94 CHRONOLOGICAL TRANSITION OF GONOCYTES TO SPERMATOGONIAL STEM CELLS DURING PREPUBERTAL AND PUBERTAL PERIODS IN DOMESTIC CATS

Abstract

The pubertal age of domestic cat (Felis catus) as defined as a complete spermatogenesis has been reported to occur around 8 months of age. During the initial phase of testicular development, the transition of gonocytes to spermatogonial stem cells (SSC) takes place within the seminiferous cords. This stage-specific transition has been demonstrated to facilitate SSC isolation and enrichment. Because information for this aspect in domestic cats is limited, this study aimed to identify the phase transition of gonocytes to SSC during newborn to puberty. Cat testes were collected and classified by age into 3 groups: group 1: 0–4 months (n = 5), group 2: 4–6 months (n = 5), and group 3: 6–12 months (n = 5). Testes were studied for conventional histology, transmission electron microscopy (TEM), and FACS analysis on GFRα-1 expression, a SSC marker. For histology, tissues were fixed, sectioned, and stained with H&E. Serial changes of germ cell development within the testes were observed using light microscopy. In addition, ultrathin sections (60 nm thickness) of testes were cut and examined with TEM for ultrastructure analysis. Immunolabelling and flow cytometry of GFRα-1 were used to identify the SSC population after testicular cell dissociation. The percentages of spermatogonia per tubule were analysed by one-way ANOVA, and data are presented as mean ± s.e. The development of testicular germ cells from gonocyte to spermatozoon was gradually demonstrated in histological sections, depending on age of the cats. For group 1, the gonocytes were clearly presented in the seminiferous cord. These gonocytes were in proliferative phase, as they frequently contained homogeneous euchromatin and less organelles. In group 2, the gonocytes transformed to spermatogonia as indicated by their small size (range 8.11–13.55 μm) with oval to flattened shape, chromatin condensation, and darkened cytoplasm. These cells migrated and settled onto the basement membrane of seminiferous cord. At this stage, mitochondria and small clumps of heterochromatin increased when compared with group 1. Some spermatogonia occasionally developed through the meiosis by 6 months of age (group 2), whereas complete spermatogenesis was first identified in 9-month testes (group 3). The percentage of spermatogonium/tubule in group 2 (15.84 ± 0.67) was significantly higher (P < 0.001) than group 1 and 3 (1.99 ± 0.22 and 6.88 ± 0.53, respectively). Because the SSC-like cells (based on their histological morphology) were predominantly found in group 2, the testes (n = 5, 4–6 months of age) were additionally digested to confirm GFRα-1 expression. Of total testicular cells, a high proportion of GFRα-1 positive cells (12.32 ± 6.31%) were identified by FACS. In conclusion, this study provides information regarding the age-dependent development of testicular germ cells in domestic cats. The findings provide the transition period of gonocytes to SSC that occurs around 4 to 6 months of age. This study can be applied for the enrichment of feline SSC upon testicular digestion.