Molecular Human Reproduction, Vol. 5, No. 9, 836-844,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular aspects of fertilization |
Sperm integrity is critical for normal mitotic division and early embryonic development*
1 The Center For Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York Presbyterian Hospital, New York, New York 10021, and 2 Department of Obstetrics and Gynecology, The Methodist Hospital, Sixth Street, Brooklyn, New York 11215, USA
Abstract
The human zygote relies on the paternal gamete to provide the centrosome component essential for the first mitotic division. It is not known whether normal centrosome function requires an intact spermatozoon, or whether donation of an isolated paternal centrosome component can result in normal zygotes and embryos. To explore this possibility, mature human oocytes were microinjected with either intact or dissected spermatozoa. Fertilization and cleavage rates were documented; nuclear and cytoskeletal changes were observed with fluorescent immunocytochemistry; and chromosomal normality was assessed with fluorescent in-situ hybridization. A pilot study was performed to identify cytoskeletal features suggestive of centrosome function. Unfertilized oocytes and tripronucleate (3PN) zygotes from in-vitro fertilization or intracytoplasmic sperm injection were assessed to confirm the sequence of the landmarks of human fertilization. Oocytes injected with mechanically-dissected spermatozoa appear to be capable of normal pronuclear formation and embryonic cleavage, but do not undergo normal mitotic division. Although decondensed, apposed nuclei are noted in combination with diffuse cytoskeleton assembly, no spindle was detected in any zygote resulting from the injection of a dissected spermatozoon. Analysis of selected embryos resulting from dissected sperm injection revealed chromosomal mosaicism in the majority of specimens. The lack of a bipolar spindle, in combination with chromosomal mosaicism, suggests abnormalities of the mitotic apparatus when sperm integrity is impaired following dissection.
abnormal fertilization/cytoskeletal assessment/ICSI/sperm head/sperm tail
Introduction
Sperm penetration is responsible for oocyte activation which effects completion of the second meiotic division, induces exocytosis of the cortical granules, and permits extrusion of the second polar body. Intracytoplasmic sperm injection (ICSI) bypasses the natural steps involved in zona pellucida penetration and obviates the need for sperm fusion with the oolemma (Palermo et al., 1995a
). Complete failure of fertilization with ICSI occurs at a rate of <2%, and abnormal fertilization involving the formation of a single pronucleus (1PN) or three pronuclei (3PN) accounts for ~12% of the resulting zygotes (Sultan et al., 1993; Palermo et al., 1995b; Moomjy et al., 1998). Parthenogenic activation has been shown to be responsible for the single pronucleus observed with ICSI (Sultan et al., 1993). The 3PN zygotes from ICSI are digynic, being characterized by one polar body. This is in contrast to the 3PN zygotes from standard in-vitro fertilization (IVF) that are dispermic (Palermo et al., 1994). The sequence of submicroscopic events influencing normal and abnormal zygote development during assisted fertilization is currently being explored.
