Suppression of chicken prolactin transcription and translation in hen anterior pituicytes by RNA interference and its effect on associated hormones

Abstract

Prolactin (PRL) is a peptide hormone synthesized and secreted by lactotroph cells in anterior pituitary of the hen. In aves, hyper secretion of PRL causes ovarian regression, ovarian dysfunction and broodiness. In addition, pituitary estrogens (E2β) play synergetic effects on both replication and synthesis of PRLin pituitary lactotrophsleading to hyperprolactinemia. The aim of thisstudy wasin vitro suppression of chPRL transcription and translation in hen anteriorpituicytes by small-interfering RNA (siRNA) targeting chPRL and assessing its effects on the mRNA levels of PRL, prolactin receptor (PRLR), follicle stimulating hormone (FSHβ), growth hormone (GH) estrogen receptor (ERα), pituitary content of estrogen (E2β) and PRL concentration in primary cultures of anterior pituitary cells obtained from broody hens. Three chPRL- siRNA’s were chemically synthesized based on turkey and chicken PRL mRNA and conducted suppression of PRL in primary cultured anterior pituicytes of hen. After 24 hours of chPRL-siRNA transfection the amount of chPRL, GH, PRLR, FSHβ, ERα, expression levels were analyzed by polymerase chain reaction (PCR). Protein content of PRL and GH in the control and siRNA transfected culture media were analysed by western blot method. Pituitary content of PRL and E2β was estimated in the cultured medium by radioimmuno assay (RIA).To investigate the effective concentration of siRNA 50nM and 60nM of PRL siRNA was introduced into primary cultured anterior pituitary cells. PRL mRNA expression was reduced to 58% at 50nM and 65% at 60nM compared with the control. Protein content of PRL was clearly suppressed in transfected cells. Conversely, the protein content of GH did not significantly different between control and treated cells. PRL concentration in the cultured medium of siRNA treated cells was significantly (P<0.05) lower than controls, whereas the pituitary E2β levels were not significantly different between the control and treated cells. Treatment of cultured cells with varying doses of E2β significantly increased the mRNA levels of PRL, GH, and ERα, protein content of PRL, GH and PRL concentration in the control group. However, mRNA levels of PRLR, FSHβ, and pituitary content of E2β levels were notsignificantly (P>0.05) increased with E2β treatment of control and siRNA transfected cells. Results suggest that, chPRL-siRNA designed in this study suppresses PRL gene expression specifically and that the level of PRLR, GH, FSHβ, ERα expression and estradiol were not associated with PRL in anterior pituitary. Our data support that chPRL- siRNA serves as a potential therapeutic tool to control hyperprolactinemia in vertebrates.