assembly of nuclear pore complexes and annulate lamellae

2841Journal of Cell Science 111, 2841-2854 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS4597

Assembly of nuclear pore complexes and annulate lamellae promotes normal

pronuclear development in fertilized mammalian oocytes

Peter Sutovsky 2, Calvin Simerly 2, Laura Hewitson 2 and Gerald Schatten 1,2,*1Departments of Obstetrics and Gynecology, and Cell and Developmental Biology, Oregon Health Sciences University, Portland,Oregon, USA2Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA*Author for correspondence (e-mail: [email protected])This article is dedicated to the memory of the late Dr Daniel Szöllosi, pioneer of mammalian fertilization research

Accepted 25 July; published on WWW 9 September 1998

In addition to functional nuclear pore complexes engagedin nucleo-cytoplasmic transport, the cytoplasmic stacks ofpore complexes, called annulate lamellae, exist in numerouscell types. Although both annulate lamellae and nuclearpore complexes are present in fertilized mammalianoocytes, their relative roles in the process of fertilizationand preimplantation development are not known. Usingepifluorescence and electron microscopy, we explored theirfate during bovine fertilization. The assembly of annulatelamellae in bovine oocytes was triggered by sperm-oocytebinding and continued concomitantly with theincorporation of the nuclear pores in the nuclear envelopesof the developing male and female pronuclei. This processwas also induced by the parthenogenetic activation ofmetaphase-II-arrested oocytes. Depletion of Ca2+,previously implicated in oocyte activation and in theinsertion of pore complexes into the nuclear envelope,prevented the formation of nuclear pore complexes, but notthe assembly of annulate lamellae in oocyte cytoplasm.Injection of the nuclear pore antagonist, wheat germagglutinin, into the cytoplasm of mature oocytes that were

subsequently fertilized caused the arrest of pronucleardevelopment, indicating the requirement of nuclear porecomplexes for normal pronuclear development. Treatmentof the fertilized oocytes with the microtubule inhibitor,nocodazole, prevented gathering of annulate lamellaearound the developing pronuclei, insertion of nuclear poresinto their nuclear envelopes, and further pronucleardevelopment. The formation of the male pronuclei wasreconstituted in Xenopus egg extracts and reflected thebehavior of nuclear pores during natural fertilization.These data suggest that nuclear pore complexes arerequired for normal pronuclear development from itsbeginning up until pronuclear apposition. Annulatelamellae may be involved in the turnover of nuclear porecomplexes during fertilization, which is in turn facilitatedby the reorganization of oocyte microtubules and influx ofCa2+ into oocyte cytoplasm.

Key words: Annulate lamellae, Nuclear pore complex, Fertilization,Pronucleus, Nuclear envelope

SUMMARY

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INTRODUCTION

The cyclic blending and separation of the nuclear acytoplasmic compartments of dividing cells is perhaps the mstriking feature of the cell cycle. The nuclear envelope (NE)mitotic cells dissolves at the entry point of the M pha(Newport and Spann, 1987) and reforms at M exit, interphase entry (Pfaller et al., 1991). This process controlsaccess of cell cycle regulators such as maturation promofactor (MPF) to their target proteins in the nucleus. Tassembly of the NE seems to occur in three distinct stewhich have specific requirements for energy substrates regulatory molecules (Boman et al., 1992; Macaulay aForbes, 1996). In the first step membrane vesicles, which mcontain receptors for the proteins of nuclear lamina (Collasal., 1996), assemble around the chromatin and eventually to form a continuous NE around it (Burke and Gerace, 19

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Pfaller et al., 1991). Next, the nuclear pore complexes (NPCassembled from the specific set of proteins called nucleopor(Davis and Blobel, 1986, 1987; Hanover et al., 1987; Snowal. 1987), are incorporated into the NE and provide thchannels for bi-directional exchange of proteins (nuclecytoplasmic transport) between the newly established nucleand cytoplasmic compartments (Benavente et al., 198reviewed by Görlich and Mattaj, 1996; Panté and Aebi, 1993Finally, nuclear lamins are imported into the nucleus and forthe scaffold of nuclear lamina underneath the NE (Newportal., 1990). In contrast to this view, some investigators suggthat lamins bind to the membrane vesicles or directly chromatin and, therefore, are among the first componentsNE to gather around the post-mitotic chromatin (Hutchinson al., 1994).

Similar to the scenario during mitosis, the assembly of thNE following mammalian fertilization starts with membrane

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free chromatin (Szollosi et al., 1972; Longo, 1973). mammalian zygotes, this is represented by two distinct entita set of maternal chromosomes that complete second meupon the sperm-triggered oocyte activation (reviewed Schultz and Kopf, 1995; Yanagimachi, 1994) and the paterchromosomes in the sperm nucleus. During the incorporaof the sperm nucleus into oocyte cytoplasm the sperm nucis stripped of its plasma membrane, cytoskeletal coat of perinuclear theca and its intrinsic NE (Snell and White, 199Sutovsky et al., 1997a; Usui, 1996; Usui et al., 199Subsequently, the assembly of the NE takes place in a fassimilar to that seen in somatic cells, regardless of whethersperm entered the oocyte during natural fertilization or whetit was introduced into the oocyte cytoplasm bintracytoplasmic sperm injection (Usui et al., 1996; Sutovset al., 1996a).

Although the assembly and fusion of membrane vesicduring NE formation have been studied extensively in licells, as well as in the cell-free system (Burke and Gera1986; Lohka and Masui, 1983; Newport, 1987; reviewed Poccia and Collas, 1996), little is known about the pathwaleading to the formation of NPCs on the NE formed de noparticularly in mammalian models. For instance, this proceseems to occur only after a continuous NE is formed arequires GTP and Ca2+ (Boman et al., 1992; Macaulay andForbes, 1996). In addition to the insertion of NPCs into the Nannulate lamellae (AL) spontaneously form in Xenopuseggextracts upon the addition of the membrane-rich fractitogether with, similar to the NPCs at the NE, hydrolysis of GT(Boman et al., 1992; Dabauvalle et al., 1991). AL have befound in the fertilized eggs of several mammalian specincluding humans (Van Blerkom et al., 1987), rhesus monk(Sutovsky et al., 1996a), cattle (Sutovsky et al., 1996Sutovsky and Schatten, 1997), pig (Laurincik et al., 199Szöllosi and Hunter, 1973), rabbit (Longo, 1975; Szollosi al., 1996) and hamster (P. Sutovsky, and J. Squirunpublished data). AL are also present in unfertilizinvertebrate and lower vertebrate oocytes and in the dividembryos of all major animal groups (see Kessel, 1983, 19for references). At present, it is not known how and when NPCs are incorporated into NEs of developing mammalpronuclei and whether NPCs and/or AL play any role durimammalian fertilization and zygotic development.

