Alteration in expression of estrogen receptor isoforms alpha and beta, and aromatase in the testis and its relation with changes in nitric oxide during aging in mice Arnab Banerjee, Shabana Anjum, Rachna Verma, Amitabh Krishna ⇑ Department of Zoology, Banaras Hindu University, Varanasi 221005, UP, India article info Article history: Received 12 August 2011 Received in revised form 6 February 2012 Accepted 6 February 2012 Available online 15 February 2012 Keywords: ER alpha and beta receptor isoforms Aromatase NO Steroidogenesis Testes abstract The aim of present study was to investigate the changes in the testicular expression of aromatase, ER alpha, ER beta and iNOS protein and correlate these with serum testosterone and nitric oxide levels, to elucidate the role of estrogen and nitric oxide in the testis during aging. This study showed localization of aromatase and ER alpha mainly in the Leydig cell and showed close correlation of testicular aromatase level with circulating testosterone level suggesting that estrogen may be modulating testicular steroido- genesis. Localization ER alpha mainly in the mitotically active germ cell suggest possible role of estrogen in germ cell proliferation. This study showed basal level of nitric oxide during reproductively active period, whereas increased serum nitric oxide coincides with decreased testicular activity in old age. This study showed inverse correlation between aromatase and NO level. Treatment with either SNP or L-NAME on testicular steroidogenic factor (3-beta HSD/ StAR) or germ cell survival factor (Bcl2) showed that increased NO causes decreased steroidogenesis and increased germ cell apoptosis. In conclusion this study suggest that estrogen modulate steroidogenesis and germ cell survival in reproductively active period whereas in old age decreased estrogen concentration causes increased nitric oxide which in turn decreases testicular steroidogenesis and germ cell apoptosis. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Estrogen is now considered as one of the important modulator of both male and female reproduction in mammals. The physiolog- ical effects of estradiol are modulated by the estrogen receptor (ER), a class I member of the nuclear receptor superfamily. The two forms of the ER are known, the classical estrogen receptor- alpha (ER alpha) and the more recently discovered estrogen recep- tor-beta (ER beta). In females, estrogen stimulates the growth and development of the follicle, uterus, fallopian tubes, cervix, vagina, labia and breasts at puberty and controls reproductive cycle [1]. In males, role of estrogen was earlier restricted to regulation of gonadotropin secretion from the pituitary. It has earlier been shown that the testis synthesizes estradiol and its concentration is fairly high in the testis [2]. Later, it has been shown that mice lacking aromatase gene, thus having no endogenous estradiol, were infertile and showed impaired spermatogenesis in adulthood [3]. Estrogen receptor (ER) alpha and beta have also been localized in the testis of human and non-human primates [4,5]. Testes of adult human, macaque and marmoset showed presence of ER beta protein in the Sertoli cells, Leydig cells and peritubular myoid cells. The intensity of immunostaining for ER beta in the germ cells was variable between species [5]. Adult ER a Knock Out (mutant) males is infertile when mated with wild type females [6,7]. Even though several studies have demonstrated the synthesis of estrogen in sig- nificant amount in the testis of several mammals via aromatase and ER alpha and beta are selectively expressed in the cells of the testis [8], clearly more studies are required to resolve the con- fusion persisting regarding the potential effects of estrogen on tes- ticular activity and its mechanism of action. Testis shows age dependent changes in the rate of steroidogen- esis and spermatogenesis [8–11]. Earlier study on rat has demonstrated decreased serum and intra testicular testosterone level resulting from impaired Leydig cells steroidogenesis during aging [12,13]. Thus, changes in serum testosterone may be considered as a good marker of testicular aging. Whether the expression of ER alpha and beta proteins also undergoes variations similar to the changes in testicular activities during aging is not yet investigated. Apart from well known effects on germ cells, emerging evidence indicates that estrogen acts to modulate nitric oxide (NO) release from vascular endothelial cells by acting on estrogen cell surface receptor [14,15]. NO release may be involved in modulating germ 0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2012.02.004 ⇑ Corresponding author. Tel.: +91 542 6702527; fax: +91 542 2368174. E-mail address: akrishna_ak@yahoo.co.in (A. Krishna). Steroids 77 (2012) 609–620 Contents lists available at SciVerse ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids