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From the * Division of Reproductive Biology,
Department of Obstetrics and Gynecology, School of Medicine and
Department of Anatomy, Physiology, and Cell
Biology, School of Veterinary Medicine, University of California, Davis,
Davis, California.
| Correspondence to: Dr Stuart Meyers, One Shields Avenue, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616 (e-mail: smeyers{at}ucdavis.edu). |
| Received for publication June 5, 2006; accepted for publication January 17, 2007. |
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Abstract |
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Key words: Macaque, spermatozoa, ICSI
Acceptable levels of success (50% recovery of motile sperm) have been reached in cryopreservation of human sperm (Brotherton, 1990; Sherman, 1990), but much of this success can be attributed to the preselection of semen donors with sperm that are relatively resistant to cryoinjury (Thyer et al, 1999). Moreover, recent studies have determined that cryopreserved human sperm play a significant role in assisted reproductive technology in clinical practice but with decreased contribution to fertility (Kelleher et al, 2001; Wright et al, 2006). Significant effort has been expended to develop cryopreservation methods for nonhuman primate sperm (Morrell and Hodges, 1998), and offspring have been reported following artificial insemination of cryopreserved sperm in chimpanzees (Gould and Styperek, 1989), cynomolgus macaques (Tollner et al, 1990), rhesus macaques (Sanchez-Partida et al, 2000; Wolf et al, 2004; Yeoman et al, 2005), and marmoset monkeys (Morrell, 1997; Morrell et al, 1998).
The sperm cell is compartmentalized in terms of structure and function, and these compartments have different sensitivities to cryoinjury. Thus, cryoprotectants that preserve the genetic material during freezing and thawing may not preserve sperm functions required for sperm transport and survival in the female, fertilization, and preimplantation embryonic development (Rall, 1992). The combination of sperm cryopreservation and reproductive technology has the potential to obviate some of the problems that currently limit efficient production of offspring from cryopreserved nonhuman primate sperm. Already offspring have been born following in vitro fertilization (IVF) with cryopreserved sperm from marmosets (Holt et al, 1994) and gorillas (Pope et al, 1997), but the concentration of frozen spermatozoa required for efficient fertilization in vitro is higher than that of fresh sperm used for artificial insemination.
Intracytoplasmic sperm injection (ICSI), which requires only 1 spermatozoon per oocyte to effect fertilization, has been performed in rhesus macaques (Hewitson et al, 1996, 1998; Sutovsky et al, 1996; Meng and Wolf, 1997) and cynomolgus macaques (Ogonuki et al, 1998). Development of macaque embryos to the blastocyst stage following ICSI of nonfrozen sperm has been demonstrated (Hewitson et al, 1998), and offspring resulting from the transfer of ICSI-produced embryos have been reported (Hewitson et al, 1999; Chan et al, 2000). Cryopreserved human epididymal and testicular sperm have been used for ICSI with no recognized adverse effects on fertilization rates, embryonic development, or pregnancy rates (Friedler et al, 1998). It has been reported that chromosomal abnormalities are not increased in human sperm that survive cryopreservation, but these data are based on karyotype analysis of sperm that were capable of fusion with zona-free hamster oocytes (Martin et al, 1991). Other investigators have reported that human sperm chromatin stability is altered by cryopreservation (Hammadeh et al, 1999).
Although the use of ICSI will simplify the requirements for sperm cryopreservation protocols, the possibility of transmitting genetic damage by ICSI must be investigated because the technique bypasses the oocyte's natural ability to bind and select sperm. Evidence of increased genetic abnormalities following human ICSI has been reported (Bonduelle et al, 1995; In't Veld et al, 1995; Bonduelle et al, 1998). This concern is greater when cryopreserved sperm are used since unprotected freezing can damage sperm DNA (Rybouchkin et al, 1996). The primary objective of this study was to investigate the genetic damage caused by macaque sperm cryopreservation. Single-cell microgel electrophoresis (comet assay) was used to assess damage to sperm DNA, and sperm chromosome damage was assessed by karyotyping after ICSI of macaque sperm into mouse oocytes.
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Materials and Methods |
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In another series of experiments, 3 ejaculates were collected from the same 5 males for sperm karyotype analysis. Motile sperm from the fresh ejaculates were injected into mouse oocytes, and the percentage of sperm with structural aberrations and aneuploidy were determined. Aliquots of these ejaculates were cryopreserved; after thawing, motile sperm were selected for ICSI and subsequent evaluation of the same cytogenetic end points.
