Review

Ultrastructural aspects of mammalian fertilization:new discoveries and inspirations from the work

of Daniel Szöllösi

Peter Sutovsky, Gerald Schatten

Departments of Obstetrics and Gynecology, and Cell and Developmental Biology,Oregon Health Sciences University, and the Oregon Regional Primate Research Center,

505 N.W. 185th Avenue, Beaverton, OR 97006, USA

(Received I October 1998; accepted 14 October 1998)

Abstract - Our current level of knowledge on mammalian fertilization would not be attained with-out the contribution of ultrastructural studies. The late Daniel Szbii6si, to whose memory this reviewis dedicated, was one of the most enthusiastic explorers of this fascinating field. In his landmarkelectron microscopic studies, he revealed the importance of nuclear envelope breakdown for oocytematuration and its reconstitution after fertilization, and predicted the era of cloning by publishing arti-cles on the remodeling of a somatic cell, thymocyte nucleus fused with an oocyte. His challenge ofBoveri’s hypothesis on the paternal inheritance of centrosomes spurred further research on this sub-ject that ultimately led to the definition of biparentally contributed mammalian zygotic centrosomes,for which the only exception is found in rodents. Very early, Sz6llbsi and his colleagues devoted theirinterest to the studies of the fate of sperm accessory structures after fertilization, an area that hasyet to be explored at the molecular level, but which may have profound implications for the swiftlyadvancing field of assisted human and animal reproduction. These studies contributed a great deal toour current understanding of mammalian fertilization and still serve as an inspiration for presentstudies on involved mechanisms. © Inra/Elsevier, Paris.

mammals / fertilization / oocyte maturation / nuclear transfer / ultrastructure

Résumé ― Aspects ultrastucturaux de la fécondation chez les Mammifères : nouvelles décou-vertes suscitées par l’oeuvre de Daniel Szdlltisi. Les connaissances actuelles sur la fécondationchez les Mammifères n’auraient pas été obtenues sans d’études ultrastructurales. Le regretté DanielSzollosi, à la mémoire duquel cette revue est dédiée, était un des explorateurs les plus enthousiastesdans ce domaine. Dans ses études marquantes de microscopie électronique, il a révélé l’importancede la rupture de l’enveloppe nucléaire pour la maturation de l’ovocyte et sa reconstitution aprèsfécondation ; il a annoncé l’ère du clonage en publiant des articles sur le remodelage du noyau d’unecellule somatique, le thymocyte, fusionné avec un ovocyte. En défiant l’hypothèse classique du

* Correspondence and reprintsE-mail: schatten@ohsu.edu

Boveri de l’héritage paternel du centrosome, il inspira de nouvelles recherches sur le sujet qui condui-sirent à la mise en évidence de l’origine biparentale du centrosome zygotique, à l’exception des ron-geurs. Très tôt, Szôllôsi et ses collègues se sont intéressés au devenir de structures accessoires des sper-matozoïdes après la fécondation chez les mammifères et sont toujours une source d’inspiration pourdes recherches sur les mécanismes impliqués. © Inra/Elsevier, Paris.

mammifères / fécondation / maturation ovocytaire / transfert nucléaire / ultrastructure

1. INTRODUCTION

In contrast to the widespread general per-ception, the paternal contributions to thefunctional zygote are not limited to intro-

ducing one half of the future embryonicgenome into oocyte cytoplasm. Several othersperm structures, including sperm mito-chondria, perinuclear theca (PT) and areduced form of the sperm centrosome, are

brought into oocyte cytoplasm where theyparticipate in the organization of zygoticdevelopment. Since the sperm cytoplasm isalmost completely eliminated during sper-miation and only certain types of cellularorganelles are retained during spermatoge-nesis, the mammalian zygote relies on theinteractions between these sperm-borne ele-ments and rich oocyte pools of organellesand molecules for its developmental suc-cess. The extensive body of work publishedby the late Daniel Sz6ll6si was propheticalin that it pointed out to many structuralevents of mammalian fertilization longbefore the molecular tools for their charac-terization became available. In this review,the new research aimed at reevaluating theimportance of these early developmentalevents for natural and assisted fertilizationand early embryogenesis, is reviewedtogether with the original works that inspiredit. The recent revival of the techniques ofnuclear transfer of somatic and germ cellselicited important questions and concernsregarding the introduction of heterogeneouscellular organelles into such in vitro recon-stituted zygotes and is likely to bring thefocus back on research of paternally inher-ited zygotic structures. It is our great plea-

sure and honor to dedicate this paper to the

bright memory of Dr Daniel Sz6ll6si, a trulyexceptional man, whose body of work willserve as an inspiration for many future gen-erations of developmental and reproductivebiologists.

2. CENTROSOME INHERITANCEAT FERTILIZATION:MATERNAL MODE IN RODENTSVERSUS BIPARENTALCONTRIBUTIONS TOTHE ZYGOTIC CENTROSOMEIN OTHER MAMMALS

Boveri’s [5] hypothesis on the paternalinheritance of centrosome during fertiliza-tion was given substantial support by Sz6l-16si’s discovery of the paucity of centriolesin unfertilized mammalian oocytes [86].Using pig zygotes, Sz6ll6si and Hunter [84]were among the first investigators to pointout that the fertilizing mammalian sperma-tozoon introduces the centriole into oocytecytoplasm. Although Sz6ll6si [83] inter-preted these findings as a loss of the centri-ole during fertilization, it later became evi-dent that during gametogenesis, sperm andoocyte organelles are reduced in a comple-mentary fashion to prevent the redundancyof cellular organelles participating in earlyembryonic development. Although oocytecytoplasm is retained and even amplifiedduring oogenesis, its microtubule organizingcenter, the centrosome composed of twocylindrical centrioles and pericentriolarmaterial, disappears completely. Conse-quently, the spindle poles are acentriolar

