Conceptus implantation and placentation: molecules related to epithelial–mesenchymal transition, lymphocyte homing, endogenous retroviruses, and exosomes

Abstract

Processes of conceptus implantation and placentation, unique to mammalian reproduction, have been extensively studied. It was once thought that processes of these events varied greatly, notably between invasive and noninvasive modes of implantation and/or placentation. Regardless of the mode of implantation, however, physiological and biochemical processes in conceptus implantation to the maternal endometrium including the kinds of gene expression and their products are now considered not to differ so much. Recent progress has identified that in addition to the hormones, cytokines, proteases and cell adhesion molecules classically characterized, epithelial–mesenchymal transition, molecules related to lymphocyte homing, the expression of endogenous retroviruses and possibly exosomes are all required for the progression of conceptus implantation to placentation. In this review, therefore, new findings related to these events are integrated into the context of conceptus implantation to the maternal endometrium.

Introduction

The uterine structures in mammalian species as we know them today are the product of a long and complex evolutionary process. A novel characteristic of mammalian reproduction is that fertilization and embryonic development proceed within the oviduct and uterus, respectively [1]. These structures must then provide an adequate environment for fertilization and embryonic growth; however, this process presents new challenges, most immediately the attachment of two epithelial layers between uterine epithelium and outer embryonic/conceptus membrane cells, trophectoderm (TE) cells. This interaction is also immunogenically complex because the conceptus carries paternal genes allogeneic to the maternal immune system. As these events proceed, the outer layer of TE cells plays a major role in the attachment and invasion to the uterine endometrium, and in the formation of placenta. All placentas with both conceptus and maternal cell structures assume the responsibility of supporting and nourishing the conceptus. However, extensive variation in TE cell types and placental structures exists across different mammalian species. In this review, new information on conceptus implantation, processes and its gene regulation, to the maternal endometrium and events proceeding to placental formation will be integrated.

Processes of implantation

It is generally accepted that there are five phases of conceptus implantation to the maternal endometrium, preceding placentation [2]. Phase 1, migration/hatching; the blastocyst/conceptus migrates and sheds from zona pellucida (ZP) in the uterus. During this phase, the blastocyst/conceptus enters and migrates within the uterus and hatching allows the expansion of the blastocyst to spherical shape, or it may migrate and change in its shape from spherical to tubular and filamentous form, as in domestic animals. Phase 2, pre‐contact; the blastocyst/conceptus reorients to be apposed to appropriate regions of the uterine lining. During this phase, the blastocyst/conceptus migrates or elongates without definitive contact between the TE cells and endometrial epithelium. In domestic animals, this is the period when the process of maternal recognition of pregnancy is initiated for the prevention of corpus luteum (CL) demise, resulting from biochemical communication between the developing conceptus and mother. Phase 3, attachment; TE cells of the blastocyst/conceptus attach to the uterine epithelium. During this phase, the outer TE cells of blastocyst/conceptus establish definitive contact with the uterine epithelium. Phase 4, adhesion; TE cells attach firmly to the uterine epithelium. In some cases, superficial glandular epithelium, during which mononucleate TE cells differentiate into binucleate and/or multinucleate syncytiotrophoblast cells. Phase 5, invasion; the blastocyst/conceptus invades the uterine endometrium. This phase is when many mammalian species begin to diverge greatly in their development as invasive TE cells cause the formation of decidualized endometrium, whereas noninvasive does not. For the first four phases, however, implantation processes among mammalian species appear fairly similar in cell–cell interactions and gene expression associated with these events [2].