Semen samples were obtained by electroejaculation from rhesus macaques (n = 5) as previously described (Sarason et al, 1991). Animals were housed at the California National Primate Research Center and maintained according to Institutional Animal Care and Use Committee protocols at the University of California.
Reagents
Fetal bovine serum (FBS) was purchased from Hyclone Laboratories Inc
(Logan, Utah), Giemsa stain solution from Merck KGaA (Darmstadt, Germany),
Dulbecco phosphate-buffered saline (DPBS) and disodium
ethylenediamine-tetra-acetic acid (Na2-EDTA) solutions from Life
Technologies (Rockville, Md), and all other chemicals from Sigma-Aldrich (St
Louis, Mo), unless stated otherwise. Chemicals used in the comet assay,
including Triton X-100, Tris base, Na2-EDTA, DPBS, protease K, and
laurylsarcosine (sodium salt), were DNase free.
Culture Media
The medium used for culturing mouse oocytes and embryos before and after
ICSI was CZB medium (Chatot et al,
1990) supplemented with 5.56 mM D-glucose. The medium
used for collection of oocytes from oviducts, for subsequent treatments of
oocytes, and for sperm microinjection was HEPES-CZB containing 20 mM HEPES and
5 mM sodium bicarbonate and 0.1 mg/mL polyvinyl alcohol
(Kimura and Yanagimachi,
1995). The medium used for slowing sperm prior to ICSI was 10%
(w/v) polyvinyl pyrrolidine (PVP, 360 kd) in HEPES-CZB.
Preparation of Oocytes
Six- to 15-week-old B6D2F1 mice were obtained from Jackson Laboratory (Bar
Harbor, Me) and induced to superovulate by intraperitoneal injection of 5 to 7
IU pregnant mare serum gonadotropin followed by 5 to 7 IU of human chorionic
gonadotropin (hCG) 48 hours later. Oocytes were collected from oviducts 13 to
15 hours after hCG injection. Cumulus were removed by treatment with 300 U/mL
bovine testis hyaluronidase in HEPES-CZB for 3 to 5 minutes. The oocytes were
rinsed and kept in CZB for up to 4 hours at 37°C under 5% CO2
in air before ICSI.
Preparation and Evaluation of Spermatozoa
All animals were housed at the California Regional Primate Research Center
in compliance with the Federal Animal Welfare Act and the National Institutes
of Health Guidelines for Care and Use of Laboratory Animals. The animals were
maintained on a 12:12-hour light:dark cycle (lights on at 0600 hours) at
25°C to 27°C and were given a diet of Purina monkey chow and water ad
libitum. The males were trained to chair restraints and were electroejaculated
with a Grass 6 stimulator (Grass Medical Instruments, Quincy, Mass) equipped
with electrocardiogram pad electrodes (Conmed Corp, Utica, NY) for direct
penile stimulation (30–50 volts, 20-ms duration, 18 pulse/s)
(Sarason et al, 1991). The
semen was collected into sterile 15-mL plastic centrifuge tubes containing 5
mL of HEPES-BWW (Irvine Scientific, Santa Ana, Calif) without bovine serum
albumin (BSA). The ejaculates were allowed to liquefy at room temperature for
1 hour, and the sperm suspensions were transferred to new centrifuge tubes.
After the suspensions were washed twice by centrifugation at 300 x
g for 10 minutes and resuspension in HEPES-BWW containing 0.3% BSA,
sperm motility was assessed subjectively at 200x magnification using a
phase-contrast microscope with warm stage (37°C). At least 200 sperm were
counted in each sample to estimate the percentage of motile sperm and
percentage of progressive sperm. Sperm with any evidence of flagellar activity
were scored as motile; if the sperm was moving in a forward direction, it was
scored as progressive. To assess sperm viability, 5 µL of sperm suspension
was mixed with 5 µL of eosin Y (5 mg/mL in DPBS) on a slide. The drop was
covered with a coverslip and examined after 30 seconds at 400x using a
bright-field microscope with warm stage (37°C). At least 200 sperm were
scored in 5 or more microscopic fields for viability. Unstained sperm were
classified as live, and sperm stained red were scored as dead.