during first and second oocyte meiosis [87].Of the two centrioles seen in mammalian

spermatogenic cells before spermiation, thedistal centriole disappears after giving rise tothe sperm axoneme, although the proximalcentriole is retained and remains intact inthe capitulum of the sperm tail connectingpiece [16, 56]. After gamete fusion, thisproximal centriole is released from oocytecytoplasm [43, 76, 77] and becomes sur-rounded by the oocyte produced, y-tubulin-rich pericentriolar material [ 18, 30, 73, 76],in which the ability to nucleate microtubulesresides [21]. Subsequently, the radial arrayof microtubules, sperm aster, is organizedaround the sperm centriole from tubulinsand centrosomal proteins. The centrioleinside this zygotic centrosome later dupli-cates and both the mother and daughter cen-trioles give rise to one spindle pole each.Thus, the sperm-borne centriole is neces-sary for the organization of microtubulesduring fertilization and first embryoniccleavage, which also requires centrosomalproteins from the oocyte cytoplasm. Suchbiparental mode of centrosomal inheritancewas described in the rabbit [59], humans[63, 64, 69], rhesus [24, 76, 104], bovine[43, 77], sheep [11, 37], pig [32] and com-mon marsupial Monodelphis domestica [6].An alternative mode of centrosomal inher-itance was discovered in the mouse, whichrelies exclusively on the maternal micro-tubule-organizing elements [66, 67]. Bothcentrioles are degraded during spermiogen-esis in the mouse [41] and rat [103], andmultiple acentriolar microtubule organiz-ing centers, entirely derived from oocytecytoplasm, control the pronuclear apposi-tion and mitotic spindle formation in themouse [66, 67] and hamster [25].

Even though the sperm centriole is nec-essary for syngamy and first mitosis in mam-mals with paternally contributed centro-somes, the ability of their oocytes toorganize microtubules independently hasbeen retained during evolution: partheno-genetically activated non-rodent mammalianoocytes organize mitotic spindles with acen-

triolar pores [32, 43, 58, 85]. Sz6ll6si andOzil [85] demonstrated the de novo forma-tion of centrioles in the blastocysts obtainedby parthenogenetic activation of rabbitoocytes. Although parthenogenetic activa-tion may generate blastocysts, this devel-opmental mode is not full term in mammals.

The sperm axoneme that houses the cen-triole is a complex structure composed of a9 + 1 arrangement of microtubule doublets,paralleled by nine outer dense fibers (ODF)in the axonemal principal piece and midpiece. In the connecting piece, which servesfor the attachment of the axoneme to thebasal plate of the sperm nucleus, the ODFare transformed into compositionally simi-lar, yet morphologically distinct striatedcolumns, caging the capitulum-embeddedproximal centriole. Both the connectingpiece and mid piece are wrapped in a helixof sperm mitochondria (for reviews, see [16,56]). During fertilization, the uppermostmitochondria are removed from the mito-chondrial sheath, thus unmasking the con-necting piece columns that are subsequentlyexcised from the sperm nucleus, and even-tually dismantled [76, 77]. This event leadsto the release of the sperm centriole in the

zygotic cytoplasm and to its transformationinto an active zygotic centrosome [37, 76,77]. Such disassembly of the connectingpiece during fertilization appears to be facil-itated by the removal of disulfide bondcross-linking from sperm proteins by oocyte-produced tripeptide glutathione [75]. Phos-phorylation/dephosphorylation events [38,59] and calcium binding by centrosomalproteins such as centrin [17, 61] may alsocontribute to this process.

3. DENUDATIONAND REMODELING OFTHE SPERM NUCLEUS INTOA MALE PRONUCLEUS

The primary binding event between thespermatozoon and an egg involves a disin-tegrin-type receptor, fertilin, on the sperm

plasma membrane and an integrin mem-brane receptor on the oolemma [2, 7, 15].Other molecules are likely to participate insperm egg fusion and provide either the sup-port for, or an alternative to the integrin-fer-tilin-mediated gamete fusion. The spermplasma membrane appears to interminglewith the oolemma during sperm-oocytefusion [39, 70]. In addition, oocyte microvillibind to perinuclear theca (PT; [10, 48-50])a cytoskeletal coat intercalated between thesperm’s plasma membrane and nuclearenvelope [78]. Subsequently, the sperma-tozoon is dragged into and engulfed by theoocyte cytoplasm and PT is removed anddissolved in it [78]. Binding of the oocytemicrovilli to PT may involve specific recep-tors on both sides, making this interaction acandidate for a gamete fusion event syner-gistic with the integrin-fertilin binding. Bullsperm with an intact PT, injected into oocytecytoplasm, does not develop into male PN[78], suggesting that the removal of PT isa vital step in the remodeling of the spermnucleus into a male PN. Earlier studiesdemonstrated the dispersion of PT in thecytoplasm of rodent oocytes [74, 83, 91,95-97]. Evidence is growing that PT har-bors the oocyte-activating oscillogenic fac-tors [4, 53] that are released into oocytecytoplasm when PT is incorporated and dis-solved [35, Sutovsky et al., unpublishedreport]. Transcriptional factor Stat4 has beenfound in murine PT [23], but the signifi-cance of this association to fertilization and

early development is not known.

The intrinsic nuclear envelope (NE) ofsperm disappears shortly after the removalof PT and sperm entry into oocyte cyto-

plasm [81, 98], suggesting that sperm NEor any other NE introduced into metaphaseII (Mll) oocytes is not compatible with highmaturation promoting factor (MPF) activ-ity in the oocytes finishing second meiosis.In line with this suggestion, Sz6ll6si et al.[93] showed NE breakdown in the red bloodcells injected into mouse oocytes within25-45 min after activation, whereas thenuclei introduced into mouse oocytes 1-7 hafter activation retained their intrinsic NE.

