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First published online November 21, 2008
doi: 10.1242/10.1242/dev.026575
1 Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada.
2 Department of Obstetrics and Gynecology (Division of Reproductive Medicine),
University of Ottawa Faculty of Medicine, Ottawa, Ontario K1H 8M5,
Canada.
3 Department of Cellular and Molecular Medicine and, University of Ottawa
Faculty of Medicine, Ottawa, Ontario K1H 8M5, Canada.
4 Department of Biochemistry, Microbiology and Immunology, University of Ottawa
Faculty of Medicine, Ottawa, Ontario K1H 8M5, Canada.
5 Biochemistry Unit, Canterbury Health Laboratories, Christchurch 8140, New
Zealand.
6 Division of Biochemistry and Molecular Biology, Australian National
University, Canberra ACT 0200, Australia.
* Author for correspondence (e-mail: jbaltz{at}ohri.ca)
Accepted 6 October 2008
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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4 hours post-egg activation and reaching a
maximum by 10 hours. One- and 2-cell embryos contained endogenous
betaine, indicating that a likely function for the transporter in vivo is the
accumulation or retention of intracellular betaine. The appearance of
transport activity after egg activation was independent of protein synthesis,
but was reversibly blocked by disruption of the Golgi with brefeldin A. We
assessed two candidates for the betaine/proline transporter: SIT1 (IMINO;
encoded by Slc6a20a) and PROT (Slc6a7). mRNA from both genes
was present in eggs and 1-cell embryos. However, when exogenously expressed in
Xenopus oocytes, mouse PROT did not transport betaine and had an
inhibition profile different from that of the embryonic transporter. By
contrast, exogenously expressed mouse SIT1 transported both betaine and
proline and closely resembled the embryonic transporter. A morpholino
oligonucleotide designed to block translation of SIT1, when present from the
germinal vesicle stage, blocked the appearance of betaine transport activity
in parthenogenotes. Thus, SIT1 is likely to be a developmentally restricted
betaine transporter in mouse preimplantation embryos that is activated by
fertilization.
Key words: Preimplantation, Betaine, Fertilization, Transport, IMINO
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INTRODUCTION |
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MATERIALS AND METHODS
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DISCUSSION
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;
Hammer and Baltz, 2002).
Previous work has shown that betaine can remarkably improve the viability of
preimplantation (PI) mouse embryos, at least under in vitro conditions. In
particular, the addition of betaine to the PI embryo culture medium protects
embryo development against stress imposed by alterations in culture medium
composition or increased osmolarity
(Biggers et al., 1993;
Dawson and Baltz, 1997).
Embryos cultured with betaine also maintain more normal intracellular
Na+ and K+ concentrations
(Biggers et al., 1993) and
their pattern of protein synthesis more closely resembles that of in vivo
embryos (Anbari and Schultz,
1993). Betaine might also function in vivo, as it is present in
mouse oviducts (Anas et al.,
2007).
One-cell embryos possess a single route for betaine transport that is
Na+- and Cl--dependent, also transports proline and is
inhibited by several methylamino acids and proline derivatives
(Anas et al., 2007). This
betaine/proline transporter has not, however, been identified at a molecular
level and is unlike any transporter previously described in 1-cell mouse
embryos (Anas et al., 2007;
Hammer and Baltz, 2002). Its
transport characteristics do not resemble those of the betaine transporter
BGT1 (encoded by Slc6a12) (Hammer
and Baltz, 2002; Yamauchi et
al., 1992), nor of the proline transporters PAT1 and PAT2
(Slc36a1 and Slc36a2, respectively)
(Broer, 2008).
One function of betaine in mammalian cells is as an organic osmolyte, one
of an array of neutral organic compounds accumulated by specialized
transporters to counter increased external osmotic pressure without the
deleterious effects of increased ionic strength
(Kwon and Handler, 1995).
Based on its osmoprotection of PI mouse embryos in culture, Biggers et al.
proposed that betaine functions as an organic osmolyte in PI embryos
(Biggers et al., 1993).
