Human Reproduction Vol.17, No.9 pp. 2344–2349, 2002

A pathology of the sperm centriole responsible for defectivesperm aster formation, syngamy and cleavage

V.Y.Rawe1, Y.Terada3, S.Nakamura3, C.F.Chillik1, S.Brugo Olmedo1 and H.E.Chemes2,3

1Center of Studies in Gynaecology and Reproduction, CEGyR, 2Laboratory of Testicular Physiology and Pathology, EndocrinologyDivision, Children’s Hospital, Buenos Aires, Argentina and 3Department of Obstetrics and Gynaecology, Tohoku University Schoolof Medicine, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan

3To whom correspondence should be addressed. E-mail: hchemes@cedie.guti.gov.ar

BACKGROUND: In the present report we analyse the structural and functional features of sperm from a patientwith severe asthenoteratozoospermia and failure of cleavage after ICSI. METHODS: Sperm were studied by phasecontrast and transmission electron microscopy and microinjected into bovine oocytes to examine aster formationusing antibodies against acetylated α- and β-tubulins. RESULTS: Acephalic sperm, headless tails and abnormalalignments of the head–tail junction were observed. Flagella evidenced the features of dysplasia of the fibroussheath. Bovine oocytes injected with patient’s sperm showed male and female pronuclei but a faulty developmentof microtubules from the sperm-derived centrosome. The first ICSI attempt using conventional sperm selectionmethods resulted in fertilized two pronuclei zygotes, but no syngamy or cleavage. Three more ICSI attempts wereperformed, carefully avoiding sperm with obvious anomalies of the connecting piece. Fertilization and cleavagetook place in all cycles, and in two of them positive βhCG plasma levels were detected but preclinical abortionsensued. CONCLUSIONS: We propose that the alterations in the head–tail junction and attachment, responsiblefor the observed sperm phenotype, result from centriolar dysfunctions that cause insufficient sperm aster formation,lack of syngamy and cleavage or defective embryos leading to early abortions.

Key words: acephalic sperm/connecting piece/dysplasia of the fibrous sheath/head–neck attachment/sperm pathology

Introduction

There is general consensus that sperm morphology correlateswith fertility in men, but the current methods of morphologicalevaluation provide only a limited insight into the physio-pathological mechanisms involved. The spermatozoon, aterminally differentiated cell with organelles specialized inparticular functions, has a pathology of its own, and the natureof most sperm abnormalities is not identified by routine semenanalysis or functional tests because these methods only revealtheir secondary manifestations.

The success of ICSI implies a controlled disassembly ofsperm components, which are lost before or during gametefusion before completion of the first embryonic cell cycle.During the last decade, the practice of ICSI has demonstratedthat the use of normal sperm is not a prerequisite for fertiliza-tion. Fertilization failure in mammals may be due to differentdysfunctions affecting the sperm chromatin, centriole or peri-nuclear theca (Tesarik and Kopecny, 1989a,b; Tesarik andTestart, 1994; Asch et al., 1995; Simerly et al., 1995; VanBlerkom et al., 1995; Sutovsky et al., 1997). Dependingon the specific characteristics of individual sperm used forinjection, ICSI outcome may change significantly.

Sperm abnormalities can involve defects in structuresessential for fertilization. A recent study of 10 patients with

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severe alterations in the head–tail junction and/or acephalicsperm demonstrated that these features derive from an abnormalbehaviour of the spermatid centriole and tail anlage that failto migrate and establish normal contact with the spermatidnucleus. A clear correlation between this pathological pheno-type and the failure of fertilization during ICSI suggested thatboth derive from sperm centriolar anomalies (Chemes et al.,1999). The use of bovine oocytes microinjected with humansperm is considered a relevant model to understand possiblesperm centrosomal function. High fertilization rates haverecently been reported in bovine oocytes using a Piezo-drivensystem (Nakamura et al., 2001).

Another severe sperm abnormality is dysplasia of the fibroussheath (DFS). This condition affects various cytoskeletalcomponents of the sperm tail and causes extreme asthenozoo-spermia or total sperm immotility (Chemes et al., 1987, 1998).ICSI is a suitable procedure because these alterations do notinterfere with normal fertilization (Brugo Olmedo et al., 1997,2000; Chemes et al., 1998; Rawe et al., 2001).

In this report we present a careful study of the structuraland dynamic characteristics of sperm from an infertile patientcarrying the two previously mentioned sperm pathologies ofpresumably genetic origin. To our knowledge, this is the firstreport of such an association in an infertile man. Sperm

Sperm centriolar abnormalities and fertilization failure after ICSI

features, microinjection into bovine oocytes and the results ofICSI provide strong evidence that the anomalies in the head–neck attachment and defective fertilization are due to theabnormal function of the sperm centriole and its centrosomalderivation in the embryo.

