**1. Introduction**

It is well known that blood testosterone level declines during the course of male aging (Feldman et al., 2002; Harman et al., 2001), a phenomena that is associated with the decreases in bone density, muscle mass and strength, sexual function and other physiological parameters (Kaufman & Vermeulen, 2005; Matsumoto, 2002; Vermeulen, 2000). Previous studies reported that serum testosterone concentrations were lower in the male patients with Alzheimer's disease in comparison to non-demented and age-matched men (Hogervorst et al., 2001; Moffat et al., 2004). Further studies observed that supplementation with testosterone in rats reduced β-amyloid peptide and hyperphosphorylation of τ-protein, two biomarkers of the disease (Gouras et al., 2000; Papasozomenos & Shanavas, 2002; Ramsden et al., 2003). The studies suggest that low blood testosterone is a possible risk factor for the development of Alzheimer's disease (Rosario & Pike, 2008). The decline in blood testosterone is a progressive process in male aging. Several longitudinal studies on the blood testosterone of aging males indicated that the incidence of hypogonadism increased with age (Feldman et al., 2002; Harman et al., 2001). In addition, many pathological and stressrelated factors may accelerate this process. Therefore, delaying the decline in blood testosterone is clinically significant for the health of aging males suffering from hypogonadism.

For delaying the decline in testosterone, understanding the mechanisms responsible for the decline is important. The studies in the last decades reported multiple factors and alterations in aging process that affect the levels of blood testosterone (Wang & Stocco, 2005). The studies further indicated that the primary reason for the decline is the decrease in testosterone biosynthesis during male aging (Chen et al., 1994). Testosterone is principally synthesized in testicular Leydig cells from the substrate cholesterol and released into the blood circulation (Miller, 1988). The rate-limiting step in testosterone biosynthesis is the transfer of cholesterol to the mitochondrial inner membrane to initiate the steroidogenic process in Leydig cells (Stocco & Clark, 1996). This step is regulated by the steroidogenic acute regulatory (StAR) protein, a critical factor in steroid hormone biosynthesis that controls the cholesterol transfer to the mitochondrial inner membrane (Clark et al., 1994; Lin et al., 1995; Wang et al., 1998). However, StAR protein also declines in Leydig cell aging and

Natural Flavonoids in StAR Gene Expression

(Tremblay et al., 2002).

et al., 2000; Tremblay et al., 2002).

and Testosterone Biosynthesis in Leydig Cell Aging 181

of its cofactor CBP (CREB binding protein) that is involved in the transcriptional activity of CREB (Chrivia et al., 1993; Mayr & Montminy, 2001; Mayr et al., 2001). SF-1 binding sites were also located on StAR promoter (Sandhoff et al., 1998). Binding of SF-1 to these binding sites is crucial for StAR gene transcription (Caron et al., 1997a; Caron et al., 1997b; Sandhoff et al., 1998; Sugawara et al., 1997). PKA-phosphorylation of SF-1 protein was detected at serine and threonine residues (Zhang & Mellon, 1996), which was further confirmed by the study with R2C rat Leydig cells (Carlone & Richards, 1997). It appears that PKAphosphorylation is needed for the maximal activity of SF-1 in the regulation of StAR gene transcription (Lopez et al., 2001; Sugawara et al., 1996). GATA-4 is another transcription factor binding on StAR promoter to regulate StAR gene expression. A GATA-4 binding site was found at -61 to -66 in StAR promoter sequences (Silverman et al., 1999; Silverman et al., 2006). Stimulation of MA-10 mouse Leydig cells with cAMP dramatically increased phosphorylated GATA-4 protein (Tremblay et al., 2002), resulting in the increase in StAR promoter activity. The essential role of PKA-phosphorylation in the activity of GATA-4 was further confirmed by over-expression of PKA catalytic subunit. While expression of PKA catalytic subunit significantly increased GATA-4-supported StAR promoter activity, the increased promoter activity was reversed by co-expression of the protein kinase inhibitor