Cleavage-stage embryos are very sensitive to moderately increased osmolarity,
and they accumulate large amounts of glycine as an organic osmolyte to balance
the osmolarity of their normal in vivo environment
(Steeves et al., 2003). Under
in vitro conditions, betaine is as effective as glycine at osmoprotection in
1-cell embryos (Anas et al.,
2007; Biggers et al.,
1993; Dawson and Baltz,
1997; Hammer and Baltz,
2002) and thus might also have a function in cell volume
homeostasis. Another established function of betaine is to serve as a donor of
methyl groups that are ultimately made available to a wide array of
methyltransferases, a function known to be important mainly in the liver
(Finkelstein and Martin, 1984;
Selhub, 1999).
Based on the dependence of betaine/proline transport in 1-cell mouse
embryos on Na+ and Cl-, we hypothesized that the
embryonic betaine/proline transporter is a member of the neurotransmitter
transporter family (NTT; the Slc6 gene family), which contains all known
mammalian Na+- and Cl--dependent transporters of organic
substrates (Chen et al., 2004;
Hoglund et al., 2005). Two
possible candidates have been proposed
(Anas et al., 2007): the brain
proline transporter (PROT; encoded by Slc6a7) and the intestinal
imino acid transporter (SIT1, also known as IMINO and XTRP3s1; encoded by
Slc6a20a). PROT is a reportedly brain-specific proline transporter
with an inhibitor profile (Fremeau et al.,
1992; Shafqat et al.,
1995) similar to that of the embryonic betaine/proline
transporter. However, to our knowledge, whether PROT actually transports
betaine is not known. SIT1 was recently identified as the long-sought
intestinal proline transporter (Kowalczuk
et al., 2005; Takanaga et al.,
2005). SIT1 has kinetic properties and an inhibitor profile very
similar to those of the embryonic transporter and it accepts betaine as a
substrate (Kowalczuk et al.,
2005; Takanaga et al.,
2005). It was unknown, however, whether either of these
transporters is expressed or active in 1-cell mouse embryos.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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),
modified by omitting glutamine and replacing BSA with polyvinyl alcohol (PVA)
(1 mg/ml; cold-water soluble, 30-70 kDa). KSOM was equilibrated with 5%
CO2 in air at 37°C. Similarly modified HEPES-KSOM (pH 7.4) was
used for oocyte and embryo collection and where specified
(Lawitts and Biggers,
1993).
Mouse oocytes and embryos
Oocytes and embryos were obtained from female CF1 mice (4-7 weeks old;
Charles River Canada, St-Constant, PQ, Canada) that had been superovulated by
intraperitoneal injection of equine chorionic gonadotropin (eCG; 5 IU). For
ovulated metaphase (M) II oocytes and PI embryos, human chorionic gonadotropin
(hCG; 5 IU) was administered by intraperitoneal injection 47 hours
post-eCG. For embryos, the female mice were caged overnight with
BDF1 males (Charles River) immediately after hCG for mating. All
procedures were approved by the Animal Care Committee of the Ottawa Health
Research Institute.
Germinal vesicle (GV) stage oocytes were obtained by mincing excised
ovaries 44-46 hours post-eCG, collecting fully grown oocytes, and
removing adherent cumulus cells by repeated pipetting. MI oocytes were
similarly obtained 4 hours post-hCG, and MII oocytes at 15 hours
post-hCG. One-cell stage embryos were collected (±2 hours) at 22
hours post-hCG (or, as specified, between 19 and 29 hours), 2-cell at 44
hours, 4-cell at 56 hours, 8-cell at 67 hours, morulae at 76
hours and blastocysts at 94 hours. MII oocytes and 1- through 8-cell
embryos were removed from excised oviducts by flushing with HEPES-KSOM
(containing 300 µg/ml hyaluronidase for MII oocytes and 1-cell embryos to
facilitate removal of cumulus), while morulae and blastocysts were flushed
from the uterotubular junction and uterus, respectively. Oocytes or embryos
were washed through four drops of HEPES-KSOM and then briefly maintained in
KSOM according to standard techniques in microdrop cultures
(Lawitts and Biggers, 1993)
under mineral oil (Sigma-Aldrich, Milwaukee, WI) at 37°C in 5%
CO2 in air until they were used.
