Induction of ATF4 and heat shock proteins by estrogen and estrogen mimics in mouse testes

Abstract

In testis Leydig cells as well as germ cells expresses aromatase and estrogen receptor, suggesting important of estrogen in steroidogenesis as well as steroidogenesis. Activating transcription factor 4 (ATF4) is a basic leucinezipper transcription factor that is a member of the ATF/cAMP responsive element-binding (CREB) protein family. ATF4 is expressed in a wide variety of organs, including the brain, heart, liver, spleen, kidney, lung, thymus, testis and regulates cell proliferation and differentiation of in a broad number of tissues. Basically, steroid hormones may have a direct effect on cellular stress because heat shock genes is activated by a number of steroids. The heat shock protein forms heteromeric complexes together with steroid-receptor. Moreover, ATF4 and molecular chaperones are candidate genes for upstream and down stream genes in the steroid response. To date however, gene battery mediating the physiological effect of estrogen has been uncovered in testis. Recently, estrogen mimicking chemicals have been reported to affect various aspects of male reproduction in animals including human. In an effort to elucidate molecular mechanism of regulation of spermatogenesis by estrogen mimics as well as natural estrogen, we examined the expression of ATF4, HSP70.1 and HSP70.3 mRNA in adult mouse testes following bisphenolA and/or 17 beta estradiol treatment. Adult male mice was treated with bisphenolA (oral administration of 20, 200, 1000 mg/kg B.W./day for 4 weeks) and/or 17beta estradiol (single i.p. injection of 300 ng/head). Testes were sampled 24h after the last dosage. Semiquantitative RT-PCR was optimized for each gene primers. Mean while immunohistochemical localization of ATF4 in testis was analyzed in control and drug treated mice. Result showed significant induction of ATF4 mRNA in estrogen or BPA-treated mouse testis. Parallel analysis of HSP70.1 and 70.3 mRNA revealed similar pattern of expression. In immunohistochemical analysis, ATF4 was found seminiferous tubule as well as interstitium of normal adult testis. Interestingly, ATF4 immunoreactivity among seminiferous tubule was heterogenous acccording to spermatogenic cycle. Among the germ cells, immunoreactivity of ATF4 was largely found in pachytene spermatocytes. Sertoli cell showed strong immunoreactivity of ATF4. These results suggest that ATF4 is a transcription factor possibly mediates the response to steroid in germ cells as well as somatic cells in testis. Similar induction of ATF4 and HSP70 by natural estrogen and BPA suggests that they may be involved in endocrine disruption by estrogen mimicking chemical such as BPA in testis.