Kessel (1992) dubbed AL ‘the last frontier in cell organellesince there is no direct evidence for any of their proposed roin either animal or plant cells. In fact, most investigators beliethat AL are the side product of nucleoporin synthesis rather tthe precursor structure required for the assembly aincorporation of NPCs into the NE (Stafstrom and Staehe1984). Here, we demonstrate that the fertilizing spermatoztriggers the assembly of AL within the cytoplasm of matubovine oocytes. These AL are predominantly found in the pepronuclear region, and drug treatments such as microtubdisruption with nocodazole or Ca2+-chelation by BAPTA causethem to spread across the oocyte cytoplasm, and preveninsertion of NPCs in the pronuclear envelopes and normpronuclear development. Similarly, pronuclear developmecan be blocked by the microinjection of the nucleoporantagonist wheat germ agglutinin (WGA) into the cytoplasmfertilization competent oocytes. We propose that tcolonization of pronuclear NE by NPCs at an early stage

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pronuclear development is required for successful fertilizatioin mammals, during which AL may be involved inNPC/nucleoporin turnover.

MATERIALS AND METHODS

Isolation and in vitro maturation of bovine oocytesOocytes were isolated by the aspiration of the contents of approx3 mm large ovarian follicles from bovine ovaries obtained from a locabattoir and then matured in vitro for 24 hours (metaphase II) in T199 medium (Gibco) supplemented with 10% fetal calf serum, 0units/ml FSH-P (Schering-Plough Animal Health Corp., KenilworthNJ), 0.2 mM pyruvate and 25 µg/ml gentamicin.

Sperm preparation and labelling with MitoTracker GreenFMStraws of frozen bull semen were thawed in warm water and tsemen centrifuged for 10 minutes at 700 g through a two-layer Percollgradient (45% and 90% in Sperm-TL medium composed of 100 mNaCl, 3.1 mM KCl, 25 mM NaHCO3, 29 mM NaH2PO4, 21.6 mMsodium lactate, 2 mM CaCl2, 4 mM MgCl2, 10 mM Hepes, 6 mg/mlbovine serum albumin, 25 µg/ml gentamicin, 1 mM pyruvate). Thesperm pellets were then resuspended and incubated for 10 minute37°C in Sperm-TL supplemented with 400 nM MitoTracker GreeFM (Molecular Probes Inc., Eugene, OR). MitoTracker-labeled spewere washed by repeated resuspension and centrifugation in SpeTL. MitoTracker is a fixable, vital, mitochondrion-selectivefluorescent probe that binds to the mitochondria, covering the mpiece of the sperm tail without affecting the motility and ability olabelled spermatozoa to fertilize an oocyte. The dye is retained aaldehyde fixation and permeabilization of zygotes, providing fluorescently tagged sperm tail associated with the male PN. Tallows us to determine which of the two pronuclei in the fertilized egis the male PN, as well as to distinguish between fertilized eggs aparthenogenotes containing two female pronuclei. At the early staof fertilization, MitoTracker tagging of the fertilizing spermatozoonallows us to distinguish the decondensing sperm chromatin from maternal chromosomes in a developing female PN and in the secpolar body (Sutovsky et al., 1996b; Sutovsky and Schatten, 1997)

In vitro fertilization of bovine oocytesFollowing a wash in Sperm-TL, MitoTracker-tagged sperm weresuspended in fertilization medium (TL; modified Tyrode-lactamedium: 114 mM NaCl, 3.2 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2,25 mM NaHCO3, 0.4 mM NaH2PO4, 10 mM sodium lactate, 6.5 i.u.penicillin, 25 µg/ml gentamicin, 6 mg/ml fatty acid-free bovine serumalbumin and 0.2 mM pyruvate) and added to 50 µl drops offertilization medium covered with mineral oil, to a final concentratioof 1×106 sperm/ml. Oocytes were incubated for various time interva(as stated in Results) at 39°C in a humidified atmosphere of 5% C2in air. For parthenogenetic activation, the mature, metaphasearrested oocytes were incubated for 5 minutes in fertilization mediucontaining 5 µM ionomycin (Sigma), washed and returned back to thfertilization medium. After 5 hours of incubation, the oocytes wertransferred for 5 minutes into the fertilization medium containing 1mM 6-dimethyl-amino-purine (6-DMAP; Sigma), washed ancultured for an additional 11 hours. To disrupt the zygotmicrotubules, 10 µM nocodazole (Sigma) was added to thefertilization drops 8 hours after insemination. To prevent the speincorporation, the oocytes were fertilized in the presence of 10 µg/mlof cytochalasin B (Sigma).

Microinjection of WGAOocytes were isolated and matured as described in above. Injectwere performed at 35°C in 100 µl drops of TL-Hepes medium

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containing 5 µg/ml cytochalasin B (CB), covered with light mineraoil. Oocytes were immobilized with the polar body in the 12 o’cloposition, the injection needle was inserted through the zona pelluinto the cytoplasm and 20 pl of FITC-WGA (Sigma) was injected a calibrated pipette in each of them. Injected oocytes were fertilias described above and processed for indirect immunofluorescwith mAb414 and DAPI as described below.

