Isolation of early stage testicular germ cells of rainbow trout by percoll gradient centrifugal method

Abstract

The purpose of this study was to isolate germ cells from rainbow trout (Oncorhynchus mykiss) testicular tissue by employing a percoll gradient centrifugation method. Therefore; testicular tissue samples from rainbow trout were incubated with 0.5% trypsin (2 h, 20°C) in phosphate-buffered saline (PBS) solution for enzymatic separation. Gradient centrifugation method was performed using percoll at different rates (45% - 10%, 50% - 10% and 55% - 10%). Following centrifugation, three layers were formed. In the uppermost layer, debris and spermatids; early stage germ cells in the middle layer and a small amount of spermatids; and in the pellet located in the bottom layer, erythrocytes and spermatids were detected intensively. In addition; both histological and immunofluorescence methods were utilized to identify testicular and somatic cells. Hematoxylin-eosin staining of histological sections from testicular tissue revealed spermatogonia types A and B, and spermatid cells. Fluorescent staining of tissue sections revealed early stage germ cells labeled with primary antibody (DDX4) and the secondary antibody (FITC).