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.
eISSN: 1529-7268 http://www.biolreprod.org
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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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present requires a large number of measurements of kinetic
properties, substrate specificities, and inhibition profiles ,
which would be difficult and very time-consuming to carry out
on different stages of growing and maturing oocytes with and
without surrounding follicular cells.
Fortunately, we now know enough about the transport
characteristics of the major classically defined amino acid
transport systems and about their molecular underpinnings that
simple tests can be devised to indicate the likely activity of
each system. Therefore, we have undertaken experiments
designed to determine whether major amino acid transport
systems (Table 1) are active in growing mouse oocytes at three
stages of growth and in fully grown GV oocytes, MI oocytes,
and MII eggs. We also assessed the effect of the presence of
granulosa cells on amino acid uptake by the enclosed oocyte
for growing oocytes and of cumulus cells for GV oocytes. This
provides the first complete picture of the likely array of amino
acid transporters present in growing and maturing oocytes of
any mammalian species and their levels of activity, and the
study findings revealed several systems that undergo substan-
tial changes in activity during meiotic maturation.
MATERIALS AND METHODS
Chemicals and Media
All chemicals and enzymes, including amino acids and analogues, were
obtained from Sigma (St. Louis, MO) unless otherwise noted. In addition to
standard a- and b-amino acids, we used cysteic acid and the amino acid
analogues 2-(methylamino)isobutyric acid (MeAIB) and 2-endoamino-bicy-
cloheptane-2-carboxylic acid (BCH) as described herein. All components of
culture media were embryo-tested grade or cell culture grade. Collagenase type
I was obtained from Worthington Biochemical Corporation (Lakewood, NJ).
The specific SLC6A9 (GLYT1) inhibitor ORG23798 was a kind gift of
Organon, Cambridge, England. ORG23798 was diluted from a stock in
dimethyl sulfoxide to a final concentration of 5 lM in medium previously
shown to completely inhibit glycine transport by the GLY transporter (GLYT1,
officially known as SLC6A9) in PI embryos .
The following radiolabeled amino acids were obtained from Amersham
Biosciences (Arlington Heights, IL):
L-[2,3,4,5-3H]arginine monohydrochloride (35–70 Ci/mmol),
partic acid (15–50 Ci/mmol), L-[35S]cystine (40–250 Ci/mmol), L-[G-3H]glut-
amine (20–50 Ci/mmol [G denotes general labeling with tritiated water]),
[3H]glycine (10–30 Ci/mmol),
L-[4,5-3H]lysine monohydrochloride (75–100 Ci/mmol), and [1,2-3H]taurine
(5–30 Ci/mmol). Henceforth, chirality and labeled groups are omitted for
brevity (e.g., [3H]alanine and [35S]cystine). All [3H]amino acids were obtained
as stocks in 2% ethanol in water and were stored at 48C until added directly to
media. [35S]cystine was obtained as a dry solid and was stored at ?808C until
the entire vial (2.09 mg) was dissolved in 1 ml of 0.1 N HCl, diluted to a final
concentration of 2.1 mM in water, aliquoted, and stored at ?808C until used.
Where the volume of radiolabeled substrate was more than 5% of the final
volume, concentrated culture medium made with less water was used so that the
final concentration of medium components remained constant within 5%.
Culture and collection media were based on potassium simplex optimized
medium (KSOM) and Hepes-KSOM mouse embryo culture media, respectively
, modified to omit glutamine and bovine serum albumin and containing
polyvinyl alcohol (1 mg/ml) as the macromolecular component (termed
modified KSOM [mKSOM]). The components of mKSOM were NaCl (95
mM), KCl (2.5 mM), KH2PO4(0.35 mM), MgSO4.7H2O (0.2 mM), Na lactate
(10 mM), glucose (0.2 mM), Na pyruvate (0.2 mM), NaHCO3(25 mM), CaCl2
(1.7 mM), edetic acid (0.1 mM), K penicillin G (0.16 mM), and streptomycin
(0.03 mM). The composition of Hepes-mKSOM was the same except that 21
mM NaHCO3was replaced by Hepes. NaOH was used to adjust the pH of
Hepes-mKSOM to 7.3–7.4. Osmolarity was confirmed using a model 5520
vapor pressure osmometer (Wescor, Logan, UT).