Following the removal of the sperm NE, thesperm nucleus is remodeled into a male

pronucleus by the action of oocyte cyto-plasm [54, 75]. The disulfide bonds in thesperm deoxyribonucleic acid (DNA)-pack-ing proteins protamines are reduced by theaction of the oocyte-generated reducingtripeptide glutathione (GSH), then removedand replaced by the oocyte-derived histones[14, 36]. The nuclear envelope is recon-structed around the decondensing spermnucleus from the oocyte-derived membranevesicles [60, 75, 81, 98]. This step in fertil-ization marks the formation of nuclear and

cytoplasmic compartments in the fertilizedoocyte and raises the question of when andhow do these two compartments communi-cate and exchange the molecules necessaryfor normal pronuclear and embryonic devel-opment. The main channel for the nucleo-cytoplasmic transport in the animal cells isthe nuclear pore complex (NPC; reviewedby Pant6 and Aebi [51]), an assembly of theO-glycosylated proteins from the nucleo-porin family. The reconstitution of the NEand the assembly of NPCs in Xenopus eggextracts was very instrumental in dissect-

ing the pathways leading to the assembly

of functional NE [40, 42, 44, 55, 60, 105].Little is known, however, about the signif-icance and timing of this process duringmammalian fertilization. Our recent stud-ies [81] in bovine suggest that NPCs areinserted into pronuclear NE at the initialstage of pronuclear development and pro-vide a vital link between the cytoplasm andthe pronuclei. Cytoplasmic stacks of NPCs,called annulate lamellae (AL), are assem-bled in oocytes activated by fertilizing sper-matozoon or by parthenogenetic stimulus[81]. AL, observed in the zygotes of mostmammalian species studied up to now [76,84, 92, 99], may be involved in the turnoverof NPCs throughout pronuclear develop-ment [81]. This NPC turnover is probablydirected by the zygotic centrosome andsperm aster microtubules that are associatedwith AL [81, 91 ]. Disruption of sperm astermicrotubules with nocodazole prevents the

assembly of NPCs at the pronuclearenvelopes, blocks pronuclear developmentand apposition [81] and induces the bleb-bing of the outer leaf of pronuclear NE.Szollosi and Sz6llbsi [86] suggested thatnuclear blebbing, an evagination of the NEcontaining granular material that was firstdescribed in mouse zygotes, is an alterna-tive pathway of nucleo-cytoplasmic trans-port in mammalian zygotes, yet this hypoth-esis remains to be confirmed.

4. ELIMINATION OF SPERMMITOCHONDRIA AND SPERMTAIL AFTER FERTILIZATION

Considering that the fertilizing mam-malian spermatozoon introduces almost 100

functional mitochondria into the cytoplasmof fertilized oocytes, the strictly maternalmode of mitochondrial DNA (mtDNA)inheritance in mammals [27] is one of themost tantalizing paradoxes of developmen-tal biology. Early observations suggestingthat the sperm mitochondria disperse acrossoocyte cytoplasm before first embryoniccleavage [22] did little to explain such a dis-proportion. Later, the ’dilution’ of paternalmitochondrial genome was taken intoaccount [3]. Sz6ll6si [82] suggested for thefirst time that sperm mitochondria are tar-

geted for destruction in oocyte cytoplasm.This view was later supported by furthermorphological [ 12, 26, 68, 72, 77] andgenetic [27, 31] studies. We conducted astudy of bovine oocytes fertilized with abull sperm that was preincubated with avital, fixable, mitochondrion-specific probeMitoTracker Green FM (Molecular ProbesInc., Eugene, OR). In these studies, thesperm mitochondrial sheath remained com-

pact throughout pronuclear developmentand traveled to one of the blastomeres dur-

ing first and second mitosis. The step-wisedestruction of the mitochondrial sheath

appeared to take place during mitotic divi-sion, suggesting a dependence on theembryonic cell cycle [77]. Accordingly, thezygotes that became arrested in the pronu-clear stage contained an intact mitochon-drial sheath event after the end of the culture

period, during which other zygotes reachedfour-cell stages and were deprived of spermmitochondria. Almost identical results werelater obtained by Cummins et al. [12], whoinjected the MitoTracker-tagged mousesperm tails into cytoplasm of mature mouseoocytes. Allen [1] suggested that sperm

mitochondria may suffer extensive oxida-tive stress and damage during the passagethrough the female reproductive tract, and assuch are eliminated by a mechanism thatrecognizes damaged mitochondria of anyorigin present in oocyte cytoplasm ratherthan by a mechanism specifically targetingpaternal mitochondria. This theory, how-ever, falls short of explaining why the mito-chondria of Mus spretus sperm are not

destroyed in the cytoplasm of Mus rreusculusoocytes in the interspecies mouse crosses[31]. Based on our observation, we predictedthe involvement in the destruction of pater-nal mitochondria of the protein scavengerubiquitin [77], an 8.5 kDa protein thatprompts metaphase-anaphase transition bydestroying the cyclin component of MPFduring meiosis and mitosis (reviewed byPines [57]). We recently confirmed thishypothesis by observing conjugation ofubiquitin to the sperm mitochondrial sheathin bovine one-, two- and four-cell embryos[79]. It appears that certain proteins of spermmitochondria are tagged with ubiquitin dur-ing spermatogenesis and immediately rec-ognized by intrinsic ubiquitin of oocyte cyto-plasm, which mediates the targeting ofsperm mitochondria towards lysosomesand/or proteasomes. The association ofoocyte-derived lysosomes and multivesic-ulated bodies with the sperm mitochondrial

sheath, previously shown by transmissionelectron microscopy [26, 82, 83] can bevisualized by labeling live oocytes fertil-ized with MitoTracker-tagged sperm by avital lysosomal probe LysoTracker [79].These observations may explain how thesuppression of the paternal mitochondrialgenome is achieved during mammaliandevelopment. Species-specificity of spermmitochondrion elimination [31] may beexplained by differences in the amino acidsequence of the individual constituents of

ubiquitin conjugation machinery and by theinability of the oocyte cytoplasm to recog-nize the proteins of the outer mitochondrialmembrane from a foreign species.

