| Objective:To investigate the promotion of erythropoietin(EPO)on the germ cell development in Cryptorchidism induced by Flutamide(Flu)in embryonic stage and the function of EPO on proliferation and differentiation as well as endocrine of primary testicular Leydig cells.Methods:1 Flu-induced offsprings randomized into three groups:Flu(n=36),Flu+EPO(n=42)and Flu+PBS(n=32)groups. Normal newborn rats were divided into control group(n=40)and EPO group(n=16).In the Flu and control groups, conventional breeding, and in the Flu+EPO,EPO and Flu+PBS groups, SD rat were treated with 1000U/(kg.d)Recombinant Rat Erythropoietin(Rr EPO) by intraperitoneal injection on Postnatal day(PD) 1,PD3,PD5 and PD7 or an equal volume of PBS. Incidence of the cryptorchidism, organ index of testis and the reproduction index were evaluated. The testicular histology, the area and the diameter of seminiferous tubules were observed. The expression of Connexin43, piwil2 and piwil4 in the testes were detected by Western blot and Immunohistochemistry, The expression of EPO and EPOR were detected by Western blot, and the testosterone level were detected by ELISA.2 The primary rat Leydig cells which obtained by differential attachment method from 1weeks, 3weeks and 7weeks old SD rats were treated with or without 2U/ml EPO. The cell proliferation index was detected by MTT and the differentiation of Leydig cell was detected by the 3β-HSD dyeing testing. and testosterone in culture medium was detected by HPLC.Results:1 The incidence of cryptorchidism in the Flu+EPO group was38.1%(16/42),which was lower than that of Flu group 55.6%(20/36)and Flu+PBS group 53.1%(17/32). Compared with the control group,the Flu-induced cryptorchidism group showed decreased testosterone level,but there is no statistical difference in the Flu+EPO induced cryptorchidism group. Compared with the control group, EPO/EPOR ratio Significantly higher in the EPO group, Flu and Flu+EPO Non-cryptorchid groups, Flu and Flu+EPO induced cryptorchidism groups.The germ cells number, The organ index,the reproduction index, the area and the diameter of seminiferous tubules in the Flu+EPO induced cryptorchidism group were greater than the Flu induced cryptorchidism group(P<0.001,P<0.05,P<0.01,P<0.01)and no significant difference from the control group(P>0.05). the expression of Connexin43 protein was normal in the testis of the Flu-induced cryptorchidism treated by Rr EPO. the expression of piwil2 and piwil4 protein was abnormal in the testis of the Flu-induced cryptorchidism group, which is normal in the Flu-induced cryptorchidism group treated by Rr EPO.2 In addition to 7W group in 72 h, the number of testicular Leydig cells in all other EPO groups were more than the non-EPO groups at the observation points.In the three age groups, the 3β-HSD dyeing cells positive rates were not statistically significant differences between EPO and non-EPO groups at the same point.In the 7W and 3W age groups,the testosterone levels in culture medium in EPO groups were significantly higher than the non-EPO groups at 24 h, 48 h and 72 h.Conclusion:1 The incidence of Flu-induced cryptorchidism can be decreased by Rr EPO treatment after birth. EPO may contribute to promoting the germ cell development of the cryptorchidism. EPO can improve testosterone levels in the cryptorchid testes, promotes the formation of blood-testis barrier, and improve reproductive potential of cryptorchid model.2 EPO can promote the proliferation and testosterone production of Leydig cell, but the differentiation of EPO on Leydig cells is not significant. |
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