L-[2,3-3H]alanine (40–60 Ci/mmol),
L-[4,5-3H]leucine (45–85 Ci/mmol),
Female CF1 mice were obtained from Charles River Canada (Saint-
Constant, QC, Canada) or from Harlan Sprague Dawley (Indianapolis, IN).
Adult CF1 mice from which fully grown oocytes were obtained were
approximately 7 wk old. CF1 neonatal female mice, from which growing
oocytes were obtained, were ordered from Charles River Canada weekly at
specific postnatal ages depending on the timing of experiments and were kept at
a ratio of 8:1 per lactating dam. Mice were maintained on a 12L:12D (light,
700-1900 h) and had unrestricted access to food and water. All animal
protocols were approved by the Animal Care Committee of the Ottawa Health
Oocyte, Follicle, and COC Isolation
In mice, a coordinated wave of follicular development begins shortly after
birth, resulting in oocyte and follicular development being directly related to
postnatal age. During Postnatal Days 5–21, a large cohort of oocytes grow to
full size, modeling oocyte growth as it happens in sexually mature mice in each
reproductive cycle. Female mice at postnatal ages of 5, 10, and 20 days were
used to obtain small, medium, and large growing oocytes, respectively. These
small, medium, and large growing oocytes had mean 6 SEM diameters of 45.5
6 0.8, 58.0 6 1.0, and 76.7 6 0.6 lm, respectively (data not shown). Only the
large growing oocytes were meiotically competent (;90% exit from GV stage
[data not shown]). Denuded small growing oocytes (Day 5) were isolated
enzymatically by incubating excised ovaries in Ca2þ- and Mg2þ-free mKSOM
with 2 mg/ml of type I collagenase and 0.01 mg/ml of DNase I for 40 min [37,
38]. Denuded medium growing oocytes (Day 10) and large growing oocytes
(Day 20) were mechanically isolated following mincing of the ovaries with a
razor blade as previously described . The different isolation protocols were
used because small oocytes could not be obtained mechanically, while large
oocytes could not be obtained enzymatically . It was confirmed in medium
growing oocytes, in which both isolation protocols are feasible, that identical
amino acid transport measurement results were obtained using both methods
(data not shown), thus validating its use with small growing oocytes. The data
for medium growing oocytes presented herein, however, were obtained using
only the mechanical isolation method because of its greater ease of use.
Intact medium preantral follicles (diameter, 95–115 lm ) containing
medium growing oocytes were mechanically isolated following mincing of
ovaries of Postnatal Day 10 mice. For some experiments, oocytes were
removed from Day 10 neonatal follicles by removing a small part of the follicle
wall with a short thin flame-pulled Pasteur pipette and then pressing on the
follicle to extrude the oocyte from it as previously described . This
produces an almost intact follicular cell shell and an intact denuded oocyte from
the same follicle.
For fully grown oocytes, adult females were primed with an i.p. injection of
5 IU of equine chorionic gonadotropin (eCG). The COCs containing fully
grown GV oocytes were collected from mechanically minced ovaries 44–48 h
after eCG administration. For denuded GV oocytes, cumulus cells were
removed by repeated pipetting through a narrow-bore pipette. Fully grown GV
oocytes had mean 6 SEM diameters of 84.6 6 0.8 lm.
To obtain in vivo-matured oocytes, an i.p. injection of 5 IU of human
chorionic gonadotropin (hCG) was administered 47 h after eCG administration.
For metaphase I (MI) oocytes, partially expanded COCs were collected from
the mechanically minced ovaries 4 h after hCG administration in 0.3 mg/ml of
hyaluronidase, and MI oocytes were isolated by repeated pipetting. The MII
oocytes were collected from excised oviducts 15 h after hCG administration
and were treated with hyaluronidase to disperse the expanded cumulus. All
were rinsed four times in collection medium before use.
