SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.
Betaine (N,N,N-trimethylglycine) added to culture media is known to substantially improve the development of preimplantation mouse embryos in vitro, and to be imported into 1-cell embryos by a transporter that also accepts proline. Here, we found that the betaine/proline transporter is active in preimplantation mouse embryos only for a short period of development, between the 1- and 2-cell stages. Betaine/proline transport was activated after fertilization, beginning approximately 4 hours post-egg activation and reaching a maximum by approximately 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.
Anas
Mohamed-Kheir Idris
MK
Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada.
Lee
Martin B
MB
Zhou
Chenxi
C
Hammer
Mary-Anne
MA
Slow
Sandy
S
Karmouch
Jennifer
J
Liu
X Johné
XJ
Bröer
Stefan
S
Lever
Michael
M
Baltz
Jay M
JM
D026921:Amino Acid Transport Systems, Neutral / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001622:Betaine / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D002970:Cleavage Stage, Ovum / Q000166:cytology / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005306:Fertilization / Q000235:genetics / Q000502:physiology; D050485:GABA Plasma Membrane Transport Proteins; D018507:Gene Expression Regulation, Developmental; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D015053:Zygote / Q000378:metabolism*
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D017931:DNA Primers; D050485:GABA Plasma Membrane Transport Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C502967:XT3 protein, mouse; C041832:proline transport protein; C072207:betaine plasma membrane transport proteins; D001622:Betaine