Amino Acid Transport Regulation and Early Embryo Development

BIOLOGY OF REPRODUCTION 64, 1–12 (2001)

M i n i r ev i ew Amino Acid Transport Regulation and Early Embryo Development Lon J. Van Winkle1 Department of Biochemistry, Midwestern University, Downers Grove, Illinois 60515 ABSTRACT

mouse embryos has been studied thoroughly, this review focuses primarily on this species. Amino acid transport is critical to early mouse embryo development. Even a brief (;5 min) exposure of fertilized mouse eggs to medium without added amino acids is detrimental [5]. Although initial culture experiments indicated no requirement for amino acids or even a fixed nitrogen source [6], the beneficial effects of amino acids on development are obscured by concomitant detrimental effects of NH41 produced from the amino acids during culture [7]. When such negative effects are prevented in vitro [8], as they would be in vivo, amino acids clearly improve preimplantation development. The various ways in which amino acid transport mediates these improvements is the major theme of this review. Our knowledge of the mechanisms of these beneficial effects should continue to expand as the regulation and function of the pertinent transport proteins are studied further. The study of amino acid transport in early embryos also has been important to the field of amino acid transport more generally. For example, the discovery of novel transport systems in early mouse embryos [9] (mainly the B and b systems in the first column of Table 1) was followed by identification of the systems in adult tissues. Such tissues include intestinal and renal epithelia, in which conspicuous systems first described in mouse blastocysts help to absorb or reabsorb amino acids [10–14]. Regulation of these transport systems in blastocysts by amino acid substrates and serine proteases also likely applies to the systems in these other epithelia [12], although such regulation has not been studied thoroughly in them. Mutations in genes encoding renal and intestinal amino acid transporters produce cystinuria and lysinuria protein intolerance in humans [15].

Amino acids are essential components of media utilized to culture fertilized human eggs to the blastocyst stage in vitro. Use of such media has led to a significant increase in the proportion of embryos that implant upon transfer to the uterus and to a decrease in the number that need to be transferred to achieve pregnancy. Little is known about the mechanisms by which amino acids foster development of healthy human blastocysts. Indications are, however, that many of these mechanisms are the same in human and mouse embryos. Both essential and nonessential amino acid transport benefit preimplantation mouse embryo development, albeit at different stages. Nonessential amino acid transport improves development primarily during cleavage, whereas essential amino acid transport supports development of more viable embryos, especially subsequent to the eight-cell stage. This review discusses likely mechanisms for these beneficial effects.

conceptus, developmental biology, female reproductive tract, gene regulation, glutamate, growth factors, implantation/early development, insulin, IVF/ART, nitric oxide, oviduct, placental transport, pregnancy, trophoblast, uterus

INTRODUCTION

Amino acids support normal preimplantation development in vivo as evidenced by studies in vitro. More than 50% of fertilized human eggs develop to the blastocyst stage in media containing amino acids, and those embryos that do not develop likely are not viable [1]. The culture of fertilized human eggs to the blastocyst stage before transfer to the uterus has led to a large increase in the proportion of embryos that implant and to a decrease in the number that need to be transferred to achieve pregnancy [2]. Although amino acids are essential ingredients in media used for these procedures [1–3], little is known about the mechanisms by which amino acids foster normal preimplantation development of human embryos. Initial indications are, however, that human and mouse preimplantation embryos contain the same amino acid transport systems [4]. Moreover, these systems are regulated both developmentally and in response to external signals and stresses [4]. Because amino acid transport by early

ESSENTIAL AND NONESSENTIAL AMINO ACID TRANSPORT PERFORMS DIFFERENT FUNCTIONS IN EARLY EMBRYOS

Both essential and nonessential amino acids benefit preimplantation development of mouse embryos, albeit at different times and by different mechanisms [16]. Nonessential amino acids improve development primarily during cleavage, whereas essential amino acids support development of more viable embryos, especially subsequent to the eight-cell stage. The following sections discuss possible mechanisms by which the transport of each group of amino acids benefits development.

Correspondence: Lon J. Van Winkle, Department of Biochemistry, Midwestern University, 555 31st Street, Downers Grove, IL 60515. FAX: 630 971 6414; e-mail: [email protected]

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Received: 14 April 2000. First decision: 23 May 2000. Accepted: 28 July 2000. Q 2001 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

Nonessential Amino Acids Beneficial to Development

Mixtures of nonessential amino acids increase the rate and frequency at which blastocysts are formed from one1

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TABLE 1. Mammalian amino acid transport protein superfamilies and likely corresponding systems in preimplantation mouse embryos.a System in embryos

Stages most highly expressed

Na1- and Cl2-dependent transporter superfamilyc Gly [23, 102] 1- to 8-cell b [22] All B0,1 [20, 21, 23, 58] Blastocyst Na1-dependent EAAT/ASC familyc XAG2 [24] Blastocyst XA2[24] Blastocyst B [21] Blastocyst Na1-dependent A/N family N? [19] A [104]

4- to 8-cell Blastocyst Inner cell mass

Na1-independent channel proteins VSOAC1 [22, 28] 1- to 2-cell VSOAC2 [22, 28] Blastocyst Na1-independent amino acid transporter superfamilyc b11a [50] 1- to 2-cell b11b [50] 1- to 2-cell b1i [50] 8-cell b12 [50] Blastocyst 0, 1 b [42, 58, 111] Blastocyst

Preferred amino acid substrate(s)

Likely transport proteinb

Accessory protein(s)

Glycine Taurine Most except Glu and Asp

GLYT1 [18] TAUT [22] *ATB0,1 [103]

None known

Glu and Asp Aspartate Zwitterionic

EAAT1 [18] *EAAT? [21, 52] *ASC2 [9, 52]

None known

Glutamine Less bulky zwitterionic

*SN1 [105–107] *SAT1?/SAT2? [108–110]

