192 AMINO ACID TRANSPORTER EXPRESSION IN BOVINE OVIDUCT EPITHELIAL CELLS

Abstract

Oviduct fluid provides the environment for the gametes and early embryo but little is known about the mechanisms underlying its formation. Components of oviduct fluid have been shown to be present at concentrations different from that in blood, indicative of selective transport by the epithelial cells lining the lumen. For example, amino acid concentrations in oviduct fluid differ from those in extracellular fluid and have also been shown to be important to preimplantation embryos in vitro, enhancing development, especially when added at physiological concentrations. However, little is known about amino acid transport systems in the oviduct, and the aim of this work was to search for mRNA transcripts for amino acid transporters in bovine oviduct epithelial cells. Contra- and ipsi-lateral oviducts were removed from abattoir-derived reproductive tracts at specific stages of the reproductive cycle. Oviducts were trimmed of surrounding tissue and fat and slit longitudinally to expose the luminal surface. Bovine oviduct epithelial cells (bOEC) were scraped from the surface using a sterile glass coverslip and washed by centrifugation. mRNA was isolated using Trizol-chloroform extraction and lithium chloride precipitation methods. PCR was used to detect cDNA encoding the amino acid transporters CAT-1, CAT-4, and LAT1. A negative control (water) and a positive control (human placental cDNA) were included in each experiment and β-actin expression was used as a positive control for cDNA library generation. Products were separated by agarose gel electrophoresis. PCR for β-actin resulted in the presence of a positive band in all samples, showing successful extraction of mRNA and generation of cDNA libraries. mRNA for CAT-1 and LAT1 was detected in bOEC from contra- and ipsi-lateral oviducts and from each cycle stage tested. There was, however, no detectable mRNA for CAT-4 in any of the samples. To our knowledge, this is the first report of amino acid transporter expression in the mammalian oviduct. CAT-1 is a ubiquitous sodium-independent uniporter of cationic amino acids that has been localized to the basolateral membrane of epithelial cells. The presence of mRNA for this amino acid transporter in all samples tested is therefore to be expected. LAT1 is a obligatory exchanger which exports glutamine and cystine and imports large uncharged branched-chain amino acids. This transporter may be partly responsible for the high concentration of glutamate in the basal compartment of in vitro cell cultures reported in our previous work (Whitear and Leese 2007 Biennial Meet. Joint Fertil. Soc., York, UK). CAT-4 shares only 40% sequence homology with CAT-1 and its function is unknown. Its expression appears to be restricted to brain, testis, and placenta, and the absence of mRNA for the oviduct was, perhaps, not surprising. Further experiments will investigate expression levels of other amino acid transporters in bOEC and transporter localization using immunohistochemistry.

This work was funded by the BBSRC and ANGLE Technology Ltd.