For parthenogenetic activation with Sr2+, ovulated MII oocytes
were incubated for 2 hours with 10 mM SrCl2 in KSOM
(CaCl2 omitted) as described
(Phillips et al., 2002). In
one experiment, eggs were instead activated using cycloheximide alone (50
µg/ml in KSOM from 1000x stock in water), which inhibits general
protein synthesis and thus activates eggs by preventing cyclin B synthesis
(Moos et al., 1996;
Phillips et al., 2002;
Siracusa et al., 1978). The
time at which Sr2+ or cycloheximide was introduced was designated
t=0.
Measurement of 3H-labeled compounds in oocytes and embryos
[3H]betaine ([methyl-3H]betaine, 85 Ci/mmol, 1
mCi/ml) and [3H]proline (L-[2,3-3H]proline,
45 Ci/mmol, 1 mCi/ml) were obtained from Amersham Biosciences (Arlington
Heights, IL). [3H]betaine was custom-synthesized by Amersham as
previously described (Anas et al.,
2007). Stocks were in 2% ethanol in water, stored at
-80°C.
For determining betaine transport activity, groups of 5-12 oocytes or
embryos were incubated with 1 µM [3H]betaine for 30 minutes,
except after parthenogenetic activation (Sr2+ or cycloheximide) or
for in vivo-maturing oocytes at defined times post-hCG, where incubation with
2 µM [3H]betaine for 10 minutes was used instead to permit more
precise timing. Proline transport was measured using 1 µM
[3H]proline for 10 minutes in the presence of 10 mM alanine to
eliminate the betaine-resistant, alanine-sensitive component of proline
transport in PI embryos (Anas et al.,
2007). Oocytes or embryos were then immediately processed for
measurement of intracellular 3H.
To determine the intracellular content of 3H, oocytes or embryos were removed from [3H]betaine- or [3H]proline-containing medium, washed in ice-cold HEPES-KSOM, and dissolved in 4 ml scintillation fluid (Scintiverse BD; Fisher Scientific, Pittsburgh, PA). 3H was detected using a 2200CA TriCarb liquid scintillation counter (Packard Instruments, Downer's Grove, IL). Background was measured in equivalent volumes of each final wash drop, and the counts obtained were subtracted from the paired embryo sample. The total amount of each compound in oocytes or embryos was calculated using standard curves constructed for each set of experiments. Transport rates were expressed as fmol/oocyte or embryo/minute (normalized to 1 µM substrate when 2 µM [3H]betaine was used).
Measurement of endogenous betaine
Groups of 50 1- or 2-cell embryos, isolated from about five females, were
washed extensively with HEPES-KSOM, placed into microcentrifuge tubes with
minimal medium, air dried and stored at -20°C. Total betaine was measured
using a modification of the high performance liquid chromatography
(HPLC)-based method described previously
(Storer et al., 2006;
Storer and Lever, 2006).
Briefly, 25 µl methanol was added to each tube, vortexed, ultrasonicated
for 10 minutes, and then 225 µl acetonitrile added prior to derivatization
with phenanthrenacyl triflate. HPLC separation was performed using a silica
column (3 µm) with a mobile phase of 5 mM dimethylbutylamine, 10 mM
succinate and 5% water in acetonitrile at a flow rate of 0.8 ml/minute for a
30-minute run time at 40°C. The sample injection volume was 50 µl.
Detection was by fluorescence. Background was determined using a similar
amount of medium from the final wash drop. Betaine was quantified by
comparison to external standards and the data are reported as pmol/embryo.
RT-PCR
RNA was extracted from two independent sets of MII oocytes and PI embryos,
with kidney and brain as control tissues, using the RNeasy Micro Kit (Qiagen,
Mississauga, ON) and reverse transcribed using the Retroscript Kit (Ambion,
Austin, TX). Primer pairs were designed (OligoPerfect, Invitrogen, Carlsbad,
CA) using mouse mRNA reference sequences spanning introns. The SIT1
(Slc6a20a) forward (sense) primer was
5'-AGCCACCAATGGCCTGATGT-3' (nt 683-702 of NM_139142) and the
reverse was 5'-AGCGATCAGGCTGCCAAAAC-3' (nt 790-809), yielding a
127 bp amplicon. The PROT (Slc6a7) forward primer was
5'-GTGGCAACTGGTGGAACACG-3' (nt 651-679 of NM_201353) and the
reverse was 5'-CACTCCTCGAACCAGCAGCA-3' (nt 957-976), yielding a
317 bp amplicon. We used, as a positive control, histone 2A family member Z
(H2afz), previously shown to have a particularly stable expression
pattern in PI embryos across different stages and culture conditions
(Mamo et al., 2007). The same
primer pair as in the original report (202 bp amplicon) was used. Each PCR
reaction contained 0.1 embryo equivalent of cDNA. PCR was carried out with
Hotstar Taq polymerase (Qiagen) in 20 µl reaction volumes in a Mastercycler
thermocycler (Eppendorf, Hamburg, Germany): 95°C for 15 minutes, followed
by 40 cycles of 94°C for 60 seconds, 60°C for 30 seconds, and 72°C
for 60 seconds. Amplicons were visualized by agarose gel (2%) electrophoresis
with ethidium bromide staining using a Typhoon 8600 Phosphorimager (Amersham,
Piscataway, NJ). Amplicons derived from MII eggs and kidney for
Slc6a20a and Slc6a7 were sequenced by the Ontario Genomics
Innovation Centre, Ottawa, Canada.
