Autonomic Biosynthesis And Functional Characteristics Of Melatonin In Thyroid In Sheep

Melatonin(MEL) and Thyroxine(TH) are two important hormones in the regulatory pathway of seasonal breeding in sheep. MEL is mainly synthesized and secreted by the pineal gland, where AA-NAT and HIOMT play important roles in the process. AA-NAT is a rate-limiting enzyme and HIOMT is a key enzyme in the process of all. TH is synthesized and secreted mainly from thyroid gland and is regulated by TRH gene and TSH gene. Recent studies have confirmed that the rats’ thyroid gland expressed the two key enzymes, AA-NAT and HIOMT which are necessary to synthesize MEL, and melatonin receptor 1A(MT1). At the same time, human and mouse’ thyroids also express TRH-β, TRHR and the alternative splicing isoforms of TSH-β. It suggestted that the synthetic MEL might be involved in the synthesis of TH. In order to verify whether the thyroid gland could independently synthesize MEL, whether the synthesized MEL could independently regulate the synthesis of TH, and even further affect the seasonal breeding regulation in sheep, the following studies were done:(1) Detecting the expression of the genes including AA-NAT, HIOMT, MT1, TRH, TRHR, TSH-β and TSHR in sheep thyroid gland by RT-PCR technology in order to determine whether the sheep thyroid can independently synthesize and secrete MEL, and potentially participate in regulating the synthesis of TH. The results showed that the sheep thyroid could express the key genes synthesizing MEL, MEL receptor gene, as well as genes regulatiing TH biosynthesis in thyroid glandincluding TRH, TRHR and TSHR, but the TSH-β gene was not detected.(2) Established the system for Primary culture of sheep thyroid of cells. By using the RT-PCR, immunofluorescence and ELISA technology, the expression of related genes including TPO, TG, TSHR, and TG, as well as the synthesis and secretion of TH T3/T4 in cultured thyroid cells were detected. The results showed that the simple and feasible method for primary culture of sheep thyroid cells was successfully established. It provides an experimental platform for studying the function of sheep thyroid gland in vitro.(3) By using immunofluorescence, Western blot, ELISA and other technologies, the expression and localization of key enzymes AA-NAT and HIOMT and MT1 in the thyroid tissue and cultured sheep primary thyroid cells were examined. The results showed that the sheep thyroid cell can independently synthesize and secrete MEL and there might exist a new pathway for regulating the synthesis of TH by MEL in sheep thyroid.(4) By using immunofluorescence method, the expression of TSH-β in thyroid tissue was investigated. The cultured primary sheep thyroid cells were treated by exogenous MEL and the levels of T3/T4 were detected by ELISA technologies. The results showed that the sheep thyroid cells might not express TSH-β. There might no direct association between the independent synthesis and secretion of MEL and that of TH in sheep thyroid gland. However, under the stimulation of exogenous MEL, the expression of TPO gene was increased. It indicated that melatonin might involve in the anti-oxidization of thyroid cells.In summary, our results showed that the sheep thyroid could independently synthesize and secrete MEL, and MT1, but not all the genes that regulate the synthesis of TH. In vitro cell experiments indicated MEL couldn’t regulate the synthesis of TH, but could stimulate the expression of TPO. The results suggested that melatonin might be involved in the anti-oxidization of thyroid cells. Whether the MEL involved in other autocrine or paracrine regulatory pathways, even participate in the regulation of seasonal breeding need to be further investigated.