Heat‐Induced Apoptosis of Mouse Meiotic Cells Is Suppressed by Ectopic Expression of Testis‐Specific Calpastatin

Abstract

Calpastatin is a naturally occurring inhibitor of calpain, a protease involved in apoptotic cell death. A testis-specific isoform of calpastatin (tCAST) has been identified that is transcribed in haploid germ cells but not in spermatocytes. To investigate the possible function(s) of tCAST, we tested the hypothesis that the ectopic expression of calpastatin in spermatocytes would suppress the death of these cells in response to an apoptosis-inducing stimulus in vivo. To this end, the 5'-flanking region of the mouse ldhc gene was linked to tCAST, and transgenic mice were generated. Immunohistochemical analysis revealed that, in contrast to control sections in which the signal for tCAST was seen in round spermatids, intense staining was visualized in pachytene spermatocytes in the transgenic animals, indicating that the strategy we used to generate the transgenic animals resulted in the ectopic expression of tCAST in spermatocytes. We then tested the effect of a short period of heating on germ cell apoptosis in the testes of wild-type and transgenic mice. Pachytene spermatocytes were the major germ cell type seen to undergo apoptosis after heat treatment. There were no differences in the number of apoptotic germ cells per seminiferous tubule between wild-type and tCAST transgenic control mice; thus, there was no apparent effect of the transgene on normal apoptosis. Heating resulted in increased numbers of TUNEL-positive germ cells in both wild-type and tCAST transgenic mice, as well as increased testicular DNA fragmentation. Heating the tCAST transgenic mouse testes resulted in significantly fewer apoptotic cells per seminiferous tubule than in wild-type mice at both 8 and 24 hours after treatment. Thus, as hypothesized, the ectopic expression of tCAST in pachytene spermatocytes suppressed germ cell apoptosis.

Ginkgo Biloba Ameliorates Subfertility Induced by Testicular Ischemia/Reperfusion Injury in Adult Wistar Rats: A Possible New Mitochondrial Mechanism

“…Somwaru et al [42] found that a high temperature induced a rapid increase in reactive oxygen in pachytene spermatocytes but not in round spermatids. Reyes et al [43] mentioned that increased testicular temperature might cause the hypoxic changes in the testicles such as increased vascularisation, decreased testicular mass, and increased interstitial space.…”

The Hypoxic Testicle: Physiology and Pathophysiology

“…As described above, many studies have been performed on experimental hyperthermia in animals, showing the triggering of apoptosis in pachytene spermatocytes [116, 117]. In contrast, few studies of hyperthermia and its associated physiological and biochemical changes have been performed in isolated cells.…”

Calpain inhibitors prevent p38 MAPK activation and germ cell apoptosis after heat stress in pubertal rat testes

“…In this model the role of the Fas system is controversial since mice harboring a loss‐of‐function mutation in FasL or Fas, show a similar degree of apoptosis, while studies in cynomolgus monkeys and mice have shown an up‐regulation of Fas at the protein level (Miura et al, 2002; Vera et al, 2004; Jia et al, 2007). Growing evidence supporting caspase‐independent cell death in testes after traumatic injury and the suggestive result showing that heat‐induced apoptosis of mouse meiotic cells is suppressed by ectopic expression of the testis‐specific calpastatin (Shiraishi et al, 2000; Somwaru et al, 2004), prompted us to evaluate the role of these enzymes in both physiological and heat‐induced apoptosis during puberty.…”