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Regular article |
a Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822
b Department of Biological Sciences, Asahikawa Medical College, Asahikawa 078-8510, Japan
c Department of Obstetrics and Gynecology, Fukushima Medical College, Fukushima 960-1295, Japan
ABSTRACT
Although sonication is a simple way to immobilize ("kill") spermatozoa prior to injection into oocytes, this has been thought to be destructive to sperm chromosomes. Mouse and human spermatozoa were immobilized by sonication and kept in various media for up to 2 h, then their nuclei were individually injected into mouse oocytes for the analysis of chromosomes at the first cleavage metaphase. In both the mouse and human, incidence of structural chromosome aberrations was much higher in the spermatozoa sonicated and stored in Biggers-Whitten-Whittingham medium for 2 h at 37.5°C than in those stored for 5 min in the same medium. We concluded, therefore, that it is not sonication per se but a prolonged exposure of sperm nuclei to extracellular milieu that is detrimental to sperm chromosomes. The incidence of structural chromosome aberrations of mouse and human spermatozoa was significantly reduced when the spermatozoa were sonicated and stored in K+-rich nucleus isolation medium containing EDTA. This suggests that sperm chromosome degradation following sperm immobilization by sonication is partly due to detrimental effects of a Na+-rich medium and of DNase on sperm chromatin. Ideally, it should be possible to prepare artificial media that maintain the integrity of sperm chromosomes for many hours after immobilization.
fertilization, IVF/ART, sperm, testes
INTRODUCTION
It is well established that sperm immobilization immediately before intracytoplasmic sperm injection (ICSI) increases the rate of successful fertilization [1–6]. The reason for this is not clear, but Yanagimachi [7] inferred that disruption or labilization of the sperm plasma membrane facilitates its disintegration and thereby directs contact of sperm components with the ooplasm. Sperm immobilization prior to ICSI has been accomplished by scoring the sperm tail with a micropipette [2, 6, 8], by sonication [9–11], by unprotected freezing [12–14], by separation of head and tail by piezopulses [11], or by detergent treatment [11, 15, 16].
It has been reported that sonication frequently induces structural aberrations in human sperm chromosomes [9] and that human spermatozoa frozen without cryoprotection have high incidence of structural chromosome abnormalities [14]. It should be noted that physical or chemical disruption of the sperm plasma membrane results in an influx of extracellular medium into the cell. Thus, all intracellular organelles (including nucleus and mitochondria) will be exposed directly to an extracellular milieu (e.g., high Na+ and low K+) that is very different from the environment inside of the cell (low Na+ and high K+). Therefore, it is possible that the chromosomal damage reported by Martin et al. [9] and Rybouchkin et al. [14] is due either to direct action of physical stresses (sonication and freezing) or the influence of extracellular milieu on sperm nucleus (chromosomes).
We report here that sonication per se is not as detrimental to mouse and human sperm chromosomes as previously believed. It is a prolonged exposure of sperm nuclei to external milieu that is primarily responsible for deterioration of sperm chromosomes after membrane disruption (immobilization) of spermatozoa.
MATERIALS AND METHODS
Reagents and Media
All inorganic and organic reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless specifically stated. The medium for culturing oocytes was Chatot-Tasca-Ziomek (CZB) medium supplemented with 5.56 mM D-glucose [17, 18]. Oocytes were collected, and microsurgery was performed in modified CZB, with 20 mM Hepes-HCl, 5 mM NaHCO3, and 0.1 mg/ml polyvinyl alcohol (PVA; cold water soluble) in place of bovine serum albumin. This was designated as Hepes-CZB and used under 100% air. The pH value of Hepes-CZB was approximately 7.4.
The media used for collection and sonication of spermatozoa were Biggers-Whitten-Whittingham medium (BWW) [19] and a slightly modified nucleus isolation medium (NIM) [11]. The latter consisted of 121.6 mM KCl, 7.8 mM NaH2PO4, 1.4 mM KH2PO4, 10 mM EDTA disodium salt, and 0.01% PVA. Its pH was adjusted to 7.2 by addition of a small quantity of 1 M KOH in distilled water.
Animals
The mice used were maintained in accordance with the guidelines of the Laboratory Animal Service at the University of Hawaii and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resource National Research Council (DHEW publication [NIH] 80–23, revised in 1985). The protocol of our animal handling and treatment was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.
Preparation of Mouse Oocytes
B6D2F1 female mice, 7–11 wk of age, were induced to superovulate by i.p. injection of 5 IU eCG and 5 IU hCG, 48 h apart. Oocytes were collected from oviducts between 14 and 17 h after hCG injection. They were freed from cumulus cells by treatment with 0.1% (w/v) bovine testicular hyaluronidase (300 UPS units/mg; ICN Pharmaceuticals, Costa Mesa, CA) for 3–5 min in Hepes-CZB. The cumulus-free oocytes were rinsed thoroughly in CZB and kept in the medium for up to 3.5 h at 37.5°C under 5% CO2 in air.
