1 Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 11 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan, 2 Pittsburgh Development Center, MageeWoman's Research Institute, Departments of Obstetrics, Gynecology and Reproductive Sciences, and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA and 3 Department of Obstetrics and Gynecology, Tohoku Kosai Hospital, 2-3-11 Kokubun-chou, Aoba-ku, Sendai, Miyagi 980-0803, Japan
4 To whom correspondence should be addressed. Email: terada{at}mail.tains.tohoku.ac.jp
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Abstract |
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Key words: cytoskeleton/drug/fertilization/infertility/sperm centrosome
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Introduction |
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Dysplasia of the fibrous sheath (DFS), a rare form of teratozoospermia, results in infertility. DFS sperm, which are immotile due to deformities from midpiece to tail (Chemes et al., 1987, 1998
) also exhibit sperm centrosomal dysfunction; both of these abnormalities may be causes of infertility (Rawe et al., 2002
). Failure of either fertilization or embryo development continued to occur in several patients, even after performing ICSI (Chemes and Rawe, 2003
). We believe that artificial methods are available to restore the sperm centrosomal dysfunction of DFS sperm.
In this study, we treated human sperm that demonstrated deficiencies in centrosomal function with two drugs. Human sperm have disulphide bonds within the head and pericentriolar regions (Seligman et al., 1994; Simerly et al., 1999
; Tateno and Kamiguchi, 1999
). Dithiothreitol (DTT), which induces the reduction of disulphide bonds, was determined to be effective for unravelling the sperm centrosome. Paclitaxel (TaxolTM), which acts as a cytoskeleton stabilizer, is frequently used for studying cytoskeletal dynamics (Hewitson et al., 1997
; Mailhes et al., 1999
). Taxol enhances microtubule polymerization within the oocyte cytoplasm and may restore the recondite function of impaired human sperm centrosomes. Centrin, a ubiquitous, calcium-sensitive, biparentally contributed centrosomal component, severs axonemal microtubules from their associated basal bodies, potentially functioning in centrosome duplication. Centrin abnormalities may lead to fertilization failure or incomplete embryonic development (Salisbury, 1995
; Schiebel and Bornens, 1995
; Levy et al., 1996
).
In this study, we attempted to restore the sperm centrosome function of aster formation, which does not occur naturally in patients with DFS. We also examined the differences in centrin expression between the sperm from a fertile donor and a patient with DFS.
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Materials and methods |
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Sperm samples
DFS sperm were collected from a 36 year old man, complaining of primary infertility, the patient whose sperm morphology and clinical progress were reported previously (Rawe et al., 2002). Normal fertile sperm were collected from a 37 year old healthy man with children.
Heterologous ICSI with human sperm into bovine oocyte
Heterologous ICSI was performed by injecting human sperm into bovine oocytes, as previously described (Nakamura et al., 2001). Briefly, bovine oocytes were recovered from small follicles and allowed to mature for 2224 h. Cyropreserved sperm from a patient with DFS sperm were thawed in a water bath at 37°C. Human sperm from a fertile donor were processed in a similar manner for use as normal controls. Human sperm centrosomal function was examined in motile and dead sperm from the fertile donor and DFS sperm. Sperm viability assessment in the fertile donor was made using eosin-Y staining (World Health Organization, 1999
). Sperm were immobilized by a brief piezo-pulse. Then, a Piezo-micromanipulator (MB-U; PRIM TECH, Japan) was used to inject the sperm into bovine metaphase II (MII) oocytes with the cumulus cells removed. The injected oocytes were then cultured for 6 h in tissue cuture medium TCM-199 supplemented with 10% fetal calf serum in 5% CO2 at 38.5°C. All of the oocytes injected with human sperm were treated with 7% ethanol in TCM-199 at 4 h post-ICSI for 5 min to artificially induce activation (Horiuchi et al., 2002
).
DTT treatment of human sperm and taxol treatment of bovine oocytes following ICSI
Both fertile donor sperm and DFS sperm were treated for 1 h with 5 mmol/l DTT (Sigma Chemical Co., USA) in modified HTF medium (Irvine Scientific Co., USA). After washing in modified HTF supplemented with 10% serum substitute supplement (SSS) (Irvine Scientific), we injected either the dead sperm from a fertile donor or the DFS sperm into bovine oocytes. Six hours after the injection, a subset of the oocytes was treated with 2 µmol/l Taxol in TCM-199 for 1 h. All oocytes were fixed at 7 h post-ICSI. Examinations were performed at each time of fixation; the experiment was repeated three times.
Immunocytochemical detection of microtubules and DNA
Zonae pellucidae were removed using M2 culture medium (Sigma) supplemented with 0.75% protease (Actinase E; Kaken Chemical Co., Japan). After a 30 min recovery period, zona-free oocytes were extracted 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% (v/v) methanol and 1% (v/v) Triton X-100 detergent for 15 min. After fixation in cold methanol for 10 min (Simerly and Schatten, 1993), microtubules were labelled with a mixture of monoclonal antibody specific for
-tubulin (clone 2-28-33; Sigma) and acetylated
-tubulin (clone 6-11-B1; Sigma). Primary antibodies were detected using a fluorescein-conjugated goat anti-mouse IgG (Zymed, USA). DNA was detected by labelling with 10 mg/ml Hoechst 33342. After mounting in anti-fade medium (Vectashield; Vector Labs, USA), oocytes were examined using an epifluorescence microscope (DMRXA/HC; Leica, Germany). Images, acquired using Leica Q550FW (Leica, UK), were digitally recorded using Adobe Photoshop software (Adobe Systems Inc., USA). These data were used to compare the rate of sperm aster formation for each group of injected oocytes using the
2-test. P<0.01 were considered to be statistically significant.
