Human Reproduction Update Advance Access originally published online on October 26, 2005
Human Reproduction Update 2006 12(2):145-157; doi:10.1093/humupd/dmi044
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System B0,+ amino acid transport regulates the penetration stage of blastocyst implantation with possible long-term developmental consequences through adulthood
1 Department of Biochemistry, 2 Department of Obstetrics and Gynecology, Midwestern University, Downers Grove, IL and 3 Department of Internal Medicine, Walter Reed Army Medical Center, Washington, DC, USA
4 To whom correspondence should be addressed at: Department of Biochemistry, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. E-mail: lvanwi{at}midwestern.edu
Submitted on July 27, 2005; resubmitted on September 13, 2005; accepted on September 26, 2005
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Abstract |
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Abstract
Introduction and scopeAmino acid transport system B0,+ was first characterized in detail in mouse blastocysts over two decades ago. Since then, this system has been shown to be involved in a wide array of developmental processes from blastocyst implantation in the uterus to adult obesity. Leucine uptake through system B0,+ in blastocysts triggers mammalian target of rapamycin (mTOR) signalling. This signalling pathway selectively regulates development of trophoblast motility and the onset of the penetration stage of blastocyst implantation about 20 h later. Meanwhile, system B0,+ becomes inactive in blastocysts a few hours before implantation in vivo. System B0,+ can, however, be activated in preimplantation blastocysts by physical stimuli. The onset of trophoblast motility should provide the physiological physical stimulus activating system B0,+ in blastocysts in vivo. Activation of system B0,+ when trophoblast cells begin to penetrate the uterine epithelium would cause it to accumulate its preferred substrates, which include tryptophan, from uterine secretions. A low tryptophan concentration in external secretions next to trophoblast cells inhibits T-cell proliferation and rejection of the conceptus. Suboptimal system B0,+ regulation of these developmental processes likely influences placentation and subsequent embryo nutrition, birth weight and risk of developing metabolic syndrome and obesity.
Key words: amino acid transport systems / embryo implantation / human development / metabolic syndrome / small for gestational age
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Introduction and scope |
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Blastocyst implantation in the mammalian uterus is a complex process with numerous, species-specific nuances. Similarly, development of obesity, type 2 diabetes mellitus and their associated chronic adult diseases are multifactorial events influenced by many genes and a multitude of environmental factors. For these reasons, it may seem at first curious to the reader to suggest that these types of long-term outcomes can all be linked to events that occur before and around the time of implantation. Specifically, we propose here that each of these adverse developmental events is tied to the relative activity of a single biological catalyst, the amino acid transport system B0,+, during the pre- and periimplantation period of blastocyst development.
System B0,+ was first described in detail in mouse blastocysts where it serves to transport amino acids across the trophectoderm apical membrane (Van Winkle et al., 1985
; Van Winkle, 2001). Although system B0,+ has broad substrate specificity, it prefers the branched chain and benzenoid amino acids, leucine, isoleucine, tryptophan and phenylalanine, over other zwitterionic and cationic amino acids (Table I; where the lower the Km value, the better the substrate). System B0,+ is also Na+-dependent (Borland and Tasca, 1974; Van Winkle et al., 1985); a characteristic that renders it able both to form gradients of its preferred substrates (Figure 1) and to change the magnitudes of such gradients with changes in the Na+ and K+ concentrations. As we shall see, an increase in the Na+ concentration of uterine secretions, and the effect of the increase on system B0,+ transport, could trigger signalling in blastocysts needed for development of the penetration stage of embryo implantation.
