Purification and characterization of primordial germ cells in male rat fetuses

The primordial germ cells in the testes of male rat fetuses at 15.5 days post coitum were purified by equilibrium centrifugation on a discontinuous Percoll gradient column after dissociation by trypsin-EDTA treatment and characterized before and after purification. Gonadal dimorphism first becomes evident at 14.5 days post coitum but the "stripey" appearance can not be observed in every fetal testis. Complete dissociation of the testes was essential for successful purification of the primordial germ cells. Using the isolation technique described here, over 50% of the primordial germ cells (around 10,000) could be harvested from each fetal testis with about 95% viability as determined by trypan blue exclusion and 90% purity, as determined by alkaline phosphatase histochemistry. Prior to and following purification, the primordial germ cells were characterized by their large size and round or oval nuclei. Their nucleoli were large and located centrally or eccentrically in the nucleus. The mitochondria were large, and round or oval, and there were a great number of ribosomes and polysomes dispersed in the cytoplasm. All these characteristics were similar to that of type A spermatogonia in postnatal testes. These morphological aspects correlate the observation that the primordial germ cells may be able to undergo normal spermatogenesis in an adult recipient testis.