The centrosomal body is of particular importance in ordering the cascade of fertilization events. Earlier this decade it was reported that centrioles in close proximity to the pole of the mitotic spindle, detected by transmission electron microscopy (TEM), were identical to sperm centrioles (Sathanathan et al., 1991). Whereas enucleation of the ICSI-derived 3PN (which are usually digynic), results in diploid embryos, enucleation of the IVF-derived 3PN (which are usually dispermic), results in mosaic embryos. These results led to the confirmation of the paternal inheritance of the human centrosome (Palermo et al., 1994, 1997). The mosaicism of embryos obtained from enucleated IVF 3PN is apparently due to an abnormal tripolar spindle generated by the presence of an additional sperm centrosome component. Further confirmation of this theory came from immunofluorescent staining of microtubules of human zygotes (Simerly et al., 1995). With the exception of the meiotic spindle, the human oocyte is devoid of centriolar structures until penetration by the spermatozoon; after which a radial microtubular array can be observed. Maternally derived 
-tubulin contributes to normal centrosome function by nucleating the paternally-derived microtubule organizing centre (Schatten, 1994). This array, the sperm aster, develops as the male pronucleus decondenses and guides the male and female nuclei into apposition at the centre of the cytoplasm (Van Blerkom et al., 1995). A bipolar spindle can be detected at the time of nuclear apposition with its focused end-points, which probably represents the replicated centrosome (Schatten et al., 1994; Sathananthan et al., 1996; Palermo et al., 1997). The critical role that the centrosomal body maintains in early embryonic development has stimulated a great deal of interest in understanding the relationship between the centriole and the nucleus within the spermatozoon. It has been reported that chemical isolation of the sperm centrosome is possible (Van Blerkom and Davis, 1995). Sperm aster formation was noted after injection into mature human oocytes of tail segments with an intact midpiece and positive mitochondrial staining. However, spindle development and embryonic competence were not assessed. Additionally, microsurgical dissection of the spermatozoon, if associated with morphologically and chromosomally normal embryos, may be more feasible than chemical processing of the spermatozoon. Centrosome defects have been considered as a possible aetiology for fertilization failure (Schatten et al., 1994; Asch et al., 1995; Sathananthan et al., 1996; Van Blerkom, 1996). A greater understanding of centrosome function should further illuminate the intimate steps of the fertilization process and early embryonic development.
This study was undertaken in order to analyse and identify specific cytoskeletal and microtubular patterns associated with the fertilization process, particularly normal and abnormal pronucleus formation. In order to study sperm aster formation and its relationship with centriolar and nuclear structures, mechanically-dissected sperm heads and/or tails were injected into oocytes after which fertilization and early embryo cleavage rates were assessed. The immunofluorescent cytoskeletal characteristics of experimentally injected zygotes were analysed. Finally, the chromosomal constituency within selected embryos was elucidated by fluorescent in-situ hybridization (FISH) of individual blastomeres.
Materials and methods
Specimen collection
Fresh and in-vitro matured oocytes were obtained from consenting couples undergoing ICSI treatment. The study was reviewed and approved by the Committee on Human Rights in Research of the New York Hospital-Cornell Medical Center (protocol numbers 0696–389 and 0495–844). These oocytes underwent experimental injection with either intact or dissected spermatozoa, as described below. As a pilot study, abnormally fertilized oocytes (following IVF or ICSI) were studied; these oocytes were also obtained from consenting couples. Immunofluorescent imaging of unfertilized oocytes was performed to ascertain the cytoskeletal and nuclear features associated with failure of fertilization or parthenogenic activation while similar imaging of 3PN zygotes served as a proximate extrapolation of the cytoskeletal and nuclear changes suggestive of active interaction of male and female gametes. The dispermic 3PN IVF zygotes were compared with the digynic 3PN ICSI zygotes in an effort reveal the cytoskeletal features of paternal centrosome function.
Injection procedure and embryological evaluation
A total of 229 mature oocytes were microinjected with intact spermatozoa (n = 69), isolated sperm heads (n = 70), separated sperm heads and tails (n = 59), or isolated sperm tails (n = 31). Of these, 86 experimental specimens were cultured for 72 h in vitro and assessed for embryonic cleavage, and 25 were subsequently karyotyped. A total of 37 zygotes resulting from experimental injections were fixed and subjected to immunofluorescent analysis. During the pilot study, 88 abnormally fertilized zygotes were also subjected to immunofluorescent analysis. ICSI was performed with intact spermatozoa, according to the standard procedure (Palermo et al., 1995). Microdissection of spermatozoa was effected by applying the pressure of the bevel of a cytoplasmic microinjection pipette at the base of the sperm head, dislocating the sperm head from the flagellum with midpiece intact. A single intracytoplasmic injection was performed to deliver the dissected sperm segment(s) into the oocyte. Assessment of PN formation was carried out 16–20 h after injection.
