Conceptus proteins' effects on luteal maintenance in the ewe

The functional lifespan of the ovine corpus luteum (CL) is prolonged when a viable conceptus is present. Infusion of unfrac-tionated day 16 ovine conceptus protein (CP) prolongs CL function up to 52 days or more, while infusion of isolated ovine trophoblast protein-1 (oTP-1) prolongs CL function to a lesser extent (3-5 days).

Studies were performed to determine whether oTP-1 is a necessary component of total CP for antiluteolysis. Ovine TP-1 was removed from total CP via repeated diethylaminoethyl-cellulose (DEAE) ion exchange column chromatography. Removal of measurable ovine trophoblast protein-1 (oTP-1) was confirmed by two dimensional polyacryl-amide gel electrophoresis (2D PAGE) and fluorography of dried gels.

Beginning on day 12 (estrus = day 0), either 3.4 ml (620 pg/ml) oTP-1 free CP or diluted sheep serum were infused daily into the uterine lumen of each of three animals for 7 days (days 12-18). Peripheral blood samples were collected daily for 13 days (days 12-24), and progesterone (P₄) concentration was determined by radio-immunoassay. On day 24, sheep infused with oTP-1 free CP were examined for maintenance of the marked CL. All sheep had ovulated and formed new CL. Results of P₄ analysis demonstrated luteal regression (P₄ < 1 ng/ml) by day 16 in all animals. Analysis by Wilcoxon's paired sample test indicated no significant difference in duration of P₄production by the CL of treated or untreated ewes. Failure of the infused fractionated protein to prolong luteal function indicates that oTP-1 may be a necessary component of the CP antiluteolysin.

A separate but related experiment determined the feasibility of increasing availability of conceptus proteins of interest by changing the culture media at 24 hour intervals. Qualitative analysis of conceptus culture media by 2D PAGE and fluorography and determination of percent incorporation of radiolabelled tracer and concentration of total proteins synthesized in vitro showed no change in the profile of proteins synthesized after prolonged incubation. Therefore, an efficient way to increase the availability of conceptus proteins of interest is to prolong the amount of time a conceptus remains in culture by changing the culture media at various intervals.