None known

Tau/Gly/Asp Tau/Gly/Asp

*CIC?/lcln ?[29–31] See text

None known

CAT2 (Fig. 2) CAT1 (Fig. 2) CAT1/CAT2 (Fig. 2) CAT1/CAT2 (Fig. 2) *b0,1 AT [54, 55] (deficient in human non-Type I cystinuria [15, 54]) *LAT1/2/? [57]

None known

*xCT [61]

L [42]

All

T [58]

All

Arginine Arginine Arginine Arginine Most bulky cationic and zwitterionic (e.g., arginine)d Branched-chain and benzenoid Benzenoid

xc [60]

1-cell

Glu and cystine

*LAT? [57]

*rBAT, *4F2hc [52–56] (deficient in Type I cystinuria [15]) *4F2hc [52, 53, 57] *4F2hc? [52, 53, 57] *4F2hc [52, 53, 61]

a

References for systems and transport-related proteins are shown in brackets. Proteins marked with * and their mRNAs (or related proteins and mRNAs) have not been detected in early embryos in most cases probably because the possibility has not as yet been studied. ASC, system ASC transport protein; ATB0,1, amino acid transporter B0,1; b0,1 AT, b0,1 amino acid transporter; CAT, cationic amino acid transporter; CIC, chloride channel protein; EAAT, excitatory (anionic) amino acid transporter; 4F2hc, heavy chain of the 4F2 cell surface antigen; GLYT, glycine transporter; Icln, chloride channel protein; LAT, system L amino acid transporter; rBAT, related to system b0,1 amino acid transporter; SAT, system A transporter; SN, system N transporter; TAUT, taurine transporter; VSOAC, volume-sensitive organic osmolyte/anion channel; xCT, system xc2 transport protein. c Van Winkle [15] for a broader summary of the amino acid transporters in this superfamily. d Though system b0,1 may transport most bulky cationic and zwitterionic amino acids depending on their relative concentrations in vivo, it selects for arginine based on the Km values for transport of various amino acids. b

cell conceptuses in culture [7, 16]. Moreover, the proportion of such blastocysts that implant in the uterus upon transfer to surrogate mothers is greater when nonessential amino acids are present during preimplantation development in vitro [16, 17]. Even brief exposure of approximately 5 min to medium not containing nonessential amino acids significantly reduces the proportion of zygotes that develop into morulae and blastocysts in vitro and the total number of cells in the blastocysts so formed [5]. To conclude that nonessential amino acid transport and its regulation contribute significantly to the beneficial effects of amino acids on development, it must be shown that embryo amino acid content (or amino acid delivery to the intracellular sites of their action or utilization) is determined, at least in part, by regulation of amino acid transport. This regulation could be accomplished by increasing or decreasing inward or outward migration of individual or groups of amino acids. Van Winkle and Campione [18] reported the correspondence between developmental changes in Na1-dependent transport activities for taurine, glycine, and aspartate and changes in the content of these amino acids in embryos developing in vivo. In a similar vein, the amino acids gly-

cine, taurine, alanine, glutamine, and glutamate are increased in quantity in blastocysts cultured in their presence from the two-cell stage in vitro [19], and systems for the Na1-dependent accumulation of each amino acid are present in preimplantation embryos [20–25]. A Na1-dependent system for glutamate transport does not appear in embryos until the blastocyst stage, however (Table 2), and neither does the ability of embryos to accumulate this amino acid above the concentration of glutamate that they otherwise contain in culture [19]. Moreover, the taurine and glycine content of embryos drops considerably [19, 26, 27] when they are incubated in hypotonic medium known to activate Na1-independent channels for their transport [22]. Hence, regulation of transport activity helps to determine the amino acid content of embryos and whether an amino acid can be beneficial to development. Developmental regulation of nonessential amino acid transport. Changes in the activities of separate Na1-dependent systems selective for glycine, taurine, or anionic amino acids in preimplantation embryos correlate with expression of mRNAs that encode proteins with the same transport characteristics (Table 1). System Gly is expressed through-

Glycine

Taurine

Taurine Glycine Aspartate

Glutamine

Aspartate Glutamate

Gly [23, 102]

b [22]

VSOAC [22, 33]

N? [18]

X2AG; X2A [24]

A. Developmental regulation owing to mRNA expression in blastocysts [18] B. Activity suppressed by uterine environment during the 10 h preceding implantation [19]

Possible transient appearance at about the 4 to 8cell stage [18]

Large changes associated with osmotic stress [22, 26, 28]

A. Developmental regulation owing to mRNA expression through the morula stage [18] B. Modest changes associated with osmotic stress [27] Large changes associated with osmotic stress [22]

Regulation

1. Cellular volume (or protein concentration) regulation and concomitant effects on metabolism throughout preimplantation development [22, 26, 39] 2. Resistance to oxidant stress and the consequences of membrane damage [112] Cellular volume (or protein concentration) regulation and concomitant effects on metabolism at 1-cell, 2-cell and blastocyst stages [22, 26, 28] Transient glutamine accumulation, cell volume (or protein concentration) regulation) and consequent metabolic effects at 4 to 8-cell stage Uptake of anionic amino acid signaling molecules and their depletion from uterine secretions if activated in vivo

Cellular volume (or protein concentration) regulation and concomitant effects on metabolism especially during early cleavage [22, 27, 37, 39]

Proposed physiological function(s)

B0,1 [20, 21, 23, 58]

b1ia; b11b; b1i; b12; b0,1 [42, 50, 58, 111]

System(s)

Most zwitterionic and cationic amino acids (e.g., leucine and tryptophan)b

Arginine

Preferred substrate(s)

A. Developmental regulation [21] B. Activation possibly via a novel proteinase activated receptor in blastocysts (see text) C. Activity strongly suppressed by uterine environment during the 10 h preceding implantation [21, 66]

