Changes in levels of immunoreactive prolactin isoforms during a reproductive cycle in turkey hens

Changes in the ratio between immunoreactive isoforms of prolactin using Wes tern blotting and in the total prolactin content using radioimmunoassay wer e measured in pituitary glands from turkey hens at different physiological stages. The type of glycosylation (N- or O-linked carbohydrates) was determ ined using endoglycosidase digestion (N-glycosidase F, O-glycosidase, and n euraminidase). Low levels of prolactin were observed in pituitary glands fr om sexually immature, out-of-lay, and molting hens. Higher levels were pres ent during the egg-laying period and the highest levels were detected in he ns which expressed incubation behavior. Two immunoreactive bands of apparen t molecular weights of 24 and 27 kDa were visualized on Western blots, corr esponding to the nonglycosylated and glycosylated forms of prolactin, respe ctively. In pituitary glands from incubating turkey hens, about 70% of the prolactin was glycosylated (27-kDa isoforms), whereas about 60% was glycosy lated in immature and in hens during the first egg-laying period. In pituit aries from out-of-lay and molting hens the percentage of glycosylated prola ctin was 38 and 33%, respectively. Thus, higher percentages of glycosylated isoforms (27 kDa) were associated with high levels of total prolactin and lower percentages were associated with low levels of prolactin content in t he pituitary gland. Digestion of the isoforms with N-glycosidase I: resulte d in a single band with an apparent molecular weight of 24 kDa. Partial deg lycosylation was achieved using neuraminidase, whereas digestion with O-gly cosidase had no apparent effect on the isoforms. Thus it appears that the g lycosylated isoforms of prolactin have N-linked carbohydrates containing si alic acid. (C) 1999 Academic Press.