First published online November 21, 2008
doi: 10.1242/10.1242/dev.026575
Development 135, 4123-4130 (2008)
Published by The Company of Biologists 2008
SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage
Mohamed-Kheir Idris Anas1,
Martin B. Lee1,
Chenxi Zhou1,
Mary-Anne Hammer1,
Sandy Slow5,
Jennifer Karmouch1,
X. Johné Liu1,2,4,
Stefan Bröer6,
Michael Lever5 and
Jay M. Baltz1,2,3,*
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.
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Fig. 1. Betaine and proline transport activity in mouse oocytes and
preimplantation embryos. (A) Total and non-saturable betaine
transport. The total rate of 1 µM [3H]betaine transport at each
stage (labeled at bottom of figure) is indicated by the black bars, and the
nonspecific, non-saturable rate (with 5 mM unlabeled betaine) by the gray
bars. Total betaine transport was significantly different from non-saturable
transport at stages indicated by asterisks (*P<0.05,
***P<0.001 within stages, by Student's
t-test). Each bar represents the mean±s.e.m. of at least three
independent measurements. (B) Specific betaine transport. Data in A
were used to obtain the rate of specific betaine transporter activity by
subtracting the mean non-saturable transport from the total transport at each
stage. Bars labeled with different letters are significantly different from
each other (P<0.05 by ANOVA and Tukey-Kramer post-hoc test).
(C) Specific proline transport. Specific transport of 1 µM
[3H]proline was calculated as for betaine. Each bar represents the
mean±s.e.m. of at least three independent measurements. nd, not
determined.
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Fig. 2. Initiation of betaine transport after parthenogenetic activation with
Sr2+. The rate of betaine transport was measured as a function
of time after activation of mouse MII oocytes by exposure to Sr2+
(exposure time indicated by bar). Activated oocytes are indicated by black
circles. Control oocytes were treated identically but not exposed to
Sr2+ (white circles). Parthenogenotes developed to the 2- and
4-cell stages (2c and 4c) at the times indicated. Asterisks indicate
significant difference from t=0 (*P<0.05,
***P<0.001; ANOVA and Tukey-Kramer post-hoc test). Each
point represents the mean±s.e.m. of three independent measurements,
except for t=0 where there were ten (s.e.m. smaller than symbol and
not shown).
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Fig. 3. Betaine transport development in vivo. The rate of betaine transport
in mouse oocytes is shown 19-29 hours post-hCG. Fertilization was assumed to
take place in the oviduct  14 hours after treatment with human chorionic
gonadotropin (post-hCG). Points not sharing the same letters are significantly
different (P<0.05; ANOVA and Tukey-Kramer post-hoc test). Each
point represents the mean±s.e.m. of three independent measurements.
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Fig. 4. Effect of inhibition of protein synthesis on development of betaine
transport activity after egg activation. The rate of betaine transport was
measured in freshly obtained mouse MII oocytes (MII 0 hr), MII oocytes
maintained in culture for the entire period (MII 12 hr),
Sr2+-activated oocytes 12 hours post-activation (Sr2+),
and oocytes that were activated with 50 µg/ml cycloheximide. Bars with
different letters are significantly different (P<0.05; ANOVA and
Tukey-Kramer post-hoc test). Each bar represents the mean±s.e.m. of
three independent measurements.
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Fig. 5. Brefeldin A (BFA) reversibly inhibits development of betaine transport
activity after MII oocyte activation. Betaine transport activity in mouse
MII oocytes parthenogenetically activated with Sr2+ was determined
in the presence or absence of BFA. Two separate experiments are shown: (a) 7
and 12 hours and MII oocytes at t=0 (triangle); (b) 10 and 30 hours
and MII oocytes (inverted triangle). The results are combined in this figure,
but statistical analysis was performed separately. Parthenogenotes were
cultured in control medium (black circles) or with BFA (white circles). The
dashed arrows indicate the transfer of parthenogenotes from BFA to BFA-free
culture at the time indicated by the arrow tail (7 or 10 hours), with betaine
transport measured at the time indicated by the arrowhead (12 or 30 hours).
