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BOR - Papers in Press, published online ahead of print July 25, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.063040
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BIOLOGY OF REPRODUCTION 77, 803–812 (2007)
DOI: 10.1095/biolreprod.107.063040
© 2007 by the Society for the Study of Reproduction, Inc.

Genomic DNA Damage in Mouse Transgenesis1

Yasuhiro Yamauchi 3, Brendan Doe 3 4, Anna Ajduk 3 5, and Monika A Ward 2 3

Institute for Biogenesis Research,3 University of Hawaii Medical School, Honolulu, Hawaii 96822 MRC Human Genetics Unit,4 Western General Hospital, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom Institute of Zoology,5 Department of Embryology, Warsaw University, 02-132 Warsaw, Poland

ABSTRACT

Creating transgenic mammals is currently a very inefficient process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In the present study, we demonstrate that this loss is due to chromosomal damage. We examined the integrity of chromosomes in embryos produced by microinjection of pronuclei, intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF)-mediated transgenesis, and correlated these findings with the abilities of embryos to develop in vitro and yield transgenic morulas/blastocysts. Chromosomal analysis was performed after microinjection of the pronuclei in zygotes, as well as in parthenogenetic and androgenetic embryos. In all the pronuclei injection groups, significant oocyte arrest and increased incidence of chromosome breaks were observed after both transgenic DNA injection and sham injection. This indicates that the DNA damage is a transgene-independent effect. In ICSI-mediated transgenesis, there was no significant oocyte arrest. The observed chromosomal damage was lower than that after pronuclei microinjection in zygotes and was dependent upon the presence of exogenous DNA. The occurrence of DNA breaks, as measured by comet assay performed on the sperm prior to ICSI, showed that DNA damage was present in the sperm before fertilization. Embryonic development in vitro and transgene expression at the morula/blastocyst stage were higher in ICSI-mediated transgenesis than after microinjection of pronuclei into zygotes. Sperm-mediated gene transfer via IVF did not affect chromosome integrity, allowed good embryo development, but did not yield any transgenic embryos. The present study demonstrates that DNA damage occurs after both the microinjection of pronuclei and ICSI-mediated transgenesis, albeit through different mechanisms.

assisted reproductive technology, embryo, gamete biology, in vitro fertilization, sperm

INTRODUCTION

Mammalian transgenesis is a powerful tool that is used in the fields of experimental and applied biology [1]. It has opened up possibilities for studying the function and regulation of genes in vivo, together with the mechanisms involved in normal and pathologic developmental processes [2–4]. Transgenic animal models that phenotypically resemble human diseases [5–7] enable scientists to understand the underlying genetic and molecular mechanisms, develop treatments, and validate drug targets [8]. Moreover, transgenesis has the potential to generate transgenic animals that could produce humanized tissues and organs for transplantation [9] or key proteins for pharmaceutical or industrial use [10].

The most common transgenesis method is microinjection of pronuclei [11]. It involves the microinjection of transgenic DNA into the pronucleus of a fertilized egg, which leads to the random insertion of exogenous DNA into the genome. An alternative method of transgenesis employs sperm as vectors to carry the transgene into the oocyte during fertilization [12]. Sperm-mediated gene transfer can take place via either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). In the IVF-based approach, sperm are incubated with exogenous DNA and then allowed to fertilize oocytes on their own [13]. In ICSI-mediated transgenesis, sperm are demembranated by freezing or by treatment with detergent, briefly incubated with exogenous DNA, and the sperm-DNA complexes are then injected into the cytoplasm of the oocyte [14]. Although all these approaches have been shown to yield transgenic offspring, they suffer from various drawbacks and their efficiency levels are far from optimal. This low efficiency results from oocyte and embryo loss at various developmental stages (i.e., low survival after manipulation, pronuclei stage arrest, impaired embryo ability to develop in vitro, and preimplantation and postimplantation embryo losses), low transgene integration, and unpredictable transgene behavior. The majority of the work in the field has focused on improving transgene transmission, whereas little attention has been paid to enhancing embryo survival [15].

Embryo loss can be caused by DNA damage. We have previously shown that in ICSI-mediated transgenesis, impaired embryo development results from paternal chromosome damage, and that the mechanisms of disruption of paternal chromosomes and integration of the foreign DNA may be closely related and depend on nucleases [16]. In sperm-mediated transgenesis, sperm are exposed to transgenic DNA when their own DNA remains bound by protamines, is highly condensed, and is not susceptible to direct mechanical damage. Transgenic DNA is then brought by the sperm into the oocyte and is present while sperm chromatin remodeling takes place. In the microinjection of pronuclei, transgenic DNA is introduced into fully formed pronuclei after the paternal DNA has already acquired a somatic cell-type structure, and often after the onset of DNA synthesis. The histone-bound DNA in male pronuclei has a more open structure and is more vulnerable to mechanical damage than protamine-bound sperm DNA.

In the present study, we focused on the analysis of genomic DNA damage in transgenesis, in order to discover a basis for future transgenesis improvements. We examined the integrity of chromosomes in embryos produced by microinjection of pronuclei, ICSI-mediated and IVF-mediated transgenesis, and evaluated the abilities of the embryos to develop in vitro and to yield transgenic morula/blastocysts. Our results indicate that the mechanisms of genomic DNA degradation in transgenesis by microinjection of pronuclei and ICSI are different.

MATERIALS AND METHODS

Reagents

Mineral oil was purchased from Squibb and Sons (Princeton, NJ), and eCG and hCG were from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. For exogenous DNA, we used the 3.1-kb SalI/BamHI fragment of plasmid pCX-EGFP (a gift from Dr. Masaru Okabe, Osaka University), which contains the gene for GFP-green expressed from the cytomegalovirus-IE-chicken-ß-actin enhancer-promoter combination but lacks the eukaryotic origin of replication. The same stock DNA solution (100 ng/µl in water) was used for all the experiments in the present study.