3H and35S measurements were performed in 4-ml volumes of scintillation
fluid using a model 2200CA TriCarb liquid scintillation counter (Packard
Instrument Co., Downer’s Grove, IL), with each sample counted for 5 min.
Conversion of counts per minute to molar amounts of labeled amino acid was
performed using a standard curve constructed for each set of experiments by
serial dilutions in water of the radiolabeled compounds.
For measurements of amino acid transport in denuded growing (small,
medium, and large) or fully grown (GV, MI, and MII) oocytes, isolated oocytes
were rinsed three times in equilibrated mKSOM medium in groups of 5–20 and
were transferred to pre-equilibrated medium containing the radiolabeled amino
acid for the specified period. They were then washed five times in ice-cold
medium and transferred to a scintillation vial, and scintillation fluid was added
essentially as previously described [19, 34]. A similar volume of the last wash
drop was transferred to a separate vial and treated identically to obtain
background that was subtracted from each paired measurement. Radiolabeled
amino acids were used at a concentration of 1 lM except for taurine, alanine,
and aspartic acid, which were used at 10 lM, and cystine at 50 lM. The
incubation period was 10 min in all cases except cystine, for which it was 30
min. The increase in concentrations for taurine, alanine, aspartic acid, and
cystine and the longer incubation time for cystine were necessary to obtain
adequate signal to noise because of their lower transport rates.
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For transport measurements on intact follicles from Day 10 neonates,
isolated follicles were cultured in mKSOM for 20 min to reduce stickiness, and
then groups of five were washed and transferred to medium containing labeled
amino acids as already described for denuded oocytes. After the incubation
period, oocytes were mechanically removed from the follicle cell shell as
already described, and the oocyte and follicle shell were processed separately
for scintillation counting. Intact follicles with enclosed oocytes were similarly
processed. Labeled amino acid concentrations were as already described for
denuded oocytes except when counts per minute were deemed too high (and
possibly detrimental), in which case radiolabeled amino acid was mixed with
unlabeled amino acid to provide the desired total concentration but at lower
specific activity. Incubation times were the same as for denuded oocytes except
for one set of experiments in which follicles were incubated for 30, 60, and 90
min as indicated.
Transport measurements on COCs were carried out identically to those on
denuded oocytes. At the end of the period of incubation with labeled amino
acids, the intact COC or the GV oocyte after mechanical removal of cumulus
cells was processed for scintillation counting.
Content of radiolabeled amino acid was expressed as femtomoles per
oocyte (after background subtraction). Rates of transport were calculated by
dividing the total molar amount of radiolabeled amino acid measured in the
sample by the number of oocytes, follicles, or COCs; the incubation time; and
the molar amount of labeled amino acid in the incubation medium. Rates were
then expressed as femtomoles per oocyte, follicle, or COC per minute. Pilot and
previous experiments had confirmed linearity over the incubation periods used
and concentrations of labeled amino acid (data not shown).
Data were expressed as the mean 6 SEM. Comparisons between means
were performed by ANOVA with Tukey-Kramer post hoc test for multiple
(.2) comparisons or two-tailed Student t-test for comparing two means using
Instat (GraphPad, San Diego, CA). Differences were assumed to be significant
at P , 0.05. Data were plotted using SigmaPlot 8.2 (SPSS, Chicago, IL). For
plots of amino acid content vs. time, data were fit by nonlinear least squares
regression (SigmaPlot 8.2) to a single exponential of the following form:
Amino Acid Content¼a(1? e?bt), where a and b are parameters determined by
the fit, and t is time.
Testing for the Presence of Amino Acid Transport Systems
As discussed in the Introduction, unequivocally establishing which amino
acid transport systems are responsible for transport of each amino acid in
oocytes at each stage of development would be prohibitive. Instead, we utilized
a series of simple assays using established substrates of major classic amino
acid transport systems to determine whether their activities are likely present.
