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Regular Article |
a Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, gamete biology, sperm, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Kuretake et al. [1] demonstrated that in ICSI, spermatozoa treated with TX could fertilize oocytes with the same efficiency as intact sperm heads. Such oocytes resulted in preimplantation and postimplantation development (i.e., live offspring) that was comparable to that of the control [2]. Treating human spermatozoa with TX before ICSI resulted in the fastest and most efficient oocyte activation and sperm head decondensation, suggesting that it is beneficial rather than detrimental [3]. As for DTT, pretreatment of spermatozoa with this reagent alone enhances the efficiency of ICSI with bovine [4, 5] or minke whale [6] spermatozoa. In addition, DTT has also been used in sperm cryopreservation [7] and long-term storage [8] and in liquefaction of nonliquefied human semen in vitro [9]. Furthermore, in at least one experimental condition, incubation of DTT with zygotes was found to prevent chromosomal aberrations that would have otherwise occurred [10].
In light of these reports, our preliminary finding that the combination of these two agents caused chromosomal damage warranted further examination. The most evident role of detergent is to solubilize proteins and to disrupt or remove sperm membranes. The latter, however, can be easily damaged (i.e., partially removed), e.g., by harsh manipulation, during different assisted reproduction technologies (ARTs). Because poor-quality sperm samples from men exhibiting severe oligo-, astheno-, or teratozoospermia are commonly used in ART, it seems reasonable to suspect that these spermatozoa already have some morphological anomalies, including membrane impairment, or can be sensitive to cell damage by external factors. It is then possible that the situation, which we attempted to mimic in our study (DTT and membrane disruption), takes place in ART laboratories. Moreover, because both TX and DTT are either currently being used [7, 8] or have been suggested to be applicable [3] in human ART, a combination of these two reagents probably will be attempted in the near future.
In the present study, we tested the hypothesis that DTT and detergent (TX or the ionic detergent mixed alkyltrimethylammonium bromide [ATAB]) act in a synergistic fashion to generate breaks in paternal chromosomes in the zygote. We confirmed that whereas neither detergent nor DTT alone generated aberrant chromosomes, the combination of both reagents resulted in a majority of the paternal karyoplates being abnormal. Furthermore, this could be partially reversed by EGTA and EDTA, suggesting a role for a sperm nuclease in this effect. These data suggest that DTT needs to be used with caution during ART procedures that manipulate spermatozoa in harsh manners and may disrupt membranes, and that the combination of TX and DTT should not be attempted in human ICSI. They also suggest that sperm nuclei have mechanisms for chromosomal degradation that can be activated by detergent and DTT.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mineral oil was purchased from Squibb and Sons (Princeton, NJ), and amino acid solutions were from Gibco BRL (Grand Island, NY). All other inorganic and organic reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.
Animals
Mice B6D2F1 (C57BL/6J x DBA/2) were obtained at 6 wk of age from the National Cancer Institute (Raleigh, NC) and were used as sperm and oocyte donors. The mice were fed ad libitum with a standard diet and maintained in a temperature- and light-controlled room (22°C, 14L:10D) in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and the Guide for the Care and Use of Laboratory Animals. The protocol for animal handling was reviewed and approved by the Institutions of Animal Care and Use Committee at the University of Hawaii.
Media
The medium used for culture of mouse oocytes was modified KSOM (mKSOMAA) [11]. The medium for ICSI and oocyte washing was Hepes-buffered CZB [12]. The mKSOMAA was maintained in an atmosphere of 5% CO2 in air, and the Hepes-CZB was maintained in air alone.
