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research-article |
Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822
ABSTRACT
Methods routinely used to preserve mouse spermatozoa require that the male be killed to recover spermatozoa from the epididymides. Here we obtained multiple samples of ejaculated spermatozoa from normal fertile C57BL/6 and infertile Hook1/Hook1 (formerly known as azh/azh) mutant males from uteri after mating, thus avoiding termination of the males. Ejaculated sperm were preserved by conventional cryopreservation or by rapid freezing without cryoprotection, and were injected into the oocytes by intracytoplasmic sperm injection (ICSI). The proportions of oocytes that survived, became activated, and developed into two-cell embryos were similar when comparing the two preservation methods in wild-type versus Hook1/Hook1 mice and tested mice versus controls (fresh and rapid-frozen epididymal and fresh ejaculated sperm). Two-cell embryos were transferred into the oviducts of pseudopregnant females, and fetal development was examined at Day 15 of gestation. A total of 39%–54% of transferred embryos produced with preserved ejaculated sperm implanted. Live, normal fetuses (11%–17%) were obtained in all examined groups and from all males included in the study. More implants (71%–82%) and fetuses (28%–31%) were noted in controls. Lower developmental potentials of embryos produced with preserved ejaculated sperm might be due to their capacitation status; the majority of sperm retrieved from the uterus were capacitated. This study bears significance for the maintenance and distribution of novel mouse strains. The method is applicable for all types of mice, including those with male infertility syndromes. The sole requirement is that the male of interest is able to copulate and its ejaculate contains spermatozoa.
assisted reproductive technology,, embryo, gamete biology, in vitro fertilization, sperm
Sperm cryopreservation has contributed greatly to animal breeding and reproductive medicine since Polge et al. reported in 1949 [1] that glycerol protected fowl spermatozoa from freezing injury. Today much of the basic research in mammalian genetics and early development is undertaken with the mouse. The numerous mutant lines surpass both the financial and physical resources available to maintain them as live populations. Efficient and dependable methods for gamete and embryo cryopreservation are needed to avoid inadvertent loss of this unique material through disease or other hazards. In addition, these methods can provide an effective means of distributing novel genetic models among the biomedical research community. Spermatozoa are produced in much larger numbers compared with oocytes and embryos. Sperm cryopreservation is more cost effective and less labor extensive than embryo freezing. Furthermore, with the advent of intracytoplasmic sperm injection (ICSI), many breeding problems exhibited by defective male reproductive function in mutant and transgenic lines (e.g., low sperm motility, concentration, and/or abnormal sperm morphology) can be overcome by this technique [2–7].
The collection of spermatozoa from mice has been limited to techniques that result in the death of the male, precluding him from further breeding. These techniques are now routinely applied by all investigators in the field. Several alternative nonlethal approaches for the collection of mouse spermatozoa were investigated. Flushing spermatozoa from the uterus of female mice bred to males seems to be the best alternative approach, in which a male is kept alive [8, 9]. This method was used to obtain spermatozoa for first in vitro fertilization (IVF) studies in the 1960s [10, 11]. This approach was successfully used recently to obtain ejaculates from hybrid mice. These spermatozoa were cryopreserved in medium containing raffinose, glycerol, and egg yolk and used for IVF, yielding live offspring after embryo transfer [12]. Injections of pernosterone and yohimbine have been used, with mixed success, to induce ejaculation in mice [13, 14]. In another approach, spermatozoa were collected repeatedly from oviduct-ligated female mice without surgery or death of male or female mice [15]. However, the latter technique was established for obtaining spermatozoa for genetic analyses, and no indication was given that retrieved sperm would able to fertilize oocytes. Techniques to electroejaculate male mice were frequently fatal as a result of coagulation factors in the semen [16] or the electrical stimulation itself [17], and only a very small number of spermatozoa could be obtained from each ejaculate [18]. These methods, although vigorously tested, did not improve over the years [19, 20].