Visualization of the pronuclei, sperm tails, AL and NPCs inbovine zygotesOocytes were removed from fertilization drops at given time poiafter insemination, attached to poly-L-lysine-coated coverslipswarm 0.1 M phosphate-buffered saline (PBS), fixed for 40 minute2% formaldehyde in 0.1 M PBS and permeabilized overnight in 0.Triton X-100 in 0.1 M PBS. Nonspecific reactions were blocked b1 hour incubation in 0.1 M PBS containing 10% normal goat ser(NGS) and the coverslips with the eggs were covered with a 1/dilution of antibody mAb414 (BabCo; Berkeley, CA; Davis anBlobel, 1986, 1987) in 0.1 M PBS containing 0.05% NaN3, 2 mMEGTA, 1% NGS and 0.1% Triton X-100 (further referred to labelling solution). This antibody recognizes a subset of NPC protein a variety of animal cells. After a short wash in labelling solutiothe coverslips were incubated for 40 minutes with a rhodamiconjugated goat anti-mouse IgG (Zymed; diluted 1/40) and 5 µg/mlof DAPI (Molecular Probes Inc., Eugene, OR), added to the solut10 minutes before the end of incubation. At the end of the incubatcoverslips were mounted on microscope slides in VectaShmounting medium (Vector Labs, Burlingame, CA). The slides weexamined using a Zeiss Axiophot epifluorescence-equippmicroscope and the triple-labeled images were acquired usingRTE/CCD 1217 camera (Princeton Instruments, Inc., Trenton, operated by Metamorph software and archived on recordable Cusing a CD recorder (Smart and Friendly). Some of the images wrecorded using a Bio-Rad 1024 or Leica TCS NT confocmicroscope. Images were contrast-enhanced, edited and printedcolor video printer (Sony VPD-8800) using Adobe Photoshop software (Adobe Systems Inc., Mountain View, CA).

Preparation of Xenopus egg extracts and permeabilizedbull spermFrogs for the extract preparation were stimulated by the injection100 units PMSG into the dorsal lymph sac on day 1 and of 500 uhCG on day 4 and allowed to lay the eggs. Eggs were rinsed modified Ringer’s solution (MMR; 10 mM NaCl, 2 mM KCl, 1 mMMgCl2, 2 mM CaCl2, 1 mM EDTA, 5 mM Hepes, pH 7.8), anddejellied by a 6-minute exposure to dejellying solution (100 mM KC0.1 mM CaCl2, 1 mM MgCl2, 2% L-cysteine, pH 7.8). After tworinses in 0.2× MMR, the eggs were rinsed four times in extract buff(XB; 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 50mM sucrose, 5 mM EGTA) and then twice more in XB containinleupeptin, chymostatin and pepstatin A (at 10 µg/ml each). Extractswere transferred in a minimal volume of XB containing proteainhibitors and CB (to prevent gelation) into centrifuge tubes acentrifuged for 2 minutes at 2000 rpm in a SW28 rotor. Excess buwas removed and extracts were stratified for 20 minutes at 20,000in an SW28 rotor. The cytoplasmic extract was fortified with 1/(v/v) of an energy mix (150 mM creatine phosphate, 20 mM ATPmM EGTA, 20 mM MgCl2), 200 mM sucrose and protease inhibitorand the samples were flash frozen in liquid nitrogen and stored −70°C.

Bull sperm were prepared by Percoll gradient separation and frfrom plasma membrane, perinuclear theca and nuclear enveaccording to a previously described protocol (Sutovsky et al., 199Briefly, the sperm were allowed to settle onto a poly-L-lysine-coa12 mm round coverslip in a drop of 37°C KMT medium (100 mKCl, 2 mM MgCl2, 10 mM Tris-HCl) at pH 7.0. The sperm-coatecoverslips were washed in KMT and incubated for 30 minutes w

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Reconstitution of bovine male pronuclei and visualizationof the NPCs and microtubules in Xenopus egg extractsThe coverslips with the permeabilized sperm were overlaid with µl of Xenopusegg extract, driven into interphase with 16 mM CaCl2and supplemented with 2 mM GTP (Sigma), 2 mM ATP, 1 µg/mlleupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin and 5 µg/mlcytochalasin B with or without the addition of 5 µg/ml WGA-FITC(Sigma), 10 µM nocodazole and 2 µM taxol, according to therequirements of individual experiments. The incubation was carriout in a humid atmosphere at room temperature for 3 hours orspecified in Results. At the end of the incubation, the coverslips wfixed for 40 minutes in 2% formaldehyde in PBS and permeabilizovernight in the labeling solution as described above for the fertilizeggs. The coverslips with the sperm incubated with WGA-FITCcontaining extracts were processed with mAb 414, followed by tgoat anti-mouse IgG-TRITC (GAM IgG/TRITC; Zymed) todemonstrate the colocalization of NPCs with WGA-FITC, whicretains its fluorescence after aldehyde fixation and permeabilizatiSimilarly, the anti-β-tubulin antibody E7, followed by GAMIgG/TRITC, was used to colocalize the WGA-FITC-labelled NPCwith the microtubules. Whenever the WGA-FITC was omitted fromthe extracts, the sperm tails were counterstained with 500 nMitoTracker Green FM added into the secondary antibody solutioIn all labeling schemes, DNA was counterstained by the addition5 µg/ml DAPI into the secondary antibody solution 10 minutes prioto the end of incubation. The slides were mounted, examined andimages recorded and processed as described for the fertilized eg

Transmission electron microscopyOocytes were individually transferred for 1 hour to a fixativcomposed of 2.5% glutaraldehyde and 0.6% paraformaldehyde0.25 M cacodylate buffer (pH 7.2), then washed in 0.1 M cacodylabuffer containing 0.2 M sucrose and post-fixed for 1 hour in 1osmium tetroxide. Following dehydratation by an ascending ethaseries (30%-100%), oocytes were infiltrated by a series of washea mixture of propylene-oxide and Polybed 812 (PolysciencWarrington, PA) EM resin and embedded in PolyBed 812. Ultrathsections were cut on a Sorval MT2B ultramicrotome, placed on 1MESH copper grids and stained in two steps with uranyl acetate alead citrate. Sections were examined and photographed in a PhiEX 120 STEM electron microscope. Negatives were scanned bKodak Leafscan 35 negative scanner or Umax Magic Scan flat bscanner, recorded on magneto-optical disc and printed using AdPhotoshop 4.0 software.