Animals

Mice B6D2F1 (C57BL/6 x DBA/2) were obtained at 6 wk of age from the National Cancer Institute (Raleigh, NC). Epididymal sperm were obtained from 12- to 16-wk-old males. Mature oocytes and fertilized oocytes were obtained from 12- to 16-wk-old females. 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 guidelines presented in National Research Council's (NCR) "Guide for Care and Use of Laboratory Animals." The protocol for animal handling and the treatment procedures were reviewed and approved by the Animal Care and Use Committee of the University of Hawaii.

Gamete Handling and Embryo Culture Solutions and Media

Sperm and oocyte collection and subsequent gamete manipulations, including sperm microinjection, were performed in Hepes-buffered CZB medium (Hepes-CZB; [17]). The T6 medium [18] was used for IVF, and the oocytes and embryos were cultured in CZB [19]. CZB and T6 were maintained in an atmosphere of 5% CO2 in air, and Hepes-CZB was maintained in air.

Collection and Preparation of Epididymal Sperm

To obtain epididymal spermatozoa for ICSI, the cauda epididymidis was removed from one male and the epididymal fluid was squeezed out and placed on the bottom of a 1.5-ml tube that contained 0.5 ml of Hepes-CZB. Spermatozoa were allowed to swim up the suspension for 5 min at room temperature, and the highly motile sperm at the top of the suspension were removed and used in the experiments.

Collection of Oocytes and Fertilized Oocytes

Females were induced to superovulate with injections of 5 IU eCG and 5 IU hCG, which were given 48 h apart. To obtain unfertilized oocytes, eCG and hCG were injected at 1800–1900 h, and the oviducts were removed 14–15 h after the injection of hCG. To obtain fertilized oocytes, eCG and hCG were injected at 0800–0900 h, females were mated with stud males ~10 h after hCG injection, and the oviducts were removed 24–26 h after hCG injection. This schedule was chosen because in preliminary testing, it resulted in the highest number of pronuclear-stage oocytes for injections in the middle of the day. Dissected oviducts were placed in PBS in a Petri dish. The cumulus-oocyte complexes were released from the oviducts into 0.1% of bovine testicular hyaluronidase (300 U/mg) in Hepes-CZB medium, to disperse the cumulus cells. The cumulus-free oocytes were washed with Hepes-CZB medium and immediately used for injection or other manipulation.

In Vitro Fertilization (IVF)-Mediated Transgenesis

The methods used for sperm capacitation and IVF using T6 medium were based on the original method of Quinn et al. [18] and have been described previously in detail by us [20]. The initial sperm suspension was prepared in T6 medium and contained 10–20 x 106 epididymal sperm. Fertilization was performed in a 0.2-ml drop of T6 medium with a final sperm concentration of 1.25–2.5 x 106/ml. The initial sperm suspension was processed differently, depending on the experiment. In all experiments (except for the testing of the effects of increased concentrations of transgenic DNA), the final concentration of transgenic DNA in the fertilization drop was 0.25 µg/ml. To test the effects of transgenic DNA concentrations, the initial sperm suspension was capacitated for 1.5 h at 37°C in a humidified atmosphere of 5% CO2 in air. Ten µl of capacitated sperm were then transferred into a 0.25-ml tube, and 50 ng (1x) or 500 ng (10x) of transgenic DNA were added. The sperm plus transgenic DNA suspension was mixed by gentle pipetting, added to the fertilization drop, and incubated for 30 min prior to the addition of oocytes. To test for the effect of capacitation, 10 µl of the initial sperm suspension were transferred into a 0.25-ml tube and 50 ng of transgenic DNA were added. The sperm plus transgenic DNA suspension was mixed by gentle pipetting, added to the fertilization drop, and incubated for 2 h (capacitation) prior to the addition of oocytes. To test for the effect of dithiothreitol (DTT), the initial sperm suspension was capacitated for 1.5 h at 37°C in a humidified atmosphere of 5% CO2 in air. A portion of the capacitated sperm was then incubated in a tube with 1 mM DTT for 15 min at 37°C. Next, 10 µl of this suspension were transferred into a 0.25-ml tube and 50 ng of transgenic DNA was added. The suspension was mixed by gentle pipetting, added to the fertilization drop, and incubated for 15 min prior to the addition of oocytes. 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 (ICSI)

ICSI was carried out as described by Szczygiel and Yanagimachi [21]. Injections were performed in Hepes-CZB within 1–2 h of oocyte collection. Sperm were randomly chosen for the 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. Oocytes that had two well-developed pronuclei and a distinct 2nd polar body were recorded as being activated. These oocytes were used for chromosome analysis or embryo culture.

ICSI-Mediated Transgenesis

The sperm suspension in Hepes-CZB (9.5 µl) was frozen in liquid nitrogen for 1 min, then thawed for 1 min to break the sperm membranes, and 50 ng of transgenic DNA (100 ng/µl) were added, followed by incubation at room temperature for 5 min. A portion of this suspension was mixed in a ratio of 1:9 with Hepes-CZB-PVP, and drops of the diluted suspension were placed on the ICSI dish. For sequential injections, drops that contained sperm only or DNA only were also prepared. The transgenic DNA concentration was the same (0.5 ng/µl) in all the DNA-containing drops. Sperm and/or DNA were injected into the oocytes. Each ICSI session was completed within 1 h of sperm thawing.

