Post-natal Developmental Changes Of NMS And Its Receptor In Male Meishan Pig Reproductive Axis

Neuromedin S (NMS), a novel neuropeptide isolated and purified as the endogenous ligand of orphans G protein coupling receptors NMUR-1(FM-3/GPR66) and NMUR-2(FM-4/TGR-1) in2005, has been shown to be widely distributed in the peripheral organs and the central nervous system(CNS). It has been documented that NMS plays a fundamental and implicated roles in the physiological regulation by its receptors, including neuroendocrine, dark-light rhythms, energy metabolism, stress responses and feeding behavior. Yet the potential contribution of NMS to the control of reproductive axis was few reported, especially in domestic animals, and there was no research about reproductive regulation of NMS to male pigs in literature. Using realtime fluorescence quantitative PCR and immunohistochemuistry, expression of NMS and NMUR-2was detected at male Meishan pig reproductive axis along post-natal development, including3,30,60,90and120days. The results are as follows:1. Clone and analysis of male Meishan pig NMS and its two receptor genesUsing gene homology cloning and RT-PCR technique, we got sequences of male Meishan pig NMS and its two receptor (NMUR-1and NMUR-2) genes. Further more, the nucleotide sequences and amino-acids sequences of the NMS and its receptors were analyzed by the bioinformatics software. The results showed that there was lack of12amino acids in C-terminal of male Meishan pig NMS amino-acid sequences compared with other animal species, but the main protein structure of prepro-NMS was high homology. In addition, the nucleotide sequence and amino acids of Meishan pig NMUR-1and NMUR-2both had high homology.2. Developmental changes of NMS and NMUR-2mRNA in post-natal male Meishan pig reproductive axisSamples of hypothalamic, pituitary and testicle were collected from male Meishan pig at3,30,60,90,120days respectively, and NMS and NMUR-2mRNA level were determined by realtime fluorescence quantitative PCR with β-actin as internal standard. In the hypothalamus, the patterns of NMS and NMUR-2mRNA level were similar. They increased with peak values on day90, and declined on day120. But60day was less than30day for NMS level, and60day more than30day for NMUR-2level, In the pituitary, the patterns of NMS mRNA level and NMUR-2mRNA level were different. For NMS mRNA level, it declined from day3to60, and increased from60to120, with peak value on day120. For NMUR-2mRNA level, it increased from day3to60, and declined from day60to120, with peak value on day60. The expression of testicular NMS mRNA showed that it increased from3to30, and90to120, but60days is the lowest. Besides, NMUR-2mRNA decreased from3day to60day, and increased sharply on day90which was a little higher than120day.3. Developmental changes of NMUR-2cells in post-natal male Meishan pig hypothalamus and testis.By immunohistochemistry, we detected the distribution and localization of NMUR-2in the hypothalamus and testis of male Meishan pigs along post-natal development. The results showed that NMUR-2positive particles were distributed in hypothalamic paraventricular nucleus and Leydig cells from3d to120d generally. In hypothalamic paraventricular nucleus, the size and number of NMUR-2positive neuron were all increased along post-natal development and the most significant change occurred in60to90days of age. In the testes, the number of Leydig cells decreased from30to60days, but NMUR-2positive particles increased greatly and in90and120days were similar to60days.Post-natal development changes of NMS and its receptor in male Meishan pig reproductive axis were investigated in the gene and protein level in this study. Finally, we found that NMS and NMUR-2showed developmental changes in reproductive axis of male Meishan pig during post-natal development, which suggested that NMS and NMUR-2might play a role in boar reproductive axis development, but its regulatory mechanism remains to be elucidated.