The signaling through cAMP-PKA pathway is able to increase the activities of a group of transcription factors that bind on StAR promoter and regulate StAR gene transcription, including C/EBPβ (Christenson et al., 1999), Sp-1 (Sugawara et al., 2000), activator protein-1 (AP-1) (Manna et al., 2004), and sterol regulatory element binding protein (SREBP) (Shea-Eaton et al., 2001), by inducing the interaction and cooperation among them. The cAMP or PKA-induced interactions among these transcription factors generated synergistic effects that increased the cAMP-induced StAR gene transcription and steroid hormone production in Leydig cells (Reinhart et al., 1999a; Silverman et al., 1999; Silverman et al., 2006; Sugawara

In addition, it was observed that cAMP stimulation of Leydig cells reduced DAX-1 (dosagesensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) protein. DAX-1 is a strong transcriptional repressor that binds to a hairpin structure of StAR promoter between -20 to -61 and depresses StAR gene expression (Zazopoulos et al., 1997). The reduction of DAX-1 by cAMP stimulation results in dramatic increases in StAR gene expression and steroid hormone production in Leydig cells (Jo & Stocco, 2004). Although it is still not clear how the signaling through cAMP-PKA pathway reduces DAX-1 protein, the observations indicate that this signaling pathway generates positive signals to increase StAR

In addition to the cAMP-PKA signaling pathway, LH induces positive signaling through AA pathway. Stimulation of Leydig cells with LH induced AA release in one minute (Cooke et al., 1991). Previous studies have described three mechanisms for the AA release by LH or hCG: 1) Binding of the trophic hormones to their receptors activates G protein. G protein directly activates phospholipase A2 (PLA2) that catalyzes AA release from phospholipids (Axelrod et al., 1988; Ronco et al., 2002); 2) G protein increases cAMP formation that induces AA release, possibly through the activation of PLA2 by PKA-phosphorylation (Piomelli & Greengard, 1991; Wang et al., 2002); 3) AA is released by the co-regulation of acyl-CoA synthetase 4 (ACS-4) and acyl-CoA thioesterase (Acot2). Trophic hormone stimulation

gene transcription by reduction of the transcriptional repressor.

**2.2 LH-induced positive signaling through AA pathway** 

the mitochondrial cholesterol transfer is defective in aged Leydig cells (Culty et al., 2002; Leers-Sucheta et al., 1999; Luo et al., 2001). Therefore, understanding the mechanism for the decline in StAR gene expression becomes an important consideration in the research on the age-related decline in testosterone biosynthesis.

Further studies observed that cyclooxygenase-2 (COX2) in Leydig cells increased in male aging, and the increase in COX2 depressed StAR gene expression and testosterone production. Inhibition of COX2 activity in aged Leydig cells reversed the declines in StAR protein and testosterone production. In addition, feeding aged rats with a COX2 inhibitor reversed the decreased StAR protein and blood testosterone concentrations (Wang et al., 2005). These observations suggest a novel mechanism involving COX2 in the age-related decline in testosterone biosynthesis. The results from the studies indicate that it is possible to delay the decline using COX2 inhibitors. However, aging is a long process and long-term application of pharmacological levels of COX2 inhibitors is limited by their potential side effects. In search for safe and practical approaches, a group of natural flavonoids in food and food supplements has been identified being able to block the COX2-dependent signaling. These flavonoids acted at the different steps of the COX2 signaling pathway and significantly enhanced StAR gene expression and testosterone biosynthesis in Leydig cells. This chapter will describe the mechanism involving COX2 in the declines in StAR gene expression and testosterone biosynthesis in Leydig cell aging. The effects of the flavonoids on this mechanism will be reviewed. The potential application of the natural flavonoids in delaying the declines in StAR gene expression and testosterone biosynthesis will be discussed.
