Implantation, Pregnancy and Parturition
(T557) MOLECULAR CLONING AND CHARACTERIZATION OF OVINE PROSTAGLANDIN TRANSPORTER: EXPRESSION AND REGULATION IN UTERUS DURING ESTABLISHMENT OF PREGNANCY.
Sontineni, Vinod1, Banu, Sakhila1, Spencer, Thomas1, Bazer, Fuller1, Arosh, Joe1, 1 Texas A&M University, College Station, TX
ABSTRACT- Prostaglandins (PGs) are central mediators in several reproductive processes that diffuse poorly through plasma membranes in spite of their lipid nature. Though PGs cross the cell membrane by simple diffusion the estimated flow rate would be too low to exert and / or maintain biological functions. Prostaglandin transporter (PGT) mediates the efflux as well as influx of PGs to effect their biological actions or cellular inactivation. Recently, we have unraveled the role of PGT in mammalian reproduction (Banu et al. PNAS 2003). In this study we have cloned ovine PGT (oPGT) and characterized its expression in endometrium and myometrium during the estrous cycle and early pregnancy (n= 4 ewes / day / status). Uteri were collected by hysterectomy on specific days of the estrous cycle (days 10-16) and pregnancy (days 10-20). Expression of PGT mRNA was studied in endometrium and myometrium by RT-PCR / Southern blot analyses. The oPGT cDNA consisted of 1935 nucleotides encodes a protein containing 644 amino acids (70 kDa). The oPGT protein is predicted to have 12 transmembrane domains (SLCO2A1 superfamily), and possesses 8.5% positively and 4.5% negatively charged amino acid residues for a net positive charge. The amino acid sequences of oPGT protein share 75-85% identity with other mammalian species. In oPGT, 3 potential glycosylation sites at positions 134, 477 and 490, and several serine and threonine phosphorylation sites in intracellular loop were predicted. Arginine and Lysine located at positions 561 and 614 respectively are considered important for functional integrity of oPGT. Valine and Alnine located at positions 611 and 612 respectively are species specific. In endometrium, the level of PGT mRNA decreased 6 fold and 4 fold on days 10-12 and 14-18 of pregnancy, respectively compared to days 10-16 of the estrous cycle. Interestingly, expression of PGT mRNA increased from day 20 of pregnancy. In myometrium, PGT mRNA was expressed at low constant level between days 10 and 16 of pregnancy and estrous cycle, but increased from day 18 of pregnancy. PGT mRNA levels were affected (P<0.01) by day, status or their interaction in endometrium but not in myometrium. Results of this study suggest the important role for PGT and the associated cellular transport of PGs in uterus during the period of luteolysis and / or peri- implantation.
KEY WORDS: prostaglandins, uterus, pregnancy, sheep