Materials and methodsA 36-year-old man, complaining of primary infertility was studied.Past history and a complete andrological examination provided noinformation on clinical problems. Semen analysis revealed severeteratozoospermia and total sperm immotility. His wife was 31 yearsold with normal ovarian function. The use of microinjection techniqueswas decided, and after ovarian stimulation and oocyte retrieval,four ICSI attempts were performed as previously described (Raweet al., 2000).

Transmission electron microscopy of semen samples and testicularbiopsies

After study under phase contrast microscopy, a fresh semen samplewas processed for transmission electron microscopy (TEM) within30 min of ejaculation, according to methods previously described(Chemes et al., 1987). In brief, sperm were washed with phosphatebuffer (0.1 mol/l, pH 7.4), pelleted by centrifugation and fixed in 3%glutaraldehyde followed by 1.3% osmium tetroxide. The pellet wasembedded in Epon Araldite (Polysciences Inc., Warrington, PA, USA)and thin sections were examined and photographed in a Zeiss 109Electron microscope (Zeiss Oberkochen, Germany) after doublestaining with uranyl acetate and lead citrate. A small aliquot of freshsemen was studied under phase contrast microscopy, and motility,viability and light microscopy morphology were investigated accord-ing to standard methods (World Health Organization, 1987).

Multiple testicular biopsies were performed under local anaesthesiausing a single mid-line scrotal raphee incision. Tissue was gentlywashed in saline solution and then placed in tubes (Falcon, Becktonand Dickinson, Lincoln Park, NJ, USA) to be sent to the laboratory.Part of this tissue was used to isolate testicular sperm for micro-injection. The rest of the sample was fixed in 3% glutaraldehyde

Figure 1. Head–tail misalignments are evident in panels (A), (C) and (D) (circles). Abnormal tail configurations, typical of dysplasia of thefibrous sheath can be seen in panels (B) and (D) (short and thick tails) and in panel (C) (rigid or stiff tails). Headless tails (arrow) and looseheads are observed in (B) and (C). Phase contrast; scale bars represent 5 µm.

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followed by 1.3% osmium tetroxide and processed for electronmicroscopy examination as described above.

Human sperm microinjection of bovine oocytes using a Piezomicromanipulator

Bovine oocytes were obtained from slaughterhouse ovarian tissueand recovered by aspiration from 2–8 mm follicles and were maturedfor 22–24 h. Oocytes that had arrested at second meiotic metaphasewere used for ICSI.

Sperm pellets from a fertile donor and the patient under studywere resuspended and added to M2 culture medium with 10%polyvinyl pyrrolidine. Sperm were injected using a Piezo micromanip-ulator (MB-U, PRIM TECH, Japan). In Piezo-ICSI, only zonapellucida was penetrated using several Piezo pulses. After a cylindricalpiece of the zona in the pipette was expelled, the immobilizedspermatozoon was positioned at the tip of the pipette. The pipettewas inserted deeply into the ooplasm without applying Piezo pulses.Then the oolema was punctured by application of one Piezo pulseand the entire sperm was expelled into the ooplasm with a minimumamount of sperm suspension medium.

After injection, oocytes were cultured in HEPES-buffered TCM-199 supplemented with 10% fetal calf serum at 38.5°C in 5% CO2in air under mineral oil until fixation and permeabilization. Forimmunofluorescence analysis, zona pellucida were removed with M2culture medium supplemented with 0.75% Protease (Sigma). After a30 min recovery at 38.5°C, zona-free oocytes and zygotes wereextracted for 15 min using buffer M [25% (v/v) glycerol, 50 mmol/lKCl, 0.5 mmol/l MgCl2, 0.1 mmol/l EDTA, 1 mmol/l EGTA,50 mmol/l imidazole hydrochloride and 1 mmol/l 2-mercaptoethanol,pH 6.8) containing 5% methanol and 1% Triton X-100 detergent andfixed in cold methanol for 10 min according to a previously describedmethod (Simerly and Schatten, 1993). Fixed oocytes were thenpermeabilized overnight with 0.1 mol/l phosphate-buffered salinecontaining 0.1% Triton X-100 detergent.

Microtubules were labelled with a mixture of monoclonal antibodyagainst β-tubulin (clone 2-28-33, diluted 1:100; Sigma) and acetylatedα-tubulin (clone 6-11-B1, diluted 1:100; Sigma). The primary anti-bodies were detected by fluorescein-conjugated goat anti-mouse IgG

V.Y.Rawe et al.