Statistical analysisOocytes for each of the treatments quantified for the frequencynuclear and cytoplasmic labelling with mAb 414 (Table 1) were fixe16 hours after sperm addition, a time point which is the hallmark nuclear apposition in bovine zygotes (Navara et al., 1994; Sutovet al., 1996b). Representative oocytes from this group are shownFigs 1F,G and 2B-O. Each experiment was repeated twice. Pexperiments designed to standardize the doses of drugs and to evainitially the effect of the described treatments are not included in Ta1. Similarly, the oocytes shown in Fig. 1, which were fixed at variotime points before/after fertilization, ranging from the germinavesicle stage up to 2-cell embryos, are not included in the calculatioFrequencies of the control and treatment group eggs displaypunctate cytoplasmic, nuclear or both types of mAb414 labelling, no labelling at all, were compared for each of the treatments descri

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Table 1. Effect of various treatments on the frequency of nuclear and cytoplasmic labelling of the nucleoporins withmAb414 16 hours after insemination or parthenogenetic activation

Treatment Cytoplasmic Nuclear Both None Total

CB treated 5 (10.9%) 2 (4.3%) 39 (84.8%) 0 (0%) 46 (100%)CB control 12 (14.8%) 0 (0%) 65 (80.3%) 4 (4.9%) 81 (100%)

Parth. act. 15 (17.0%) 10 (11.4%) 21 (23.9%) 42 (47.7%) 88 (100%)Control fert. 10 (24.4%) 0 (0%) 30 (73.2%) 1 (2.4%) 41 (100%)

+BAPTA 38 (63.3%) 1 (1.7%) 9 (15.0%) 12 (20.0%) 60 (100%)Control 6 (19.4%) 0 (0%) 25 (80.6%) 0 (0%) 31 (100%)

WGA injected* 1 (7.7%) 2 (15.4%) 6 (46.2%) 4 (30.7%) 13 (100%) Sham injected 6 (54.5%) 0 (0%) 4 (36.4%) 1 (28.1%) 11 (100%)

+Nocodazole 36 (67.9%) 1 (1.9%) 3 (5.7%) 13 (24.5%) 53 (100%)Control 15 (24.2%) 0 (0%) 45 (72.6%) 2 (3.2%) 62 (100%)

Oocytes were fixed 16 hours after insemination or parthenogenetic activation with ionomycin/6-DMAP and assessed by indirect immunofluorescence. Only thefertilized oocytes with a MitoTracker-labelled sperm tail in their cytoplasm or parthenogenetically activated oocytes (CB and Parth. act. lines only) were taken intoaccount.

CB, cytochalasin B; Parth. act., parthogenetically activated; Control fert., control fertilized; WGA, wheat germ agglutinin.*Numbers (%) with abnormal pronuclear development in the WGA-injected and sham-injected oocytes were as follows:

Normal PN Aberrant PN

WGA-injected 2 (15.4%) 11 (84.6%) Sham-injected 10 (90.9%) 1 (9.1%)

above and in Results. The diffuse cytoplasmic labelling with mAb41observed in both fertilized and unfertilized oocytes, probably reflethe existence of soluble cytoplasmic pool of antigens recognizedthis antibody and was not taken into consideration in theexperiments. The significance of the differences induced by individual treatments was assessed using the chi-squared (χ2) test.

RESULTS

Assembly and redistribution of annulate lamellaeand nuclear pore complexes during bovinefertilization and early embryonic developmentThe nuclei (germinal vesicles) of fully grown bovine oocytedisplayed a continuous ring of NPC labelling on their NEs ano labelling was found in their cytoplasm (Fig. 1A). Thnuclear labelling disappeared following the resumption oocyte meiosis (germinal vesicle breakdown) and neithcytoplasmic nor nuclear labelling other than diffusbackground-like stain in the cytoplasm was seen in the oocyreaching the metaphase of second meiosis, during whfertilization occurs in bovine (Fig. 1B). A distinct punctatpattern of NPC labelling appeared throughout the ooccytoplasm upon the binding of the fertilizing sperm to thoocyte plasma membrane within 6-8 hours after inseminat(Fig. 1C). The nuclear labeling, accompanied by cytoplasmlabelling, first appeared early after sperm incorporation on surface of the sperm nuclei undergoing the initial swelling (F1D,E), as well as around the decondensing female chrom(Fig. 1F). Enlargement of the PNs and pronuclear appositiseen in most fertilized oocytes within 16 hours aftinsemination, was accompanied by gathering of NPCs in peri-pronuclear region of the fertilized oocytes (Fig. 1G,H). the individual trials, 72.6-80.6% of oocytes fixed 16 hours afinsemination contained both nuclear and cytoplasmic labellwith mAb414 and up to 24.4% of those oocytes containcytoplasmic labelling only (Table 1). After completing

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pronuclear apposition, the chromosomes in both PNs begancondense and the NPCs disappeared approximately 20 hoafter gamete mixing (Fig. 1I). The NPCs reappeared on thsurface of the nuclei in each daughter cell after the first mitotcleavage (Fig. 1J). Interestingly, very little cytoplasmiclabelling with mAb414 was found in two cell embryos (Fig.1J).

At the ultrastructural level (Fig. 2), numerous AL werefound in oocyte cytoplasm 8 hours after insemination, at whicpoint the more or less continuous NE formed on the surface sperm nuclei undergoing initial swelling and decondensatio(Fig. 2A). Concomitantly, the paracrystaline bodies containingthe annulate lamellae (PAL) were found predominantly in thevicinity of the developing male and female PN (Fig. 2B-F)These structures were composed of a central paracrystallcore, from which the annulate lamellae emanated (Fig. 2Band an adjacent symmetrical array of round membrane ciste(Fig. 1C). The decondensation of the maternal and paternchromatin and the formation of the NE around the developinPN were accompanied by the gathering of the PALs, AL, awell as numerous membrane vesicles in the peri-pronucleregion (Fig. 2D). The PALs (Fig. 2E,F) and the stacks of AL(Fig. 2G-I) were often found adjacent to the pronuclear NEIn some cases, the individual sheaths of AL were continuowith the NEs of the growing pronuclei (Fig. 2J-N).Occasionally, the intranuclear AL were observed inside thpronuclei (Fig. 2O).