Cytoskeletal analysis
Serial fixation of the zygotes was performed at 24–36 h. The fixation technique initially involved attachment to a poly-L-lysine coverslip followed by treatment with cold methanol, but this was abandoned due to a 40% rate of damage or loss and results from this technique were not included. The subsequent fixation method which reduced the specimen loss rate to <3% involved attachment of the zygote to a poly-L-lysine slide via a fibrin clot (Czaban and Forer, 1985; Hunt et al., 1995). Only unfertilized and 3PN IVF specimens underwent the additional step of zonae removal in acidic human tubal fluid (HTF) as removal of zona-bound spermatozoa was necessary for clearer imaging. As zona removal contributed to specimen damage, all ICSI specimens and experimental specimens were analysed with their zona intact. Penetration of the zona can be demonstrated by immunofluorescent staining of the polar body. The final pictures include polar bodies when located laterally. Polar bodies located in a superior or inferior position were not included in the final immunofluorescent pictures. The fixation step was followed by a 30 min incubation (at 37°C) in a microtubule stabilization buffer containing 0.1 mol/l PIPES at pH 6.9, 5 mmol/l MgCl2 6H2O, 2.5 mmol/l EGTA, 2.0% formaldehyde, 0.5% Triton X-100, 1 µmol/l taxol, 10 IU/ml aprotinin and 50% deuterium oxide (Sigma Chemical Co, St Louis, MO, USA) (Messinger and Albertini, 1991). Three 15 min washes utilized a blocking solution containing phosphate-buffered saline (PBS) with 2% bovine serum albumin (BSA), 2% normal goat serum, 0.1 mol/l glycine and 0.01% Triton X-100. Immunofluorescence staining of the cytoskeleton (Hunt et al., 1995) was performed using ß-tubulin antibody, (N 357, diluted 1:100, Amersham, Arlington Heights, IL, USA; and E7, undiluted, Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA), and acetylated 
-tubulin antibody (clone 6–11 B-1, diluted 1:1000, Sigma Chemical Co). Antibodies to maternally-inherited centrosomic -tubulin were not used. Specimens were rinsed again in blocking solution, with the addition of RNAase. The microtubules were labelled with fluorescein isothocyanate (FITC)-conjugated anti-mouse antibody and the nuclei with propidium iodide. The ability of this assay to reliably detect microtubules and the mitotic spindle was confirmed by the fixation and staining of actively dividing cells from a pituitary tumour cell line. Cytoskeletal and chromatin imaging of both experimentally-injected specimens, and abnormally fertilized specimens, were carried out with a fluorescence microscope (Olympus B Max 50, New York/New Jersey Scientific, NJ, USA) and with an argon laser scanning confocal microscope (Molecular Dynamics, Sunnyvale, CA, USA). Adobe photoshop software was employed to combine different planar images obtained from a single specimen under the fluorescence microscope (Adobe Systems Inc, Mountain View, CA, USA). The final composite pictures within this text are from conventional fluorescence imaging.
Cytogenetic analysis
FISH was carried out using probes for chromosomes X, Y, 18, 13/21. Blastomere biopsy and FISH were performed as previously described (Grifo et al., 1992; Munné et al., 1993; Palermo et al., 1995, 1997). Each blastomere was placed in hypotonic solution (1% sodium citrate in water with BSA) for 5 min. Under the stereomicroscope, 10 µl of fixative (acetic acid in methanol at a concentration of 1:3) was then dropped on top of the cell and, with gentle puffing, cell membrane rupture was effected during evaporation. The slide was stored at –20°C until analysis. DNA probes for -satellite repeat clusters in the centromeric region of X and 18 chromosomes, and the satellite-III DNA on the long arm of the Y chromosome were used (Imagenetics, Naperville, IL, USA). The probe for Y was directly labelled with a red fluorochrome, the probe for 18 with a green fluorochrome (CEP Spectrum Orange and Green respectively; Imagenetics). The probe for X was labelled with a 1:1 mixture of red and green fluorochromes. The probe for chromosomes 13/21 was labelled with digoxigenin and rodamine-labelled anti-digoxigenin antibodies. Using a dual wavelength filter (Chroma Technology, Brattleboro, VT, USA), the Y chromosome appeared red, chromosome 18 green, and the X chromosome yellow. Chromosomes 13/21 gave a larger red signal than the Y chromosome. FISH chromosomal probes were evaluated with the Olympus fluorescence microscope.
