Saccharin Sodium Regulated Testosterone Synthesis In The Testicular Leydig Cells Of Congjiang Xiang Pigs Through T1R3

Although the synthesis of testosterone is regulated by the classical hypothalamicpituitary-gonadal axis-mediated cAMP/PKA signaling,numerous factors were also affected.Saccharin sodium is an artificial sweetener commonly used in everyday life and production.The male reproductive activity could be influenced by the expression of Taste receptor type 1 subunit 3(T1R3),which was encoded by the Tas1r3 gene and largely expressed not only in taste cells but also in Leydig cells and spermatocytes.T1R3 can perceive saccharin sodium.Latest studies from us revealed that Saccharin sodium by T1R3 regulation of testosterone synthesis might be related to intracellular cAMP in the Xiang pig’s Leydig cells.Whereas cAMP/PKA signaling pathway to be considered as the classical signal for testosterone synthesis in Leydig cells,it is therefore hypothesized that T1R3 regulates the synthesis of testosterone through cAMP/PKA signaling in Leydig cells of Xiang pig testes.To verify the hypothesis,this study was focused on cAMP/PKA signaling pathway,by activating or inhibiting the T1R3 in Leydig cells,the upregulation or downregulation effects of T1R3 on testosterone synthesis-related factors,cAMP/PKA signaling and their downstream transcription factors were explored;Metabolomics technique was applied to clarify the relevant signaling pathways and metabolites profile shifting during the regulation of testosterone synthesis by T1R3.cAMP/PKA upstream and downstream signaling inhibitors were consequently applied to investigate the role and mechanism of this signaling pathway of T1R3 in the regulation of testosterone synthesis.Ultimately,the specific mechanism of T1R3 regulation of testosterone synthesis was revealed to better understand the reproductive characteristics of Xiang pigs,improve fertility,and promote the exploitation of Xiang pig resources.The main results are as follows:1.Developing the inhibition/activation model of T1R3 in testicular Leydig cellThe Leydig cells of Congjiang Xiang pig were under the incubation and stimulation of Lactisole and saccharin sodium for 2 h and 4 h respectively,the expression of T1R3 receptor was significantly decreased with 5 m M Lactisole treatment and increased with 1 m M saccharin sodium treatment at both m RNA and protein levels.2.Activating or inhibiting the T1R3 down-regulated/up-regulated testosterone levels and the expression of testosterone synthesis related factors in testicular Leydig cellsInhibition of T1R3 decreased the concentration of testosterone,the expression of steroidogenic enzymes(3β-HSD and CYP17A1),and the level of intracellular cAMP and PKA,as well as downregulating the phosphorylation level of the transcription factor CREB that involving in the downstream signaling pathway of cAMP/PKA.In contrast,activation of T1R3 receptors significantly increased the testosterone level and the expression of the testosterone synthesis related factors,as well as intracellular cAMP and PKA concentrations.3.T1R3 altered concentration profiles of the steroidogenic related metabolites and its cAMP-PKA signaling pathway in Leydig cellsIn testicular Leydig cells of the Congjiang Xiang pig,the concentrations of progesterone,17a-hydroxyprogesterone,androstenedione,testosterone and deoxycorticosterone were significantly decreased under the T1R3 inhibition group comparing with the control.The five steroidogenic metabolites levels: progesterone,17a-hydroxyprogesterone,11-hydroxyandrosterone,deoxycorticosterone,and testosterone,were increased significantly after activation of T1R3 receptors.Combining with the pathway analysis demonstrated that T1R3 regulated testosterone synthesis presumedly via a cAMP-PKA signaling pathway,and progesterone was ultimately converted into testosterone by the catalytic action of steroid hormone synthases.4.Blocking the upstream/downstream signaling of cAMP-PKA inhibited T1R3 agonist that inducing testosterone synthesis in Leydig cellsIn Leydig cells,AC inhibitor and PKA inhibitor were applied to intervene the T1R3 agonist-treated group,it was found that the T1R3 agonist treatment could significantly elevated the concentration of testosterone and the expressions of testosterone synthesis-related factors when comparing with the control.However,the application of either AC inhibitor or PKA inhibitor alone merely decreased the expressions of the steroidogenic related factors and the testosterone levels.Furthermore,combining AC inhibitor with PKA inhibitor significantly inhibited the increase of testosterone concentration and steroidogenic enzymes’ expressions,as well as the rise of intracellular cAMP and PKA contents and CREB phosphorylation levels that induced by T1R3 agonist(saccharin sodium),inducing no effect on T1R3 expression.In summary,the results suggested that Saccharin sodium can mediate cAMPPKA signaling pathway via T1R3 to regulated the testosterone synthesis in testicular Leydig cells from the Congjiang Xiang pig.