Stress response of the rat testis: in situ hydridization and immunohistochemical analysis of heme oxygenase-1 (HSP32) induction by hyperthermia.
By using in situ hybridization and immunohistochemistry, the distribution patterns of heme oxygenase (HO)-1 (HSP32) transcript and protein were studied, and their response to thermal stress was examined. And, by 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. The digoxigenin-labeled probe detected a strong signal for HO-1 transcript 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 in a defined pattern. Thermal stress (42 degrees C, 20 min) did not change 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) increased in testicular 1.8-kb HO-1 mRNA. Immunohistochemical analysis of normal and heat shock patterns of testicular HO-1 expression showed robust staining of Sertoli ad leydig cells after heat shock; in normal tissue, immunoreactivity was low in these cell populations. As with the transcript distribution, hyperthermia did not affect the pattern of HO-1 immunoreactivity, and the protein was not detected in spermatogenic cells under control or stress conditions. In the Leydig cells, hyperthermia led to a more than 3-fold increase in the intensity of cytoplasmic staining for HO-1 protein. Consistent with the selective expression of HO-1, the level of the single HO-1 immunoreactive protein (approximately 32 kDa) detected in total testis microsomes showed a modest (1.5-fold) increase 6 h after heat shock. Data are consistent with te suggestion that differential distribution of HO-1 protein in the germ cell line and Sertoli cells reflects differential HO-1 mRNA processing in 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 radical formation. We propose that induction of HO-1 protein in Sertoli and Leydig cells may function to protect the spermatogenic cells under conditions of thermal stress.
Maines
M D
MD
University of Rochester School of Medicine, Department of Biophysics, New York 14642, USA.
Ewing
J F
JF
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004076:Digoxigenin; D015870:Gene Expression; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis* / Q000235:genetics; D006358:Hot Temperature; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007985:Leydig Cells / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012708:Sertoli Cells / Q000201:enzymology; D013312:Stress, Physiological / Q000201:enzymology*; D013737:Testis / Q000201:enzymology*
D012333:RNA, Messenger; D006419:Heme Oxygenase (Decyclizing); D004076:Digoxigenin