ORIGINAL ARTICLE Immunolocalization of steroidogenic enzymes and their expression during the breeding season in the testes of wild raccoon dogs (Nyctereutes procyonoides) Qiang WENG, 1,4 Toshio TSUBOTA, 3 Mingdao DAI, 1 Jiaju WENG, 2 Yang TIAN, 1 Meiyu XU, 1 Gen WATANABE 4,5 and Kazuyoshi TAYA 4,5 1 College of Biological Science and Technology, Beijing Forestry University and 2 School of Basic Medical Sciences, Peking University, Beijing, China; 3 Laboratory of Wildlife Biology, Graduate School of Veterinary Medicine, Hokkaido University, Kita, Sapporo, 4 Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo and 5 United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan ABSTRACT The objective of this study was to investigate immunolocalization of steroidogenic enzymes 3bHSD, P450c17 and P450arom and their expression during the breeding season in wild male raccoon dogs. The testicular weight, size and seminiferous tubule diameters were measured, and histological and immunohistochemical observations of testes were performed. The messenger RNA expression (mRNA) of 3bHSD, P450c17 and P450arom was measured in the testes during the breeding season. 3bHSD was found in Leydig cells during the breeding and non-breeding seasons with more intense staining in the breeding season. P450c17 was identified in Leydig cells and spermatids in the breeding season, whereas it was present only in Leydig cells in the non-breeding season. The localization of P450arom changed seasonally: no immunostaining in the non-breeding season; more extensive immunostaining in Leydig cells, Sertoli cells and elongating spermatids in the breeding season. In addition, 3bHSD, P450c17 and P450arom mRNA were also expressed in the testes during the breeding season. These results suggested that seasonal changes in testicular weight, size and seminiferous tubule diameter in the wild raccoon dog were correlated with spermatogenesis and immunoreactivity of steroidogenic enzymes and that steroidogenic enzymes may play an important role in the spermatogenesis and testicular recrudescence and regression process. Key words: immunohistochemistry, mRNA, raccoon dog, steroidogenic enzymes, testes. INTRODUCTION Reproductive strategies of seasonal breeders are adap- tations to annual changes in the environment and they minimize the animals’ energetic efforts for repro- duction. Mature males show synchronized cycles of testicular growth and involution between breeding and non-breeding periods (Bronson & Heidemann 1994). Seasonal changes in spermatogenesis, mor- phology of the testis and peripheral testosterone con- centrations have been reported in numerous species (Komatsu et al. 1997; Tsubota et al. 1997, 2001; Hay- akawa et al. 2010; Zhang et al. 2010). In these animals, immunohistochemical studies on testicular tissues have demonstrated that the distributions of immu- noreactivities for steroidogenic enzymes in testicular tissue are associated with seasons and species. Steroid biosynthesis is accomplished by the actions of two major families of enzymes. The first are the hydroxy- lase enzymes, encoded by genes belonging to the cytochrome P450 superfamily. The second family, the steroid dehydrogenase enzymes, belong to one of two distinct groups, the shortchain alcohol dehydrogenase/reductase family or the aldo-keto reductase superfamily. Together, the P450 and dehy- drogenase enzymes are necessary for steroidogensis in the gonads, placenta and adrenals (Hinshelwood 1998). Correspondence: Qiang Weng, Laboratory of Animal Physiology, College of Biological Science and Technology, Beijing Forestry University, Beijing 100083, China. (Email: qiangweng@bjfu.edu.cn) Received 20 April 2011; accepted for publication 26 Septem- ber 2011. Animal Science Journal (2012) doi: 10.1111/j.1740-0929.2011.00990.x © 2012 The Authors Animal Science Journal © 2012 Japanese Society of Animal Science