Cadmium accumulation and binding characteristics in intact Sertoli/germ cell units, and associated effects on stage‐specific functions in vitro: insights from a shark testis model

Abstract

The increased human use of cadmium (Cd) and its increased occurrence in the environment is of concern. The testis is sensitive to Cd because of the steroid-mediated regulation of spermatogenesis, high levels of DNA synthesis and gene transcription, all of which varies in a stage-related manner. Validated techniques (acridine orange vital staining to detect apoptosis and dextran-rhodamine exclusion to assess blood-testis barrier function) were recently developed and the shark testis was proposed as an alternative model for assessing stage-specific functions in living testicular tissue and to study toxicant actions on spermatogenesis. The present paper shows that 109Cd accumulation and binding in vitro was stage-dependent (premeiotic, PrM > meiotic, M > postmeiotic, PoM), rapid and persisted in spermatocysts (intact germ cell/Sertoli cell units) 49 h after washout. In competitive binding analyses of all three spermatocyst stages, Hg, but not Zn, could replace bound 109Cd, suggesting that Cd binding was specific. These findings were associated with a biphasic apoptotic response in the PrM spermatocysts, which was maximal at 10 microm CdCl2 and 1 microm CdCl2 after 2 and 4 days in culture, respectively. Although Cd uptake in PoM cysts was more than 2-fold less than uptake in PrM cysts, the percentage dextran-rhodamine permeant PoM cysts was approximately 8-fold greater than in controls in the presence of both 10 microm CdCl2 and 30 microm CdCl2 after 4 days culture, indicating that blood-testis barrier function in PoM spermatocysts was compromised. These findings demonstrate that this model has utility for use in screening assays of environmental toxicants.

Fetal bovine serum simultaneously stimulates apoptosis and DNA synthesis in premeiotic stages of spermatogenesis in spiny dogfish (Squalus acanthias) in vitro: modulation by androgen and spermatogenic activity status

“…Using a 109 objective, 6-10 fields of view of each triplicate AO-and PI-stained well of dispersed spermatocysts (*50 cysts per field of view), were randomly captured and stored on computer for later analysis. A cyst was designated AO-positive if it contained three or more bright green punctate fluorescent spots, a standard previously used [14][15][16]. A cyst was designated PI-positive if at least half of the cyst in the plane of focus was filled with bright red round fluorescent nuclei.…”

“…A curious finding though in both studies was thus the relative resistance of immature premeiotic stages (PrM, stem cell and early-stage spermatogonia) to apoptosis in the normal testis and after spermatoxicant exposure in vivo. However, this was not maintained in vitro as the immature spermatogonial clones spontaneously entered apoptosis during 2 days culture [16].…”

“…For at least 7 days in culture, spermatocysts retain their closed spherical shape, normal Sertoli-germ cell relations, and continue to express many stage-related growth and differentiated traits (e.g. DNA, protein and steroid synthesis), which are responsive to added growth factors, nutrients and pharmacologic agents [19][20][21] and even spermatotoxicants [16].…”

Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells

“…In toxicological studies, S. acanthias testis preparations in vitro were used to study cadmium effects on spermatogenesis (McClusky, 2008 a ). Cadmium accumulated in spermatocysts in culture, and experiments indicated that the binding was specific.…”

“…Cadmium accumulated in spermatocysts in culture, and experiments indicated that the binding was specific. Micromolar concentrations of cadmium caused apoptosis over 2–4 days in culture, and evidence indicated that the elements of the blood‐testis barrier function were negatively influenced (McClusky, 2008 a ). Foetal bovine serum produced both apoptosis and thymidine incorporation in spermatocyst cultures, and the apoptotic effect was decreased by addition of androgen (McClusky, 2008 b ).…”