Biochemical analysis of programmed cell death during premeiotic stages of spermatogenesis in vivo and in vitro.

Abstract

Control points of regulator action during spermatogenesis are not completely known. Using the shark testis model, which facilitates analysis of spermatogenesis stage-by-stage in vivo and in vitro, an early biochemical marker of programmed cell death (PCD) was detected. Nucleosomal oligomers were seen in DNA extracts of testis and isolated spermatocysts (clonal germ cell/Sertoli cell units) at premeiotic (PrM), but not meiotic (M) or postmeiotic (PoM), stages. Cell nuclei isolated from M stages of development were susceptible to cleavage by micrococcal nuclease, suggesting that developmental control of factors other than a nuclease-insensitive chromatin structure may account for stage specificity. Cytological features of apoptosis were seen in germ cells, but not Sertoli cells, of a subset of isolated PrM spermatocysts and appeared to be all-or-none in affected clones. In culture, DNA fragmentation occurred on schedule with or without various additives, but the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased accumulation of DNA breakdown products. Identification of the apoptotic form of PCD as a major, variable component of normal spermatogenesis and the use of PrM spermatocysts as an in vitro test system will allow further definition of mechanisms and developmental and physiological controls.