Preparation of Mouse and Human Spermatozoa
Spermatozoa of B6D2F1 mice, 8–12 wk old, were collected from the cauda epididymides and suspended in approximately 5 ml of BWW or in NIM with (+) or without (-) 0.5 mM PMSF and 0.5 µg/ml leupeptin, both protease inhibitors. The sperm concentration in each suspension was adjusted to approximately 1 x 106 per milliliter. Regardless of media used, over 90% of spermatozoa displayed high motility.
Human spermatozoa were obtained by masturbation from two men of proven fertility. Semen samples were allowed to liquefy at 37.5°C for 1 h, mixed with 5 ml of BWW, and centrifuged at 500 x g for 5 min. The sperm pellet was resuspended in 0.5 ml of BWW, the sperm suspension was placed in the bottom of a 15-ml sterile centrifuge tube containing 10 ml of BWW, and motile spermatozoa were allowed to swim up at 37.5°C under 5% CO2 in air for 1 h. The upper 8 ml of the medium containing actively motile spermatozoa was carefully retrieved, transferred to another tube, and centrifuged again at 500 x g for 5 min. Sperm pellet was resuspended in approximately 5 ml of BWW or NIM(+), and the sperm concentration was adjusted to 1 x 105–6 per milliliter.
Sonication of Spermatozoa
The sperm suspension (5 ml) was transferred to a 10-ml beaker in ice water (0°C) and sonicated for 5 sec at 60% output of a sonicator (Brinwell Scientific, Rochester, NY; model BP-II, with 12-mm diameter horn). The beaker was shaken constantly during sonication. This treatment rendered all the spermatozoa motionless. More than 95% of mouse spermatozoa had their heads and tails separated. More than 95% of human spermatozoa had broken tails. Spermatozoa sonicated in BWW were kept in the same medium at 37.5°C under 5% CO2 in air; those sonicated in NIM(+) or NIM(-) were kept at 37.5°C in air.
Microinjection of Mouse Sperm Heads and Human Spermatozoa into Mouse Oocytes
Immediately before injection, each sperm suspension was centrifuged at 500 x g for 5 min. Each sperm pellet was resuspended in 5 ml NIM free of EDTA, PMSF, and leupeptin and centrifuged again. A small amount of sperm suspension (1 µl) was finally suspended in 5 µl of EDTA- and protease inhibitor-free NIM medium containing 12% polyvinyl pyrrolidone (PVP; Mr 360 000). Mouse sperm heads and human spermatozoa with broken tails in the NIM medium were individually injected into mouse oocytes according to the method of Kimura and Yanagimachi [20], except that all operations were carried out at room temperature (24–26°C) rather than at 16–17°C. The sperm injection was carried out within 1 h of the transfer to the NIM medium. Control mouse spermatozoa were unsonicated spermatozoa whose heads were separated from the tails immediately before injection into oocytes. A single motile spermatozoon was drawn, tail first, into the injection pipette until the head-midpiece junction was at the opening of the pipette. Upon application of a few piezopulses, the head was separated from the tail. Only the head was injected into an oocyte. Control human spermatozoa were prepared by drawing a single motile spermatozoon, tail first, into the injection pipette until the middle of tail was at the opening of the pipette. Several piezopulses were applied to the tail until the spermatozoon became completely immotile. The entire body of the spermatozoon was immediately injected into a mouse oocyte. Isolated heads or immobilized spermatozoa in experimental groups were left in test media for 5 min to 2 h after sonication before they were transferred into EDTA- and protease inhibitor-free NIM for injection into oocytes.
Activation and Culture of Injected Oocytes
Sperm-injected oocytes were kept in Hepes-CZB at room temperature for 10 min, then transferred into a droplet (50 µl) of CZB under mineral oil (Squibb, Princeton, NJ) and cultured at 37.5°C under 5% CO2 in air. Preliminary experiments revealed that the rate of oocyte activation is greatly influenced by the time interval between sperm sonication and injection. Although more than 90% of the oocytes were activated when fresh human spermatozoa were used, only about 30% were activated by the spermatozoa sonicated ("killed") 2–3 h previously. Therefore, all the oocytes injected with spermatozoa, regardless of sperm's age after immobilization, were activated by 30 min of treatment with 5 mM SrCl2 in Ca2+-free CZB [21]. Six to 8 h later, eggs were examined with an inverted microscope for the presence or absence of pronuclei. Those with two distinct pronuclei were transferred into 0.2 ml CZB containing 0.006 µg/ml vinblastine and cultured until they reached metaphase of the first cleavage division.
