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a Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, gamete biology, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Successful cryopreservation of mouse spermatozoa was achieved relatively recently. Many techniques have been described in the literature during the last 10 years, claiming varying degrees of success [2–5]. Okuyama et al. [2] showed that mouse sperm preservation with a cryoprotective solution consisting of raffinose and skim milk resulted in a postthawing sperm survival ranging between 30 and 50%. Later, a variety of combinations of raffinose and skim milk were tested and a mixture of 18% raffinose and 3% skim milk was found to be optimal for sperm preservation [6]. Nakagata and coworkers published several articles describing the successful cryopreservation of spermatozoa from a variety of strains and transgenic stocks with this combination of cryoprotective agents [7–11]. Although some laboratories have adopted what is now generally known as Nakagata's freezing method [12–14], the technique is not universally successful for all strains of mice. While the spermatozoa from some strains appear undamaged by exposure to cryoprotectant and the freezing procedure, those of other (mostly inbred strains) are seriously damaged, as shown by very low rates of fertilization. To increase the efficiency of fertilization with cryopreserved spermatozoa, Nakagata employed partial dissection of the zona pellucida (PZD) before IVF [10]. Recently, we reported that the separation of live spermatozoa by Sephadex filtration prior to cryopreservation significantly improves the success of fertilization after cryopreservation [15].
Another approach, which can be used in conjunction with sperm preservation, is intracytoplasmic sperm injection. Already, it has been reported that live offspring can be produced after ICSI with freeze-dried spermatozoa or spermatozoa frozen rapidly without cryoprotection [16, 17], but these techniques need to be improved to minimize genetic damage to spermatozoa.
In this study, we have examined and compared a) the efficiency of ICSI and IVF in generating embryos from epididymal spermatozoa frozen conventionally with 18% raffinose and 3% skim milk for cryoprotection; b) a hybrid strain B6D2F1 (C57BL/6J x DBA/2J) and an inbred strain (C57BL/6J) commonly used in mutagenic and transgenic studies (the spermatozoa of the former are relatively resistant to freezing, whereas those of the latter are known to be extremely venerable to freezing) [4]; c) the genetic integrity of spermatozoa by the analysis of the chromosome complement of oocytes prior to the first cleavage division; and d) the developmental potential of preimplantation embryos generated by IVF and ICSI from fresh and frozen spermatozoa.
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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), nonessential and essential amino acids (NEAA and EAA) from GIBCO BRL (Grand Island, NY), and eCG and hCG from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
Animals
Mice were obtained at 6 wk of age from the following sources: B6D2F1 (C57BL/6J x DBA/2) and CD-1 from the National Cancer Institute (Rayleigh, NC) and C57BL/6J from Jackson Laboratory (Bar Harbor, ME). 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 Committee on Care and Use of Laboratory Animal of the Institute of Laboratory Resources National Research Council (DHEW publication 80-23, revised in 1985).
Media
T6 medium [18] was used for IVF and HEPES-buffered CZB medium (HEPES-CZB [19]) for gamete handling and ICSI. Medium KSOM containing NEAA and EAA (mKSOMAA [20]) was chosen for embryo culture because preliminary observations showed that a significantly higher proportion of two-cell embryos from C57BL/6J mice developed into blastocysts in mKSOMAA compared with CZB medium [21]. The mKSOMAA and T6 were maintained in an atmosphere of 5%CO2 in air and HEPES-CZB and Dulbecco phosphate buffered saline (PBS) in air.
Sperm Collection
Epididymal spermatozoa were obtained from males 8–16 wk of age. The caudae epididymides were removed from each animal and placed in 100µl of cryoprotectant solution in an organ tissue culture dish (cat. no. 3513037; Falcon, Bedford, MA). The epididymal contents were expressed from the cauda epididymis with needles and the tissue discarded. Spermatozoa were allowed to disperse in the medium for 2–5 min at room temperature. Samples of the sperm suspension were removed for IVF and ICSI and the remainder loaded into straws for freezing.
Sperm Freezing
Spermatozoa were frozen by a method described by Nakagata [9], with some minor modifications.
Cryoprotectant The cryoprotectant solution containing 18% D-(+)-raffinose pentahydrate (w/v) and 3% skim milk (w/v) was prepared by dissolving 3.6 g of D-(+)-raffinose pentahydrate and 0.6 g of skim milk in 20 ml of 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. Immediately before use, an aliquot was thawed and warmed to 37°C.
