Original articleIs matrix metalloproteinase required in postnatal testicular tubules for germ cell maturation?☆
Section snippets
Animals
Sprague-Dawley (SD) rats were purchased from a commercial supplier and housed in the institute's Animal Research Laboratory in standard shoebox cages, and they were maintained in a temperature-controlled atmosphere with a 12-hour light-dark cycle and fed commercial rat chow and water ad libitum. All studies were approved by the institutional animal experimentation ethics committee (A644).
Immunohistochemistry
Time-mated dams were killed at E19, with E0 defined as the day a vaginal plug was found and the male fetuses
Immunoblotting
Protein was extracted from testes (n = 4-12/group), and immunoblotting was performed. Briefly, SD rats were killed as described earlier, and testes were dissected, immediately frozen on dry ice, and stored at −70°C. Frozen testes were homogenized in 1% (vol/vol) Triton X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 2 mM sodium vanadate, 10 mM NaF, and complete protease inhibitor mixture (1 tablet per 50 mL; Roche Applied Bioscience, Mannheim, Germany). After homogenization by
Mouse VASA homologue
The mouse germ cell marker, MVH, identified a large number of gonocytes in the center of the SD rat testicular cords at E19 (Fig. 1, Fig. 2A) . On P2, the MVH-labeled gonocytes were still centrally located in the cords, with the first evidence of migration between the Sertoli cells to reach the basement membrane on P4. This was conspicuous at P4 to P6 with the MVH+ germ cells no longer round but with visible cell processes extending between the Sertoli cells (Fig. 2B). By P8, nearly all the
Discussion
These results show that there are high levels of MT1-MMP protein in germ cells in the testis at birth in male SD rats. Around P4 to P6, when the gonocytes were migrating between the Sertoli cells to reach the basement membrane of the testicular cords, MT1-MMP was increased on the Sertoli cells and other somatic cells as well as on the basement membrane itself. Matrix metalloproteinase 2, which is required for ECM remodeling, was present weakly between the testicular cords. The protein levels of
Acknowledgment
We sincerely thank Shirley D'Cruz for her help on the manuscript; Silverton Buraundi, Pam Farmer, and Daniela Bodemer for laboratory technical assistance; Priscilla Soo for her help on the immunoblotting graph; and Dr Cong Sun for germ cell statistical analysis.
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Supported in part by NH&MRC Grant 607365 and the Victorian Government's Operational Infrastructure Support Programme. J.-G.Z is supported by NHMRC Program Grant 461219.