Results
Five criteria were employed for cytoskeletal assessment: (i) diffuse cytoskeleton assembly is a diffuse lattice of interlacing fibres with either a granular or smooth texture; (ii) flecks of microtubules are small fluorescent irregularities, usually <10 in number, which are randomly dispersed throughout the cell. This finding could be made in addition to another noted pattern of cytoskeletal microtubules; (iii) central clearing of the cytoskeleton is a lattice of microtubules similar to diffuse cytoskeleton assembly, however, there is an absence of 
20% of the microtubules in the centre of the cell; (iv) absence of microtubules is a pattern lacking any microtubule immunofluorescence with the presence of microtubule staining of the polar body to serve as the positive control for the technique; and (v) aster or spindle formation includes the identification of the aster as a centripetal arrangement of microtubules emanating from one nucleus. The spindle is identified by the presence of either an aster type formation emanating from both nuclei (an unfocused spindle) or the presence of contiguous microtubules between both nuclei in a barrel type formation (a focused spindle). When an aster or spindle was documented, the pattern of microtubules throughout the cell was also recorded.
Nuclear features of all specimens were categorized as follows: (i) nuclear absence requires the identification of nuclear staining of the polar body to serve as a positive control; (ii) dispersed karyomeres is the presence of small irregular nuclear fragments randomly located within the cell; (iii) condensed nuclei are densely stained small nuclei with distinct margins usually round or oval in appearance; (iv) decondensed nuclei are noted when there is heterogeneous nuclear staining of a nucleus, up to four times larger than the size of a condensed nucleus. Nuclear margins are soft, irregular and not distinct; (v) mixed nuclei are a combination of condensed and decondensed nuclei; (vi) appposed nuclei exist when the nuclei are equivalent in size and the distance separating them is less than one nuclear diameter; (vii) formed chromosomes are densely stained, wavy condensed chromosomes which may be clustered together or in a linear array.
Pilot study: cytoskeletal analysis of abnormal fertilization after IVF or ICSI
Comparison of unfertilized eggs and abnormally fertilized zygotes revealed many qualitatively different features of the cytoskeleton and of the nucleus (Tables I and II). Immunofluorescent analysis of cytoskeletal and nuclear features was performed on 33 unfertilized eggs and 55 abnormally fertilized zygotes from IVF and ICSI.
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Microtubules were distinctly absent for 41% of the unfertilized oocytes from IVF but present in all other specimens (Figure 1c
). Flecks of microtubules were observed in 67% of the 1 PN from ICSI and 27% of the unfertilized oocytes from ICSI (Figure 2a). This feature was infrequently found in IVF specimens. Diffuse cytoskeleton assembly detection (Figures 2c, 3a,b) ranged from 32% of unfertilized IVF oocytes to 88% of ICSI tripronucleates. Central clearing (Figures 1d,e, 2b,d, 3c) was noted for all specimens, but the greatest rate of 50% was noted for the 1PN from ICSI. Aster or spindle formation was primarily detected in 3PN from IVF at a rate of 29% (Figures 3c–h). Of note, no 3PN from ICSI revealed aster or spindle formation (Figure 2c,d) (Table I
).
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Condensed nuclei (Figures 1b,c, 2b
) occurred in 45–55% of unfertilized IVF and ICSI oocytes and 1PN from ICSI. Decondensed nuclei (Figures 2a,c,d, 3a,b) occurred in 88% of the 3PN from ICSI and 50–59% of all other specimens except for unfertilized oocytes from ICSI. Mixed nuclei (Figure 2b) were uncommon but did affect up to 33% of 1PN and unfertilized oocytes. Apposed nuclei (Figures 2c, 3a,b) were generally confined to 3PN from ICSI and IVF with rates of 25 and 46% respectively. Formed chromosomes (Figures 2b, 3c,d,e,h) were detected in a minority of all specimens, <18% of any group. Dispersed karyomeres (Figure 1e) occurred in 27–55% of the unfertilized IVF and ICSI specimens respectively (Table II). Pituitary tumour cells used as assay controls uniformly showed diffuse cytoskeleton assembly only; and large nuclei with some occasional decondensed nuclei associated with spindle formation (Figure 1f). There was negligible non-specific binding of antibodies to the slide.