In this study we describe the results of preservation of spermatozoa from C57BL/6 mice that were obtained from the uteri after mating. We preserved ejaculated sperm from normal fertile C57BL/6 males and infertile mutant Hook1/Hook1 males by conventional cryopreservation and by rapid freezing without cryoprotection, and we used ICSI as a fertilization method. We demonstrated that normal viable fetuses can be obtained with a similar efficiency using sperm from both fertile and infertile mice, regardless of the preservation method used.
Mineral oil was purchased from Squibb and Sons (Princeton, NJ), and equine chorionic gonadotropin (eCG) and human chorionic gonadotrophin (hCG) were purchased from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.
All mice were obtained at 6 wk of age from the following vendors: B6D2F1 (C57BL/6J x DBA/2) and C57BL/6 from the National Cancer Institute (Raleigh, NC), and CD-1 mice from Charles River (Wilmington, MA). Homozygous Hook1/Hook1 mutant males were produced in house from Hook1/+ heterozygous breeders purchased from Jackson Laboratory (Bar Harbor, ME). Ejaculates were obtained from 12- to 16-wk-old C57BL/6 and Hook1/Hook1 males. Mature oocytes for ICSI were obtained from 8- to 12-wk-old C57BL/6 females. CD-1 females 8–16 wk old were used for mating with examined males and as surrogate mothers for embryo transfer. The mice were fed ad libitum with a standard diet and were 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 guidelines presented in National Research Council's Guide for Care and Use of Laboratory Animals published in 1996 by the Institute for Laboratory Animal Research of the National Academy of Science (Bethesda, MD). The protocol for animal handling and treatment procedures was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.
Oocyte collection and subsequent manipulation, including microinjection, was done in HEPES-buffered CZB medium (HEPES-CZB; [21]). Phosphate-buffered solution (PBS) was used for sperm dispersion preceding preservation. Culture of sperm-injected oocytes and embryos was done in CZB medium [22]. The CZB was maintained in an atmosphere of 5% CO2 in air, and HEPES-CZB and PBS in air.
Collection of Epididymal Sperm
Epididymal spermatozoa were obtained from males 8–16 wk of age. Caudae epididymides were removed from one male, and the epididymal fluid was squeezed out and placed on the bottom of a 1.5-ml tube containing 0.4 ml HEPES-CZB. Spermatozoa were allowed to swim up for 5 min at room temperature, and then they were taken for injections.
Collection of Ejaculated Sperm
Sperm donors (C57BL/6 and Hook1/Hook1) were caged individually. Females (CD-1) were examined in the evening on the day preceding mating, and those in estrus were separated. We chose CD-1 females for pairing because they have large, convenient-to-manipulate uteri, and it is easy to recognize estrus on the basis of vagina color and swelling. We preferred to select females for mating based on their vaginal appearance rather than use hormones to synchronize the estrous cycle, because when mating did not take place nonstimulated females could be immediately reused, whereas those with hormonally induced estrus required at least 10 days of shelf rest. Early in the morning (0700–0800 h) on the next day, females were placed with males. The females were examined for the presence of vaginal plugs as an indication of successful mating. The first examination was done 30 min after pairing and at 30-min intervals thereafter. Positive females were killed 1 h after finding the plugs, usually 0900–1000 h. The female reproductive tracts were excised and freed from residual adipose tissue and adhering blood. Each uterine horn was cut at its cervical end, and the uterine contents (0.2–0.4 ml) were released into the well of an Organ Tissue Culture Dish (catalog no 3513037; Falcon, Bedford, MA) containing 1 ml warm PBS. Sperm were allowed to swim out from the dense uterine mass into PBS for 10 min at 37°C. Motile spermatozoa in PBS (
0.4 ml) were collected from the periphery of the well, transferred into a 1.5-ml tube containing 1 ml PBS, and mixed gently by rotating the tube. Half of the sperm suspension was transferred to another 1.5-ml tube. Sperm suspensions were centrifuged (720 x g, 10 min, 25°C). The supernatant was removed, and the spermatozoa were resuspended in 50 µl of either cryoprotectant for conventional cryopreservation (tube 1) or EGTA-containing buffer for rapid freezing without cryoprotection (tube 2).