Maternal recognition of pregnancy

In mammalian species, the continued secretion of a steroid hormone, progesterone (P4), by functional CL is a prerequisite for the establishment and continuation of pregnancy. It has been well established that P4 is involved directly and/or indirectly in various gene expressions in utero, which regulate numerous uterine functions through endometrial secretions, alteration of blood flow at implantation sites and promotion of physiological and/or immune environments suitable for normal embryonic development. Despite similar requirements, the biochemical as well as molecular mechanisms by which CL is maintained for continued P4 production differ from species to species. In higher primates including humans, CL is maintained by a luteotrophic factor, chorionic gonadotropin (CG), produced by the TE cells as they face and invade the uterine epithelium during implantation [3]. In rodents, CL is prolonged through the release of copulation‐induced pituitary prolactin surges [4]. Whichever the molecules associated with the maintenance of CL life span, they must be produced long before CL regression begins, and the period during which CL is protected from a luteolytic signal is known as the period of maternal recognition of pregnancy [5]. During such period, trophoblast and uterine endometrium under the influence of P4 must communicate with each other, resulting in the establishment of the proper uterine environment necessary for conceptus survival, implantation, and subsequent placental formation. It should be noted, however, that as the administration of human CG (hCG) does not prevent CL regression in non‐pregnant women, hCG may not be the only factor maintaining P4 production [6]. However, to date, no factors other than hCG have been definitively identified for CL maintenance in humans.

In the ruminant ungulates including cows, sheep and goats [5], interferon tau (IFNT), a major cytokine produced by TE cells during the peri‐implantation period, is the anti‐luteolytic factor essential for the prolongation of CL life span [7, 8, 9, 10, 11]. IFNT exhibits structural and functional similarities to those of type I IFNs such as IFNA and IFNB [12]. These include antiviral and anti‐proliferative activities, but IFNT shows much less cytotoxic activity than do IFNA or IFNB [13, 14, 15, 16, 17]. Type I IFNs are known to bind to a common receptor complex with two polypeptide subunits (IFNAR1 and IFNAR2) [18], both of which are present in ovine uterine epithelial cells [19]. It has been thought that the luminal epithelium of the uterine endometrium is the primary target for IFNT [11, 20]. Identification of the receptor or receptor subunits suggests that IFNT can reach the stroma, and even the uterine myometrium [21, 22, 23]. Indications are that IFNT likely reaches the circulating immune cells and the ovaries as well [24]. Upon binding to the receptor, type I IFNs activate the Janus kinase‐signal transducer and activator of transcription‐interferon regulatory factor (JAK‐STAT‐IRF) signaling pathway [25, 26], causing the activation of a group of interferon‐stimulated genes (ISGs) [27, 28]. In addition to ISGs, wingless‐type MMTV integration site family (WNTs) and LGALS gene expression [29, 30], IFNT induces several chemokines in endometrial tissues including chemokine ligand 10 (CXCL10) and CXCL9 [31, 32]. Endometrial CXCL10 in turn attracts immune cells, particularly NK cells, to the caruncular regions of the endometrium [33], and by acting through the CXCL10 receptor, CXCR3, this chemokine regulates TE cell migration and integrin expressions [32]. Together with P4, IFNT regulates endometrial gene expression necessary for the establishment of the proper uterine environment during the implantation period [28]. These changes result in conceptus migration, apposition and initial attachment to the uterine epithelial cells in mammals including ruminant species [33, 34] (Fig. 1).

Figure 1.

Processes of conceptus implantation to the maternal endometrium; hatching, attachment, invasion, and syncytialization or cell fusion. During the implantation period, molecules related to lymphocyte homing and EMT are used in both human and bovine species. Upper human implantation. Cell structures of cytotrophoblast and syncytiotrophoblast are shown in the black box. Lower Bovine (cattle) implantation. Unique to the ruminant ungulates, the trophoblast after hatching elongates (conceptus) and the elongated conceptus attachment to the maternal luminal epithelium occurs, but the TE cell invasion is kept minimal. During conceptus elongation, binucleate cells form in the TE cell layer, which take on nuclei from the epithelial cells of maternal endometrium, resulting in the formation of trinucleate cells

