| Background:Cryptorchidism is one of the most common congenital urogenital malformations and the high incidence shows a rising trend year by year. The treatment plan of cryptorchidism has been mature, but there are still long-term complications such as male infertility. Cryptorchidism leads to infertility for many reasons, such as testicular pathological damage, germ cell development defect, spermatogonia cell proliferation, differentiation and meiosis impairment.The current treatment for cryptorchidism infertility includes testicular organ transplantation, tissue transplantation and stem cell transplantation. However, all the above methods are restricted by ethics, which make it difficult to apply in clinical.Recent studies have found that retinoic acid is essential for the development and maintenance of male reproductive system, plays an important role in spermatogenesis microenvironment and spermatogenic cells development. However, there is no research about the role of retinoic acid in cryptorchidism to restore their spermatogenic functioncan. Objective: The purpose of this study is to clear the retinoic acid on spermatogenic cells development plays a promoting role in cryptorchidism, and study the spermatogenesis microenvironment to elucidate the mechanism of RA in promoting the development of spermatogenic cells in cryptorchidism.Method: Thirty pregnant SD rats were randomly divided into three groups: control group, cryptorchidism group and RA intervention group. In the control group, rats were fed normally without any treatment. In the cryptorchidism group, rats received continuous subcutaneous injection of Flu at the dose of 25mg?kg-1?d-1 from Day 12 to Day 20 after pregnancy. In the RA intervention group, on the basis of Flu cryptorchidism group, male newborn rats received continuous subcutaneous injection of RA at the dose of 1mg?kg-1?d-1 from Day 1 to Day 10 and Day 28 to Day 30 after birth.The incidence of cryptorchidism was recorded and the testis weight was weighed, the RA concentration of testicular tissues was analyzed by HPLC; at the same time, TUNEL apoptosis test was performed, and HE staining was carried out to observe its pathological changes; the expression of key factors for proliferation and differentiation(C-kit, PLZF, Stra8, Scp3) and testicular connexin 43 were detected by immunohistochemistry, WB, q-PCR. The tight junctions of the testis were observed by TEM. The epididymis sperms in mature rats were collected in all groups, to count them and observe their morphological changes. 15 mature male rats were reserved in each group for mating experiments, to observe their pregnancy rates.Results: The cryptorchidism models of SD rats were successfully induced by Flu. The incidences of cryptorchidism in cryptorchidism group and RA group were high and showing no significant difference between them, but the testis weight in RA group was significantly higher than that in the cryptorchidism group. The retinoic acid concentration in the cryptorchidism group was significantly lower than that in the control group and RA group(P <0.05). In the cryptorchidism group, seminiferous tubule lumen of testis was apparently narrowed, and the lumen gap became large, the spermatogenic cells disordered with less amount and growth retardation; no sperm cells and sperms in central lumen were observed. In the control group and RA group, spermatogenic cells were arranged neatly and a large number of sperm cells and sperms could be observed.The TUNEL results showed that, the spermatogenic cell apoptosis in the cryptorchidism group was significantly higher than that in the control group and RA group. In SD rats with cryptorchidism, the expression of spermatogonia proliferation and differentiation marker C-kit, PLZF and meiosis key factor Stra8, Scp3 were down regulated, and after RA intervention, the expressions were close to normal. The tight junction between Sertoli cells in undesceaded testicle was destroyed and the expression and location of gap junction protein Cox43 was abnormal, but after RA treatment, the tight junctions and gap junction can be improved. And in RA intervention group, the sperm count(4.60±0.27)×108/ml was higher than Flu cryptorchidism group(1.99±0.13)×108/ml, but similar to normal control group(5.53±0.17)×108/ml, meanwhile, the sperm deformity rate was lower than Flu cryptorchidism group. The final mating experiment found that cryptorchid rats interfered by RA, the rate of normal female pregnancy increased from 46.7% to 80%.Conclusion: In the Flu induced cryptorchidism in rats, obvious pathological changes occurred in testis, the proliferation and differentiation of spermatogonia stem cell was blocked, the spermatogenesis microenvironment was destructed, the spermatogenesis process was blocked and the sperm count and quality was declined sharply. RA intervention on the rats with cryptorchidism can not reduce the incidence of cryptorchidism, but it can work on the key processes of impaired spermatogenesis, to restart spermatogenesis process and reconstruct reproductive functions, finally restore the fertility of rats with cryptorchidism. |
PDF Full Text Request