Spermatozoa were cryopreserved according to the method of Nakagata [23], with modifications. Briefly, the cryoprotectant solution containing 18% D-(+)-raffinose pentahydrate (w/v) and 3% skim milk (w/v) was prepared by dissolving 3.6 g D-(+)-raffinose pentahydrate and 0.6 g skim milk in 20 ml glass-distilled water at 60°C. The solution was centrifuged at 10 000 x g for 20 min. The supernatant was collected, filtered through a sterile 0.45-µm Millipore filter, and stored at –20°C in 0.5-ml aliquots in sterile 1.5 ml polystyrene Eppendorf tubes. An aliquot was thawed and warmed to 37°C immediately before use. Aliquots of 10 µl spermatozoa suspended in the cryoprotectant were loaded immediately into 0.25-ml straws (Edwards Innovations, Spring Valley, VA). Each straw was sealed with Critoseal (Oxford Labware, St. Louis, MO). Straws were placed inside the body of a 60-ml syringe. The tip of the syringe had been heat sealed, and a piece of Styrofoam filled the bottom 15 mm of the syringe. The syringe body was wired onto a 50-cm-long acrylic rod that facilitated placing, plunging, and removing samples in the Dewar (model XC35/12; MVE Biological Systems). The samples were frozen by placing the syringe body inside the Dewar so that it floated on liquid nitrogen. The samples floated 10 min in the Dewar before being plunged into the liquid nitrogen. Three parameters that were found important for maintaining the correct cooling rates [24] were: 1) Dewar opening diameter of 100 mm, 2) liquid nitrogen height of 300 mm, and 3) syringe float depth of 45 mm. Samples were stored for no less than 24 h and up to 6 mo. For thawing, the straws were removed from the storage container and immediately immersed in a water bath at 37°C for 10 min. The contents of a straw were expressed into a Petri dish, and spermatozoa were used immediately for ICSI.
Rapid Freezing Without Cryoprotection
The solution for rapid freezing without cryoprotection consisted of 50 mM EGTA (ethylene glycol-bis [beta-aminoethyl ether]-N,N,N',N'-tetraacetic acid), 50 mM NaCl, and 10 mM Tris-HCl buffer [25]. The pH was adjusted to 8.2–8.5 by adding a small quantity of 1 M NaOH. This final solution, referred to as EGTA Tris-HCl-buffered solution (ETBS) [26], was stored at 4°C for no more than 1 wk before use. Aliquots of 10 µl sperm suspension in ETBS were loaded in 0.25-ml straws. Each straw was sealed with Critoseal and placed in a plastic holder. In preliminary experiments the straws were transferred directly from room temperature to –196°C by plunging into liquid nitrogen. This resulted in the total disintegration of many straws due to rapid pressure changes occurring during the procedure. This problem was overcome by placing the straws in a plastic holder on the surface of the LN2 for 10 min before immersion. Immediately before ICSI a straw was removed from the storage container and thawed at room temperature (25°C) for 5 min before expressing the contents into a Petri dish.
Females were induced to superovulate with injections of 5 IU eCG and 5 IU hCG that were given 48 h apart. Oviducts were removed 14–15 h after the injection of hCG and placed in PBS in a Petri dish. The cumulus-oocyte complexes were released from the oviducts into 0.1% bovine testicular hyaluronidase (300 USP U/mg) in HEPES-CZB medium to disperse cumulus cells. The cumulus-free oocytes were washed with HEPES-CZB medium and used immediately for ICSI.