Preparation of Parthenogenetic and Androgenetic Pronuclear-Stage Embryos

To prepare parthenogenetic embryos, mature oocytes were incubated in Ca2+-free CZB that contained 5 mM Sr2+ for 4 h to achieve activation. Activated oocytes (i.e., those that had extruded a 2nd polar body) were washed and cultured in CZB until the pronuclei were clearly visible (1–2 h). To prepare androgenetic embryos, mature oocytes were first enucleated and then injected with sperm. Enucleation was carried out in Hepes-CZB that contained 5 µg/ml cytochalasin B using Eppendorf Micromanipulators. The metaphase II chromosome-spindle complex, which was recognized as a translucent spot in the ooplasm under 20x Hoffman optics, was drawn into the enucleation pipette (10-µm internal diameter) with a small amount of accompanying ooplasm, and then gently pulled away from the oocyte until the cytoplasmic bridge was severed. After enucleation, the oocytes were transferred into cytochalasin-free Hepes-CZB, allowed to rest for 5–10 min, and then injected with sperm. After sperm injection, the oocytes were cultured in CZB until the pronuclei became visible.

Pronuclei Microinjection

Exogenous DNA was prepared for injection by diluting the DNA stock (100 ng/µl in water) to a final concentration of 2 ng/µl in microinjection buffer (10 mM Tris [pH 8.0], 1 mM EDTA), and centrifuging at 20 800 x g for 1 min through a 0.22-µm spin-x filter (Costar, Corning Inc., New York, NY). Three µl of DNA were loaded into a Femtotip microinjection needle (Femtotip I, defined opening with 0.5-µm inner diameter and 1.0-µm outer diameter [± 0.2 µm]; Eppendorf, Germany) using a microloader tip (Eppendorf). The microinjection needle was then attached to a holder, which was connected to an air-filled 60-ml syringe. Microinjections were performed in a 50-µl drop of FHM (Chemicon, Temecula, CA) that was overlaid with liquid paraffin (VWR, Poole, UK). Up to 50 pronuclear-stage oocytes were injected per 30-min session. During injection, the pronuclei were brought into focus at 400x magnification. The larger of the two pronuclei (presumably male) was positioned in the hemisphere closest to the microinjection needle, and as close to the central axis of the holding pipette as possible. The tip of the injection needle was then placed close to the oocyte and brought into the same focal plane as the pronucleus. Once in the same focal plane, the tip of the injection needle was introduced into the pronucleus and the DNA was injected, as evidenced by visible swelling of the pronucleus, and the needle was then withdrawn rapidly. It is estimated that 1–3 pl of DNA solution could be injected with this approach [22, 23]. Thus, the estimated total amount of DNA injected into each pronucleus was 2–6 x 10–6 ng. DNA delivery was achieved by applying positive pressure to the air-filled syringe, thereby expelling the DNA from the microinjection needle. Injected oocytes were washed and cultured in CZB.

Chromosome Analysis

Chromosome analysis was performed as described previously [24, 25]. All of the oocytes for chromosome analysis, including parthenogenotes, androgenotes, and metaphase II oocytes, were incubated in CZB that contained 0.006 µg/ml vinblastine overnight prior to chromosome spread preparation. Zygotes obtained after mating were transferred into CZB plus vinblastine shortly after pronuclei injection (at approximately 1500 h), to prevent syngamy. The remaining oocytes were transferred into CZB plus vinblastine later in the day (1800–1900 h). The chromosomal pattern of a spermatozoon assessed in a zygote was considered to be normal when an oocyte contained 40 normal metaphase chromosomes. It was not always possible to distinguish between chromosomes of paternal and maternal origin. However, since oocyte chromosomes rarely show structural aberrations at the first-cleavage metaphase after parthenogenetic activation, any abnormal chromosomes within the zygote were considered to be of sperm origin.

Comet Assay

Chromatin fragmentation in sperm was assessed using the Comet Assay kit (Trevigen, Gaithersburg, MD) under neutral conditions, as described recently by us [26]. One-hundred DNA tails were photographed and analyzed per slide and each experiment was repeated three times. The length of each tail was measured from the center of the comet head to the end of the tail using the Image J software [27].

Air-Dried Giemsa-Stained Sperm Preparations for Assessment of Sperm Chromatin Remodeling

Sperm chromatin remodeling after ICSI was examined as described in our recent study [28]. Fertilized oocytes were air-dried on microscope-slides starting from "time 0" and at 30-min intervals for up to 4.5 h (i.e., 10 time-points). Fertilization was achieved within 5 min for each oocyte group. The 0-min time-point was defined as the end of injection of one group of oocytes. The preparations were fixed, stained and examined using a light microscope at 1000x magnification. More than 10 (range, 11–19) oocytes were examined at each time-point for each group. The following sequential remodeling stages were differentiated: unchanged sperm head, chromatin decondensation, chromatin recondensation, beginning of pronuclei formation, and developed pronuclei. In addition, their frequencies at the specific time-points were noted. In the chromatin decondensation and recondensation groups, both partially and fully decondensed or recondensed sperm, respectively, were included. The developed pronuclei group contained early and fully developed pronuclei.

Experimental Design

The experiments were designed to assess the extent and origin of chromosomal damage in embryos produced by ICSI-mediated transgenesis and transgenesis via microinjection of pronuclei. Chromosome analysis was performed for: 1) zygotes, parthenogenotes, and androgenotes after pronuclear transgenic DNA injection; 2) oocytes and zygotes after transgenic DNA and/or sperm cytoplasmic injection; 3) zygotes obtained after in vitro fertilization with sperm exposed to transgenic DNA. Moreover, the comet assay for sperm and paternal chromatin remodeling after fertilization were also performed after sperm exposure to transgenic DNA. Embryo culture was performed for zygotes from all the analyzed groups. When both chromosome analysis and embryo culture were carried out, a group of oocytes injected on a given day was divided in two and half of the sample was used for each test. The same pool of oocytes was used for pronuclei injection, sham injection, and noninjected controls on each day. The comet assay was performed on the same sperm samples that were used for ICSI. All experiments were repeated at least three times.