Freezing After dispersion in the cryoprotectant, aliquots of 10 to 15 µl sperm suspension were loaded immediately into 250-µl straws (Edwards Innovations, Spring Valley, VA). Each straw was sealed with Critoseal (Oxford Labware, St. Louis, MO) and placed in a plastic holder, which was then floated on the surface of liquid nitrogen (LN2) in a LN2 storage container for 15 min before immersing in LN2. Samples were stored for up to 1 mo.
Thawing
The straws were removed from the storage container and immersed in a water bath at 37°C for 10–15 min. The contents of a straw were expressed into a Petri dish and 2.5–5.0 µl of the sperm suspension was either added to a small drop (
10 µl) of T6 medium for ICSI or to 200 µl of T6 medium for IVF.
Oocyte Collection
Mice, 8–12 wk old, were induced to superovulate with injections of 5 IU eCG and 5 IU hCG given 48 h apart. Oviducts were removed 14–15 h after the injection of hCG and placed in PBS in a Petri dish. For IVF, oviducts were transferred beneath the mineral in the plastic dish (cat. no. 351007; Falcon) close to the fertilization drop (T6 medium plus spermatozoa). The cumulus-oocyte complex was released from the ampullary region of each oviduct into the oil by rupturing the oviduct with the aid of a 25-gauge needle. The oviduct was discarded and the cumulus-oocyte complex moved into the fertilization drop. For ICSI, the cumulus-oocyte complexes were released from the oviducts into 0.1% of bovine testicular hyaluronidase (300 USP units/mg) in HEPES-CZB medium to disperse cumulus cells. The cumulus-free oocytes were washed with HEPES-CZB medium and used immediately for ICSI. In each replicate, oocytes were obtained from two females for ICSI and another two females for IVF.
In Vitro Fertilization
Gametes of the same strain were used for IVF. The method for sperm capacitation and IVF using T6 medium has been described elsewhere [18]. Briefly, 200-µl drops of T6 medium (fertilization drops) were overlaid with mineral oil in a plastic culture dish (60-mm diameter) and equilibrated overnight at 37°C in an humidified atmosphere of 5% CO2 in air. The volume of sperm suspension added to the fertilization drop was dependent on the concentration of spermatozoa after dispersion in the cryoprotectant solution. Generally, between 2.5 and 5.0 µl of sperm suspension was added to each fertilization drop to give the final sperm concentrations of approximately 2 x 106/ml. Spermatozoa were incubated in the T6 medium for about 1 h before the addition of oocytes. The contents of four oviducts were released into each fertilization drop. After incubation for 4 h, the oocytes were washed several times with HEPES-CZB medium followed by at least one wash with mKSOMAA medium. Only morphologically normal oocytes were selected for culture.
Intracytoplasmic Sperm Injection
ICSI was carried out according to Kimura and Yanagimachi [19], with some modifications. A small drop of sperm suspension incubated for 1 h at 37°C 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, Germany) with a piezo-electric actuator (PMM Controller, model PMAS-CT150; Prima Tech, Tsukuba, Japan). A single motile 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. ICSI was done in HEPES-CZB within 1–2 h after oocytes collection. Only motile spermatozoa were used for injection in this study because earlier observations indicated that the incidence of abnormal sperm karyotypes increased when spermatozoa were not selected for motility. Sperm-injected oocytes were transferred into mKSOMAA medium and cultured at 37°C. The oocytes were examined 6 h after ICSI for survival and activation.
Preimplantation Culture
After IVF and ICSI, the oocytes were placed in 50-µl drops of mKSOMAA medium preequilibrated overnight with humidified 5%CO2 in air. The culture drops were contained in plastic culture dishes (cat. no. 351007; Falcon) and overlaid with mineral oil. The survival of ICSI oocytes was scored 1–2 h after the commencement of culture. The number of two-cell embryos with a second polar body (fertilized) was recorded after 24 h in culture. Progress through preimplantation to the blastocyst stage was subsequently examined up to 96 h after the commencement of culture.