Intracytoplasmic Sperm Injection
ICSI was carried out as described recently by Szczygiel and Yanagimachi [27]. Briefly, a small drop of sperm suspension was mixed thoroughly with an equal volume of HEPES-CZB containing 12% (w/v) polyvinyl pyrrolidone (PVP; Mr 360 kDa) immediately before ICSI. ICSI was performed using Eppendorf micromanipulators (Micromanipulator TransferMan; Eppendorf) with a piezo electric actuator (PMM controller, model PMAS-CT150; PrimeTech, Tsukuba, Japan). A single spermatozoon was drawn, tail first, into the injection pipette and moved back and forth until the head-midpiece junction (the neck) was at the opening of the injection pipette. The head was separated from the midpiece by applying one or more piezo pulses. After discarding the midpiece and tail, the head was redrawn into the pipette and injected immediately into an oocyte. Injections were done in HEPES-CZB within 1 h after oocyte collection and sperm reconstitution. Sperm motility after cryopreservation was low. Whenever possible, motile spermatozoa were used, because our earlier observations indicated that the incidence of abnormal sperm karyotypes increased when spermatozoa were not selected for motility. After rapid freezing all sperm were immotile and were randomly chosen for injections. Sperm-injected oocytes were transferred into CZB medium for culture. The oocytes were examined 6 h after ICSI to assess their survival and activation. The oocytes with two well-developed pronuclei and the distinct second polar body were recorded as activated. They were allowed to develop further.
Embryos reaching the two-cell stage were transferred to the oviducts (5–10 per oviduct) of CD-1 females mated during the previous night with vasectomized CD-1 males. The number of implantation sites and the number of fetuses were recorded at Day 15 of gestation. Examination of fetuses at Day 15 of gestation was chosen to provide information on the extent of early embryonic loss after implantation. Normal fetuses at Day 15 rarely fail to develop to full term.
Chlortetracycline Fluorescence Assay
Chlortetracycline (CTC) staining was performed for freshly obtained epididymal, capacitated epididymal, and ejaculated spermatozoa retrieved from the uteri. Epididymal and ejaculated spermatozoa were obtained as described above. To obtain capacitated sperm the caudae epididymides from one male were removed and placed in a 0.4-ml drop of T6 medium [28] under oil. The epididymal contents were teased out from each cauda epididymis with needles, and the tissue was discarded. Spermatozoa were capacitated for 1.5 h at 37°C, 5% CO2 in air.
The CTC assay was performed according to the procedure described previously [29]. CTC was dissolved at 500 µM in a chilled buffer of 20 mM Tris, 130 mM NaCl, and 5 mM cysteine, and the pH was adjusted to 7.8. The solution was kept in a light-shielded container at 4°C at all times. At the time of assay, 10 µl of the CTC solution was added to a warmed (37°C) slide, immediately after which an equal volume of sperm suspension was added, with mixing. The CTC-sperm suspension was incubated for 10 sec, and 12.5% glutaraldehyde in 1 M Tris buffer (pH 7.8) was added to a final concentration of 0.1% glutaraldehyde. The suspension was stirred thoroughly, and a cover glass was attached. The slides were examined using a fluorescence microscope (Nikon Eclipse E600) at x400 magnification. The fluorescence patterns of a minimum of 100 sperm per group were scored, and the experiment was repeated three times.
The experiments were designed to assess fertilization, implantation, and fetal development after the injection of oocytes with preserved ejaculated spermatozoa from wild-type and Hook1/Hook1 C57BL/6 males. Three C57BL/6 and three Hook1/Hook1 males were used as sperm donors. At least three (three to five) ejaculates were obtained from each male for each preservation method. The same males were used as sperm donors for conventional cryopreservation and rapid freezing without cryoprotection. In the majority of cases, the same ejaculate was used for both cryopreservation and freezing without cryoprotection. ICSI and embryo transfer also were performed with sperm from three controls: fresh epididymal sperm, epididymal sperm preserved by rapid freezing, and fresh ejaculated sperm.
