BIOLOGY OF REPRODUCTION 81, 1041–1054 (2009)
Published online before print 15 July 2009.
Amino Acid Transport Mechanisms in Mouse Oocytes During Growth
and Meiotic Maturation1
Ame ´lie M.D. Pelland, Hannah E. Corbett, and Jay M. Baltz2
Ottawa Hospital Research Institute and Departments of Obstetrics and Gynecology (Division of Reproductive
Medicine) and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
Amino acids are transported into cells by a number of
different transport systems, each with their own specific range of
substrates. The amino acid transport systems active in preim-
plantation embryos and the amino acids required by embryos for
optimal development have been extensively investigated. Much
less is known about amino acid transport systems active in
growing and meiotically maturing oocytes or about develop-
mental changes in their activity. As a first step in determining the
array of amino acid transporters active in oocytes, the transport
characteristics of nine amino acids were measured in small,
medium, and large growing oocytes; in fully grown germinal
vesicle (GV)-stage oocytes; in metaphase I oocytes; and in
metaphase II eggs. Whether each of 11 classically defined amino
acid transport systems was likely active in oocytes at each stage
was determined using assays based on measuring the transport of
radiolabeled amino acids into oocytes and the effect of a limited
set of potential competitive inhibitors. Six amino acid transport
systems were found to be active during oocyte growth or
maturation. L, b0,+, and ASC/asc were active throughout oocyte
growth and maturation, increasing during growth. In contrast,
GLY, beta, and xc
became activated during meiotic maturation. Surprisingly, the
presence of follicular cells surrounding medium growing oocytes
or cumulus cells surrounding GV oocytes did not confer amino
acid transport by additional transport systems not present in the
oocyte. In some cases, however, follicular cells coupled to the
oocyte enhanced uptake of amino acids by the same systems
present in the oocyte.
?had little or no activity during growth but
amino acid transport, cumulus cells, granulosa cells, meiosis,
The availability of amino acids substantially enhances
mammalian preimplantation (PI) embryo development and
developmental potential [1–3], and amino acids are now a key
component of most mammalian embryo culture media .
Some amino acids and combinations of amino acids have been
shown to stimulate PI embryo development from the 1-cell
stage in culture, while others were inhibitory . In addition,
the effects of amino acids are stage dependent, with different
amino acid requirements, for example, in postcompaction vs.
cleavage-stage embryos [2, 3].
Virtually all significant effects of amino acids in the external
environment on PI embryo development are due to amino acid
transport into embryos via selective transport systems . It
has long been known that mammalian cells possess an array of
specialized transport systems for amino acids [3, 6]. These
amino acid transporters generally do not transport only single
amino acids but rather accept groups of related compounds as
defined by the transporter’s substrate binding site structure.
Amino acid transport systems were defined classically by the
range of their substrate specificities and by the presence or
absence of obligate Naþcotransport [7, 8] (Table 1). Many of
these systems exist in several variants with differences in the
array of substrates accepted, kinetics, or regulation. In most
cases, the more recent identification of the genes encoding the
proteins for each transport system revealed that these variants
represented products of distinct genes, usually within the same
gene family (Table 1).
The amino acid transporters active in PI embryos,
particularly of the mouse (Table 1), have been extensively
studied . A striking feature of virtually all of these is that
they are developmentally regulated during the PI period and are
active only during specific PI embryo stages. Thus, the classic
glycine transport system GLY is highly active during the early
cleavage stages but is inactive after compaction [16, 17]. In
contrast, system B0,þ, a transporter that accepts a wide array of
cationic and neutral amino acids, is reportedly active only in
blastocysts . These expression patterns reflect PI embryo
physiology because glycine is required for cell volume
regulation at the early cleavage stages but not later [17, 19],
while B0,þactivity regulates trophoblast motility during
blastocyst implantation in the uterus .
There is substantially less information about amino acid
transport and transporter activity during the growth and meiotic
maturation of oocytes. Several studies [21–26] have shown that
amino acids can be taken up by fully grown germinal vesicle
(GV)-stage oocytes. However, in most cases, it had not been
established that transport was by a saturable transporter, and
later work in some of these cases showed that it was not (e.g.,
proline ). The identity of the specific transport system
responsible for uptake has been determined in even fewer
cases. Colonna et al.  reported that leucine uptake by GV
oocytes had the characteristics of system L transport, while
alanine transport was by system L and by transport resembling
ASC activity. Haghighat and Van Winkle  similarly
identified the glycine transport system in GV oocytes as
GLY, while the small amount of cystine and glutamate
transport in GV oocytes was identified by Van Winkle et al.
 as xc
1This work was part of the Program on Oocyte Health funded under the
Healthy Gametes and Great Embryos Strategic Initiative of the
Canadian Institutes of Health Research (CIHR) Institute of Human
Development, Child and Youth Health (IHDCYH), grant number
2Correspondence: Jay M. Baltz, Loeb Research Centre, Ottawa Hospital
Research Institute, Ottawa, ON K1Y 4E9, Canada. FAX: 613 761 5196;
Received: 22 May 2009.
First decision: 10 June 2009.
Accepted: 23 June 2009.
? 2009 by the Society for the Study of Reproduction, Inc.
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More studies [16, 26, 28, 29] of amino acid transport have
included unfertilized metaphase II (MII) eggs, with systems b,
L, GLY, xc
amino acid transporters found in MII eggs were the same as in
1-cell embryos, and their activities were comparable [16, 26,
28, 29]. An exception is the proline and betaine transporter
SLC6A20 (system IMINO), which is quiescent in MII eggs but
is activated several hours after fertilization .