Cryopreservation of Spermatozoa
The method of Tollner et al
(1990) was used for sperm
cryopreservation. Macaque sperm preserved by this method have been used for
artificial insemination to produce normal offspring
(Tollner et al, 1990). TEST
buffer (4.325% Tes, 1.027% Tris, 1.0 % dextrose [pH 7.4]) was supplemented
with egg yolk obtained from freshly laid chicken eggs (30%, v/v) and
commercial fluid skim milk (20%, v/v). The solution was inactivated by
incubation at 56°C for 30 minutes and then centrifuged at 10 000 x
g for 15 minutes. The supernatant was transferred to another tube and
centrifuged again at 1000 x g for 30 minutes. The upper two
thirds of the volume was collected, and 0.025% streptomycin sulfate and 0.015%
penicillin G were added. The freezing medium was filtered through a 0.45-µm
syringe filter and stored at –20°C before use.
Freezing medium was thawed in a 37°C water bath, and washed sperm were resuspended in the medium at a concentration of 1 x 108/mL. Then glycerol was added drop by drop to the sperm suspension and gently mixed until the final concentration of glycerol was 3%. For freezing without cryoprotectants, washed sperm were resuspended in DPBS. Aliquots (0.5 mL) of processed sperm suspension were transferred into sterile 1.2-mL cryogenic vials (Nalge Nunc International, Rochester, NY). Cryovials were sealed tightly and loaded onto canes, which were placed in the freezing chamber of a computer-controlled freezer (model 1010; Cryomed Ltd, Mansfield, United Kingdom). The sperm were cooled to 8°C at a rate of 0.2°C/min. Then the sperm sample temperature was rapidly lowered to –110°C in 6.8 minutes. After freezing, the canes were transferred immediately to a liquid nitrogen storage tank and were stored in liquid nitrogen for at least 3 days. Frozen samples were removed from liquid nitrogen and immediately thawed in a 37°C water bath. The percentages of motile sperm, progressive sperm, live sperm, and dead sperm in the extended semen were determined before and after freezing, as described above. The recoveries of motile and viable sperm were calculated as percentages of prefreeze motility and prefreeze viability.
ICSI
ICSI was carried out according to Kimura and Yanagimachi
(1995) with some
modifications. Holding pipettes and micro-injection needles were prepared from
borosilicate glass capillary tubes (Sutter Instrument Co, San Rafael, Calif)
using a horizontal micropipette puller (model P-97; Sutter Instrument Co) and
a Narishige microforge (Narishige Scientific Instrument Lab, Tokyo, Japan).
The holding pipette had an external diameter of 50 to 60 µm and an internal
diameter of 10 to 15 µm. The injection pipette was beveled at an angle of
20 degrees and had an internal diameter of 6 to 8 µm. Injection procedures
were performed with an inverted Olympus IX70 microscope equipped with Hoffman
modulation contrast optics, Narishige micromanipulators, and Piezo impact
drive (PrimeTech, Ibaraki, Japan). The procedures were carried out at room
temperature in 5-µL drops of CZB-HEPES medium covered with light mineral
oil (Sigma-Aldrich) in the lid of a 100 x 15 mm tissue culture dish (BD
Falcon, Franklin Lakes, NJ).
An aliquot of fresh semen or frozen-thawed extended semen was transferred to a drop of 10 % PVP in HEPES-CZB, and only progressively motile sperm were selected for ICSI. Sperm were picked up, tail first, into the injection pipette and then immobilized by applying a few piezo-pulses (intensity 2–4, speed 2–3) to the sperm tail. A mouse oocyte was fixed by the holding pipette at the 9-o'clock position, and the metaphase II spindle-chromosome complex was positioned either at the 12- or 6-o'clock position. Several piezo-pulses (intensity 2–4, speed 2–3) were applied to penetrate through the zona pellucida while a light negative pressure was applied to the zona. After the zona fragment was expelled from the injection pipette into the perivitelline space, the sperm was pushed forward and the tip of the pipette was advanced against the oolemma to the opposite side of the oocyte cortex. The oolemma was punctured by applying 1 or 2 piezo-pulses (intensity 1–2, speed 1–2), and the sperm was expelled into the ooplasm with a minimum amount of medium. After retrieving as much medium as possible, the pipette was gently withdrawn, leaving the sperm within the ooplasm. Ten to fifteen oocytes were injected with spermatozoa within 30 minutes after transfer of the oocytes onto the operation dish. Sperm-injected oocytes were rinsed with CZB medium and incubated in this medium at 37°C under 5% CO2 in air.
