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Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, embryo, gamete biology, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The majority of children born from ICSI are still under reproductive age; therefore, their reproductive health remains unknown. It was reported that some chromosomal anomalies known to cause infertility were transmitted from fathers to sons via ICSI [11–13]. When these children become adults, some of them likely will have the same types of infertility that their parents had, leaving them with ICSI as their only possibility for reproduction. What is not yet known is whether this heritable impairment of fertility will fully match paternal infertility or whether ICSI will cause its enhancement in the next generation of men conceived with this method.
The azh (abnormal spermatozoon head shape) mutation (now officially designated Hook1 mutation [14]) in the mouse was first described in 1984 [15]. Although many mutations affecting sperm head shape were shown to have pleiotropic effects on spermatogenesis and other tissues, azh mutation was reported as specific for sperm head shape: All spermatozoa from mice homozygous for azh mutation displayed abnormal head morphology [16]. The azh mutation also was found to cause tail abnormalities, resulting in coiled tails or decapitation of the sperm head from the flagellum [14, 17]. The azh mutation was demonstrated to display an autosomal recessive pattern of inheritance, and the azh locus was mapped to mouse chromosome 4 [18]. Recently, a gene responsible for the azh phenotype was isolated and characterized [14]. The azh mice were found to carry a mutated version of the Hook1 gene in which two exons were deleted, leading to a putative truncated protein. It was suggested that Hook1 gene function is necessary for the correct positioning of microtubular structures within the haploid germ cell [14]. The homozygosity for azh mutation has a dramatic effect on male fecundity, yielding males that are almost completely infertile [15, 16]. It was reported that spermatozoa from azh/azh mice failed in binding to the zona pellucida [19], which suggested that the major cause of infertility in azh/azh mice is at the level of binding and penetration of the zona pellucida. It was later shown that mice homozygous for azh mutation were unable to fertilize intact oocytes but were able to fertilize zona-free eggs [20].
The present study examines the efficiency and effects of ICSI in successive generations of azh mice generated with this method. The azh mutant mice were chosen because 1) males are infertile and, therefore, are good candidates for modeling ICSI in humans; 2) males demonstrate characteristic phenotype, teratozoospermia (increased frequency of morphologically abnormal sperm), that is easily measurable; and 3) earlier works done with these mice indicated negative effects of ICSI [21, 22]. Three successive generations of azh mice were produced by ICSI, and ICSI-derived progeny were evaluated with an emphasis on their fecundity and sperm morphology. No negative effects of ICSI were found.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mineral oil was purchased from Squibb and Sons (Princeton, NJ), and eCG and hCG were purchased from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO) unless otherwise stated.
Animals
Three breeding pairs of azh/+ mice on C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, ME) at 6 wk of age and used to establish an azh colony. Other mice (B6D2F1 [C57BL/6J x DBA/ 2], C57BL/6N, and CD-1) were obtained at 6 wk of age from the National Cancer Institute (Rayleigh, NC). The mice were fed ad libitum with a standard diet and maintained in a temperature- and light-controlled room (22°C, 14L:10D photoperiod) in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and the guidelines presented in the National Research Council 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).
Media
Medium T6 [23] was used for in vitro fertilization (IVF) and Hepes-buffered CZB medium (Hepes-CZB) [24] for gamete handling and ICSI. Medium CZB [25] was used for embryo culture. Both CZB and T6 were maintained in an atmosphere of 5% CO2 in air, and Hepes-CZB was maintained 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 a 0.4-ml drop of T6 medium (capacitation drop) under oil. The epididymal contents were expressed from the cauda epididymis with needles, and the tissue was discarded. Spermatozoa were allowed to disperse for 2–3 min at room temperature. A sample of sperm suspension for ICSI was taken immediately after sperm dispersion. For IVF, a sample of sperm suspension was taken after 1.5 h of capacitation.
Analysis of Sperm Morphology
Spermatozoa suspended in T6 medium as described above were spread over clean glass slides and air-dried. The slides were fixed and stained using Diff-Quick Stain Kit (BDH, Poole, UK). The morphology of spermatozoa was examined with light microscopy at x2700 magnification. All preparations for analysis of morphology were coded and scored blind.