Chi-square, likelihood ratio, and Fisher exact probability tests were used for analyzing all responses. Lack of statistical significance was reported when all tests gave P > 0.05. The presence of statistical significance was noted when at least one of the three tests showed P 
0.01 or P 0.05. The computations were done using KyPlot version 2.0 beta 13 software (developed by Koichi Yoshioka and available online at http://www.woundedmoon.org/win32/kyplot.html).
Repeated Retrieval of Ejaculated Spermatozoa from the Uteri
Young mature C57BL/6 and Hook1/Hook1 males were trained for morning mating. They were first exposed to females in estrus in the evening and allowed to mate during the night. After they had achieved mating competency, they were mated with females during early morning hours. Such training was necessary because under normal conditions mice mate in the middle of the night light cycle, and we wanted them to mate in the morning. Several males were successfully trained and became efficient in morning matings. Three C57BL/6 and three Hook1/Hook1 males that produced the highest number of ejaculates were used in this study. Each male delivered at least three (three to five) ejaculates. There were no significant differences in mating efficiency between males, with the exception of one C57BL/6 male that for unknown reasons stopped mating prematurely.
Ability of Ejaculated Preserved Spermatozoa to Fertilize Oocytes by ICSI
Spermatozoa obtained from the uteri and preserved by cryopreservation or rapid freezing without cryoprotection were able to fertilize oocytes by ICSI. We did not observe differences in the proportions of oocytes that survived, became activated, and reached the two-cell stage (a measure of successful fertilization) when comparing the two preservation methods and wild type versus Hook1/Hook1 mice (P > 0.05). The obtained results were not different (P > 0.05) from those obtained when control groups of fresh and rapid-frozen epididymal and fresh ejaculated sperm were injected (Table 1). There were statistically significant differences in fertilization rates (fertilization rate = proportion of two-cell embryos obtained from oocytes injected) between individual males within cryopreservation and rapid-freezing groups (Fig. 1, A and B). However, all but one male (hh2; P < 0.001) gave similar fertilization rates in both preservation methods.
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In this study most embryos were transferred at the two-cell stage. Occasionally, when there were not enough surrogates on the day following ICSI, four-cell embryos were transferred on the second day after ICSI. There were no differences when embryo transfer was done with two-cell and four-cell embryos. It is known that not all pseudopregnant females become pregnant following embryo transfer. This can be caused by technical difficulties during procedure (embryo loss) or by mistakes in evaluating estrus prior to mating with a vasectomized male. When embryos produced with ejaculated sperm from C57BL/6 were transferred, 76% (13 of 17) and 88% (15 of 17) of recipients became pregnant in the rapid-freezing and cryopreservation groups, respectively. The same tendency was noted with sperm from Hook1/Hook1 males (79%, 15 of 19; and 89%, 16 of 18, for rapid freezing and cryopreservation, respectively). Preimplantation embryo loss observed after embryo transfer with embryos produced with preserved ejaculated sperm was not far off from what we normally obtain during transfer using various types of embryos (M. Ward, unpublished results). All recipient females became pregnant in control groups in this study (fresh epididymal sperm, 8 of 8; rapid frozen epididymal sperm, 4 of 4; and fresh ejaculated sperm, 7 of 7; Table 1), but the number of embryos transferred (and the number of recipients) in controls was lower than in tested groups, which could account for a higher success rate.
Postimplantation Embryo Development
The implantation rate (proportion of embryos transferred that implanted) ranged from 39% to 54% in preserved ejaculated sperm groups and from 71% to 82% in controls (Table 1). When embryos produced with rapidly frozen spermatozoa were considered, those derived from ejaculated sperm implanted at lower rates compared with those produced with epididymal sperm (P < 0.05). The implantation rates also were lower for cryopreserved (P < 0.01) and rapid-frozen (P < 0.05) ejaculated sperm compared with fresh ejaculated sperm. There were differences in implantation rates between embryos produced with sperm from different males preserved in the same manner (Fig. 1, C and D; example: wt1 vs. wt2, rapid freezing, P < 0.001) and between embryos produced with sperm from the same male preserved differently (Fig. 1, C and D; example: wt2, P < 0.001 and hh2, P < 0.05).