A. Developmental regulation owing in some cases to mRNA expression (Table 1 and Fig. 2). B. Activation of b12 and b0,1 possibly via a novel proteinase activated receptor in blastocysts [12] C. Substrate inhibition/activation of system b0,1 ([42] and see text) D. Activity of system b12 apparently stimulated by uterine environment during the ten hours preceding implantation [21]

Regulation

1. Regulation of polyamine and NO production possibly through direct delivery of arginine to sites of synthesis 2. Net amino acid uptake for protein synthesis and accumulation in blastocysts in conjunction with system B0,1 3. Regulation of protein kinases involved in protein synthesis regulation. 4. Depletion of arginine from uterine secretions possibly resulting in suppression of T-cell proliferation if system B0,1 is activated in vivo at implantation 1. Net amino acid uptake for protein synthesis and accumulation in blastocysts 2. Regulation of protein kinases involved in protein synthesis regulation in blastocysts 3. Depletion of leucine and tryptophan from uterine secretions possibly resulting in suppression of T-cell proliferation if system B0,1 is activated in vivo at implantation

Proposed physiological function(s)

Systems selective primarily for essential amino acids

b

References for systems, regulation of their activities, and their proposed physiological functions are shown in brackets when they have been studied or proposed previously. Though system B0,1 may transport a broad scope of amino acids depending on their relative concentrations in vivo, it selects for leucine and tryptophan based on the Km values for transport of various amino acids.

a

Preferred substrate(s)

System(s)

Systems for nonessential amino acids

TABLE 2. Regulation and proposed physiological functions of amino acid transport systems in early embryos.a

AMINO ACID TRANSPORT AND EARLY EMBRYO DEVELOPMENT

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out cleavage, and so is mRNA encoding glycine transporter 1 (GLYT1). Neither is, however, expressed in blastocysts [18]. Similarly, system XAG2 and mRNA encoding excitatory (anionic) amino acid transporter (EAAT) 1 are both expressed in blastocysts but not in embryos at earlier stages of development [18]. In contrast to these dramatic developmental changes, taurine transport by system b and mRNA encoding the taurine transporter (TAUT) are expressed conspicuously throughout preimplantation development [22]. Each of these correlations is consistent with a causal relationship between the presence of an mRNA encoding a protein and expression of the pertinent transport activity, although the identities of the proteins actually catalyzing transport need to be verified. Similarly, several transport proteins have characteristics resembling those of transport systems/channels in early embryos (Table 1), but it remains to be shown that the systems/channels actually contain these or related proteins. For example, volume-sensitive organic osmolyte/anion channels (VSOAC) apparently transport taurine, glycine, aspartate, and chloride in one- and two-cell embryos and in blastocysts [22, 28]. Moreover, several chloride channel proteins that may catalyze such transport have been identified [29–31] (Table 1). None of these proteins has, however, been shown to transport all of these substrates, nor have the proteins or their mRNAs been detected in preimplantation conceptuses. Moreover, pharmacological agents that inhibit VSOAC transport activity in other tissues are only very weak or noninhibitors of the VSOAClike transport in blastocysts [12]. In this regard, the EAAT4 and EAAT5 proteins (which are in the EAAT subfamily of anionic amino acid transporters) appear to serve better as anion channels than for Na1/amino acid cotransport [32, 33]. Moreover, the pharmacological agents that inhibit other anion channels do not inhibit EAAT4 channels [32]. It has apparently not been determined whether EAAT4 and EAAT5 also serve as channels for the organic osmolytes taurine, glycine, and aspartate, although such transport seems to be feasible in light of the ability of EAAT proteins to catalyze anionic amino acid symport with Na1 [9]. For these reasons, it will be interesting to learn whether the channel activity of EAAT4, EAAT5, or a related protein begins to help regulate cell volume when embryos form blastocysts. Similarly, a transport system for glutamine appears to be developmentally regulated in early embryos. The glutamine content of embryos in vivo increases dramatically, but transiently, at the four- to eight-cell stage of development, whereas the content of other amino acids does not increase in this way [19]. These data support the hypothesis that system N, which selectively transports glutamine, appears transiently at approximately this stage of development [18]. System N actively has, however, not as yet been detected in early embryos, possibly because it is expressed only briefly. The Na1-dependent transport of glutamine through a weak interaction with system Gly [25] also could account for concentrative glutamine uptake by embryos at the fourto eight-cell stage, at least in vitro. In vivo, however, the concentration of glycine in oviductal secretions appears to be approximately twice that of glutamine [34], so glycine would likely inhibit virtually all such glutamine transport. It is also conceivable that glutamine is produced metabolically at this stage of development, as appears to be the case in blastocysts [19]. Such a source of glutamine seems unlikely, however, because this amino acid does not accumulate to a high level in four- to eight-cell conceptuses that