Chromosome Examination
Between 6 and 8 hours after sperm injection, oocytes in CZB medium were
examined using an inverted phase-contrast microscope. Those oocytes with 2
distinct pronuclei and a second polar body were considered normally fertilized
and were transferred to CZB medium containing 0.006 µg/mL vinblastine to
prevent spindle formation and syngamy. The incubation was continued for an
additional 12 to 15 hours when the oocytes were arrested at metaphase of the
first cleavage. Oocytes with persistent pronuclei following incubation were
counted and not treated further. Other oocytes were treated with 0.5 % (w/v)
actinase (Kaken Pharmaceuticals, Tokyo, Japan) for 5 minutes to remove the
zona pellucida. The oocytes were then treated with a hypotonic solution (1:1
mixture of 1% [w/v] sodium citrate and 30% FBS) for 10 minutes at room
temperature.
Fixation and spreading of chromosomes were carried out according to the gradual fixation-air drying method of Mikamo et al (1994). Briefly, 5 to 10 oocytes with a small amount of hypotonic solution were placed gently on the bottom of a hollow glass slide filled with fixative I (methanol:acetic acid:water, 5:1:4). When the oocytes became transparent and began to move slowly, they were aspirated onto a clean glass slide. As the oocytes settled onto the slide, they were covered immediately by a gentle flow of fixative II (methanol:acetic acid, 3:1) so that the oocytes were tightly bound to the slide. The slide was placed into a jar filled with fixative II for 2 minutes. Finally, the slide was dipped into fixative III (methanol:acetic acid:water, 3:3:1) for 1 minute and then dried by warm air blown from a hair dryer. The chromosomes on the slides were stained with 2% (v/v) Giemsa solution in PBS (pH 6.8) for 10 minutes and analyzed with bright-field microscopy at 1000x. Mouse and rhesus monkey chromosome spreads were easily distinguished by their chromosome sizes (more uniform in mouse) and numbers of chromosomes (20 in mouse and 21 in rhesus monkey). Chromosome gaps and chromatid gaps were distinguished from chromosome breaks and chromatid breaks by the width of the staining discontinuity according to Brandriff et al (1984).
Comet Assay
The alkaline comet assay for DNA damage in spermatozoa was carried out
according to Hughes et al
(1996,
1997) with some modifications.
Normal melting point agarose and low melting point agarose (Sigma-Aldrich)
were dissolved in DPBS by boiling for 5 minutes. A 50-µL aliquot of 0.5%
normal melting point agarose was pipetted onto a fully frosted slide (Fisher
Scientific, Waltham, Mass) and spread by moving the end of another slide
against it before air drying. A spermagarose mixture was prepared by mixing 10
µL of sperm suspension in HEPES-BWW with 90 µL of 0.5% low melting point
agarose at 37°C to give a final sperm concentration of 1 million/mL. A
50-µL aliquot of sperm-agarose mixture was pipetted onto the agarose-coated
slide and spread by covering with a No. 1 coverslip. The slide was maintained
at 4°C for 10 minutes to allow the agarose to solidify, and then the
coverslip was gently removed. Seventy-five µL of 0.5% low melting point
agarose was added to the agarose gel and spread by covering with a No. 1
coverslip. After the agarose gelled, the coverslip was gently removed, and the
slide was placed in freshly prepared lysing buffer (2.5 M NaCl, 100 mM
Na2EDTA, 10 mM Tris, pH 10, 1% Triton X-100, 1% laurylsarcosine
sodium salt) for 1 hour at 4°C in the dark. Then the slides were treated
at 37°C with protease K (200 µg/mL in lysis buffer) for 20 hours in the
dark. After the protease K solution was drained from the slides, they were
placed in a horizontal electrophoresis unit (Sub-Cell; Bio-Rad, Hercules,
Calif) filled with freshly prepared alkaline electrophoresis buffer containing
300 mM NaOH and 10 mM EDTA for 20 minutes in the dark to allow the DNA to
denature. Electrophoresis was performed at 20 V and 160 to 180 mA for 15
minutes. The slides were then neutralized in 0.4 M Tris-HCl (pH 7.5) and
stained with 50 µL of 20 µg/mL ethidium bromide and mounted with a
coverslip.
Two hundred sperm were scored per slide at 400x magnification using an epifluorescence microscope. Sperm nuclei with mobile fragments of DNA were classified as "comets" (Singh et al, 1988, 1989) (Figure 1). Overlapping sperm and sperm at the agarose edge were not scored. Fluorescence images of sperm processed in the comet assay were acquired using an AxioCam digital camera (Carl Zeiss Vision GmbH, Oberkochen, Germany) with an Olympus BX60 fluorescence microscope.