Statistics

The chi-square, Likelihood Ratio, Fisher exact probability, and Student t-tests were used for analyzing all the responses. Lack of statistical significance was reported when all the tests gave P values > 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 the KyPlot version 2.0 beta 13 software (available online at http://www.woundedmoon.org/win32/kyplot.html).

RESULTS

Transgenic DNA Injection into Pronuclei Results in Oocyte Arrest, Chromosome Clumping, Increased Incidence of Chromosome Breaks, and Impaired Embryo Development

Transgenic DNA was injected into the pronuclei of naturally fertilized pronuclear stage 1-cell embryos (zygotes), activated oocytes (parthenogenotes), and enucleated, sperm-injected oocytes (androgenotes). In all the examined groups, a high number of injected oocytes arrested and did not progress beyond the pronuclear stage (Fig. 1). The arrest was more pronounced in adrogenotes than in zygotes (P < 0.001) or parthenogenotes (P < 0.01). Sham-injected oocytes arrested in a similar manner to their transgenic DNA-injected counterparts in all the examined groups (P > 0.05). Both transgenic DNA and sham-injected oocytes in all groups arrested more frequently than their noninjected controls (P < 0.001). The highest level of oocyte arrest was observed in oocytes in which both pronuclei were injected with DNA (Fig. 1, 2 PN; P < 0.001 compared with all other oocytes). The oocyte arrest observed in DNA-injected zygotes was within the range of what is normally seen for microinjection of pronuclei. In the Transgenic Facility of the Medical Research Council (Western General Hospital, Edinburgh, UK), the rate of oocyte arrest after injection of pronuclei has ranged from 1% to 31% (mean ± SD, 18.7143 ± 10.7665% for a total of 1523 zygotes injected; seven different plasmids used; data collected during the last few years).


Figure 01
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  		FIG. 1 Oocyte arrest after transgenic DNA injection into pronuclei. Transgenic DNA was injected into the pronuclei of zygotes, parthenogenotes, and adrogenotes. In each examined group, controls of sham-injected oocytes and noninjected oocytes were included. In addition, a group in which two pronuclei in each zygote were injected with DNA is shown (2 PN). Each bar represents a mean of at least thee replicates ± SD. Statistical significance is indicated as: a, different than others within group (P < 0.001); b, different than the same treatment in other groups (P < 0.01 or P < 0.001); and c, different from all others (P < 0.001). n, number of oocytes injected in all replicates.


Figure 02
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  		FIG. 2 Chromosome analysis after injection of pronuclei. A, B, D) Transgenic DNA-injected zygotes. C) Sham-injected zygotes. E, F) Transgenic DNA-injected parthenogenotes. G, H) Transgenic DNA-injected androgenotes. A) Thirty-three separate chromosomes and a clump (arrowhead), without visible fragments. B) Twenty-seven separate chromosomes and a clump (arrowhead), with visible fragments (long arrows). C) Two separate chromosome plates, one with 20 normal chromosomes (N) and one with 11 separate chromosomes and a clump (arrowhead). D) Chromosomes from two plates are mixed but can be differentiated by different morphology, one set has 20 normal chromosomes, shorter and curvy (short double arrows) and one set has 13 separated, longer and straighter chromosomes (short arrows) and a clump (arrowhead). E, G) Twenty normal chromosomes, without visible fragments. F, H) Abnormal karyoplates with visible fragments (long arrows) and abnormal chromosome configurations (two small arrowheads). Bar = 10 µm.

In all the examined groups, significantly more transgenic DNA-injected oocytes exhibited chromosomal breaks than their noninjected controls (Table 1, Fig. 2). There were no differences in the incidences of chromosome breaks between transgenic DNA and sham-injected oocytes in all the groups. Overall, the chromosome analysis revealed that the microinjection of pronuclei results in an increased incidence of chromosomal breaks, and that genomic DNA damage is transgene-independent.


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  		TABLE 1 Chromosome analysis after pronuclei injection.

During the chromosome analysis, we observed an interesting phenomenon. A high number of karyoplates contained clumped chromosomes after pronuclei injection (Table 1, Fig. 2). Usually, ~7–9 chromosomes clumped, while the others remained separated (Fig. 2, A, C, and D), although karyoplates with more clumped chromosomes were also noted occasionally (Fig. 2B). Although it was not always possible to differentiate between maternal and paternal karyoplates, in cases where it was feasible, the chromosomes from one set only, presumably originating from injected pronuclei, were affected by clumping (Fig. 2, B and C). Chromosome clumping was noted most often in transgenic DNA-injected zygotes (54%) and was decreased in parthenogenotes (27%) and androgenotes (11%). There were no statistically significant differences in the incidence of karyoplates with clumped chromosomes between the transgenic DNA-injected and sham-injected oocytes in each group, and clumping was not present or was negligible in the noninjected controls. Thus, this effect appeared as a result of microinjection and was not related to the presence of transgenic DNA.