develop in vitro in the absence of glutamine [19]. Regardless of the mechanism by which glutamine may accumulate, it appears to benefit embryos, at least in part, by protecting against osmotic stress in vitro and in vivo. In this regard, the glycine content of embryos in vivo decreases at the four- to eight-cell stage [19, 35], so glutamine accumulation at this stage may partly replace glycine to protect embryos from volume decreases and the concomitant effects on metabolism discussed below. Nonessential amino acids benefit development through osmoregulation and cellular signaling. Most mammalian cells probably require mechanisms for regulatory volume increases or decreases in response to hormonal (e.g., insulin and glucagon) and other stimuli known to alter cellular volume [36]. Additionally, preimplantation mouse embryos appear to develop in a somewhat hypertonic environment in oviductal fluid [37]. In vitro, glycine, alanine, glutamine, and taurine protect preimplantation embryos from the otherwise detrimental effects of hypertonic media [37–39]. Hence, it has become important to measure the osmolarity (or osmolality) of oviductal secretions during development. (Because the osmolarity and osmolality of the media described here are nearly equal, these terms are used interchangeably depending on their use in the original literature.) Collins and Baltz [40] reasoned that one- or two-cell embryos could themselves be utilized as osmometers by observing whether their cells shrink or swell when removed from oviductal secretions. They compared the volumes of such embryos over time in media of various measured osmolalities to their initial volumes and to the volumes of the embryos in oviductal secretions. Their results were consistent with the conclusion that the osmolality of oviductal fluid is approximately 290–305 mOsmol/kg [40]. Collins and Baltz did not include amino acids in their media lest they complicate their volume measurements. Such amino acids are, however, present in oviductal fluid [19], and their uptake could influence the volumes of one- and two-cell embryos significantly. In support of this possibility, we found that 1.0 mM glycine increases the volume of onecell embryos by approximately 10% after 15 min in medium of 310 mOsmol/kg (P , 0.02 for the volumes of 42 embryos in the presence of 1.0 mM glycine vs. the volumes of 50 embryos in medium not containing an added amino acid; Kruskal-Wallis H test [41]). Similarly, Dawson et al. [27] detected the same effect of glycine on embryo cell volume in medium of 310, but not of 250 or 340, mOsmol/ kg. We conclude provisionally from these data that the results of Collins and Baltz [40] would have been influenced slightly, but significantly, had the media also contained amino acids. In our estimation, such an influence probably would have raised the osmolality at which no volume change was detected from 290–305 to approximately 310– 320 mOsmol/kg. Media of 310–320 mOsM significantly impairs early embryo development in the absence of added amino acids [38, 39]. Only amino acids (or their less abundant analogues) for which Na1-dependent systems for their transport are known to be present in cleavage-stage conceptuses are able to improve development in vitro in somewhat hypertonic medium. Hence, glycine, taurine, and other substrates of the Na1-dependent systems Gly and b protect embryos from hyperosmotic media (Table 2), whereas lysine and leucine, for which transport is virtually entirely Na1-independent in cleavage-stage conceptuses [42, 43], do not [37, 39]. The small amount of mediated alanine transport that can be de-


tected in cleavage-stage conceptuses is Na1-dependent but otherwise only poorly characterized [21]. We attribute the modest ability of alanine to protect embryos from hypertonic stress [37] to this low but concentrative [19] transport activity. Similarly, circumstantial evidence discussed above indicates that a glutamine-selective Na1-dependent system such as N appears transiently during cleavage. Such a system could account for the beneficial effects of glutamine in somewhat hypertonic media [39]. Alternatively, glutamine could be concentrated [19] by the Na1-dependent, but otherwise poorly characterized, system for alanine transport in cleavage-stage conceptuses [21] or by system Gly [25] in the absence of good competing substrates such as glycine. Hence, glycine, taurine, alanine, and glutamine support preimplantation embryo development in somewhat hypertonic environments such as oviductal secretions, probably by maintaining a larger cellular volume (or a lower cellular protein concentration) [22, 37–39]. The larger cell volume resulting from concentrative amino acid accumulation likely helps to maintain proper balance between anabolic and catabolic processes in early embryos [22]. The signaling mechanisms by which an increase in cellular volume promotes anabolism are emerging [36, 44] but beyond the scope of this review. Similarly, growth factors may promote development of preimplantation embryos [45] by influencing the balance between anabolism and catabolism. Most of these growth factors have not been shown consistently [46] to act by increasing cellular volume either in cleavage-stage conceptuses or in other mammalian cells. For this reason, their mechanism of action appears to be unlike the mechanism involving cellular volume change because of nonessential amino acid transport. In this regard, the effects of nonessential amino acids and growth factors on embryos in somewhat hypertonic media do not appear to be additive and may even be competitive, at least for glycine and insulin-like growth factor I or II (Fig. 1). Hence, signaling by growth factors and by nonessential amino acid transport may be incompletely redundant mechanisms to ensure development of healthy preimplantation conceptuses. As for glycine and taurine, aspartate appears to be transported by the VSOAC expressed in one- and two-cell conceptuses [28]. Aspartate is, however, present in early embryos at too low a concentration to contribute much to the required regulatory volume decreases, at least in vitro. Moreover, a Na1-dependent system for accumulation of anionic amino acids [19] does not appear in conceptuses until the blastocyst stage [24], so uptake of aspartate likely would not promote preimplantation development in somewhat hypertonic media. We propose instead that a system develops in blastocysts to regulate the concentrations of aspartate and glutamate in uterine secretions at sites of implantation. In particular, system XAG2 activity appears to come under regulation by the uterine environment just before blastocyst implantation [19]. If uterine or blastocyst tissues express receptors for anionic amino acids at this time, then glutamate/aspartate transport (or its inhibition) could influence the signaling among these tissues that is needed for implantation. Glutamate/aspartate receptors have not as yet been detected in the uterus or blastocyst, but their location in the periphery is well documented [47]. Moreover, the Cl2 channels in blastocysts discussed above that may contain the transport protein EAAT4 or EAAT5 are believed to serve for signaling in the cerebellum and retina [48]. Hence, they could also be involved in signaling in blastocysts as a result of a change in the concentrations

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FIG. 1. Glycine (1.0 mM) and insulin-like growth factors (IGF) I and II (100 ng/ml) each increase the proportion of two-cell embryos that develop into blastocysts in somewhat hypertonic medium. Oviductal fluid-like medium [37] was adjusted to a total osmolality of 310–320 mOsmol/kg with water and then used to culture two-cell embryos to the blastocyst stage as described previously [37]. The proportion of 85 embryos in each group that developed into blastocysts after 3 days was determined in three separate experiments (60 embryos and two experiments in the case of glycine plus IGF-II). Groups marked with different letters are significantly different (P , 0.05 for a vs. b; P , 0.01 for b vs. c and for c vs. d) as determined using contingency tables [37].

