PROBLEM: During spermatogenesis, it has been suggested that the number of germ cells to be matured is regulated and restricted through the apoptotic mechanism. In the present study, we investigated the expression and apoptotic role of Fas and Fas ligand (L) in the murine testis. METHOD OF STUDY: The expression of Fas-FasL in the murine testis was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR)-Southern blot hybridization, in situ hybridization, and Western blot methods. The terminal deoxynucleotide transferase mediated dUTP-nick end label (TUNEL) and DNA fragmentation methods were applied to detect the generation of apoptosis in germ cells. RESULTS: By means of RT-PCR-Southern blot hybridization, we demonstrated the positive expression of Fas in testicular germ cells, and of FasL in testicular cells. supporting the findings with in situ hybridization that Fas was localized in germ cells, whereas FasL was localized in Sertoli cells of murine testis. A specific band at 45 kDa was obtained in the lysates from testis and germ cells with Western blot analysis. Then, the co-incubation of germ cells with Spodoptera frugiperda (Sf9)-FasL cells in vitro resulted in the induction of apoptosis in germ cells detected by the TUNEL method. Furthermore, DNA fragmented ladders were also demonstrated in germ cells co-incubated with Sf9-FasL cells. CONCLUSION: Fas-FasL system seemed to play an apoptotic role in spermatogenesis by the molecular interaction between FasL on Sertoli cells and Fas on germ cells.
PROBLEM: During spermatogenesis, it has been suggested that the number of germ cells to be matured is regulated and restricted through the apoptotic mechanism. In the present study, we investigated the expression and apoptotic role of Fas and Fas ligand (L) in the murine testis. METHOD OF STUDY: The expression of Fas-FasL in the murine testis was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR)-Southern blot hybridization, in situ hybridization, and Western blot methods. The terminal deoxynucleotide transferase mediated dUTP-nick end label (TUNEL) and DNA fragmentation methods were applied to detect the generation of apoptosis in germ cells. RESULTS: By means of RT-PCR-Southern blot hybridization, we demonstrated the positive expression of Fas in testicular germ cells, and of FasL in testicular cells. supporting the findings with in situ hybridization that Fas was localized in germ cells, whereas FasL was localized in Sertoli cells of murine testis. A specific band at 45 kDa was obtained in the lysates from testis and germ cells with Western blot analysis. Then, the co-incubation of germ cells with Spodoptera frugiperda (Sf9)-FasL cells in vitro resulted in the induction of apoptosis in germ cells detected by the TUNEL method. Furthermore, DNA fragmented ladders were also demonstrated in germ cells co-incubated with Sf9-FasL cells. CONCLUSION: Fas-FasL system seemed to play an apoptotic role in spermatogenesis by the molecular interaction between FasL on Sertoli cells and Fas on germ cells.
Authors: A D'Alessio; A Riccioli; P Lauretti; F Padula; B Muciaccia; P De Cesaris; A Filippini; S Nagata; E Ziparo Journal: Proc Natl Acad Sci U S A Date: 2001-02-27 Impact factor: 11.205