Asterisks indicate significant difference from MII oocytes at t=0
(**P<0.01, ***P<0.001; ANOVA and
Tukey-Kramer post-hoc test). The rates after transfer from BFA (arrows) were
not significantly different at 12 hours (white diamond) or 30 hours (white
square) from rates after culture in the continuous absence of BFA at the same
times (black circles at 12 and 30 hours). Each point is the mean±s.e.m.
of three independent measurements of the total rate of betaine transport
(non-saturable rate was not subtracted in this set of experiments).
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Fig. 6. RT-PCR detection of mRNA for SIT1 (Slc6a20a) and PROT
(Slc6a7). PCR was performed on cDNA from mouse MII eggs (MII),
1-cell (1c), 2-cell (2c), 4-cell (4c), 8-cell (8c), morula (M) and blastocyst
(B) stage embryos, with kidney (K) as a positive control to indicate the
position of amplicon (the band intensity from kidney relative to those of the
embryos is of no physiological relevance). Slc6a20a and
Slc6a7 mRNAs were most strongly detected in MII eggs and 1-cell
embryos. H2afz was used as a positive control for the presence of
cDNA. The expected amplicon sizes (bp) are indicated on the left and by arrows
in each marker lane (m, a 100-bp ladder, the largest visible band of which is
sized). Only portions of each gel are shown, but no other bands were visible
below or above. The example shown is one of two independently collected sets
of RNA. In the other, intense bands were evident at the MII and 1-cell stages
as shown here, but faint bands were also visible at other stages.
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Fig. 7. Proline and betaine transport by Xenopus oocytes expressing
mouse SIT1 or PROT. (A,B) Transport characteristics of
Xenopus oocytes expressing mouse SIT1. Proline (A) and betaine (B)
transport were measured in oocytes expressing SIT1 or in control
water-injected oocytes (H2O) as indicated. The effect of 5 mM
betaine (+Bet), histidine (+His) or 2-methylaminoisobutyric acid (+MeAIB) is
shown. Bars with different letters (a, b) are significantly different within
each panel (P<0.05 by ANOVA and Tukey-Kramer post-hoc test). Each
bar represents the mean±s.e.m. of four to seven individual oocytes,
except for the water-injected control (n=10). (C,D)
Transport characteristics of Xenopus oocytes expressing mouse PROT.
Proline (C) and betaine (D) transport were measured in oocytes expressing PROT
(labels as in A,B). Since there was no saturable betaine transport (D),
competitive inhibitors were not tested for betaine. Each bar represents the
mean±s.e.m. of five to eight oocytes, except the water-injected control
(n=10). One additional set of injections of PROT was performed (not
shown), which confirmed that proline but not betaine uptake was increased in
PROT-expressing oocytes as compared with water-injected oocytes
(n=5-7 each).
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Fig. 8. Morpholino against Slc6a20a suppresses betaine transport in
mouse parthenogenotes. (A) Effect of morpholinos on betaine
transport in mouse parthenogenotes. Rates of betaine transport were determined
after injection of MOs into GV oocytes followed by in vitro maturation and
parthenogenetic activation with Sr2+. The MO designed to block
translation of SIT1 (Slc6a20a) significantly decreased the rate of
betaine transport (a versus b, P<0.01 by ANOVA and Tukey-Kramer
post-hoc test) relative to uninjected parthenogenotes (none) or those injected
with a control MO. Each bar represents the mean±s.e.m. of three
independent experiments, each containing 8-12 oocytes. (B) Effect of
MOs on functional expression of exogenous mouse SIT1 in Xenopus
oocytes. Xenopus oocytes were co-injected with Slc6a20a cRNA
and MOs (labeled as in A). Induced proline uptake was significantly decreased
by the Slc6a20a MO (a versus b, P<0.001 by ANOVA and
Tukey-Kramer post-hoc test). Each bar represents the mean±s.e.m.
proline uptake in seven to eight individual oocytes after subtraction of the
mean uptake in water-injected controls.
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© The Company of Biologists Ltd 2008