Routinely, we score karyoplates as normal only when all the chromosomes are clearly separated and no fragmentation or other aberration is observed. In the initial analysis, we excluded the karyoplates with clumped chromosomes. However, a significant number of these karyoplates had obvious chromosomal breaks (Table 1, Fig. 2B), and it could be anticipated that those without visible fragments had chromosomal breaks hidden within the clumps. To address this possibility, we present the proportions of normal chromosomes in two ways (Table 1): 1) calculated from the clump-free karyoplates (our routine protocol); and 2) calculated from all the karyoplates analyzed (including those with clumps, and with the assumption that karyoplates with clumps were abnormal). The differences within and between the examined groups remained similar with both methods of scoring, although the incidences of normal karyoplates in transgenic DNA-injected and sham-injected oocytes decreased when all the karyoplates were included in the analysis. This decrease was most pronounced in the zygotes. The proportion of normal karyoplates decreased from 76% to 35% and from 83% to 38% when calculated for the clump-free karyoplates and from all karyoplates in transgenic DNA-injected and sham-injected zygotes, respectively. Since ~25% of the injected zygotes arrested and only ~35% of the remaining nonarrested zygotes were chromosomally normal, the total rate of embryo loss resulting from DNA damage after the microinjection of pronuclei was 75% of the manipulated oocytes.

Embryo development in vitro was assessed to provide an indication of the overall embryo developmental potency and success of transgene transmission. The analysis was carried out only for zygotes (Table 2) because parthenogenotes and androgenotes have limited abilities to develop in vitro and do not yield live offspring. Embryonic development was impaired in transgenic DNA-injected zygotes as compared to noninjected controls (P < 0.001). The development of sham-injected zygotes was similar to that of transgenic DNA-injected zygotes (P > 0.05) and worse than that of noninjected controls (P < 0.05 and P < 0.01 at the morula and blastocyst stages, respectively). The proportion of green (transgenic) embryos after pronuclear transgenic DNA injection was 21%.


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  		TABLE 2 Embryo development and transgene expression after zygote pronuclei injection.

Due to the high incidence of oocyte arrest in zygotes after the injection of both pronuclei (Fig. 1), only a few (n = 13) chromosome plates were analyzed in this group; six (46%) contained clumped chromosomes, and five were normal (71%, 5/7 and 39%, 5/13, in the two scoring systems). Embryonic developmental potential was not assessed. The injection of two pronuclei resulted in a similar chromosome analysis outcome as that seen for single pronucleus injection, in spite of the extreme oocyte arrest at the pronuclear stage.

Paternal Chromosome Integrity and Embryo Development after ICSI with Sperm Exposed to Transgenic DNA Is Impaired and Depends upon the Presence of Transgenic DNA

Transgenesis can be achieved by coinjecting sperm and exogenous DNA into the oocyte cytoplasm (ICSI-mediated transgenesis) [14]. Previously, we have shown that sperm exposure to exogenous DNA results in an increased incidence of paternal chromosome breaks in the zygote [16]. In the present study, we wanted to test if it was necessary for sperm to be exposed to exogenous DNA before injection or if sequential injections of sperm and transgenic DNA would have the same effect. We also wanted to check if injection of exogenous DNA into the ooplasm affected maternal DNA integrity.

Three groups of oocytes were analyzed after sperm and DNA injection: 1) oocytes injected with a mixture of sperm and DNA; 2) oocytes injected with DNA 30 min prior to sperm injection; and 3) oocytes injected with DNA 30 min after sperm injection. In all the groups, frozen-thawed sperm were used for the injections and oocytes injected with frozen-thawed sperm were used as controls. Chromosome analysis revealed that 39% of the oocytes injected simultaneously with sperm and DNA had normal paternal chromosomes (Table 3). This percentage was significantly less than that observed for oocytes injected with control sperm (57%, P < 0.05; Table 3). The severity of DNA damage, as judged by aberrations per sperm, was relatively mild (< 2.0) in both groups. When DNA and sperm were injected sequentially, 30 min apart, the incidence of normal paternal karyoplates when DNA was injected prior to sperm was the same as in the control. However, when sperm were injected first, followed by DNA injection, significantly fewer normal chromosomes were noted, as compared to the control (38% vs. 57%, P < 0.05; Table 3). Embryos from all the examined groups developed to the blastocyst stage, but transgene expression was observed only when sperm and DNA were mixed prior to injection and injected jointly; 54% of the morula/blastocyst-stage embryos were green (Table 3). These results suggest that sperm DNA damage observed after simultaneous injection of sperm and DNA into the oocytes during ICSI-mediated transgenesis is transgene-dependent. The lack of transgene expression after sequential injection of sperm and exogenous DNA indicates that the interaction of these two components prior to fertilization is essential for transgene transmission.


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  		TABLE 3 Chromosome integrity, embryo development, and transgene expression in ICSI-mediated transgenesis.

To test the effect of exogenous DNA on maternal DNA integrity, transgenic DNA was injected in the same manner as described above into the cytoplasm of metaphase II (MII) oocytes or into the cytoplasm of pronuclear stage activated oocytes (Act). Some MII oocytes were chemically activated immediately after transgenic DNA injection (MII -> Act), to mimic maternal DNA changes that normally take place after fertilization. All of the injected oocytes were incubated overnight in CZB plus vinblastine, and subjected to chromosome analysis on the following morning. Of the analyzed karyoplates, all were normal in the MII and Act groups (100%, 44/44, and 100%, 35/35, respectively) and almost all were normal in the MII -> Act group (96%, 45/47). Thus, the injection of transgenic DNA into the ooplasm did not impair maternal DNA integrity.