Embryological and cytoskeletal analysis of experimental fertilization by sperm segment injection
The results of injections of different sperm components are summarized in Table III. There were 2PN in 67% of the oocytes injected with an intact spermatozoon; in 44% injected with a sperm head; and in 46% injected with a separated sperm head and tail. Only 16% of the oocytes injected with an isolated tail segment resulted in activation. Subsequent to dissected sperm injections, 3PN developed in up to 14% of the oocytes and 1PN development occurred in 9–19%. Fertilization failed to occur in 27–32% of the oocytes injected with head or head and tail components and in 71% of oocytes injected with sperm tail only. Oocytes injected with intact spermatozoa had the lowest rate of fertilization failure (16%). Other than those injected with an isolated tail segment in which the cleavage rate was a mere 24%, a significant proportion (52–64%) of experimentally injected oocytes displayed regular cleavage despite chromosomal constituency (Table III).
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With regard to microtubular and nuclear organization (Tables IV and V
), the experimental injections of sperm head or sperm head and tail revealed diffuse cytoskeleton assembly (Figure 4b,e,g) to at a rate of 40–60%, similar to injections of intact spermatozoa (Figure 4a). Isolated tail injections yielded only a 25% rate of cytoskeletal assembly. Absence of microtubules affected 20% of oocytes injected with head fragment only, while not being detected in any other specimens. Flecks of microtubules were quite common for experimental zygotes (Figure 1a) affecting 50, 25, and 25% of isolated head fragment, isolated tail fragment, and combined head and tail fragment injections respectively. Central clearing (Figure 4c,d) was observed in 75, 38, and 30% of the zygotes resulting from injections with tail segment only, combined head and tail segment, and isolated head segment injections respectively. Aster or spindle formation was noted in 20% of intact sperm-injected oocytes (Figure 4a), for 13% of zygotes from isolated head segment injection (Figure 4e), and in none of the remaining experimental zygotes.
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With regard to nuclear assessment, experimentally-injected oocytes revealed that decondensation of nuclei (Figure 4b,c
) was achieved by 40, 75, and 53% of intact spermatozoa, combined separated head and tail fragment, and isolated head fragment injections respectively. None of the tail only injection resulted in nuclear decondensation. Apposition of male and female PN (Figure 4b,c,d) occurred in 75% of the injections with separated sperm head and tail, in 30% with intact spermatozoa and in 20% with isolated sperm head. Dispersed karyomeres (Figure 4f,h) occurred at the greatest frequency in tail only injections. Only one head and tail injected specimen formed chromosomes (Figure 4g).
Chromosomal analysis of embryos resulting from experimental injection of sperm segments
FISH analysis was successful in 25 out of 29 cleaved embryos. Chromosomal mosaicism was detected in 11 out of 12 embryos derived from isolated sperm head, 10 out of 11 embryos from separated sperm head and tail, and two out of two embryos from isolated tail segments. A normal diploid chromosomal constituency was detected for one embryo derived from isolated sperm head injection and for one embryo derived from separated head plus tail injection (Table VI).
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Discussion
Unfertilized IVF specimens may have any type of nuclear arrangement from dispersed karyomeres to formed chromosomes. This is congruent with a previous cytoskeletal and nuclear study of failed fertilization of human oocytes (Asch et al., 1995) Similarly, unfertilized ICSI specimens have all stages of nuclear progression except apposed nuclei. The lower incidence of nuclear decondensation noted amongst the unfertilized ICSI oocytes in comparison to the unfertilized IVF oocytes may reflect late fertilization of the IVF oocytes or, alternatively, intrinsic sperm nuclear dysfunction of the ICSI spermatozoon. However, given the high rate of nuclear decondensation for digynic ICSI tripronucleates, lack of nuclear decondensation may represent a dysfunction of the oocyte. Diffuse cytoskeletal assembly was noted for one third of the unfertilized IVF oocytes and for two-thirds of the unfertilized ICSI oocytes. This difference may be explained by increased activation in the process of aspiration of the cytoplasm during ICSI. Absence of microtubules is a feature found only in unfertilized IVF specimens and is, therefore, to be interpreted as complete lack of sperm function or penetration and possibly oocyte degeneration. This is in agreement with previously published images showing a lack of microtubules only for the unfertilized oocyte (Simerly et al., 1995).
