Effect of injected CAII-protein on NBCe1 transport activity at different bicarbonate concentrations and constant CO₂.

<p>Original recordings of membrane current (A) in NBCe1-expressing oocytes with or without injection of 50 ng CAII-protein after changing from a HEPES-buffered, bicarbonate-free saline (pH 7.0) to a 5% CO₂/10 mM HCO₃⁻-buffered saline (pH 7.0) as well as after increasing the HCO₃⁻ concentration from 10 mM (pH 7.0) to 77 mM HCO₃⁻ (pH 7.9) in a 5% CO₂-equilibrated saline, before or during application of EZA (10 µM). Statistical analysis of membrane current (B) revealed an increase in NBCe1 transport activity after injection of CAII-protein in both salines. Original recordings of the change of intracellular proton concentration (C) and statistical analysis of rate of rise of proton concentration (D) of native oocytes and oocytes injected with CAII-protein. The asterisks above the bars correspond to the control cells without CA (−CA) before (−EZA) or during EZA (+EZA) application.</p

Fluorescent staining of CA isoforms and mutants.

<p>Optical slices of oocytes, labeled via primary antibodies and Alexa Fluor 488-linked secondary antibodies against CAI (A), CAII (B), CAIII (C), CAII-Y7F (D), -H64A (E) and -V143Y-expressing oocytes (F). As control, a staining of native, uninjected oocytes or oocytes injected with H₂O with primary antibodies against CAI (G), CAII (H) and CAIII (I) as well as corresponding secondary antibodies, respectively, was performed.</p

Characteristics of candidate genes associated with embryonic development in the cow: Evidence for a role for <i>WBP1</i> in development to the blastocyst stage

<div><p>The goal was to gain understanding of how 12 genes containing SNP previously related to embryo competence to become a blastocyst (<i>BRINP3</i>, <i>C1QB</i>, <i>HSPA1L</i>, <i>IRF9</i>, <i>MON1B</i>, <i>PARM1</i>, <i>PCCB</i>, <i>PMM2</i>, <i>SLC18A2</i>, <i>TBC1D24</i>, <i>TTLL3</i> and <i>WBP1</i>) participate in embryonic development. Gene expression was evaluated in matured oocytes and embryos. <i>BRINP3</i> and <i>C1QB</i> were not detected at any stage. For most other genes, transcript abundance declined as the embryo developed to the blastocyst stage. Exceptions were for <i>PARM1</i> and <i>WBP1</i>, where steady-state mRNA increased at the 9–16 cell stage. The SNP in <i>WBP1</i> caused large differences in the predicted three-dimensional structure of the protein while the SNP in <i>PARM1</i> caused smaller changes. The mutation in <i>WBP1</i> causes an amino acid substitution located close to a P-P-X-Y motif involved in protein-protein interactions. Moreover, the observation that the reference allele varies between mammalian species indicates that the locus has not been conserved during mammalian evolution. Knockdown of mRNA for <i>WBP1</i> decreased the percent of putative zygotes becoming blastocysts and reduced the number of trophectoderm cells and immunoreactive CDX2 in the resulting blastocysts. <i>WBP1</i> is an important gene for embryonic development in the cow. Further research to identify how the SNP in <i>WBP1</i> affects processes leading to differentiation of the embryo into TE and ICM lineages is warranted.</p></div

Models of the three-dimensional structure of WBP1.

<p><b>A</b>: variant T, corresponding to the major allele; <b>B</b>: variant P corresponding to the minor allele; <b>C</b>: Superimposed models of both isoforms of the protein. In each panel, the light color side chains represent the position of the AA affected by the SNP.</p

Quantification of CAII mutants.

<p>Western blot of the different CAII mutants (CAII-Y7F, CAII-H64A and CAII-V143Y) as well as wild-type CAII (A; 12 µg total protein/lane), β-tubulin was used as loading control, and quantification of the expression of the CAII mutants in oocytes, as compared to wild-type by determination of the density of intensity (B; Density INT/mm²). Western blot of CAII-expressing and NBCe1+CAII-coexpressing oocytes, with β-tubulin used as loading control (C; 15 µg total protein/lane) and quantification of effect of NBCe1 coexpression on CAII expression rate (D).</p

Activity of CAI, II and III.

