Effects of chicken prolactin siRNA on pituitary insulin like growth factor-1 and prolactin receptor in in vitro cultured hen anterior pituicytes

Abstract

Up-regulation of the chicken prolactin (chPRL) has been implicated in incubation behavior in galliforms that induces broodiness and gonadal involution. In this study, we evaluated the knock down of PRL in hen anterior pituicytes cultured in vitro by small-interfering RNA (siRNA) targeting of chPRL, as well as the efficacy of this treatment on PRL gene expression, prolactin receptor (PRLR) and insulin like growth factor I (IGF-I).The chPRLtargeted siRNA were chemically synthesized and constructed based on chicken and turkey PRL mRNA. Expression of chPRL was analyzed by polymerase chain reaction (PCR) with designed primers. Culture media were assayed for IGF-I content utilizing the immuno radio metric assay (IRMA) Kit. Results indicate that, significant (P<0.05) reduction (56%) in PRL mRNA was observed following chPRL siRNA transfection into primary cultured anterior pituicytes. Protein content of PRL was clearly suppressed in siRNA transfected cells. Levels of IGF1 and PRLR mRNA were not significantly different between treated and non-treated cells. This analysis was carried out to study the effects of chPRL siRNA on the expression profiles of pituitary IGF-I and PRLR which are similar to the biological actions of PRL. Treatment of cultured cells with varying doses of vasoactive intestinal peptide (VIP) stimulated PRL levels in the control group. Our results may suggest that the siRNA designed in this study suppresses PRL gene expression specifically and that the levels of IGF1 and PRLR mRNA expression are not associated with PRL in anterior pituitary. We found that RNA interference (RNAi), targeting chPRL specifically suppressed PRL without showing any effects on pituitary IGF1 and PRLR. Our data support that siRNA against chPRL serves as a potential therapeutic tool to control broodiness in domestic hens.