Expression of murine transporters in Xenopus laevis oocytes and transport activity measurements
Mouse SIT1 (Slc6a20a) cDNA was cloned into a pGEM-He-Juel vector
as described previously (Kowalczuk et al.,
2005). Mouse PROT (Slc6a7) in a pYX-Asc vector was
purchased from Open Biosystems (Huntsville, AL). Slc6a7 and
Slc6a20a cRNA were prepared from NotI-linearized cDNA
templates (Qiagen T7 mMessage Machine). Stage VI Xenopus laevis
oocytes were obtained as previously described
(Liu and Liu, 2006). Briefly,
sexually mature female Xenopus (NASCO, Fort Atkinson, WI) were
stimulated with eCG (50 IU) 3-7 days before oocyte retrieval. Oocytes were
released from excised ovaries by incubation in OR2 medium (82.5 mM NaCl, 2.5
mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 5 mM HEPES,
pH 7.8; CaCl2 omitted) with 1.5 mg/ml collagenase and 1 mg/ml
soybean trypsin inhibitor, and then maintained in OR2 containing 0.1 mg/ml
gentamicin. Protocols were approved by the Animal Care Committee of the Ottawa
Health Research Institute. Oocytes (in CaCl2-free OR2) were
injected with cRNA (625 µg/ml in water) into the cytoplasm, using injection
volumes of 20 nl for SIT1 and 10 nl for PROT, or control injections of water.
Injected oocytes were incubated in OR2 for 48 hours (PROT) or 72 hours (SIT1)
at 16°C before use.
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Morpholino design and injection
A morpholino antisense oligonucleotide (MO) (Gene Tools LLC, Philomath, OR)
of the sequence 5'-CACTGAGGCCGTGCCTTCTCCATGT-3' (start
codon underlined) directed against mouse Slc6a20a (NM_139142.2;
confirmed unique in mouse with BLASTN) and a standard control MO (human
β-globin β-thalassemia mutation) were used. GV mouse oocytes were
injected with 5 pl of Slc6a20a or control MO (0.5 mM in water). Where
indicated, Xenopus oocytes were injected with 10 nl of MO (1 mM; MO
was co-injected with Slc6a20a cRNA, above, where indicated; owing to
inclusion of plasmid sequence, the final MO base and cRNA were mismatched).
Final MO concentrations in both mouse GV and Xenopus oocytes were
10 µM. Injected and uninjected mouse GV oocytes from the same cohort
were cultured overnight (20 hours) in Minimal Essential Medium 
with L-Gln (MEM; Invitrogen) with 1 mg/ml PVA added, during
which time they spontaneously matured to MII eggs, and then were
parthenogenetically activated using Sr2+ and maintained in culture
in KSOM for a further 8.5 hours. The rate of [3H]betaine transport
was measured as described above. Injected Xenopus oocytes were
incubated for 72 hours and [3H]proline measured as described
above.
Data analysis
Data are expressed as mean±s.e.m. Graphs were produced using
SigmaPlot 8.02 (SPSS, Chicago, IL). Statistical analyses were performed using
InStat (GraphPad, San Diego, CA).
