Effect Of Testicular Denervation On Male Germ Cells

Objective:To investigate distribution pattern of spermatic nerve fibers in testes and effect of testicular denervation on apoptosis and differentiation of male germ cells.Methods:Immunohistochemical ABC staining method was used to observe PGP9.5,CPON,NPY and VIP immunopositive nerve of testes in adult males.Mature SD rats(350-375g)received bilateral surgical removal of the superior spermatic nerve(SSN)and the inferior spermatic nerve(ISN). The animals were killed 1d,4d and 8d after operation.Terminal deoxynuleotidyl tansferase-mediated dUTP nick end-labeling(TUNEL)were applied to detect apoptosis.Forty-eight mature SD male rats were randomized into a sham operation group(SO)and three experiment groups, and the latter underwent bilateral surgical removal of the superior spermatic nerve(SSN)or/and the inferior spermatic nerve(ISN).Blood samples were obtained before and after operation.The serum levels of testosterone(T), luteinizing hormone(LH),follicle stimulating hormone(FSH),prolactin (PRL)and estrogen(E2)were assayed.Adult SD rats received a single intraperitoneal injection of EDS at a dose of 40mg/kg,60mg/kg,75mg/kg, 90mg/kg,130mg/kg respectively.The vehicle control received injection of DMSO,and the blank control was injected nothing.Rats were killed on days 3,7 and 14 after initial treatment.The testis were collected for stereology, immunohistochemistry of P450scc.Results:1)PGP9.5 and CPON positive fibers are mainly observeded in the testicular interstitium,in close association with seminiferous tubules. CPON immunopositive nerves were also found in perivascular area.The density of distribution of CPON nerves is less than that of PGP9.5.NPY immunopositive nerves were only observed in perivascular area.VIP immunopositive nerve was absent in the testicular parenchyma.2)TUNEL showed that spermatogonia and Leydig cells underwent apoptosis in denervated testes.Peak of spermatogonia apoptosis occurred 1 day after operation.Levels of Leydig cell apoptosis were same at the three time points. 3)After operation the T levels decreased and there were significant differences between preoperative levels and postoperative levels in the SSN, ISN and SSN+ISN groups(P＜0.05).The LH levels in the SSN,ISN and SSN+ISN group rose severely after operation and they were significantly different compared to the preoperative levels(P＜0.05).The serum levels of FSH also elevated sharply at 1 day following operation in all groups(P＜0.05), but they soon after returned to the levels before operation(P＞0.05).The PRL levels rose gradually after operation except in the SO group.Compared to preoperative levels,significant differences were found at 21 days,28 days following surgery in the ISN group(P＜0.05),at 14 days,21 days,28 days following surgery in the SSN group(P＜0.05)and also at 1 day,7 days,14 days,21 days,28 days following surgery in the SSN+ISN group(P＜0.05).Including the SSN,ISN and SSN+ISN groups,the serum levels of E2 dramatically decreased from 1 day after operation to 14 days after operation(P＜0.05),then sustained the low levels(P＜0.05).4)days after EDS treatment,Leydig cells were almost killed,and there were no cells stained for P450scc in testis interstitial.14 days after EDS treatment,we discovered a new Leydig cell population in the group of 60mg/kg.The changes in P450scc enzyme activities coincided with the results of stereology. 3 day and 7 day after EDS treatment,the number of positive cells staining P450scc reduced in all EDS treating group when compared to the blank control(P＜0.05);In the group of 60mg/kg,the number of positive cells staining P450scc 14 days after EDS treatment was higher than 3 day and 7 day(P＜0.05),and was not significant different with the blank control(P＞0.05).Conclusion:1)Adult male gonads exhibit rich innervation of peptidergic nerves,which may be involved in regulation of spermatogenesis. 2)Testicular denervation causes significant apoptotic death of spermatogonia and Leydig cells a few days after operation.Apoptosis of the two cells inevitably leads to regressed tubular epithelium.3)Contribution of Testicular Denervation to Gonadal Hormone in the Adult Rats is high.Spermatic nerves play an important role in spermatogenesis.4)The EDS can kill the Leydig cells of adult rats actually.The dose of 60mg/kg could modify the process of Leydig cells development at pubertal best.