1 Center of Studies in Gynaecology and Reproduction, CEGyR, 2 Laboratory of Testicular Physiology and Pathology, Endocrinology Division, Childrens Hospital, Buenos Aires, Argentina and 3 Department of Obstetrics and Gynaecology, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan
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Abstract |
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Key words: acephalic sperm/connecting piece/dysplasia of the fibrous sheath/head-neck attachment/sperm pathology
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Introduction |
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The success of ICSI implies a controlled disassembly of sperm components, which are lost before or during gamete fusion before completion of the first embryonic cell cycle. During the last decade, the practice of ICSI has demonstrated that the use of normal sperm is not a prerequisite for fertilization. Fertilization failure in mammals may be due to different dysfunctions affecting the sperm chromatin, centriole or perinuclear theca (Tesarik and Kopecny, 1989a,b
; Tesarik and Testart, 1994
; Asch et al., 1995
; Simerly et al., 1995
; Van Blerkom et al., 1995
; Sutovsky et al., 1997
). Depending on the specific characteristics of individual sperm used for injection, ICSI outcome may change significantly.
Sperm abnormalities can involve defects in structures essential for fertilization. A recent study of 10 patients with severe alterations in the headtail junction and/or acephalic sperm demonstrated that these features derive from an abnormal behaviour of the spermatid centriole and tail anlage that fail to migrate and establish normal contact with the spermatid nucleus. A clear correlation between this pathological phenotype and the failure of fertilization during ICSI suggested that both derive from sperm centriolar anomalies (Chemes et al., 1999). The use of bovine oocytes microinjected with human sperm is considered a relevant model to understand possible sperm centrosomal function. High fertilization rates have recently been reported in bovine oocytes using a Piezo-driven system (Nakamura et al., 2001
).
Another severe sperm abnormality is dysplasia of the fibrous sheath (DFS). This condition affects various cytoskeletal components of the sperm tail and causes extreme asthenozoospermia or total sperm immotility (Chemes et al., 1987, 1998
). ICSI is a suitable procedure because these alterations do not interfere 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 structural and dynamic characteristics of sperm from an infertile patient carrying the two previously mentioned sperm pathologies of presumably genetic origin. To our knowledge, this is the first report of such an association in an infertile man. Sperm features, microinjection into bovine oocytes and the results of ICSI provide strong evidence that the anomalies in the headneck attachment and defective fertilization are due to the abnormal function of the sperm centriole and its centrosomal derivation in the embryo.
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Materials and methods |
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Transmission electron microscopy of semen samples and testicular biopsies
After study under phase contrast microscopy, a fresh semen sample was processed for transmission electron microscopy (TEM) within 30 min of ejaculation, according to methods previously described (Chemes et al., 1987). In brief, sperm were washed with phosphate buffer (0.1 mol/l, pH 7.4), pelleted by centrifugation and fixed in 3% glutaraldehyde followed by 1.3% osmium tetroxide. The pellet was embedded in Epon Araldite (Polysciences Inc., Warrington, PA, USA) and thin sections were examined and photographed in a Zeiss 109 Electron microscope (Zeiss Oberkochen, Germany) after double staining with uranyl acetate and lead citrate. A small aliquot of fresh semen was studied under phase contrast microscopy, and motility, viability and light microscopy morphology were investigated according to standard methods (World Health Organization, 1987
).
Multiple testicular biopsies were performed under local anaesthesia using a single mid-line scrotal raphee incision. Tissue was gently washed in saline solution and then placed in tubes (Falcon, Beckton and Dickinson, Lincoln Park, NJ, USA) to be sent to the laboratory. Part of this tissue was used to isolate testicular sperm for microinjection. The rest of the sample was fixed in 3% glutaraldehyde followed by 1.3% osmium tetroxide and processed for electron microscopy examination as described above.
Human sperm microinjection of bovine oocytes using a Piezo micromanipulator
Bovine oocytes were obtained from slaughterhouse ovarian tissue and recovered by aspiration from 28 mm follicles and were matured for 2224 h. Oocytes that had arrested at second meiotic metaphase were used for ICSI.
