Leydig cell–derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress
Nobuaki Ozawa, Nobuhito Goda, Nobuya Makino, Tokio Yamaguchi, Yasunori Yoshimura, Makoto Suematsu
Nobuaki Ozawa, Nobuhito Goda, Nobuya Makino, Tokio Yamaguchi, Yasunori Yoshimura, Makoto Suematsu
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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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Demonstration of apoptotic cells in seminiferous tubules in the 12-hour ...
Demonstration of apoptotic cells in seminiferous tubules in the 12-hour CdCl2–treated rats assessed by in situ 3′ end labeling of single-strand DNA. (a and b) Representative pictures of testes in the CdCl2-untreated and -treated groups, respectively. Note that peritubular germ cells constitute a major cellular component displaying apoptosis. (c) Effects of pretreatment with ZnPP on the CdCl2-elicited apoptosis of germ cells. (d) Restoration of CdCl2-induced germ cell apoptosis by CO supplementation with dichloromethane under blockade of the HO reaction by ZnPP. Bar, 100 μm. (e) Differences in histograms showing the density of apoptotic cells in individual seminiferous tubules among groups. Data were indicated by relative frequency (%) as a function of the density of apoptotic cells per a single tubule. (f) Differences in percentages of TUNEL-positive seminiferous tubules (percentage of apoptosis-positive tubules) among groups. Two different doses of CdCl2 (10 and 20 μmol/kg) were examined. Effects of other interventions were examined in rats treated with 20 μmol/kg CdCl2. Tubules containing at least one TUNEL-positive cell were considered to be positive in this analysis. Bars represent the mean ± SE of measurements from four to eight separate experiments. In an individual experiment, at least 200 different tubules were examined to calculate a single data set. DCM, dichloromethane; Test, testosterone, DFO, desferrioxamine. *P < 0.05 as compared with the control. **P < 0.05 as compared with the group treated with 20 μmol/kg CdCl2. ***P < 0.05 as compared with the group treated with ZnPP + CdCl2.