Use of lymphocyte homing molecules in conceptus attachment

It is well documented that cell–cell interactions and integrin (ITG)‐mediated signaling between the conceptus and endometrium are critical for successful implantation in humans and murine species [35, 36]. Specifically, the extracellular domain of ITGs acts as a receptor for extra‐cellular matrix components (ECMs) such as fibronectin, vitronectin, laminin, collagen‐type IV, and osteopontin (SPP) [37, 38]. In goats, sheep, and cattle, constituents of uterine histotroph such as IP‐10 (CXCL10), galactoside‐binding soluble 15 (LGALS 15), and insulin‐like growth factor binding protein (IGFBP)‐1 have been characterized to activate ITGs through their RGD domain during the period of TE cell attachment to the uterine epithelium [35, 36, 37]. In the bovine species, the expression of ITGs has been characterized at the uteroplacental interface during the periods of TE attachment [38, 39] and placentation [40]. During the stages of bovine TE binucleate cell migration and fusion with the uterine epithelial cells, five α subunits (ITGA2B, ITGA3, ITGA5, ITGA8 and ITGAV) and two β subunits (ITGB1 and ITGB3) have been characterized [40]. In the previous investigation [41], we found that integrin subunits α (ITGAV, ITGA5) and β (ITGB1, ITGB3 and ITGB5) are constitutively expressed in bovine peri‐attachment TE cells, whereas the expression of ITGA4 and ITGA8 is induced after attachment of TE cells to uterine epithelial cells is initiated.

Vascular cell adhesion molecule (VCAM‐1), a trans‐membrane glycoprotein member of the immunoglobulin gene superfamily [42], is well known as a cell adhesion mediator during the process of lymphocyte homing [43], angiogenesis [44] and allantoic membrane fusion to the chorion [45]. In VCAM‐1 gene ablation study [45], the allantois fails to fuse with the chorion, resulting in abnormal placental development and embryonic losses at 9.5–11.5 days of gestation, although a minority of VCAM‐1‐deficient mice with abnormal distribution of allantoic mesoderm over the chorionic surface survives. In humans, VCAM‐1 is present on the endometrial side, specifically localized on decidual stromal cells in the areas where migrating TE cells are present, but not on vascular endothelial cells in decidua parietalis. Endometrial expression of VCAM‐1 at the peri‐implantation stage of patients with unexplained infertility was significantly lower than in control patients [46], suggesting that the expression of VCAM‐1 might be essential for the preparation of the endometrium for invasive mode of implantation. In the study of early pregnancy in sheep, VCAM‐1 is first found in endothelial cells on days 17–19 in both caruncular and intercaruncular areas of the endometrium, and becomes strongly induced in endothelial cells on days 26–27 [47].

VCAM‐1, induced by various cytokines in different tissues or organs in mice [48], functions through integrin α4β1 (ITGA4/ITGB1), also known as very late antigen‐4 (VLA4) [49]. Homozygous loss of ITGB1 expression in mice was lethal during early post‐implantation development, resulting in inner cell mass failure [50]. It was also identified that homozygous ITGA4 null knockout mice fail to complete fusion of the allantois with the chorionic membrane during placentation period [51], the cellular event similar to that of VCAM‐1 gene ablation. In our previous investigation on bovine conceptuses, ITGA4 mRNA was found at elevated expression levels on day 22, 2–3 days after the initiation of trophoblast attachment to the endometrial epithelium [41, 52]. We also found that changes in TE cells’ gene expression including ITGs were seen when bovine TE (CT‐1) cells were cocultured with endometrial epithelial cells (EECs), which was further enhanced with the addition of uterine flushings from pregnant animals [52, 53]. These results suggest that components of uterine flushings/histotroph including ECMs and/or various cytokines, as well as cell–cell interactions are important in the progression of conceptus attachment to the uterine epithelium in the bovine and other mammalian species.

Epithelial and mesenchymal transition

The outer layer of TE cells possesses epithelial characteristics, including apicobasal cell polarity, lateral junctions with neighboring cells and basal contact with the basement membrane proteins [54, 55, 56]. Despite the fact that the apical plasma membranes of simple epithelia normally lack adhesive properties, TE cells still manage to adhere to the uterine epithelium through its apical domains as part of the pre‐implantation process. Thus, the adhesion between TE cells and uterine epithelial cells has long been considered a cell biological paradox [57]. With the exception of rodents, in which the conceptus enters a receptive uterus and attaches immediately to the uterine epithelium, primates and most domestic animals have a prereceptive phase during which the conceptus does not physically interact with the uterine epithelium. In the bovine species, attachment between TE cells and endometrial epithelium is first seen on day 20 of gestation, and subsequent stable adhesion occurs between days 20 and 22 [58].

