Expression of functional aromatase in the epididymis: Role of androgens and LH in modulation of expression and activity
Introduction
Epididymal development and function is governed by a complex interplay of hormones and testicular growth factors, among which testosterone is crucial for epididymal functioning (Ezer and Robaire, 2002). However, recent studies unfold compelling evidence regarding the role of the ‘female hormone’ estrogen, in modulating functions of the efferent ductules and the epididymis (Hess, 2003, Shayu et al., 2005). The epididymis and the efferent ductules express moderate to abundant amount of estrogen receptors (Hess et al., 1997, Mowa and Iwanaga, 2001) and E2 is one of the key hormones that regulates the functioning of these tissues (Hess et al., 2001, Lee et al., 2001). The source of E2 for the excurrent ducts, is mainly the testis which has three cell types capable of E2 biosynthesis. In the immature rat, the Sertoli cells are the main sites of E2 production, subsequent to which, in the adult, the Leydig cells take over the function of E2 biosynthesis (Carreau et al., 1999). Besides these two cell types, the germ cells are a unique source of E2 in the testis and synthesize E2 in measurable quantities (Janulis et al., 1998).
E2 biosynthesis is catalyzed by a microsomal enzyme complex, called aromatase that is responsible for the irreversible transformation of androgens into estrogens. The aromatase enzyme complex is comprised of two proteins, the ubiquitous NADPH cytochrome P450 reductase and cytochrome P450 aromatase (P450AROM). The P450AROM component contains the heme and the steroid binding pocket. Aromatase can utilize either androstenedione or testosterone as androgenic substrates, which are aromatized to estrone (E1) and E2, respectively. The further conversion of E1 to the highly potent E2 is catalyzed by the enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD)-I. The 17β-HSD family consists of multiple isoforms, of which type III catalyzes the interconversion of androstenedione to testosterone, and type I catalyzes the interconversion of E1 to E2 (Luu-The et al., 1995, Mindnich et al., 2004).
The expression of P450AROM is principally regulated at the level of transcription (Simpson et al., 1994). P450AROM is transcribed from a single gene CYP19, which contains multiple promoters that are utilized in a tissue-specific manner. Thus, while P450AROM expression in human adipose tissue is driven by promoter I.4 that is subject to regulation by glucocorticoids and cytokines (Mahendroo et al., 1993, Zhao et al., 1996), in the ovary and testis, a proximal promoter PII governs P450AROM expression under the influence of the gonadotropins LH and FSH (Donadeu and Ascoli, 2005, Jenkins et al., 1993, Lanzino et al., 2001). LH mediates its functions in the target tissues by binding to LHR, resulting in increased intracellular cAMP concentration. The cAMP generated phosphorylates target proteins and activates gene transcription by binding to cAMP responsive elements present on the promoters of several genes, including aromatase (Bulun et al., 1993, Simpson, 2000). Interestingly, besides LH, androgens are also strong modulators of P450AROM expression and influence P450AROM expression in ovarian granulosa cells, testicular germ cells and Leydig cells (Bourguiba et al., 2003, Genissel and Carreau, 2001, Hamel et al., 2005).
Intriguingly, despite the proximity of the testicular E2 sources, the efferent ductules and the epididymis also express P450AROM mRNA and protein (Carpino et al., 2004, Pereyra-Martinez et al., 2001, Wiszniewska, 2002). A recent report described E2 biosynthesis from epididymal cells cultured in vitro (Wiszniewska, 2002), and since cell isolations excluded the presence of sperm, which possess aromatase (Janulis et al., 1998), it was evident that the epididymal cells themselves were capable of E2 synthesis. In light of these recent reports, the study of P450AROM expression and regulation in the epididymis gains significance. In the present investigation, the functional and regulatory aspects of epididymal P450AROM were studied. As mentioned earlier, LH and androgens are strong modulators of P450AROM expression (Bourguiba et al., 2003, Genissel and Carreau, 2001). Considering that androgens are crucial for the expression of several epididymal genes, the role of androgens in regulation of P450AROM expression was examined.
Until recently, it was believed that LHR expression was confined to the gonads; however, interestingly several extra-gonadal tissues, including the epididymis have been identified to possess LHR (Lei et al., 2003). Although the epididymis expresses LHR, the segmental distribution of LHR in the epididymis or their precise biological role in the epididymis remains elusive. Hence, the expression of LHR in the caput and cauda regions of the epididymis was studied, and the ability of epididymal LHR to bind the ligand hCG and transduce downstream signaling was also assessed. Finally, the modulation of epididymal P450AROM expression and activity by LH was examined.
Section snippets
Animals and Leydig cell isolation
Adult male rats unless indicated, were of age 40 days. Animals were obtained from the Central Animal Facility of the Institute and all the procedures carried out on the animals were approved by the Ethics Committee of Indian Institute of Science (Protocol Number 21). The anterior and posterior poles of the epididymis, namely caput and cauda regions were dissected free from fat. The caput and cauda regions were excised carefully to exclude the efferent ductules and vas deferens, respectively.
P450AROM and 17β-HSDI mRNA are expressed in the caput and cauda regions of the epididymis
P450AROM mRNA was expressed in both the caput and cauda regions of the epididymis. A single band of 290 base pair was obtained by RT-PCR analysis as shown in Fig. 1A. Interestingly, the level of P450AROM expression did not differ between the two regions. The enzyme aromatase can utilize either testosterone or androstenedione as substrates. In addition to P450AROM, the mRNA for 17β-HSDI could also be detected in the caput and cauda (Fig. 1B). Ontological analysis of P450AROM expression revealed
Discussion
Recent literature on the diverse roles of E2 in the male reproductive tract has resulted in a renewed interest in studying the E2 source(s) for these tissues. The present study elucidates the presence of P450AROM mRNA in both the caput and cauda regions of the epididymis and in accordance with aromatase expression, both regions were capable of E2 biosynthesis. Among the few methods available to study aromatase activity, RIA and the tritiated water based assay are perhaps the most prevalent. The
Acknowledgements
The authors would like to gratefully acknowledge DBT (Indo-U.S. Program), Mellon Foundation, CONRAD U.S.A., ICMR, ICMR Advanced Centre, CSIR, DST, Government of India and Indian Institute of Science, Bangalore for providing financial assistance. The authors thank Mr. Vijay, Ms. Deepika and Mrs. Hemalatha for technical assistance. Author AJR is thankful to CSIR for award of Emeritus Scientistship and SD is grateful for the award of CSIR-SRF fellowship.
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