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Two routes for proline transport were previously identified at the 1-cell
stage: one that is inhibited by alanine and the other being the
betaine/proline transporter (Anas et al.,
2007). We determined the rate of saturable proline transport in
the presence of 5 mM alanine at key developmental stages to reveal proline
transport by the betaine/proline transporter. The pattern of proline transport
activity (Fig. 1C) was similar
to that of betaine (Fig. 1B),
with a peak at the 1-cell stage, decline during the 2-cell stage, and no
activity in oocytes or 8-cell stage embryos. Thus, the activity of the
betaine/proline transporter was essentially restricted to the 1- and 2-cell
stages.
Appearance of betaine transporter activity at egg activation
We used parthenogenetic activation with Sr2+ to obtain precisely
timed activation of mouse MII oocytes with which to investigate the appearance
of betaine/proline transport after egg activation. Betaine transport initially
appeared at 4 hours after Sr2+-induced activation and reached
a maximum by 10 hours (Fig. 2).
Parthenogenotes in the early 2-cell stage (24 hours post-activation) exhibited
maximal betaine transport, but activity declined somewhat by 30 hours and was
lost by the 4-cell stage. MII oocytes maintained under identical culture
conditions exhibited only low betaine transport activity, indicating that egg
activation was required.
To confirm that betaine transport was initiated with a similar time course in fertilized eggs, and thus that Sr2+-activated parthenogenotes provided an adequate model, we measured the rate of betaine transport by fertilized oocytes immediately after removal from the oviduct at the specified times post-hCG. At 19 hours post-hCG (2-4 hours after fertilization, when fertilization can be first confirmed by second polar body emission), the rate of betaine transport was still low (Fig. 3). By 21 hours, an increased rate was observed, reaching a plateau by 23 hours. Thus, betaine transport appears with a similar time course in vivo.
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90% inhibition of
35S-methionine incorporation into the TCA-insoluble fraction (not
shown). Cycloheximide-activated parthenogenotes maintained for 12 hours in the
continuous presence of cycloheximide developed betaine transport activity, the
rate of which was not significantly different from that of
Sr2+-activated parthenogenotes
(Fig. 4). This indicated that
the appearance of betaine transport activity after oocyte activation did not
require protein synthesis, and showed that an independent method of
parthenogenetic activation also resulted in the appearance of betaine
transport.
We used the fungal toxin brefeldin A (BFA), which has been widely employed
to inhibit Golgi-based membrane vesicle fusion
(Dinter and Berger, 1998), to
determine whether the appearance of betaine transport requires an intact
Golgi. We previously confirmed that BFA causes the physical disruption of
Golgi aggregates in mouse oocytes (Wang et
al., 2008), consistent with previous findings
(Moreno et al., 2002). Betaine
transport was measured in fresh MII oocytes (t=0) and in
parthenogenotes (Sr2+-activated) cultured in the continuous
presence or absence of BFA for up to 30 hours. A sample was removed from BFA
at 7 hours and then cultured to 12 hours in its absence
(Fig. 5, arrow a), and another
was removed from BFA at 10 hours and then cultured to 30 hours
(Fig. 5, arrow b). The effect
of BFA was reversible, as transport activity developed in parthenogenotes
after removal from BFA (Fig. 5,
arrows). BFA also did not directly inhibit betaine transport because the rate
of transport in 1-cell embryos was not significantly decreased upon 4 hours
exposure to BFA (not shown). Since, in many systems, Golgi-derived vesicles
are transported by cytoskeleton-dependent mechanisms, we assessed the effect
of several cytoskeleton-perturbing agents. However, there was no effect on
betaine transport activity at 10 hours post-Sr2+ activation of the
continuous presence of demecolcine or nocodozole (microtubule
depolymerization), cytochalasin D (F-actin depolymerization), or
jasplakinolide (F-actin stabilization) (not shown).
Endogenous betaine content of 1-cell and 2-cell mouse embryos
To determine whether mouse embryos contain endogenous betaine at the
developmental stages at which we have found betaine transport activity, in
vivo-derived 1- and 2-cell embryos were isolated from oviducts, washed
extensively, and then groups of 50 placed in tubes and dried for betaine
measurements. The total endogenous betaine contents of three independent
samples each of 1- and 2-cell embryos were determined. Very little betaine
(0.05±0.03 pmole per embryo equivalent of medium, mean±s.e.m.,
n=6) was found in wash drop samples, indicating that there was no
significant contamination from extracellular betaine (not significantly
different from 0 by one-sample Student's t-test, P=0.20).
The embryo samples (after background subtraction), by contrast, contained
betaine at 0.98±0.23 pmole/1-cell embryo and 1.30±0.23
pmole/2-cell embryo (both significantly different from 0,
P<0.05).
Expression of candidate betaine/proline transporters in mouse eggs and PI embryos
Two candidates had been identified that might mediate betaine/proline
transport activity in 1-cell embryos: the brain proline transporter (PROT,
encoded by Slc6a7) and the intestinal imino acid transporter (SIT1,
encoded by Slc6a20a). To determine whether mRNA for either
transporter was present in PI embryos, we carried out RT-PCR on RNA isolated
from MII eggs, PI embryos from the 1-cell to blastocyst stages, kidney and
brain, with primers designed to amplify Scl6a7, Slc6a20a and
H2afz (control). Surprisingly, we found that both Scl6a7 and
Slc6a20a mRNAs were present in MII eggs and 1-cell embryos
(Fig. 6). Amplicons derived
from MII eggs and kidney for Slc6a20a and Slc6a7 were
excised from gels and sequenced to confirm their identity. We had previously
confirmed the H2afz amplicon by sequencing. Slc6a7 and
H2afz, but not Slc6a20a, were detected in brain as expected
(not shown). We also used independent sets of primers for each gene
(Slc6a20a and Slc6a7) in 1-cell embryos and confirmed their
expression (not shown). Thus, both PROT and SIT1 remained as possible
candidates for the betaine/proline transporter, as their mRNAs were present
before transport activity appears.
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), we expressed mouse SIT1 and PROT in Xenopus
oocytes, and determined the characteristics of betaine and proline transport
by each. Expression of SIT1 in Xenopus oocytes resulted in a
9-fold increase in the uptake of 1 µM [3H]proline over 20
minutes (Fig. 7A). Induced
proline transport was reduced to the level in control (water-injected) oocytes
by 5 mM unlabeled betaine, and thus was saturable. Proline transport was also
eliminated in the presence of 5 mM 2-methylaminoisobutyric acid (MeAIB) but
was unaffected by 5 mM histidine. SIT1-expressing Xenopus oocytes
similarly showed an 8-fold increase in [3H]betaine uptake,
which was reduced to the level in control oocytes by 5 mM unlabeled proline,
was eliminated in the presence of MeAIB, but unaffected by histidine
(Fig. 7B).
Expression of PROT in Xenopus oocytes resulted in a 4-fold
increase in proline uptake (Fig.
7C). In contrast to that in SIT1-expressing oocytes, proline
transport in PROT-expressing oocytes was not inhibited by the presence of
either 5 mM unlabeled betaine or 5 mM MeAIB, but was reduced to the level in
control oocytes by 5 mM histidine. Uptake of betaine by PROT-expressing
Xenopus oocytes was not greater than that of control oocytes
(Fig. 7D), indicating that PROT
does not transport betaine at detectable levels.
Effect on betaine transport in 1-cell mouse embryos of a morpholino directed against Slc6a20a
To determine whether knockdown of SIT1 expression affected endogenous
betaine transport in mouse parthenogenotes, we used a morpholino (MO) designed
to block Slc6a20a translation. De novo protein synthesis is
apparently not required for the appearance of betaine transport activity after
egg activation (see above, Fig.
4). Therefore, we injected GV mouse oocytes and then in vitro
matured and parthenogenetically activated them, hypothesizing that the
presence of the Slc6a20a MO for the 28 hours required for oocyte
maturation and activation might interfere with the maintenance of SIT1
protein. GV oocytes that were in vitro matured and then parthenogenetically
activated transported betaine at a rate similar to parthenogenetically
activated in vivo matured oocytes, indicating that in vitro maturation itself
did not affect the post-activation development of betaine transport. In
oocytes injected with the Slc6a20a MO, however, transport was
significantly reduced (to approximately the level of non-specific transport;
see Fig. 1A), whereas the
control MO had no effect (Fig.
8A). Therefore, the appearance of betaine transport after egg
activation is substantially inhibited when SIT1 protein translation is
specifically blocked in mouse oocytes.