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Blastocyst implantation in the uterus has been divided into three stages (Enders and Schlafke, 1967; Enders and Schlafke, 1969). The apposition stage is followed by attachment of the embryo in a manner that renders it unable to be isolated simply by flushing the uterine lumen with culture medium. In mice and probably other species, these first two stages of implantation are regulated in the embryo, in part, by 4-hydroxy-17ß-estradiol (4-OH-E2) (Paria et al., 1998) and trafficking of ß1 integrins to the apical trophectoderm membrane (Sutherland et al., 1993; Schultz et al., 1997), respectively. Many mammals, including mice, rats and humans, also exhibit the third stage of implantation; namely, blastocyst penetration of the uterine epithelium (Schlafke and Enders, 1975). Such penetration requires differentiation of trophectoderm cells into motile trophoblast cells (Sutherland, 2003). Mouse and rat trophoblast cells do not appear to migrate much in vivo but instead use their protrusive activity to invade by phagocytizing apoptotic decidual cells (Welsh and Enders, 1987; Bevilacqua and Abrahamsohn, 1989). Not only is development of trophoblast motility regulated by amino acid transport system B0,+ initiated signalling beginning about 20 h before implantation (Martin et al., 2003) but the onset of motility also reactivates system B0,+ during the penetration stage of implantation to protect blastocysts from immunologic rejection (see below). Interestingly, porcine blastocysts, which do not penetrate the uterine epithelium (e.g. Lee and DeMayo, 2004), do not express the amino acid transport system B0,+ (Prather et al., 1993).
Both trophoblast motility and suppression of immunologic rejection are needed successfully to establish placental function and pregnancy. More than half of all pregnancies appear to end during the periimplantation period, and implantation abnormalities likely help to cause miscarriage, pre-eclampsia, placental accreta and ectopic pregnancy (Nayak and Guidice, 2003). The significance of amino acid transport system B0,+ in regulating development does not, however, cease with blastocyst implantation and its abnormalities.
Rather, some alleles of the SLC6A14 gene, which encodes the amino acid transporter B0,+, are associated with an increased risk of obesity in human adults and probably children (Suviolahti et al., 2003; Durand et al., 2004). As for humans, the same gene in mice maps to a region on the X chromosome (Kawai et al., 2001) previously shown to be linked to body weight (Dragani et al., 1995). Interestingly, this body weight gene in mice influences body mass only on the right genetic background (Dragani et al., 1995), and it appears to be influenced by diet (West et al., 1992), as is the case for most human obesity. The gene encoding the system B0,+ amino acid transporter is, however, expressed at the protein level predominantly in lung, colon and eye in adults (Sloan et al., 2003; Hatanaka et al., 2004). Hence, it is somewhat difficult to relate expression of the gene in adults to tissues and to metabolic abnormalities possibly causing obesity.
When one considers the importance of amino acid transport system B0,+ in establishing placental nutrition of the embryo, however, the connection between some alleles of the gene encoding amino acid transporter B0,+ and adult obesity appears clearer. Placental nutrition influences the size of offspring, and small for gestational age offspring have an increased risk of developing obesity, type 2 diabetes mellitus and other chronic adult conditions associated with the metabolic syndrome (Kanaka-Gantenbein et al., 2003; Levy-Marchal and Jaquet, 2004; Ten and Maclaren, 2004). We discuss in detail below the evidence that amino acid transport system B0,+ regulates blastocyst implantation and immunologic rejection and thus may influence subsequent placentation, embryo nutrition and fetal size at birth. Such a scenario is consistent with the known association of obesity with some alleles of the system B0,+ gene, SLC6A14, in humans (Suviolahti et al., 2003; Durand et al., 2004) and probably mice.
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System B0,+ amino acid transport activity is regulated to appear in abundance at the blastocyst stage of preimplantation mouse and rat embryo development |
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Transcripts encoding the amino acid transporter B0,+ (ATB0,+) were detected at all but the one cell stage of preimplantation mouse embryo development using microarrays (Zeng et al., 2004
). However, we detected the ATB0,+ transcript only at the blastocyst stage in mouse preimplantation embryo cDNA libraries using PCR (Martin et al., 2003). More importantly, system B0,+ transport activity is expressed abundantly in blastocysts but not at earlier stages of preimplantation mouse (Van Winkle et al., 1990a) and rat (Van Winkle et al., 1990b) embryo development. Interestingly, system B0,+ transport activity can be detected in porcine oocytes but not blastocysts (Prather et al., 1993), and porcine blastocysts do not display the penetration stage of blastocyst implantation (Lee and DeMayo, 2004). Hence, system B0,+ transport activity is regulated to appear in abundance in mouse and rat blastocysts when they need it to control development of trophoblast motility and invasion of the uterine epithelium. |
