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Now in its third decade of mechanistic investigation, testicular injury caused by 2,5-hexanedione (2,5-HD) exposure is a well-studied model with a rich database. The development of this model reflects the larger changes that have moved biology from a branch of chemistry into the molecular age. Critically examined in this review is the proposed mechanism for 2,5-HD-induced testicular injury in which germ cell maturation is disrupted owing to alterations in Sertoli cell microtubule-mediated functions. The goal is to evaluate the technical and conceptual approaches used to assess 2,5-HD-induced testicular injury, to highlight unanswered questions, and to identify fruitful avenues of future research.

Hsp70B 0 is a unique member of the human Hsp70 family of chaperones about which information is scarce. Unlike the major inducible Hsp72 protein, Hsp70B 0 is strictly inducible having little or no basal expression levels in most cells. We observed that Hsp70B 0 appears transiently in response to heat stress whereas Hsp72 levels persist for many days. Also, Hsp70B 0 is optimally induced when cell numbers are low, whereas Hsp72 levels are greatest at higher cell number. Hsp70B 0 promoter activation was measured by flow cytometry using an Hsp70B 0 promoter-driven GFP construct. In heat stressed cells, promoter activation is cell number independent over a broad range. However, when cell number increases beyond a certain population size, cells are less stress inducible for Hsp70B 0 and induction becomes highly cell number-dependent. Cell number differences in Hsp70 activation cannot be explained by changes in Hsf-1 DNA-binding activity or hyperphosphorylation. Cells with few or no cell matrix attachments (laminin-coated and low attachment plates, respectively) appear to be more sensitive to cell number-dependent inhibition. Medium conditioned by the low cell number (LCN) populations supports increased Hsp70B 0 promoter activation in high cell number (HCN) cultures. Likewise, medium conditioned in HCN culture conditions causes decreased activation of Hsp70B 0 promoter in LCN cultures. As HCN-conditioned medium has all the components necessary for cell growth, two possibilities for the activation of Hsp70B 0 gene expression exist: an inhibitory component that accumulates in culture medium at HCN, or an activator that accumulates at LCN.