To confirm that the MO we used could inhibit functional expression of SIT1, we injected it into Xenopus oocytes expressing exogenous mouse SIT1 (as described above). Induced [3H]proline transport was significantly inhibited by the Slc6a20a MO, but not the control MO (Fig. 8B), confirming that the Slc6a20a MO decreased functional expression of mouse SIT1.
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DISCUSSION
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;
Van Winkle et al., 1988;
Van Winkle et al., 1990).
Indeed, to our knowledge, none of the diverse array of transporters of
biologically important organic compounds has been found to be activated (or
inactivated) shortly after fertilization in mammalian eggs, although several
ion channels and intracellular pH-regulatory mechanisms change in activity
during the immediate post-fertilization period
(Day et al., 1998;
Lane et al., 1999;
Phillips and Baltz, 1999), and
a cysteine transporter is reportedly activated shortly beforehand, during
meiotic maturation (Van Winkle et al.,
1992). Here we have found that the betaine/proline transporter
previously identified in mouse 1-cell embryos is quiescent in oocytes prior to
fertilization and then becomes activated within several hours of egg
activation, providing the first example of such a transporter that is
activated upon fertilization in mammals. Once betaine/proline transport is
activated, it persists for only a little more than one cell cycle before
becoming inactive again. We found high transporter activity only at the 1-cell
and 2-cell stages, both in embryos that had developed in vivo and in
parthenogenotes that developed in vitro. We are not aware of any other
transporters, the functional expression of which is restricted to so short a
developmental period of very early PI embryogenesis.
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), unlike the betaine/proline transporter. A better candidate
is a distinct process of protein trafficking that has been reported to be more
slowly induced by egg activation in mammals and is blocked by BFA but
insensitive to disruption of F-actin or microtubules
(Clayton et al., 1995), similar
to our findings here for betaine/proline transport. This mechanism mediates
the appearance of the cell adhesion molecule uvomorulin (E-cadherin) in the
plasma membrane over a period of 6 hours following egg activation
(Clayton et al., 1995), and
occurs during the same period that the fragmented, inactive Golgi in MII
oocytes becomes reorganized following fertilization
(Payne and Schatten,
2003).
Identification of the betaine/proline transporter in mouse embryos
It was previously proposed (Anas et al.,
2007) that the betaine/proline transporter in 1-cell embryos was
most likely a member of the neurotransmitter transporter (Slc6) gene family,
and two candidates were identified based on their acceptance of proline as a
substrate:PROT (Slc6a7) and SIT1 (Slc6a20a). We unexpectedly
found mRNA for both in eggs and 1-cell embryos. In addition, gene array data
for PI embryo development including MII oocytes
(Zeng et al., 2004) deposited
in the Gene Expression Omnibus database
(http://www.ncbi.nlm.nih.gov/projects/geo)
show similar expression patterns for each gene to those we report here (GEO
accession/array ID/gene name: GDS813/1422899_at/Slc6a20a and
GDS814/1455469_at/Slc6a7). Thus, we could not rule out either
candidate based on mRNA expression.
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). The results appear to rule out PROT because
although Xenopus oocytes expressing PROT showed increased transport
of proline, there was no increase in betaine transport. Also, proline
transport was blocked by histidine but not inhibited by MeAIB and, most
importantly, it was not inhibited by excess betaine. Although we cannot rule
out the possibility that accessory proteins required for normal PROT function
are absent from Xenopus oocytes, the close correspondence of proline
transport characteristics in PROT-injected Xenopus oocytes to those
previously reported in mammalian cells
(Fremeau et al., 1992) makes
this unlikely. By contrast, the transport characteristics of mouse SIT1 in
Xenopus oocytes closely matched those of betaine/proline transport in
1-cell mouse embryos (Anas et al.,
2007). Both betaine and proline were transported, inhibited by
MeAIB, and resistant to histidine. In addition, the rate of transport of 1
µM proline was approximately twice that of 1 µM betaine, which is the
same relationship as that found in 1-cell mouse embryos
(Anas et al., 2007). Taken
together, these results implicate SIT1 as the transporter that is likely to
mediate betaine and proline transport in 1-cell mouse embryos. This conclusion was supported by the results obtained with a MO targeted against Slc6a20a, which showed a substantial loss of betaine transport by 1-cell mouse embryos in which the Slc6a20a MO had been present from the GV stage. Since inhibition of global protein synthesis with cycloheximide did not block the appearance of betaine/proline transport activity at 12 hours post-egg activation, the efficacy of the MO when present from the GV stage implies SIT1 synthesis during meiotic maturation. Further investigations into the mechanism underlying the appearance of transport activity after fertilization are clearly needed. Immunolocalization of the transporter protein in mouse oocytes and early embryos would help address this question. However, thus far we have not been able to successfully use the available antiserum raised against SIT1 (a kind gift of Dr F. Verrey) for immunocytochemistry in mouse oocytes.
