Detection of novel molecules recognized by anti-placental lactogen antibody in rat amniotic fluid

Amniotic fluid contains various bioactive substances including the placental PRL family. In the present study, it was elucidated that rat amniotic fluid contained immunoreactive proteins which had different molecular sizes and pI values from the authentic placental lactogens (PLs) in the rat, recognized by antipeptide antibody to the N-terminal peptide of rat PL-I. Immunoreactive PLs residing in the amniotic fluid were characterized further by two-dimensional sodium dodecyl sulfate gel electrophoresis (2DE), immunoblotting and anion-exchange chromatography. Amniotic fluid collected from rats on day 12 of pregnancy contained two PL-like molecules, tentatively called A1 (MW 75 kDa, pI 4.6) and A2 (MW 99-102 kDa, pI 5.3-5.4). A1 and A2 are specific to the amniotic fluid, because no such molecules were found in the serum or placental extracts. Immunoblot analysis of amniotic fluid revealed that A1 levels increased, whereas those of A2 decreased to an undetectable level up to day 16 of pregnancy. When the A1 concentrations from days 12 to 20 were monitored intensively, they increased from day 12 to 14, were maintained until day 18, and then decreased dramatically by day 20. The expression pattern for A1 was therefore completely different from those of authentic placental PRL family members found in serum and placental tissue, indicating that the A1 is distinct from them. Partial purification by anion-exchange chromatography and 2DE revealed that A1 consisted of 5 isoforms.