Regulatory mechanism of Toona sinensis on mouse leydig cell steroidogenesis

Abstract

Toona sinensis (TS), a kind of arbor, widely distributes nowadays in Asia. The leaves of TS have been used as an effective nutritious food in Chinese society for a long time. It was reported that Toona sinensis can induce apoptosis of cancer cells, reduce plasma glucose in diabetic rats, and improve lipolysis of differentiated 3T3-L1 adipocyte and its uptake of glucose. It has also been shown that TS may increase dynamic activity of human sperm. Thus, we are interested to investigate whether Toona sinensis has any effect on mouse Leydig cell testosterone production, which correlates to sperm activity. Primary mouse Leydig cells were purified to conduct the in vitro experiments. Different concentrations of crude Toona sinensis were added to primary mouse Leydig cells and the testosterone production was determined. The results showed that crude TS significantly inhibited both basal and human chorionic gonadotropin (hCG)-stimulated testosterone productions in dose dependent manner, respectively (P<0.05). Crude TS also reduced the forskolin- and dibutyryl-cAMP (dbcAMP)-stimulated testosterone production (P<0.05), which indicated that crude TS might affect protein kinase A (PKA) signal transduction pathway at the site after the formation of cyclic AMP. Moreover, TS inhibited Leydig cell steroidogenesis by suppressing the activity of steroidogenic enzymes including P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase, 20α-hydroxylase and 17β-hydroxysteroid dehydrogenase (P<0.05). In summary, these results suggested that TS inhibited steroidogenesis by suppressing the cAMP-PKA signaling pathway and the activities of steroidogenic enzymes in normal mouse Leydig cells.

Introduction

Toona sinensis Roem belongs to the family Meliacceae. It is widely distributed in China where it is commonly known as Chinese Mahogany (Chuen tien shu) (Edmonds and Staniforth, 1998). The leaves and young shoots are used as vegetable in China and Malaysia. In fact, the trees are also widely used medically. The bark is used as astringent and depurative, the powdered root is used as a corrective, and the fruits are used as an astringent and for the treatment of eye infection (Perry, 1980, Stuart, 1911). It is previously reported that extract from the leaves of Toona sinensis Roem exerts potent antiproliferative effect on human lung cancer cells (Chang et al., 2002). Moreover, TS can improve the secretion of insulin in diabetic rats, and increase the expression of GLUT4 (glucose transpoter) protein in adipose tissue of Alloxan induced diabetic rats (Yu, 2002). Reports have also demonstrated that extract of Toona sinensis can enhance the uptake of glucose in 3T3-L1 adipocytes (Hsu et al., 2003a, Hsu et al., 2003b, Yang et al., 2003). Furthermore, it was shown that the extract of Toona sinensis can improve the dynamic activity of human sperm (Yang, 2003). Thus, we are interested to investigate whether Toona sinensis has any effect on mouse Leydig cell testosterone production, which correlates to sperm activity.

In male reproductive system, gonadotropin-releasing hormone (GnRH) from hypothalamus stimulates anterior lobe of pituitary to release luteinizing hormone (LH). LH will be transported to the testis to stimulate Leydig cells, which activates adenylate cyclase through G protein. The activation results in an increase in intracellular cyclic AMP, which in turn activates protein kinase A (PKA) (Moger et al., 1991). PKA will phosphorylate some proteins and/or induce de novo synthesis of proteins, which play important roles during steroidogenesis (Stocco and Clark, 1996). These proteins will facilitate the transfer of cholesterol into the inner mitochondria membrane, where cholesterol will be converted to pregnenolone (Stocco, 2000). Steroidogenic enzymes in smooth endoplasmic reticulum will then process pregnenolone to testosterone, an inevitable hormone for male reproduction (Saez, 1994).

To determine the effect of curde Toona sinensis in male reproductive function, purified Leydig cells was treated with TS at different dosages with or without hCG for different time scales. Since testosterone production with human chorionic gonadotropin (hCG) treatment was suppressed by TS in a dose dependent manner, we continued to examine whether TS exerts direct action on sites along the cAMP-PKA pathway and/or on steroidogenic enzymes in Leydig cells. Cells were treated with dibutyryl cAMP (dbcAMP) with or without TS to examine the potential site where TS exerted on. Also, maximal doses of forskolin (stimulator of adenylate cyclase), 22R-hydroxycholesterol (substrate for P450scc), pregnenolone (substrate for 3β-HSD), progesterone (substrate for 17α-hydroxylase), 17α-hydroxyprogesterone (substrate for 20α-hydroxylase), and androstenedione (substrate for 17β-HSD) were added to cells with or without TS-crude to determine whether TS affected enzyme activity.