of their ligands, glutamate and aspartate, in uterine secretions. Whereas such signaling by nonessential amino acids is speculative, essential amino acid transport in blastocysts almost certainly helps to regulate the signaling needed for embryo growth, communication with the uterus, and even suppression of T-cell proliferation. Essential Amino Acids Beneficial to Development

Essential amino acids increase the cleavage rate in embryos after the eight-cell stage and, thus, promote development of blastocysts with more cells in their inner cell masses. Such blastocysts more frequently give rise to viable fetuses upon transfer to surrogate mothers than blastocysts that did not develop in the presence of essential amino acids [16]. The fetuses are also larger when blastocysts develop in medium containing essential amino acids. Whereas an obvious function of essential amino acids in this case is to promote cellular growth and development as nutrients, these substances also likely perform important signaling functions, and their transport may help to regulate other peri-implantation processes (Table 2). To conclude that these processes depend on transport rather than simply the presence of exogenous essential amino acids, it is important

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FIG. 2. Expression of mRNA encoding the CAT1 protein for arginine transport in preimplantation mouse conceptuses. Polymerase chain reaction (PCR) was used to amplify cDNA segments of the indicated size in libraries produced from oocyte, two- and eight-cell embryo, and blastocyst mRNA [93, 113, 114]. A PCR product was not obtained in the blank lane for the oocyte library, although a product was detected for this library in several other experiments. Similar results were obtained for amplification of a portion of the sequence encoding CAT2 (data not shown). In addition, reverse transcription (RT)-PCR was used to detect both CAT1 and CAT2 mRNA in extracts from one-cell conceptuses (not shown). The nucleotide sequences of the amplified segments were determined to verify that they did, in fact, encode portions of CAT1 and CAT2. The procedures used for RT-PCR are described more completely by Van Winkle et al. [22]. 100bp, 100 Base pair ladder; 2, two-cell embryo; 8, eight-cell embryo; B, blastocyst; C, control PCR from a known CAT1 cDNA clone.

to show that transport processes help to regulate the amounts of essential amino acids that conceptuses contain. Incubation of mouse eggs and blastocysts for 1 or 2 h with the essential amino acid methionine (100 mM) greatly increases the amount of this amino acid they contain [49]. Although blastocysts were incubated with methionine for twice as long as eggs [49], we attribute the 10-fold greater accumulation of this amino acid against its total chemical potential gradient in blastocysts to their conspicuous Na1dependent system B0,1 activity [20, 21] (Table 1). Only very small amounts of Na1-dependent transport activity capable of accumulating methionine are present in embryos before the eight-cell stage [21]. Moreover, most of the methionine taken up by eggs appears to have been in exchange for other system L substrates [49]. Little or none of the cationic amino acids in eggs (i.e., lysine, arginine, and histidine) are lost in exchange for methionine, whereas these amino acids are greatly depleted by methionine in blastocysts [49]. These observations can be accounted for by the dramatic, 30-fold increase in system b0,1 activity that occurs as blastocysts form [42]. System b0,1 appears to function primarily for exchange of zwitterionic amino acids (e.g., methionine) for cationic amino acids under physiological conditions [9]. Whereas these data relating transport activity with the essential amino acid content of early embryos are not extensive, they seem adequate to support the conclusion that essential amino acids improve development

largely because they are transported into preimplantation embryos. Developmental regulation of essential amino acid transport. As for nonessential amino acid transport, developmental regulation of essential amino acid transport likely depends on expression of pertinent transporter or accessory protein mRNAs. Embryos have, however, been tested for only two of these mRNAs so far (Table 1). We detected sequences encoding cationic amino acid transporter 1 (CAT1) and CAT2 in cDNA libraries produced from mouse oocyte, two- or eight-cell embryo, or blastocyst mRNA (Fig. 2). Moreover, we used RT/PCR to detect mRNA encoding CAT1 and CAT2 in fertilized mouse eggs (L.J. Van Winkle and A.L. Campione, unpublished data). Hence, these transporters likely account for cationic amino acid transport by systems b11 and b12 in one- to two-cell embryos and blastocysts, respectively [50]. A system with characteristics intermediate to these two systems selectively transports cationic amino acids at the eight-cell stage [50]. In adult tissues, CAT1 and CAT2 display characteristics of the better-known system y1 [51–53]. Because the characteristics of systems y1, b11, and b12 have been carefully distinguished from one another [50], as-yet-unidentified accessory proteins may modify the transport characteristics of CAT1 and CAT2 expressed in embryos at different stages of preimplantation development. The existence of such accessory proteins could account for our findings that CAT1 or CAT2 expression in Xenopus oocytes produces multiple components of transport [9]. Additionally, CAT1 and CAT2 could be expressed selectively in different embryo cell types, especially after blastocysts form. At the one-cell stage, expression of both CAT1 and CAT2 likely means that system b11 actually is composed of at least two transport activities, which are provisionally designated as systems b11a and b11b (Table 1). Developmental regulation of other Na1-independent transport systems selective for essential amino acids in preimplantation embryos also probably depends on expression of mRNAs encoding the pertinent transport proteins (Table 1). System b0,1, which increases in activity in embryos by approximately 30-fold as they form blastocysts, likely contains a transport protein similar to the b0,1 amino acid transporter (b0,1AT) [54, 55]. Mutations in the gene encoding b0,1AT abolish transport activity and cause non-type I cystinuria in humans [54]. We propose that mRNA encoding b0,1AT or a related protein increases greatly in amount in mouse embryos as they form blastocysts. It is also possible that b0,1AT and its mRNA are expressed more or less equally throughout development, but that b0,1AT transport activity is altered by changes in expression of either or both of the two accessory proteins, rBAT (related to b0,1 amino acid transporter) and the heavy chain of the cell surface antigen 4F2 (4F2hc) [12, 56]. Changes in 4F2hc or rBAT expression could function to regulate not only amino acid transport but also cellular proliferation, differentiation, adhesion, and fusion in early embryos [57]. Systems L and T in embryos also may be composed of transport and accessory proteins at all stages of preimplantation development (Table 1). Members of the system L amino acid transporter (LAT) family [57] likely are the transport proteins in these systems. The substrate selectivity of transport system L in early embryos [42, 58] resembles that of LAT1 [59]. No such protein has been identified for system T, but its characteristics resemble system L, though with a more restricted amino acid substrate selectivity (Table 1). Modulation of systems L and T transport activities


