First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00784
Preferentially localized dynein and perinuclear dynactin associate with nuclear pore complex proteins to mediate genomic union during mammalian fertilization
Christopher Payne1,2,
Vanesa Rawe2,
João Ramalho-Santos2,3,
Calvin Simerly2 and
Gerald Schatten2,*
1 Program in Molecular and Cellular Biosciences, Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97201, USA
2 Pittsburgh Development Center, Magee-Womens Research Institute, Departments of Obstetrics, Gynecology and Reproductive Sciences, and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
3 Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal
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Fig. 1. Sperm-aster microtubules and dynein distribute to the female pronucleus. (A) Fluorescent micrographs of microtubules (MTs) and DNA in bovine zygotes during pronuclear migration and apposition. The male pronucleus (M) occupies a central position throughout these processes, whereas the female pronucleus (F) initially occupies a cortical position (left) and then migrates to the center to meet the male pronucleus (right). During pronuclear migration, sperm-aster microtubules radiate out from a region next to the male pronucleus (asterisk) and extend to the surface of the female pronucleus. The arrow indicates the direction of female pronuclear movement. (B) During pronuclear migration and apposition, dynein intermediate chain concentrates around the female pronucleus (left and center). Dynein also distributes as dim cytoplasmic foci but is absent from the surface of the male pronucleus. Pre-absorption of anti-dynein antibodies with their antigens results in a loss of detection in zygotes (right). Dynein localization is observed in both rhesus-monkey zygotes (B, left) and bovine zygotes (B, center). Scale bars, 10 µm.
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Fig. 2. Dynactin subunits localize around both female and male pronuclei, whereas dynein distributes only to the female. (A) Dynactin subunits p150Glued, p50 and p62 distribute around both female (F) and male (M) pronuclei during pronuclear migration and apposition in bovine zygotes. Additional diffuse staining is detected in the cytoplasm for both p150Glued (far left) and p50 (center left). Pre-incubation of anti-dynactin p150Glued antibodies with human endothelial cell lysates results in a loss of detection in zygotes (far right). (B) Double-staining of dynein 74 kDa (green) and dynactin p150Glued (red) shows co-localization around the female, but not the male, pronucleus (Merge; top). Staining of calreticulin (red) shows distribution throughout the cytoplasm but no localization to pronuclear surfaces (Merge; bottom). Scale bars, 10 µm.
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Fig. 3. Dynein and dynactin are required for pronuclear migration and apposition. (A) Pronuclear apposition is normal when control IgG antibodies are transfected into bovine zygotes, but female pronuclear migration is inhibited following transfection of antibodies against either dynein intermediate chain (70.1) or dynactin p150Glued. Dynein shows normal distribution around the female (F), but not the male (M), pronucleus in controls (left) and localizes to the female pronuclear surface proximal to the sperm aster after anti-dynein antibody transfection (center). Dynactin concentration around both pronuclei is reduced following anti-dynactin antibody transfection (right). Microtubule (MT) organization is unperturbed in zygotes transfected with anti-dynein and anti-dynactin antibodies, with sperm asters clearly visible. Scale bars, 10 µm. (B) Quantification of the effects of antibody transfection on pronuclear apposition. Zygotes were transfected 12 hours post-insemination (HPI) and developed until 24 HPI, when they were then fixed and analysed by immunocytochemistry. Pronuclear apposition was scored by measuring the distance between pronuclei, with 10 µm representing the average diameter of a pronucleus. Pre-absorbed antibodies refer to anti-dynein and anti-dynactin antibodies incubated with their antigens or cell lysates prior to transfection.
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Fig. 4. Dynein does not localize to the female pronucleus in the absence of a sperm aster. Parthenogenetic activation of bovine oocytes induces female (F) pronuclear formation and microtubule polymerization without aster formation. Dynactin p150Glued (green; left) concentrates around the female pronucleus (blue), whereas dynein intermediate chain (green; right) distributes near the cortex, enriched with unfocused microtubules (red). Dynein is not detected at the female pronuclear surface. The arrow shows dynactin surrounding the female pronucleus. Scale bar, 10 µm.