Effects of sperm incorporation block, Ca 2+ depletion,parthenogenetic activation and WGA injection onpronuclear development and the assembly anddistribution of AL and NPCsThe incorporation of the fertilizing sperm into the oocytecytoplasm, but not oocyte activation, can be blocked bthe addition of the microfilament inhibitor CB in thefertilization medium. One or two parthenogenetic femalepronuclei develop as a result of such sperm-induced oocy

2845N

uclear pore assembly during fertilization

Fig. 1. Dynamics of the nuclear pore complexes (NPC) in fertilized and unfertilizedbovine oocytes, as demonstrated by the triple labelling of a subset of NPC antigens withantibody mAb414 (A-J), and by the DNA stain DAPI (A′-J′), combined with the labellingof the sperm tail mitochondria with a vital probe MitoTracker Green FM (C′-J′; arrows).Oocytes were fixed at the germinal vesicle stage prior to the resumption of first meioticdivision (A,A′), at the second metaphase meiotic arrest (B,B′), at the time of sperm-oolemma binding 8 hours after insemination (C,C′), shortly after sperm incorporation intooocyte cytoplasm (D-E′), at the initial stage of pronuclear development (F,F′), at the time

of pronuclear apposition (G-I′) and after the first embryonic cleavage (J,J′). Diffusecytoplasmic labelling with mAb414 (A,B,F,J) probably reflects the presence of a solublecytoplasmic pool of antigens recognized by this antibody in the cytoplasm of bovineoocytes. The arrow in D points to the ring of NPC around the sperm nucleus undergoingthe initial swelling in oocyte cytoplasm. Arrows in E point to the putative annulatelamellae found in the vicinity of decondensing sperm nuclei shortly after spermincorporation into oocyte cytoplasm. m, male PN; f, female PN; arrows in C′-I′, spermtails. Bars, 10 µm.

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Fig. 2.Ultrastructural characterization of the NPCs and AL infertilized bovine eggs. (A) Annulate lamellae (al) found in thevicinity of the sperm nucleus (n) shortly after the spermincorporation into oocyte cytoplasm. Arrows point to the membranvesicles undergoing fusion on the surface of the demembranatedsperm chromatin. (B) A paracrystalline annulate lamellae-containingbody (PAL) composed of a dense paracrystalline core (asterisk) astack of annulate lamellae (al). (C) Distal end of a PAL featuring aparacrystalline core (asterisk) and an array of elongated cisternae(arrows), shown here in cross sections. (D) Advanced stage ofpronuclear development: annulate lamellae (al), endoplasmicreticulum (er) and Golgi (g) gather around the developing PN (pn);some of the AL (arrows) are continuous with the nuclear envelope(NE). (E,F) Association of PALs with the NE of the developing maand female pronuclei (pn). Note the ribosome-like particles (arrows)decorating the surface of AL. (G-I) Stacks of AL adjacent to the Nand fusing membrane vesicles. Arrows in I point to the ribosome-particles intercalated between AL and NE; pn, pronucleus. (J) Asagittal section of AL (al) associated with the developing pronucleu(pn). (K-N) Apposition and continuity of AL with the NE. Arrowspoint to the fused stretches of AL and NE; pn, pronucleus.(O) Internalization of AL (al) in the pronucleus (pn); ne, nuclearenvelope. Bars, 500 nm (A,C-I,O); 200 nm (B,K-N).

activation (Sutovsky et al., 1996b, 1997a). We used treatment to determine whether sperm-oolemma binding alis sufficient to induce the assembly of AL and the insertionNPC into the NEs of the parthenogenetic female pronuclei. observed both the nuclear NPC signal and the cytoplaslabelling in 84.8% of eggs activated by sperm binding (Ta1), which was similar to the frequency of nuclear-ancytoplasmic labelling in the control (80.3%; Table 1). Thoocytes activated by sperm binding contained one or two femPN with a distinct ring of NPCs on their surface, a loose focof AL in the perinuclear region and a sperm nucleus devoidNPCs on the surface of the oolemma (Fig. 3A,A′). EM studyconfirmed the presence of NPCs on the nuclear envelopethe normal appearance, although somehow smaller in sizeAL and PALs in oocyte cytoplasm (Fig. 3C-E). It also showthat the sperm, bound to the cortex of those oocytes, didbecome incorporated in the oocyte cytoplasm (Fig. 3B).

Ca2+ has been implicated in both oocyte activation (reviewby Schultz and Kopf, 1995) and in the assembly of NPCsNE (Macaulay and Forbes, 1996). Depletion of Ca2+ ions bythe cell-permeant chelator BAPTA-PM did not affect thassembly of AL in the cytoplasm (Fig. 3F,I), but prevented tincorporation of NPCs into NE (Fig. 3G,H). Although thmorphology of AL in the cytoplasm was not substantiaaltered by BAPTA treatment (Fig. 5I-K), their gathering in thperinuclear region did not occur (Fig. 3F). As a result, the PAand AL were found scattered throughout the cytoplasmpronuclear stage-oocytes and often associated with the inface of the oolemma (Fig. 3J,K). This distribution pattern wnot seen in the control fertilized oocytes.

Parthenogenetic activation of bovine oocytes wiionomycin and 6-DMAP (Fig. 3L-O) induced both AL anNPC assembly in 23.8% of activated oocytes (Table 1).47.7% of oocytes, no mAb414 staining could be detectedeither the nuclear or the cytoplasmic compartments. In 11.4the parthenogenetic female pronuclei displayed labelling wmAb414 without any detectable labelling present in tcytoplasm and 17% of parthenogenotes contained cytoplassignal only.

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The lectin wheat germ agglutinin (WGA), which specificallyrecognizes the O-linked sugar moiety of nucleoporins, ispowerful blocking agent for nucleo-cytoplasmic transpo(Finlay et al., 1987; Panté and Aebi, 1996). The injection inoocytes of 20 pl of FITC-conjugated WGA prior to fertilizationprevented normal pronuclear development, normally indicatby the apposition of a large male PN and a smaller female Pin 84.6% of pre-injected, fertilized oocytes (Table 1). WGAtreatment, however, had no effect on the fertilization-induceAL assembly in oocyte cytoplasm (Fig. 4A-B′,D,E) in 53.9%of oocytes and the formation of NPCs on the pronuclear N(Fig. 4C) in 61.6%. Occasionally, the accumulation of denmaterial underneath the NPCs was observed in tnucleoplasm by electron microscopy (Fig. 4C).

