Kooistra Megan, Trasler Jacquetta M, Baltz Jay M
Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
Biol Reprod. 2013 Sep 19;89(3):63. doi: 10.1095/biolreprod.113.111146. Print 2013 Sep.
Endogenous folate stores are required in preimplantation embryos of several species, but how folates are accumulated and whether they can be replenished has not been determined. Folates are generally taken up into cells by specific transporters, mainly the reduced folate carrier RFC1 (SLC19A1 protein) and the high-affinity folate receptors FOLR1 and FOLR2. Quantitative RT-PCR showed that Slc19a1 mRNA was expressed in mouse cumulus-oocyte complexes (COCs) and oocytes, whereas Folr1 showed expression only in preimplantation embryos, increasing from the 2-cell stage onward. The mRNAs encoding Folr2 and the intestinal folate transporter Slc46a1 were not detected. Methotrexate (MTX), an antifolate often used as a model substrate for folate transport, exhibited saturable transport in COCs and in preimplantation embryos starting at the 2-cell stage. However, folate transport characteristics differed between COCs and embryos. In COCs, transport of MTX and the reduced folate leucovorin was inhibited by the anion transport inhibitor SITS that blocks RFC1 but was insensitive to dynasore, a specific dynamin inhibitor that instead inhibits folate receptor-receptor mediated endocytosis, whereas the opposite was found in 2-cell embryos and blastocysts. The inhibitor profile and transport properties of MTX and leucovorin in COCs correspond to established transport characteristics of RFC1 (SLC19A1), whereas those in 2-cell embryos and blastocysts correspond with those of FOLR1, consistent with the mRNA expression patterns. Considerable folate was accumulated in COCs via RFC1, but the presence of cumulus cells did not enhance folate accumulation in the enclosed oocyte, indicating a lack of transfer from cumulus to oocyte.
几种物种的植入前胚胎需要内源性叶酸储备，但叶酸如何积累以及是否可以补充尚未确定。叶酸通常通过特定转运蛋白进入细胞，主要是还原型叶酸载体RFC1（SLC19A1蛋白）以及高亲和力叶酸受体FOLR1和FOLR2。定量逆转录聚合酶链反应显示，Slc19a1 mRNA在小鼠卵丘-卵母细胞复合体（COC）和卵母细胞中表达，而Folr1仅在植入前胚胎中表达，从2细胞阶段开始增加。未检测到编码Folr2和肠道叶酸转运蛋白Slc46a1的mRNA。甲氨蝶呤（MTX）是一种常用作叶酸转运模型底物的抗叶酸药物，在COC和从2细胞阶段开始的植入前胚胎中表现出饱和转运。然而，COC和胚胎之间的叶酸转运特性有所不同。在COC中，MTX和还原型叶酸亚叶酸的转运受到阻断RFC1的阴离子转运抑制剂SITS的抑制，但对dynasore不敏感，dynasore是一种特异性发动蛋白抑制剂，反而抑制叶酸受体介导的内吞作用，而在2细胞胚胎和囊胚中则发现相反的情况。MTX和亚叶酸在COC中的抑制剂谱和转运特性与RFC1（SLC19A1）已确定的转运特性相对应，而在2细胞胚胎和囊胚中的特性与FOLR1的特性相对应，这与mRNA表达模式一致。相当数量的叶酸通过RFC1在COC中积累，但卵丘细胞的存在并未增强封闭卵母细胞中的叶酸积累，表明缺乏从卵丘到卵母细胞的转移。