Chromosome Preparation and Analysis
Between 19 and 21 h after sperm injection, metaphase eggs were treated with 1% pronase (1000 tyrosine units/mg; Kaken Pharmaceuticals, Tokyo, Japan) for 5 min at room temperature to soften zonae pellucidae. They were then treated with a hypotonic solution (1:1 mixture of 1% sodium citrate and 30% fetal bovine serum) for 10 min at room temperature. Chromosomes were spread on slides by the gradual-fixation/air-drying method [22]. The preparations were stained with 2% Giemsa (Merck, Darmstadt, Germany) in buffered saline solution (pH 6.8) for 8 min for conventional chromosome analysis. Subsequently, chromosomes were C-banded according to Sumner [23], with some modifications to detect dicentric chromosomes and acentric fragments. Briefly, the slides were treated with 0.2 N HCl for 30 min at room temperature, 5% Ba(OH)2 for 2 min at 50°C, and 2x saline sodium citrate (2x SSC) for 5 min at 50°C, followed by staining in 4% Giemsa for 10 min.
Statistical Analysis
Fisher's exact probability test and chi-square test were used to analyze data on percentage of eggs and sperm with chromosome aberrations, respectively. Differences in the number of aberrant chromosomes per cell were analyzed by Student's t-test or Welch's t-test. Differences at P < 0.05 were considered significant.
RESULTS
Chromosome Analysis of Mouse Spermatozoa after Sonication
Of a total of 533 mouse oocytes injected with mouse sperm heads, 438 survived, and 388 (89%) were fertilized. Out of the fertilized eggs, 369 (95%) developed to the first cleavage metaphase, and 366 eggs were subjected to chromosome analysis. The remainder were unsuitable for chromosome analysis because their chromosomes were underspread. The incidence of chromosome aberrations and the number and types of structural chromosome aberrations in the mouse zygotes are summarized in Table 1. When the spermatozoa were kept in BWW after sonication, the incidence of structural chromosome aberrations in zygotes increased sharply with increasing time between sonication and injection (9% in the control, 35% in 5-min storage after sonication, and 92% in 2-h storage after sonication). Many of the zygotes created with 2-h-stored sperm heads had multiple chromosome aberrations (Fig. 1, A and B), their incidence being highest (8.61 per zygote) when sperm heads were stored in BWW for 2 h. Storing sperm heads in NIM seems to be less damaging to sperm chromosomes. The presence of protease inhibitors in NIM did not reduce the time-dependent increase in the incidence of structural chromosome aberrations.
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In this study, we classified structural chromosome aberrations into four categories [24]: 1) chromosome-type break, 2) chromosome-type exchange, 3) chromatid-type break, and 4) chromatid-type exchange. Breaks and exchanges of chromosome-type were predominant in all experimental groups (Table 1). No significant increase in incidence of zygotes showing aneuploidy or polyploidy was detected in any of the experimental groups.
Chromosome Analysis of Human Spermatozoa after Sonication
Of 372 mouse oocytes injected with human spermatozoa, 312 survived, and 239 (77%) were fertilized. Of the fertilized eggs, 219 (92%) reached the first cleavage metaphase. It was possible to distinguish human chromosomes from mouse chromosomes in the interspecies hybrid zygotes because of their marked structural differences. A total of 216 human sperm chromosome sets were analyzed (Table 2). The incidence of structural chromosome aberrations and the number of chromosome aberrations per sperm in the spermatozoa sonicated and stored for 5 min in BWW and for 2 h in NIM(+) were similar to those in the control spermatozoa. The incidence of structural chromosome aberrations increased sharply when sonicated spermatozoa were stored in BWW for 2 h. As in the mouse spermatozoa, most structural chromosome aberrations were of chromosome type (Table 2 and Fig. 1C). There was no significant increase in the incidence of aneuploidy or polyploidy in any of the experimental groups.
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DISCUSSION
During transport through the epididymis, the nuclei of mammalian spermatozoa are physically and chemically stabilized by extensive cross-linking of nuclear protamines by disulfide bonds [25–27]. We have previously reported that sperm nuclei are able to decondense and transform into normal-looking pronuclei even after harsh treatments such as heating at 90°C [28], dehydration in alcohol [29], or sonication [11]. Recently, Wakayama and Yanagimachi [30] obtained normal mouse offspring by injecting freeze-dried spermatozoa into oocytes.