These assays (diagrammed in Fig. 1) are based on first showing that an
established substrate for a given transport system is taken up; second, showing
that its transport is saturable by competition with excess (10 mM) unlabeled
substrate; and third, showing that a characteristic competitive inhibitor or set of
inhibitors eliminates the saturable component of transport. In the case of
cystine, the substrate was not sufficiently soluble to reach 10 mM. Therefore,
we used cysteic acid at 10 mM instead to establish saturable cystine transport.
In some cases, obtaining clear results was straightforward. For example,
taurine transport inhibited by excess b-alanine is very likely attributable to the b
transport system because it is the only transport mechanism that fits this pattern.
In most cases, however, somewhat more complex assessments are needed.
Thus, for example, lysine transport inhibited by leucine could indicate activity
of either b0,þ- or B0,þ-mediated transport. A second set of measurements using
BCH (which competitively inhibits B0,þbut not b0,þ) was then used to
differentiate between these two possibilities. We were also able to somewhat
reduce the number of measurements needed by utilizing information obtained
using one substrate to reduce the possibilities that needed to be assessed for
another. Thus, if lysine transport was found to be BCH insensitive at all oocyte
stages examined, therefore eliminating the possibility of B0,þactivity, it would
be unnecessary to test lysine as a potential competitive inhibitor of leucine to
similarly test for B0,þ.
We used nine radiolabeled amino acids (Fig. 1) and measured the rate of
transport and inhibition profile for each in small, medium, and large growing
oocytes; in GV, MI, and MII oocytes; and in medium growing follicles and
COCs and oocytes isolated from these after amino acid uptake. Using the
resulting information, we assigned transport to the likely classic amino acid
transport systems active at each stage of development.
Amino Acid Transport in Growing and Maturing Oocytes
We measured the rate of transport of each of nine
radiolabeled amino acid substrates in small, medium, and large
growing oocytes and in fully grown GV, MI, and MII oocytes.
Saturable transport of each substrate, determined as the
difference between the rate of transport in the absence and
presence of excess (10 mM) unlabeled substrate, was found to
be present during at least some stages of oocyte development for
all amino acid substrates tested except aspartate (Figs. 2 and 3).
For three amino acids, glycine, taurine, and cystine,
saturable transport was very low or absent during all stages
of growing oocytes and in fully grown GV oocytes but
increased during meiotic maturation (Fig. 3, A–C). For glycine,
saturable transport was still increasing in MI oocytes (4 h after
hCG administration) and became much greater in MII oocytes
(Fig. 3A), while saturable taurine transport was already
maximal in MI oocytes (Fig. 3B). An unusual feature of
taurine transport was that there was a comparatively large
component of nonspecific transport in large growing and GV
oocytes, which decreased progressively during meiotic matu-
ration (Fig. 2B) as saturable transport appeared (Fig. 3B).
Saturable cystine transport was low in growing and maturing
oocytes through the MI stage and then increased significantly
at the MII stage (Figs. 2C and 3C).
The remaining five amino acids that exhibited saturable
transport in oocytes (alanine, lysine, leucine, glutamine, and
system activities. Measurements of transport rates of the nine radiolabeled
substrates were performed in the absence of any addition and in the
presence of the putative competitive inhibitors listed. The classic amino
acid transport system (at the right) whose substrate transport is blocked by
a given inhibitor is indicted by arrows. Details are given in the text.
Schematic diagram of tests used to reveal amino acid transport
PELLAND ET AL.
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Amino acid transport characteristics in growing and maturing oocytes. The rates of transport of each radiolabeled amino acid as indicated by the labels in A–I were measured for small, medium, and
large growing oocytes and for GV, MI, and MII oocytes as indicated at the bottom. Substrate and potential competitive inhibitors used (10 mM each) are indicated by inset keys in each panel. Each bar indicates the mean 6 SEM of at least three independent measurements. Means within each stage (i.e., within the same bracket above the bars) were tested for difference by ANOVA with Tukey-Kramer post
hoc test. Bars not sharing the same letter within a given stage are significantly different (P , 0.05). NS, no significant difference.
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