Chromosomal Analysis
Oocytes from ICSI and IVF treatments were transferred after a 6–8-h culture into mKSOMAA containing 0.006 µg/ml vinblastine. Vinblastine was added to inhibit spindle formation and syngamy. Between 19 and 21 h after ICSI or IVF, oocytes were treated with 1% pronase (1000 tyrosine units/mg; Kaken Pharmaceuticals, Tokyo, Japan) for 5 min at room temperature to soften zonae pellucidae. Then the oocytes were treated with hypotonic solution (1:1 mixture of 1% sodium citrate and 30% fetal bovine serum) for 5 min at 37°C or 10 min at 25°C. Chromosomes were spread on glass slides by the gradual fixation/air-drying method [22]. The preparations were stained with 2% Giemsa (Merck, Darmstadt, Germany) in PBS (pH 6.8) for 10 min for conventional chromosome analysis. The chromosomes of a spermatozoon were considered normal when an egg contained normal 40 metaphase chromosomes. It was not always possible to distinguish between chromosomes of paternal and maternal origin. However, because oocyte chromosomes rarely show structural aberrations at the first cleavage of metaphase after parthenogenetic activation (unpublished observations), abnormal chromosomes within fertilized oocytes were considered of sperm origin.
Determination of Blastocyst Cell Number
Blastocysts were treated with 1% pronase for 5 min at room temperature to soften zonae pellucidae. Then they were treated with the hypotonic solution described above before spreading on glass slides by the gradual fixation/air-drying method [22]. The slides were stained with 2% Giemsa (Merck) in buffered PBS (pH 6.8) for 10 min before counting the nuclei.
Experimental Design
Experiments were designed to compare the rates of fertilization and development to the blastocyst stage after ICSI and IVF with fresh and frozen spermatozoa from the inbred and hybrid strains. A single male was the source of spermatozoa for fresh and frozen spermatozoa for ICSI and IVF in each experimental replicate in order to reduce the possible variation in fertility of spermatozoa between males within a strain. For ICSI and IVF, oocytes and spermatozoa were from the same strain. Oocytes from the same group of superovulated females were used for ICSI and IVF each day. The overall efficiency of fertilization and preimplantation development in vitro after ICSI and IVF was expressed as the proportion of two-cell embryos and blastocysts developing from the original number of oocytes injected or inseminated.
To determine the frequency of sperm chromosome abnormalities, samples of oocytes were taken from some of the replicates and arrested with vinblastine prior to the first cleavage. Blastocysts were chosen randomly for determining cell number. All preparations for chromosome analysis and counting cell nuclei were coded and scored blind.
Statistics
Chi-square, likelihood ratio, and Fisher exact probability tests 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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It is possible that traces of cryoprotectant are introduced into the oocyte during ICSI even though the spermatozoa are washed several times prior to ICSI. To determine whether the cryoprotectants have a deleterious effect on the development of ICSI-produced embryos in vitro, a comparison was made between the development of embryos produced from B6D2F1 oocytes fertilized by ICSI with B6D2F1 epididymal spermatozoa exposed or not exposed to raffinose/skim milk. The data for three replicates were homogeneous within each treatment, and the combined data are summarized in Table 1. Fertilization of oocytes and embryonic development in vitro were not affected by exposure of spermatozoa to the cryoprotectants before ICSI.
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Fertilization of Oocytes after IVF or ICSI with Fresh and Frozen Epididymal Spermatozoa and the Subsequent Development of the Embryos In Vitro
No differences were observed between the quality of fresh and frozen-thawed B6D2F1 and C57BL/6J spermatozoa. The motility of the frozen-thawed spermatozoa decreased approximately twofold when compared with fresh samples in both strains.
B6D2F1 Hybrid
Data for the fertilization and preimplantation development of B6D2F1 oocytes fertilized by ICSI or IVF with fresh and frozen B6D2F1 epididymal spermatozoa are presented in Table 2. The fertilization rate was expressed as the proportion of oocytes developing to the two-cell stage 24 h after injection or insemination. ICSI yielded high rates of fertilization (87%) with both fresh and frozen spermatozoa. A similar high fertilization rate was obtained following IVF with fresh spermatozoa (89%), but the rate decreased significantly with frozen spermatozoa (56%, 
2 [1] = 66.89; P < 0.001). Development of two-cell embryos to the blastocyst stage appeared slightly higher for IVF-produced embryos (94–98%) than for ICSI-produced embryos (84–88%), but the difference was not statistically significant. Sperm freezing did not seem to affect the development of embryos produced either by ICSI or IVF embryos. However, the overall proportion of blastocysts obtained from the number of oocytes inseminated or injected was significantly higher for fresh spermatozoa after IVF (87%) than for any other treatment, but the percentage of blastocysts produced after ICSI was similar for fresh and frozen spermatozoa (72% and 77%).