Effect of the inhibition of microtubule-based motilityon the dynamics of NPCs and AL, and pronucleardevelopmentWith the exception of some rodents, the rearrangement oocyte cytoplasm and pronuclear apposition in fertilizemammalian oocytes is guided by the sperm aster microtubu(Schatten, 1994). Consequently, the disruption of sperm aswith nocodazole prevents pronuclear development aapposition (Sutovsky et al., 1996b). In the present studnocodazole treatment was employed to explore the role sperm aster microtubules in the assembly of AL and NPduring bovine fertilization. In contrast to the perinuclear focuof AL seen in the control pronuclear oocytes (Fig. 5B), the Ain the oocytes treated with 10 µg/ml of nocodazole werescattered throughout the cytoplasm and often accumulaunderneath the oolemma (Fig. 5A). Only 16.7% of nocodazotreated oocytes displayed pronuclear labelling of NPCs (Tab1) and pronuclear development, measured by the size of mand female PN, was severely impaired by this treatment (F5A). TEM revealed that the sperm centriole failed to nucleamicrotubules in the nocodazole-treated oocytes (Fig. 5C) ano NPCs were found in the NEs of such oocytes (Fig. 5D). Ain those oocytes were of normal appearance (Fig. 5E,F), were rarely found in the peri-pronuclear region. Frequently, tstacks of AL were found attached to the inner face of thoolemma (Fig. 5E) or to the lipid droplets in the oocytcytoplasm (Fig. 5F). The NEs frequently displayed numeronuclear blebs (Fig. 5G-H) and abnormal appearance (Fig. 5Association of the microtubules with PAL was regularly seein control fertilized oocytes (Fig. 5J).

Assembly of nuclear pore complexes around thebull sperm nuclei exposed to Xenopus egg extractsTo lend more support to the data provided by the observatioof fertilized oocytes, the dynamics of NPCs during pronucledevelopment were further explored using interphase Xenopusegg extracts and bull sperm deprived of their plasmmembrane, perinuclear theca and nuclear envelope. At onset of incubation in extracts, the NPCs could not be seeither in the extracts or on the surface of the permeabilizsperm nuclei (Fig. 6A). After 3 hours of incubation, a distincring of NPCs appeared on the surface of the swollen spenuclei and large patches of NPCs formed in the extracts aclumped around the sperm nuclei and axonemes (Fig. 6B). Tpattern of NPC assembly was also visualized in live cells labelling with FITC-conjugated WGA, added to the extract 3

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Fig. 3. Visualization of AL and NPC following oocyte activation.Microfilament disruption with cytochalasin B (A-E) prevented speincorporation, but not the assembly of AL and NPCs. Numerouscytoplasmic AL labelled with mAb414 (A) were assembled arounthe parthenogenetic female pronucleus (f; A′) upon the activation bya sperm nucleus (sn)bound to the surface of this oocyte; arrow,sperm tail mitochondria pre-labelled with MitoTracker prior tofertilization. (B) Failed sperm incorporation during fertilization inthe presence of cytochalasin B. (C) An oddly shaped parthenogefemale PN in the same egg as shown in B; numerous AL were fooutside the PN (arrowheads) as well as inside in the nucleus(arrows). (D,E) Normal AL (D) and PAL (E) were found in theoocytes activated by the sperm in the presence of CB. Depletion Ca2+ ions with BAPTA AM (F-I) prevented the insertion of NPCsinto the pronuclear NEs without impairing the assembly ofcytoplasmic AL and the NE itself. (F,F′) AL became scatteredthroughout the cytoplasm upon the treatment of the fertilized oocwith BAPTA AM; f, female PN; m, male PN; arrow, sperm tailmitochondria. (G) An unusually small female PN in a BAPTA-treated oocyte. (H) Detail of an NE in such an oocyte showing noNPCs. (I-K) Normal AL (I) and PAL (J,K) were found scatteredthroughout the cytoplasm and often associated with the inner leathe oolemma in BAPTA-treated oocytes (J,K); ps, perivitelline space.NPCs (L-O) and DNA (L′-O′) were visualized in the eggs activatedparthenogenetically with ionomycin and 6-DMAP. Some of thoseeggs displayed only diffuse cytoplasmic labelling (L), while otherscontained nuclear (M), cytoplasmic (N) or nuclear-and-cytoplasm(O) dot-like labelling with mAb414. Bars, 5 µm (A′,F′,O′); 2 µm(C,G); 500 nm (B,D,E); 200 nm (H-K).

Fig. 4. Impaired pronuclear development in an egg injected with 2oocyte DNA; lower part, sperm DNA, insert shows both at low macontrol egg (B,B′). Note the difference in PN size between control tail mitochondria. (C) Two NPCs (arrowheads) at the site of accufertilized, WGA-injected oocyte; pn, pronucleus. (D,E) AL found in t5 µm (B), 200 nm (C), 500 nm (D,E).

minutes prior to the end of incubation (Fig. 6C). The additioof 5 µl of the FITC-conjugated WGA at the onset of incubatioappeared to accelerate the assembly of NPCs and thclumping around the sperm nuclei, although the enlargemeof nuclei was less obvious, when compared to the control (F6D). Interestingly, FITC-conjugated WGA can be fixed bformaldehyde and the fluorescence of the NPC-boumolecules was retained even after permeabilization aprocessing for indirect immunofluorescence with mAb41(Fig. 6D) or another antibody (Fig. 6C, anti-β-tubulin). Theresulting double labelling of AL and NPCs yielded the overlaof nucleoporin and WGA signals. Similar to the sperm astmicrotubules in the fertilized eggs, the addition of 10 µMnocodazole into extracts at the onset of incubation partiareduced the assembly of NPCs around the sperm nuclei as was their enlargement after 3 hours of incubation (Fig. 6EFurthermore, the examination of the coverslips witpermeabilized sperm incubated for 3 hours in the extrasupplemented with 5 µl of WGA-FITC and processed forindirect immunofluorescence with anti-tubulin antibody E7demonstrated that the WGA-labelled structures mostcolocalized with the E7-labelled microtubules (Fig. 6F,G). Thassociation became more obvious in the extracts treated w2 µM taxol, a microtubule-stabilizing drug, after 3 hours oincubation (Fig. 6H,I).

