Characterization of glycosylation of prolactin in galliformes

Prolactin (PRL) is a highly versatile hormone in terms of its biological functions. In Galliformes, high levels of PRL are associated with incubation behaviour and hatching. It has been shown that these high levels of PRL were associated with an increased ratio of glycosylated PRL (G-PRL) versus non-glycosylated PRL (NG-PRL) in turkey (Bedecarrats et al., 1999a). This suggests that glycosylation of PRL is related to its proper function during those stages of life. However, the mechanism(s) controlling post-translational modification of PRL are unknown. In order to investigate genes associated with the glycosylation of PRL, an in vitro study was undertaken. The pituitary glands of day 24 turkey embryos (n=60) were collected and pooled into two groups which were incubated in medium 199 for 4 hours in the absence or presence of 10-7 M vasoactive intestinal peptide (VIP). Western blot analysis of PRL was used to assess the response of the pituitary glands to the VIP stimulation. As expected, the absolute level of PRL as well as the percentage of glycosylated PRL isoform increased following stimulation with VIP. The mRNA of both stimulated and non-stimulated samples were extracted and reverse transcribed into cDNA. Using suppression subtractive hybridization (SSH), a cDNA subtractive library which only contained differentially expressed cDNAs between VIP stimulated and non-VIP stimulated turkey pituitary glands was constructed. Seventeen percent of the genes from the library are related to cell proliferation and apoptosis. Ten genes were selected for real-time PCR analysis. The functions of these genes and their potential roles in response to the stimulation by VIP and glycosylation of PRL are discussed