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C57BL/6J
Data for the fertilization and preimplantation development of C57BL/6J oocytes fertilized by ICSI or IVF with fresh or frozen C57BL/6J epididymal spermatozoa are presented in Table 3. ICSI resulted in consistently high rates of fertilization regardless of whether the spermatozoa were fresh or frozen (90% and 85%). In contrast, fertilization after IVF was significantly lower with fresh spermatozoa (55%) and it was reduced drastically after freezing (9%), the rate of fertilization varying considerably between replicates (0–25%).
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Both ICSI- and IVF-fertilized oocytes developed in similar proportions to the blastocyst stage, irrespective of whether the spermatozoa were fresh or frozen (Table 3). Although the proportion of two-cell stage embryos that developed to blastocysts appeared lower in the ICSI groups (42–46%) when compared with the IVF groups (56–61%), the only comparison where a small but significant reduction in development occurred was between ICSI and IVF with frozen spermatozoa (42% vs. 61%: 2[1] = 4.41; P < 0.05). However, the overall developmental rate, i.e., the proportion of blastocysts developing from the total number of oocytes originally injected or inseminated, was higher after ICSI (35% and 41%) than after IVF (6% and 31%).
Comparison of Blastocyst Cell Numbers in Hybrid (B6D2F1 x B6D2F1) and C57BL/6J Embryos
The total number of cells contained in blastocysts obtained from the culture of oocytes fertilized after ICSI or IVF with fresh or frozen spermatozoa was determined by counting the nuclei in stained preparations. The data are presented in Table 4. There were significantly more cells in hybrid blastocysts compared with C57BL/6J blastocysts (approximately twice as many) irrespective of whether they were derived by ICSI or IVF from fresh or frozen spermatozoa. The delayed development of C57BL/6J embryos may result from suboptimal culture conditions that interfere with oocyte activation and/or the length of the cell cycle. For hybrid embryos, the blastocyst cell number was significantly lower in embryos produced by ICSI than IVF for both fresh and frozen spermatozoa. This difference was only apparent in C57BL/6J blastocysts generated from frozen spermatozoa. Overall, slower development of the C57BL/6J embryos and the smaller sample sizes may have reduced the difference in the developmental rates between ICSI and IVF.
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Chromosome Analysis
Chromosome analysis of fertilized B6D2F1 and C57BL/6J oocytes generated by ICSI and IVF from fresh and frozen spermatozoa is presented in Table 5. Because the incidence of structural chromosome abnormalities in the maternal complement of chromosomes is very low, it was assumed that any increase would be associated with the sperm or paternal complement of chromosomes. Most of the fertilized oocytes had normal chromosomes (87–100%). The proportion of fertilized oocytes with normal chromosomes after ICSI and IVF with fresh spermatozoa was similar in both strains, but with frozen spermatozoa, the frequency of normal karyotypes was significantly lower in the ICSI fertilized group. The incidence of chromosome aberrations, when expressed per oocyte, was significantly higher in oocytes fertilized by ICSI with fresh and frozen spermatozoa of both strains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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High proportions of hybrid embryos derived from fresh and frozen spermatozoa by ICSI or IVF developed to the blastocyst stage in vitro (Table 2), but development of C57BL/6J embryos was severely compromised (Table 3), with many of the embryos arresting at the morula stage and failing to cavitate. There were twice as many cells in hybrid embryos at the blastocyst stage, indicating that the inbred embryos were delayed in developmental time by about one cell division (Table 4). This reflects differences commonly observed in the ability of inbred strains to develop in vitro throughout the whole preimplantation period in routine mouse embryo culture media. The balance between media constituents such as glucose and phosphate are known to be crucial for the development of preimplantation mouse embryos, and this varies between strains [24, 25]. Further work is needed to define optimal conditions for the culture of embryos from different strains.
The majority of spermatozoa that fertilized oocytes by ICSI and IVF had normal chromosomes (Table 5), but the incidence of chromosome aberrations was higher in ICSI-derived oocytes irrespective of whether the spermatozoa were fresh or frozen. However, the frequency was similar to that reported previously for ICSI with fresh spermatozoa [26]. It is unlikely that the small increase in chromosome aberrations was responsible for the poor development of the inbred embryos.
