Stage-Dependent Expression of Protein Gene Product 9.5 in Donkey Testes

SIMPLE SUMMARY: Spermatogenesis and steroidogenesis are key functions of the testes. Molecular markers that identify each stage of germ cells and Leydig cells can identify and isolate specific germ or Leydig cells. Protein gene product (PGP)9.5 is observed in neuroendocrine cells and tumors; it is also used for the immunohistochemical detection of spermatogonial stem cells (SSCs) in various species of animals. It was found that the immunolabeling of PGP9.5 in testicular tissue was not observed in the seminiferous tubules in the pre-pubertal stage. However, in the post-pubertal stage, spermatogonia were immunolabeled with PGP9.5. Interestingly, some Leydig cells were immunolabeled with PGP9.5 in both pre- and post-pubertal stages. This study reflects that the PGP9.5 antibody can be used as a tool to identify and isolate spermatogonia from seminiferous tubules in the post-pubertal stage of donkey testes. ABSTRACT: Molecular markers can be used to identify and isolate specific developmental stages of germ cells and Leydig cells. Protein gene product (PGP)9.5 expression in spermatogonia and Leydig cells has been reported in several species. The stages of spermatogonia and Leydig cells expressing PGP9.5 vary depending on the species and reproductive stages. Thus, the objectives of this study were (1) to identify the localization of PGP9.5 in donkey testicular cells, and (2) to compare the expression patterns of PGP9.5 in donkey testicular cells between pre- and post-pubertal stages. Testes samples were collected following the routine field castration of six donkeys. Western blotting was performed to verify the cross-reactivity of the rabbit anti-human PGP9.5 antibody to donkey testes. Immunofluorescence was performed to investigate the expression pattern of PGP9.5 in testicular tissues at different reproductive stages. In Western blotting, the protein band of the PGP9.5 antibody appeared at approximately 27 kDa, whereas the band was not observed in the negative control treated with normal mouse IgG. In the pre-pubertal stage, the expression of deleted in azoospermia-like (DAZL) was found in some spermatogonia in pre-pubertal testicular tissues. However, the immunolabeling of PGP9.5 in testicular tissue was not observed in the seminiferous tubules. In stages 1 and 2, spermatogonia were immunolabeled with either PGP9.5 or DAZL. In contrast, PGP9.5 and DAZL were co-immunolabeled in some of the spermatogonia in stages 3 to 8. Interestingly, some Leydig cells were immunolabeled with PGP9.5 in both pre- and post-pubertal stages. In conclusion, the PGP9.5 antibody can be used as a tool to identify and isolate spermatogonia from seminiferous tubules.