Other sperm tail accessory structures,

including fibrous sheath, outer dense fibersand microtubule doublets are eliminated byoocyte cytoplasm at various stages ofembryonic development. Although the FSdisappears within a few hours after thesperm is incorporated into bovine oocyte[77], the ODF and axonemal microtubulesdegenerate slowly and are still seen at thetwo-cell stage [26, 68, 76, 77].

5. EFFECT OF PATERNALLYCONTRIBUTED ZYGOTICCOMPONENTS ON THE OUTCOMEOF ASSISTED FERTILIZATION

Mammalian and human oocytes can be

successfully fertilized and brought to termby the intracytoplasmic injection of a maturespermatozoon (ICSI; [29, 52, 100]), or bythe injection or fusion of an immature sper-matogenic cell such as primary [47, 62] orsecondary [33] spermatocyte, or a roundspermatid [45, 46]. Consequently, injectionsof mature spermatozoa and elongated orround spermatids are now used to treathuman infertility [ 19, 52, 94]. Normal devel-opment was achieved using the injection orfusion of spermatogenic cells with theoocytes in the mouse [33, 46, 62], hamster[45] and rabbit [71]. In addition to germcells, nuclear transfer of somatic cells iso-lated from fetal, juvenile or even adult tis-sues resulted in the production of live off-spring [8, 9, 101, 102]. Predicted by thearticles on the reprogramming of thymocytenuclei in oocyte cytoplasm [13, 88-90], theidea of cloning from somatic cells, or at leastof using it for nuclear transfer has beenaround for more than a decade. However,the overall success rate of methods such astransfer of nuclei of somatic cells remains

very low. Since the injection of isolatedsomatic cell nuclei gave similarly low suc-cess rates [102], one possible reason is thecollision of organelles and molecules fromthe donor cell with those of the recipientooplasm after fusion. For instance, it is not

known whether the foreign, i.e. somatic (orimmature germ cell) mitochondria, broughtinto oocyte cytoplasm by nuclear transferor intracytoplasmic injection, are eliminatedby the oocyte in a manner identical to that ofnatural fertilization. The persistence of twodifferent mitochondrial genomes in the cyto-plasm of a single cell, a condition referred toas heteroplasmy, may interfere with thedevelopment of such embryos and result insevere anomalies. Similarly, sperm acces-sory structures introduced into an oocyte byICSI or round spermatid injection (ROSI)may collide with normal zygotic develop-ment. It was shown previously that the acro-some and subacrosomal layer of perinucleartheca often persist on the surface of rhesussperm injected into mature rhesus oocytes,and cause a heterogeneous decondensationof sperm chromatin and abnormal pronu-clear development [24, 76]. The persistenceof subacrosomal PT was recently shown inhuman ICSI zygotes [65] and may explainthe higher rate of chromosomal abnormali-ties seen in human fetuses conceived byICSI [20, 28], which may be mainly due tochromosome anomaly of the patient, as com-pared to those from conventional in vitrofertilization. In contrast to a mature humanand primate spermatozoon carrying a sin-gle, proximal centriole destined to organizethe sperm aster after fertilization [24, 69,76], the round spermatid contains an addi-tional, distal centriole, which appears to beinvolved in the formation of sperm axoneme

[80]. It is not clear whether either of thesecentrioles is competent to organize the spermaster after ROSI, and whether the additionalcentriole present in the ROSI-conceived

zygote does not interfere with pronucleardevelopment and formation of the firstmitotic spindle. More research is necessaryto address these important questions relatedto assisted fertilization.

6. CONCLUSION

The ultrastructural studies of Sz6ll6si andothers demonstrated that the fate of various

sperm accessory structures after fertiliza-tion is precisely determined by their inter-actions with the oocyte. In the past fewyears, the use of molecular tools for fertil-ization studies culminated in a string of newdiscoveries, including that of the biparent-ally contributed centrosome in the zygotes ofnon-rodent mammals and the eliminationof sperm mitochondria by an ubiquitin-dependent proteolytic pathway duringpreimplantation development. Perinucleartheca, once thought to play a marginal, ifany role in fertilization, now appears to con-tribute the oocyte activating factor, whichassures the initiation of the embryonic cellcycle and the activation of anti-polyspermydefense. The reduction of oocyte centro-somes and the ability of oocyte cytoplasm toremodel the nucleus of a somatic cell, pre-viously described by Szoll6si and his col-leagues, appear to assure the developmentalsuccess of embryos procreated by assistedfertilization methods such as ICSI, ROSIand nuclear transfer. It is necessary to

emphasize, however, that the remodeling ofgametes after assisted fertilization may notnecessarily mirror that seen during naturalfertilization. Focused research into molecularand cellular mechanisms of assisted fertil-

ization, using relevant animal models, isnecessary to substantiate their clinical use.

ACKNOWLEDGEMENTS

We are pleased to acknowledge our colleaguesand collaborators for stimulating discussions andfor sharing their unpublished data: Dr A. Chan,Dr T. Dominko, Dr L. Hewitson, Dr M. Luet-jens, Dr G. Manandhar, Dr R. Moreno, DrR. Oko, Dr J. Ramalho-Santos, Dr C. Simerlyand Dr Y. Terada. The technical and clericalassistance of Ms M. Emme, Ms C. Martinovich,Ms D. Takahashi, Mr M. Webb, and staff mem-bers of the Oregon Regional Primate ResearchCenter, Beaverton, OR, is sincerely appreciated.Special thanks belong to Dr J.E. Fléchon forinspiring this review and to Dr C. Thibault forreading the manuscript. Original researchreviewed in parts of this paper was supported bygrants from NIH and USDA to G.S.