We obtained live, normal fetuses in all examined groups and from all males included in the study (Table 1). Fetal rate (proportion of normal fetuses obtained from embryos transferred) was 11% for both rapidly frozen and cryopreserved ejaculated sperm from C57BL/6 males, and 11% for rapidly frozen and 17% for cryopreserved ejaculated sperm from Hook1/Hook1 males. This was significantly less than in controls (28%, 29%, and 31% in fresh ejaculated, rapid frozen epididymal, and fresh epididymal sperm, respectively). Cryopreserved sperm from Hook1/Hook1 males yielded more fetuses than rapid-frozen Hook1 or C57BL/6 sperm (P < 0.05; Table 1). There were no differences in fetal rates obtained with sperm from different wild-type C57BL/6 males, regardless of the preservation method used. One Hook1/Hook1 male (hh3) yielded a lower number of fetuses when rapid-frozen sperm were used for ICSI compared with both cryopreserved sperm and with rapid-frozen sperm from other Hook1/Hook1 males (P < 0.05; Fig. 1F).
CTC Staining of Ejaculated Sperm
Ejaculated sperm retrieved from the uteri are exposed to the environment of the female reproductive tract. We performed CTC staining to test whether ejaculated sperm retrieved from the uteri differed from epididymal sperm in their capacitation status. Four types of fluorescence patterns were differentiated (Fig. 2). Diffused fluorescence over the entire head with (pattern F1; Fig. 2A) or without (pattern F2; Fig. 2B) a brighter line of fluorescence across the equatorial segment is characteristic of noncapacitated sperm. Bright fluorescence in the anterior segment of the head and absence of fluorescence over the equatorial and postequatorial segment (pattern B; Fig. 2C) is a hallmark of capacitated, acrosome-intact sperm. Dull or absent fluorescence over the entire head (pattern AR; Fig. 2D) is characteristic for acrosome-reacted sperm [29]. When comparing three types of sperm analyzed—fresh epididymal, epididymal capacitated in vitro, and fresh ejaculated—significant differences (P < 0.001) in the frequencies of F and B patterns between groups were noted (Table 2). Only 12% of ejaculated sperm were classified as noncapacitated compared with 85% of fresh epididymal sperm. Ejaculated sperm had more capacitated sperm than did the epididymal sperm capacitated in vitro (77% vs. 45%; P < 0.001), but the occurrence of a spontaneous acrosome reaction was lower (11% vs. 19%; P < 0.01).
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In this study we evaluated the capacity of ejaculated sperm from C57BL/6 mice to fertilize oocytes and yield live offspring after preservation and injection into the oocytes. Ejaculated sperm were obtained from the uterus after mating and did not require killing the male.
We chose C57BL/6 inbred strain because it is likely to become a common genetic background of choice in all future research [30], and therefore there is demand for developing successful sperm preservation methods. The majority of known mutations exist on this background, and many molecular biology tools, such as BAC libraries and DNA probes, are derived from this strain [30]. Moreover, the public genome sequencing effort used C57BL/6J. This strain also is known to have high vulnerability to freezing [31] and low success rates following IVF with cryopreserved sperm [32–35], making it an ideal candidate strain for fertilization via ICSI. In addition to normal wild-type C57BL/6 mice we included in the study one exemplary infertile strain with abnormalities in the male reproductive system, the Hook1/Hook1 mutant mouse (also on C57BL/6 genetic background). The Hook1 mutation [36] (previously known as azh, abnormal spermatozoon headshape mutation) is specific for sperm head shape: all spermatozoa from mice homozygous for Hook1 mutation display abnormal head morphology [37]. The homozygosity for the Hook1 mutation has a dramatic effect on male fecundity, yielding males that are almost completely infertile [37]. We have shown recently that Hook1 mice can be reproduced by ICSI, yielding live offspring with high efficiency [5].
