Externally applied 17,20β-DHP induced natural spawning.

<p>(A) Fertilization rates were compared between eggs squeezed from females treated with <i>in vivo</i> 17,20β-DHP and eggs resulting from normal pairing. Three spawning pairs of zebrafish were selected by standard pairing techniques and checked for fertilization rate. Several days after spawning, when ready for their next spawning event, females were treated with 17,20β-DHP and artificial fertilization conducted upon squeezed eggs using sperm obtained from the paired males. (B) Treatment of more than thirty female zebrafish with 17,20β-DHP <i>in vivo</i> began at time zero. During incubation, three females were selected and artificial fertilization conducted for eggs squeezed at each time point. Fertilization rates were assessed by counting the proportion of eggs dividing into the 4-cell stage after fertilization. Each value represents the mean of data from three different females. Vertical lines indicate standard deviation.</p

Characterization of 17,20β-DHP- and DES-induced oocyte maturation <i>in vivo</i>.

<p>(A) 17,20β-DHP and DES induce accumulation of cyclin B. Extracts were prepared from twenty oocytes after incubation with ethanol, 17,20β-DHP or DES for four hours. Extracts of each treatment were separated by electrophoresis under denaturing conditions (10.0% gel) and stained with Coomassie Brilliant Blue (CBBR), or immuno-stained with anti-goldfish cyclin B polyclonal antibody after electroblotting (α-cycB). The arrow indicates a band representing cyclin B. Time-course change of GVBD and ovulation induced by 17,20β-DHP and DES. Oocyte maturation induced by 0.01 µM 17,20β-DHP (B), 5 µM DES (C) <i>in vivo</i>. At each time point, ovarian fragments were dissected from sacrificed females and %GVBD (closed column) and %ovulation (open column) was determined by scoring oocytes that had become transparent and formed fertilization membranes. Each value represents the mean of data from three different females. Vertical lines indicate standard deviation.</p

Environmental agents induce and prevent fish oocyte maturation and ovulation.

<p>Oocytes were prepared from female zebrafish after incubation with each compound for four hours. The morphology of oocytes was photographed (A); 0.01% ethanol, 5 µM DES, 0.01 µM 17,20β-DHP, 0.5 µM PCP with 0.01 µM 17,20β-DHP, 1 µM Tes and 1 µM Prog. Oocytes became transparent after treatment with 17,20β-DHP, DES, Prog or Tes but remained opaque following treatment with ethanol or PCP with 17,20β-DHP. A fertilization membrane developed in oocytes ovulated by 17,20β-DHP or Prog treatment, as indicated by the arrow. (B) Effect of various compounds on the <i>in vivo</i> induction of GVBD and ovulation. Each compound was added into the water at a final concentration of 1 mM except 17,20β-DHP (0.001, 0.01 or 0.1 µM) and DES (1, 2 or 5 µM). After four hr incubation, %GVBD (closed column) and %ovulation (open column) was determined by scoring oocytes that became transparent and formed a fertilization membrane. (C) Inhibition of <i>in vivo</i> oocyte maturation by PCP. PCP was added to the indicated concentrations, and then maturation induced by 0.01 µM of 17,20β-DHP. After further four hr incubation, %GVBD and %ovulation was assessed by scoring the oocytes. Each value represents the mean of data from three different females. Vertical lines indicate standard deviation.</p

Effect of the length of treatment time of 17,20β-DHP and DES treatment during induction of oocyte maturation <i>in vivo</i>.

<p>(A, B) Each agent was added at time zero. Agents were then washed-out by changing the water three times at indicated times. After four hr incubation, %GVBD and %ovulation was assessed by scoring the oocytes. Each panel shows 17,20β-DHP (A) and DES treatment (B). (C) Effect of serial treatment with DES and 17,20β-DHP. Incubation was started by the addition of DES. Then, 17,20β-DHP was added at the indicated times. %GVBD (closed column) and %ovulation (open column) was assessed by scoring the oocytes after four hours from addition of 17,20β-DHP. Each value represents the mean of data from three different females. Vertical lines indicate standard deviation.</p

Scatchard plot analysis of purified hmPRα.

<p>Saturation curves and Scatchard plots of specific [³H]1,2,6,7-progesterone binding to purified recombinant hmPRα.</p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -0

50 mM Tris-HCl, pH 7.6, 1 μg/ml 20S proteasome, and 10 μM peptidyl substrates (: Suc-LLVY-MCA for chymotrypsin-like activity; : Z-LLE-MCA for PGPH activity; : Boc-LRR-MCA for trypsin-like activity) and increasing concentrations of SDS at 37°C for 1 h. The reactions were started and stopped as described in methods. Symbols represent the wild-type strain (○) and the mutant (●). Values are means ± SD of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -5

Matched in the w>database search indicated with asterisks. Panels correspond to a spot from the 20S() and 20S() proteasomes, respectively.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -9

50 mM Tris-HCl, pH 7.6, 1 μg/ml 20S proteasome, and 10 μM peptidyl substrates (: Suc-LLVY-MCA for chymotrypsin-like activity; : Z-LLE-MCA for PGPH activity; : Boc-LRR-MCA for trypsin-like activity) and increasing concentrations of SDS at 37°C for 1 h. The reactions were started and stopped as described in methods. Symbols represent the wild-type strain (○) and the mutant (●). Values are means ± SD of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Summary of the purification of recombinant human mPRα from <i>Pichia pastoris</i>.

<p>^a The activities of each fraction were measured with 4 nM of [³H] 1,2,6,7 progesterone as described in Materials and Methods.</p><p>^b The yield of total progesterone binding activities of each fraction are presented as percentages of the crude extract, which was assumed to have a binding activity of as 100%.</p><p>Summary of the purification of recombinant human mPRα from <i>Pichia pastoris</i>.</p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -7

Erred to a PVDF membrane. The α1- and α7-subunits were detected using their respective antibodies as described in the methods. () Cells from wild-type and the mutant (wt, ) grown at 25°C (OD= 1.0) were harvested and the cell extracts were applied to a Q-sepharose column. Active Q-sepharose fractions were concentrated and treated with (AP+) or without (AP-) alkaline phosphatase as described in the methods. The samples were then subjected to SDS-PAGE in a 12% polyacrylamide gel. The α7-subunit was detected by immunostaining with anti-α7 antibody [,].<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p