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Raw RT-PCR gels images.
No full textAquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.</div
Metabolic fuels in developing cod and turbot embryos and larvae
No full textAerobic metabolic rates of embryos and larvae of Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) were measured and used stoichiometrically to balance the substrates, in the form of free amino acids (FAAs), proteins and lipids, that quantitatively disappeared during development. Calculations reveal that cod embryos and larvae predominantly fuel their metabolism with FAAs (73%) throughout development; in addition, following closure of the blastopore, lipid is also steadily catabolized (27%). Turbot, however, utilize different substrates at separate stages of development. FAAs are predominantly burned during the embryonic stage, while lipids are burned during the yolk-sac stage. From the time of first-feeding, proteins are recruited from body stores and catabolized. Thus, for turbot, free and protein-bound amino acids together contribute 40% to the energy metabolism, while lipids contribute 60%.</p
The dominant-negative activity of teleost Aqp1ab1 and -1ab2 splice forms.
No full text(A-C) Changes in Pf of X. laevis oocytes injected with water or expressing SaAqp1ab1-WT (A), HhAqp1ab1-WT (B) or SsAqp1ab2-WT (C) alone or in combination with different amounts of the isoforms of each paralog. For SsAqp1ab2-WT and splice forms, oocytes were co-injected with halibut YwhazLb and exposed to FSK. (D) Effect of the inhibition of the oocyte Pf by the different Aqp1ab1 splice forms, at a concentrations that produced approximately half-maximal reduction (as observed in A-C), in SaAqp1ab1 or HhAqp1ab1 expressing oocytes in the presence or absence of YwhazLa, and exposed to FSK. The percentage of Pf inhibition elicited by each isoform under the two conditions is indicated in each plot. In all panels, data are the mean ± SEM (n indicated above each bar) and were statistically analyzed by an unpaired Student’s t-test (*, P P P < 0.001; with respect to oocytes injected with the WT form alone).</p
Raw Western blot images from Fig 6B, 6D and 6F.
No full textAquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.</div
Functional characterization and subcellular localization of teleost wild-type Aqp1ab1 and -1ab2 and their splice variants.
No full text(A-C) Pf of X. laevis oocytes injected with water (W, controls) or expressing seabream SaAqp1ab1-WT, SaAqp1ab1_v1 or SaAqp1ab1_v2 (A), Atlantic halibut HhAqp1ab1-WT or HhAqp1ab1_v1 (B), or sole SsAqp1ab2-WT, SsAqp1ab2_v1 or SsAqp1ab2_v2 (C). Oocytes injected with SsAqp1ab2-WT or splice forms were co-injected with halibut YwhazLb and exposed to FSK for 1 h prior to the swelling assay. The data are the mean ± SEM (n indicated above each bar) and were statistically analyzed by an unpaired Student’s t-test (***, P D-F) Double immunostaining of oocytes expressing untagged SaAqp1ab1-WT or HA-tagged SaAqp1ab1_v1 or HA-SaAqp1ab1_v2 (D), Flag-tagged HhAqp1ab1-WT or HA-tagged HhAqp1_v1 (E), or Flag-tagged SsAqp1ab2-WT or HA-tagged SsAqp1ab2_v1 or HA-SsAqp1ab2_v2 (F), and the ER marker protein disulfide isomerase (PDI). The plasma membrane is indicated by an arrowhead, whereas the co-localization of aquaporin channels and PDI in the cytoplasm is indicated by arrows. Scale bars, 10 μm (insets 5 μm).</p
Production of Senegalese sole total Aqp1ab2 and Aqp1ab2_v1 specific antisera and immunoblot analysis of Aqp1ab2 splice variants protein expression in developing ovarian follicles.
