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BOR - Papers in Press, published online ahead of print October 29, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023044
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BIOLOGY OF REPRODUCTION 70, 616–624 (2004)
DOI: 10.1095/biolreprod.103.023044
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Paternally Transmitted Chromosomal Aberrations in Mouse Zygotes Determine Their Embryonic Fate1

Francesco Marchetti2,3, Jack B. Bishop4, Lidia Cosentino3, Dan Moore, II5, and Andrew J. Wyrobek3

Biology and Biotechnology Research Program,3 Lawrence Livermore National Laboratory, Livermore, California 94550 National Institute of Environmental Health Sciences,4 Research Triangle Park, North Carolina 27709 Comprehensive Cancer Center,5 University of California San Francisco, San Francisco, California 94120


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The developmental consequences of chromosomal aberrations in embryos include spontaneous abortions, morphological defects, inborn abnormalities, and genetic/chromosomal diseases. Six germ-cell mutagens with different modes of action and spermatogenic stage sensitivities were used to investigate the relationship between the types of cytogenetic damage in zygotes with their subsequent risk of postimplantation death and of birth as a translocation carrier. Independent of the mutagen used, over 98% of paternally transmitted aberrations were chromosome type, rather than chromatid type, indicating that they were formed during the period between exposure of male germ cells and initiation of the first S phase after fertilization. There were consistent one-to-one agreements between the proportions of a) zygotes with unstable aberrations and the frequencies of dead embryos after implantation (slope = 0.87, confidence interval [CI]: 0.74, 1.16) and b) zygotes with reciprocal translocations and the frequency of translocation carriers at birth (slope = 0.74, CI: 0.48, 2.11). These findings suggest that chromosomal aberrations in zygotes are highly predictive of subsequent abnormal embryonic development and that development appears to proceed to implantation regardless of the presence of chromosomal abnormalities. Our findings support the hypothesis that, for paternally transmitted chromosomal aberrations, the fate of the embryo is already set by the end of G1 of the first cell cycle of development.

developmental biology, embryo, fertilization, sperm, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well established that spontaneous and induced numerical and structural chromosomal aberrations cause abnormal reproductive outcomes [1–3], yet their etiology remains largely unknown. The developmental consequences of chromosomal aberrations in early embryos include spontaneous abortions, developmental and morphological defects, inborn abnormalities, and genetic/chromosomal diseases [2–8]. Understanding the mechanism of induction and the embryonic consequences of transmissible damage is important for assessing the genetic risk associated with exposures of human germ cells to environmental mutagens [6, 9]. The mouse has been the long-standing model for investigating the induction and transmission of gene mutations and chromosomal aberrations after parental exposures to germ-cell mutagens [10]. Among the standard methods used for assessing transgenerational transmission of chromosomal damage are the dominant lethal (DL) and the heritable translocation (HT) tests [1, 11]. DL measures the induction of genetic damage that causes the death of the developing embryo, usually after implantation, while HT measures the transmission of chromosomal rearrangements to liveborns. Brewen et al. [12] proposed that postimplantation DL seen after paternal exposure to methyl methanesulphonate (MMS) was caused by transmitted chromosomal aberrations.

Chromosomal aberrations can be first detected after fertilization by standard chromosome analysis at the first-cleavage (1-Cl) metaphase [13, 14]. The 1-Cl assay distinguishes between maternal and paternal damage because the two sets of parental chromosomes do not join until the metaphase plate of the first mitotic division [15] and maternal chromosomes show a higher degree of condensation with respect to paternal chromosomes [13, 16–18]. We recently modified this methodology combining the use of the dye 4,6-diamidino-2-phenylindole (DAPI) for detecting unstable chromosomal aberrations such as dicentrics and acentric fragments and chromosome painting (PAINT) for detecting stable aberrations such as translocations and insertions [19, 20].

In the present study, we have applied this methodology to address the hypothesis that, independent of the mutagen used, dosing regimen, and spermatogenic stage sensitivity characteristic of the mutagen, stable and unstable aberrations in zygotes are fully predictive of the risk of HT and DL, respectively. We tested six mutagens that induced both DL and HT but in different relative proportions and with different peaks of sensitivity during spermatogenesis: acrylamide (AA), cyclophosphamide (CP), etoposide (ET), ionizing radiation (IR), MMS, and melphalan (MLP). Because DL and HT studies require several thousands of animals for every mutagen to be tested, we compared the frequencies of zygotes with unstable and stable aberrations versus published DL [21–26] and HT data [23, 25–30]. We found a strong correlation between the presence of unstable and stable chromosomal aberrations in mouse zygotes and the risk of embryonic lethality and birth of offspring with reciprocal translocations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals, Treatments, and Metaphase Preparation

These animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Lawrence Livermore National Laboratory Institutional Animal Care and Use Committee. B6C3F1 males, 8–12 wk old, were treated with one of the following mutagens: 125 mg/kg AA (CAS No. 79-06-1; Fisher Scientific, Fair Lawn, NJ) or five daily injections of 50 mg/kg AA dissolved in sterile distilled water, 120 mg/kg CP (CAS No. 50-18-0; Sigma-Aldrich, St. Louis, MO) dissolved in PBS, 80 mg/kg ET (CAS No. 33419-42-0; Sigma) dissolved in dimethyl sulfoxide, whole-body irradiation dose of 4 Gy with a delivery rate of 0.61 Gy/min using a 137Cesium Mark 1 Irradiator (J. L. Shepherd and Assoc., Glendale, CA); 40 mg/kg MMS (CAS No. 66-27-3; Sigma) dissolved in PBS; 7.5 mg/kg MLP (CAS No. 148-82-3; Sigma) dissolved in 60% methanol. All chemicals were administered i.p. at the final volume of 0.01 ml/g body weight (BW), except MLP, which was given at the final volume of 0.01 ml/30 g BW. Treated males were allowed to mate with untreated females at different times from exposure (Table 1) to investigate the effects on specific germ cell types. These doses and mating times were selected to produce a wide range of effects (from ~2% to 70%, Table 1) and to allow comparisons with published DL and HT data. When the available DL and HT data for a given mutagen were obtained with different doses, preference was given to the dose used in the DL. Zygotes were collected from superovulated females according to the mass harvest method [31] and classified into one of the following five groups according to their appearance [20]: unfertilized oocytes-oocytes with meiotic chromosomes or degenerating chromatin without a sperm head or tail; meiotic eggs-zygotes showing female meiotic chromosomes and a sperm head or tail; degenerated zygotes-zygotes with degenerating chromatin and a sperm head or tail, or fragmented pronuclei; pronuclei-zygotes with two well-defined pronuclei showing the difference in size between paternal (larger) and maternal (smaller) pronuclei; and zygotes-zygotes with mitotic chromosomes. For each mutagen and time point, three to four repetitions, each using a group of 12 treated males, were performed.


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  		TABLE 1. Frequencies of dominant lethals (DL) and heritable translocations (HT) after paternal exposure to six signature germ cell mutagens

Fluorescence In Situ Hybridization

Ten DNA composite painting probes were used: five probes labeled with fluorescein isothiocyanate (FITC), each specific for chromosomes 1, 3, 5, X, or Y and five biotin-labeled probes, each specific for chromosomes 2, 4, 6, X, or Y. The probes were purchased as concentrated DNA probes from CAMBIO (Cambridge, UK). For each slide to be hybridized, 1.5 µl of each DNA probe was used. Total DNA was precipitated using 1.5 µl of 3 M sodium acetate and 41.25 µl of ethanol. After ~5 h at -80°C, the DNA was spun down at 15 000 rpm for 30 min, the supernatant removed, and the pellet allowed to dry for 15 min at room temperature. The pellet was then resuspended in 15 µl of hybridization buffer (CAMBIO) and kept overnight at 4°C. The following day, the hybridization mixture was denatured in 70% formamide at 75°C for 5 min and subsequently placed at 37°C for 60 min to renature preferentially repeated DNA sequences. Slides were denaturated in 70% formamide at 80°C for 5 min and then dehydrated in 70% (twice), 90% (twice), and 100% cold ethanol for 3.30 min each. Slides were kept on a slide warmer set at 42°C for 2 min, the hybridization mixture was then applied to the slide under 22-mm2 coverslip, which was sealed with rubber cement. The slides were then kept at 37°C for ~48 h. Posthybridization washes were performed twice in washing solution (50% formamide, 2x SSC, pH 7) and twice in 0.1x SSC for 5 min each at 45°C. Amplification of the signals was obtained using the CAMBIO Dual Color Painting Kit (Biotin-Texas Red and FITC) following manufacturer specifications. DAPI at 0.25 µg/ml diluted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) was used as counterstaining.