Approximately half of the 1PN from ICSI had nuclear condensation and half had nuclear decondensation, none had dispersed karyomeres. Flecks of microtubules, while present in most of these specimens, may either represent disorganization of the cytoskeleton related to breakage of the oolemma during ICSI (Palermo et al., 1996) or an inability to generate a mitotic spindle secondary to the haploid status. It has been shown that 90% of the 1PN from ICSI are haploid, while those from IVF are diploid at a rate of 60–80% (Sultan et al., 1995).
The 3PN ICSI zygote shown to have a triploid constitution and a single functioning centrosome (Palermo et al., 1994) represents a failure of extrusion of the second polar body, probably related to excessive disruption of the oolemma (Palermo et al., 1996). It would be anticipated that immunofluorescent results should be similar to a normal zygote with 2PN. As it is not possible to fix and assess the normal zygote, the experimental injection of an intact spermatozoon into an in-vitro matured oocyte should have similar cytoskeletal and nuclear features. This similarity between the ICSI 3PN and the intact sperm-injected oocyte was observed. The vast majority of ICSI 3PN had decondensed nuclei, which could be interpreted as enhanced nuclear decondensation by this digynic zygote. Diffuse cytoskeletal assembly was noted for the majority of ICSI 3PN and the majority of intact spermatozoa injected into in-vitro matured oocytes.
A marked variation of aster and spindle formation was noted amongst the IVF 3PN. In this category, the dispermic, monogynic, IVF zygote has two sperm centrosome components and permits inferences regarding sperm-related centrosomal function (Palermo et al., 1994). The fact that spindles or asters were detected so frequently in the IVF 3PN may be secondary to a dysfunction of the spindle in these states that delays the rapid progression through mitosis, since cleavage of the IVF 3PN is known to be delayed (Sathanathan et al., 1991). According to the differences presented herein between 3PN and unfertilized oocytes and 1PN, we suggest that apposition of the nuclei or polarity of the spindle fibres should be interpreted as evidence of sperm centrosome function. This is in agreement with an extensive study of the spatial and temporal nuclear and cytoplasmic changes associated with sperm penetration (Van Blerkom et al., 1995). This is also in agreement with a cytoskeletal analysis of unfertilized oocytes for a few couples unable to achieve fertilization with ICSI, which documented small condensed, unapposed nuclei with truncated or absent sperm asters, interpreted as evidence of sperm centrosome dysfunction (Van Blerkom, 1996). It has been concluded from human and mammalian studies that apposition of pronuclei, and of the nucleoli or chromatin, is indicative of sperm centrosome function (Edwards and Beard, 1997). The importance of juxtaposed pronuclei has also been demonstrated in a clinical study to be positively associated with higher implantation rates for patients undergoing pronuclear embryo transfers (Scott and Smith, 1998).
Sperm nuclear decondensation has been shown by time-lapse video cinematography to be the last morphological change to precede extrusion of the second polar body (Payne et al., 1997). If decondensed apposed nuclei truly represent progress in early fertilization, then the experimental specimens resulting from isolated sperm head injections and from combined separated sperm head and tail injections did indeed illustrate significant submicroscopic progress in early fertilization. Diffuse cytoskeleton assembly was similar among experimental groups except for isolated tail fragment injections. Diffuse cytoplasmic assembly may be the immunohistochemical feature that is correlated with the cytoplasmic flare, which has been shown to immediately precede male pronuclear formation in time-lapse video cinematography (Payne et al., 1997). Aster or spindle formation was detected for isolated sperm head injections at a similar rate as intact sperm injections. This present study suggests that association of the centrosome component with the tail segment is far less functional than association of the centrosome component with the head segment given the impaired activation, cleavage, cytoskeleton assembly, and nuclear decondensation for tail segment injections. Flecks of microtubules, which seem to suggest damaged or disorganized cytoskeleton, was most prevalent for head only injections. Evidence at many levels supports the presence of an active centrosome component in some oocytes injected with a sperm head. This evidence includes formation of 2PN; morphologically normal embryo cleavage; apposition of the nuclei; an occasional aster or atypical spindle; and an occasional diploid constitution. The sperm tail-only injections did not display any of these features thereby suggesting insufficient or absent centrosome function. The similar findings of sperm head injections in comparison to head and tail injections suggest that the isolated sperm head resulting from mechanical dissection may deliver a functional centrosome component. Coincident to this study, centrosomal immunofluorescent labelling revealed a similar frequency of association of the centrosome component with the dissected sperm head as with the dissected tail (Palermo et al., 1997).
