Isolation of choriomammotropin from the ovine placentome

Placentomes, taken from ewes at 101-141 days gestation, were homogenized and subjected to differential and density gradient centrifugation. In the presence or absence of protease inhibitors, choriomammotropin (ovine chorionic somatomammotropin, oCS) was detected by radio-immunoassay, and banded at two positions in 30-60% w/v sucrose gradients. The first band (soluble form) eluted in the sample zone of the gradient and contained 33 +/- 6.1% (mean +/- s.e.m., n = 10) of the total oCS present in the gradient. The remainder (48 +/- 7.6%) of the oCS (particulate form) sedimented to 37.5-43% w/v sucrose. Both these bands became sharper with decreasing protein loaded onto the gradients. 125I-oCS, pre-equilibrated with a placentome preparation, eluted in the same position as the first band of endogenous oCS. When loaded onto a second gradient the second band eluted at 38-41% w/v sucrose, but after being subjected to ultrasonic disruption or treatment with Triton X-100 this oCS eluted in the sample zone. There was no correlation between gestational age of the ewe and the percentage oCS in either the soluble or particulate form, the concentration of oCS, or enrichment by protein. We have shown that following homogenization, differential and density gradient centrifugation, oCS exists in two forms. We suggest that the particulate form of the hormone is contained within the electron-dense granules of placentome cells. The soluble form may represent oCS in the process of secretion and hormone which has leached out of the granules during homogenization and centrifugation of the placentomes.