during development [42, 58] might occur as a result of changes in expression of the types and amounts of LAT family members and their accessory proteins. One other Na1-independent system in embryos also likely contains a member of the Na1-independent amino acid transporter superfamily. System xc2 for cystine (here considered essential) and glutamate transport has relatively high activity in unfertilized and fertilized mouse eggs, but its activity is greatly reduced in two-cell conceptuses and appears to be lost by the time that blastocysts form [60]. System xc2 in macrophages contains a transport protein termed system xC transporter (xCT) [61]. We propose that system xc2 in mouse eggs contains xCT or a related protein, but that this protein is lost from embryos because the mRNA encoding it is not produced from the early embryo genome. As for the cases discussed above, it is also conceivable that system xc2 transport activity is regulated during development by changes in expression of 4F2hc or a related accessory protein (Table 1). Finally, expression of Na1-dependent systems selective for essential amino acids may be regulated by expression of mRNAs encoding their transport proteins. The dramatic increase in system B0,1 activity in embryos at the blastocyst stage [21] probably results from transcription of the gene encoding amino acid transporter B0,1 (ATB0,1) or a related protein (Table 1). Similarly, accumulation of mRNA encoding a member of the Na1-dependent EAAT/ASC family, such as the system ASC-like transporter 2 (ASC2), may account for the increase in system B activity when embryos form blastocysts (Table 1). Developmental regulation of essential amino acid transport system expression likely helps to ensure that the systems are present when they are needed in embryos. Further regulation of the systems (probably by other means) appears, however, to be needed for them to fully perform their physiological functions. Protein synthesis and accumulation. Essential amino acid transport and its regulation may have several interdependent functions in embryos after the eight-cell stage of development. First, essential amino acids likely are needed for the net protein accumulation that occurs in blastocysts in vivo during the 10 h preceding implantation [62]. In vitro, essential amino acids appear to support net protein accumulation somewhat earlier in development in association with more rapid cell division and development of blastocysts with more cells in their inner cell masses [16]. Such blastocysts are more likely to form fetuses when transferred to surrogate mothers than are those that do not develop in the presence of essential amino acids, and the fetuses are larger when the blastocysts develop in the presence of these nutrients [16]. Moreover, each essential amino acid, except perhaps isoleucine, increases the proportion of mouse blastocysts that form trophoblast outgrowths in vitro [63]. The growth of blastocysts appears further to be stimulated by chymotrypsin and chymotrypsin-like enzyme activity. Exposure of blastocysts to chymotrypsin in vitro for 24 h results subsequently in their more rapid attachment and outgrowth in culture [64]. Moreover, chymotrypsin-like enzyme activity increases transiently by approximately fourfold in mouse uterine washings between 6 and 12 h before blastocyst implantation [65]. Significantly, essential amino acid transport by systems B0,1, b12, and probably, b0,1 also is stimulated by chymotrypsin in vitro [12] (unpublished data showing a twofold stimulation of system B0,1, but not of system L, by chymotrypsin; P , 0.01). In vivo, the potential of blastocysts to express system B0,1 activity increases dramatically just after the rise in chy-

7

motrypsin-like enzyme activity in uterine secretions, although this system B0,1 activity may remain, at first, largely latent [66]. The greater capacity of blastocysts to transport essential amino acids after exposure to chymotrypsin undoubtedly supports their more rapid growth. We propose that chymotrypsin acts to stimulate essential amino acid transport and, probably, other events in embryos via a novel serine protease-activated receptor [12], although this receptor and the full effects of its activation remain to be identified. Arginine transport via system b0,1 in blastocysts also is more rapid when these embryos develop in vitro in the presence of this preferred b0,1 substrate (P ,0.01; unpublished results), and the more rapid transport cannot be attributed to transtimulation. Regulation of essential amino acid transport by chymotrypsin and arginine could conceivably have broader implications, because it also seems likely to occur in the intestine, where systems similar to B0,1 and b0,1 are conspicuous [11–14]. Dietary essential amino acids, including arginine, are, of course, also present in the lumen of the intestine, as are serine proteases after a meal. Hence, amino acids and proteases might increase the absorptive capacity of the intestine as they do the embryo. Preimplantation blastocysts, however, also need to grow. More rapid transport of essential amino acids may support this growth not only as nutrients but also as signaling molecules that influence protein kinases involved in protein synthesis regulation. Essential amino acids act as novel signaling elements in insulin-sensitive tissues [67–71]. Because early embryos contain insulin-sensitive tissues [45], they probably also respond to essential amino acid signaling molecules. Essential amino acids appear to stimulate protein synthesis and accumulation via the serine-threonine protein kinase, target of rapamycin (TOR), and its downstream targets, p70 S6 kinase and 4E-BP1. The 4E-BP1 (also called PHAS-I) is a translational repressor whose association with eukaryotic initiation factor, eIF-4E, is decreased by phosphorylation. Similarly, phosphorylation of ribosomal protein S6 by p70 S6 kinase may result in preferential translation of mRNAs encoding proteins involved in protein synthesis. Thus, signaling by essential amino acids leads to an increase in the amount of protein synthetic machinery in cells and to an increased in the utilization of this machinery. Such a mechanism could explain how amino acids may help to maintain mRNAs at their normal levels in blastocysts [72]. In this regard, 200 nM rapamycin delays the onset of mouse blastocyst outgrowth in vitro (P , 0.01; unpublished data), probably by slowing or preventing an increase in the rate of protein synthesis. Furthermore, leucine and, probably, arginine are among the most effective essential amino acid signaling molecules [67–71]. Whereas system B0,1 and, to a lesser extent, system b0,1 transport a wide variety of zwitterionic and cationic amino acids in blastocysts, their preferred substrates based on Km values are leucine (ùtryptophan) and arginine, respectively [23, 42, 58]. Moreover, leucine, arginine, and glucose deprivation together inhibits mouse blastocyst outgrowth in vitro [73], and arginine is maximally able to support outgrowth at a concentration (1.0 mM) [74] approximately equal to its Km value for transport by system b0,1 [42]. For these reasons, chymotrypsin- and substrate-stimulated system b0,1, B0,1, and b12 transport of leucine and arginine should promote growth of blastocysts both by an increased nutrient supply and by signaling that results in an increased capacity for and rate of protein synthesis. Moreover, arginine is a substrate for the synthesis of polyamines

