| The morphological and functional integrity of the epididymis is dependent on androgens,and Dihydrotestosterone（DHT）is one of the most potent androgens.DHT plays an important role in maintaining secondary sex characteristics and sperm maturation and storage.Two reductases,5α-red1 and 5α-red2,are involved in and regulated the secretion of DHT.Melatonin is a kind of indole hormone which is mainly synthesized and secreted by pineal gland.It can regulate the reproductive physiological function of testis and epididymis of many male animals.However,the synthesis and expression patterns of melatonin,its synthetase and membrane receptors in sheep epididymis and the regulation of physiological functions of epididymis are still unclear.In this study,the epididymis of sheep was used as the research object.The methods of liquid phase mass spectrometry,real-time fluorescence quantitative PCR（RT-q PCR）,Western blotting,immunohistochemistry,tissue and cell immunofluorescence were used to explore the synthesis and expression of melatonin and its membrane receptors MT1 and MT2 in the epididymis of sheep;the effects of melatonin on dihydrotestosterone synthesis and its synthase in sheep cauda epididymal epithelial cells;Regulation of melatonin on epididymis function in sheep at proteomics level;Regulation of melatonin on inflammatory response in sheep cauda epididymal epithelial cells.The results are as follows:1.Synthesis of melatonin and AANAT,HIOMT,MT1 and MT2 in sheep epididymisThe results of immunohistochemistry and immunofluorescence showed that AANAT,HIOMT,MT1,and MT2 were distributed in epididymal epithelial cells and smooth muscle cells,mainly in epididymal epithelial cells;liquid mass spectrometry detected that melatonin levels in the cauda epididymis was significantly higher than that in the caput,corpus epididymis;the results of RT-q PCR and Western blotting were similar to those of liquid mass spectrometry,the m RNA and protein levels of AANAT,HIOMT,MT1 and MT2were the highest in the cauda epididymal;meanwhile,the immunofluorescence and Western blotting showed that MT1 and MT2 were mainly localized in the neck and tail of spermatozoa,and their expression levels increased with the increase of sperm maturation.2.Effect of melatonin on DHT synthesis in sheep cauda epididymal epithelial cellsThe sheep cauda epididymal epithelial cells were cultured by enzyme digestion in vitro,and the cauda epididymal epithelial cells were treated with different concentrations of melatonin,luzindol（MT1 and MT2 non-selective antagonists）and 4P-PDOT（MT2selective antagonist）.ELISA,q PCR and Western blotting showed that 10-9,10-8 and 10-7 M melatonin significantly inhibited the synthesis of DHT in cauda epididymal epithelial cells,and 10-10-10-7 M melatonin significantly inhibited the m RNA and protein expressions of DHT synthase 5α-red1 and 5α-red2.In addition,immunofluorescence detection revealed that MT1 and MT2 were expressed in cauda epididymal epithelial cells.The combined treatment of melatonin and 4P-PDOT or luzindole blocked the inhibitory effect of melatonin on DHT synthesis and 5α-red1 and 5α-red2 expression in cauda epididymal epithelial cells,and the inhibition effect of luzindole was more significant.3.iTRAQ proteomic analysis of caput epididymal epithelial cellsIn this study,proteomics iTRAQ technique was used to analyze and identify the differentially expressed proteins in sheep caput epididymal epithelial cells after 10-7 M melatonin treatment.A total of 69 differentially expressed proteins were found by biological analysis,among which 41 proteins were up-regulated and 28 proteins were down-regulated in the caput epididymal epithelial cells treated with melatonin.In addition,the expressions of SOD1,COL1A1,COL1A2,PRM1,NQO2,and FN1 were verified by q PCR and Western blotting.The results showed that the expression trend of these proteins was consistent with the results of iTRAQ analysis,which indicated that the results of iTRAQ analysis were reliable.At the same time,through bioinformatics analysis,it was found that melatonin could regulate the function of epididymal epithelial cells by regulating the levels of related proteins in the process of antioxidant and anti-inflammatory response.4.Effect of melatonin on inflammatory response of sheep cauda epididymal epithelial cellssheep cauda epididymis were cultured by enzyme digestion in vitro,and Lipopolysaccharide（LPS）was used to establish the inflammatory model in vitro.q PCR and Western blotting were used to detect the regulation effect of melatonin on LPS-induced inflammatory response of sheep cauda epididymal epithelial cells.The results showed that LPS could significantly increase the expression of related proteins and inflammatory factors in TLR4/NF-κB signaling pathway in sheep cauda epididymal epithelial cells;however,melatonin treatment significantly decreased the expression of TLR4/NF-κB pathway related proteins and inflammatory factors in cauda epididymal epithelial cells.In addition,melatonin MT1 and MT2 antagonists 4P-PDOT and luzindole co-treated cauda epididymal epithelial cells,and significantly blocked the inhibitory effect of melatonin on the expression of TLR4/NF-κB pathway-related proteins and inflammatory related factors in cauda epididymal epithelial cells.In conclusion,melatonin synthesis pathway and melatonin-related protein expression are found in the caput,corpus and cauda epididymis of sheep,and melatonin can regulate the synthesis of DHT in epididymal epithelial cells through MT1 and MT2.Moreover,melatonin can participate in the regulation of sheep caput epididymal epithelial cell function through antioxidant and anti-inflammatory pathways,and melatonin effectively inhibits the inflammatory response of LPS induced sheep cauda epididymal epithelial cells,and the possible mechanism is that melatonin downregulates the expression of TLR4/NF-κB signaling pathway related proteins through MT1 and MT2,thus reducing the expression of inflammatory factors.The results provide a basis for further study of the regulation mechanism of melatonin on reproductive mechanism of male animals. |
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