System B0,+-catalyzed leucine uptake regulates development of trophoblast motility and, thus, the penetration stage of blastocyst implantation |
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It has been known for many years that amino acid deprivation prevents formation of trophoblast outgrowths by mouse blastocysts in vitro (Gwatkin, 1966
; Naeslund, 1979). More recently, such deprivation has been shown to limit development of protrusive activity in the blastocyst trophectoderm, and this protrusive activity immediately precedes the more obvious expression of trophoblast motility and outgrowth (Martin and Sutherland, 2001). Specifically, leucine and arginine deprivation completely inhibits trophoblast outgrowth (Naeslund, 1979), whereas deprivation of other essential amino acids impairs but does not prevent this process (Spindle and Pedersen, 1973; Van Winkle et al., 2003). Similarly, in previously unpublished studies we showed that leucine and, apparently to a lesser extent, arginine each alone support development of trophoblast motility and outgrowth (Figure 2). The proportion of blastocysts eventually forming outgrowths in the presence of leucine is similar to the proportion forming outgrowths when all 20 amino acids are present, although the time at which outgrowth begins in vitro is delayed a day or two when leucine is the only amino acid supplied in the culture medium (Van Winkle et al., 2003). Because concentrations above 50 µM arginine are toxic to blastocysts when present as the only amino acid supplied in the medium (Van Winkle et al., 2003), we could not determine whether it supports development of trophoblast motility at the same rate as leucine (Figure 2). Interestingly, leucine protects embryos from this arginine toxicity (Van Winkle et al., 2003).
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Leucine uptake triggers development of trophoblast motility selectively: it does not regulate other aspects of trophectoderm differentiation |
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Although system B0,+ leucine uptake triggers development of trophoblast motility, it is not needed to foster other aspects of trophectoderm differentiation. For example, blastocysts begin to express placental lactogen-I (a marker for the giant-cell transformation in the trophectoderm) regardless of whether amino acids are supplied in the culture medium (Martin and Sutherland, 2001
). Similarly, these embryos do not need amino acids to exhibit ß1 integrin-dependent (Sutherland et al., 1993; Schultz et al., 1997) attachment to the substratum in culture (Martin and Sutherland, 2001). Finally, delayed implantation blastocysts normally increase their ability to take up amino acids during in vitro culture (Van Winkle, 1981), but this increase in amino acid transport system activity occurs regardless of whether amino acids had been supplied in the culture medium before measuring transport activity (Figure 3A). Hence, leucine selectively fosters development of trophoblast motility and the penetration stage of implantation and not other aspects of trophectoderm differentiation or the attachment stage of implantation.
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How does leucine accumulation selectively regulate development of trophoblast motility in mouse blastocysts? |
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We reported that rapamycin inhibits development of trophoblast motility and outgrowth (Van Winkle, 2001
). Since mammalian target of rapamycin (mTOR) signalling fosters an increase in the amount of protein synthetic machinery (Fox et al., 1998; Hara et al., 1998; Patti et al., 1998; Xu et al., 1998; Shigemitsu et al., 1999; Kimball and Jefferson, 2000), we postulated that rapamycin inhibits outgrowth owing to inhibition of an increase in the rate of protein synthesis in blastocysts (Van Winkle, 2001). In such a scenario, however, leucine alone should not support development of trophoblast motility and outgrowth as we observed it to do (Figure 2). In this case, the absence of other essential amino acids would limit any net increase in protein synthesis and accumulation. Leucine is also much more effective than other amino acids at triggering mTOR signalling in most other tissues (Fox et al., 1998; Xu et al., 1998; Shigemitsu et al., 1999; Proud, 2002). Although an increase in the rate of protein synthesis undoubtedly accompanies normal development of trophoblast motility in blastocysts, another mTOR-dependent process likely causes the trophoblast cells to become motile.
Martin and Sutherland (2001) not only verified that rapamycin blocks development of trophoblast motility but also showed that amino acid (leucine) uptake by the trophectoderm fosters phosphorylation of the mTOR substrate, p70S6 kinase, in blastocysts. Conversely, both amino acid deprivation and rapamycin prevent this phosphorylation. mTOR also phosphorylates PHAS/4EBP proteins and, thus, frees eIF4E selectively to initiate translation of mRNAs encoding proteins, such as ornithine decarboxylase (ODC), that help to regulate cellular growth and differentiation (Kimball et al., 1999; Martin et al., 2003). In part, because mTOR signalling regulates ODC expression and, hence, polyamine synthesis, we propose that polyamines are the downstream signalling molecules more directly regulating development of trophoblast motility (see next section).
