Enrichment of haploid spermatids in mice by flow sorting / 中华男科学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To establish an effective method for haploid spermatid enrichment by Hoechst 33342 staining and flow sorting.</p><p><b>METHODS</b>Mouse testicular monoplast suspension was prepared by two-step enzyme digestion, and the cells were incubated in the medium containing Hoechst 33342 and Verapamil. Haploid spermatids were separated and enriched according to their DNA content by flow sorting. The gene expressions in the spermatids of several histone-modified enzymes, including the histone acetylases (HAT) and histone deacetylases (HDAC), were examined by RT-PCR and compared with that in the HAT-inhibitor curcumin-treated counterparts.</p><p><b>RESULTS</b>We successfully enriched the haploid spermatids with high purity and further purified the round and elongated spermatids. RT-PCR results indicated the specificity of the expression of the HAT gene in the spermatids, and that it was influenced by curcumin.</p><p><b>CONCLUSION</b>Flow sorting can efficiently improve the purity of haploid spermatid enrichment, which helps a lot to elucidate the mechanisms of spermiogenesis.</p>