Sperm Preparation
Cauda epididymes were removed from one male; the epididymal fluid was squeezed out and placed in chilled Hepes-CZB supplemented with additions as noted (0.5% [w/v] ATAB with or without 2 mM DTT or 0.5% [v/v] TX with or without 2 mM DTT). In all buffers, the pH remained constant (at 7.5) regardless of the detergent or DTT that was added. Spermatozoa were vigorously pipetted to disperse evenly in solution at room temperature, and 1 ml of this sperm suspension was then carefully layered over a 0-5 ml cushion of 1 M sucrose and 25 mM Tris (pH 7.4) in a microfuge test tube. This step gradient was centrifuged at 3000 x g in a swing bucket rotor for 10 min at 4°C. The pellet was resuspended in 100 µl of Hepes-CZB and used for ICSI. In some experiments, 50 mM EGTA or 50 mM EGTA + 50 mM EDTA were added to the Hepes-CZB solution before the addition of spermatozoa. Again, in these buffers, the pH was kept constant (at 7.4). In these experiments, the same concentration of EGTA or EGTA + EDTA was also added to the final suspension of spermatozoa after centrifugation.
Swim-up spermatozoa were prepared by gently placing a drop of epididymal fluid on the bottom of a microfuge tube containing 500 µl of Hepes-CZB and incubating for 15 min at 37°C. After 15 min, 200 µl were gently taken off the top of the suspension and incubated for an additional 15 min with or without 2 mM DTT.
Sperm Decondensation Assay
To test whether the protamines were reduced in all the types of sperm preparation described above, 10 µl of the final suspension was added to 10 µl of 4 M NaCl (to make a final concentration of 2 M NaCl) and incubated for 10 min at room temperature. This suspension was then added to an equal volume of 2 M NaCl containing 200 µg/ml of ethidium bromide and was examined under a fluorescent microscope. For samples in which the protamines were reduced, the 2 M NaCl was able to extract the protamines, and the sperm nuclei either decondensed completely or formed nuclear halos [13]. For the samples in which the protamines were not reduced, the sperm nuclei remained condensed even in the presence of 2 M NaCl.
For swim-up spermatozoa treated with DTT, the decondensation assay was modified slightly, because the final suspension of spermatozoa that was used for ICSI still had 2 mM DTT in the solution. It was therefore possible that when 4 M NaCl was added, the DTT would enter the sperm nucleus, because the membranes were damaged by the high salt concentration. Thus, to test for protamine reduction in swim-up spermatozoa, the sperm suspension was layered over 1 M sucrose and centrifuged at 3000 x g for 10 min at 4°C to remove the DTT before the salt extraction. The pellets were then resuspended in 50 µl of Hepes-CZB, an equal volume of 4 M NaCl was added, and the sperm nuclei were examined under a fluorescent microscope.
Oocyte Collection
Female mice were superovulated by i.p. injection of 5 IU of eCG (Calbiochem, San Diego, CA), followed 48 h later by an i.p. injection of 5 IU of hCG (Calbiochem). Ova were collected between 14 and 15 h post-hCG administration by removing entire oviducts and placing them into 10 ml of warm PBS. Oviducts were then transferred into 1% bovine testicular hyaluronidase (300 USP units/mg) in Hepes-CZB. Cumuli were released from the oviduct at the site of the cumulus bulge by tearing with a 25-gauge needle, and cumulus cells were removed. Oocytes were then transferred into a droplet of Hepes-CZB under mineral oil and used immediately for ICSI.
Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection was carried out according to the technique described by Kimura and Yanagimachi [12], except that all operations were performed at room temperature (25°C) instead of at 16–17°C. Treated spermatozoa (see above) were used for ICSI immediately after treatment. A small volume of sperm was mixed thoroughly with Hepes-CZB containing 12% (w/v) polyvinyl pyrrolidone (Mr 360 kDa). In addition, ICSI was performed with the use of an Olympus Inverted Microscope (model IX70; Olympus, Tokyo, Japan) equipped with an Eppendorf micromanipulation system (Micromanipulator Transfer-Man; Eppendorf, Hamburg, Germany) and a Piezo (PMM Controller, model PMAS-CT150, PMP; Prima Tech, Tsukuba, Japan). A single spermatozoon was drawn, tail first, into the injection pipette and moved back and forth until the head-midpiece junction (i.e., the neck) was at the opening of the injection pipette. The head was then separated from the midpiece by applying one or more Piezo pulses; the tails of the ATAB-treated spermatozoa had already been removed. After discarding the midpiece and tail, the head was redrawn into the pipette and injected into the oocyte. The whole procedure was repeated for all oocytes available. Then, ICSI was performed in Hepes-CZB within approximately 1 h. Only morphologically normal oocytes were used for injections. The injected oocytes were transferred into mKSOMAA medium and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Spermatozoa treated with ATAB were devoid of activation factor and the oocytes had to be activated after injection. Immediately after sperm injection, those oocytes were transferred into Ca2+-free CZB containing 5 mM Sr2+ and incubated for 4–6 h [14].
Approximately 6 h after ICSI, activation was estimated by scoring ova with pronuclei. Oocytes with two distinct pronuclei and the second polar body were considered to be normally fertilized. No delay in activation was observed in any treatment group. Fertilized oocytes were transferred into mKSOMAA containing 0.006 µg/ml of vinblastine and cultured until they reached metaphase of the first cleavage division.
Chromosomal Analysis
Injected oocytes were cultured in mKSOMAA for 6–8 h before being transferred into mKSOMAA containing 0.006 µg/ml of vinblastine. Vinblastine was added to prevent spindle formation and syngamy. Between 19 and 21 h after sperm injection, eggs arrested at the metaphase of the first cleavage were treated with 1% pronase (1000 tyrosine units/mg; Kaken Pharmaceuticals, Tokyo, Japan) for 5 min at room temperature to soften zonae pellucidae. The zygotes were then treated with hypotonic solution (1:1 [v/v] mixture of 1% sodium citrate and 30% fetal bovine serum) for 5 min at 37°C or 10 min at room temperature. Chromosomes were spread on slides by the gradual fixation/air-drying method [15]. The preparations were stained with 2% Giemsa (Merck, Darmstadt, Germany) in buffered saline solution (pH 6.8) for 10 min for conventional chromosome analysis. A spermatozoon was considered to be chromosomally normal when the examined oocyte contained 40 structurally normal chromosomes at the metaphase of the first mitotic division. We looked at the percentage of normal and abnormal metaphases (differentiating between metaphases with minor and multiple aberrations). When more than nine aberrations per karyoplate were observed, we called them multiple and scored as 10. The number of aberrations per spermatozoon, which was calculated by dividing the total number of aberrations by the total number of karyoplates examined in one treatment, expressed the intensity of the chromosomal damage. From three to six replicates of each experiment were performed. Cytogenetic analysis was performed on coded samples in a blind manner. At least 100 metaphase plates were examined for each treatment.
Statistics
In examination of sperm genetic stability, the percentage of sperm with normal chromosomes (from karyoplates examined) in all subgroups was analyzed and compared. Experiments within each treatment were repeated from three to six times. The variation between experiments within the same treatment was determined by calculating the percentage of normal karyoplates in each experiment, taking the mean, and calculating the standard deviation. Also, homogeneity test was performed for all treatments to confirm that it was correct to calculate and compare the means. Three tests (chi-square, likelihood ratio, and Fisher exact probability test) were used for analyzing all responses. The computations were done using KyPlot version 2.0-beta 13 software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the effect of DTT in combination with detergent on mouse sperm chromatin stability, we treated spermatozoa three different ways, each with and without DTT: 1) membrane disruption by vigorous pipetting, 2) treatment with the nonionic detergent TX, or 3) treatment with the ionic detergent ATAB. Live, swim-up spermatozoa (with or without DTT) were used as controls. We first verified that DTT reduced the protamine disulfides in each treatment. In each experiment, a small aliquot of treated spermatozoa was incubated in 2 M NaCl, then subjected to fluorescence microscopy with ethidium bromide staining. Without DTT, even in the presence of 2 M NaCl, the sperm nuclei remained condensed, because the protamines still contained intact disulfide cross-links (Fig. 1A). When the protamine disulfides were reduced, the salt was able to extract the protamines, and the sperm nuclei decondensed (Fig. 1B). In all four treatments, even with live, swim-up spermatozoa incubated with DTT, the sperm nuclei decondensed when DTT was included, indicating that the protamines were reduced in all cases.
