Leydig cell–derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress
Nobuaki Ozawa, … , Yasunori Yoshimura, Makoto Suematsu
Nobuaki Ozawa, … , Yasunori Yoshimura, Makoto Suematsu
Published February 15, 2002
Citation Information: J Clin Invest. 2002;109(4):457-467. https://doi.org/10.1172/JCI13190.
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Article

Leydig cell–derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress

Abstract

Stress-induced downregulation of spermatogenesis remains poorly understood. This study examined the induction of heme oxygenase-1 (HO-1), a carbon monoxide–generating inducible enzyme, in modulation of spermatogenesis. Rats were exposed to cadmium chloride (CdCl2), a stressor causing oligozoospermia, and HO-1–induction was monitored by following HO isozyme expression. CdCl2-treated testes increased HO-1 activity and suppressed microsomal cytochromes P450, which are required for steroidogenesis. CdCl2-elicited HO-1 occurred mostly in Leydig cells and coincided with CO generation, as judged by bilirubin-IXα immunoreactivity. Under these circumstances, germ cells in peripheral regions of seminiferous tubules exhibited apoptosis; laser flow cytometry revealed that these apoptotic cells involve diploid and tetraploid germ cells, suggesting involvement of spermatogonia and primary spermatocytes in CdCl2-elicited apoptosis. Pretreatment with zinc protoporphyrin-IX, an HO inhibitor, but not copper protoporphyrin-IX, which does not block the enzyme, attenuated the CdCl2-induced apoptosis. Such antiapoptotic effects of zinc protoporphyrin-IX were repressed by supplementation of dichloromethane, a CO donor. Upon CdCl2-treatment, both Sertoli cells and the germ cells upregulated Fas ligand; this event was also suppressed by zinc protoporphyrin-IX and restored by dichloromethane. Thus, Leydig cells appear to use HO-1–derived CO to trigger apoptosis of premeiotic germ cells and thereby modulate spermatogenesis under conditions of stress.

Authors

Nobuaki Ozawa, Nobuhito Goda, Nobuya Makino, Tokio Yamaguchi, Yasunori Yoshimura, Makoto Suematsu

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CdCl2-induced disruption of cell phase–specific DNA contents in testicul...
CdCl2-induced disruption of cell phase–specific DNA contents in testicular germ cells and effects of the HO blockade by ZnPP and CO supplementation by dichloromethane (DCM). (a) Characterization of HO-2 expression in testicular germ cells. s, subhaploid; h, haploid; d, diploid; t, tetraploid. Note that the cells in subgroups s and h exhibited greater expression of HO-2 than those in d and t. The right panel exhibits two-color dot-plot analysis using mouse IgG as a negative control. (b) Differences in histogram showing DNA contents in specific cell phases among groups. The ratio of DNA contents as judged by PI fluorescence is 1:2:4 for subgroups h, d, and t, respectively. Note that variations of DNA contents become evident in the CdCl2-treated group (asterisks in b). Such variations of DNA contents were also evident in the ZnPP + CdCl2–treated group supplemented with DCM (arrows in b). (c) Differences in CV values of each cell population among groups. *P < 0.05 as compared with the control. Data indicate mean ± SE of six to seven separate experiments for each group. **P < 0.05 as compared with the CdCl2-treated group. ***P < 0.05 as compared with the group treated with ZnPP and CdCl2.