Apoptosis as a Normal Mechanism of Growth Control and Target of Toxicant Actions during Spermatogenesis

Abstract

Production and development of mature male gametes is essential for normal reproductive efficiency and survival of species. A5 part of a program of research to elucidate factors and mechanisms that regulate spermatogenesis, we have identified the spiny dogfish shark (Squalus acanthias) as an advantageous experimental model (Callard et al. In function of Somatic Cells in the Testis, Bartke A. (ed.), Springer, Ny; pp. 27-54, 1994). In contrast to the complexities of testicular organization in common laboratory species, Squalus and other sharks have a unique combination of features that facilitate stage-by-stage analysis: (a) a cystic mode of spermatogenesis; (b) a simple linear arrangement of succeeding stages across the testis diameter; and (c) reliance on Sertoli cells exclusively for somatic cell support. After seasonal spermatogenic arrest, or following hypophysectomy, a band of degenerating sperrnatocysts (ZD) appears between premeiotic (PrM) and meiotic (M) stages, suggesting withdrawal of hormonal support. As measured by detection of nucleosomal fragmentation products in DNA extracts, acridine orange (AO) labeling of condensed nuclei of 1Mng spermatocysts, and terminal transferase mediated nick end-Iabeling (TUNEL) of double-stranded DNA breaks in fixed tissue sections, the apoptotic form of programmed cell death was identified as the mechanism of degeneration occurring in PrM cysts and leading to formation of ZD. Apoptosis affected spermatogonia, not Sertoli cells, but Sertoli cells of ZD cysts contained phagocytosed germ cell corpses. Apoptosis was rare in the germinal one but increased progressively in succeeding spermatogonial generations (1-13) to a maximum of 300% of the cysts entering meiosis. Apoptosis was never seen in M or postmeiotic (PoM) cysts. In vivo exposure to cadmium (Cd), a known spermatotoxicant, increased apoptosis in mid-to late-spermatogonial generations but stem cells and early generations were unaffected. In vitro, apoptosis continue on schedule, but Cd increased and agents known to regulate DNA synthesis (insulin, IBMX, estradiol) decreased apoptosis. Results imply that apoptosis is a normal mechanism of growth control in proliferative stages of spermatogenesis and a possible target of hormone and sperrnatotoxicant action.