Advanced methods of isolation and identification of porcine primordial follicles

Abstract

An effective isolation and identification method of primordial follicles would greatly benefit the animal production practice, transgenic animal production and endangered species conservation in the future. This study has not only advanced the isolation method but also developed an identification marker of primordial follicles. After enzymatic digestion, Percoll gradient centrifugation and mesh filtrations, the obtained follicular separations were then subjected to a cell sorter in order to collect primordial follicles. The study greatly improved the yield of primordial follicles (from about 1.85 × 10 5 to 7.79 × 10 5 per prepubertal ovary) by means of increasing cell layer number from 1 ml to 2.5 ml after Percoll gradient centrifugation. Based on traditional morphological criteria, the purity of recovered primordial follicles was averagely about 82.43 ± 9.41% ( n = 5) because of their similar size and appearance with somatic cells. To further exactly appreciate the purity of sorted primordial follicles, a germ cell-specific protein, MSY2, was used to recognize the oocytes of primordial follicles. The results of repeat experiments showed that about 98.31 ± 0.73% ( n = 4) of the primordial follicles cluster was MSY2-positive, which indicated the identification method of primordial follicles was more effective and high-yielded than the previous methods because of its higher purity.