Bovine conceptuses removed from the uterus during the peri- and postattachment periods of placentation (Days 17-24 and 26-38, respectively) were cultured in a modified minimum essential medium in the presence of L-[3H]leucine to characterize in vitro synthesis of proteins released into the medium. Patterns of protein production were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography of dried gels. Four groups of low molecular weight acidic proteins (LMWAP) were observed to be synthesized during the peri- and postattachment periods. The number and relative concentrations of these changed with development. One group (A) consisted of three major and two or more minor isoelectric species (pI approximately equal to 5.8-6.8); these were the major synthesized proteins observed from Days 17-22. The major polypeptides of Group A were present at all time points examined through Day 38 and, in several preparations, appeared as doublets (Mr approximately equal to 22,000 and 24,000) through Day 29 but not thereafter. Group A polypeptides from Day 19 and 36 conceptus cultures were demonstrated by immunoblot analysis to cross-react with antiserum produced against ovine trophoblast protein-1 (oTP-1). A second group of proteins (A1) and a single protein (B) in the 20,000-24,000 Mr range were observed between Days 17 and 22. These were acidic relative to Group A and were not detected after Day 22. A fourth group (C) of LMWAP (Mr approximately equal to 14,000-18,000) was first observed around Day 21 and appeared to increase relative to Group A through Day 29. One protein from this group, C3, was the predominant LMWAP at Day 38.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine trophoblast protein-1 (bTP-1) was isolated to 90% purity from culture medium of Day 18-20 conceptuses incubated in vitro (in the presence of L-[3H]leucine) by a combination of Sephacryl S-200 gel filtration chromatography and O-(diethylaminoethyl) (DEAE) anion-exchange high-performance liquid chromatography (DEAE-HPLC). The radiolabeled protein had an Mr of 21,200 +/- 800 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had three isoelectric variants (pI 5.7-6.5) by two-dimensional SDS-PAGE. DEAE-HPLC-enriched bTP-1 cross-reacted with anti-o TP-1 serum on Western blots. A monospecific antiserum against bTP-1 was produced by excising the bTP-1 polypeptide band from preparative SDS-PAGE gels. Antiserum reacted with a single polypeptide with an Mr of 21,200 as determined by Western blotting of SDS-PAGE-separated conceptus medium proteins and by immunoprecipitation from L-[35S]methionine-labeled medium proteins followed by SDS-PAGE and autoradiography. Bovine trophoblast protein-1 was localized by immunocytochemistry in the cytoplasm of both mono- and binuclear trophectoderm cells of Day 20 bovine conceptuses, indicating that it is a product of the trophoblast.

Bovine allantoic (A) and chorionic (C) membranes from Days 29, 32, 36, and 40 of pregnancy were isolated by dissection and cultured in a modified minimum essential medium in the presence of L-[35S]methionine to characterize in vitro synthesis and release of proteins. Membranes were also cultured in the presence of the glycosylation inhibitor tunicamycin. Proteins synthesized and released into the medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography of dried gels. Stained gels were used to analyze protein from allantoic fluids. Percent incorporation of the radiolabeled amino acid into nondialyzable protein was higher for A than for C (A = 8.0 +/- 1.2 vs. C = 6.4 +/- 0.5 per 200 mg tissue) but not significantly different. C released significantly more total protein (nonradioactive) into the medium (57.0 +/- 3 vs. 9.6 +/- 0.6 micrograms/ml). Of the 25 proteins analyzed, 19 appeared to be produced primarily by one membrane or the other. In general, C was the source of a number of basic-to-neutral glycosylated proteins and A was the source of a number of more acidic glycosylated proteins. Many but not all proteins synthesized by A were present in allantoic fluid. The present study is the first to characterize protein production by isolated chorionic and allantoic membranes and to demonstrate that both tissues contribute to the production of secretory conceptus proteins.

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