Another surprising finding was that changes in gene expression associated with the epithelial–mesenchymal transition (EMT) occurred not before attachment, but rather on day 22, 2–3 days after the initiation of conceptus attachment to the uterine epithelium [41]. Positive signals for both the epithelial marker cytokeratin and the mesenchymal marker vimentin were seen in the elongated TE on day 22. Increased transcripts of N‐cadherin, vimentin, matrix metalloproteinase 2 (MMP2), and MMP9 were also found on day 22, concurrent with E‐cadherin mRNA and protein down‐regulation [41]. These observations indicate that after the conceptus‐endometrium attachment, EMT‐related transcripts as well as cytokeratin are present in the bovine TE, and suggest that in addition to extracellular matrix expression, partial EMT is required for proper adhesion of elongated conceptus to the maternal endometrium.

In that study, we also identified that transcription factor SNAI2, ZEB1, ZEB2, TWIST1, TWIST2, and KLF8 transcripts were up‐regulated concurrent with cytokeratin expression in the TE cells [41]. It has been characterized that SNAIL, ZEB, and KLF8 factors bind to and repress E‐cadherin promoter activity [59, 60], whereas TWIST1 and TWIST2 repress E‐cadherin transcription indirectly [61]. In addition, SNAIL and ZEB factors are known to induce the expression of MMPs that can degrade the basement membrane, thereby favoring invasion [62]. Although the bovine conceptus does not penetrate into the endometrium, the confirmation that MMP2 and MMP9 transcripts are up‐regulated not only suggests that they play a role in noninvasive trophoblasts, but also confirms the similarity between invasive and noninvasive modes of implantation.

Placenta: structural diversity

Fertilized eggs differentiate into an inner cell mass (ICM) and an outer TE in the early development of the mammalian trophoblast. ICM develops into the embryo as well as the amnion, yolk sac and allantois whereas the TE forms chorionic membrane and later becomes a major part of the conceptus placenta. To receive nutrients and gases from the mother in utero, the conceptus forms the placenta; however, its cell types as well as structures vary considerably among mammalian species. Various cell types that form a barrier between the fetal and maternal blood in epitheliochorial placentation (pigs and horse) are: (1) the endothelium of the maternal capillary, (2) uterine endometrium (stroma and/or decidua), (3) the epithelial layer of the uterine endometrium, (4) the layer or layers of TE cells that make up the chorionic epithelium, (5) fetal connective tissues, and (6) the endothelium of the fetal capillary [63, 64, 65, 66]. In hemochorial placentation (rodents and primates), as maternal blood directly reaches the TE cells, only three layers exist between maternal‐fetal circulations. In any form of placentation, maternal nutrients and gases must traverse these cell layers to reach the fetus, and waste materials must be expelled back to the maternal circulatory system.

In an epitheliochorial placenta, the uterine epithelium is in direct contact with the chorionic TE cells. This type of placentation is found in several orders including even‐toed ungulates, whales and dolphins and lower primates. In an endotheliochorial placenta, the endothelium of the maternal capillaries is located close to the TE cells, resulting from stromal thinning and a loss of uterine epithelium. This type of placenta is seen in carnivores, but is also found in the distantly related elephants [67]. In hemochorial placentation, by contrast, maternal blood is directly in touch with the trophoblast, functioning without the capillary endothelium. This type of placentation is seen in many rodents and in higher primates including humans. In this type of placentation, the multinucleate TE, syncytiotrophoblast, serves the function of efficient nutrient and gas transfer. However, molecular mechanisms by which this cell type forms have not been definitively elucidated.