(diluted 1:40; Zymed, San Francisco, CA). DNA was detected afterlabelling with 10 mg/ml Hoechst 33342.

Coverslips were mounted in anti-fade medium (Vectashield; VectorLabs, Burlingame, CA, USA) and were examined using conventionalepifluorescence microscopy (OPTIPHOT-2, Nikon, Japan). Theimages were recorded digitally and archived on magnet optical disksprocessed using Adobe PhotoShop software (Adobe Systems Inc.,Mountain View, CA, USA). Percentages of pronuclear formation andsperm aster formation were recorded for oocytes injected with spermfrom a fertile donor or the patient under study.

Figure 2. (A) and (B), elongating spermatids in testicular biopsy. Acrosomal development, nuclear elongation and beginning of chromatincondensation are normal. The sperm centriole and tail anlagen are not associated with the caudal pole of the nucleus (asterisks), or show aneccentric implantation (white arrowhead). (C) Cross-section of the proximal centriole in an ejaculated spermatozoon showing structuralpreservation of centriolar triplets. A testicular late spermatid (D) and an ejaculated spermatozoon (E) show implantation of the tail at thecaudal pole of nuclei and hyperplasia of the fibrous sheath. (F) A testicular late spermatid with head–tail dissociation. (G) Headless tail withfeatures typical of dysplasia of the fibrous sheath. (H) Loose head in semen. Scale bars represent (A, B) 2 µm, (C) 0.1 µm, (D–H) 0.5 µm.

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All procedures were performed with the approval of an internalreview board in Tohoku University School of Medicine.

Results

Semen analysis

Recently ejaculated semen samples were studied under phasecontrast microscopy. Sperm motility ranged between 0 and0.02% (progressive motility 0% in all cases). Sperm showed

Sperm centriolar abnormalities and fertilization failure after ICSI

an abnormal development of the neck region with headsattached either to the tip or to the sides of the mid-piecewithout a linear alignment with the sperm axis. The anglesbetween heads and tails were up to 90–180°. Completelydetached heads and acephalic flagella were also visualized(Figure 1). A few sperm with heads normally aligned wereidentified. Sperm flagella were short, rigid and thick, frequentlydisplaying irregular contours and/or coiled tails (Figure 1).

Electron microscopy studies

Testicular biopsyEarly round spermatids did not show abnormalities. Duringmaturation, acrosome development, nuclear elongation andchromatin compaction proceeded normally, but the centriolaranlagen and developing tail failed to attach to the caudal poleof the spermatid nucleus or established abnormal lateralassociations with it (Figure 2). This resulted either in anindependent development or in abnormal connections betweenheads and tails. In a few spermatids, the alignment of theconnecting piece (head–neck junction) was preserved(Figure 2). There was also an abnormal development of thefibrous sheath in late spermatids. Redundant fibres of randomorientation were seen disrupting the axoneme with resultingthickening and irregular contours. These two types of altera-tions co-existed in most spermatids that showed thecombination of abnormal head–tail attachments and thick andshort flagella with hyperplasia of the fibrous sheath (Figure 2).

Semen sampleUnder TEM, most sperm evidenced severe alterations in theneck region. Sperm nuclei and acrosomes showed normalfeatures. The fine structure of the proximal centriole wasstudied. In particular, centriolar triplets evidenced normalorganization (Figure 2C). Centriolar abnormalities were notobserved in any case. The connecting piece appeared structur-ally normal, but in most sperm it was not linearly oriented inrelation to the head (Figure 2). This misalignment ranged froma complete lack of connection between heads and tails to alateral positioning of the head at a 90–180° angle to the mid-piece. Loose heads and acephalic sperm were frequently found(Figure 2). When heads and tails were separated, the centriolesremained with the tails. A second abnormality was observedsimultaneously. Sperm flagella had marked hypertrophy andhyperplasia of the fibrous sheath, and complete disorganizationof the mid-piece with absent or translocated mitochondria(Figure 2).

Injection of bovine oocytes: sperm aster and male pronuclearformation

Bovine oocytes were fixed at 6 h post-ICSI and were examinedfor male pronuclear and sperm aster formation. When bovineoocytes were microinjected with sperm from a fertile donor,a radial microtubule array was observed originating from thesperm centrosome with microtubules extending throughout thecytoplasm to establish contact with the developing femalepronucleus (Figure 3). Pronuclei were formed in all injectedoocytes (100%) and well-developed sperm asters were observedin 68% (Table I). These data are similar to those previously

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reported (Nakamura et al., 2001). When sperm from thepatient under study were used for microinjection, microtubulesoriginating from the sperm centrosome at the site of associationwith the male pronucleus were few in number and truncated,resulting in arrested sperm asters (Figure 3). Male pronuclearformation, and especially the percentage of well-developedsperm asters (60 and 16% respectively), was much lower thanthat obtained from oocytes injected with sperm from the fertiledonor (Table I).