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Results |
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Effects of Cryopreservation on Sperm DNA and Chromosome Structure
As shown in Table 2, fresh
sperm from the 5 males had little evidence of DNA damage, with 0% to 3% of the
sperm heads appearing as comets after electrophoresis
(Table 2;
Figure 1A). After
cryopreservation and thawing, the percentage of comets increased greatly and
ranged from a mean of 25% to 44% depending on the male
(Table 2;
Figure 1B). The semen samples
frozen without cryoprotectants had even higher percentages of comets, with
mean values ranging from 53% to 92% for individual males
(Table 2).
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The frequency of abnormal karyotypes was low in motile sperm from fresh ejaculates. The percentages of sperm with structural chromosomal abnormalities ranged from 0% to 6%, and the percentages of sperm with aneuploidies ranged from 0% to 8% (Table 3). The incidence of abnormal karyotypes was not significantly higher in motile frozen-thawed sperm than in fresh motile sperm (Tables 4 and 5). The structural abnormalities observed in fresh and frozen-thawed motile sperm included chromosome gaps, chromatid gaps, chromosome breaks, chromatid breaks, and chromosome exchanges (Figure 2). Chromosome breaks were the most common type structural abnormality encountered.
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Discussion |
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Two methods were used in the present study for assessing cryopreservation damage to the genetic material of macaque spermatozoa. Damage to sperm DNA was assessed by single-cell microgel electrophoresis. This technique, known as the comet assay, was developed to detect DNA strand breaks induced by radiation (Ostling and Johanson, 1984). In this assay, nuclei from single cells are electrophoresed at alkaline pH and nuclei with DNA damage are recognized as "comets," with mobile, fragmented DNA forming the comet tail (Singh et al, 1988, 1989). Studies of various cell types have indicated that the comet assay primarily detects single-strand breaks of DNA (Singh et al, 1988, 1989; Olive et al, 1990, 1992; Klaude et al, 1996). The comet assay has been applied previously in studies of human sperm (Singh et al, 1989; Aravindan et al, 1997), and in the present study we found that unprotected freezing resulted in 52% to 92% of sperm with evidence of DNA damage. Even when cryoprotectants were used, as many as 40% of sperm had DNA damage.
Damage to sperm chromosomes was evaluated by cytogenetic analysis after injection of motile macaque sperm into mouse oocytes. This method has been used previously for analysis of chromosomes in mouse (Kusakabe et al, 2001) and human sperm (Lee et al, 1996). The results of the present study showed that the percentage of sperm with chromosome damage was not increased in frozen-thawed sperm when motile sperm were selected for ICSI. It is not known whether the sperm with normal karyotypes were also free of other DNA damage because the comet assay cannot discriminate between viable and nonviable sperm.
Most studies that have evaluated frozen sperm in the nonhuman primate have used end points such as motility of sperm or fertilization rates in vitro following ICSI or IVF. With these methods, subpopulations of motile sperm within an ejaculate cannot be assessed with the exception of ICSI, and with the latter technique only very small numbers of sperm can be observed. In our study, we were able to evaluate specifically the motile sperm population for chromosomal damage using a heterologous ICSI method and cytogenetic analysis. Our results are in general agreement with those in mouse sperm reported by Kusakabe et al (2001) in that cryopreservation in the presence of cryoprotectants does not cause significant chromosomal damage to motile sperm to an extent greater than that of fresh motile sperm, although cryopreservation results in overall increases in DNA damage, aneuploidy, and chromosome fragmentation.
Mechanisms for cryopreservation-induced DNA damage to sperm are likely to be multifactorial, but studies in other species have demonstrated that production of reactive oxygen species resulting from membrane lipid peroxidation may be a significant factor (Ball et al, 2001; Fraser and Strzezek, 2005; Jiang et al, 2005). Lipid peroxidation can also contribute directly to chromatin cross-linking, base changes, and DNA strand breaks (Hughes et al, 1996; Kodama et al, 1997; Twigg et al, 1998). Studies in equine sperm have demonstrated that cryopreservation contributes to DNA fragmentation as detected by the comet assay (Baumber et al, 2003).
In conclusion, the results of the present experiments demonstrated that protocols for cryopreservation of macaque sperm result in damage to sperm DNA. Our findings suggest that genetically competent frozenthawed macaque sperm can be selected for fertilization by using only motile sperm for ICSI.
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