The combination of probes used in this study hybridizes with ~42.8% of the genome for those zygotes in which the paternal pronucleus contained an X chromosome (Y-) and with ~40.8% of the genome for the paternal pronuclei containing a Y chromosome (Y+) [32]. The proportion of exchanges that can be seen is described by the polynomial expansion (p + q + r + s)2, where p is the fraction of the genome painted in green (chromosomes 1, 3, and 5), q is the fraction of chromosomes painted in red (chromosomes 2, 4, and 6), r is the fraction of the genome painted in yellow (chromosomes X and Y), and q is the remaining unpainted fraction (chromosomes 7–19). Solution of the polynomial expansion shows that 60.2% of all potential chromosome exchanges are detected in pronuclei Y- (57.7% in pronuclei Y+). This means that ~1.7 metaphases have to be analyzed to obtain the amount of information equivalent to one banded cell. The data for AA (multiple daily injections) and ET were obtained with a chromosome painting probe combination that allowed the detection of ~37% of all potential chromosomal exchanges [33, 34].

A Zeiss Axioplan2 fluorescent photomicroscope (Carl Zeiss Inc., Oberkochen, Germany) was used for cytogenetic analysis. The microscope was equipped with a double-bandpass excitor (81P102; Chroma Technology, Brattleboro, VT) for visualizing the red (Texas-Red) and green (FITC) signals; a triple-bandpass filter set (61002; Chroma Technology) for the simultaneous detection of the red, green, and blue (DAPI) signals; and another DAPI filter set (487901; Zeiss) for visualizing DAPI fluorescence only. Metaphases were analyzed as previously described [19]. Briefly, the double-bandpass filter was used to detect chromosome aberrations involving painted chromosomes. The triple-bandpass filter, which allowed the observation of bright DAPI staining of the heterochromatic region near the centromere, was used to discriminate between translocations and dicentrics. Scoring of these aberrations was done according to the PAINT nomenclature system [35]. The separate filter visualizing DAPI fluorescence only was used to determine the total number of chromosome aberrations regardless of whether it involved painted or unpainted chromosomes. This analysis, referred to as DAPI analysis, gives information similar to that obtained by C banding. Images of normal and aberrant metaphases were taken using the CytoVision Imaging Analysis System (Applied Image Biosystems Inc., Santa Clara, CA) and assembled using the Adobe Photoshop 4.0 software (Adobe System Incorporated, San Jose, CA).

Statistical Analyses

A chi-square test with adjustment for overdispersion [36] was used for comparing the frequencies of zygotes with unstable and stable aberrations versus dead implants and translocation carriers because the observations within the treatment groups did not follow a Poisson distribution. For regression analyses, we fit a linear functional relation to the data [37] with the assumption that the variance was the same for both variables. To obtain confidence regions for the slope, intercept, and DL or HT predictions for fixed 1-Cl values, we performed 1000 bootstrap resamplings of the data and used 2.5 and 97.5 percentiles.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pre- and Postfertilization Toxicity

Exposure of male mice to AA, MLP, and ET (25 day) produced a significant reduction in the number of eggs that developed to the zygotic metaphase stage because of pre- and/or postfertilization toxicities (Table 2). Prefertilization toxicity was measured as a reduction in the percentages of eggs that were fertilized (columns 5–6, Table 2) and it is an indication that paternal exposure impaired some aspects of the sperm physiology. Postfertilization toxicity was measured as a reduction in the percentages of fertilized eggs that reached the metaphase stage of the first cleavage division (column 15, Table 2) and it is an indication that paternal exposure affected the proper progression of the first cell cycle of development. These endpoints are of particular interest because it has been shown that several agents can induce similar effects in humans and that chemicals that affect mouse spermatogenesis can affect also human spermatogenesis [38]. The data in Table 2 show that the overall reduction in the number of 1-Cl metaphases was due mostly to postfertilization toxicity for ET (25 day), and to a combination of pre- and postfertilization toxicity for AA and MLP.


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  		TABLE 2. Pre- and postfertilization toxicities: frequencies and types of eggs recovered after paternal exposures to six signature germ cell mutagens