DNA Integrity in Sperm Exposed to Transgenic DNA Is Impaired Prior to Fertilization and Is Transgenic DNA-Dependent

Sperm DNA integrity in frozen-thawed spermatozoa exposed to transgenic DNA was analyzed by the comet assay. As a control, frozen-thawed sperm that were not exposed to transgenic DNA were used. The severity of DNA damage can be expressed as comet tail length and type. Four types of comet tails expressing different levels of DNA damage were differentiated: 1) short tail; 2) long tail, with the majority of the DNA still in the head; 3) long tail, with the DNA evenly distributed throughout; and 4) long tail, with most of the DNA at the distal portion (balloon shape). The severity of DNA damage increased proportionately as the tail type changed from type 1 to 4, and with increasing tail length. The results are shown as the frequencies of comets with different tail lengths and with different tail types (Fig. 3). The mean length of comet tail was significantly longer in frozen-thawed sperm exposed to transgenic DNA than in control sperm (192.29 ± 54.74 µm vs. 176.59 ± 46.27 µm; P < 0.001). The distribution of tail lengths was different in both groups (Fig. 3A). Sperm exposed to transgenic DNA had significantly fewer 100–150-µm tails (P < 0.01) and more 200–250-µm (P < 0.05), 250–300-µm (P < 0.05), and 300–350-µm tails (P < 0.01), as compared to the control. The comparison of tail types (Fig. 3B) revealed that sperm exposed to transgenic DNA had fewer type 1 tails (P < 0.001) and more type 2 (P < 0.01) and type 3 (P < 0.05) tails than the control.


Figure 03
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  		FIG. 3 DNA fragmentation in sperm exposed to transgenic DNA and frozen-thawed, as assessed by the comet assay. A) Tail lengths. B) Tail types. Two groups of sperm were examined: 1) the group exposed to transgenic DNA and subsequently frozen-thawed (DNA+FT); and 2) the frozen-thawed control (FT) group. Controls included sperm prepared in exactly the same way as the tested sample but without exposure to the transgenic DNA.

Overall, the results of the comet assay demonstrated more DNA damage in frozen-thawed sperm exposed to transgenic DNA than in control sperm, which indicates a transgene-dependent effect. DNA damage was present before fertilization, which suggests the involvement of sperm-specific nucleases.

Sperm Exposure to Transgenic DNA Does Not Affect Paternal Chromatin Remodeling

Recently, we have shown that paternal chromatin remodeling differs when sperm are subjected to various manipulations [28]. In ICSI-mediated transgenesis, sperm are exposed to transgenic DNA prior to fertilization and then carry it into the oocytes. In the present study, we examined whether the presence of transgenic DNA affected sperm chromatin remodeling after fertilization. We tested frozen-thawed sperm exposed to transgenic DNA and their frozen-thawed only controls. In the comet assay analyses, both of these treatments were found to cause DNA damage to chromosomes, with more severe DNA impairment after exposure to transgenic DNA.

Previously reported differences in paternal chromatin remodeling relate mainly to its synchrony. Each remodeling stage was defined as synchronous when more than 80% of the oocytes with chromatin at this stage were accumulated in at least a single time-point. There were no differences in the number of synchronous remodeling stages between sperm exposed to transgenic DNA and their nonexposed controls; both had four stages out of five synchronous (Fig. 4). There were no differences between the tested and control groups in terms of chromatin decondensation. In both groups, chromatin decondensation was rapid and synchronous. Only the very early stages of decondensation and fully decondensed sperm heads, and almost no partially decondensed sperm, were observed. In sperm exposed to transgenic DNA, the recondensation peak was seen earlier (2 h) than in the controls (2.5 h), while some recondensing sperm were still seen at 4.5 h. This suggests that this remodeling stage is slightly prolonged after exposure to transgenic DNA. However, the differences in recondensation between the transgenic DNA-exposed group and the control group were not statistically significant. Pronucleus formation was initiated earlier in the transgenic DNA-exposed group than in the frozen-thawed control group (43% and 7% of sperm were forming or had formed pronuclei at 2.5 h postinjection, respectively; P < 0.05). Overall, the pattern of chromatin remodeling was not significantly affected by sperm exposure to transgenic DNA prior to fertilization.


Figure 04
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  		FIG. 4 Sperm chromatin remodeling. Paternal chromatin remodeling was examined in oocytes injected with sperm exposed to the transgenic DNA and subsequently frozen without cryoprotection (DNA+FT) and unexposed, frozen-thawed sperm (FT), at 10 different time-points. At least 10 oocytes (range, 11–19) were examined at each time-point in each group.

Paternal Chromosome Integrity and Embryonic Development Are Not Impaired after In Vitro Fertilization with Sperm Exposed to Transgenic DNA

It has been shown before that live, motile sperm can act as vectors, and that transgenic offspring can be obtained after IVF with sperm exposed to exogenous DNA [13]. We performed chromosome analysis of zygotes produced with sperm exposed to transgenic DNA. We also looked at embryonic development in vitro and transgene expression at the morula/blastocyst stage. Our protocol for IVF-mediated transgenesis was based on that developed by Lavitrano et al. [13], and the conditions of IVF (sperm concentration, transgenic DNA concentration, sperm to transgenic DNA ratio) were essentially the same as those published previously. Under these conditions, sperm exposed to transgenic DNA fertilized oocytes efficiently and yielded chromosomally normal and developmentally competent embryos (Table 4). However, none of the embryos of the morula/blastocyst stage appeared green, suggesting a lack of successful transgene transmission.


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  		TABLE 4 Chromosome integrity, embryo development, and transgene expression after in vitro fertilization with sperm exposed to transgenic DNA (IVF-mediated transgenesis).

