These studies revealed that subtle modifications of
lactosaminoglycan carbohydrates 12), sulfated glycolipids 15), fucosylated carbohydrates 12,16) and
heparan sulfate and heparan sulfate binding protein3) are related to the hormonally induced
preparative and receptive phases of uterine epithelium. These studies, however, have not identified
the cell adhesion molecule unique to embryo implantation.

HT-H AND SNG-M CELL LINES MAY SERVE AS AN IN VITRO MODEL FOR IMPLANTATION

Cell lines established from mouse teratocardnomas have been used for an in vitro model for early
embryogenesis, as these cell lines often exhibit cells at early embryos. HT-H cells established from
testicular teratocarcinoma form syncytia in vitro and synthesize and secrete progesterone and
hCG17). They also exhibit phagocytic activity and express a colony-stimulating factor-1 receptor (c-
fms product). HT-H cells express cytokeratin, but not vimentin, indicative of the epithelial nature of
this cell line. These properties of HT-H cells suggested that HT-H cell line represents
syncytiotrophoblasts at early embryonic stage.

SNG-M cell line has been established from a metastatic region of endometrial adenocarcinoma 8).
Various properties suggested that this cell line represents endometrial epithelial cells. When HT-H
cells were added to a monolayer of SNG-M cells, HT-H cells immediately adhered to the upper
surface of SNG-M cells 22). Electron micrographs of the contact site between HT-H and SNG-M cells
showed numerous microvilli on both cell types (Figure 1 A & B). After co-culturing for 6 hours, the
microvilli flatten (Figure 1C), and after 4 days co-culturing, formation of junctional complexes
consisting of desmosomes was seen which is indicative of stronger adhesion between these two cell
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types (Figure 1-D). These morphological features resemble those that have been reported in the
literatures for embryo implantation site19), suggesting that the Hr-H and SNG-M cells are useful
models for analyzing adhesive interaction between trophoblast and endometrial epithelial cells at the
time of implantation.

IDENTIFICATION OF THE MOLECULES RESPONSIBLE FOR THE ADHESION BETWEEN THE

HT-H AND SNG-M CELLS BY EXPRESSION cDNA CLONING20)

Molecules responsible for the initial attachment of HT-H cells to a monolayer of SNG-M cells were
identified by expression cDNA cloning, as illustrated in Figure 2. Two clones were identified; one is
named "trophinin" and the other named "tastin".

Trophinin is predicted to be an intrinsic membrane protein composed of 749 amino acids. The amino
terminal region is predicted to form a cytoplasmic domain. The most striking structural feature of
trophinin is that more than 90% of this molecule is made of tandem repeats of a decapeptide.
Trophinin's decapeptide repeats is predicted to form repeated -turn, which may be the structural
element of self-binding. Antibodies raised against the predicted cell surface domains of trophinin
inhibited the adhesion between HT-H and SNG-M cells. When trophinin polypeptides are added to
trophinin expressing cells, such as HT-H and SNG-M cells, trophinin peptides adhered to the surface
of these cells.

Tastin is predicted to be a cytoplasmic protein made of 778 amino acids. When trophinin was
expressed sigly, they were seen as a regularly distributed antigen on the cell surface. In contrast,
when trophinin and tastin are expressed together in COS-1 cells, trophinin are seen as clustered
patches on the cell surface. Thus tastin appears to induce clustering of trophinin. Association of
trophinin with tastin would restrict the distribution of trophinins, creating multivalent and efficient
cell adhesion sites on the plasma membranes.

POTENTIAL INVOLVEMENT OF TROPHININ AND TASTIN IN EMBRYO IMPLANTATION20)

Immunohistology of various human tissues revealed that neither trophinin nor tastin are expressed in
normal adult human cells except macrophages. In human en-dometrium, trophinin and tastin were
not detected in the proliferation stage, at the ovulation stage and late secretary phases. Trophinin is
strongly expressed on the apical plasma membranes of the surface epithelium at an early secretory
phase (Figure 3). Tatsin was also detected in the endometrial epithelial cells where trophinin was
expressed. These observations suggest that trophinin and tastin are expressed in surface epithelia at
the time of the "implantation window"20).

Strong expression of trophinin was detected at the apical plasma membranes of trophectoderm of
monkey blastocysts (Figure 4). Trophinins are elevated at the embryonic pole and are relatively
weakly expressed at the mural pole. Such polarized distribution of trophinin is consistent with the
observations that, in both primates and humans, a blastocyst attaches to endometrial epithelium at
its embryonic pole1,2,19). The specific and restricted in vivo expression of trophinin and tastin in the
cells involved in implantation strongly suggests that trophinin and tastin are the molecules mediating
the initial attachment of embryo to the uterus.
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FUTURE PROSPECT OF THE RESEARCH OF EMBRYO IMPLANTATION

The unique cell adhesion process of the trophoblast to endometrium epithelia at the time of
implantation and subsequent invasion of the trophoblast into the maternal tissue forms an essential
element of embryo implantation. This process is often compared to metastasis and invasion of
malignant tumors. Therefore, understanding the mechanisms underlying the adhesive cell-to-cell
interactions between trophoblast and endometrial epithelial cells will allow us to better understand
both implantation and tumor metastasis and invasion.

ACKNOWLEDGEMENTS

This article is based on the lecture that was given on September 6th 1995 at Keio University School of
Medi-cine, hosted by Professor Shiro Nozawa, Department of Gynecology and Obstetrics. Detailed
version has been given as a review in Keio Journal of Medicine, 45 (1) 37-43, 1996.

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Received on November 5, 1996 and accepted on February 25, 1997

The Burnham Institute
La Jolla, California 92037, U.S.A.

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