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0 pl of nuclear pore antagonist WGA prior to fertilization (A,A′; upper part,gnification), as opposed to normal pronuclear apposition in a sham-injected(B) and WGA-injected (A) oocytes; f, female PN; m, male PN; arrow, spermmulation of the electron-dense material (arrows) in the nucleoplasm of thehe WGA-injected oocytes after in vitro fertilization. Bars, 10 µm (A),

2850P. S

utovsky and others

Fig. 5. Effect of microtubule disruption with nocodazole on the pronuclear developmentand distribution of AL and NPCs. Accumulation of AL in oocyte cortex in a nocodazole-treated fertilized oocyte (A) as opposed to the assembly of AL and NPCs in the peri-pronuclear region of a control oocyte (B). Note the sub-oolemmal labeling with mAb414in A and difference in PN size between A′ and B′; m, male PN; f, female chromatin;arrow, sperm tail mitochondria. A′ and B′ show pronuclei stained with DAPI; male PNare tagged with a MitoTracker-labelled sperm tail (arrows). (C) Absence of microtubulesaround the sperm-contributed centriole-turned zygotic centrosome (asterisk) in a

nocodazole-treated, fertilized oocyte; arrows, remnants of the outer dense fibers of thesperm axoneme. (D) An NPC-free pronuclear NE in such an oocyte. (E,F) Association ofAL with the plasma membrane (E) or lipid droplets (ld; F) in the nocodazole-treatedoocytes; ps, perivitelline space. ‘Blebbing’ of the NE (G,H) and deviant NE formation (I)were frequently seen in the nocodazole-treated, fertilized oocytes. pn, pronucleus.(J) Association of the microtubules (arrowheads) with a PAL in a control fertilizedoocyte. Bars, 25 µm (A), 5 µm (B,D), 10 µm (C), 500 nm (F-L).


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Fig. 6. Reconstitution of the NPC assembly in a cell-free system composed of permeabilized bull sperm and Xenopusegg extracts.(A) Control sperm incubated for 1 hour with WGA (green) and labelled with DAPI (blue) and mAb414 (red). (B) Sperm incubated for 3hours with the egg extracts and labelled with mAb414 (red) and DAPI (blue). Sperm tails were counterstained with MitoTracker Green FM.(C) These sperm were incubated for 3 hours with egg extracts, then labelled for 30 minutes with WGA (green) added to the extracts, fixed andlabelled with anti-tubulin antibody E7 (red) and DAPI (blue). (D) Colocalization of WGA (green) with NPCs (red) in the sperm incubated for3 hours with 5 µg/ml WGA added to the extract, yielded the overlap of WGA and mAb414 labelling (orange); blue DNA stain, DAPI.(E) Sperm incubated for 3 hours with the extracts supplemented with 10 µM nocodazole and labelled with mAb414 (red), MitoTracker GreenFM (green) and DAPI (blue). (F-I) Colocalization of microtubules (red) with the WGA-labelled NPCs (green) in the sperm samples incubatedfor 3 hours with the WGA-containing extract only (F,G), or followed by a 2 hour incubation with WGA extract supplemented with 2 µM taxol(H,I). C,F-I were recorded by a confocal microscope. G and I are enlarged versions of the areas boxed in F and H, respectively. Bars, 10 µm(A), 5 µm (B-F,H), 2 µm (G,I).

DISCUSSION

Intrinsic sperm NE is removed during mammalian fertilizatio(Usui et al., 1997; Sutovsky et al., 1996a, 1997a) in ordemake the sperm chromatin accessible to oocyte cytoplasfactors (Ecklund and Levine, 1975; Krzanovska, 198Perreault et al., 1984; Sutovsky and Schatten, 199Subsequent remodeling of the sperm nucleus requnucleoplasmin, histones, nuclear lamins and probably mother oocyte-produced proteins, which are transported throNPCs (reviewed by Poccia and Collas, 1996). Our stusuggests that the demembranation of the sperm chromduring bovine fertilization is almost immediately followed bthe de novo assembly of pronuclear NE and NPCs, trestricting the free influx of cytoplasmic molecules into thnucleus. NPCs are probably involved in selective control of tpronuclear uptake and their plugging with WGA therefoseverely impairs pronuclear development (this stud

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Furthermore, the disruption of zygotic microtubules bynocodazole or the depletion of the oocyte Ca2+ pool withBAPTA prevents both the gathering of AL in the peri-pronuclear region and the insertion of NPCs into the pronucleNE. All of the above treatments prevent normal pronucleadevelopment, defined as the apposition of a larger male ansmaller female PN in the cytoplasm of a fertilized oocyte.

The structural characteristics of AL have been known foseveral decades and recent research has finally providedinsight into the molecular composition of AL, suggesting thaAL share many nucleoporins with the NPCs and lack laminand some of the membrane components of the NE (Chen aMerisko, 1988; Cordes et al., 1996; Ewald et al., 1996). Icontrast, the role of AL in animal and plant cells has beesubjected to an ongoing debate since there is a lack experimental data supporting any of the proposed functions AL, ranging from the precursor of NPCs to the microtubuleorganizing center (reviewed by Kessel, 1992; Merisko, 1989

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Consequently, most investigators have agreed that AL in mcell types are merely a byproduct of nucleoporin synthesisat most, their cytoplasmic storage (Stafstrom and Staehe1984; Merisko, 1989). Our study suggests that the assembAL is a vital process during mammalian fertilization. Althougnot detected prior to fertilization in bovine, AL are rapidassembled in the oocyte cytoplasm upon oocyte activation (study). The presence of a soluble cytosolic pool of nuclear pcomponents recognized by mAb414 is suggested by the difcytoplasmic labeling of both fertilized and unfertilized oocytin this study. Similarly, nucleoporins migrate from cytosol the membrane fraction and form AL in the activated Xenopusegg extracts (Dabauvalle et al., 1991; Meier et al., 199During bovine fertilization, AL appear first scattered randomthroughout the oocyte cytoplasm, but later become confito the peri-pronuclear region. This fertilization-inducereorganization may be a process restricted to the AL,perhaps part of a more general reorganization of cytoplasmembranous structures including all subtypes of Epreviously shown in sea urchin eggs (Terasaki and Jaffe, 19Association of AL with NE in fertilized bovine oocytesuggests that they may be involved in the assembly of theand in the incorporation of NPCs in it. This is also supporby our finding that neither NPCs are inserted into pronuclNE, nor does normal PN development occur when gathering of AL around PN is prevented by nocodaztreatment. At this moment, however, we cannot rule out possibility that AL are only/also involved in the removal oNPCs from pronuclear NE prior to entry of an embryo into fimitotic division. In vivo studies using fluorescent fusioproteins will be necessary to determine the exact role of ALthis process.

