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

Changes in the ratio between immunoreactive isoforms of prolactin using Western blotting and in the total prolactin content using radioimmunoassay were measured in pituitary glands from turkey hens at different physiological stages. The type of glycosylation (N- or O-linked carbohydrates) was determined using endoglycosidase digestion (N-glycosidase F, O-glycosidase, and neuraminidase). Low levels of prolactin were observed in pituitary glands from sexually immature, out-of-lay, and molting hens. Higher levels were present during the egg-laying period and the highest levels were detected in hens which expressed incubation behavior. Two immunoreactive bands of apparent molecular weights of 24 and 27 kDa were visualized on Western blots, corresponding to the nonglycosylated and glycosylated forms of prolactin, respectively. In pituitary glands from incubating turkey hens, about 70% of the prolactin was glycosylated (27-kDa isoforms), whereas about 60% was glycosylated in immature and in hens during the first egg-laying period. In pituitaries from out-of-lay and molting hens the percentage of glycosylated prolactin 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 the pituitary gland. Digestion of the isoforms with N-glycosidase F resulted in a single band with an apparent molecular weight of 24 kDa. Partial deglycosylation was achieved using neuraminidase, whereas digestion with O-glycosidase had no apparent effect on the isoforms. Thus it appears that the glycosylated isoforms of prolactin have N-linked carbohydrates containing sialic acid.