Sperm pellets from a fertile donor and the patient under study were resuspended and added to M2 culture medium with 10% polyvinyl pyrrolidine. Sperm were injected using a Piezo micromanipulator (MB-U, PRIM TECH, Japan). In Piezo-ICSI, only zona pellucida was penetrated using several Piezo pulses. After a cylindrical piece of the zona in the pipette was expelled, the immobilized spermatozoon was positioned at the tip of the pipette. The pipette was inserted deeply into the ooplasm without applying Piezo pulses. Then the oolema was punctured by application of one Piezo pulse and the entire sperm was expelled into the ooplasm with a minimum amount 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% CO2 in air under mineral oil until fixation and permeabilization. For immunofluorescence analysis, zona pellucida were removed with M2 culture medium supplemented with 0.75% Protease (Sigma). After a 30 min recovery at 38.5°C, zona-free oocytes and zygotes were extracted for 15 min using buffer M [25% (v/v) glycerol, 50 mmol/l KCl, 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 and fixed in cold methanol for 10 min according to a previously described method (Simerly and Schatten, 1993). Fixed oocytes were then permeabilized overnight with 0.1 mol/l phosphate-buffered saline containing 0.1% Triton X-100 detergent.
Microtubules were labelled with a mixture of monoclonal antibody against ß-tubulin (clone 2-28-33, diluted 1:100; Sigma) and acetylated -tubulin (clone 6-11-B1, diluted 1:100; Sigma). The primary antibodies were detected by fluorescein-conjugated goat anti-mouse IgG (diluted 1:40; Zymed, San Francisco, CA). DNA was detected after labelling with 10 mg/ml Hoechst 33342.
Coverslips were mounted in anti-fade medium (Vectashield; Vector Labs, Burlingame, CA, USA) and were examined using conventional epifluorescence microscopy (OPTIPHOT-2, Nikon, Japan). The images were recorded digitally and archived on magnet optical disks processed using Adobe PhotoShop software (Adobe Systems Inc., Mountain View, CA, USA). Percentages of pronuclear formation and sperm aster formation were recorded for oocytes injected with sperm from a fertile donor or the patient under study.
All procedures were performed with the approval of an internal review board in Tohoku University School of Medicine.
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Results |
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Injection of bovine oocytes: sperm aster and male pronuclear formation
Bovine oocytes were fixed at 6 h post-ICSI and were examined for male pronuclear and sperm aster formation. When bovine oocytes were microinjected with sperm from a fertile donor, a radial microtubule array was observed originating from the sperm centrosome with microtubules extending throughout the cytoplasm to establish contact with the developing female pronucleus (Figure 3). Pronuclei were formed in all injected oocytes (100%) and well-developed sperm asters were observed in 68% (Table I
). These data are similar to those previously reported (Nakamura et al., 2001
). When sperm from the patient under study were used for microinjection, microtubules originating from the sperm centrosome at the site of association with the male pronucleus were few in number and truncated, resulting in arrested sperm asters (Figure 3
). Male pronuclear formation, and especially the percentage of well-developed sperm asters (60 and 16% respectively), was much lower than that obtained from oocytes injected with sperm from the fertile donor (Table I
).
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First attempt
A total of six metaphase II oocytes (MII) were injected with sperm selected using conventional criteria. Three oocytes fertilized, but when observed 24 h later, none of them showed pronuclear syngamy and/or cleavage.
Second attempt
In view of the previous results, and after a thorough re-evaluation of sperm pathology, a second ICSI was attempted using a strict selection of the sperm to be injected. The main selection criterion was a headneck alignment as normal as possible. Three oocytes fertilized (out of five injected) and cleaved resulting in good quality embryos for transfer. A positive ßhCG plasma level was detected after 12 days of transfer and a preclinical abortion later took place.
Third attempt
During the third ICSI attempt, four oocytes were injected with strictly selected sperm. After 15 h of injection, two oocytes showed two pronuclei (2PN) and two oocytes remained unfertilized. Two good quality embryos were obtained and transferred, but 12 days later no pregnancy was obtained.
Fourth attempt
At the last ICSI attempt, a total of 13 oocytes were obtained. Following the same selection criteria for sperm, 10 MII oocytes were injected. After 15 h of injection, eight oocytes showed 2PN and two oocytes remained unfertilized. Eight good quality embryos were obtained and three of them were transferred. A positive ßhCG plasma level was detected 12 days later followed by a preclinical abortion. Five embryos were cryopreserved.
