RESUMEN
Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of â¼227 µM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Betaína/metabolismo , Blastocisto/metabolismo , Células del Cúmulo/metabolismo , Oocitos/metabolismo , Aminoácidos/metabolismo , Animales , Betaína/farmacocinética , Transporte Biológico/fisiología , Blastocisto/citología , Proteínas Portadoras/metabolismo , Células del Cúmulo/citología , Femenino , Fertilización/fisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Uniones Comunicantes/metabolismo , Iones/metabolismo , Ratones , Ratones Endogámicos , Oocitos/citología , Embarazo , TritioRESUMEN
Radiolabeled compounds that are substrates for transmembrane transporters can be used to study transport and metabolism in mammalian oocytes and preimplantation embryos. Because even very small amounts of radioisotopes can be detected, these techniques are feasible to use with only a few oocytes or embryos, even down to the level of single oocytes or embryos. Here, we describe the methods for determining the transport and accumulation of radiolabeled compounds into oocytes and preimplantation embryos and the determination of the rate of saturable transport via specific transporters in the plasma membrane.
Asunto(s)
Embrión de Mamíferos/metabolismo , Oocitos/metabolismo , Animales , Transporte Biológico , Calibración , Membrana Celular/metabolismo , Implantación del Embrión , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Marcaje Isotópico , Ratones , Oocitos/citologíaRESUMEN
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 preimplantation 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 developmental 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, b(0,+), and ASC/asc were active throughout oocyte growth and maturation, increasing during growth. In contrast, GLY, beta, and x(c)(-) had little or no activity during growth but 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.