REFERENCES

[ 1 ] Allen J.F., Separate sexes and the mitochondrialtheory of ageing, J. Theor. Biol. 180 (1996)135-140.

[2] Almeida E.A.C., Huovila A.-P.J., Sutherland A.E.,Stephens L.E., Calarco P.G., Shaw L.M., Mer-curio A.M., Sonnenberg A., Primakoff P.,Myles D.G., White J.M., Mouse egg integrin(x6pi functions as a sperm receptor, Cell 81 1(1995) 1095-1104.

[3] Ankel-Simons F., Cummins J.M., Misconcep-tions about mitochondria and mammalian fer-tilization: implications for theories on humanevolution, Proc. Natl. Acad. Sci. USA 93 ( 1996)13859-13863.

[4] Battaglia D.E., Koehler J.K., Klein N.A.,Tucker M.J., Failure of oocyte activation afterintracytoplasmic sperm injection using round-headed sperm, Fertil. Steril. 68 (1997) 118-122.

[5] Boveri T., Zellen-studien: Ueber die Natur derCentrosomen, IV Fisher, Jena, Germany, 1901. 1 .

[6] Breed W., Simerly C., Navara C.S., Vanderberg J.,Schatten G., Distribution of microtubules ineggs and early embryos of the marsupial, Mon-odelphis domestica, Dev. Biol. 164 (1994) 230-240.

[7] Bronson R.A., Fusi F.M., Integrins and humanreproduction, Mol. Hum. Rep. 2 (1996) 153-168.

[8] Campbell K.H., Loi P., Cappai P., Wilmut J.,Improved development to blastocyst of ovinenuclear transfer embryos reconstructed duringthe presumptive S-phase of enucleated activatedoocytes, Biol. Reprod. 50 (1994) 1385-1393.

[9] Campbell K., McWhir J., Ritchie W., Wilmut J.,Sheep cloned by nuclear transfer from a cul-tured cell line, Nature 380 (1996) 64-66.

[ 10] Courtens J.L., Courot M., Flechon J.E., The per-inuclear substance of boar, bull, ram and rabbitspermatozoa, J. Ultrastruct. Res. 57 (1976) 54-64.

[11] ] Crozet N., Behavior of sperm centriole duringsheep fertilization, Eur. J. Cell Biol. 53 (1990)326-332.

[ 12] Cummins J.M., Wakayma T., Yanagimachi R.,Fate of microinjected sperm components in themouse oocyte and embryo, Zygote 5 (1997)301-308.

[13] Czolowska R., Modlinski J.A., Tarkowski A.K.,Behaviour of thymocyte nuclei in non-activatedand activated mouse oocytes, J. Cell Sci. 69(1984) 19-34.

[14] Ecklund P.S., Levine L., Mouse sperm basicnuclear protein. Electrophoretic characteriza-tion and fate after fertilization, J. Cell Biol. 66(1975)251-262.

[IS] Evans J.P., Schultz R.M., Kopf G.S., Mousesperm-egg plasma membrane interactions: anal-ysis of roles of egg integrins and the mousesperm homologue of PH-30 (fertilin) (3, J. CellSci. 108 (1995) 3267-3278.

[ 16] Fawcett D.W., The mammalian spermatozoon,Dev. Biol. 44 (1975) 394-436.

[ 17J Fechter J., Schbneberg A., Schatten G., Theexcision and disassembly of sperm tail micro-tubules during sea urchin fertilization: require-ments for microtubule dynamics, Cell Motil.Cytoskel. 35 (1996) 281-288.

[ 18] Felix M., Antony C., Write M., Maro B., Cen-trosome assembly in vitro: role of y-tubulinrecruitment in Xenopus sperm aster formation,J. Cell Biol. 124 (1994) 19-31. 1.

[ 19] Fishel S., Green S., Bishop M., Pregnancy afterintracytoplasmic injection of spermatid, Lancet345 (1995) 1641-1642.

[20] Flaherty S.P., Payne D., Swann N., Mathews D.,Aetiology of failed and abnormal fertilizationafter intracytoplasmic sperm injection, Hum.Reprod. 10 (1995) 2623-2629.

[21 Gould R.R., Borisy G.G., The pericentriolarmaterial in Chinese hamster ovary cells nucleatesmicrotubule formation, J. Cell Biol. 73 ( 1977)601-615.

[22] Gresson R.A.R., Presence of the sperm middle-piece in the fertilized egg of the mouse (Musmusculus), Nature 145 ( 1940) 425.

[23] Herrada G., Wolgemuth D.J., The mouse tran-scription factor Stat4 is expressed in haploidmale germ cells and is present in the perinucleartheca of spermatozoa, J. Cell Sci. 110 (1997)1543-1553.

[24] Hewitson L.C., Simerly C.R., Tengowski M.W.,Sutovsky P., Navara C.S., Haavisto A.J., Schat-ten G., Microtubule and DNA configurationsduring Rhesus intracytoplasmic sperm injec-tion: successes and failures, Biol. Reprod. 55(1996)271-280.

[25] Hewitson L.C., Haavisto A., Simerly C., Jones J.,Schatten G., Microtubule organisation and chro-matin configurations in hamster oocytes duringfertilization and parthenogenetic activation, andafter insemination with human sperm, Biol.

Reprod. 57 (1997) 967-975.[26] Hiraoka J., Hirao Y., Fate of sperm tail compo-

nents after incorporation into the hamster egg,Gamete Res. 19 (1988) 369-380.

[271 Hutchinson C.A., Newbold J.E., Potter S.S.,Edge] M.M., Maternal inheritance of mam-malian mitochondrial DNA, Nature 251 ( 1974)536-538.

