Protein secretion by the endometrium during pregnancy in the ewe

Abstract

Mature Merino ewes were surgically prepared so that after mating they became unilaterally pregnant. Ewes were killed between days 91 and 124 of pregnancy and the uterine milk was collected from the ligated uterine horn. Explants of intercaruncular and intercotyledonary endometrium were collected from the ligated and unligated uterine horns, respectively, and incubated in the presence of L-[4,5-3H]leucine to determine in vitro rates of protein synthesis and secretion. Protein:DNA and DNA:wet tissue ratios were also determined in the explants from some ewes. The proteins secreted in vitro and those present in uterine milk were partially characterized, using several procedures. Antisera raised in rabbits against uterine milk and absorbed against plasma from an ovariectomized ewe were used to detect uterine milk antigens in a variety of tissues and plasma. An average of 150 +/- 26 mL of creamy coloured uterine milk containing 30.7 +/- 4.6 mg mL-1 protein was recovered from 14 ewes. The rates of protein synthesis and secretion in vitro and the protein:DNA ratio were all higher in intercotyledonary than in intercaruncular endometrium, but the predominant radiolabelled protein(s) secreted in vitro by both types of endometrium was shown by gel filtration to have a molecular weight of approximately 60 kDa. This 'uterine pregnancy protein' was not secreted by endometrial explants from a non-pregnant ewe. Uterine milk contained a substantial amount of 60 kDa protein. The molecular weight and lack of sub-unit structure of the 60 kDa fraction of proteins secreted in vitro was confirmed by SDS-acrylamide electrophoresis and density gradient centrifugation. Pronase treatment confirmed the proteinaceous nature of the 60 kDa fraction, and heparin-Sepharose affinity chromatography showed that the predominant secreted protein(s) in this fraction was a glycoprotein. In immuno-double-diffusion tests antisera prepared against uterine milk produced a number of precipitation bands against both uterine milk and culture medium (after incubation of endometrial explants from a pregnant ewe) and a single band against allantoic fluid, but did not react with plasma from an ovariectomized ewe or a day-124 pregnant ewe or its fetus. The antisera did not react with lung, kidney or liver from the same pregnant ewe or with uterine flushings from a non-pregnant ewe. In immuno-electrophoresis tests, at least eight distinct antigens were detected in uterine milk and three of these were also present in allantoic fluid.