No full text(A) Amino acid sequence alignment of SsAqp1ab2-WT, SsAqp1ab2_v1 and SsAqp1ab2_v2 splice forms highlighting the sequences employed for the production of the sole-specific Aqp1ab2 and Aqp1ab2_v1 antibodies (α-Aqp1ab2-Nt and α-Aqp1ab2_v1, in red and blue color, respectively). The N- and C-termini of the WT channel, the six transmembrane helices (TM1-TM6) and the two conserved NPA motifs are also indicated. (B) Immunoblots of X. laevis oocytes injected with water or expressing Aqp1ab2-WT, SsAqp1ab2_v1 or SsAqp1ab2_v2 and probed with the α-Aqp1ab2-Nt or α-Aqp1ab2_v1 antisera separately. (C) Representative photomicrographs of sole ovarian follicles at different developmental stages during oocyte growth and maturation. PG, primary growth stage; CA, cortical alveoli stage. Scale bars, 50 and 250 μm. (D) Immunoblots of Aqp1ab2-WT, Aqp1ab2_v1 and Aqp1ab2_v2 in protein extracts from previtellogenic (Pv), vitellogenic (Vt), hydrating (H) and mature (Mt) follicle-enclosed oocytes, as well as from ovulated oocytes (Ov), using the generated antisera. Alpha tubulin (Tuba) was used as loading control. Duplicated blots (right) were run in parallel and incubated with the primary antibodies preadsorbed by the antigenic peptide to test for specificity. The brackets indicate potential post-translational modifications. (E) Hoechst staining of intact vitellogenic follicles and defolliculated oocytes. Scale bar, 100 μm. (F) Immunoblots of Aqp1ab2 isoforms in total protein extracts from intact or defolliculated vitellogenic follicles (+/- follicle cells, Fc). In B, D and E, the arrowheads indicate aquaporin monomers, whereas the asterisks in D and E indicate cross-reactive polypeptides revealed with the Aqp1ab2_v1 antiserum. In all blots, molecular mass markers (kDa) are on the left.</p
Raw Western blot images from Fig 5G–5I.
No full textAquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.</div
Identification of <i>aqp1ab1</i> and <i>aqp1ab2</i> alternative splicing in teleosts.
No full text(A-C) Genomic organization of tandemly arranged aqp1ab2 and aqp1ab1 genes in seabream (A) and Atlantic halibut (B), and of the single aqp1ab2 gene in Senegalese sole and Common sole (C). The four exons (1–4) of both genes are represented by blue and red boxes, and the start codons are indicated by upper arrows. Lower arrows indicate the position of the oligonucleotide primers used for RT-PCR. (D-E) Representative RT-PCR analysis of aqp1ab1 and aqp1ab2 expression in different adult tissues from seabream (D) and halibut (E). A single aqp1ab2 mRNA species is expressed in seabream and halibut, whereas two or one splice variants (aqp1ab1_v1 and aqp1ab1_v2) in addition to aqp1ab1 are detected, respectively, as indicated. (F-G) RT-PCR of aqp1ab2 expression in Senegalese sole (F) and Common sole (G) where two splice forms (aqp1ab2_v1 and aqp1ab2 _v2) in addition to aqp1ab2 are amplified in the ovary and testis. In D-G, the sizes (kb) of molecular markers are indicated on the left.</p
Teleost Aqp1ab1 and -1ab2 splice forms generate distinct mechanisms of dominant-negative repression.
No full text(A-C) Double immunolocalization of SaAqp1ab1-WT, Flag-tagged HhAqp1ab1-WT or Flag-tagged SsAqp1ab2-WT (green) with the respective HA-tagged splice forms in X. laevis oocytes as indicated. Oocytes expressing SsAqp1ab2-WT and splice forms were co-injected with halibut YwhazLb and exposed to FSK. In all panels, the plasma membrane is indicated by an arrowhead, whereas the co-localization of WT aquaporins and splice forms is indicated by arrows. Scale bars, 10 μm. (D-F) Representative immunoblots of total (TM) and plasma (PM) membrane protein extracts from oocytes injected with water (W) or co-expressing the different aquaporin WT channels with increasing amounts of the splice forms as indicated. (G-I) Illustrative immunoblots of precipitated proteins from oocytes treated as in A-C using anti-SaAqp1ab1 or anti-Flag (for HhAqp1ab1 and SsAqp1ab2) antibodies, and revealed with antibodies against SaAqp1ab1, Flag or HA. In D-I, upper blots were probed with SaAqp1ab1 (D and G) or Flag (E, F, H and I) antibodies, whereas in all panels the lower blots were probed with HA antibodies. Molecular mass markers (kDa) are on the left. The arrowhead in F and I indicate potential post-translational modifications of the SsAqp1ab1_v2 isoform.</p
Raw Western blot images from Fig 5D–5F.
No full textAquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.</div
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