[28] In’t Veld P., Brandenburg H., Verhoeff A.,Dhont. M., Los F., Sex chromosomal abnor-malities and intracytoplasmic sperm injection,Lancet 773 (1995) 346.

[29J Iritani A., Micromanipulation of gametes for invitro assisted fertilization, Mol. Reprod. Dev.28 (1991) I 99-207.

[30] Joshi H.C., !y-tubulin: the hub of cellular micro-tubule assemblies, BioEssays 15 (1993) 637-643.

[ 3 1] Kaneda H., Hayashi J.-L, Takahama S., Taya C.,Fischer-Lindahl K., Yonekawa H., Eliminationof paternal mitochondrial DNA in intraspecificcrosses during early mouse embryogenesis, Proc.Natl. Acad. Sci. USA 92 (1995) 4542-4546.

[32] Kim N.-H., Simerly C., Funahashi H., Schatten G.,Day B.N., Microtubule organization in porcineoocytes during fertilization and parthenogenesis,Biol. Reprod. 54 (1996) 1397-1404.

[33] Kimura Y., Yanagimachi R., Development ofnormal mice from oocytes injected with sec-ondary spermatocyte nuclei, Biol. Reprod. 53( 1995) 855-862.

[34] Kimura Y., Yanagimachi R., Intracytoplasmicsperm injection in the mouse, Biol. Reprod. 52( 1995) 709-720.

[35] Kimura Y., Yanagimachi R., Kuretake S.,Bortkiewicz H., Perry A.C.F., Yanagimachi H.,Analysis of mouse oocyte activation suggeststhe involvement of sperm perinuclear material,Biol. Reprod. 58 (1998) 1407-1415.

[36] Kopecny V., Pavlok A., Autoradiographic studyof mouse spermatozoan arginine-rich nuclearprotein in fertilization, J. Exp. Zool. 191 (1975)85-96.

[37] Le Guen P., Crozet N., Microtubule and cen-trosome distribution during sheep fertilization,Eur. J. Cell Biol. 48 (1989) 239-249.

[38] Long C.R., Duncan R.P., Robl J.M., Isolationand characterization of MPM-2-reactive spermproteins: homology to components of the outerdense fibers and segmented columns, Biol.Reprod. 57 (1997) 246-254.

[39] Longo F.J., Incorporation and dispersal of spermsurface antigens in plasma membranes of insem-inated sea urchin (Arbacia punctulata) eggs andoocytes, Dev. Biol. 131 (1989) 37-43.

[40] Macaulay C., Forbes D.J., Assembly of thenuclear pore: biochemically distinct stepsrevealed with NEM, GTPYS, and BAPTA,J. Cell Biol. 132 (1996) 5-20.

[41 ] Manandhar G., Sutovsky P., Joshi H.C., Steams T.,Schatten G., Centrosome reduction duringmouse spermiogenesis, Dev. Biol. 203 (1998)424-434.

[42] Meier E., Miller B.R., Forbes D.J., Nuclear porecomplex assembly studied with a biochemicalassay for annulate lamellae formation, J. CellBiol. 129 (1995) 1459-1472.

[43] Navara C.S., First N.L., Schatten G., Micro-tubule organisation in the cow during fertiliza-tion, polyspermy, parthenogenesis, and nucleartransfer: the role of the sperm aster, Dev. Biol.162 ( I 994) 29-40.

[44J Newport J.W., Nuclear reconstitution in vitro:stages of assembly around protein-free DNA,Cell 48 (1987) 205-217.

[45] Ogura A., Yanagimachi R., Round spermatidnuclei injected into hamster oocytes form pronu-clei and participate in syngamy, Biol. Reprod. 48(1993) 219-225.

[46] Ogura A., Matsuda J., Yanagimachi R., Birthof normal young after electrofusion of mouseoocytes with round spermatids, Proc. Natl. Acad.Sci. USA 91 (1994) 7460-7462.

[47] Ogura A., Wakayama T., Suzuki 0., Shin T.-Y.,Matsuda J., Kobayashi Y., Chromosomes ofmouse primary spermatocytes undergo meioticdivision after incorporation into homologousimmature oocytes, Zygote 5 (1997) l77-182.

[48] Oko R., Clermont Y., Isolation, structure andprotein composition of the perforatorium of ratspermatozoa, Biol. Reprod. 39 (1988) 673-687.

[49] Oko R., Maravei D., Protein composition of theperinuclear theca of bull spermatozoa, Biol.Reprod. 50 (1994) 1000-1014.

[50] Oko R., Maravei D., Distribution and possiblerole of perinuclear theca proteins during bovinespermiogenesis, Microsc. Res. Tech. 32 (1995)520-532.

[5) 1 Pante N., Aebi U., Sequential binding of importligands to distinct nuclear pore regions duringtheir nuclear import, Science 273 (1996) 1729-1732.

!52! Palermo G., Joris H., Devroey P., Van Steirte-ghem A.C., Pregnancies after intracytoplasmicsperm injection of single spermatozoon into anoocyte, Lancet 340 (1992) 17-18.

[531 Parrington J., Swann K., Schevchenko V.I.,Sesay A.K., Lai F.A., Calcium oscillations inmammalian eggs triggered by a soluble spermfactor, Nature 379 (1996) 364-368.

[54] Perreault S.D., Wolf R.A., Zirkin B.R., The roleof disulfide bond reduction during mammaliansperm nuclear decondensation in vivo, Dev.Biol. 101 (1984) 160-167.

[55] Pfaller R., Smythe C., Newport J.W., Assem-bly/disassembly of the nuclear envelope mem-brane: cell cycle-dependent binding of nuclearmembrane vesicles to chromatin in vitro, Cell65(1991)209-217.

[56] Phillips D.M., Spermiogenesis, Academic Press,New York, 1974.

[57] Pines J., Ubiquitin with everything, Nature 371(1994)742-743.