Formation of the microtubule sperm aster around the speborne centriole in the oocyte cytoplasm appears to placentral role in pronuclear apposition (Schatten, 1994), yet liis known about the involvement of sperm aster microtubulethe recruitment of organelles and molecules into pronuclregion. Our data on nocodazole treatments and colocalizaof AL with microtubules in cell-free extracts and bovinoocytes suggest that the AL may be pulled along the spaster microtubules towards the pronuclei and thus bring nucleoporins or preassembled NPCs into the peri-pronucregion. Before studying bovine fertilization, we observed tgathering of AL and PALs in the peri-pronuclear region fertilized rhesus oocytes (Sutovsky et al., 1996a). Szöllosal. (1996) observed the association of microtubules with ‘striated rootlets’, which appear to be identical to bovine Aand PALs, in rabbit zygotes, and attributed to them the funcof a microtubule-organizing center. Although this may not the case, the association of AL with microtubules seems towell established (reviewed by Kessel, 1992). The dependeof AL on a microtubule-based transport mechanism may aexplain why Ca2+ depletion with BAPTA causes AL to bescattered throughout the oocyte cytoplasm and preventsinsertion of NPCs into the pronuclear NEs at the same ticalcium plays a central role in the activation of the zygocentrosome and microtubule polymerization durinfertilization (Schatten, 1994). It is also of interest to note high incidence of nuclear blebbing in nocodazole-treafertilized eggs. Szöllosi and Szöllosi (1988) suggested tnuclear blebbing may be an alternative pathway for nucl

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cytoplasmic transport. It is not ruled out that some of the nospecific effects of nocodazole may contribute to the alteremembrane dynamics in the fertilized eggs.

Ca2+ oscillations are one of the main paradigms omammalian fertilization, where they appear to be triggered bthe release of a sperm-borne inductor peptide into the ooccytoplasm (Parrington et al., 1996; Sette et al., 1997). Oocyactivation can be divided into several relatively independeprocesses, including the release of intrinsic calcium frocytoplasmic stores in oocyte ER, resumption of the cell cycand the exocytosis of cortical granules (reviewed by Schuland Kopf, 1995). The assembly of AL and NPCs can now badded to the list of typical features of oocyte activation. Amuch as Ca2+ ions appear to be a ubiquitous messengemolecule during oocyte activation, some of these events aless sensitive to Ca2+ than others. For instance, corticalgranule exocytosis and the recruitment of maternal mRNAcan be stimulated by Ca2+ injections although pronuclearformation and histone H1-kinase activity, reflecting that oMPF, are relatively independent of Ca2+ concentrations inmouse eggs (Xu et al., 1996). Our study demonstrates thCa2+ depletion with BAPTA-AM does not affect the assemblyof AL in bovine oocytes, although it does block therecruitment of NPCs to the pronuclear NE. This is not likelto be due to the effect of Ca2+ depletion on the formation ofpronuclear NE: the BAPTA-treated eggs contain PN witcontinuous NEs (this study), and the depletion of Ca2+ ionsonly affects the processes downstream of membrane vesifusion during NE formation in vitro (Macaulay and Forbes1996). In contrast, calcium chelation may affect the dynamiof microtubules or the activity of the zygotic centrosome, suggestion consistent with the scattering of AL throughout thcytoplasm of the BAPTA-treated oocytes. Although Ca2+ ionsappear to be indispensable during the insertion of NPCs inNE (Macaulay and Forbes, 1996), they may not be necessfor the assembly of AL.

Our previous studies showed that a substantial portion human eggs, fertilized in vitro for the purposes of infertilitytreatment, do not develop normal male and/or female Pfollowing the successful penetration of the zona pellucida anincorporation into the oocyte cytoplasm (Asch et al., 1995Simerly et al., 1995). This pattern of fertilization failure ismirrored by in vitro fertilization and intracytoplasmic sperminjection (ICSI) of rhesus monkey oocytes (Hewitson et al1996; Sutovsky et al., 1996a). In the latter study, we observonly a limited, if any, number of NPCs being inserted in thmale pronuclear NEs in some of the ICSI-fertilized rhesueggs, which was in sharp contrast to the large number of PAand AL found in their cytoplasm. Therefore it seems feasibto speculate that failure of these sperm to attract nucleoporand/or preassembled NPCs from the cytoplasm of activatoocytes may cause some of the fertilization arrests observedprimates. On the other hand, it would be interesting to assethe presence and availability of the cytoplasmic pool of nuclepores and nucleoporins in the eggs of female patienundergoing infertility treatment.

In conclusion, the present study documents the importanof NPCs for normal pronuclear development duringmammalian fertilization. It also implies that calcium ionssperm aster microtubules and possibly the AL, are involved the regulation of NPC turnover during this process.


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Our thanks belong to Dr Brian Miller of the University oCalifornia, San Diego for inspiring discussion and provision antibody samples, and to Drs Tanja Dominko and João RamaSantos for the stimulating discussions and manuscript readAdministrative and technical assistance of Michelle Emme, Dr AnCornea, Carol Gabel, Randal Massey, Diana Myers, Dr Grayson SDiana Takahashi, Michael Webb and Kari Weber, the use of Electron Microscopy Facilities of the University of WisconsinMadison, and of the Oregon Regional Primate Research Ce(ORPRC), Beaverton, OR, and the use of the Confocal MicroscoLaboratory of the ORPRC Beaverton, OR, are gratefuacknowledged. Bovine sperm samples were provided by AmeriBreeders Service, DeForest, WI. This work was supported by grafrom NIH and USDA to G. S. The ORPRC is funded as an NRegional Primate Research Center (RR00163). P. S. was in supported by the fellowship No. 5FO5 TWO5183-02 from the FogaInternational Center, NIH.

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assembly of nuclear pore complexes and annulate lamellae

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