[Isolation, purification and immunochemical characteristics of spermatogonia of rat].
OBJECTIVE
To explore the approach of isolation and purification of spermatogonia and its immunochemical characteristics.


METHODS
Compound enzymatic digestions were used to prepare germ cell suspensions of Sprague-Dawley rats aged 10 days, and velocity sedimentation and discontinuous Percoll density gradient centrifugation were used to isolate and purify the spermatogonia. Using c-kit and alpha(6)-integrin multiclone antibodies as markers respectively, the immunochemical characteristics of the spermatogonia in the testicular tissue were observed and the c-kit and alpha(6)-integrin expression rates of the purified cells were detected by flow cytometry.


RESULTS
The spermatogonia uniquely expressed c-kit and alpha(6)-Integrin in the testicular tissue. C-kit and alpha(6)-integrin were positively expressed in the purified cell suspensions. Using c-kit as the cell marker, the positive rate was 1.59% +/- 0.04% in the unpurified group, significantly lower than that of the purified group (68.33% +/- 2.45%, P < 0.01). Using alpha(6)-integrin as the cell marker, the positive rate of the unpurified group was 2.38% +/- 0.60%, significantly lower than that of the purified group (72.04% +/- 3.65%, P < 0.01). Trypan blue staining showed that the cell viability of the purified cell suspensions was more than 95%.


CONCLUSIONS
c-kit and alpha(6)-integrin can be used as the molecular markers of spermatogonium at special stage. Spermatogonia with high purity and viability can be obtained via the steps including digestions with enzymes, velocity sedimentation and discontinuous percoll density gradient centrifugation.
Ma
Liang-hong
LH
Department of Urology, Huashan Hospital, Fudan University, Shanghai 200040, China.
Ding
Qiang
Q
Wang
Xiang
X
Wang
Yang
Y
Zhang
Yuan-fang
YF
D000818:Animals; D000831:Animals, Newborn; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D008297:Male; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013093:Spermatogonia / Q000166:cytology* / Q000276:immunology*; D013737:Testis / Q000166:cytology*
D019009:Proto-Oncogene Proteins c-kit