The Effect of Ovine Trophoblast Protein-One on Endometrial Protein Secretion and Cyclic Nucleotides1

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Abstract
The effect of ovine trophoblast protein-one (oTP-1) on endometrial protein secretion was examined by using a dual radioisotope technique in which 3H- and 35S-methionine were employed to measure relative rates of protein release into the medium by endometrial explant cultures (Exp. I). Endometrium (200 mg) from Day (D) 12 of the cycle was cultured with either 5 .mu.g/ml oTP-1, 5 .mu.g/ml bovine serum albumin (BSA) or 1 mM dibutyryl cyclic adenosine 3'',5''-monophosphate (DbcAMP). Culture media from control BSA and treated explant cultures were mixed. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and detected by fluorography. Individual protein spots were punched from gels, extracted, and their radioactive content measured. Ratios of 3H:35S were used to determine treatment effects. In Experiment II, 3H- and 14C-leucine were used for the dual radiolabel, and the DbcAMP treatment was omitted. In both experiments, a protein having a molecular weight (Mr) of about 70,000 and a pI .simeq. 4 was increased (p < 0.01) 200-400% by oTP-1. Secretion of several other endometrial proteins was also a amplified in the presence of oTP-1. The polypeptides that increased in response to oTP-1 were inhibited by DbcAMP, and vica versa. In Experiment III, endometrial explants from D12 cyclic ewes were cultured for 4 h with either 5 .mu.g/ml oTP-1 or 5 .mu.g/ml BSA to determine whether oTP-1 influenced concentrations of 3'',5''-cyclic adenosine monophosphate (cAMP) and 3'',5''-cyclic guanosine monophosphate (cGMP). Concentrations of cAMP in oTP-1-treated endometrium were lower (p < 0.1) than in BSA-treated endometrium (0.29 vs. 0.41 pmoles/mg tissue, respectively). Levels of cGMP were unaffected by oTP-1. In Experiment IV, endometrium from D14 of the cycle was incubated in medium alone or in medium containing either 2 .mu.g/ml oTP-1, 1 .mu.g/ml oxytocin (OXY), or oTP-1-plus-OXY. None of the treatments significantly affected cAMP levels. In Experiment V, D16 endometrium was collected from pregnant and nonpregnant ewes that had received either 0 or 10 IU OXY i.v. cAMP was higher (p < 0.01) in endometrium from pregnant ewes compared to nonpregnant ewes (27.9 vs. 13.0 pmoles/mg tissue, respectively), but OXY had no detectable effect on endometrial content of cAMP in either nonpregnant or pregnant ewes. Data from these experiments indicated that 1) oTP-1 amplifies secretion of at least 11 endometrial proteins, 2) oTP-1 decreases cAMP and has no effect on cGMP, 3) DbcAMP and oTP-1 have opposite effects on secretion rates of specific endometrial proteins, and 4) oxytocin does not stimulate an increase in cAMP in endometrial tissue of pregnant or nonpregnant ewes.