Detection of prolactin inhibitory activity in uterine epithelial cell secretions and rat serum.

We have shown that the uterus of the rat contains a substance that diminishes the release of the luteotropic hormone PRL by acting directly at the anterior pituitary gland. This study was designed to determine which cell type(s) within the uterus secretes this PRL inhibitory activity (PIA) and if PIA of uterine origin appears in circulation. Enzymatically dispersed cells from uteri of ovariectomized (OVX) rats were cultured for 24 h in serum-free Dulbecco's Modified Eagle's Medium. Estimation of PIA in the spent media from heterogeneous uterine cell cultures was evaluated after 24 h by the ability to suppress PRL release from confluent monolayers of cultured pituitary cells. Spent media from uterine cell cultures containing 0.125 X 10(6) or 0.25 X 10(6) cells/well failed to significantly suppress PRL secretion. However, media from 0.5 X 10(6) and 1 X 10(6) uterine cells/well induced 36% and 85% inhibition of PRL release, respectively, without affecting basal LH release. To determine which cell type(s) in the uterus is responsible for PIA secretion, whole uteri were partitioned into epithelial, stromal, and myometrial cell fractions by differential enzymatic dissociation. Varying numbers of cells from each fraction were cultured for 24 h. PIA in the spent media from the homogeneous uterine cell cultures was estimated by its ability to suppress PRL release from cultured anterior pituitary cells. Media from epithelial cell cultures suppressed PRL secretion in a dose-dependent fashion. Media obtained from cultures of a comparable number of stromal or myometrial cells had no significant effects on PRL secretion. LH secretion was unaffected by media obtained from any concentration of the various uterine cell types. Since cultured anterior pituitary cells treated directly with crude uterine extract for 24 h recover and secrete PRL at the same rate as untreated controls during a 72-h posttreatment interval, it is unlikely that the uterine PIA is either proteolytic or cytotoxic. To determine if PIA is detectable in peripheral circulation, sera obtained from OVX or OVX hysterectomized (OVX-HYST) donor rats were incubated with cultured anterior pituitary cells for 24 h. In response to the OVX sera, there was significant inhibition of PRL release. However, upon replacement of OVX sera with OVX-HYST sera, the inhibition was significantly reduced. Thus, PIA is higher in sera of rats bearing uteri. Taken together, these data suggest that the epithelial layer of the uterus secretes a PIA which probably reaches the hypothalamo-pituitary axis through the circulation.