[Isolation and identification of spermatids from mouse testis].
OBJECTIVE
To develop a simple and effective method by which spermatids can be isolated from mouse testis.


METHODS
Combination of enzymatic digestion was used to prepare suspension of spermatogenic cells from adult mouse testis, and then a modified discontinuous Percoll gradient (15%, 22%, 30%, 40%, 50%, 60%) centrifugation method was introduced to isolate spermatids from the cellular suspension. The content of spermatids in each isolated fraction by Percoll method was determined by morphology (Wright-Giemsa staining) and flow cytometry analysis, and the viability of spermatogenic cells was assessed using Eosin Y exclusion test.


RESULTS
More than 97% of the testicular cells remained their viability after enzymatic digestion. After Percoll centrifuged, six fractions were formed. In each isolated fraction, the 22% fraction contained mostly spermatids(mean 86.7%) and cell viability was more than 85.5%. While in the 30% fraction, immature spermatogenic cells were present, and more than 92% of the cells remained their viability.


CONCLUSIONS
A large of relatively purified spermatids can be isolated from mouse testis by enzymatic digestion combined discontinuous Percoll gradient centrifugation method.
Zhu
Pei-Yuan
PY
Department of Microbiology & Immunology, Nanjing Medical University, Nanjing, Jiangsu 210029, China.
Huang
Yu-Feng
YF
Xu
Jian-Ping
JP
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D008297:Male; D051379:Mice; D013087:Spermatids / Q000166:cytology*; D013737:Testis / Q000166:cytology*