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Although vasoactive intestinal peptide (VIP) is a well characterized physiological PRL-releasing factor in avian species, its regulated expression is not fully understood. We cloned complementary DNAs encoding the prepro-turkey VIP (prepro-tVIP) molecule from an adult turkey hypothalamic complementary DNA library. When the amino acid sequence of the prepro-tVIP was compared to chicken and mammalian sequence, it was found that the isolated tVIP molecules lacked the 27-amino acid peptide histidine isoleucine (PHI) portion of the precursor protein. Several tissues showed an alternatively spliced tVIP transcript that lacked the PHI sequence. Only in the hypothalamus did tVIP-specific primer pairs and reverse transcription-polymerase chain reaction produce two alternatively spliced fragments. The larger hypothalamus-specific fragment was subjected to nucleotide sequence analysis and identified as containing the alternatively spliced PHI-containing exon, which encoded a 27-amino acid PHI peptide in addition to the 8 amino acids that flanked the peptide. Hypothalamic tVIP expression was shown to be up-regulated during the incubation phase of the reproductive cycle. The increased steady state level of tVIP messenger RNA appears to be regulated by nesting behavior, because nest deprivation dramatically suppressed its expression. Levels of the minor tVIP transcript containing both the PHI- and VIP-encoding exons did not significantly change between reproductive stages and were maintained at approximately 4-6% of the total tVIP transcript level. Our findings provide further evidence that VIP is the most important PRL-releasing factor in birds. Our study should serve as a useful model for determining whether PHI contributes in any way to the physiological role of PRL regulation. Revealing the tissue distribution of VIP and PHI gene expression and tissue-specific alternative splicing could contribute to an understanding of the physiological functions of the two peptides as well as their relative roles in PRL regulation.

Vasoactive intestinal peptide (VIP) has potent PRL-releasing activity, but its physiological role in the regulation of PRL release during the avian reproductive cycle is not known. We used indirect immunofluorescence to determine if changes in hypothalamic VIP are associated with the shifts in circulating PRL during the reproductive cycle of the domestic turkey. In the naturally hyperprolactinemic incubating hen, the majority of VIP immunoreactivity (VIP-IR) existed within neurons of the infundibular nuclear complex (INF) and fibers in the external layer of the median eminence. Within the INF, the numbers of VIP-IR cells increased during the cycle, paralleling increases in serum PRL. In the reproductively inactive, nonphotostimulated hen with low serum PRL, essentially no positive cells were noted, whereas the incubating hen exhibited 32.1 +/- 2.2 cells/pair of adjacent sections in the anterior INF and 59.6 +/- 2.0 cells in the posterior INF. Exposure of inactive hens to a stimulatory photoperiod resulted in a 2.6-fold increase in serum PRL with the appearance of VIP-IR cells in the INF. During laying and incubation, further increases were observed in the number of positive cells in the INF and serum PRL as well as a greater fiber density in the median eminence. To further examine the association between changes in VIP-IR and serum PRL, circulating PRL was artificially lowered by depriving incubating hens of their nests for 0, 2, 5, and 10 days. On day 2 of nest deprivation, serum PRL declined markedly to 12% of day 0 levels, with VIP-IR cell numbers at 64% and 46% in the anterior and posterior INF, respectively. By day 10, birds exhibited cell numbers in the INF averaging 20% of those observed in the day 0 incubating hens, with serum PRL at 6% of day 0 levels. The results of these studies indicate a possible causal relationship between hypothalamic VIP and changes in PRL secretion during the avian reproductive cycle, providing a basis for further research on the importance of this peptide as well as factors responsible for the modulation of its expression in hypothalamic INF neurons.