ICSI attempts

After a careful comparison of the quality of testicular andejaculated sperm it was decided to use the latter for micro-injection of oocytes. Ejaculated sperm were processed usingdiscontinuous Percoll gradients (50–95%) as previouslydescribed (Rawe et al., 2000).

First attemptA total of six metaphase II oocytes (MII) were injected withsperm selected using conventional criteria. Three oocytesfertilized, but when observed 24 h later, none of them showedpronuclear syngamy and/or cleavage.

Second attemptIn view of the previous results, and after a thorough re-evaluation of sperm pathology, a second ICSI was attemptedusing a strict selection of the sperm to be injected. The mainselection criterion was a head–neck alignment as normal aspossible. Three oocytes fertilized (out of five injected) andcleaved resulting in good quality embryos for transfer. Apositive βhCG plasma level was detected after 12 days oftransfer and a preclinical abortion later took place.

Third attemptDuring the third ICSI attempt, four oocytes were injected withstrictly selected sperm. After 15 h of injection, two oocytesshowed two pronuclei (2PN) and two oocytes remainedunfertilized. Two good quality embryos were obtained andtransferred, but 12 days later no pregnancy was obtained.

Fourth attemptAt the last ICSI attempt, a total of 13 oocytes were obtained.Following the same selection criteria for sperm, 10 MII oocyteswere injected. After 15 h of injection, eight oocytes showed2PN and two oocytes remained unfertilized. Eight good qualityembryos were obtained and three of them were transferred. Apositive βhCG plasma level was detected 12 days later followedby a preclinical abortion. Five embryos were cryopreserved.

Discussion

The present study describes fertilization failure after ICSI thatmost likely results from a deficiency in the function of thesperm-derived centrosome of the zygote. The patient reportedsuffered from a severe form of teratozoospermia characterizedby defective head–mid-piece attachment and acephalic sperm.During the first ICSI attempt, oocytes reached the pronuclearstage but failed to undergo syngamy and cleavage, which isin accordance with previous reports by Chemes et al. in asimilar patient (Chemes et al., 1999). Bovine oocytes injectedwith sperm from the patient evidenced a severe arrest in

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Figure 3. Bovine oocytes injected with human sperm. A well formed sperm aster that followed the injection of sperm from a fertile donorcan be seen in panel (A). (B) One oocyte microinjected with sperm from the patient shows blue male (upper, note sperm tail) and femalepronuclei, but the sperm aster is not formed. Green immunofluorescence with anti-tubulin antibodies, blue fluorescence: nuclear contrastwith Hoechst 33342. Scale bars represent 25 µm.

Table I. Rate of pronuclear formation and sperm aster organization inbovine oocytes injected with sperm from a fertile donor or the patient understudy

Pronuclear formation Sperm aster organizationn (%) n (%)

Fertile donor (n � 22)a 22 (100) 15 (68)Patient (n � 25)a 17 (60) 4 (16)

an � number of bovine oocytes injected.

the development of microtubules from the sperm-derivedcentrosome of the zygote. The lack of sperm aster formationwhen sperm from our patient were injected into bovine oocytessuggested a defect of paternal rather than maternal origin,because the centriole within the nascent zygotic centrosomeis sperm-derived in humans (Schatten, 1994; Hewitson et al.,1997; Sutovsky et al., 1999). This was in sharp contrast withthe high percentage of fully developed sperm asters obtainedwhen sperm from a fertile donor were injected in parallel(Nakamura et al., 2001; and this paper). This indicates thatbovine oocytes can provide the components that are recruited bynormally functional sperm centrioles to form the zygotic aster.

Alterations similar to those reported here have also beenattributed to failures in the microtubular systems of the zygote(Van Blerkom et al., 1995; Hewitson et al., 1997; Sutovskyet al., 1999). Various sperm abnormalities have been consideredresponsible for unsuccessful fertilization. The lack of removalof the perinuclear theca from the apical region of the spermhead probably precludes normal oocyte activation (Tesarik andTestart, 1994; Sutovsky et al., 1997). Alternatively, the spermchromatin may fail to decondense, preventing the access ofoocyte factors required for male pronuclear development(Tesarik and Kopecny, 1989a,b). Finally, failure of the microtu-bules to assemble around the paternal centriole results inabnormal sperm aster formation with failed pronuclei apposi-tion and arrest of development (Van Blerkom, 1996). Most of

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these unfertilized arrested oocytes have been observed afterIVF or ICSI. This is the first report of arrested zygotedevelopment linked to a deficient behaviour of the spermcentrosome in a human–bovine ICSI system, in a patient withabnormal head–mid-piece attachments, probably derived fromdefective centrioles during spermiogenesis (Chemes et al.,1999).