Oocyte Collection
At 8–12 wk of age, mice were induced to superovulate with injections of 5 IU of eCG and 5 IG of 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 (catalog no. 351007; Falcon, Bedford, MA) 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% 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 Vitro Fertilization
The method for sperm capacitation and IVF using T6 medium has been described elsewhere [23]. Briefly, 200-µl drops of T6 medium (fertilization drops) were overlaid with mineral oil in a plastic culture dish (diameter, 60 mm) and equilibrated overnight at 37°C in a 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 capacitation drop. Generally, 10 µl of sperm suspension from the capacitation drop were added to each fertilization drop to give the final concentrations of approximately 2 x 106 sperm/ml. The contents of four oviducts were released into each fertilization drop. After gamete coincubation for 4 h, the oocytes were washed several times with Hepes-CZB medium followed by at least one wash with CZB medium. Only morphologically normal oocytes were selected for culture.
Intracytoplasmic Sperm Injection
The ICSI was carried out as described recently by Szczygiel and Yanagimachi [26]. Briefly, a small drop of sperm suspension was mixed thoroughly with an equal volume of Hepes-CZB containing 12% (w/v) polyvinyl pyrrolidone (Mr, 360 kDa) immediately before ICSI, which 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. The ICSI was done in Hepes-CZB within 1–2 h after oocyte collection. Only motile spermatozoa were used for injection in the present 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 CZB medium and cultured at 37°C. The oocytes were examined approximately 6 h after ICSI for survival and activation.
Embryo Culture and Transfer
After IVF and ICSI, the oocytes were placed in 50-µl drops of CZB medium pre-equilibrated overnight with humidified 5% CO2 in air. The culture drops were contained in plastic culture dishes and overlaid with mineral oil. The survival of ICSI oocytes was scored 1–2 h after the commencement of culture. The number of 2-cell embryos (fertilized) was recorded after 24 h in culture.
Embryos reaching the 2-cell stage were transferred to the oviducts (n = 5–10 per oviduct) of CD-1 females mated during the previous night with vasectomized CD-1 males. Surrogate mothers were allowed to deliver and raise their offspring. The progeny were genotyped at weaning (age, 21 days) and subsequently used for breeding or as sperm donors for IVF and the next series of ICSI.
Genotyping for azh Mutation
Genomic DNA was obtained from ear punch at weaning and isolated using Qiagen DNeasy Tissue Kit (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) analysis was performed using three primers: azh-for, 5'-gCC AgA TgT Tgg TCA gAg gCA gTA A-3'; azh-rev, 5'-ggC CAA ATC TAg gAg AgC ggA gCA T-3'; and azh-rev2, 5'-CAg ggA AAC CTg AAg AgC TCA gTA A-3'. The PCR conditions were as follows: initial denaturation at 96°C for 15 min; followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, and extension at 72°C for 45 sec; and a final elongation at 72°C for 10 min. The amplification products were analyzed on ethidium bromide-stained agarose gels.
Experimental Design
Experiments were designed to evaluate the efficiency of serial (successive generations) ICSI to reproduce azh mice and the fecundity and sperm morphology of ICSI-derived azh mice. The efficiency of ICSI was measured as fertilization rate (proportion of 2-cell embryos obtained from oocytes injected), embryo transfer rate (proportion of live offspring from 2-cell embryos transferred), and live offspring rate (proportion of live offspring from oocytes injected). Fecundity of males was measured as sperm potential to fertilize oocytes in vitro and was reflected as fertilization rate in IVF (proportion of 2-cell embryos from oocytes inseminated). Evaluation of sperm morphology was done for at least three males (n = 3–5), and at least 100 spermatozoa were examined per male. All preparations for analysis of morphology were coded and scored blind.
Statistics
Chi-square, likelihood ratio, and Fisher exact probability tests were used for analyzing all responses. Lack of statistical significance was reported when all three tests gave P > 0.05. 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 (software developed by Koichi Yoshioka and available online: http://www.woundedmoon.org/win32/kyplot.html).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three azh/+ breeding pairs purchased from Jackson Laboratory were used to establish an azh mice colony. These pairs were later supplemented by three additional breeding pairs (azh/azh females and azh/+ males) produced in-house. Over the period of 10 mo, 108 progeny were obtained and genotyped. Fourteen azh/azh (9 females and 5 males), 31 azh/+ (15 females and 16 males), and 16 +/+ (6 females and 10 males) mice were obtained when heterozygotes were bred. Seventeen azh/azh (6 females and 11 males) and 30 azh/+ (13 females and 17 males) mice were obtained when homozygous females were bred with heterozygous males. Homozygous mice were used as sperm and oocyte donors. All breeding crosses done in the present study are shown in Table 1.
