Search

Spermatogonial stem cells (SSC) have the capacity for self-renewal and the potential of producing progenitor spermatogonia that will differentiate into spermatozoa. SSC transplantation may be a valuable alternative for the propagation of genetically important males and preservation of endangered wild felids, as recently demonstrated by the production of ocelot spermatozoa after the xeno-transplantation of a mixed germ cell population into the testis of a domestic cat (Silva et al. 2011 J. Androl.). SSC are in low numbers in the testis and have been isolated in different mammalian species by using specific cell surface markers; however, the expression of SSC-surface markers in feline species has not been characterised. In the present study, testes of domestic cats were obtained from veterinary clinics. The selected testes (n = 4) ranged in size from 1.3 to 2.0 cm in length. To obtain a suspension of a mixed population of spermatogonial cells, seminiferous tubules were enzymatically dissociated using two digestion steps followed by dual filtration through 100-μm and 40-μm nylon mesh filters and a final separation over a 5-layer Percoll™ density gradient (35, 30, 27.5, 25 and 20%). Spermatogonial cells were morphologically identified by their characteristic large round nucleus with a homogenous appearance in the bands formed at the 30%, 27.5% and 25% layers. The mean number of cells/testis collected was ~13 × 106 ± 12.2, with an overall percentage of live cells of 97%. After isolation, cells were fixed with 4% PFA, blocked overnight and stained with primary antibodies specific for SSC-surface markers (CD49f, CD9, C-Kit, GFRα1, GPR125 and Thy-1) and pluripotent stage-specific embryonic antigen markers (SSEA-1 and SSEA-4). Fluorescence microscopy showed a positive expression of GFRα1, GPR125 and C-Kit, but not for CD49f, CD9, or Thy-1. It also revealed that a low number of cells were positive for SSEA-1 and SSEA-4. For further characterisation, molecular detection of the pluripotent gene Oct-4 and the germ cell-specific genes BOLL, DAZL and VASA was performed in germ cells isolated from one testis of four individuals. For RT-qPCR, the Cells-to-cDNA™ II kit (Ambion) was used to produce cDNA from an aliquot of ~30 000 cells directly after isolation. RT-qPCR showed that none had detectable levels of Oct-4 within the range of the standard. Three of the four testes expressed all three germ cell-specific genes, BOLL, DAZL and VASA, while only VASA was detected in the remaining testis. These results suggest that cat SSCs and spermatogonial cells express some of the SSC markers tested. However, the positive expression of SSEA-1 and SSEA-4 in a low number of cells further supports the stem cell-like state of cat SSCs and that these markers can be used in dual staining for purifying cat SSCs from a mixed germ cell population by fluorescence-activated cell sorting. [ABSTRACT FROM AUTHOR]

Differences in the stability of commonly used reference genes have been discovered between species and among different tissue types. These inconsistencies underscore the importance of validation and selection of an appropriate reference for normalization of gene expression to an endogenous control in RT-qPCR experiments. Three different reference transcripts (GAPDH, RPS19 and 18S rRNA) of 3 different functional categories were selected for evaluation during pre-implantation embryonic development and in spermatogonial cells in the domestic cat. Amplification efficiency was done by standard curve analysis with 5-fold template dilutions. For pre-implantation analysis, transcripts were assayed at 4 developmental stages: 2- to 6-cell (2-6C), 8- to 16-cell (8-16C), morula (M) and blastocyst (BL). Embryos were from pools of 4 to 10 in vitro matured and fertilized domestic cat embryos. For spermatogonia, isolates of 20 000 to 30 000 cells were assayed from single testis isolates following a collagenase and trypsin with DNase digestion and Percoll gradient separation. Total mRNA was isolated using the Cells-to-cDNA II Kit, with a minimum of 2 biological replicates for each sample type. The RNA quantitation was done by RiboGreen analysis and 47 ng of RNA was used for cDNA synthesis. Transcript abundance was detected in 2 technical replicates per sample by SYBR Green chemistry and data were analysed with NormFinder and one-way ANOVA. Each transcript showed varying levels of expression throughout development and among embryonic stages and in spermatogonial cells. The GADPH and 18S rRNA transcripts were expressed at higher levels in BL than in 2-6C and 8-16C, respectively. Contrarily, transcript levels of RSP19 were lower in BL compared with levels at 2-6C. Even though differences were observed among embryonic stages, analysis with NormFinder indicated that the most stable reference gene throughout early pre-implantation development was RPS19. When each stage was analysed separately, 18S rRNA was the most stable at 2-6C, whereas RPS19 was found to be the most stable at 8-16C, M and BL stages. In spermatogonial samples, GAPDH was the most stably expressed gene. Our results support the selection of an appropriate reference gene based on the needs of the experimental design. We conclude from these findings that when gene expression throughout the duration of early embryonic development is examined, RPS19 is the preferred selection. If gene expression is analysed within discrete time points of development only, it is appropriate to select 18S rRNA at the 2-6C stage and RPS19 for 8-16C, M and BL stage embryos. For spermatogonial samples, if comparing only different biological replicates, GAPDH should be selected for normalization of expression; however if the purpose of the experiment is to assay genes that are also expressed in BL, the most stably expressed reference gene is RPS19. [ABSTRACT FROM AUTHOR]