Immunocytochemical detection of centrin expression in human sperm
Fertile donor and DFS sperm samples, mounted on coverslips, were fixed using 2% formaldehyde in modified HTF for 1 h at 37°C. Centrin was detected with a polyclonal antibody (anti-centrin-1; Sigma). Primary antibody was detected with an affinity-purified goat anti-rabbit immunoglobulin (whole molecule) conjugated to tetramethylrhodamine isothiocyanate (TRITC; Sigma). Microtubules and DNA were detected as described for the fixed bovine oocytes. Sperm samples on coverslips were mounted in anti-fade medium and were examined on an epifluorescence microscope. A total of 1000 sperm was analysed in each sample.
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Results |
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Discussion |
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In cases of human necrozoospermia, oocytes injected with dead sperm exhibit fertilization failure (Tournaye et al., 1996). In this study, male pronuclear formation and sperm centrosomal function in bovine oocytes were absent from dead human sperm. While sperm from fertile donors activated bovine MII oocytes at a high rate of
80% without artificial activation (Nakamura et al., 2001
), dead sperm could not activate bovine MII oocytes. Therefore, artificial activation of injected oocytes was achieved by ethanol treatment. However, artificial oocyte activation did not improve sperm centrosomal function. Normally, the reducing environment of the mammalian oocyte facilitates the breaking of disulphide bonds, allowing pronuclear decondensation and possibly centrosomal decondensation (Mellon and Rebhun, 1976
; Oliver et al., 1976
; Sanders and Salisbury, 1994
). The DTT priming of dead sperm promoted the decondensation of the sperm nucleus, but was ineffective to restore the deficient sperm centrosomal function. Thus, we attempted to reinstate the centrosomal function of dead sperm using a cytoskeletal accelerant. Taxol treatment alone induced microtubule organization in the bovine oocyte cytoplasm; however, the organization of microtubules by the dead sperm centrosome was not observed. We then attempted the priming of sperm by DTT in conjunction with Taxol treatment of injected bovine oocytes. This treatment was effective to restore the centrosomal function of dead human sperm. We could observe the sperm aster, an astral microtubule array, at a significantly higher rate than that seen with the other treatments. We speculated that DTT induced the decondensation of the sperm nucleus and centrosome, accelerating the separation of the tail microtubule from the basal body after ICSI. Upon oocyte activation, the naked sperm centrosome is easily affected by Taxol for use as an accelerator of microtubule organization. Dead sperm may have some alterations in their centrosomes that make the release of their disulphide bonds difficult, thereby affecting centrosomal function. It appears likely, however, that these defects are not severe alterations of the sperm centrosome, because these structures could introduce sperm aster formation following treatment with DTT and Taxol. Interestingly, bovine oocytes treated with Taxol after ICSI exhibited astral microtubule organization within the oocyte cytoplasm. It is possible that bovine oocytes have a few MTOC in their cytoplasm that may function in parthenogenetic development (Navara et al., 1994
). Such structures, however, are not observed in normal fertilization. Taxol may induce the microtubule organization from maternal MTOC, as observed in this study.
DFS sperm centrosome did not display any function, even after combined treatment with DTT and Taxol. DFS sperm exhibit abnormal centrin expression, indicating severe sperm centrosomal dysfunction. Rawe et al. (2002) reported the abnormal alignment of the headtail junction in DFS sperm, suggesting this abnormality as the cause of the fertilization failure. The combination of alterations in the necktail junction and the aberrant expression of centrin in DFS sperm may inhibit the release of the sperm centrosome from the basal body. If this is the case, these sperm would exhibit severe centrosomal dysfunction, despite DTT treatment of the sperm and Taxol treatment of the injected oocytes.
In conclusion, we examined human sperm centrosomal function using the heterologous ICSI system in an attempt to restore deficient sperm centrosomal function by treatment with DTT and Taxol. Although untreated dead sperm exhibited centrosomal dysfunction, they responded well to DTT treatment before ICSI in combination with the Taxol treatment of oocytes after ICSI. Sperm from a patient with DFS, a teratozoospermia resulting in infertility, displayed centrosomal dysfunction and abnormal centrin expression. DTT and Taxol treatments were not effective at restoring DFS sperm centrosomal function, indicating that the deficiency of DFS sperm in centrosomal function may be too severe to be saved by this treatment method. The heterologous ICSI system of sperm priming by DTT followed by oocyte Taxol treatment after sperm injection, however, may be a new method to assess sperm centrosomal function. Needless to say, this assessment system cannot be a tool for clinical rescue of sperm centrosomal dysfunction, because we are presently unable to assess the safety of this cytoskeletal manipulation.
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Acknowledgements |
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References |
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Submitted on December 16, 2004; resubmitted on February 13, 2005; accepted on March 4, 2005.
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