371 EVALUATION OF ENRICHMENT STRATEGIES FOR PORCINE SPERMATOGONIA BY EXPRESSION OF PROTEIN GENE PRODUCT 9.5, A SPERMATOGONIA-SPECIFIC MARKER IN THE PIG TESTIS

Abstract

Transplantation of genetically altered male germ cells is under investigation as a novel route to generate transgenic animal models. Identification and isolation of spermatogonial stem cells are a prerequisite for this strategy. The objectives of this study were to validate a marker for identification of undifferentiated porcine spermatogonia, and to use this marker to develop a practical enrichment strategy for spermatogonia from pig testis. We established that expression of protein gene product (PGP) 9.5 is a spermatogonia-specific marker in porcine testis through analysis of its expression pattern in testis cells, by comparison with the expression of the cell-type specific proteins GATA-4 (expressed in Sertoli cells) or PLZF (expressed in undifferentiated mouse spermatogonia) in seminiferous tubules at different ages, and by comparison of expression levels of PGP 9.5 and the germ cell-specific protein VASA in different cell fractions after differential plating. Using expression of PGP 9.5 as a marker, we characterized enrichment of porcine spermatogonia from two-week-old (2wo) and 10-week-old (10wo) pigs by immunofluorescence either after differential plating only or after velocity sedimentation at unit gravity followed by differential plating. After differential plating with overnight culture to deplete testicular somatic cells that firmly attach to culture dishes, spermatogonia (mean ± SEM per 1000 cells) were 5-fold enriched (P < 0.05) in cells remaining in suspension (fraction I) (2wo: 54.0 ± 9.1; 10wo: 162.7 ± 30.5) and in populations slightly attached to the culture plate (fraction II) (2wo: 92.7 ± 8; 10wo: 159.5 ± 22.5) compared to the initial samples (2wo: 12.3 ± 2.7; 10wo: 27.2 ± 2.9). Slightly attached spermatogonia appear to be superior for future experiments due to higher viability (>90%) than spermatogonia remaining in suspension (<50%). Cell populations containing up to 70% spermatogonia with good viability (>80%) were achieved by velocity sedimentation isolation followed by differential plating. These results indicate that expression of PGP 9.5 is a useful marker for identification of undifferentiated porcine germ cells. Simple differential adhesion culture of testis cells harvested from pre-pubertal boars can supply cell populations enriched in spermatogonia for subsequent genetic manipulation and transplantation.

This work was supported by 1 R01 RR17359-01.