CHARACTERIZATION AND CULTURE OF SPERMATOGONIAL STEM CELLS IN PRE- AND POST-PUBERTAL CATS

Abstract

Study 1 : aimed to culture and characterize feline SSCs. In Exp. 1, testes from pubertal domestic cats were immunolabeled to examine the expression of markers (GFRalpha-1, c-kit and DDX-4). In Exp. 2 and 3, testicular cells were digested and cultured in vitro using a modified SSC culture system. The resultant presumptive SSC colonies were then collected for SSC identification (Exp. 2), or further cultured in vitro on feeder cells (Exp. 3). Morphology, gene expression and immunofluorescence were used to identify the SSCs. Exp. 1 demonstrated that varying types of spermatogenic cells exist and express different germ cell/SSC makers. A rare population of SSC located at the basement membrane of the seminiferous tubules was specifically identified by co-expression of GFRalpha-1 and DDX-4. Following enzymatic digestion, grape-like colonies formed by 13-15 days of culture. These colonies expressed GFRA1 and ZBTB16, but did not express KIT. Although we successfully isolated and cultured feline SSCs in vitro, the SSCs could only be maintained for 57 days. In conclusion, this study demonstrates for the first time, that SSCs from testes of pubertal domestic cats can be isolated and cultured in vitro. These cells exhibited SSC morphology and expressed SSC specific genes Study 2 : aimed to identify G/SSC transition and also to improve the SSC establishment in vitro. The testes were divided into 3 groups according to donor age (I:<4 months, II:4-6 months and III:>6 months). Exp. 1 studied the development and morphology of testicular cells by histology, transmission electron microscopy, immunohistochemistry. Exp. 2 determined expression of GFRalpha-1, DDX-4 and c-kit. The numbers of GFRalpha-1+ cells were also analyzed using flow cytometry. The established SSCs isolated from group II and III were characterized by mRNA expression and TEM (Exp. 3). Chronological changes of testicular cells, in terms of morphology, proliferation and apoptosis markers at G/SSC transition were demonstrated. The size and morphology including the ultrastructure of SSCs were apparently distinguishable from the gonocytes. The results demonstrated that group II testes contained highest numbers of SSC per seminiferous cord/tubule (17.66±2.20%) and GFRalpha-1+ cells (14.89±5.66%) compared with other groups. The findings coincided with an increased efficiency of SSC derivation in group II when compared to group III (74.33±2.64%vs.23.33±2.23%, p<0.001). The resulted colonies expressed mRNA essentially importance for SSCs (GFRA1, ZBTB16, RET and POU5F1). This study concludes that the G/SSC phase occurs at 4-6 months of age. Study 3 : aimed at purifying SSC-like cell using different types of extracellular matrixes and the discontinuous gradient density. In Exp. 1, the testes (n=6) were analyzed for histology and protein expressions of laminin, SSEA-4, DDX-4 and GFR alpha-1. Cell suspension after enzymatic digestion was plated onto either laminin or gelatin coated dish. The number of SSC like cells in relation to total attached cells were determined at 15, 30 and 60 min of culture (Exp. 2). The Exp. 3 was performed to test whether or not the additional step of PercollTM gradient density could really improve purification of SSCs. Testicular histology represented the complete spermatogenesis with laminin expression at the extracellular matrixes surrounded SSC and basal lamina of the seminiferous tubules. SSEA-4 and GFRalpha-1 co-localized with DDX-4 essentially expressed in spermatogonia. The relative percentage of SSC-like cells, as determined by SSEA-4 (59.42±2.18%) and GFRalpha-1 (42.70±1.28%) expressing cells revealed that the highest SSC-like cell purity was obtained for the 15-min incubation on laminin coated dish compared with other incubation times and gelatin treatment (p<0.05). The PercollTM treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33±0.14%, p<0.001) and purity (83.82±2.05% for SSEA-4 and 64.39±1.51% for GFRalpha-1, p<0.05). These attached cells demonstrated a typical SSC morphology and also expressed POU5F1, RET and ZBTB16 mRNA. The double SSC selection by PercollTM treatment and laminin plating is a simplified technique for SSC purification.