Chromosomal Aberrations in Zygotes

The frequencies of zygotic metaphases with unstable chromosomal aberrations as detected by DAPI staining (Fig. 1D) are shown in Table 3. Of the 10 zygotes with chromosomal aberrations in the control group, 9 were paternal while only 1 was maternal in origin. This is consistent with data in humans that show that the majority of spontaneous structural rearrangements are paternal in origin [5, 39–43]. In all treated groups, chromosomal aberrations were present exclusively in the paternal chromosomes. Across all mutagens used, essentially all aberrations were of the chromosome type, i.e., affecting both sister chromatids [44, 45], suggesting that the mutagen-induced sperm lesions were converted to chromosomal aberrations before the first S-phase after fertilization. Chromatid-type aberrations, in which only one sister chromatid was affected (Fig. 1F), were very rare (Table 4). The majority of the metaphases recovered after paternal exposures had no more than 4 chromosomal aberrations per metaphase (column 5, Table 3). In this analysis, a zygote with a dicentric and an acentric fragment was considered as having two chromosomal aberrations. However, after exposure to AA (7 day), MMS, and MLP, a considerable fraction of metaphases had five or more chromosomal aberrations with some having nine or more. An example of such a highly damaged zygote is shown in Figure 1E. Additionally, for the three mutagens for which comparable data are also available in somatic cells (AA [46], IR [47], and MMS [48]), we found significantly more damage per cell after exposure of male germ cells (200-, 2-, and 12-fold increases for AA, IR, and MMS, respectively). This higher sensitivity of germ cells and in particular postmeiotic germ cells may be related to reduced DNA repair capacity of these spermatogenic stages [49].



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  		FIG. 1. Photographs of first-cleavage (1-Cl) zygote metaphases after hybridization with chromosome-specific painting probes for chromosomes 1, 2, 3, X, and Y labeled with FITC and chromosomes 2, 4, 6, X, and Y labeled with biotin and signaled with Texas Red. Bars = 10 µm. A) Normal 1-Cl zygote metaphase with the X-bearing sperm-derived chromosomes on the right. Paternal chromosomes can be identified because of their lower degree of condensation with respect to maternal chromosomes. B) 1-Cl zygote with X-bearing sperm-derived chromosomes showing a dicentric (arrow) and two unrejoined fragments (arrowheads). This category of zygote was used to predict the frequencies of dead implants. C) 1-Cl zygote with X-bearing sperm-derived chromosomes showing a reciprocal translocation (arrows). This category of zygotes was used to predict the frequencies of translocation carriers among viable offspring. D) DAPI image of paternal chromosomes with multiple chromosomal abnormalities. There are two dicentrics, which can be identified because of the two centromeric heterochromatin regions with bright DAPI staining (arrowheads) and a centric fragment in which the bright centromeric heterochromatin region is bigger that the rest of the chromosome (arrow). E) Paternal chromosomes from a 1-Cl zygote with extensive chromosomal damage. There are at least nine chromosomal aberrations. This category of zygote is predicted to result in the loss of the embryo before implantation. F) Paternal chromosomes with an incomplete interchromatid exchange.


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  		TABLE 3. Predicted versus observed frequencies of embryonic lethality (DL) by DAPI analysis of mouse first cleavage zygote metaphases (1-Cl) after paternal exposures to six signature germ cell mutagens


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  		TABLE 4. Chromosome structural aberrations detected by DAPI and PAINT analysis of mouse zygotes after paternal exposures to six signature germ cell mutagens

As shown in Table 4, over 90% of the aberrations detected by DAPI were unstable (i.e., had two or more centromeres [dicentrics]) or lacked a centromere (acentric fragments). Relatively common were Robertsonian fusion-like, i.e., chromosomes originated from breaks within the heterochromatin region near the centromere [50] and centric fragments, i.e., chromosomes in which the bright DAPI heterochromatin region was as long as the rest of the chromosome (Fig. 1D), suggesting the presence of an extensive chromosomal deletion. As expected, the use of chromosome painting allowed the detection of stable aberrations such as translocations and insertions. In general, the number of chromosomal aberrations per metaphase as determined by DAPI and PAINT analyses were in agreement, suggesting that painted chromosomes were not preferentially involved in the aberrations.

Unstable Aberrations in Zygotes Are Associated with Dominant Lethality

Zygotes with unstable aberrations (i.e., dicentrics, acentric, and centric fragments, etc.) are expected to result in embryonic lethality because of the loss of genetic material that is associated with such aberrations. We used the frequencies of zygotes with unstable aberrations to calculate a predicted frequency of dead implants for comparison with actual DL data (Table 3). As shown in Figure 2A, the frequencies of zygotes with unstable aberrations were related to the DL frequencies in a linear one-to-one relationship that explained 88% of the variance between these endpoints (ß = 0.87, R2 = 0.88, P < 0.001). Using a bootstrap approach, regression analysis showed that the data are consistent with a slope of one (95% CI: 0.74, 1.16) but with a Y-axis intercept significantly different from zero (P < 0.05). All DL predictions based on the frequencies of zygotes with unstable aberrations fall within the 95% CI of this regression line suggesting that a) the frequencies of zygotes with unstable aberrations after paternal exposure are fully predictive of the frequencies of dead implants, independent of the mutagen, dose regimen and germ cell sensitivity pattern and b) a fraction of embryonic deaths are due to causes other than chromosomal aberrations or to types of chromosomal aberrations that are not detected by DAPI (i.e., small deletions, duplications, and inversions).