Endogenous retroviruses and pregnancy

Numerous analyses on mammalian genomic DNAs revealed that endogenous viral elements (EVEs) make up at least 45 and 40 % of human and mouse genomes, respectively (Human and mouse genomic sequencing consortium, Nature 2001, 2002). Among EVEs, endogenous retroviruses (ERVs) and long terminal repeat (LTR) retrotransposons make up 8 and 10 % of human and murine genomes, respectively. ERVs, as parts of an organism's genome, can potentially exhibit functions, although their nucleotide structures largely consist of deletions and/or mutations. In general, the genome of retroviruses contain gag, pro, pol, and env genes, and 5′‐ and 3′ LTRs, some of which are still active in their transcription and could code for proteins. Extensive studies of mammalian ERVs have provided insight into their env proteins, which enable ERV infection to the host cells through specific receptors and induce cell–cell fusion. In humans, 18 ERV‐env nucleotide structures have been identified as having actual protein expression, three of which possess fusogenic activity [68, 69].

Evidence has accumulated that ERVs are now realized as factors implicated in development and differentiation of TE cells in mammalian species such as humans, rodents, dogs/cats, rabbits, sheep, cattle, tenrecs and opossums [70, 71, 72, 73, 74, 75, 76, 77, 78]. During the course of evolution, all vertebrates have been exposed to multiple waves of cross‐species infection by exogenous retroviruses, some of which infected germ cells and have been inherited in an integrated, proviral form [79]. Despite this prevalence in the mammalian genome, these were once considered non‐functional junk DNAs. However, it is now realized that ERVs play biological roles in protection against retroviral infection [80] and in placental development [81, 82]. Recently, it was found that high levels of transcripts found in ES cells, most of which are expressed in two‐cell stage embryos, are induced by ERVs’ LTRs, suggesting the possibility that the foreign sequences have helped to drive cell‐fate regulation of early embryos in placental mammals [83].

Similar to malignant cells, TE cells possess the ability to invade non‐TE cells. If unchecked, therefore, TE cells have the potential to damage or destroy uterine structures, and this aggression must be regulated for the protection of uterine endometrium. When the cell cycles of TE cells are restricted, these cells go through endoreduplication, resulting in the formation of giant trophoblast cells in murine species. In other mammalian species, TE cells become multinucleate cells through cell fusion. These multinucleate cells do not go through cell cycles, and thereby their invasiveness is held under control [84]. For example, syncytin 1 and 2 are products of the two human ERV envelope (env) genes, and are involved in the fusion of trophoblast cells, resulting in multinucleated syncytiotrophoblast formation [70, 71]. It was determined that syncytin 2 entered the primate lineage more than 40 million years ago (MYA) while syncytin‐1 entered the lineage 25–40 MYA [71]. Under physiological conditions, syncytin 1 possesses stronger fusogenic activity than that of syncytin 2. In addition, syncytin 2, not syncytin 1, has immunosuppressive activity.

From murine genome analysis, two env genes, syncytin A and B, with fusogenic activity in vitro were found and are homologous to human syncytin‐1 and ‐2, respectively [70, 85]. There are three layers in mice trophoblasts, and syncytin A is found in the second layer, syncytiotrophoblast layer‐I (ST‐I) whereas syncytin B is localized in the third layer, syncytiotrophoblast layer‐II (ST‐II) [72]. Ablation of syncytin A results in the failure of ST‐1 formation, and these embryos die between days 11.5 and 13.5 of pregnancy. Because syncytin A exhibits fusogenic activity in Green monkey Vero and human 293T cells, it is also thought to be involved in trophoblast cell fusion. Similar to human syncytins, GCM1 functions as a transcription factor bound to the upstream region of syncytin A gene, regulating its gene transcription [86]. This is in agreement with the observation that GCM1 gene ablation precludes the development of labyrinth zone in mice placenta [87]. On the other hand, syncytin B gene ablation does not result in embryonic death, although ST‐II layer formation is insufficient and the number of pups born is smaller than that for the control mice possessing syncytin B function. In in vitro assay, syncytin B exhibits fusogenic activity only in canine MDBK cells; however, it possesses strong immunosuppressive activity [88]. These findings for syncytin B, found in murine trophoblasts, closely resemble those for syncytin 2 in human cytotrophoblasts.