mTOR gene-knockout experiments support the conclusion that system B0,+ leucine transport fosters development of trophoblast motility through mTOR. mTOR (–/–) conceptuses fail to develop significantly into the penetration stage of implantation (Gangloff et al., 2004; Murakami et al., 2004). Only a small amount of motility develops in the trophectoderm of mTOR (–/–) blastocysts possibly owing to the presence in oocytes of some maternal mTOR mRNA that survives to the blastocyst stage (Gangloff et al., 2004). (But see other possible explanations below.) We are studying whether mouse blastocysts lose this surviving maternal mTOR mRNA when the preimplantation period is prolonged through delay of implantation. Upon resumption of development of delayed implantation blastocysts, mTOR (–/–) conceptuses should neither penetrate the uterine epithelium in vivo nor form outgrowths in vitro.
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mTOR (–/–) embryos also will be useful for studying the signalling processes needed for implantation that are downstream from mTOR |
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In a previous report (Martin et al., 2003), we discussed evidence supporting the hypothesis that polyamines serve as downstream signalling molecules of system B0,+ leucine transport-stimulated mTOR. Polyamines likely foster development of trophoblast motility in blastocysts in a manner analogous to the mechanism by which these signalling molecules promote development of motility in intestinal epithelial cells (Rao et al., 2003
; Ray et al., 2003). In the latter tissue and cell lines, polyamine accumulation owing to physical injury increases Kv channel gene expression and consequently causes plasma membrane hyperpolarization, increased cytosolic Ca2+ concentration, greater GTP-Rho-A and Rac 1 activity, Rho-kinase activation, myosin phosphorylation, myosin/F-actin stress fiber formation and cell migration. Polyamine-dependent development of motility in intestinal epithelial cells depends ultimately on Rac 1 activation, and we suggest that mTOR signalling initiates the same series of polyamine-dependent events in the blastocyst trophectoderm (Martin et al., 2003).
We showed previously that blastocysts fail to form outgrowths in vitro in the presence of inhibitors of polyamine synthesis (Van Winkle LJ and Campione, 1983, 1984). Similarly, embryos deficient in enzymes required for polyamine synthesis perish at about the time of implantation (Pendeville et al., 2001; Nishimura et al., 2002). The block to development of trophoblast motility by inhibitors of polyamine synthesis can, however, be overcome by polyamines in the medium. Hence, leucine transport-dependent mTOR signalling could conceivably stimulate downstream polyamine accumulation and signalling by promoting both polyamine synthesis and uptake by the trophectoderm.
In this regard, we found that polyamines partially reverse rapamycin inhibition of blastocyst outgrowth (P <0.01, Van Winkle and Campione, unpublished data). Polyamines are taken up by blastocysts through a process that appears to select the polyamines, spermidine (Spd) and spermine (Spm), over putrescine (Put) (Figure 4). Interestingly, amino acid-deprivation impairs development of polyamine transport activity in the blastocyst trophectoderm (Figure 3B). Hence, amino acid (leucine) transport promotes mTOR signalling and development of polyamine transport activity in the trophectoderm, but amino acid transport is not needed for other aspects of differentiation in this tissue (recall the above section concerning selective regulation of development of trophoblast motility by leucine uptake). For these reasons, we conclude that system B0,+-catalysed leucine transport triggers mTOR signalling and consequently polyamine accumulation. The polyamines then signal development of trophoblast motility ultimately owing to Rac 1 activation, as is the case for intestinal epithelial cells (Ray et al., 2003).
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mTOR (–/–) embryos will allow us more definitively to study the relationship between polyamine and mTOR signalling during development of trophoblast motility. The studies showing polyamine reversal of inhibition of blastocyst outgrowth by rapamycin, described above, need verification because it is never certain that the effects of an inhibitor are exclusively on the intended target (in this case, mTOR). Because polyamines partially overcome inhibition of trophoblast outgrowth by rapamycin, it is likely that these substances are taken up in quantities adequate to support development of trophoblast motility even when mTOR is inhibited or inactive. Moreover, mTOR (–/–) blastocysts should not develop an increased capacity to take up polyamines like wild-type blastocysts do (Figure 3B). Thus, it should be possible to use mTOR (–/–) embryos to study the signalling processes emanating from mTOR that are needed to stimulate development of increased polyamine transport activity and, subsequently, trophoblast motility. Since most of the studies described here and in the preceding sections have been or will be performed in vitro, however, it is also necessary to learn what regulates system B0,+ leucine uptake, mTOR signalling and development of trophoblast motility in blastocysts in vivo.
