| The Congjiang Xiang pig is one of the unique germplasm resources in China and not much research has been done on its reproductive characteristics.Taste receptor type 1 subunit 3(T1R3)is responsible for the perception of sweet and umami tastes in the oral cavity,but is also widely present in various non-taste tissues and organs(testis,kidney,liver,lung,adipose tissue,etc.)to perform non-taste functions,and the expression of T1R3 in testis tissue is only lower than that of the tongue.At the cellular level,the group has previously identified the involvement of T1R3 in the regulation of androgen synthesis using Congjiang Xiang pig Leydig cells.In fact,c AMP is a second messenger for testosterone synthesis and a downstream component of T1R3 transduction of taste,and it is of interest to know whether T1R3 influences steroid hormone synthase expression via c AMP-PKA-regulated transcription factors.Testosterone,an important androgen,is mostly secreted in the testicular Leydig cells and has important roles in male genital organogenesis/development,spermatogenesis,and the stimulation and maintenance of libido.In this thesis,the effects of activation and inhibition of T1R3 taste receptors by sweeteners on testosterone synthesis in Leydig cells were firstly investigated using testicular Leydig cells from the Xiang pig,and then the role of the c AMP-PKA signalling pathway in T1R3-mediated testosterone synthesis was analysed,and the potential association of transcription factors downstream of c AMP-PKA signalling on steroid synthase was explored.Finally,saccharin sodium combined with lactisole was used to treat Leydig cells to further validate the effect of saccharin sodium on testosterone synthesis,while mice were injected intra-testicularly in vivo to verify the mechanism by which taste receptors affect testosterone synthesis via c AMP-PKA signalling,thus clarifying the specific mechanism by which T1R3 regulates testosterone synthesis in mesenchymal cells of testis from Congjiang Xiang pigs.The main findings are as follows:1.Effect of T1R3 receptor activation model on testosterone synthesisThe levels of testosterone,second messenger c AMP and PKA were significantly increased in Leydig cells treated with 1 m M saccharin sodium for 4 h compared to the control group(P<0.05).Western blotting and q PCR results showed that the protein and m RNA levels of the taste receptor T1R3 were significantly increased in Leydig cells after saccharin sodium treatment(P<0.05).In addition,the phosphorylation levels of transcription factors SP1,total protein levels of SP1 and protein expression levels of St AR and 3β-HSD were significantly elevated(P<0.05).Similar to the results of saccharin sodium,testosterone levels were also significantly increased after0.5 m M,5 m M and 10 m M sucralose and 0.1 m M acesulfame-K treatment of testicular Leydig cells(P<0.05).In addition,protein expression of steroid hormone synthase 3β-HSD and CYP17A1 was also significantly increased in the sucralose and acesulfame-K-treated groups(P<0.05).2.Role of c AMP-PKA in T1R3-mediated testosterone synthesisThe c AMP,PKA levels and p-SP1,SP1,CYP17A1 and 3β-HSD protein expression were significantly reduced in the saccharin sodium + AC inhibitor group compared to the saccharin sodium group(P<0.05).Thus,AC inhibitor could inhibit the high expression of p-SP1,SP1,CYP17A1 and 3β-HSD proteins induced by saccharin sodium.Compared with the saccharin sodium group,the expression of p-SP1,St AR,CYP17A1 and 3β-HSD proteins was significantly lower in the saccharin sodium + PKA inhibitor group(P<0.05).Taken together,the role of c AMP,PKA and SP1 in T1R3-mediated testosterone synthesis was confirmed.The above results suggest that T1R3 regulates transcription factor SP1 via c AMP-PKA signalling to affect testosterone synthesis in Leydig cells.3.Effect of transcription factor SP1 on the activity of the promoter region of steroid hormone synthase CYP17A1In this section,eukaryotic expression vectors for transcription factor SP1 and promoter region vector for steroid hormone synthase CYP17A1(-1814 bp～-10 bp)were successfully constructed.After co-transfection into 293 T cells,the experimental results showed that the relative activity of luciferase was significantly higher in the p EGFP-C1-SP1 group compared with the control group(P<0.05),indicating that transcription factor SP1 can make steroid hormone synthase CYP17A1 promoter activity was significantly higher(P<0.05).4.Cellular and in vivo experiments validate the signaling mechanism of T1R3 regulation of testosterone synthesis via c AMP-PKAThe results showed that the phosphorylation level of transcription factor SP1 and the protein expression level of 3β-HSD and CYP17A1 were significantly lower in the Leydig cells treated with 5 m M lactisole in combination with saccharin sodium for 4 h(P<0.05)compared to the group treated with saccharin sodium alone.In addition,to clarify the effect of sweeteners on the steroid hormone synthesis process,we conducted in vivo experiments with mice injected with saccharin sodium in the testis.The results showed that the testis of mice injected with saccharin sodium had significantly higher levels of testosterone and c AMP than the DMEM control group(P<0.05),as well as significantly higher levels of phosphorylation of transcription factors SP1(P<0.05).This was accompanied by a significant increase in the expression of total SP1 protein(P<0.05).In addition,we also examined the expression of steroid hormone synthase and showed that the expression of CYP17A1 was significantly higher in the saccharin sodium injection group(P<0.05).The above study shows that T1R3 regulates the transcription factor SP1 via c AMP-PKA signalling to influence the process of testosterone synthesis in the Congjiang Xiang pig Leydig cells. |