The ability of round spermatids to achieve fertilization has been disappointing. While there have been a few pregnancies and births after ICSI of round spermatids or secondary spermatocytes (Tesarik et al., 1995; Antinori et al., 1997; Vanderzwalmen et al., 1997; Kahraman et al., 1998; Sofikitis et al., 1998), our unpublished data include 15 patient cycles with no normally fertilized oocytes for round cell injection in the setting of complete spermatogenic arrest. The need to precisely isolate a round spermatid from other round cells including lymphocytes and diploid germ cells remains the current challenge. Flow cytometry coupled to cell sorting may prove a reliable method for round spermatid isolation (Ziyyat et al., 1999). Nevertheless, poor to absent fertilization rates with round spermatid injection may reflect a dysfunctional or non-functional centrosome. The development of a technique which could isolate a functional paternal centrosome from the chromatin material could be applied in clinical research to assist the vast majority of patients with spermatogenic arrest who have not experienced fertilization with round spermatid injection.
The high rate of chromosomal mosaicism with this mechanical sperm dissection technique supports the need to investigate other avenues of dissection or separation of the sperm's chromosomal contents from its other functional elements. Sonication to effect dissection of sperm head from tail has been reported to result in sperm aster formation following ICSI (Van Blerkom and Davis, 1995). Chromosomal normality with this method remains to be tested. Further exploration towards effective microsurgical and/or chemical methods of sperm dissection is needed. The positive embryological and nuclear and cytoskeletal findings presented herein suggests that normal diploid fertilization following sperm dissection might be achievable.
Acknowledgments
The authors are greatly indebted to A.K.Rajasekaran, Department of Cell Biology and Anatomy, Weill Medical College, for technical assistance with confocal microscopy and for donation of the pituitary tumour cells. The authors also express gratitude to Richard S.LaRocco for preparation of the photos.
Notes
* Presented at the Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, 1996 
3 To whom correspondence should be addressed at: Center for Reproductive Medicine and Infertility, HT-360, 505 E70th Street, New York, NY 10021, USA
References
Antinori, S., Versaci, S., Dani, G. et al. (1997) Fertilization with human testicular spermatids: four successful pregnancies. Hum. Reprod., 12, 286–291.
Asch, R., Simerly, C., Ord, T. et al. (1995) The stages at which human fertilization arrests: microtubule and chromosome configurations in inseminated oocytes which failed to complete fertilization and development in humans. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, 1897–1906.
Czaban, B.B. and Forer, A. (1985) The kinetic polarities of spindle microtubules in vivo, in crane-fly spermatocytes. Kinetochore microtubules that reform after treatment with colcemid. J. Cell Sci., 79, 1–37.[Abstract]
Edwards, R.G. and Beard, H.K. (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol. Hum. Reprod., 3, 863–905.
Grifo, J., Tang, Y.X., Cohen, J. et al. (1992) Ongoing pregnancy in hemophilia carrier by embryo biopsy and simultaneous amplification of X and Y chromosome specific DNA from single blastomeres. J. Am. Med. Assoc., 6, 727–729.
Hunt, P., LeMaire, R., Embury, P. et al. (1995) Analysis of chromosome behavior in intact mammalian oocytes: monitoring the segregation of a univalent chromosome during female meiosis. Hum. Mol. Genet., 4, 2007–2012.
