In vitro release of isoforms of prolactin from pituitary glands of turkey hens at different physiological stages

To study the in vitro release of PRL isoforms, anterior pituitary glands fr om medium white turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Pituitaries were cut into hemi-pituitaries aft er collection and placed into separate perifusion chambers. Medium (M199) w as continuously perifused through the system and pituitaries were stimulate d with cVIP (10(-7) M). Total PRL content was monitored by RIA and, the rat io of immunoreactive PRL isoforms in the perifusate was estimated by Wester n blotting. After exposure to X-ray film for autoradiography, the relative intensity of the bands was analyzed by densitometry. All the perifused pitu itaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands with relative molecular weights of 24 and 27 kDa were detected by Western blotting. The immunoreactive band corresponding to the glycosylated isoforms of PRL (27 kDa) was predominant in samples from egg- laying and incubating hens and the band corresponding to the nonglycosylate d isoform (24 kDa) was predominant in samples from out-of-lay and molting s tages. No changes in the ratio of isoform released were detected during cVI P stimulation. Our data clearly show that glycosylated and nonglycosylated PRL isoforms are released by the pituitary gland in vitro in the same relat ive proportion that was previously observed in pituitary extracts and thus are likely to reflect the secreted forms of PRL in the blood during various physiological stages. In addition, the PRL-releasing activity of VIP does not affect the ratio of isoforms secreted by the pituitary gland in vitro. (C) 1999 Academic Press.