Because IVF-mediated transgenesis is known to be inefficient [29, 30], we modified the original procedure and tested new conditions. Initially, we tested the effects of increased transgenic DNA concentration. When sperm were incubated with a 10-fold amount of transgenic DNA, the results were similar to those obtained with the 1-fold concentration of transgenic DNA, except that the fertilization rate was slightly lower (Table 4). No green embryos were present. Next, we tested for the effect of capacitation in the presence of transgenic DNA. Since sperm undergo several membrane modifications during capacitation, we speculated that capacitation in the presence of transgenic DNA might increase the chances of transgenic DNA binding/integration. When sperm were capacitated in the presence of transgenic DNA, no green embryos were observed, and no changes were noted in any of the other examined parameters. Finally, we tested whether modification of sperm DNA made it more accessible and facilitated transgene transmission. We incubated sperm with DTT prior to exposure to transgenic DNA. In the initial experiments, we tested the conditions that would allow efficient reduction of disulfide bridges in sperm protamines while maintaining sperm motility at a high level. Protamine reduction was tested by the halo assay, as described previously [31]. We tested five DTT concentrations (20, 2, 1, 0.5, and 0.1 mM) and chose the concentration of 1 mM, which was the lowest concentration that resulted in protamine reduction in all sperm. Sperm motility was only slightly reduced after incubation with 1 mM DTT. Chromosomal integrity was maintained in sperm exposed to DTT plus transgenic DNA, DTT alone, and in the control. The obtained 2-cell embryos developed well in vitro (Table 4). The fertilization rate was slightly lower after incubation with DTT, presumably due to decreased sperm motility. None of described modifications resulted in the presence of green fluorescence in morula/blastocyst-stage embryos.

Overall, our results demonstrate a lack of DNA damage after IVF with sperm exposed to transgenic DNA. However, as we did not observe any transgenic embryos, our conclusion at this point is that under the conditions tested, sperm-mediated transgene transmission in IVF did not occur.

DISCUSSION

To the best of our knowledge, the present study is the first direct demonstration of genomic DNA damage in transgenesis via microinjection of pronuclei. Our results indicate that the mechanism of genomic DNA damage in transgenesis via microinjection of pronuclei is different than that in ICSI-mediated transgenesis. Our study also supports the claim that ICSI-mediated transgenesis is more efficient than the microinjection of pronuclei.

The lower efficiency of microinjection of pronuclei resulted from embryo loss at various time-points. The first negative effect of microinjection of pronuclei is the inability of injected oocytes to progress beyond the pronuclear stage. Microinjection of pronuclei is known to induce oocyte arrest. Previously, we have observed that increased oocyte arrest usually correlates with chromosomal damage detected in the sperm used for injection, and it is not overcome by chemical oocyte activation. The oocyte arrest observed in the present study corresponds well with the results of the chromosome analysis. It is likely that it originated directly from DNA damage that was too severe to bypass the checkpoints that prevent the initiation of the M phase [32]. The second level at which embryos are lost during microinjection of pronuclei is related to chromosome clumping, a phenomenon that we have not seen previously when performing zygote chromosome analysis [16, 24, 25, 31, 33, 34]. One possible explanation for this phenomenon is the presence of EDTA in the buffer used as the carrier for the injected transgenic DNA. Divalent ions, especially the divalent cation Mg2+, maintain proper condensation of chromatin in the nucleus [35]. Ion chelators may decrease the Mg2+ level within nuclei and interfere with DNA condensation, rendering the chromatin more susceptible to clumping. It is also possible that chromosome clumping is induced by physical forces during pronuclear swelling after injection. Such an extreme manipulation could rearrange the DNA positioning within the pronucleus, thereby forcing interactions between the individual chromosomes that would otherwise not take place. The third factor that contributes to embryo loss during the microinjection of pronuclei is DNA degradation, as evidenced by the presence of chromosome fragments in a significant proportion of the embryos. Due to the presence of chromosome clumping, we analyzed the karyoplates in two ways: 1) including all karyoplates with the assumption that karyoplates with clumps are abnormal; and 2) calculating clump-free karyoplates only. We believe that the former approach better reflects the actual damage, as the majority of karyoplates with clumped chromosomes had visible chromosome breaks and clumping would be expected to impair proper chromosome segregation during cleavage.

In our previous study, we have shown that paternal chromosome damage in ICSI-mediated transgenesis is dependent upon the presence of transgenic DNA [16]. In the present study, oocyte arrest, chromosome clumping, chromosome breaks, and impaired development in vitro were observed after sham injection, in which transgenic DNA-free medium was injected into the pronuclei. This clearly indicates that in transgenesis via pronuclei microinjection, the manipulation itself rather than the introduced exogenous DNA is the cause of DNA damage and developmental impairment. In the microinjection of pronuclei, DNA-containing injection buffer is injected into the pronucleus until visible swelling is observed. The mechanical force to which the genomic DNA is exposed is extreme. Since the paternal DNA in pronuclei has a somatic cell-type structure and is vulnerable to stress, it is possible that the DNA damage observed during microinjection of pronuclei is caused by mechanical trauma and does not involve any enzymatic degradation. Alternatively, the mechanical stress evokes a cell reaction that ends in the release of a nuclease(s). Our finding that oocyte arrest and the incidence of chromosome breaks are similar after sham injection supports the hypothesis that DNA damage is caused by the manipulation rather than the response to exogenous DNA.

By injecting pronuclei in both parthenogenetic and androgenetic embryos, we show that the chromosomal damage observed in transgenesis via microinjection of pronuclei is not mediated by a factor that originates from sperm. The more pronounced oocyte arrest in androgenotes is presumably due to the more excessive manipulations to which the oocytes were subjected (i.e., enucleation, sperm injection, and microinjection of pronuclei). The finding that chromosome clumping was less prominent and more karyoplates with normal chromosomes were noted in the parthenogenotes and adrogenotes than in the zygotes may be due to the fact that the pronuclei were injected into the zygotes at a later stage, shortly before syngamy. It is possible that this difference in pronucleus maturity affects chromosome stability. If this is true, then injecting early developed pronuclei rather than late pronuclei should increase the efficiency of transgenesis via microinjection of pronuclei by eliminating, at least partially, chromosome clumping. In addition, early pronuclei injection is more likely to deliver the transgene at the onset of DNA replication, which can enhance its integration. The presence of DNA damage in parthenogenotes further substantiates our conclusion that DNA degradation in microinjection of pronuclei is sperm-independent.