We were able to obtain ejaculated sperm in a repeated manner from both wild-type and infertile mice. The quality of ejaculates was similar among males and depended on the length of exposure to the female reproductive tract. In the preliminary experiments we mated mice in the evening and retrieved ejaculates in the morning. The quality of samples was low, and only few sperm were motile. Moreover, there was an excess of somatic cells in the uterine content, presumably leukocytes that are known to infiltrate the uterus [13], that increased sperm aggregation. When we changed the protocol to morning mating, sample quality improved significantly. Spermatozoa retrieved from the uterus were actively motile, and there were only a few cells other than sperm in the contents of the uteri.
It is known that mouse spermatozoa are more vulnerable to freezing than sperm from other species, such as bovine and human. Many approaches for mouse sperm preservation were described in the literature during the last 15 years, claiming varying degrees of success [31, 38–40]. Currently, the most popular approach to cryopreserve mouse sperm is based on a method developed by Okuyama et al. [38] (later improved and promoted by Nakagata [41, 42], Nakagata and Takeshima [43], and Takeshima et al. [44]) that uses a cryoprotective solution consisting of 18% raffinose and 3% skim milk. Although many laboratories have adopted what is now generally known as "Nakagata's freezing method" [34, 35, 45], the technique is not universally successful for all strains of mice, with C57BL/6 being one of the most troublesome backgrounds. Several different approaches were attempted to overcome low fertilization rates in IVF with cryopreserved mouse sperm [46–48], with the highest efficiency provided by ICSI [33]. Successful introduction of mouse ICSI enabled the development of alternative methods for mouse sperm preservation, such as freeze drying [25, 49, 50], rapid freezing without cryoprotection [25, 50, 51], and drying [52, 53]. Spermatozoa preserved with these methods are physiologically dead, but when injected into the oocytes they yield normal fetuses and live offspring. Freeze drying and rapid freezing was successful with sperm from both hybrid [25, 49, 51] and inbred [25, 50] strains. When rapid freezing was used in C57BL/6 mice, normal fetuses were obtained with the same efficiency as for fresh sperm [25].
In all the aforementioned reports on mouse sperm preservation, sperm were collected from the epididymides after the male was killed. To the best of our knowledge there is only one study in which cryopreservation was applied to ejaculated sperm [12]. In this elegant work the authors compared epididymal and ejaculated sperm from hybrid mice. They showed that two types of spermatozoa had similar initial motility and viability, and fertilized oocytes in vitro with a comparable efficiency. When ejaculated spermatozoa were cryopreserved in raffinose, glycerol, and egg yolk, their motility and viability decreased, but they were still capable of fertilizing oocytes in vitro, and the method was shown to be feasible in obtaining embryos and live offspring [12]. The drawback of this approach is that it is IVF based, and therefore it might be applicable only for robust hybrid mice. Spermatozoa from inbred strains frequently show poor resistance to cryopreservation, and low fertilization rates are obtained with cryopreserved epididymal sperm [32–35]. Similar, if not worse, IVF outcome is likely to be seen with ejaculated sperm.
