Studys On Expression Of MRNA For Mouse PrRP And PrRP-R In Hypothalamus, Pituitary And Periphery Glands

ABSTRACT To investigate PrRP and PrRP-R mRNA expression in hypothalamus, pituitary and periphery glands during pregnancy and lactation, Firstly, total RNA was extracted from mouse hypothalamus and amplified by RT-PCR. The PCR products were purified and ligated into the pMD-19T vector, then transformed into competent cells of E. coli DH5a.The positive cloning was sequenced to gain mouse prolactin-releasing peptide cDNA. The nucleotide and amino acid sequence of mouse PrRP were analyzed and blasted to those of rat, human, cattle, sheep by DNAstar software. Then 36 female mouses were divided randomly into six groups (n=6). hypothalamus, pituitary, thyroid, adrenal and ovary were sampled for mRNA extraction. Expression levels of PrRP and PrRP-R mRNA in these tissues were measured by simi-RT-PCR with GAPDH as control. At the same time, blood were sampled on 6,12 and 18 d of pregnancy and lactation respectively to determine plasma concentration of progestin(P), estrogen(E2) and prolactin(PRL) by RIA. Corelation between PrRP, PrRP-R mRNA and plasma P, E2 and PRL were analyzed by SPSS 13.0 software to investigate the relation among them.As cloning result showed mouse PrRP consisted of a 276 bp open reading frame encoding 92 amino acids. The nucleotide sequences alignment result indicated that mouse PrRP nucleotide for the coding region showed 88.5%,74.8%,73.3%,78.2% identity with that of rat, sheep,cattle, human respectively. The amino acid sequences alignment result showed that deduced amino acids from mouse PrRP had 87.1%, 71.6%,70.5%,71.6% identity with that of rat, sheep,cattle, human respectively. Further analysis showed that deduced amino acid sequence for mouse PrRP-31 had 100.0%,93.8%,93.8%,84.4% identity with that of rat, sheep,cattle, human respectively. Mouse PrRP-20 had 100.0%,95.0%,95.0%,90.0% identity with that of rat, sheep,cattle, human respectively. The key amino acid residues segments RGIRPVGRFGRRRA (45-58) of PrRP, which were conservative and characteristic to mammal PrRP and indispensible for further modification to RF peptide, were identical among these species.Study on PrRP mRNA indicated that:during pregnancy and lactation, hypothalamus, pituitary, thyroid, adrenal and ovary all showed PrRP mRNA expression. Ovary had highest level and hypothalamus followed. During pregnancy, adrenal PrRP mRNA had significant positive correlation with those of pituitary and thyroid, at the same time, ovary PrRP mRNA had significant positive correlation with those of hypothalamus.Results indicated that hypothalamus and pituitary PrRP may regulated pregnancy by modulation of thyroid, ovary and adrenal respectively.During lactation, correlation between hypothalamus PrRP mRNA and pituitary, pituitary PrRP mRNA and thyroid, adrenal, ovary, all showed positive relation, which indicated that target gland PrRP mRNA might be positive regulated by hypothalamus-pituitary during lactation. The data that tissue PrRP mRNA expression levels changed on different phases indicated that PrRP might take part in relative physiological regulation during pregnancy and lactation by action on hypothalamus, pituitary and periphery glands.Study on PrRP-R mRNA indicated that:PrRP-R mRNA expressed in hypothalamus, pituitary, thyroid, adrenal and ovary during pregnancy and lactation. During pregnancy, thyroid and hypothalamus had significant higher PrRP-R level than pituitary. Hypothalamus PrRP-R had significant positive correlation with pituitary PrRP and negative correlation with ovary PrRP, which suggested that pituitary PrRP might action on hypothalamus and ovary PrRP action on non-hypothalamus to participate in physiological regulation. During lactation, Hypothalamus PrRP-R had significant positive correlation with throid PrRP, however throid PrRP-R had negative correlation with hypothalamus PrRP, which suggested that, among hypothalamus, pituitary and throid, throid PrRP might feedback on hypothalamus and hypothalamus PrRP action on non-throid to participate in physiological regulation. Pituitary PrRP-R had significant positive correlation with ovary PrRP indicated that ovary PrRP might feedback on pituitary to modulate lactation.Analysis for plasma hormones showed plasma P had significant negative correlation with PrRP mRNA in pituitary during pregnancy, which suggested that higher plasma P might inhibit PrRP expression in pituitary, then by which to regulate pregnancy. During lactation, plasma E2 had significant negative correlation to PrRP mRNA in ovary, which indicated that ovary PrRP might directly or indirectly inhibit the secretion of E2 from ovary. Plasma PRL had no signifant correlation with PrRP, PrRP-R of hypothalamus, pituitary and periphery glands provided further evidence that tissue PrRP maybe did not regulated plasma PRL.