Elsevier

Journal of Pediatric Surgery

Volume 47, Issue 9, September 2012, Pages 1724-1729
Journal of Pediatric Surgery

Original article
Is matrix metalloproteinase required in postnatal testicular tubules for germ cell maturation?

https://doi.org/10.1016/j.jpedsurg.2012.03.062Get rights and content

Abstract

Background/Aim

Cryptorchidism may cause infertility by failed transformation of neonatal gonocytes into adult dark spermatogonia, the putative stem cells for spermatogenesis. Gonocytes migrate centrifugally to the tubular basement membrane to become adult dark spermatogonia. Regulation of this transformation remains unknown. We aimed to investigate neonatal rodent testis matrix metalloproteinase (MMP) production to see whether MMPs loosen extracellular matrix between Sertoli cells to facilitate gonocyte movement.

Methods

Sprague-Dawley rat testes (n = 4-6 per group) were collected at embryonic day 19 (E19) and postnatal (P) days P0 to 10 for immunohistochemistry. Immunofluorescent confocal images were captured for presence of membrane type 1 MMP (MT1-MMP), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 2 (TIMP2), mouse VASA homologue, anti-Müllerian hormone, and androgen receptor in tissue sections. Testicular proteins were analyzed by immunoblotting.

Results

Membrane type 1 MMP was strongly present in gonocytes at E19 then decreased, whereas it increased in testicular somatic cells from P0 to P10. Testicular protein levels of MT1-MMP, MMP2, and androgen receptor were constant from E19 to P10. Anti-Müllerian hormone protein sharply decreased after P2, whereas TIMP2 gradually increased from E19 to P10. Gonocytes migrated to basement membrane at P2 to P6.

Conclusion

Membrane type 1 MMP, MMP2, and TIMP2 were present in testis from E19 to P10 during gonocyte migration and transformation into spermatogenic stem cells. Increased knowledge about germ cell development may aid efforts to improve fertility in cryptorchidism.

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.

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