Our results with ICSI-mediated transgenesis are in agreement with our previous findings [16]. Although the freezing of unprotected sperm per se causes chromosome fragmentation, we observed a clear increase in the incidence of abnormal karyoplates after sperm exposure to transgenic DNA. These results support the notion that paternal DNA damage in ICSI-mediated transgenesis is transgene-dependent. The lack of transgene transmission in sequential transgenic DNA and sperm injections suggests that sperm and transgenic DNA interactions prior to fertilization are needed for transgenic DNA internalization. The slightly lower incidence of normal karyoplates observed in the paternal complements after sequential sperm and DNA injections may be the result of more extensive manipulation (double oocyte injection) or the activities of nucleases inside the oocyte. It is interesting to note that under all conditions involving cytoplasmic transgenic DNA injections, the maternal chromosomes were unaffected, which suggests that maternal DNA is protected from this DNA damage.

Our comet assay results show that sperm DNA damage is significantly more severe in sperm exposed to transgenic DNA, and that DNA damage is present in sperm prior to injection. In the comet assay, all histones and protamines are extracted using a high concentration of salt and disulfide-reducing agents [36, 37], which rendered the entire DNA fully accessible to analysis. The comet assay detects all DNA breaks, regardless of their chromosomal position. On the other hand, chromosomal analysis detects only double-stranded breaks that are associated with breaks in the proteinaceous chromatin scaffold and that are probably located only at the sites of DNA loop attachment to the scaffold. The latter analysis would not detect DNA breaks within loop domains that are not associated with the scaffold. Thus, the two assays complement each other in terms of defining DNA damage. The breaks detected by chromosomal analysis are likely to be much more important for embryonic development because the DNA loop domain attachment sites are known as the sites of DNA replication in somatic cells [38]. Taken together, the results of the chromosome analysis and comet assay show that genomic damage in ICSI-mediated transgenesis is transgene-dependent and takes place before fertilization.

It has been reported that live mouse spermatozoa incubated with exogenous DNA yield transgenic offspring after IVF and embryo transfer [13]. However, the method did not prove to be successful in other hands [29], and its applicability remains a subject of debate [1]. In the present study, sperm DNA damage was not observed in embryos produced according to the protocols for IVF-mediated transgenesis, and all of the embryos developed very well in vitro. However, although we tested several different conditions, we could not obtain even a single green embryo that would indicate that the approach used allowed successful transgene incorporation into the genome. We conclude that, at least in our hands, IVF-mediated transgenesis does not work. More important for the present study is the consistent correlation between exogenous DNA integration and paternal chromosome damage.

Our results show that DNA damage in ICSI-mediated transgenesis is dependent upon transgenic DNA, while the DNA damage associated with the microinjection of pronuclei is not. Therefore, the mechanisms of DNA degradation in transgenesis via the microinjection of pronuclei and ICSI are different. In ICSI-mediated transgenesis, the transgene is acquired by sperm prior to fertilization and then brought into the oocyte. After fertilization, the sperm chromatin undergoes intensive remodeling, which may promote transgene integration. However, our comet assay results reveal a transgene-dependent effect before fertilization, suggesting that at least the initiation of the process that potentially culminates in transgene integration occurs before chromatin reorganization. The fact that we did not observe significant differences in the pattern of chromatin remodeling in transgenic DNA-exposed sperm and controls supports this line of thinking. Considering the complexity of sperm chromatin structure [39], it is reasonable to suspect that enzymatic activity to unwind and break condensed sperm DNA is present. Previous reports have suggested that several nucleases are responsible for the digestion of sperm DNA, with different locations and modes of action [16, 26, 31, 33, 40–42]. Although in the present study, we did not test directly for the presence of nucleases, for example by including ion chelators and other inhibitors, our previous work has indicated the involvement of nucleases in DNA damage and transgene integration [16]. Therefore, we believe that DNA damage in ICSI-mediated transgenesis is nuclease-dependent. We are currently characterizing some sperm-specific nucleases and hope to learn more about their mechanisms of action in the near future.

When comparing genomic DNA damage after microinjection of pronuclei and ICSI-mediated transgenesis, it is clear that the former method is much more detrimental. In our hands, ICSI-mediated transgenesis yielded more viable embryos with higher transgene transmission rates. Judging the efficiency of transgene transmission by observing its expression in preimplantation embryos has limitations, but it can nevertheless be considered as an indication of transgenesis efficiency. Taken together, our results indicate that ICSI-mediated transgenesis is more efficient than microinjection of pronuclei.

The present study demonstrates that for the development of transgenic animals, maintaining chromosome stability is a significant concern. As we have previously shown, some genomic DNA damage is important for transgene integration [16]. A future challenge is the establishment of conditions that ensure efficient transgene integration while maintaining genomic DNA stability sufficient for proper embryo development. The recently developed modification of ICSI-mediated transgenesis, named ‘active transgenesis,' which utilizes fresh sperm and employs recombinases [43] and transposases [44, 45] to facilitate transgene insertion, is an important advance towards achieving this goal. The maintenance of genomic DNA integrity in these approaches remains to be evaluated.

ACKNOWLEDGMENTS

The authors thank Dr. Stefan Moisyadi for the preparation of the transgenic DNA.

FOOTNOTES

1Supported by NIH HD048446 and HD048845 grants to M.A.W. Back

Correspondence: 2Monika 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; e-mail: mward{at}hawaii.edu

Received: 22 May 2007.

First decision: 20 June 2007.

Accepted: 18 July 2007.

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