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Discussion |
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Alterations similar to those reported here have also been attributed to failures in the microtubular systems of the zygote (Van Blerkom et al., 1995; Hewitson et al., 1997
; Sutovsky et al., 1999
). Various sperm abnormalities have been considered responsible for unsuccessful fertilization. The lack of removal of the perinuclear theca from the apical region of the sperm head probably precludes normal oocyte activation (Tesarik and Testart, 1994
; Sutovsky et al., 1997
). Alternatively, the sperm chromatin may fail to decondense, preventing the access of oocyte factors required for male pronuclear development (Tesarik and Kopecny, 1989a
,b
). Finally, failure of the microtubules to assemble around the paternal centriole results in abnormal sperm aster formation with failed pronuclei apposition and arrest of development (Van Blerkom, 1996
). Most of these unfertilized arrested oocytes have been observed after IVF or ICSI. This is the first report of arrested zygote development linked to a deficient behaviour of the sperm centrosome in a humanbovine ICSI system, in a patient with abnormal headmid-piece attachments, probably derived from defective centrioles during spermiogenesis (Chemes et al., 1999
).
As shown by our ultrastructural studies, the alterations of the attachment of heads and tails are due to an abnormal migration of the tail anlage during testicular spermiogenesis. The centrioles are an essential part of this structure and are paternally inherited in humans. We propose that the lack of syngamy and cleavage in our patient are probably a consequence of abnormal centrioles in sperm with alterations in the headtail attachment. We have previously documented a large series of 10 patients with this kind of anomaly (Chemes et al., 1999). This series included two brothers, which strongly suggests familial transmission of this phenotype, as was also shown by Baccetti et al. (Baccetti et al., 1989
). Sperm with headtail misalignment show an increased fragility of the neck region that results in separation of heads and tails in semen and arrest of zygote development at the pronuclear stage after ICSI (Chemes et al., 1999
). This represents pathology of the sperm centriole with characteristic structural abnormalities and the corresponding anomalous behaviour during fertilization. This hypothesis was confirmed in the patient under study by the results of the second, third and fourth ICSI attempts, in which the use of sperm strictly selected by the regular alignment of heads and tails (a possible indication of a better connecting piece attachment to sperm heads) resulted in progression to syngamy and morphologically normal cleavage. However, the fact that these three attempts led to lack of implantation or preclinical abortions may indicate that the quality of the embryos was suboptimal. As embryo quality is assessed basically by morphological means, better methods are needed, which may allow the identification of hitherto non-reported embryonic abnormalities responsible for faulty implantation.
The cause of the observed centriolar dysfunction is difficult to identify. Previous ultrastructural studies and the present report have confirmed morphologically intact centrioles in acephalic sperm/abnormal spermtail attachments (Perotti et al., 1981; Chemes et al., 1987
; Baccetti et al., 1989
). In these cases, sperm centrioles give rise to normal axonemes during spermiogenesis, but are unable to migrate towards the sperm nucleus, and to act as microtubule organizing centres in the zygote. Since these minute structures can be assessed in only few sperm, the possibility that they could be structurally abnormal cannot be ruled out completely. However, to date there has not been a single report of structural centriolar abnormalities in acephalic sperm. This suggests that the alteration involved is not structural. A deviation in the mechanism of centriolar duplication/reproduction may be responsible for defective embryo cleavage. Centrosome reproduction and the release of the sperm centriole after fertilization seem to be regulated by the phosphorylation of nucleophosmin NO38/B23 by Cdk2-E and ubiquitin-mediated proteolysis of selected targets by 26S proteasomes localized near the centrosome in the neck region of human sperm (Wojcik et al., 2000
; Hinchcliffe and Sluder, 2001
). An in-depth study of the molecular mechanisms of centriolar function may shed some light into the nature of abnormal sperm centriolar behaviour in the pathology under study.
An unfortunate yet scientifically intriguing feature of this patient is the association of two serious defects in the same sperm. It has been shown that the alterations in the headtail junction herein reported and DFS are two uncommon sperm phenotypes of possible genetic origin (Chemes, 2000). As previously reported (Chemes et al., 1998
, 1999
), these phenotypes are completely unresponsive to medical or hormonal therapies. Alterations of the headtail junction seem to be deleterious for normal fertilization (Chemes et al., 1999
; and this report), while patients with DFS have a standard outcome after ICSI in terms of fertilization and pregnancy (Brugo Olmedo et al., 1997
, 2000
; Chemes et al., 1998
). This indicates that a normal sperm centriole is essential for successful fertilization after ICSI, while tail abnormalities do not play a significant role in fertilization failure. The present findings emphasise the need for a careful morphological evaluation of sperm to fully understand the pathophysiology of abnormal fertilization and as a useful prognostic tool in assisted reproduction.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 2, 2002; resubmitted on March 12, 2002; accepted on May 3, 2002.