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Results of PCR Analysis
The azh mutation results from a deletion of exons 10 and 11 in the murine Hook1 gene, leading to a nonfunctional protein [14]. The PCR analysis was performed using one primer located in a region of intron 9 that is not deleted (azh-for) in the azh allele and one primer in intron 9 that is absent in the azh allele (azh-rev). A third primer, located in intron 11, also was included (azh-rev2). With the combination of primers azh-for and azh-rev, a 505-base pair (bp) fragment was obtained in DNA from +/+ and azh/+ mice, whereas no amplification occurred in DNA from azh/ azh mice. The combination of the primers azh-for and azh-rev2 yielded 2364 bp (not shown in the gel) in DNA from +/+ mice and a PCR fragment reduced to 343 bp when a deletion in the azh allele (both azh/+ and azh/azh) was present (Fig. 1).
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Production of Successive Generations of azh ICSI Offspring
All breeding crosses done in the present study are shown in Table 1. Three generations of ICSI offspring were produced successfully (Table 2). To produce first-generation ICSI offspring (azh/+ ICSI1), spermatozoa from homozygous, infertile azh/azh mice were injected into the oocytes from C57BL/6 females. The second (azh/+ ICSI2) and third (azh/+ ICSI3) generations were produced by injecting spermatozoa from azh/+ males derived by the first and second round of ICSI, respectively, into the oocytes from C57BL/ 6 females. The efficiency of fertilization with ICSI, measured by the proportion of 2-cell embryos obtained from oocytes injected, was high. In all three series of ICSI, more than 80% of 2-cell embryos were obtained. When 2-cell embryos were transferred into the oviducts of pseudopregnant females, more than 35% gave rise to normal live offspring. No statistically significant differences were observed between ICSI efficiency (measured both as the proportion of 2-cell embryos and the proportion of live offspring obtained from oocytes injected) in all three generations.
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The ICSI with both oocytes and spermatozoa obtained from homozygous azh/azh mice also was attempted. Although live offspring were obtained successfully (azh/azh ICSI1 and azh/azh ICSI2), the results were not yet optimal. The azh/azh females did not respond well to hormonal stimulation. The number of oocytes retrieved from the oviducts was low, and from those obtained, many were immature and/or of poor quality and were not used for injections. On two occasions, no oocytes were retrieved from the oviducts pooled from stimulated azh/azh females (three females in each attempt). Overall, only nine azh/azh females provided oocytes for ICSI, which was the reason for the low number of oocytes injected and the lack of experimental replicates. As many as 79% of azh/azh ICSI1 embryos transferred developed to live offspring, but from the seven live offspring obtained, only three survived to weaning (Table 2). This was unusual, because our norm in embryo transfer, regardless of the types of embryos transferred, is 80–100% of live-born offspring surviving to weaning. One of azh/azh ICSI1 males was used as a sperm donor for the next ICSI round with the oocytes from azh/azh females. Although sperm quality was not different than that of azh/azh males produced by mating (data not shown), spermatozoa from azh/azh ICSI1 males appeared to be less efficient in ICSI than expected. Some of the oocytes injected did not cleave, a low number of 2-cell embryos transferred developed to live offspring, and the offspring, on delivery via cesarean section on day 21 p.c., appeared to be developmentally retarded (abnormally small) and were cannibalized by a foster mother within 2 days after delivery. Before any clear conclusions can be drawn on the efficiency of ICSI with both gametes derived from homozygous mice, more experiments need to be done.
Fecundity
The ICSI-derived azh/+ males reproduced successfully by mating (data not shown), ICSI (Table 2), and IVF (Table 3). Spermatozoa from all three generations of ICSI-derived males did not show any impairment of fecundity in IVF. They successfully fertilized oocytes from both C57BL/6 and B6D2F1 females, with fertilization rates ranging from 70% to 92%. The difference in fertilization rates with two types of oocytes was statistically significant only with spermatozoa from azh/+ ICSI1 males (P 0.05).
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As a direct test for the effects of the fertilization method on the fecundity of azh/+ males, the progeny derived by three methods (ICSI, IVF, and mating) were evaluated (Table 4). To reduce the possible variation between individual males, the same azh/+ ICSI2 male was used for mating and as the source of spermatozoa for ICSI and IVF. Thus, male offspring whose fecundity was evaluated were derived from the same father. Oocytes from the same group of superovulated females were used for IVF with spermatozoa from males from three groups (ICSI, IVF, and mating) each day. A high fertilization rate (72–94%) was obtained in all groups, with ICSI- and IVF-derived males giving results higher than those of males derived by mating (P 0.001).