As shown by our ultrastructural studies, the alterations ofthe attachment of heads and tails are due to an abnormalmigration of the tail anlage during testicular spermiogenesis.The centrioles are an essential part of this structure and arepaternally inherited in humans. We propose that the lackof syngamy and cleavage in our patient are probably aconsequence of abnormal centrioles in sperm with alterationsin the head–tail attachment. We have previously documenteda large series of 10 patients with this kind of anomaly (Chemeset al., 1999). This series included two brothers, which stronglysuggests familial transmission of this phenotype, as was alsoshown by Baccetti et al. (Baccetti et al., 1989). Sperm withhead–tail misalignment show an increased fragility of the neckregion that results in separation of heads and tails in semenand arrest of zygote development at the pronuclear stage afterICSI (Chemes et al., 1999). This represents pathology of thesperm centriole with characteristic structural abnormalities andthe corresponding anomalous behaviour during fertilization.This hypothesis was confirmed in the patient under study bythe results of the second, third and fourth ICSI attempts, inwhich the use of sperm strictly selected by the regular alignmentof heads and tails (a possible indication of a better connectingpiece attachment to sperm heads) resulted in progression tosyngamy and morphologically normal cleavage. However, thefact that these three attempts led to lack of implantation orpreclinical abortions may indicate that the quality of theembryos was suboptimal. As embryo quality is assessedbasically by morphological means, better methods are needed,which may allow the identification of hitherto non-reportedembryonic abnormalities responsible for faulty implantation.

Sperm centriolar abnormalities and fertilization failure after ICSI

The cause of the observed centriolar dysfunction is difficultto identify. Previous ultrastructural studies and the presentreport have confirmed morphologically intact centrioles inacephalic sperm/abnormal sperm–tail attachments (Perottiet al., 1981; Chemes et al., 1987; Baccetti et al., 1989). Inthese cases, sperm centrioles give rise to normal axonemesduring spermiogenesis, but are unable to migrate towards thesperm nucleus, and to act as microtubule organizing centresin the zygote. Since these minute structures can be assessedin only few sperm, the possibility that they could be structurallyabnormal cannot be ruled out completely. However, to datethere has not been a single report of structural centriolarabnormalities in acephalic sperm. This suggests that thealteration involved is not structural. A deviation in themechanism of centriolar duplication/reproduction may beresponsible for defective embryo cleavage. Centrosome repro-duction and the release of the sperm centriole after fertilizationseem to be regulated by the phosphorylation of nucleophosminNO38/B23 by Cdk2-E and ubiquitin-mediated proteolysisof selected targets by 26S proteasomes localized near thecentrosome in the neck region of human sperm (Wojcik et al.,2000; Hinchcliffe and Sluder, 2001). An in-depth study of themolecular mechanisms of centriolar function may shed somelight into the nature of abnormal sperm centriolar behaviourin the pathology under study.

An unfortunate yet scientifically intriguing feature of thispatient is the association of two serious defects in the samesperm. It has been shown that the alterations in the head–tailjunction herein reported and DFS are two uncommon spermphenotypes of possible genetic origin (Chemes, 2000). Aspreviously reported (Chemes et al., 1998, 1999), these pheno-types are completely unresponsive to medical or hormonaltherapies. Alterations of the head–tail junction seem to bedeleterious for normal fertilization (Chemes et al., 1999; andthis report), while patients with DFS have a standard outcomeafter ICSI in terms of fertilization and pregnancy (BrugoOlmedo et al., 1997, 2000; Chemes et al., 1998). This indicatesthat a normal sperm centriole is essential for successfulfertilization after ICSI, while tail abnormalities do not play asignificant role in fertilization failure. The present findingsemphasise the need for a careful morphological evaluation ofsperm to fully understand the pathophysiology of abnormalfertilization and as a useful prognostic tool in assisted repro-duction.

AcknowledgementsThe authors wish to express their appreciation to Peter Sutovsky,PhD for his technical and intellectual contributions. This work hasbeen supported by Grants from CONICET (PICT 0900) and ANPCyT(PICT 0450) and by CEGyR Foundation.

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Submitted on January 2, 2002; resubmitted on March 12, 2002; accepted onMay 3, 2002