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What regulates system B0,+-catalysed leucine uptake in blastocysts in vivo? |
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Wild-type blastocysts that develop from the two-cell stage in vitro in the absence of amino acids are poised to take up leucine (and isoleucine) but not other essential and non-essential amino acids (Van Winkle and Dickinson, 1995
; Martin et al., 2003). Net leucine uptake then stimulates mTOR signalling when these in vitro-grown blastocysts are transferred to medium containing amino acids (Martin and Sutherland, 2001). In contrast, leucine and other amino acids appear to be present in uterine secretions, and their concentrations in this environment do not appear to change as blastocysts and the uterus develop towards implantation (Gwatkin, 1969). We have used the delay of implantation model to show that the uterine environment can stimulate leucine accumulation through existing system B0,+ activity in blastocysts in vivo (see below). Such system B0,+-catalysed leucine accumulation would trigger mTOR signalling and development of trophoblast motility.
Normally, a surge of estrogen secretion about 3.5 days after female mice mate initiates blastocyst development towards implantation. Implantation can be delayed, however, if the mice are ovariectomized before the estrogen surge. Blastocysts remain in delay of implantation in ovariectomized, progesterone-maintained mice until estrogen is administered along with the progesterone. Blastocyst implantation then begins about 25 h after estrogen administration (Yoshinaga and Adams, 1966; Weitlauf and Greenwald, 1968; Van Blerkom et al., 1978; Van Winkle and Campione, 1987; Mead, 1993; Renfree and Shaw, 2000; Dey et al., 2004; Lee and DeMayo, 2004).
Blastocysts in delay of implantation will form outgrowths in vitro in the absence of prior estrogen administration if the culture medium contains leucine or a mixture of all 20 amino acids (Figure 2). However, these same blastocysts have no such requirement for amino acids to form outgrowths in vitro when the blastocysts are removed from the uterus 24 h after estrogen administration (Van Winkle and Campione, unpublished data). Hence, net leucine accumulation by blastocysts through system B0,+ likely occurs in vivo some time between 0 and 24 h after estrogen administration. This leucine accumulation by blastocysts in vivo triggers mTOR signalling and development of trophoblast motility regardless of whether amino acids are present subsequently in the blastocysts’ environment.
In this regard, the Na+ and K+ concentrations in uterine secretions increase significantly within 6 h after estrogen administration to progesterone-maintained, ovariectomized rats (Figure 5) and probably mice (Van Winkle et al., 1983). In fact, since the increases in the Na+ and K+ concentrations are accompanied by a similar rise in the Cl– concentration in uterine secretions (Nilsson and Ljung, 1985), the environment of blastocysts likely changes from hypo- or isotonic to hypertonic after estrogen administration (i.e. from less than 300 to over 400 mOsmolar assuming equal concentrations of monovalent anions and cations, Figure 5). The latter change might negatively influence mTOR since cell shrinkage inhibits such signalling (Fumarola et al., 2005).
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Cell shrinkage would be counteracted, however, and mTOR signalling triggered by the net accumulation of leucine and other amino acids in the trophectoderm at higher extracellular Na+ and K+ concentrations (Figure 1). All amino acid substrates of system B0,+ would serve as osmolytes, whereas leucine alone would trigger mTOR signalling. The stoichiometry of Na+ : amino acid cotransport through system B0,+ appears to be about 2:1 (Sloan and Mager, 1999). Hence, the increase in the Na+ concentration in uterine secretions can be calculated to increase the intracellular amino acid concentration nearly three-fold if the trophectoderm is able to maintain the new Na+ concentration (or more precisely, the new Na+ total chemical potential) gradient (Van Winkle, 1999).
