Localization of Five Steroidogenic Enzyme mRNAs in Japanese Black Bear (Ursus thibetanus japonicus) Testes During the Mating Season by In Situ Hybridization

Abstract

Abstract. The Japanese black bear (Ursus thibetanus japonicus) is a typical seasonal breeder that has a mating season in early summer. Spermatogenesis and testicular steroidogenesis are known to develop and regress annually; however, its molecular mechanism has not yet been investigated. In the present study, we clarified the mRNA sequence of 5 steroidogenic enzymes (P450scc, 3βHSD, P450c17, 17βHSD3 and P450arom) using RT-PCR and RACE methods and the localization of these gene expressions in the bear testis using an in situ hybridization technique. The amino acid sequence deduced from each mRNA sequence had high homology with the corresponding sequences of other species and possessed a motif typical of the P450 family or short chain alcohol dehydrogenase family. Expression of P450scc, 3βHSD and P450c17 mRNA in interstitial tissue indicated that conversion from cholesterol to androstenedione occurs in Leydig cells. On the other hand, the mRNA of 17βHSD3, which plays a central role in synthesizing testosterone, was detected not only in the interstitium but also inside the seminiferous tubules, along the basement membrane. P450arom mRNAs were distributed in the seminiferous tubules. These results suggest the possibility of testosterone and estradiol-17β synthesis inside the seminiferous tubules in the bear testis. We expect that the results of this study will be useful for further investigation of the molecular mechanism of steroidogenic seasonality in the bear testis. Key words: In situ hybridization, Japanese black bear, Steroidogenic enzyme, Testis (J. Reprod. Dev. 56: [236][237][238][239][240][241][242] 2010) n the majority of mammalian species, there is a seasonal variation in gonadal activity. In males, the testicular function of spermatogenesis and steroidogenesis develops before the mating season, and regression occurs when this period is over. The Japanese black bear (Ursus thibetanus japonicus), a subspecies of the Asiatic black bear living in Japan, is a typical seasonal breeder that has a mating season in early summer. The changes in the histological appearance of the testicular seminiferous tubules and the concentration of testosterone in peripheral blood have been described for some species of bears, including the Japanese black bear [1][2][3][4][5]. However, the molecular mechanisms that produce reproductive seasonality in the testis have not yet been elucidated in bears.Spermatogenesis is primarily regulated by sex steroid hormones, such as testosterone and estradiol-17β. The biosynthesis of sex steroid hormones in animal tissues is catalyzed by three forms of cytochrome P450 and two forms of hydroxysteroid dehydrogenases [6]. The cholesterol side-chain cleavage P450 enzyme (P450scc) converts cholesterol into pregnenolone as a first step. Then, 3β-hydroxysteroid dehydrogenase (3βHSD) and 17α-hydroxylase/C17-20 lyase (P450c17) convert pregnenolone into androstenedione through progesterone (Δ4 pathway) and dehydroepiandrosterone (Δ5 pathway), respectively. Subsequently, androstenedione is converted...