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Intracytoplasmic Sperm Injection
We next determined whether the different treatments had any adverse effects on the ICSI procedure (Table 1). After all treatments, with the exclusion of swim-up controls, spermatozoa were immotile and physiologically "dead." However, when used for ICSI, they were able to participate in the next stages of the fertilization process: decondensation, pronuclei formation, and condensation into metaphase chromosomes. In membrane-damaged, "dead" spermatozoa, DNA disintegration occurs within time [16, 17]. Our previous observations with spermatozoa that were freeze-dried or frozen without cryoprotection proved that if injection was performed within 1 h, the chromosomal integrity of "dead" (i.e., membrane-disrupted) spermatozoa was retained [18]. All injections in the present study were completed within 1 h after sperm treatment. Injections were done from three to six times per treatment. After washing with ATAB ± DTT, sperm tails separated from the heads. After treatment with TX + DTT, heads and tails remained together but were softened. This made it difficult to detach sperm heads for ICSI and became a reason for the slight decrease in the egg survival rate in this group compared to all others (63% vs. 81–92%).
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DTT with Detergent Causes Chromosomal Damage
In each treatment, sperm nuclei were injected into oocytes for cytogenetic analysis, and paternal and maternal chromosomes of the zygote before the first cleavage were visualized and examined (Table 2 and Fig. 2). As expected, treatment of spermatozoa with DTT alone (swim-up + DTT) or with either TX or ATAB alone did not cause chromosomal damage of the paternal chromosomes more frequently than in the control swim-up spermatozoa without DTT. However, when these two reagents were combined, either detergent in the presence of DTT generated a substantial number of paternal karyoplates with visible chromosomal breaks. Treatment with the nonionic detergent TX + DTT caused the most significant damage, with only 22% of the karyoplates having normal chromosomes and 39% having severe aberrations. This treatment also gave the highest number of aberrations per sperm nucleus (5.1). Membrane disruption by vigorous pipetting did not cause a statistically significant difference in the number of normal karyoplates as compared to controls. Examples of chromosomal damage are shown in Figure 3.
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One of the causes of chromosomal breakage could be the release of endogenous nucleases from plasma membrane-damaged spermatozoa. Most endonucleases are dependent on calcium and/or magnesium ions. To address this possibility, we tested whether the presence of chelating agents (EGTA and EDTA) in the sperm isolation medium could contribute to the improvement of chromosome integrity. We examined the effect of EGTA and a mixture of EGTA and EDTA on chromosome stability in sperm treated with TX + DTT. When one or both chelators were present, we observed a slight increase in the frequency of normal karyoplates and a reduced number of aberrations per sperm (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It was tempting to speculate that the main role of detergent was to allow DTT to enter the cell, because DTT can decondense the sperm nucleus in vitro when the spermatozoon has been treated with a plasma membrane-permeabilizing agent [19, 20]. However, we revealed that in swim-up spermatozoa incubated with DTT, the chemical entered the nucleus spontaneously and reduced disulfide bonds in the protamines. This is supported by the study of Tateno and Kamiguchi [10], who also demonstrated that DTT could enter intact hamster spermatozoa and reduce the protamines. Because we obtained mostly normal karyoplates in this treatment, we concluded that the reduction of protamines alone was not sufficient for causing chromosomal breakage.
