Localization of PGP9.5 in equine testes

Abstract

Spermatogonial stem cells (SSCs) are the origination of spermatogenesis. The SSCs undergo self-renewing division to sustain stem cell population and differentiating division to produce spermatozoa (Oatley JM, Brinster RL. Methods in enzymology. 2006;419:259-82). Protein Gene Product (PGP) 9.5 is used as a putative marker for SSCs in other species including boar (Luo J, et al. Mol Reprod Dev 2006;73:1531-40), ram (Sosa JR, et al. Theriogenology. 2006;66:2091-103), and buck (Heidari B, et al. J Assist Reprod Gen. 2012;29:1029-38). The objectives of this study were 1) to identify the expression of PGP9.5 in stallion testes by immunohistochemistry and Western blot and 2) to evaluate age and seasonal effects on the number of PGP9.5 positive cells per cross section of seminiferous tubules. Testes were obtained during routine field castration of stallions of several breeds including Warmblood, Throughbred and Quarter Horses. The reproductive stage of stallions were classified as pre-pubertal (younger than 1 yr, n1⁄43), pubertal (1w1.5 yr, n1⁄47), post-pubertal (2 w 3 yr, n1⁄44), adult (4w13 yr, n1⁄45), and aged (older than 13 yr, n1⁄41). The primary antibody for PGP9.5 (rabbit-anti human, Serotec) was used at 1:1,500 dilution for immunohistochemistry. For double staining, antibody for the depleted in azoospermia-like (DAZL) or GATA4 was used as a marker for germ and Sertoli cells, respectively. Western blot procedure was carried out to verify the specificity of