Viable Cell Preparations and In Situ 3-End Labeling of DNA (TUNEL Analysis)
After isolation, each 1-mm tubular segment was transferred in 10 |xl of PBS onto a microscope slide. After determining its diameter under the microscope, the segment was then carefully squashed under a coverslip. Squashing was monitored under a phase-contrast microscope and stopped when single cell layers were obtained.
The slides thus obtained were first examined under a phase-contrast microscope to identify the stages of the cycle, the most developed germ cell type, and apoptotic cells. Apoptosis of type A spermatogonia was seen to proceed in a manner similar to the corresponding process in somatic cells, with accumulation of heterochromatin followed by condensation of DNA at the periphery of the nucleus to form bright, phase-negative apoptotic bodies. In the case of spermatocyte apoptosis, elevated amounts of heterochromatin were present just inside the nuclear envelope and deeper within the nucleus, giving rise to bright, phase-negative spheres. read only
The numbers of apoptotic cells per 1 mm of tubule length and per 1 l^m3 of calculated volume were determined, and these values were confirmed by in situ end labeling of DNA strands (TUNEL analysis) in the same samples. After being rapidly frozen in liquid nitrogen and having the coverslip removed, the slides were dipped for a short period in ice-cold ethanol, fixed for 10 min in 4% formalin, washed twice with PBS for 5 min each time, postfixed in ethanol:acetic acid (2:1) at —20°C for 5 min, washed again with PBS as before, and finally dehydrated and stored at —70°C until analysis. The DNA 3′-end labeling was performed employing the standard procedure as described previously by Billig et al.. Using distinct staining and an obviously apoptotic nucleus as criteria, the numbers of apoptotic cells per 1 mm of tubule length and 1 |xm3 of calculated volume were quantitated.