The present results indicate a clear synergy between detergent and DTT in generating chromosomal damage. Both tested detergents (ATAB and TX) gave high rates of chromosomal abnormalities when combined with DTT. That TX was more harmful than ATAB to sperm DNA could be explained by suggesting that the strength of ATAB may actually help to prevent damage. In fact, ATAB does remove the membrane and many other cellular components more effectively than TX and, therefore, may inactivate (at least partially) or remove some factors responsible for chromosomal breakage. This would result in the frequency of chromosomal aberrations and their intensity being less severe.
Mouse spermatozoa are extremely sensitive to mechanical damage [21], so the vigorous shaking, pipetting, and centrifugation we used in all procedures could easily have caused sperm membrane disruption. As revealed by Live/Dead assay (data not shown), we did disrupt sperm membranes during our procedure. Thus, if the major action of the detergent was membrane removal, we might have expected chromosomal breaks in procedure + DTT treatments. However, the frequency of abnormal metaphases was less than 50%, and most of them were minor. This was far less chromosomal damage than occurred with either ATAB + DTT or TX + DTT. We do not yet know if this difference between chemical and mechanical membrane disruption is purely quantitative, in that mechanical disruption is more gentle than detergent treatment. We also do not yet know if a qualitative component exists, because detergents remove more than just membranes. Nevertheless, the data suggest that at least part of the effect of detergent on chromosome stability is membrane disruption. The possibility that membrane disruption alone in the presence of DTT can result in chromosomal damage has implications for current ART practices, because human sperm are mechanically manipulated in the presence of DTT in some cases.
It is still not clear when breaks in chromosomes are induced: in the oocyte, after injection of the sperm nucleus into the cytoplasm of the oocyte, or when the DNA is still highly condensed in the sperm head. One possible mechanism of DNA degradation within the sperm nucleus could be endonucleases being released from plasma membrane-damaged spermatozoa. The existence of Ca2+-dependent endonucleases in mouse spermatozoa has already been reported [22]. It has been suggested that the presence of EDTA and the absence of Ca2+ and Mg2+ from media for sperm head isolation could improve chromosome stability [1, 23]. We showed that chelating reagents could offer partial protection to chromosomes, but some DNA damage was still observed. The simultaneous action of EGTA and EDTA provided better protection than EGTA alone, probably because a wider spectrum of ions to be chelated was covered. This supports the idea that one or more endogenous nucleases caused the breaks. However, because not all the karyoplates were normal, it is possible that the endonucleases were not inhibited entirely, that other factors were involved, or that some DNA degradation took place after injection in the oocyte as well.
The present results indicate that it is not the reduction of disulfide bonds alone that generates chromosomal damage but, rather, the combination of DTT and detergent. The mechanism that causes this chromosomal damage is unclear. It is possible, for example, that detergent releases an endonuclease from an internal vesicle, and that this endonuclease is activated by a pathway that is initiated by the presence of reduced protamines in mature spermatozoa. Alternatively, an enzyme that breaks sperm chromosomes could be activated by DTT independently of protamine disulfide reduction. The mechanism may also be as simple as the detergents removing some of the protamines after disulfide reduction, thereby exposing DNA to existing endonucleases. A final possibility is that one or more of these mechanisms initiate an undefined activity in the oocyte that results in chromosomal damage after the sperm head is injected into the egg.
Our data demonstrate that the sperm cell and/or the embryo contain a mechanism to disrupt paternal chromosomes. These results also suggest that even inadvertent disruption of the sperm membrane may contribute to chromosomal damage and, therefore, should be avoided. Finally, this work also demonstrates that TX and DTT, two reagents that have been proposed to increase the efficiency of ART, should not be used together. Future experiments will focus on the mechanism of DNA damage.
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FOOTNOTES |
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2 Correspondence: Monika A. Szczygiel, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. FAX: 808 956 7316; szczygie{at}hawaii.edu
Received: 17 December 2001.
First decision: 14 January 2002.
Accepted: 13 June 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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