Unlike in primates and rodents, syncytiotrophoblasts are not formed in TE cells of ruminant ungulates. However, bovine TE cells form binucleate cells (BNCs) as well as trinucleate cells (TNCs). While it has not been definitively determined whether BNCs result from cell fusion or endoreduplication, it is clear that TNCs are products of fusion between binucleate cells and uterine epithelial cells [89, 90, 91, 92]. In addition, TE cells of ruminants are not invasive, and thus do not penetrate deep into uterine stroma or spiral arteries; however, BNCs from bovine placenta possess one ERV, BERV‐K1 [91], with fusogenic activity [92]. It should be noted that multi‐nucleate and syncytiotrophoblast cell formation results from homologous cell fusions, however, TNC formation results from heterologous cell fusion between TE cells and maternal endometrial epithelia. TNCs are only located in the endometrium [93], suggesting that this cell fusion may strengthen the adhesion between conceptus and uterine endometrium at the placentomes. Similar to syncytiotrophoblasts of primates, these cells are in closest proximity to maternal immune cells, possibly suggesting that TNCs may play a role in the protection of the allogeneic embryo during the course of pregnancy.

Recently, a bovine ERV, BERV‐K1, with strong fusogenic activity was considered to be the main factor involved in TNC formation and was therefore named Fematrin‐1 [92]. It was also reported that syncytin‐Rum1 has been integrated into ruminant genomes, including cattle and sheep, and was possibly involved in fetomaternal cell–cell fusion in both species [77]. It should be noted however that Fematrin‐1 integrated into the bovine genome, but not in the sheep genome. Current hypothesis supports that syncytin‐Rum1 was integrated into ruminant genomes 20 MYA while Fematrin‐1 was integrated into the bovine genome 11 MYA [94]. As for bERVE‐A, its mRNA is found only in binucleate cells throughout the gestation period, and this gene contains syncytin 1‐like SU domain and ASCT2 binding domain; however, it does not possess fusogenic activity due to a loss of the fusion peptide [76]. Furthermore, the integration of BERV‐P was recently found in ruminants [95]. Nucleotide structures of BERV‐P closely resemble that of syncytin found in dogs/cats, syncytin‐Car1 [75]; however, BERV‐P does not possess fusogenic activity. Although several ERVs are found in ruminants, syncytin‐Rum1 and Fematrin‐1 are unique in possessing fusogenic activity. Integration of syncytin‐Rum1, followed by Fematrin‐1 into the bovine genome suggests that a two‐step process could be required for viral particle integration into the genome. First, unless viral activity is lost, viral genomes may not be endogenized into the genome. Second, if in fact, a part of integrated viral genes could be used by the host animals, it is possible that the host may utilize them for more efficient cellular and/or genomic functions (Fig. 2).

Figure 2.

Exosome production. At endosomes of various cell types, cytosolic proteins and nucleic acids are packaged into exosomes. Exosome‐filled cytosolic structures are either fused with plasma membrane to release their contents in the extracellular spaces or send their contents to lysosomes for degradation. Released exosomes are often used for intercellular communications, including cell–cell fusion

Exosomes promote TE cell fusion?

