Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/ c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ER␣, a mutant ER␣ (E207A/G208A) that selectively lacks ERE binding, or ER␣ null. In the compound heterozygote (AA/؊) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17␤-estradiol (E 2 ). The uterine epithelial proliferative response to E 2 and 4-hydroxytamoxifen was retained in the AA/؊ females, and uterine luminal epithelial height increased commensurate with the extent of ER␣ signaling. This proliferative response was confirmed by 5-bromo-2-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathwayresponsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ER␣ (E207A/G208A) mutant. These results indicate that nonclassical ER␣ signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ER␣ plays a critical physiologic role in the uterine response to estrogen.The uterus responds to cyclical changes in estrogen and progesterone levels in preparation for embryo implantation. Estrogen mediates the principal proliferative response of the uterus through two related but distinct estrogen receptors (ER␣ 2 and ER␤) (1-3). ER␣ is the predominant form in the murine uterus (4), but ER␤ transcripts are also detected at very low levels (4). After estrogens bind to the ligand domain of the ER, it undergoes conformational changes that allow interactions with coactivator molecules (5-7). In the classical pathway of estrogen action, the ER binds as a dimer to EREs in the promoter regions of target genes (8). However, it is now clear that the ER can regulate genes that lack a canonical ERE, suggesting additional pathways for estrogen action (9 -11). For example, the ER may activate genes via protein-protein tethering with c-Fos/c-Jun B (AP-1) and Sp1 (12, 13) or suppress expression mediated through NF-B (14). This alternate response mechanism has been referred to as the tethered or nonclassical pathway.We previously generated an ER␣ mutant that selectively functions via the nonclassical pathway. Substitution of two amino acids in the DNA recognition sequence of the fir...

Estrogen sulfotransferase (EST, encoded by SULT1E1) catalyzes the sulfoconjugation and inactivation of estrogens. Despite decades of biochemical study and the recognition that high levels of estrogen sulfates circulate in the blood of pregnant and nonpregnant women, the physiological role of estrogen sulfation remains poorly understood. Here we show that ablation of the mouse Sult1e1 gene caused placental thrombosis and spontaneous fetal loss. This phenotype was associated with elevated free estrogen levels systemically and in the amniotic fluid, increased tissue factor expression in the placenta and heightened platelet sensitivity to agonist-induced activation ex vivo. Treatment of pregnant Sult1e1-null mice with either an anticoagulant or antiestrogen prevented the fetal loss phenotype. Our results thus identify Est as a critical estrogen modulator in the placenta and suggest a link between estrogen excess and thrombotic fetal loss. These findings may have implications for understanding and treating human pregnancy failure and intrauterine growth retardation.

Elicitation of biological responses by estrogen in target tissues requires the presence of ER as well as receptor-active ligand in the local microenvironment. Though much attention has been devoted to the study of the receptor in estrogen target tissues, the concept is emerging that tissue estrogen sensitivity may also be regulated by ligand availability through metabolic transformation in situ. Here, we show that targeted disruption, in the mouse, of an estrogen metabolic enzyme, estrogen sulfotransferase (EST), causes structural and functional lesions in the male reproductive system. EST catalyzes the sulfoconjugation and inactivation of estrogen and is expressed abundantly in testicular Leydig cells. Although knockout males were fertile and phenotypically normal initially, they developed age-dependent Leydig cell hypertrophy/hyperplasia and seminiferous tubule damage. Development of these lesions in the testis could be recapitulated by exogenous E2 administration in younger knockout mice, suggesting that they arose in older knockout mice from chronic estrogen stimulation. Older knockout mice were also found to have reduced testis and epididymis weights but increased seminal vesicle/coagulating gland weight because of tissue swelling. Furthermore, total and forward sperm motility of older knockout mice was reduced by 60% and 80%, respectively, and these mice produced smaller litters compared with age-matched wild-type males. These findings establish a role for EST in the male reproductive system and indicate that intracrine and paracrine estrogen activity can be modulated by a ligand transformation enzyme under a physiological setting. Thus, inhibition of estrogen metabolic enzymes by environmental chemicals, as has been demonstrated recently for the human EST, may constitute a novel mechanism of endocrine disruption in vivo.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens. It is expressed abundantly in the mammalian testes in which it may modulate the activity of locally produced estrogen. We demonstrate here that testicular Leydig cells from mice rendered deficient in EST expression by targeted gene deletion acquire a phenotype of increased cholesterol ester accumulation and impaired steroidogenesis with natural aging or in response to estrogen challenge. Abnormal accumulation of cholesterol ester in the mutant Leydig cells correlated with induced expression of the scavenger receptor type B class I, and cultured EST-deficient but not wild-type Leydig cells avidly uptook high-density lipoprotein cholesterol ester ex vivo. EST-deficient Leydig cells in culture produced 50-70% less testosterone than wild-type cells. This deficiency was reversed by androstenedione but not progesterone supplementation, indicating that reduced activities of 17-alpha-hydroxylase-17, 20-lyase were responsible. This conclusion was corroborated by decreased expression levels of 17-alpha-hydroxylase-17, 20-lyase but not of other key steroidogenic enzymes in the mutant cells. These results suggest that EST plays a physiologic role in protecting Leydig cells from estrogen-induced biochemical lesions and provide an example of critical regulation of tissue estrogen sensitivity by a ligand-transformation enzyme rather than through estrogen receptors.