Function of the betaine/proline transporter SIT1 in PI mouse embryos
The activation of betaine/proline transport after fertilization and its
presence only during a very short period of development implies that it serves
an important function during PI embryo development. Although, in vitro, SIT1
transports betaine and proline, we propose that its physiological function in
the early embryo is to mediate betaine transport. Betaine is apparently
accumulated to relatively high levels in vivo. Assuming a volume of
cleavage-stage embryos of 180 pl, the measured amounts of endogenous
betaine in 1- and 2-cell embryos correspond to intracellular concentrations of
6-7 mM. This is comparable to the average concentration of 4 mM in
liver, which is the tissue with the highest known endogenous betaine level,
and much higher than the 0.1 mM in rodent blood plasma
(Slow et al., 2008) or
0.5 mM in mouse oviducts (Anas et al.,
2007). The apparent accumulation of betaine in vivo would appear
to imply that a major function of SIT1 is to mediate betaine accumulation or
retention by early PI embryos.
The betaine/proline transporter in PI embryos that we have identified here
as SIT1 is the sole route of betaine transport in PI embryos, whereas there
are at least two routes of proline transport
(Anas et al., 2007). The second
proline transport route in 1-cell embryos does not correspond to PROT despite
the presence of its mRNA, but instead resembles the classical transport system
ASC (Anas et al., 2007).
Although proline is likely to be transported into early embryos, the
importance of SIT1-mediated uptake relative to other routes is not known.
The mechanism by which betaine exerts a beneficial effect on PI embryo
development is not yet known. As discussed above, two established functions
for betaine in mammals are as an organic osmolyte and as a methyl group donor.
We (Dawson and Baltz, 1997;
Hammer and Baltz, 2002) and
others (Biggers et al., 1993)
have established the ability of betaine to protect PI embryo development
against increased osmolarity in vitro, although it is not yet certain that it
performs this role in vivo. Previous work has established glycine as a major
organic osmolyte in cleavage-stage PI mouse embryos. Glycine is transported by
the specific transporter GLYT1 (SLC6A9)
(Steeves et al., 2003), and
endogenous glycine is present in 1-cell mouse embryos at high levels [25
mM free glycine in freshly obtained 1- and 2-cell embryos; our unpublished
data, measured as described by Steeves et al.
(Steeves et al., 2003)]. Thus,
betaine present at 6-7 mM might play an important role along with glycine
in osmoprotection in vivo.
An alternative hypothesis is that intracellular betaine might instead serve
as a methyl pool during PI embryogenesis. One crucial set of events occurring
during PI embryogenesis is the global DNA demethylation and remethylation
between fertilization and implantation, which mediates the switch from
parental to embryonic epigenetic marking, while maintaining DNA methylation of
uniparentally imprinted genes (Lucifero et
al., 2004). Although the folate pathway is usually thought to
provide the methyl groups for the large array of methyltransferases in most
cells outside liver, it might be that betaine participates in PI embryos
because late PI embryos appear to express betaine-homocysteine
methyltransferase (BHMT), the key enzyme in liver that mediates the transfer
of methyl groups from betaine (our unpublished data). Further work, however,
is needed to explore this possibility, including determining whether betaine
accumulated at the 1- to 2-cell stage can be retained long enough to be
utilized in the peri-implantation period.
In summary, we have found that the betaine/proline transporter is expressed for only a restricted period of development starting a few hours after fertilization and continuing through the 2-cell stage. This transport activity is likely to correspond to SIT1, arising by activation of pre-existing transporter proteins after fertilization. Although betaine has been clearly shown to have beneficial effects on PI embryos, acting as an organic osmolyte and promoting in vitro development past the 2-cell stage, further work is needed to fully establish the physiological functions of the betaine that is accumulated by early PI mouse embryos.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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