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Fig. 5. Microtubules are required to retain dynein, but not dynactin, at pronuclear surfaces. (A) Dynein-intermediate-chain localization to the female pronucleus is no longer detected in bovine zygotes following nocodazole treatment (left). Microtubules (MTs), observed around both female (F) and male (M) pronuclei in controls, are not seen in nocodazole-treated zygotes. Dynactin p150Glued, however, localizes to both pronuclei in nocodazole-treated bovine zygotes, even when microtubules are no longer detected (right). Scale bars, 10 µm. (B) Quantification of the effects of nocodazole treatment on dynein and dynactin association with pronuclei. Results represent the average ± s.d. of four sets of experiments, with a total of 512 zygotes examined for dynein staining; P<0.001 compared with controls (Student's t test). A total of 504 zygotes were examined for dynactin staining, with the differences not statistically significant compared with controls (Student's t test).
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Fig. 6. Dynactin interacts with nucleoporins, vimentin and dynein during female pronuclear migration. (A-C) Western blots following immunoprecipitation (IP) of dynactin p150Glued from either pronucleate-stage zygotes or unfertilized oocytes. Lanes on the blots are beads only (B), IP pellets (P), IP supernatants (S) and whole cell lysates from either zygotes (Z) or oocytes (O). (A) Five nucleoporins of molecular mass 270 kDa, 175 kDa, 62 kDa, 35 kDa and 18 kDa are detected in the zygote IP pellet and whole zygote lysate, as well as in the oocyte IP supernatant and whole oocyte lysate. Nucleoporins in the IP pellet show modest enrichment. (B) Vimentin, identified at 58 kDa, is enriched in the zygote IP pellet and detected in both zygote and oocyte IP supernatants, as well as in whole zygote and oocyte lysates. (C) Dynein (74 kDa) and dynactin (150 kDa) are enriched in zygote and oocyte IP pellets and detected in whole zygote and oocyte lysates. (D) Dynactin p150Glued (green) and nucleoporin p62 (red) co-localize around the female (F) and male (M) pronuclei, and at cytoplasmic foci (merged channels appear yellow). (E) Dynactin p150Glued (green) and vimentin (red) co-localize around the pronuclei (merged channels appear yellow), with vimentin showing additional branching in the region between and surrounding the pronuclei. Scale bar, 10 µm.
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Fig. 7. Nucleoporins and vimentin are required for pronuclear migration and apposition. (A) Pronuclear apposition is normal when control IgG antibodies are transfected into bovine zygotes, but female pronuclear migration is inhibited following transfection of antibodies against either nuclear pore complex proteins (NPCs) or vimentin. NPCs show normal distribution around the female (F) and the male (M) pronucleus in controls (left), and similar localization is seen following anti-nucleoporin antibody transfection (center). Vimentin branching around and between the distant pronuclei is detected after anti-vimentin antibody transfection (right). Dynactin p150Glued shows normal localization to pronuclei in control IgG-transfected and anti-nucleoporin-transfected zygotes, but its distribution around pronuclear surfaces appears reduced following transfection with anti-vimentin antibodies. Scale bar, 10 µm. (B) Quantification of the effects of antibody transfection on pronuclear apposition. Zygotes were transfected 12 hours post-insemination (HPI) and developed until 24 HPI, when they were then fixed and analysed by immunocytochemistry. Pronuclear apposition was scored by measuring the distance between pronuclei, with 10 µm representing the average diameter of a pronucleus. Pre-absorbed antibodies refer to anti-nucleoporin and anti-vimentin antibodies incubated with their antigens prior to transfection.
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Fig. 8. A model for pronuclear assembly, motility and union. Sperm entry activates the mature oocyte, leading to second polar body extrusion (A; first polar body not shown). Formation of pronuclei is accompanied by sperm-aster-independent microtubules (B, left), which bring dynactin (black rectangle with side arm) and vimentin filaments (green lines) to the cytoplasmic face of the nuclear pore complex (basket structure) (B, right). Disassembly of microtubules and inhibition of nucleoporins and dynactin block proper complex formation at the outer surfaces of both pronuclei. Growth of sperm-aster microtubules, nucleated by the centrosome attached to the male pronucleus, extends microtubule plus ends away from the male pronucleus, some of which then reach the female pronuclear surface (C, left). These microtubule plus ends could deliver dynein (red wishbone) preferentially to the surface of the female pronucleus, allowing dynein to bind to dynactin and vimentin at nuclear pores, and enabling the dynein-dynactin complex to transport the female pronucleus to the minus ends along the sperm aster (C, right). The inhibition of either dynactin or dynein blocks migration and prevents apposition (D).
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© The Company of Biologists Ltd 2003