8

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FIG. 3. Propagation of total chemical potential gradients of leucine, tryptophan, and other zwitterionic amino acids by system b0,1 to form a similar gradient of arginine. Assuming a 1:1 stoichiometry for the antiport catalyzed by system b0,1, the maximum gradient of arginine would approach that of the combined gradients of its countersubstrates (see Van Winkle [9] for theoretical considerations). In the scheme presented, cells in the embryo accumulate good substrates of system B0,1 against their gradients by symport with Na1 along its gradient (maintained by Na1K1ATPase). The good B0,1 substrates, leucine and tryptophan, are preferred substrates for heteroexchange for arginine (and lysine) by system b0,1. Such heteroexchange of cationic for zwitterionic amino acids appears to be the usual way that system b0,1 functions under physiological conditions [9].

and nitric oxide (NO), and each of these substances is needed for normal pre- and peri-implantation development [75– 83]. Polyamine and NO synthesis from arginine. Blastocysts contain conspicuous amounts of three of the six or so known transport systems for cationic amino acids (i.e., B01, b0,1, and b12), and arginine is the preferred substrate of two of these systems (i.e., b0,1, and b12) [42, 50]. Moreover, arginine transport appears to limit and, perhaps, to regulate NO production in endothelial cells [84], macrophages [85], and probably, other types of cells. Similarly, thrombin-stimulated vascular smooth muscle cell polyamine synthesis results, in part, from the induction of expression of the arginine transporters CAT1 and CAT2 [86]. In blastocysts, arginine transport system activities likely are stimulated via a novel chymotrypsin-activated receptor both in vitro and in vivo (Table 2 and see above). Polyamine and NO production from arginine are needed in early mouse and rat embryos both for their development and for their implantation in the uterus [75–83]. Probably, NO signals local vasodilation in the uterus and the increased capillary permeability needed for implantation [80–83]. Too much NO production appears to be detrimental to these processes, however [87], so it is reasonable to suppose that arginine transport is carefully regulated. Particularly at the time of implantation, arginine accumulation by blastocysts might be highly susceptible to regulation by the uterine environment. During the few hours immediately preceding blastocyst implantation, system B0,1 transport activity appears to be greatly suppressed by the uterine environment [21, 66]. Such inhibition of system B0,1 activity would slow arginine

accumulation not only directly by inhibition of its transport via this Na1-dependent transport system but also indirectly by concomitant inhibition of system b0,1-catalyzed exchange. In fact, the latter mechanism is probably the main way that arginine accumulation is suppressed, because arginine is the preferred substrate of system b0,1 [42]. Arginine would be concentrated in cells when good substrates of system B0,1 (e.g., leucine and tryptophan) are taken up against their total chemical potential gradients because of cotransport with Na1 along its gradient (Fig. 3). The Na1 is, of course, extruded to maintain its gradient by Na1K1-ATPase [88]. The gradients of good substrates of system B0,1 could then be used to concentrate cationic amino acids against their gradients through exchange via system b0,1 [9, 12]. The best substrates of system B0,1 are also among the best zwitterionic substrates of system b0,1. Moreover, system b0,1 appears to function under physiological conditions primarily for heteroexchange of zwitterionic amino acids for cationic ones [9]. Because arginine is the preferred substrate of system b0,1 based on Km values, system b0,1 should serve to concentrate arginine inside cells against its total chemical potential gradient to the same degree that system B0,1 concentrates its best substrates (Fig. 3). Nearly complete inhibition of system B0,1 activity by the uterine environment [66] could, therefore, reduce or prevent accumulation of leucine, tryptophan, and other zwitterionic amino acids and, thus, limit the supply of arginine to the trophoblast during the 6 h before implantation. We propose that removal of this inhibition in blastocysts at approximately the time of implantation would increase the supply of arginine to NO synthase (NOS) and, hence, might result in a burst of NO production. Moreover, this rapid and large increase in the ability of systems B0,1 and b0,1 together to concentrate leucine, tryptophan, and arginine in trophoblast cells and, hence, to deplete the amino acids from uterine secretions may also help to suppress T-cell proliferation and, thus, to protect the trophoblast from rejection. Suppression of T-cell proliferation. Munn et al. [89] caused T cell-induced rejection of all allogeneic conceptuses by Day 9.5 postcoitus (p.c.) in mice, apparently by preventing T-cell tryptophan deprivation. They administered an inhibitor (1-methyl-DL-tryptophan) of the rate-limiting enzyme in tryptophan catabolism, indoleamine 2,3dioxygenase (IDO), in slow-release pellets placed under the dorsal skin on Day 4.5 p.c. Allogeneic, but not syngeneic, conceptuses were rejected after this treatment to inhibit tryptophan catabolism. T-cell proliferation is inhibited in medium depleted of tryptophan by macrophages induced to express IDO [90], and IDO is present in the syncytiotrophoblast, at least in humans [91]. Moreover, interferon-g (IFN-g) induces expression of IDO in many cultured cell lines [92] as well as in macrophages [90], and IFN-g likely is produced by mouse blastocysts [93]. For these reasons, IDO in the trophoblast appears to prevent T cell-induced rejection of conceptuses by lowering the local tryptophan concentration to below that needed for T-cell proliferation. For its concentration to be lowered, however, tryptophan must first be transported across the plasma membrane to the cytosolic location of IDO. In this regard, system B0,1 for plasma membrane tryptophan transport persists in the rodent trophoblast after implantation [94], and 1-methyl-DLtryptophan inhibits system B0,1 (unpublished data) as strongly as it inhibits IDO [95]. Moreover, the rate of tryptophan catabolism in hepatocytes depends on both the rate of its biomembrane transport and the rate of its catabolism


