Earlier studies have shown that the efferent ductules (ED) of the male mouse are a target for estrogen. The loss of estrogen receptor (ER) function through either knockout technology (alpha ERKO mouse) or chemical interference (pure antagonist, ICI 182 780) results in a failure of a major function of the ED, the reabsorption of testicular fluids. The purpose of this study was to test the hypothesis that estrogen controls fluid (water) reabsorption in the ED by modulating ion transporters important for passive water movement through a leaky epithelium such as the ED. Northern blot analysis was used to detect the mRNA levels for key ion transporters in the following experimental groups: 1) wild-type (WT) control for the 14-day experiment, 2) ER alpha knockout (alpha ERKO) control for the 14-day experiment, 3) WT treated with ICI 182 780 (ICI) for 14 days, 4) alpha ERKO treated with ICI for 14 days, 5) WT control for the 35-day experiment, and 6) WT treated with ICI for 35 days. Estrogen differentially modulated the mRNA levels of key ion transporters. ER alpha mediated carbonic anhydrase II mRNA abundance, and there was a decrease in Na(+)/H(+) exchanger 3 mRNA levels in the alpha ERKO that appeared to be a cellular effect and not a direct estrogen effect. The loss of ER alpha control resulted in an increase in mRNA abundance for the catalytic subunit of Na(+)-K(+) ATPase alpha 1, whereas an increase in the mRNA abundance of the Cl(-)/HCO(3)(-) exchanger and the chloride channel cystic fibrosis transmembrane regulator was significantly ER beta mediated. Our results indicate for the first time that estrogen acting directly and indirectly through both ER alpha and ER beta probably modulates fluid reabsorption in the adult mouse ED by regulating the expression of ion transporters involved in the movement of Na(+) and Cl(-).

Estrogen regulates thymic development and involution and modulates immune function. Despite its critical role in thymus, as well as in autoimmune disorders, the mechanism by which estrogen affects the thymus is not well understood. We previously reported that the estrogenic soy isoflavone genistein, as well as 17beta-estradiol (E2), could induce thymic involution, but genistein effects were only partially mediated through estrogen receptors. To provide insights into mechanisms of estrogenic effects in the thymus, we investigated thymic gene expression changes induced by E2 (125 ng/day) and genistein (1500 ppm in feed) in weanling mice using high-density DNA arrays. We identified several E2-responsive genes involved in thymic development and thymocyte signaling during selection and maturation. Functional characterization indicated effects on genes involved in transcription, apoptosis, and the cell cycle. This study also identified changes in several E2-regulated transcripts essential to maintain immune self-tolerance. E2 upregulated more genes than genistein, while genistein downregulated more genes than E2. Though each treatment regulated several genes not altered by the other, there was considerable overlap in the genes regulated by E2 and genistein. Changes in transcription factors and cell cycle factors were consistent with decreases in cell proliferation induced by both genistein and E2. As indicated by the regulation of non-E2-responsive genes, genistein also induced unique effects through non-estrogenic mechanisms. The specific downregulation of the CD4 coreceptor transcript by genistein was consistent with the decline of CD4+ thymocytes in genistein-treated mice in our previous study. This is the first study identifying E2 and genistein target genes in the thymus. These findings provide new mechanistic insights toward explaining estrogen action on thymocyte development, selection, and maturation, as well as the effects of genistein on prenatal and neonatal thymic development and function.

Plasminogen activator (PA) is hypothesized to be important in the remodeling of the extracellular matrix during follicular growth. The granulosa layer produces high amounts of PA in response to a stimulatory factor, produced by the theca layer, that is inhibited by LH. To determine the site and mechanism by which LH inhibits PA production, we asked 1) whether LH acts on the granulosa layer and/or the theca layer to inhibit PA production by the largest preovulatory follicle (F1), and 2) whether LH affects PA production by acting at the mRNA or protein level. Sections (10 mm in diameter) of granulosa layers obtained from the F1 follicle before (14 h before ovulation) or after (2 h before ovulation) the LH surge were incubated (24 h at 37 degrees C) in theca-conditioned medium; this medium had been prepared by incubation of 10-mm-diameter sections of theca layers, obtained before (14 h before ovulation) or after (2 h before ovulation) the LH surge, in Dulbecco's Modified Eagle's Medium for 24 h at 37 degrees C. PA production in culture medium was measured with use of the chromogenic substrate S-2251. PA production was high when granulosa layers obtained before the LH surge were incubated in theca-conditioned medium obtained before the LH surge; it was also high when granulosa layers obtained before the LH surge were incubated in theca-conditioned medium obtained after the LH surge. PA production was low when granulosa layers obtained after the LH surge were incubated in theca-conditioned medium obtained before the LH surge, and was also low when granulosa layers obtained after the LH surge were incubated in theca-conditioned medium obtained after the LH surge. Northern and Western blots and activity assays performed on granulosa layer homogenates indicated that PA mRNA, protein, and activity were high before the LH surge and low after the LH surge. Production of the stimulatory factor by the theca layer is apparently unaffected by LH. After exposure to LH, the granulosa layer is no longer capable of producing PA, even in the presence of the theca-derived stimulatory factor. We conclude that the granulosa layer is the site of mRNA and/or protein regulation of PA production by LH.

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