Mouse ICSI has been used to overcome male infertility due to poor sperm motility [27, 28] and abnormal sperm structure [29]. It was also used to fertilize oocytes with freeze-dried spermatozoa with disrupted plasma membranes [17, 30]. Although intact sperm plasma membrane is not essential for successful ICSI, its presence is important in keeping the sperm nucleus physiologically intact. Upon disruption of the sperm plasma membrane, sperm nuclei are exposed directly to extracellular components (including ions). It is not the disruption of sperm plasma membrane per se but the contact with an extracellular milieu that is detrimental to sperm nuclei [26]. Thus, if freeze-thawed spermatozoa are to be used for ICSI, the use of motile ones is preferable.
To date, ICSI technique has been performed on oocytes from a limited number of hybrid and inbred strains with variable degrees of success. Recently, the first full-term development of inbred mouse embryos produced with ICSI was reported [31]. The authors observed differences between an inbred (C57BL/6) and two hybrid strains (B6D2F1, B6C3F1) in the post-ICSI survival rate of the oocytes and development to term. They suggested that oocytes of inbred mice may be more sensitive to mechanical trauma during ICSI and that the healing capacity of plasma membrane might be inferior to the hybrid strains. In this study, we did not encounter any unexpected procedural difficulties with the survival of C57BL/6J oocytes after ICSI. Oocyte survival as well as the fertilization rate were similar to those obtained with B6D2F1 oocytes. Furthermore, we showed recently that survival and fertilization of oocytes after ICSI with freeze-dried and rapidly frozen spermatozoa were similar for several different inbred mouse strains (BALB/c, 129/SvJ, C57BL/6J) and the B6D2F1 hybrid [30].
Nakagata et al. [10] achieved higher rates of fertilization with frozen-thawed C57BL/6J spermatozoa (up to 70%) when the zonae pellucidae of C57BL/6J oocytes were partially dissected (PZD) with a glass needle before insemination. Dissecting zonae, however, increases the risks of polyspermic fertilization, premature escape of the embryo, and infection with viruses or bacteria. Exposure of oocytes to a high concentration of sucrose (0.3 M) during PZD may also have a deleterious effect on embryonic development. In an attempt to avoid these potential problems, Kawase et al. [32] made smaller incisions in the zonae pellucidae of C57BL/6J oocytes with a piezo manipulator. Sucrose was also omitted from the medium for the manipulation. However, fertilization rates did not differ from what they obtained with PZD oocytes (50%). The results for fertilization were similar to those we obtained recently by IVF with intact C57BL/6J oocytes and cryopreserved C57BL/6J spermatozoa selected for high motility by Sephadex separation before freezing [15], but the proportion of inseminated oocytes developing into fetuses was significantly lower.
The direct comparison of ICSI and IVF shows that there are considerable savings in the number of oocytes and spermatozoa required to generate embryos. The advantage of ICSI over IVF is that it requires only a single spermatozoon to fertilize an oocyte, while IVF needs hundreds of thousands of spermatozoa in the fertilization medium even for a few oocytes. Sperm cryopreservation may not work well every time. If the number of motile spermatozoa in a particular sample is too low for IVF, hundreds of thousands of spermatozoa in the sample are lost. The use of the ICSI technique can solve this problem. Theoretically, with the ICSI technique, thousands of offspring can be produced from the cryopreserved spermatozoa of a single male provided the spermatozoa are stored in many straws. Where spermatozoa of some mutant and transgenic mice are vulnerable to cryopreservation, ICSI should be able to rescue them as long as few spermatozoa survive freezing and thawing. ICSI requires specialized equipment, but these capabilities are closely related to those already in place in many laboratories for generating genetically altered mouse strains. Also, it is by no means a difficult technique, as evidenced by its routine use in many human infertility clinics worldwide. For the species whose spermatozoa tolerate the freeze-thawing procedures well, routine IVF is recommended, but if spermatozoa respond to freeze-thawing erratically or almost all spermatozoa are found motionless or dead after thawing, ICSI is the only alternative to IVF.
In conclusion, ICSI is a more efficient and effective technique than IVF for generating embryos from frozen mouse spermatozoa. More importantly, ICSI can be especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.
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ACKNOWLEDGMENTS |
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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
3 Current address: Department of Biological Sciences, Asahikawa Medical College, Asahikawa, Hokkaido 078-8510, Japan
Received: 2 April 2002.
First decision: 29 April 2002.
Accepted: 27 May 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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