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Sperm Morphology
Analysis of sperm morphology was performed for at least three males (n = 3–5) from each examined group, and 100 spermatozoa were analyzed per male. In addition to counting normal versus abnormal spermatozoa, three types of spermatozoa were differentiated within the abnormal sperm group: spermatozoa with abnormal head only, spermatozoa with abnormal tail only, and spermatozoa with abnormal head and abnormal tail. When examining sperm heads, the following categories were counted: N, normal head; 1, thin and elongated head; 2, club-shaped head; 3, mild defects of head shape; and 4, other head anomalies. When examining sperm tails, the following categories were scored: N, normal tail; 1, bent head; 2, looping neck-midpiece; 3, folded midpiece and principal piece; 4, lasso type; 5, incorrect head/neck connection; and 6, combined defects or other anomalies (Fig. 2).
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All spermatozoa from azh/azh males were morphologically abnormal (Table 5). When comparing azh/azh males derived from mating and those produced by ICSI with both gametes from azh/azh mice (azh/azh ICSI1), no differences in the proportions of various types of anomalies were observed (Table 6 and Fig. 3).
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The proportion of morphologically normal spermatozoa in azh/+ males was similar for all three generations of ICSI males (azh/+ ICSI1, azh/+ ICSI2, and azh/+ ICSI3), and no differences were noted when comparing ICSI-derived males with azh/+ males derived by mating and wild-type controls (+/+) on the same genetic background (Table 5). Although some differences were noted among examined groups in three major categories of anomalies (head only, tail only, and head plus tail) (Table 6) as well as in specific types of head and tail abnormalities (Fig. 3), these differences were not significant. Spermatozoa from azh/+ males from all groups (and from wild-type controls) showed the same pattern in frequency of anomalies. The only case in which ICSI-derived males differed from males derived by mating was in the proportion of combined head plus tail abnormalities (P 0.05) (Table 6).
Enhancement in anomalies of sperm morphology in azh/+ males produced by ICSI reported previously [21] was observed in males on mixed genetic background, produced by injecting spermatozoa from azh/azh males on C57BL/6 background into the oocytes from B6D2F1 hybrid females. In the present study, when analysis of sperm morphology was performed in such males (azh/;plICSI1 Mix), the proportion of normal spermatozoa was high (67.7%) (Table 5), and the frequency of major types of anomalies (Table 6) was not different from that in azh/+ males derived by mating.
To look directly at the effect of the fertilization method on anomalies of sperm shape, analysis of morphology was performed in progeny derived from the same male (azh/+ ICSI2) by three methods: ICSI, IVF, and mating (Table 4). No differences were found in the proportion of normal spermatozoa between ICSI- and IVF-derived males, but the males produced with ART scored higher than did the males produced by mating (ICSI vs. mating, P 0.05; IVF vs. mating, P 0.01). The same trend was observed when comparing proportion of normal heads (ICSI and IVF vs. mating, P 0.05) and of normal tails and mild defects in head shape (IVF vs. mating, P 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of ICSI in successive generations of mice were evaluated by examining their fecundity in vitro and performing analysis of sperm morphology. Fecundity was measured by sperm ability to fertilize oocytes in vitro. Because previous works suggested that the fecundity of azh/ azh males was dependent on the phenotype of the female [20], and because it is commonly known that gametes from hybrid mice are more robust than those from inbred mice, oocytes (cumulus- and zona-intact) from both C57BL/6 inbred and B6D2F1 hybrid females were used in the IVF system in the present study. No fertilization in IVF was achieved with sperm from homozygous mutant mice, regardless of the oocytes used. It is worth noting that spermatozoa from the same pool were used successfully for ICSI. Spermatozoa from azh/+ ICSI-derived males, from all generations, were able to fertilize oocytes in vitro, as expected for heterozygous azh/+ mice and with an efficiency comparable to that published previously for azh/+ mice derived by mating [20]. Interestingly, the fertilization rate in IVF with spermatozoa from azh/+ ICSI-derived males and oocytes from C57BL/6 females was higher than those published previously by our group (58% [30] and 55% [31]) using IVF with both gametes from wild-type C57BL/6 mice. The data from the present study show that heterozygous mice produced with ICSI do not exhibit impaired fecundity.
