This result also indicates that PGP9. 5 is usually expressed in the cytoplasmic area of spermatogonia A and B. Interestingly, Leydig cells Trichostatin-A (TSA) of donkeys were also immunolabeled with PGP9.5 at both pre- and post-pubertal stages in this study. 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 Trichostatin-A (TSA) PGP9.5 antibody can be used as a tool to identify and isolate spermatogonia from seminiferous tubules. strong class=”kwd-title” Keywords: PGP9.5, donkey, testes, spermatogenesis 1. Introduction 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. Using tools such as molecular markers, biological processes for the proliferation and differentiation of germ and Leydig cells can be explored. These markers can also be applied to develop advanced assisted reproductive techniques such as spermatogonial stem cell (SSC) or Leydig cell transplantation and diagnose subfertility or infertility in donkeys. Previous studies have examined molecular markers for germ cells. Glial cell-derived neurotrophic factor family receptor alpha-1, promyelocytic leukemia zinc finger, and colony-stimulating factor 1 receptor were found to be markers for SSCs . In a previous study, our laboratory reported that a co-immunolabeling system with undifferentiated embryonic cell transcription factor 1 (UTF1) and deleted in azoospermia-like (DAZL) could be used to identify undifferentiated (UTF1 only), differentiating (UTF1 and DAZL), or differentiated spermatogonia (DAZL only) . Thus, the ability to more efficiently detect Trichostatin-A (TSA) more molecular markers that can identify various stages of germ and Leydig cells would be beneficial. Protein gene product (PGP)9.5, also known as ubiquitin C-terminal hydrolase L1 (UCHL1), was initially found to be expressed in neuroendocrine cells and tumors . The function of PGP9.5 is to Mouse monoclonal antibody to SMAD5. SMAD5 is a member of the Mothers Against Dpp (MAD)-related family of proteins. It is areceptor-regulated SMAD (R-SMAD), and acts as an intracellular signal transducer for thetransforming growth factor beta superfamily. SMAD5 is activated through serine phosphorylationby BMP (bone morphogenetic proteins) type 1 receptor kinase. It is cytoplasmic in the absenceof its ligand and migrates into the nucleus upon phosphorylation and complex formation withSMAD4. Here the SMAD5/SMAD4 complex stimulates the transcription of target genes.200357 SMAD5 (C-terminus) Mouse mAbTel+86- increase the available pool of ubiquitin that can attach to the proteins which are about to be degraded by the proteasome . The expression of PGP9.5 was also reported in reproductive systems such as the testes in several animals, which include mice , cattle , pigs , sheep , and goats . The expression of PGP9.5 was detected in spermatogonia and Leydig cells, but the immunolabeling pattern varies depending on the species. In addition, the PGP9.5 was immunolabeled in a reproductive stage-dependent manner. Thus, to confirm the localization of PGP9.5 in testes, the expression patterns of the marker must be tested in each species at different reproductive stages. We recently reported that PGP9.5 was expressed in the germ cells adjacent to the seminiferous tubule membrane and cytoplasmic area of Leydig cells in stallions . Based on the results of our study and other previous studies, we hypothesized that PGP9.5 is expressed in donkey testes, and it can be used as a marker for specific stages of germ cells and Leydig cells. 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.