Three different mechanisms of cell–cell communication have been recognized: (a) by the use of soluble factors such as hormones, cytokines, chemokines, bioactive ions and lipids in an autocrine, paracrine or endocrine manner, (b) through direct adhesion contacts between cells such as receptor‐ligand signaling and trogocytosis, and (c) by shuttling of information through intercellular nanotubes [96, 97]. Recently, a fourth way of intercellular communication through release and uptake of extracellular membrane‐bound microvesicles (EMV) has been identified. This type of intercellular exchange relies on the transport of membranally bound molecules, containing cytosolic proteins and nucleic acids such as mRNA and microRNA (miRNA), and delivers information in the near vicinity and/or at a distance [98]. EMVs have been found in all living organisms, including plants, and produced by both prokaryotic and eukaryotic cells. Evidence has been accumulated that EMVs comprise a heterogeneous group of vesicles, and are present in blood and all kind of bodily fluids, including plasma and serum, ocular effluent and aqueous humor, cerebrospinal fluid, saliva, breast milk, synovial fluid, nasal and bronchial secretions, bile, urine, amniotic fluid, semen, and from pleural and peritoneal effusions in pathological conditions [98]. Although an official definition has not been finalized yet [99], the current definition of “exosome” among EMVs is as follows: (a) secreted membrane‐bound nanovesicles of endosomal origin, (b) size from 30 to 100 nm or approximately 150 nm, (c) cup‐shaped form, (d) tetraspanin‐, cholesterol‐ and sphingophospholipid‐rich detergent‐resistant membrane, and (e) buoyant density of 1.13–1.19 g/ml on sucrose gradient [100]. In addition, the specific pathway of biogenesis of exosomes separates them from all other known EMVs. Exosomes are produced in the late endosomal compartment by inward budding of the limiting membrane of multi‐vesicular bodies (MVBs) [101]. The membrane invagination process fills the MVBs with intraluminal microvesicles (ILVs). Cytosolic proteins and nucleic acids are packaged and carried inside exosomes. Exosome‐filled MVBs are either fused with the plasma membrane to release their contents in the extracellular space or send their contents to lysosomes for degradation [98].

In humans, syncytin‐1 triggers a cell fusion process, resulting in the formation of syncytiotrophoblasts. To initiate the cell fusion processes, the fusion‐active domain of the protein must bring the phospholipid bilayer of two cells into close proximity [102], allowing the next steps of the fusion processes to proceed. These steps involve the protein‐free areas on the cell surface, allowing lipid mixing between the two cell membranes. This lipid interdigitation proceeds favorably in cell surface areas enriched in fusogenic lipids such as phosphatidic acid in the presence of calcium. Calcium is known to bind to the phosphate moiety of the phosphatidic acid molecule and disrupts the thermodynamic barrier made by the water molecules bound to the membrane phospholipids, from which lipid interdigitation is initiated. Exosomes are enriched in phosphatidic acid [103], and also contain the endosomal phospholipid Bis(Monoacylglycero)Phosphate (BMP) [104] which is fusogenic [103]. It has been thought that fusion between exosomes and another membrane might occur more readily at acidic pH since BMP is fusogenic at endosomal pH [103, 105]. However, the presence of syncytin in the exosome membrane could enable BMP to function even at neutral pH. More importantly, exposure of phosphatidylserine on the outer layer of cell membranes has been reported to favor fusion [106], because exosomes expose this phospholipid on their external membranes [107]. These recent observations favor that together with syncytin‐1, exosomes could well be involved in the promotion of intercellular fusion, resulting in the initiation of syncytiotrophoblast formation.

Conclusions

The placentas as we see them today are considered to be a fairly recent development in mammals, and the conceptus side consists of mononucleate TE cells and fused TEs, syncytiotrophoblasts. Until recently, processes of conceptus implantation to the maternal endometrium had been studied from the standpoint of attachment and invasion through extracellular matrices, cell adhesion molecules, cytokines, and/or proteinases and their inhibitor expression. Recent progress suggests that implantation processes should be analyzed as a whole as well as in specific events; however, each event and/or their gene expression must be studied on a continuum sequence of events. In particular, any implantation study must include ERV genes and their specific expression as well as other components and/or players such as exosomes and EMT related molecules. ERV and exosome research in reproduction represents fairly new directions and with various ERV genes and other components yet to be found, our current understanding of implantation and placental formation may be far from finalized. We must then treat these processes as a work still in progress, and, therefore, prepare for much work ahead in the elucidation of molecular mechanisms associated with implantation and placentation, all of which result in reproductive advantages in mammalian evolution.

Acknowledgments

This was supported by the Program for Promotion of Basic research Activities for Innovative Bioscience (BRAIN) and by Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry. The authors would like to thank Mr. Robert Moriarty for his thorough evaluation of the manuscript.