Expression And Localization Of Transforming Growth Factor-β1 And Its Signal Transducer Smad2 And Smad4 In Alpaca Testis

Objective: Transforming growth factor -β1 (TGF-β1) plays important roles during testicular development and spermatogenesis, Smad2 and Smad4 are downstream mediators for TGF-β1. The main aim of the present study is to determine the expression and localization of TGF-pi and its signal transducer Smad2 and Smad4 in alpaca testis.Methods: The whole testes were obtained from alpacas aged 12 months, 24 months (n=3) at the Scientific Research Base of Shanxi Agriculture University. The protein expression of TGF-β1, Smad2 and Smad4 in alpacas testes was examined by Western blotting. The cellular localization and developmental changes were examined by immunohistochemistry SABC. Quantitative analysis of the immunostaining was measured by the Computer Image Analysis System (product of Sichuan University. China, Mias-2000).The results were analysed with one-way ANOVA, and expressed as means ±SE.Results: TGF-pi , Smad2 and Smad4 were present throughout testicular development, the most pronounced changes in TGF-β1, Smad2 and Smad4 immunoreactivity occurred during spermatogenesis. The immunostaining of TGF-pi was present in Leydig cells, Sertoli cells, Myoid cells and spermatogenic cells at the testis of alpacas aged 12 months and 24 months, the optical density (OD) of TGF-β1 was respectively 0.7305±0.0115, 0.5626±0.0122 in Leydig cell: 0.4207±0.0130. 0.3257±0.0082 in sertoli cells, 0.6455 + 0.0820, 0.6569 + 0.0230 in myoid cells, 0.4579±0.0092, 0.3743±0.0158 in Spermatogonia ( p<0.01 ); Smad2 was immunolocalized at testis of alpacas aged 12 months, there was positive immunoreaction in Leydig cell, sertoli cells, spermatogenic cells, the density of Smad2 was respectively 0.54460±01034, 0.5712±0.0086 in sertoli cells(p>0.05 ), 0.1741 ±0.0088, 0.3794±0.01066 in sertoli cells, 0.30160±.01440. 0.5604±0.0078 in Spermatogonia (P<0.01); Smad4 was immunolocalized at testis of alpacas aged 12 months and 24 months, there was positive immunoreaction in Leydig cells, Sertoli cells, spermatogenic cells, the density of Smad4 was respectively 0.7425±0.0113, 0.6599±0.0107 in Leydig cells (p>0.05), 0.2552±0.0129> 0.4699±0.0083 in Sertoli cells, 0.4417±0.0138, 0.5136±0.0089 in Spermatogonia (p<0.01 ), No expression was detected in the Myoid cells at the 12 months testis. The positive immunoreactivity was located at the cytoplasm, not in the nucleus; No positive immunoreactivity was observed in the negative control.Conclusion: The distribution of TGF-β1, Smad2 and Smad4 in the testes of alpacas aged 12 months, 24 months was demonstrated. From the changes in pattern of localization of these peptides, we conclude that they are strictly regulated in Leydig cells, Sertoli cells, germ cells. It suggests their involvement in the regulation of spermatogenesis, and provides direct evidences for the moleculer mechnism of TGF- β1 action in the alpaca testis during postnatal development and spertogensis.