[58] Pinto-Correia C., Collas P., Ponce de Leon A.,Robl J.M., Chromatin and microtubule organi-sation in the first cell cycle in rabbit parthenotesand nuclear transplant embryos, Mol. Reprod.Dev. 34 (1993) 33-42.

[59] Pinto-Correia C., Poccia D.L., Chang T.,Robl J.M., Dephosphorylation of sperm mid-piece antigens initiates aster formation in rabbitoocytes, Proc. Natl. Acad. Sci. USA 91 (1994)7894-7898.

[60] Poccia D., Collas P., Transforming sperm nucleiinto male pronuclei in vivo and in vitro, in: Ped-ersen R.A., Schatten G.P. (Eds.), Current Top-ics in Developmental Biology, vol. 34, Aca-demic Press Inc., New York, 1996, pp. 25-88.

[61 1 Sanders M.A., Salisbury J.L., Centrin-mediatedmicrotubule severing during flageller excision inChlamydomonas reinhardtii, J. Cell Biol. 108( 1989) 1751-1760.

[62] Sasagawa I., Kuretake S., Eppig J.J., Yanagi-machi R., Mouse primary spermatocytes com-plete two meiotic divisions within the oocytecytoplasm, Biol. Reprod. 58 (1998) 248-254.

[63] Sathananthan A.H., Kola L, Osborne J., Troun-son A., Ng S.C., Bongso A., Ratnam S.S., Cen-trioles at the beginning of human development,Proc. Natl. Acad. Sci. USA 88 (1991) 4806-4810.

[641 Sathananthan A.H., Ratnam S.S., Ng S.C.,Tarin J.J., Gianaroli L., Trounson A., The spermcentriole: its inheritance, replication and per-petuation in early human embryos, Hum.Reprod. 1 (1996)345-356.

165] Sathananthan A.H., Szell A., Ng S.C., Kausche A.,Lacham-Kaplan 0., Trounson A., Is the acro-some reaction a prerequisite for sperm incorpo-ration after intra-cytoplasmic sperm injection’?,Reprod. Fert. Dev. 9 (1997) 703-709.

[661 Schatten G., Simerly C., Schatten H., Micro-tubule configurations during fertilization, mito-sis, and early development in the mouse and therequirement for egg microtubule-mediated motil-ity during mammalian fertilization, Proc. Natl.Acad. Sci. USA 82 ( 1985) 4152-4156.

[67] Schatten H., Schatten G., Mazia D., Balczon R.,Simerly C., Behavior of centrosomes during fer-tilization and cell division in mouse oocytes andsea urchin eggs, Proc. Natl. Acad. Sci. USA 83(1986) 105-109.

[68] Shalgi R., Magnus A., Jones R., Phillips D.M.,Fate of sperm organelles during early embryo-genesis in the rat, Mol. Reprod. Dev. 37 ( 1994)264-271.

[69] Simerly C., Wu G., Zoran S., Ord T., Rawlins R.,Jones J., Navara C., Gerrity M., Rinehart J.,Binor Z., Asch R., Schatten G., The paternalinheritance of the centrosome, the cell’s micro-tubule-organizing center, in humans and theimplications for infertility, Nature Med. I ( 1995)47-53.

170] Snell W.J., White J.M., The molecules of mam-malian fertilization, Cell 85 (1996) 629-637.

1711 Sofikitis N.V., Toda T., Miyagawa L, ZavosP.M., Pasyianos P., Mastelou E., Beneficialeffects of electrical stimulation before round

spermatid nuclei injections into rabbit oocytes onfertilization and subsequent embryonic devel-opment, Fertil. Steril. 65 (1996) 176-185.

[721 Soupart P., Strong P.A., Ultrastructural obser-vations on polyspermic penetration of zona pel-lucida-free human oocytes inseminated in vitro,Fertil. Steril. 26 ( 1975) 523-537.

[73] Stearns T., Evans L., Kirschner M., y-tubulin isa highly conserved component of the centro-some, Cell 65 (1991) 825-836.

[74] Stefanini M., Oura C., Zamboni L., Ultrastruc-ture of fertilization in the mouse. 2. Penetrationof the sperm into the ovum, J. Submicr. Cytol. 1(1969) 1-23.

[75] Sutovsky P., Schatten G., Depletion of glu-tathione during oocyte maturation reversiblyblocks the decondensation of the male pronu-cleus and pronuclear apposition during fertil-ization, Biol. Reprod. 56 ( 1997) 1503-1512.

[761 Sutovsky P., Hewitson L., Simerly C.R., Ten-gowski M.W., Navara C.S., Haavisto A., Schat-ten G., Intracytoplasmic sperm injection (ICSI)for Rhesus monkey fertilization results inunusual chromatin, cytoskeletal, and membraneevents, but eventually leads to pronuclear devel-opment and sperm aster assembly, Hum. Reprod.11 (1996) 1703-1712.

1771 Sutovsky P., Navara C.S., Schatten G., The fateof the sperm mitochondria, and the incorpora-tion, conversion and disassembly of the, spermtail structures during bovine fertilization in vitro,Biol. Reprod. 55 (1996) 1195-1205.

1781 Sutovsky P., Oko R., Hewitson L., Schatten G.,The removal of the sperm perinuclear theca andits association with the bovine oocyte surfaceduring fertilization, Dev. Biol. 188 (1997) 75-84.

[791 Sutovsky P., Moreno R., Schatten G., Spermmitochondrial ubiquitination and a modelexplaining the strictly maternal mtDNA inheri-tance in mammals, Mol. Biol. Cell Suppl. 9( 1998) 309a.

[80] Sutovsky P., Ramalho-Santos J., Moreno R.,Oko R., Hewitson L., Schatten G., On-stageidentification and selection of single round sper-matids using a vital, mitochondrion-specific flu-orescent probe MitoTrackerTM, Hum. Reprod.(1999) (in press).

