Estrogen regulation of ion transporter messenger RNA levels in mouse efferent ductules are mediated differentially through estrogen receptor (ER) alpha and ER beta.

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(-).

Evaporative microdialysis: an effective improvement in an established method of protein crystallization.

Evaporative dialysis is a simple variant of conventional microdialysis in which the reservoir solution is allowed to evaporate slowly. The slow increase in precipitant concentration allows crystals to grow without increasing nucleation. The method is useful for proteins that have a very narrow metastable zone (the range of solution conditions under which crystals grow but nuclei do not form at an appreciable rate). The method is demonstrated with the coat protein of potato virus X.

Morphology and function of rooster efferent ductule epithelial cells in culture.

Regulation of the excurrent ducts of the testis is not well understood, particularly in avian species. To investigate the role of steroid hormones in the male reproductive tract, we developed a primary cell culture of epithelia isolated from rooster ductuli efferentes (efferent ductules). Efferent ductules of the avian testis comprise 77% of the epididymal region and form a mass of tubules containing a heavily folded epithelium enmeshed in connective tissue. The epididymal region was separated by microdissection and small epithelial plaques isolated by serial digestion with collagenase, elastase and repeated pipetting. Isolated cell plaques were cultured in a bicameral chamber on Millicell-CM inserts coated with two layers of basement membrane matrix, consisting primarily of laminin and Types I and IV collagen. Active ciliary beat was observed before plating and this activity was maintained for 14 days in culture. Cell plaques attached within 24 h and outgrowths formed a confluent monolayer by 5-6 days. The epithelial nature of cultured cells was demonstrated by immunocytochemical staining for cytokeratin. Light and electron microscopy confirmed that morphology and polarity of the original epithelial cells were maintained in culture. Cultured efferent ductal epithelium was cuboidal in shape and maintained many of the cytoplasmic organelles typical of these cells in vivo. The uptake of cationic ferritin indicated the endocytotic activity of these cultured cells was maintained. Estrogen receptor mRNA expression was maintained in cultured cells. These data demonstrate avian efferent ductal epithelium can be isolated and grown in defined culture medium for the purpose of determining the role of hormones and other factors in regulating the function of the epididymal region in the bird.

Granulosa layer: primary site of regulation of plasminogen activator messenger ribonucleic acid by luteinizing hormone in the avian ovary.

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.