Study On Testicular T1R3 Involved In Testosterone Synthesis Of Congjiang Xiang Pig At Puberty

Taste receptor type 1 subunit 3(T1R3),is encoded by Tas1r3 in mice.As a sweet or umami taste receptor,T1R3 is widely expressed in extra-oral cells,especially in testicular Leydig cells(LC)and spermatozoa.It was reported that the model mice with deletion of Tas1r3 and gnat3 was displayed the poor semen quality,abnormal testicular morphology and male sterility.Our previous studies showed that mice fed with saccharin sodium contributed to change the testicular T1R3 expression,and a higher expression of T1R3 could increase the testosterone level,while a lower expression of T1R3 led to an inferior semen quality and a disordered spermatogenic.Given that testicular LC is the main site of testosterone synthesis,it raises a possibility that T1R3 can exert an effect on animal reproductive performance by regulating the process of testosterone synthesis and the relationship between T1R3 and testosterone synthesis was still need to be clarified.Congjiang Xiang pig is a rare kind of local minipig breed pig in Guizhou,which is characterized by favarite meat and similar to human on Physiology and biochemistry.Collectively,in present study,we first determined the method of testicular fixation in Congjiang Xiang pig.Then,comprehensively consider the testis index and morphological features on a variety of ages to decide whether the pig is arriving at puberty.Next,the expression of T1R3 was describe in different stage during testis development and spermatogenic epithelium cycle by RT-qPCR and Western blotting.Finally,in vitro study,the association between T1R3 and testosterone systhesis-related factors was investigated by using saccharin sodium to activate T1R3.The main results are as follows:1.The results showed that the mDF-fixed testis was discolored and weight reduction compared with 4%PFA-fixed testis.Furthermore,the intercellular space between spermatogenic epithelial cells increased,the cytoplasm vacuolized and the lumen interstitial area atrophic which fixed with 4%PFA.Intensity of immune response of marker proteins of germ cells,Sertoli cells and Leydig cells increased by 39.8%,27.8% and 76.4% compared with mDF-fixed testis,the mDF-fixed testis was suitable for HE staining,while the 4%PFA-fixed testis was more suitable for IHC.2.The testicular weight,long axis and short axis at 15-30 d increased 298.05%,66.42% and 65.45% respectively(P<0.05),the area of seminiferous tubules and the thickness of seminiferous epithelium increased by 136.12% and 40.19% respectively(P<0.05),while the above indexes was immutable after 60 days old.Microscopically,a small number of deformed spermatozoa were found in the seminiferous tubules of Xiang pig for the first time in 30 days old.Although the number of SC did not change with age,we still observed a rapid increase in the number of germ cells(P<0.05).Indepth analysis showed that the increase of the number of germ cells was positively correlated with the increase of area of seminiferous tubules and thickness of seminiferous epithelium.We divided the pre and post spermatogenic cycle into three or eight stages according to morphological changes of spermatogenic epithelium.3.The mRNA and protein levels of the two proteins at 30-day old were significantly higher than that of at 15-day old but lower than at 60-day old,while there was no significant difference between 60,90 and 120-day old,and the expression pattern of above proteins is consistent with plasma testosterone level.IHC analysis showed that T1R3 was mainly located in LC and extended sperm cells,with a small amount in SC;PLCβ2 was located in all types of germ cells,SC and LC.T1R3 and PLCβ2 were strong expressed in stage Ⅱ-Ⅵ than in stage Ⅰ,Ⅶ and Ⅷ in the seminiferous epithelial cycle.Intensity of immune response of T1R3 and PLCβ2 in their cytoplasm was higher than that of other types of cells in the same period when round spermatozoa transformed into extended spermatozoa and extended spermatozoa in stage Ⅱ-Ⅵ.T1R3 was continuously expressed in Ⅶ -Ⅷ pachytene spermatocytes and round spermatozoa,but the expression of PLCβ2 decreased.4.The results showed that both 100IU and 50IUhCG could up regulate the mRNA and protein expression of steroid hormone synthesis key enzyme,and increased the level testosterone and intracellular second messenger cAMP.The activation model showed that the T1R3 expression of LC in Xiang pig could be significantly activated by saccharin(1 mM,0.1 mM for 4 h).T1R3 activation will increase the mRNA expression of 3β-HSD,STAR,CYP17A1 and 17β-HSD,which is consistent with the Western blotting results of 3β-HSD and CYP17A1.We further observed that the ability of secrete testosterone was consistent with the change of cAMP content and the expression of key enzymes of steroid hormone synthesis and T1R3.Furthermore,T1R3 activation by 1mM-dose saccharin sodium promoted the testosterone synthesis of LC which has been induced by 50IUhCG,and accompanied overexpression of T1R3 and steroid hormone synthetase and increased intracellular cAMP,3β-HSD,CYP17A1 and17β-HSD,which eventually led to testosterone synthesis.