STRESS-RESPONSE OF THE RAT TESTIS - IN-SITU HYBRIDIZATION AND IMMUNOHISTOCHEMICAL ANALYSIS OF HEME OXYGENASE-1 (HSP32) INDUCTION BY HYPERTHERMIA

By using in situ hybridization and immunohistochemistry, the distribut ion patterns of heme oxygenase (HO)-1 (HSP32) transcript and protein w ere studied, and their response to thermal stress was examined. And, b y using an HO-1 cDNA probe and polyclonal antibody, the levels of HO-1 mRNA and protein in normal and heat-shocked testis were quantified, T he digoxigenin-labeled probe detected a strong signal for HO-1 transcr ipt in Leydig cells, and in the Sertoli cells, spermatogonia, primary spermatocytes, and spermatids of the seminiferous tubules. In all cell types, the transcript was predominantly concentrated in the nucleus i n a defined pattern. Thermal stress (42 degrees C, 20 min) did not cha nge the cell population pattern of HO-1 transcript expression; however , it did cause distortion of the nuclear pattern and diffusion of the transcript signal in cells, Hyperthermic treatment of rats resulted in a modest (2- to 2.8-fold), time-dependent, and sustained (1-16 h) inc rease in testicular 1.8-kb HO-1 mRNA. Immunohistochemical analysis of normal and heat shock patterns of testicular HO-1 expression showed ro bust staining of Sertoli and Leydig cells after heat shock; in normal tissue, immunoreactivity was low in these cell populations. As with th e transcript distribution, hyperthermia did not affect the pattern of HO-1 immunoreactivity, and the protein was not detected in spermatogen ic cells under control or stress conditions. In the Leydig cells, hype rthermia led to a more than 8-fold increase in the intensity of cytopl asmic staining for HO-1 protein. Consistent with the selective express ion of HO-1, the level of the single HO-1 immunoreactive protein (simi lar to 32 kDa) detected in total testis microsomes showed a modest (1. 5-fold) increase 6 h after heat shock. Data are consistent with the su ggestion that differential distribution of HO-1 protein in the germ ce ll line and Sertoli cells reflects differential HO-1 mRNA processing i n these cell types. The increase may be essential for the catalysis of the heme moiety of denatured hemoproteins such as cytochrome P450 and hemoglobin heme and hence may protect against heme-catalyzed free rad ical formation. We propose that induction of HO-1 protein in Sertoli a nd Leydig cells may function to protect the spermatogenic cells under conditions of thermal stress.