It was reported previously that the progeny derived by ICSI with spermatozoa from azh/azh males injected into the oocytes from normal females exhibited a higher incidence of anomalies in sperm morphology than that expected in heterozygous offspring [21, 22]. Here, sperm morphology in three generations of ICSI-produced azh/+ mice was evaluated and compared with that of azh/+ and wild-type males derived by mating. No significant differences in the proportion of normal spermatozoa were observed. In the previous work, an increased frequency of morphological anomalies reflected sperm tail abnormalities [21]. Here, ICSI-derived males did not differ from those derived by mating when looking at head and tail abnormalities independently. A statistically significant difference was noted between azh/+ males derived by mating and azh/+ males from first and second-generation ICSI only in the proportion of spermatozoa, in which both head and tail were scored as abnormal.
A lasso-like configuration of the tail was found to be predominant in epididymal sperm from azh/azh mice [17] and also was observed in azh/+ mice derived by ICSI [21]. Mochida et al [17] proposed a model illustrating the major steps in formation of the abnormal tail in the azh/azh mice, in which initial bending of the midpiece proximal to the head is followed by formation of the loop, initial coil, and finally, lasso type. In the present study, the distribution of various subtypes of anomalies of sperm morphology, both within the head and the tail, was similar in males from all groups (Fig. 3). Only a few lasso-type sperm were observed in homozygous azh mice, and almost none was observed in ICSI-derived heterozygotes. The highest incidence among tail abnormalities occurred in the "bent head" category, in which the top part of the sperm head was bent back onto the tail (Fig. 2, T1). This type of bending is different from that proposed in the model of Mochida et al. [17].
The increase in anomalies of sperm morphology after ICSI reported previously [21, 22] was shown in azh/+ males that were produced with spermatozoa from azh/azh males on C57BL/6 background and oocytes from B6D2F1 females. The progeny were 75% C57BL/6 and 25% DBA. Our three generations of azh/+ ICSI offspring were produced on "clean" C57BL/6 background. It is possible that an effect of haplosufficiency of the azh gene product existed that was seen only on mixed genetic background. To determine if the discrepancy between results from the present study and those reported earlier is caused by differences in the genetic background of the mice that were examined, one generation of males was produced exactly as was done before. No increase in the frequency and quality of sperm morphological abnormalities was noted when compared to data from azh mice on C57BL/6 background. It is not clear what the cause is for the disparity in results between previous reports and the present study. The possible reason may be a low number of males examined previously and significant differences between individual males both in the incidence of sperm tail anomalies and in the type of anomaly that dominated [21]. In the present study, normal tail morphology was observed in the majority (70%–78%) of spermatozoa from three successive generations of ICSI males, and no statistically significant differences in the distribution of different categories of sperm tail abnormalities were recorded.
As a final test to demonstrate whether ICSI has some negative effects on the well-being of the progeny derived with this method, males derived from the same father by three methods (ICSI, IVF, and mating) were compared. Two hallmarks of the azh mutation were evaluated: fecundity in vitro, and sperm morphology. No impairment was found in ICSI progeny. On the contrary, spermatozoa from males derived by mating tended to be less efficient in IVF, and less of them were morphologically normal.
The measured differences between mice from successive generations of ICSI in their fecundity (Table 3) and in effects of fertilization method on sperm morphology (Table 4) appear to favor successive ICSI generations and ART techniques (ICSI and IVF) versus mating. The fact that these methods affect fecundity and sperm morphology raise concerns relevant to clinical applications of ART. Although the present study shows that ART and repetitive ICSI bring no negative effects to the mice, ICSI in humans may carry additional risks, and more work is needed to fully determine its safety.
In the present study, the efficacy and effects of mouse ICSI were tested over successive generations. The results demonstrated that ICSI does not pose any obvious risks or negative effects when it is continuously applied and that ICSI allows valuable mice to be maintained with high efficiency. This report bears significance for human assisted reproduction and for the preservation and maintenance of medically important mouse strains with fertility problems.
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FOOTNOTES |
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2 Correspondence: Monika A. Ward, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. FAX: 808 956 7316; mward{at}hawaii.edu
3 Monika A. Ward published previously under the name Monika A. Szczygiel
Received: 3 February 2005.
First decision: 4 March 2005.
Accepted: 18 March 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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