Significantly, the free energy needed to maintain a greater Na+ concentration gradient would be available to Na+K+ATPase in blastocysts (Figure 1). The K+ concentration increases enough in uterine secretions (Figure 5) to reduce the free energy needed to transport it against its total chemical potential gradient by about the same amount of free energy as is needed to maintain the new Na+ concentration gradient (Van Winkle, 1999). Moreover, Na+K+ATPase is present in the apical as well as the basolateral membrane of the trophectoderm (Van Winkle and Campione, 1991), and, of course, the apical membrane faces the cations in uterine secretions.
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Somewhat surprisingly then, the penetration stage of implantation appears to be regulated in blastocysts primarily if not exclusively by a single signalling pathway. Estrogen stimulates increases in the Na+ and K+ concentrations in uterine secretions, and these changes in the Na+ and K+ concentrations power an increase in the trophectoderm leucine concentration (Figure 1). Only about a 10% increase in the intracellular leucine concentration can trigger some mTOR signalling, so the nearly three-fold increase in leucine that should occur in the blastocyst trophectoderm is more than enough to stimulate mTOR signalling. The greater increase in intracellular amino acid concentrations than needed to initiate mTOR signalling likely counteracts trophectoderm cell shrinkage which would otherwise impair mTOR signalling (Fumarola et al., 2005
). mTOR signalling then leads to development of trophoblast motility in blastocysts approximately 25 h after the estrogen surge or after estrogen administration in the case of delay of implantation. Polyamines likely serve as signalling molecules downstream from mTOR that are needed for development of trophoblast motility ultimately owing to Rac 1 activation.
Estrogen, of course, has other effects on blastocyst implantation. For example, it leads to production of the 4-OH-E2 apparently needed for blastocysts to undergo the apposition stage of implantation (Paria et al., 1998). Apposition precedes blastocyst attachment and penetration of the uterine epithelium. Similarly, estrogen is needed for leukemia inhibitory factor (LIF) production, and LIF signalling is needed for the uterus to develop a phenotype that is receptive to implantation (Stewart et al., 1992; Stewart and Cullinan, 1995; Chen et al., 2000). Nevertheless, development of trophoblast motility and penetration of the uterine epithelium on the part of the blastocyst appear to be regulated not by 4-OH-E2, LIF or numerous other signalling molecules that are involved in blastocysts implantation (Dey et al., 2004; Lee and DeMayo, 2004). (See also the section below concerning other signalling molecules.) Rather, system B0,+ leucine transport triggers the mTOR signalling needed for differentiation of motile trophoblasts and penetration of the uterine epithelium by the blastocyst (Figure 1).
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What functions may other amino acid transporters play in blastocyst implantation? |
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Another amino acid transport system first described in mouse blastocysts (Van Winkle et al., 1988a) also may function in implantation. This system is Na+ independent, so by convention (Bannai et al., 1984
), a lower case ‘b’ was used in its designation, system b0,+, to distinguish it from the Na+-dependent system B0,+ (Van Winkle et al., 1985). System b0,+ is a heterodimer composed of a light chain transporter, termed b0,+ amino acid transporter (b0,+AT), and a heavy chain accessory protein (Broer and Wagner, 2002; Van Winkle, 2002; Palacin et al., 2005). The accessory protein is required for migration of the transporter to the plasma membrane, and it helps to determine the amino acid substrate selectivity of the transporter (Rajan et al., 2000) (Table II).
The system b0,+ heavy chain accessory protein is usually rBAT (Palacin et al., 2005) but apparently sometimes the related protein CD98 (also termed 4F2hc) (Rajan et al., 2000). The Km values for transport are in general lower when CD98 rather than rBAT is associated with b0,+AT (Rajan et al., 2000). Transcripts encoding each of these amino acid transport-related proteins are present in mouse (Hamatani et al., 2004; Qian, 2004; Zeng et al., 2004; Maekawa et al., 2005) and human (Van Winkle et al., 2001) blastocysts although the transcript encoding CD98 appears to be much more abundant in blastocysts than those encoding the other two proteins (National Center for Biotechnology Information BLAST Data Base). Consistent with the latter findings, CD98 also serves as the accessory protein for several other transporters expressed in mouse and human blastocysts including y+LAT1 (Zeng et al., 2004), y+LAT2 (Van Winkle et al., 2001; Qian, 2004; Zeng et al., 2004), LAT1 (Hamatani et al., 2004; Qian, 2004; Maekawa et al., 2005) and LAT2 (Qian, 2004; Hamatani et al., 2004). Based on the substrate selectivity of system b0,+ for arginine versus other amino acids (Table II), we conclude provisionally that system b0,+ in blastocysts is composed of the b0,+AT light chain and the CD98 heavy chain.
