BIOCHEMICAL-ANALYSIS OF PROGRAMMED CELL-DEATH DURING PREMEIOTIC STAGES OF SPERMATOGENESIS IN-VIVO AND IN-VITRO

Control points of regulator action during spermatogenesis are not comp letely known. Using the shark testis model, which facilitates analysis of spermatogenesis stage-by-stage in vivo and in vitro, an early bioc hemical marker of programmed cell death (PCD) was detected. Nucleosoma l oligomers were seen in DNA extracts of testis and isolated spermatoc ysts (clonal germ cell/Sertoli cell units) at premeiotic (PrM), but no t meiotic (M) or postmeiotic (PoM), stages. Cell nuclei isolated from M stages of development were susceptible to cleavage by micrococcal nu clease, suggesting that developmental control of factors other than a nuclease-insensitive chromatin structure may account for stage specifi city. Cytological features of apoptosis were seen in germ cells, but n ot Sertoli cells, of a subset of isolated PrM spermatocysts and appear ed to be all-or-none in affected clones. In culture, DNA fragmentation occurred on schedule with or without various additives, but the phosp hodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased ac cumulation of DNA breakdown products. identification of the apoptotic form of PCD as a major, variable component of normal spermatogenesis a nd the use of PrM spermatocysts as an in vitro test system will allow further definition of mechanisms and developmental and physiological c ontrols. (C) 1995 Wiley-Liss, Inc.