Martin et al. [9] and Rybouchkin et al. [14] reported that human sperm nuclei (chromosomes) are vulnerable to physical stresses, such as sonication and unprotected cryopreservation. This may not be the case in the mouse because we obtained normal offspring from the oocytes injected with sperm heads that were sonicated [11] or frozen-thawed without any cryoprotection [31]. The present study revealed that it is not sonication per se that is detrimental to sperm chromosomes but rather a prolonged exposure of membrane-damaged spermatozoa to storage media. Because the sperm plasma membrane has no self-repairing ability, even a local disruption of the membrane is terminal to the spermatozoon. Both sonication and unprotected cryopreservation must damage sperm plasma membrane irreversibly, resulting in the influx of medium components into the cells. Sperm organelles (including the nucleus) are then directly exposed to the medium, the ionic composition of which is very different from that of the sperm's intracellular compartment. The longer the exposure of sperm nuclei to medium components, the more lesions to their chromosomes. Neither Martin et al. [9] or Rybouchkin et al. [14] described how long they kept sonicated or frozen-thawed spermatozoa in media before injection into oocytes. Therefore, it is very likely that the increased incidence of structural chromosome aberrations they reported is due presumably to a prolonged sojourn of membrane-damaged ("killed") spermatozoa in the medium prior to injection.
We found that both mouse and human sperm chromosomes are damaged more when sperm nuclei are kept in BWW medium (a cell culture medium) than when they are kept in NIM medium (an artificial medium with high K+ and low Na+ concentrations; Tables 1 and 2). In all cells, the cytosolic concentration of K+ is much higher than that of Na+. The intracellular concentrations of K+ and Na+ in bovine epididymal spermatozoa have been estimated to be 120 ± 5 mM and 14 ± 2 mM, respectively [32]. Thus, when sperm plasma membrane is damaged (e.g., by sonication), sperm nuclei are inevitably exposed to the medium with significantly higher concentration of Na+ and lower concentration of K+. This may cause deleterious changes in chromatin (DNA and associated proteins), resulting in structural chromosome aberrations. Kuretake et al. [11] and Suzuki et al. [33] reported that the ability of isolated mouse sperm heads (nuclei) and spermatid nuclei to participate in normal development is maintained significantly longer in a K+-rich medium than in Na+-rich cell culture media. Perhaps the nuclei (chromosomes) of plasma membrane-damaged spermatozoa and of spermatids are less damaged by K+-rich (NIM) media than by Na+-rich (BWW) media.
There is another possibility: that the presence of EDTA and the absence of Ca2+ and Mg2+ in NIM medium reduce the digestion of sperm DNA by endogenous nucleases because the enzyme activity is detected in murine spermatozoa [34, 35]. It is unlikely that proteases released from acrosomes and other compartments of the spermatozoa are involved in the deterioration of sperm chromatin because the presence of protease inhibitors in NIM medium did not prevent the deterioration of sperm chromatin (Table 1). Chromosome study of spermatozoa stored at 4°C after sonication would be needed in order to understand the involvement of endogenous enzymes in deterioration of sperm chromatin.
The present study revealed that mouse sperm chromosomes deteriorate more quickly than human sperm chromosomes after the spermatozoa are immobilized (Tables 1 and 2). Before experiments, we expected the opposite result because human sperm chromatin is known to be less stabilized by disulfide bonds than that of many other species (e.g., chinchilla, mouse, hamster, rat, guinea pig, rabbit, and bull) [36, 37]. Moreover, when viewed with an electron microscope, chromatin of human spermatozoa appear to be less dense [38, 39] than that of most other species. In this study, differences in chromatin structure between human and mouse sperm nuclei do not appear to account for their differential sensitivity to DNA damage during storage in BWW or NIM medium.
In ICSI technique, removing the tails is particularly helpful when trying to inject sperm with long tails, and disruption of the sperm plasma membrane facilitates contact of sperm components with the ooplasm. Sonication is a simple and effective way to separate sperm heads from tails or to damage the sperm plasma membrane. It was somewhat disappointing that chromosomes in isolated sperm heads, in particular those of the mouse, did not remain intact for many hours in K+-rich NIM medium as we had hoped (Tables 1 and 2). However, a delay in sperm chromosome deterioration in the NIM medium is encouraging for our search for ideal media that maintain the integrity of sperm chromosomes for many hours after sperm nucleus isolation and/or sperm immobilization.
ACKNOWLEDGMENTS
We thank Dr. J. Michael Bedford, Department of Obstetrics and Gynecology, Cornell Medical College, for reading the original manuscript and giving us invaluable advice.
FOOTNOTES
First decision: 2 February 2000.
1 This study was supported by grants (HD-34362 and HD-38205 to R.Y.) from the National Institutes of Health, the Victoria S. and Bradley L. Geist Foundation, and The Kosasa Family Foundation. H.T. was a research fellow of the Ministry of Education, Science, Sports and Culture of Japan. 
2 Correspondence: Ryuzo Yanagimachi, Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, 1951 East-West Road, Honolulu, HI 96822. FAX: 808 956 5474; yana{at}hawaii.edu
Accepted: February 29, 2000.
Received: January 1, 2000.
REFERENCES
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