Although there is some information about amino acid
transport in GV oocytes, and considerably more in MII eggs, it
was obtained separately and under different conditions. Very
few reported investigations have systematically assessed amino
acid transport characteristics over the course of meiotic
maturation, including the demonstration by Colonna et al.
 that system L activity decreased somewhat from the GV to
MII stages and a report of unpublished data by Van Winkle et
al.  showing that system xc
fold during oocyte maturation. Thus, there is little information
available about specific amino acid transport or amino acid
transporters in GV oocytes and especially about any changes
they undergo during meiotic maturation.
Even less information is available about amino acid
transport in growing oocytes. It was shown that leucine,
alanine, and glycine uptake, attributed to systems L, ASC, and
GLY, respectively, was present in medium (diameter, ;55–65
lm) growing oocytes [21, 25], and L activity was also reported
in small (;40–50 lm) oocytes . However, whether the
array or activity of amino acid transporters changes over the
course of oocyte growth has essentially not been determined.
One area that has received more attention is the question of
whether follicular granulosa cells provide the enclosed oocyte
with amino acids. During growth, the oocyte is coupled to the
surrounding granulosa cells via gap junctions, a connection that
?, and b0,þshown to be present in eggs. In general,
?activity increased by about 5-
is required for both oocyte and follicle viability and growth
. After ovulation and cumulus expansion, this oocyte-
cumulus coupling is lost, and compounds taken up by cumulus
cells can no longer be directly transferred into the enclosed
oocyte [31, 32]. It is widely accepted that cumulus cells take up
a number of compounds that are transported at low rates by
oocytes and transfer them to the enclosed oocyte via gap
junctions, including uridine, choline, and pyruvate [31–33].
Amino acids whose uptake by oocytes was similarly increased
several-fold by the presence of coupled cumulus cells include
glycine, alanine, proline, histidine, and serine, with modest
stimulation of lysine, glutamate, and tyrosine [22, 24, 25].
However, this effect is selective, with no stimulation of leucine,
valine, or phenylalanine uptake by oocytes within the cumulus-
oocyte complex (COC) vs. denuded oocytes [22, 24, 25].
Eppig et al.  recently found that Slc38a3 mRNA encoding
a subtype of the system N transporter is present in cumulus
cells but not in oocytes and that substrates accepted by this
transporter (histidine and alanine) are accumulated by enclosed
oocytes at a higher rate when cumulus is present.
By analogy with advances in PI embryo culture media,
growing and maturing oocytes should also benefit from
inclusion of stage-appropriate amino acids in culture medium.
Also, elucidation of the developmental pattern of amino
transport in PI embryos has helped reveal key features of their
physiology, including cell volume regulatory systems [19, 27,
34], protective mechanisms against oxidative stress [3, 26], and
signaling of trophoblast implantation [20, 35]. Similar insights
may be expected with growing and maturing oocytes.
However, the current extensive knowledge of amino acid
transport in PI embryos has resulted from many years of very
detailed work on each system or amino acid substrate. To
unequivocally show that a given amino acid transport system is
TABLE 1. Amino acid transport systems assessed.
Preferred substrates PI embryo activityb
Ion dependenceGene(s) Accessory protein genec
A Small aliphatic (e.g., Ala), MeAIBNone; poss. ICMNaþ
asc Ala, Ser, CysUnknown None Slc3a2
ASC Ala, Ser, Cys
SLC1A5 broader specificityd
Cationic and large neutral
Neutral, cationic, bicyclic, BCH
Cationic (e.g., Arg, Lys)
1c-Bl; Bl highest
Large branched (e.g., Leu), BCH
N Gln, His, AsnUnknown Naþ
Cystine, Glu 1cNone
aThe first three columns are adapted from information compiled by Wan Winkle et al.  and references therein, with the addition of published
information on substrates, cotransported ions, and genes [9–13].
cReferences [3;15]; proteins are also known as 4F2hc (Slc3a2) and rBAT (Slc3a1) .
dSLC1A5 may underlie the neutral amino acid transport system designated B that is an inconspicuous component of transport in blastocysts .
eA number of different CAT-related transporters with differing specificities are designated yþin somatic cells and bþvariants in blastocysts .
PELLAND ET AL.
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will need to be investigated further (e.g., by assessing the effect
of inhibition of protein synthesis, or the effect of inhibition of
metabolism by decreasing temperature, on measured CIs).
In conclusion, we have found herein that oocytes exhibit at
least six of the classic amino acid transport systems (Fig. 7).
Three (GLY, b, and xc
and appeared or were strongly upregulated during meiotic
maturation (Fig. 7B), while the remaining three (b0,þ, L, and
asc/ASC) seemed to be constitutively active throughout oocyte
growth and maturation. Surprisingly, no additional transport
activities were identified in follicles or COCs that were not
already present in denuded oocytes, although apparent
metabolic coupling to follicular cells enhanced amino acid
uptake in several cases (Fig. 7A). We have thus determined the
likely array of major amino acid transport systems available to
the growing and maturing mouse oocyte. These transport
systems likely contribute to the uptake of amino acids needed
for protein synthesis during oocyte growth. In addition, the
three systems that become activated during meiotic maturation
likely provide amino acids for specific physiological functions
such as cell volume regulation and protection against oxidative
stress. Future work, building on the identification of active
transport systems in oocytes and follicles, will be needed to
determine the full array of amino acids taken up by oocytes at
each stage, their physiological functions, and the role of follicle
cells under physiological conditions.
?) had low activity in growing oocytes
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