<p>Original recordings of changes of intracellular proton concentration (ΔH⁺; A) and statistical analysis of the rates of rise of proton concentration (ΔH⁺/t; B) after application of 5% CO₂/24 mM HCO₃⁻-buffered solution before or during application of EZA (10 µM). The asterisks above the bars correspond to the control cells without CA (−CA) before (−EZA) or during application of EZA (+EZA). (C) Original recordings of the log enrichment of 20 oocytes expressing CAI, CAII and CAIII, respectively, either alone or together with the NBCe1, or 20 CAII-V143Y, native or just NBCe1-expressing oocytes as measured by mass spectrometry (MS). The arrows indicate the application of 20 intact oocytes, which were rapidly lysed inside the cuvette by stirring. (D) Statistical analysis of CA activity (U/ml) after subtraction of either native (4 U/ml; 20 oocytes, n = 3) or NBC-expressing oocytes (5 U/ml; 20 oocytes, n = 3), as obtained by mass spectrometry. Oocytes expressing the catalytically inactive mutant CAII-V143Y were used as control. Calibration curve of different CAI- and CAII-protein concentrations (E) to determine the amount of CA-protein expressed in oocytes (F). The asterisks above the bars for NBCe1-coexpressing oocytes (+NBCe1, 13.8 ng NBCe1-RNA) correspond to those without NBCe1-coexpression (−NBCe1). A significance level of p≤0.05 is marked with *, p≤0.01 with ** and p≤0.001 with ***.</p

Activity and effect of CAII mutants on NBCe1 transport activity.

<p>Changes of the intracellular proton concentration as recorded in native, CAII-, CAII-H64A- and CAII-Y7F-expressing oocytes (A) to determine rates of rise of the proton concentration (B). Original recordings of the changes in membrane current (C) and intracellular sodium concentration (E) in NBCe1 and CAII-, CAII-H64A- or CAII-Y7F- coexpressing oocytes after application of 5% CO₂/24 mM HCO₃⁻-buffered solution before or during application of EZA (10 µM). Statistical analysis of membrane current (D) and rates of rise of intracellular sodium concentration (F) revealed an increase in NBCe1 transport activity after coexpression of either CAII or one of the two mutants. The asterisks above the bars correspond to the control cells without CA (−CA) before (−EZA) or during EZA (+EZA) application.</p

Phylogenetic tree of <i>WBP1</i>.

<p>The reference allele is indicated in red. All species distal to the common ancestor had the same reference allele unless indicated by placement of a distinct letter. For <i>B</i>. <i>taurus</i>, both alleles are presented (major/minor). The G allele (underlined) was previously associated with superior development to the blastocyst stage.</p

Effect of CAI-protein injection (0–200 ng) on the catalytic activity and NBCe1 transport activity.

<p>Original recordings of the changes of intracellular proton concentration of CAI-injected oocytes (A) and of membrane current of CAI-injected, NBCe1-expressing oocytes (D) after application of 5% CO₂/24 mM HCO₃⁻-buffered solution in the absence and presence of EZA (10 µM). Rates of rise of proton concentration (B) and changes of membrane current (E) before (filled squares) or during (open squares) EZA application in CO₂/HCO₃⁻-buffered solution. EZA-sensitive rates of rise of proton concentration (C) and changes of membrane current were plotted (F). The asterisks correspond to the control cells without CA-injection (0 ng CAI).</p

Potential binding motif (bold amino acids) of hCAII (Swiss-Prot.: P00918) against NBCe1 is incompletely conserved in N-terminus of intracellular hCAI (Swiss-Prot.: P00915) or hCAIII (Swiss-Prot.: P07451).

<p>Potential binding motif (bold amino acids) of hCAII (Swiss-Prot.: P00918) against NBCe1 is incompletely conserved in N-terminus of intracellular hCAI (Swiss-Prot.: P00915) or hCAIII (Swiss-Prot.: P07451).</p