Kahraman, S., Polat, G., Samli, M. et al. (1998) Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum. Reprod., 13, 104–110.
Messinger, S.M. and Albertini, D.F. (1991) Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J. Cell Sci., 100, 289–298.
Moomjy, M., Sills, E.S., Rosenwaks, Z. and Palermo, G.D. (1998) Implications of complete failed fertilization after intracytoplasmic sperm injection for subsequent fertilization and reproductive outcome. Hum. Reprod., 13, 2212–2216.
Munné, S., Lee, A., Rosenwaks, Z. et al. (1993) Diagnosis of major chromosomal aneuploidies in human preimplantation embryos. Hum. Reprod., 8, 2185–2191.
Palermo, G.D., Munné, S. and Cohen, J. (1994) The human zygote inherits its mitotic potential from the male gamete. Hum. Reprod., 9, 1220–1225.
Palermo, G.D., Cohen, J., Alikani, M. et al. (1995a) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil. Steril., 63, 1231–1240.[Web of Science][Medline]
Palermo, G.D., Munné, S., Colombero, L.T. et al. (1995b) Genetics of abnormal human fertilization. Hum. Reprod., 10, 120–127.
Palermo, G.D., Alikani, M., Bertoli, M. et al. (1996) Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic injection. Hum. Reprod., 11, 172–176.
Palermo, G.D., Colombero, L.T. and Rosenwaks, Z. (1997) The human sperm centrosome is responsible for normal syngamy and early embryonic development. Rev. Reprod., 2, 19–27.[Abstract]
Payne, D., Flaherty, S.P., Barry, M. and Matthews, C.D. (1997) Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum. Reprod., 12, 532–541.
Sathananthan, A.H., Kola, I., Osborne, J. et al. (1991) Centrioles in the beginning of human development. Proc. Natl. Acad. Sci. USA, 88, 4806–4810.
Sathananthan, A.H., Ratnam, S.S., Ng, S.C. et al. (1996) The sperm centriole: its inheritance, replication and perpetuation in early human embryos. Hum Reprod., 11, 345–355.
Schatten, G. (1994) The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev. Biol., 165, 299–335.[Web of Science][Medline]
Scott, L.A. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod., 13, 1003–1013.
Simerly, C., Wu, G.-J., Zoran, S. et al. (1995) The paternal inheritance of the centrosome, the cell's microtubule-organizing center, in humans, and the implications for infertility. Nat. Med., 1, 47–52.[Web of Science][Medline]
Sofikitis, N., Yamamoto, Y., Miyagawa et al. (1998) Ooplasmic injection of elongating spermatids for the treatment of non-obstructive azoospermia. Hum. Reprod., 13, 709–714.
Sultan, K., Munné, S., Palermo, G. et al. (1995) Chromosomal status of uni-pronuclear human zygotes following in-vitro fertilization and intracytoplasmic sperm injection. Hum. Reprod., 10, 132–136.
Tesarik, Y., Mendoza, C. and Testard, J. (1995) Viable embryos from injection of round spermatids into oocytes. N. Engl. J. Med., 333, 525.
Van Blerkom, J. (1996) Sperm centrosome dysfunction: a possible new class of male factor infertility in the human. Mol. Hum. Reprod., 2, 349–354.
Van Blerkom, J. and Davis, D. (1995) Evolution of the sperm aster after microinjection of isolated human sperm centrosomes into meiotically mature human oocytes. Hum. Reprod., 10, 2179–2182.
Van Blerkom, J., Davis, P., Merriam, J. and Sinclair, J. (1995) Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human. Hum. Reprod. Update, 1, 429–461.
Vanderzwalmen, P., Zech, H., Birkenfeld, A. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type or selected spermatids and oocyte activation. Hum. Reprod., 12, 1203–1213.
Ziyyat, A., Lassalle, B., Testart, J. et al. (1999) Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients. Hum. Reprod., 14, 379–387.
Submitted on January 18, 1999; accepted on June 1, 1999.
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