Poor fertilization rates can be significantly improved by ICSI. We have previously shown that the ICSI technique provides an efficient means of generating embryos from cryopreserved [33] and rapid-frozen [50] mouse spermatozoa. We and others also have demonstrated that this method yields live offspring with high efficiency when used with sperm from infertile mice [5–7, 54]. Here for the first time we used ICSI to inject preserved ejaculated spermatozoa. We obtained normal viable fetuses after ICSI with ejaculated preserved sperm from C57BL/6 mice. Similar proportions of fetuses were obtained with sperm preserved by cryopreservation and by rapid freezing, suggesting that both methods are equally efficient. Moreover, the results obtained with sperm from Hook1 mutant mice were comparable with those obtained with wild-type C57BL/6 mice, implying that the approach is universal for both fertile and infertile mice. Embryo development after ICSI with preserved ejaculated sperm was lower (11%–17%) when compared to controls (28%–31%), regardless of the preservation method or the type of mouse used. One of the most striking differences between the epididymal and ejaculated sperm retrieved from the uterus is that the latter are exposed to the female reproductive tract. During copulation, spermatozoa are deposited in the vagina, where they undergo several significant changes and become capacitated (i.e., capable of fertilizing oocytes). Here we have demonstrated that the majority of ejaculated sperm retrieved from the uterus were indeed capacitated (Table 2). In our recent study on sperm chromatin remodeling after IVF and ICSI we showed that when capacitated sperm were injected into the oocytes by ICSI, early postfertilization events were affected. Chromatin remodeling was less synchronous than in ICSI with fresh sperm, and pronuclei formation and maturation and initiation of DNA synthesis were significantly delayed compared with IVF and fresh sperm ICSI [55]. Thus, it could be speculated that poorer development of embryos produced with preserved ejaculated sperm observed in this study resulted from sperm acquiring capacitation status prior to injection. Developmental rates were lower for preserved ejaculated sperm compared with fresh ejaculated sperm. This might be due to further enhancement of capacitation status by low temperatures [56]. In the course of our ongoing investigations on the mechanism of sperm DNA damage we have found that ejaculated sperm exhibit different nuclease activity compared with epididymal sperm (M. Ward, unpublished results). We also know that nuclease-dependent sperm DNA degradation may be enhanced by sperm freezing [25, 57]. These two findings also may explain lower developmental rates after ICSI with frozen ejaculated sperm.
The significance of this study is multifold. First, we have shown that spermatozoa can be successfully obtained from the same male in a repeated manner while keeping him alive and able to breed. Previous reports have demonstrated feasibility of obtaining ejaculated sperm from hybrid mice [8, 12]. This is the first report describing the use of this method for inbred and infertile mice. Second, we have shown that sperm retrieved from the uterus and subsequently preserved are fully functional in ICSI. IVF with sperm from inbred mice is frequently inefficient, especially with cryopreserved sperm, and it is reasonable to expect that ICSI will be a method of choice for strain reconstitution in the future. Here we took a first step in introducing the use of ICSI for ejaculated mouse sperm. Third, we have shown that two preservation methods, cryopreservation and rapid freezing, are equally efficient when applied to ejaculated sperm. This supports our previous conclusion [50] that rapid freezing without cryoprotection is as efficient as the conventional methods of sperm cryopreservation. Because of its simplicity, we recommend it as a preferred method of sperm preservation when used with ICSI. Finally, we have demonstrated that our approach (ICSI with preserved ejaculated sperm retrieved from the uterus) can be applied successfully to infertile mice with a spermatogenesis defect.
More than 10 000 mouse strains and more than 20 000 mouse embryonic stem cells containing gene-trapped or targeted mutations, many of which will be made into live mice, already exist, and many more will be created in the near future [30]. Our approach is of importance for maintenance and distribution of novel mouse strains. It is applicable for all types of mice, and the sole requirement is that a male of interest is able to copulate and that his ejaculate contains spermatozoa.
ACKNOWLEDGMENTS
The authors thank Bayard Storey from University of Pennsylvania for providing a protocol for CTC staining, and Robert Taft from Jackson Laboratory for insightful discussion about obtaining mouse sperm from the uteri.
FOOTNOTES
1Supported by National Institutes of Health grant HD048446 to M.A.W. 
Correspondence: 2Monika A. Ward, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Rd., Honolulu, HI 96822. FAX: 808 956 7316; e-mail: mward{at}hawaii.edu
Received: 28 December 2006.
First decision: 30 January 2007.
Accepted: 19 February 2007.
REFERENCES
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