System b0,+ functions in the apical membrane of the blastocyst trophectoderm mainly to take up its preferred substrate, arginine (Figure 1). Arginine serves as a precursor for the synthesis of polyamines which are likely downstream signalling molecules of an mTOR signalling pathway (see above). In addition, arginine is a substrate of nitric oxide (NO) synthase, and NO appears also to help regulate pre- and periimplantation blastocyst development (Chwalisz and Garfield, 2000; Khorram, 2002; Martin et al., 2003; Thaler and Epel, 2003). Similarly, arginine is probably needed as a precursor for proline and thus extracellular matrix synthesis in blastocysts (Biggers et al., 2000; Martin et al., 2003). Perhaps because an arginine supply is essential to early development, other novel arginine-selective transport systems also operate in blastocysts (Van Winkle and Campione, 1990; Van Winkle, 2001). The latter systems appear largely to compensate for an absence of system b0,+ activity in blastocysts, since b0,+AT deficient mice are in general healthy and fertile (Feliubadalo et al., 2003). As for humans with similar mutations, however (Feliubadalo et al., 1999; Palacin et al., 2005), b0,+AT (–/–) mice develop cystinuria (Feliubadalo et al., 2003; Palacin et al., 2005).
Somewhat surprisingly then, CD98 deficient embryos fail to develop beyond the periimplantation period (Tsumura et al., 2003). We are currently determining the precise time and mechanism by which development ceases in early CD98 (–/–) embryos. In this regard, CD98 modulates ß1 integrin function in the plasma membrane (Cai et al., 2005; Feral et al., 2005), and it fosters migration of ß1 integrins to the basolateral membranes of polarized epithelial cells (Cai et al., 2005). Contrariwise, blastocyst attachment to the substratum in vitro (Sutherland et al., 1993; Schultz et al., 1997) and probably the attachment stage of implantation in vivo (Martin et al., 2003) require ß1 integrin trafficking to the trophectoderm apical membrane. We suggest that a yet to be identified signal may redirect some CD98 from the basal to the apical membrane and consequently also send both ß1 integrins and b0,+AT to the latter membrane. As discussed above, ß1 integrins and b0,+AT have been observed to function in the trophectoderm apical membrane for attachment to the substratum (Sutherland et al., 1993; Schultz et al., 1997) and amino acid (arginine) transport (Van Winkle et al., 1988a), respectively.
CD98 also has been shown to be essential for normal migration and spreading of embryonic stem cells, fibroblasts and polarized epithelial cells (Cai et al., 2005; Feral et al., 2005). Interestingly, this CD98-mediated ß1 integrin function depends on ß1 integrin signalling that activates Rac (Feral et al., 2005). The reader should recall that mTOR-dependent polyamine signalling appears to lead to development of trophoblast motility by fostering Rac 1 activation in the blastocyst trophectoderm (see above). For these reasons, we are currently investigating whether both CD98- and mTOR-mediated signalling are needed for optimal Rac 1 activation and subsequent development of trophoblast motility in blastocysts (Figure 6). Conceivably either CD98- or mTOR-mediated signalling alone could lead to some, albeit, insufficient Rac 1 activation in blastocysts. Both CD98- and mTOR-mediated signalling may, however, be needed for fully effective Rac 1 activation, development of trophoblast motility and penetration of the uterine epithelium during implantation. In the absence of either CD98- or mTOR-mediated signalling in CD98 (–/–) (Tsumura et al., 2003) or mTOR (–/–) (Gangloff et al., 2004) blastocysts, respectively, only limited motility may develop in trophoblast cells. We are currently working to determine whether such is the case for CD98 (–/–) embryos, and such is known to be the case for mTOR (–/–) embryos in vitro and in vivo (Gangloff et al., 2004). (Other explanations are, of course, possible for the latter observation, as discussed in preceding sections.) Regardless of the explanations for these observations, however, both amino acid transport-related CD98 and amino acid transport-dependent mTOR-mediated signalling are essential for full development of trophoblast motility and the penetration stage of implantation. Many other signalling molecules contribute to but are not essential for development of these and other aspects of implantation.