1811 Sutovsky P., Simerly C., Hewitson L., Schat-ten G., Assembly of nuclear pore complexesand annulate lamellae promotes normal pronu-clear development in fertilized mammalianoocytes, J. Cell Sci. I I I ( 1998) 2841-2854.

1821 Szollosi D., The fate of the sperm middle-piecemitochondria in the rat egg, J. Exp. Zool. 159( I 965) 367-378.

[ 83 J Szollosi D., Oocyte maturation and paternal con-tribution to the embryo in mammals, Curr. Top.Pathol. 64 ( 1976) 9-27.

[841 Sz6ll6si D., Hunter R.H.F., Ultrastructuralaspects of fertilization in the domestic pig: spermpenetration and pronucleus formation, J. Anat.116(1973) 181-206.

[ 85 j Sz6ll6si D., Ozil J.P., De novo formation of cen-trioles in parthenogenetically activated,diploidized rabbit embryos, Biol. Cell 72 (1991)61-66.

[861 Sz6ll6si M.S., Sz6ll6si D., ’Blebbing’ of thenuclear envelope of mouse zygotes, earlyembryos and hybrid cells, J. Cell Sci. 91 ( 1988)257-267.

[87 ! Sz6ll6si D., Calarco P., Donahue R.P., Absenceof centrioles in the first and second meiotic spin-dles of mouse oocytes, J. Cell Sci. I I ( 1972)521-541.

[88] Sz6ll6si D., Czolowska R., Soltynska M.S.,Tarkowski A.K., Ultrastructure of cell fusionand premature chromosome condensation (PCC)of thymocyte nuclei in metaphase II mouseoocytes, Biol. Cell 56 (1986) 239-250.

[89] Sz6llbsi D., Czolowska R., Sz6ll6si M.S.,Tarkowski A.K., Remodeling of thymocytenuclei in activated mouse oocytes: an ultra-structural study, Eur. J. Cell Biol. 42 (1986)140-151. 1 .

[90] Sz6ll6si D., Czolowska R., SzOll6si M.S.,Tarkowski A.K., Remodeling of thymocytenuclei depends on the time of their transfer intoactivated, homologous oocytes, J. Cell Sci. 91 1(1988) 603-613.

[91] Sz6llbsi D., Sz6ll6si M.S., Czolowska R.,Tarkowski A.K., Sperm penetration into imma-ture mouse oocytes and nuclear changes duringmaturation: an EM study, Biol. Cell 69 (1990)53-64.

[92] Sz6ll6si M.S., Adenot P., Sz6ll6si D., Centro-somes with striated rootlets in rabbit zygotes,Zygote 4 (1996) 173-179.

[93] Szollosi D., Czolowska R., Borsuk E., Sz6ll6siM.S., Debey P., Nuclear envelope removal/maintenance determines the structural and func-tional remodelling of embryonic red blood cellnuclei in activated mouse oocytes, Zygote 6(1998)65-73.

[94] Tesarik J., Mendoza C., Testart J., Viableembryos from injection of round spermatids intooocytes, N. Engl. J. Med. 333 (1995) 525.

[95] Thompson R.S., Moore Smith D., Zamboni L.,Fertilization of mouse ova in vitro: an electron

microscopic study, Fertil. Steril. 25 (1974) 222-249.

[96] Usui N., Morphological differences in nuclearmaterials released from hamster sperm heads atan early stage of incorporation into immatureoocytes, mature oocytes, or fertilized eggs, Mol.

Reprod. Dev. 44 (1996) 132-140.

[97] Usui N., Yanagimachi R., Behavior of hamstersperm nuclei incorporated into eggs at various

stages of maturation, fertilization and earlydevelopment, J. Ultrastruct. Res. 57 (1976) 276-288.

[98] Usui N., Ogura A., Kimura Y., Yanagimachi R.,Sperm nuclear envelope: breakdown of intrinsicenvelope and de novo formation in hamsteroocytes or eggs, Zygote 5 (1997) 35-46.

[99] Van Blerkom J., Bell H., Henry G., The occur-rence, recognition and developmental fate ofpseudo-multipronuclear eggs after in vitro fer-tilization of human oocytes, Hum. Reprod. 2(1987) 217-225.

[100] Van Steirteghem A., Nagy S., Joris H., Liu J.,Staessen C., Smitz J., Wisanto A., Devroey P.,High fertilization and implantation rates afterintracytoplasmic sperm injections, Hum. Reprod.8(1993) 1061-1066.

[ 101 ] Vignon X., Chesné P., Le Bourhis D., Flechon J.E.,Heyman Y., Renard J.P., Developmental poten-tial of embryos reconstructed from enucleatedmatured oocytes fused with cultured somaticcells, C. R. Acad. Sci. Paris 321 (1998) 735-745.

[102] Wakayama T., Perry A.C.F., Zucotti M., John-son K.R., Yanagimachi R., Full-term develop-ment of mice from enucleated oocytes injectedwith cumulus cell nuclei, Nature 394 (1998)369-374.

[103] Woolley D.M., Fawcett D.W., The degenera-tion and disappearance of the centrioles duringthe development of the rat spermatozoon, Anat.Rec. 177 (1973) 289-302.

[104) Wu G., Simerly C., Zoran S.S., Funte L.R.,Rawlins R., Binor Z., Schatten G., Microtubuleand chromatin configurations during fertiliza-tion and early development in rhesus monkeys,and regulation by intracellular calcium ions,Biol. Reprod. 55 (1996) 260-270.

[ 105] Xu Y.S., Overton W.R., Marman J.L., Leonard J.C.,McCoy S.P., Buttler G.H., Li H., Complete repli-cation of human sperm genome in egg extractsfrom Xenopus laevis, Biol. Reprod. 58 (1998)661-667.