initiated by dioxygenase [96]. Hence, 1-methyl-DL-tryptophan likely promotes T-cell proliferation by inhibiting both tryptophan transport and catabolism. T-cell proliferation might also normally be inhibited earlier in development, at approximately the time of implantation, if system B0,1 and, consequently, system b0,1 (Fig. 3) are activated to accumulate arginine, leucine, and tryptophan in blastocysts and out of the surrounding uterine secretions. The initial intimate contact between mother and conceptus occurs at implantation, so T cell-induced rejection might otherwise begin at this relatively early time during development. Because the volume of uterine secretions is very small at the time of implantation [97], blastocysts likely would decrease significantly the concentrations of essential amino acids in neighboring secretions if their largely latent system B0,1 [21, 66] became fully active at this time. Moreover, the Km values for leucine, tryptophan, and arginine transport via systems B0,1 and b0,1 are in the low micromolar range [23, 42, 58], and they are lower than the Km value of IDO for tryptophan [95, 98]. Hence, systems B0,1 and b0,1 together might be as able as IDO to lower the local extracellular concentrations of their substrates to approximately 0.1 mM (i.e., the tryptophan concentration at which T-cell proliferation is maximally inhibited [90]) depending on the intracellular amino acid concentrations in blastocysts [9]. In this regard, the INF-g produced by blastocysts [93] could conceivably induce transient IDO expression [90, 92] in the trophectoderm before implantation. Because 1-methyl-DL-tryptophan inhibits both system B0,1 and IDO with Ki values in the micromolar range, it will be interesting to learn whether this inhibitor also promotes rejection of peri-implantation blastocysts before Day 9.5 p.c. when administered in a way that inhibits system B0,1 and possible IDO in blastocysts at the time of implantation on Day 4.5 p.c. SUMMARY AND FUTURE DIRECTIONS System B0,1 and other amino acid transport systems in early mouse embryos likely are regulated at the genetic level to appear during development when the transport they catalyze is physiologically beneficial. The transport is further regulated by various conditions and substances in the female reproductive tract to optimize the functions of the systems. The precise mechanisms of regulation and their physiological consequences are, however, yet to be fully described. Hence, studies are needed to determine whether known or new isoforms and subisoforms of transport proteins are present in most transport systems in early embryos. It will also be interesting to determine the mechanisms by which genes are regulated in cases where such regulation underlies the dramatic changes in transport system expression that occur during preimplantation development. Similarly, it remains to be determined how components of the uterine environment regulate transport system activities in blastocysts. Identification of a novel chymotrypsinactivated receptor in blastocysts would help to explain how chymotrypsin activity in vitro and chymotrypsin-like enzyme activity in vivo increase transport by systems B0,1, b12, and b0,1. Moreover, identification of arginine receptor sites that are separate from its sites of transport would help to explain how this amino acid stimulates its own transport by system b0,1 in blastocysts. Similar sites for the stimulation of protein synthesis via protein kinases are known for leucine as well as for arginine in other cells, and such could be the mechanism for stimulation of system b0,1 transporter or accessory protein expression in blastocysts.

9

The increased capacity to transport these and other essential amino acids likely contributes to the accumulation of protein in preimplantation blastocysts in vitro and in vivo, both by supplying nutrients and by signaling via the target of rapamycin pathway. Increased uptake of leucine, arginine, and tryptophan from uterine secretions via systems B0,1 and b0,1 at approximately the time of implantation also could help to protect peri-implantation blastocysts from rejection by inhibiting T-cell proliferation. Amino acid uptake in the latter case likely is in excess of requirements for protein synthesis, and the amino acids are metabolized to NO and polyamines in the case of arginine and, possibly, beginning with IDO in the case of tryptophan. Additionally, IDO, inducible nitric oxide synthase, and amino acid transport all are induced by INF-g in one cell type or another [99, 100], so INF-g production by blastocysts may have multiple, overlapping paracrine and autocrine functions around the time of implantation. In this regard, NO production is doubled on exposure of the implanting mouse trophoblast to INF-g [101]. For all these reasons, it will be interesting to learn whether NO promotes implantation by interacting with the IDO pathway [99] in addition to promoting vasodilation and increased capillary permeability. Signaling by glutamate and aspartate also could conceivably contribute to these processes. Earlier in development, metabolism appears to be regulated by changes in NO production, growth factor levels, and cell volume, but the details of these signaling processes and their effects on metabolism in preimplantation conceptuses are only now emerging. Nevertheless, nonessential amino acid transport likely is needed for much of the latter signaling. Hence, nonessential and essential amino acid transport supports pre- and peri-implantation development, respectively, by a variety of signaling processes as well as by supplying important nutrients to embryos. ACKNOWLEDGMENTS The author thanks Drs. Philip Iannaccone, Richard Tasca, and Susan Viselli for useful comments about earlier versions of the manuscript and Allan Campione and Barb Le Breton for helping to prepare it. Dr. Barbara Knowles generously supplied the cDNA libraries used to produce Figure 2.

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