It is known that the fetal placenta, via the production of an unidentified """"""""placental luteotrophin"""""""" (PL), plays an important role in the maintenance of corpus luteum function in the pregnant rabbit. The primary specific aim of the studies proposed in this grant is to isolate, purify, and identify rabbit """"""""placental luteotrophin"""""""" (PL). For these studies, the following in vitro bioassay for PL will be used to test the presence of PL bioactivity during the isolation and purification process; granulosa lutein cells taken from corpora lutea taken on day 1 of pseudopregnancy will be dissociated with collagenase and maintained in monolayer cell culture for 5 days in medium 199 (+ 0.1% BSA), in an atmosphere of 5% carbon dioxide in air at 37C. These cell cultures respond readily, with increased progesterone secretion, to hormones such as LH, and also to a factor/hormone in fetal placental-conditioned medium (FPI), which is presumed to be PL. Preliminary studies using dialysis have shown that the active component in FPI is probably a protein of greater than 8,000-10,000MW. The source of PL used for subsequent isolation and purification studies will be FP1, produced by incubating fetal placental tissue (Day 21 of pregnancy) in medium 199. Initially, FP1 will be subjected to acetone precipitation to concentrate it and to remove steroids and prostaglandins. APFPI will then be subjected to ion-exchange (DEAE and CM-cellulose) and Sephadex G-100 chromatography and HPLC. SDS polyacrylamide gel electrophoresis will be performed to determine the molecular weight and the subunit structure of the PL-bioactive protein. Throughout these purification steps, fractions will be examined for PL bioactivity in granulosa lutein cell cultures. Also, frequent comparisons of PL- bioactive material will be made with chorionic gonadotrophin and placental lactogen, which are known placental luteotrophins in other species. A monoclonal antibody will be prepared against PL and used in affinity chromatography to purify large quanitites of PL from fetal placental conditioned medium. The monoclonal antibodies and purified PL obtained will be used in subsequent studies to investigate further the physiological role of PL and to understand the mechanism by which PL acts to promote luteal function.
| Weerasinghe, K M; Gadsby, J E (1992) The presence of glandular kallikrein in rabbit fetal placental conditioned medium. Endocrinology 131:1777-81 |
| Weerasinghe, K M (1992) Prekallikrein activation in human, bovine, and rabbit plasmas: presence of an inhibitor in bovine plasma. Inflammation 16:205-13 |
