Electrophoretic and immunocytochemical analysis of Hsp72 and Hsp73 expression in heat-stressed mouse testis and epididymis

Objective: One of the most profound events in stressed cells is the synthesis of a highly conserved family of proteins, the 'heat shock proteins' (Hsp). The Hsp70 family is the most diverse, and includes constitutive as well as stress-inducible proteins with overlapping or unique functions in different cell compartments. Elucidation of Hsp70 expression during different stages of spermatogenesis and maturation of germ cells is of particular interest due to their high sensitivity to heat treatment. Study design: Expression of the main isoforms of the Hsp70 family (constitutive Hsp73 and stress-inducible Hsp72) was determined in normal and heat-stressed mouse testes and epididymis from sexually mature (60-day-old) mice during spermatogenesis and maturation of germ cells. Immunocytochemical analysis and one- and two-dimensional gel electrophoresis were used to separate mouse testicular and epididymal proteins from saline extracts, followed by Western blotting. Results: Using a polyclonal anti-Hsp70 antibody that recognizes both isoforms, inducible Hsp72 expression, was demonstrated immunocytochemically only in heat-stressed tissues, while a high level of constitutive Hsp73 isoform expression was found in both normal and heat-stressed mouse male reproductive tissues. Morphological studies have shown that round and elongated spermatids in the testes, as well as all segments of the epididymis, are most sensitive to heat stress. In the epididymis, the reaction was localized in different cell compartments. Conclusion: In heat stress conditions, Hsp73 is mobilized to prevent apoptosis in the testes and epididymis, and assists Hsp72 in the repair of stress-altered protein conformations. (C) 2013 Elsevier Ireland Ltd. All rights reserved.