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If amino acid transport-related CD98 and amino acid transport-dependent mTOR-mediated signalling regulate development of trophoblast motility and the penetration stage of blastocyst implantation, then what role do numerous growth factors, cytokines and other more conventional signalling molecules play in these and related processes? |
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Although the actions of a few signalling molecules on blastocysts are required for implantation, many other such molecules are involved in but not essential for nidation (Aplin and Kimber, 2004
; Dey et al., 2004; Imakawa et al., 2004; Red-Horse et al., 2004). One frequent explanation for these observations is that the non-essential signalling molecules are so important to implantation and successful reproduction that several such redundant and therefore ‘non-essential’ signalling molecules accomplish the same end.
In contrast, we liken the impaired implantation sometimes accompanying knockout of expression of a signalling process in mice (Aplin and Kimber, 2004; Dey et al., 2004; Imakawa et al., 2004; Red-Horse et al., 2004) to the impaired but not prevented outgrowth of blastocysts deprived of essential amino acids other than leucine and arginine in vitro (Spindle and Pedersen, 1973; Van Winkle et al., 2003) (Figure 2). In our view, leucine uptake triggers mTOR signalling and development of trophoblast motility in vitro and in vivo, whereas other essential amino acids and most other signalling molecules support primarily the growth of the conceptus needed simultaneously with the penetration stage of implantation. Even if the latter nutrients and signalling molecules are needed specifically to support implantation, they do not in most cases appear to be a single signalling process that alone regulates development of trophoblast motility. In contrast, system B0,+ leucine transport triggers the mTOR signalling that is required for development of enough trophoblast motility to establish pregnancy (Martin et al., 2003; Gangloff et al., 2004) during the penetration stage of implantation (Figure 1). The resultant trophoblast motility also likely regulates system B0,+ activity further to foster successful implantation and embryo nutrition, as described in the following section.
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While system B0,+ leucine transport triggers mTOR signalling in blastocysts about 20 h before implantation, system B0,+ activity is suppressed by the uterine environment as the time of implantation draws nearer |
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Increases in the Na+ and K+ concentrations in uterine secretions should cause leucine accumulation through system B0,+ in the trophectoderm no more than 6 h after estrogen administration to progesterone-maintained, ovariectomized rats (Nilsson and Ljung, 1985) and mice (Van Winkle et al., 1983) (Figure 1). The uterine environment suppresses blastocyst system B0,+ activity, however, between 12 and 25 h after estrogen administration (Figure 7A, filled symbols). The relatively inactive system B0,+ can then be activated by physical stimuli, such as removing periimplantation blastocysts from the uterus (Figure 7A, open symbols) or gently massaging the uterus containing the embryos (Figure 7B, open squares). No such activation occurs, however, in blastocysts one day before implantation (Van Winkle and Campione, 1987; Van Winkle et al., 1990a,b; Figure 7). The physiological physical stimulus most likely activating system B0,+ in blastocysts approaching implantation in vivo is the onset of trophoblast motility and initiation of the penetration stage of implantation.
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The penetration stage of implantation is associated with the need for a new function of system B0,+ in blastocysts; namely, its activation in the trophoblast helps to deplete surrounding secretions of another of its preferred substrates, tryptophan (Table 1). A few days after initial penetration of the uterine epithelium, the rate-limiting enzyme in tryptophan catabolism in trophoblast cells, indoleamine 2,3-dioxygenase (IDO), also begins to contribute to tryptophan depletion from the environment of the mouse conceptus (Munn et al., 1998
; Baban et al., 2004). Tryptophan depletion suppresses T-cell proliferation and the resultant immunologic rejection of the conceptus that would otherwise occur (Munn et al., 1998; Baban et al., 2004). This mechanism suppressing rejection persists to term in humans (Hönig et al., 2004; Kudo et al., 2004), and even at term, tryptophan transport into trophoblast cells is a rate-limiting step for IDO-mediated tryptophan degradation (Kudo and Boyd, 2001). T-cell suppression through tryptophan depletion should, however, be needed at the time when the trophoblast first penetrates the uter