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  		FIG. 2. Linear regression analyses of the relationships between the frequencies of zygotes with unstable aberrations and dominant lethality (A) and between the frequencies of reciprocal translocation in zygotes and heritable translocations (B). The 95% confidence interval is shown (dotted lines). The HT data for cyclophosphamide was not plotted because of the large difference in the doses used (see Table 1). Graph legend: 1, Controls; 2, acrylamide; 3, acrylamide (3 day); 4, acrylamide (7 day); 5, acrylamide (10 day); 6, acrylamide (13 day); 7, cyclophosphamide; 8, etoposide (7 day); 9, etoposide (25 day); 10, etoposide (35 day); 11, etoposide (42 day); 12, ionizing radiation; 13, melphalan; 14, methyl methanesulfonate

Stable Aberrations in Zygotes Are Predictive of Translocation Carriers at Birth

Next, we used PAINT analysis to examine the association between the frequencies of zygotes with reciprocal translocations and the frequencies of translocation carriers among livebirths. Estimates of the predicted frequencies of offspring with reciprocal translocations at birth are shown in Table 5. These predicted frequencies were calculated using the frequencies of zygotes with reciprocal translocations (Fig. 1C) divided by the total number of zygotes that were expected to reach birth, i.e., cytogenetically normal zygotes plus zygotes with reciprocal translocations. We then compared our predictions with the actual HT data for the same paternal mutagen regimen (Table 5, Figure 2B). Reciprocal translocations in zygotes and heritable translocations in offspring showed a linear one-to-one relationship that explained 78% of the variance between these endpoints (ß = 0.74, R2 = 0.78, P = 0.008; Figure 2B). Regression analysis showed that data were consistent with a slope of one (95% CI: 0.48, 2.11) with a Y-axis zero intercept (95% CI: -3.28, 7.91). All HT predictions based on the frequencies of zygotes with reciprocal translocations fall within the 95% CI, except for CP, which had a 3-fold difference in dose between the HT and zygote studies.


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  		TABLE 5. Predicted versus observed frequencies of offspring with reciprocal translocations (HT) by PAINT analysis of mouse first cleavage zygote metaphases (1-Cl) after paternal exposures to six signature germ cell mutagens

We then compared the distribution of chromosomes involved in the reciprocal translocations as detected by PAINT analysis in zygotes with that observed in a list of 175 spontaneous and induced reciprocal translocations in live mice reported at the Medical Research Council web site (http://www.mgu.har.mrc.ac.uk/anomaly/anomaly-intro.html; [51]) and by Adler [28]. For translocations involving autosomes, a subset of 77 translocations detected in offspring that involved two autosomes differentially painted by our probe combination (i.e., chromosome 1, painted green, and chromosome 2, painted red; or chromosomes 3, painted green, and chromosome 7, stained with DAPI) was used. As shown in Table 6, there was a good agreement between the distributions of autosomal chromosomes involved in the translocations as detected in zygotes and offspring. Unlike for autosomes, all translocations involving sex chromosomes could be detected by our probe combination; therefore, the entire set of 175 translocations was used to compare the data in zygotes and offspring, and in this case also, a good agreement between the distributions of chromosomes involved in the translocations as detected in zygotes and offspring was found (Table 6). These findings suggest that reciprocal translocations induced after paternal exposure to mutagens are already present at the zygotic metaphase and that essentially all zygotes with reciprocal translocations will survive through implantation and embryogenesis to produce viable translocation carriers.


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  		TABLE 6. Distributions of chromosomes among translocations.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A total of 14 mutagen dosage regimens involving 6 mutagens were tested. The results showed that exposure of male germ cells to these mutagens produced both pre- and postfertilization toxicities as well as stable and unstable chromosomal aberrations at 1-Cl metaphases. The results suggest that chromosomal aberrations in zygotes are highly predictive of subsequent abnormal embryonic development.

Analyses of zygotes that did not reach the metaphase stage suggest several mechanisms of toxicity. Across all mutagens, the majority of fertilized eggs that did not reach the metaphase stage were arrested before pronuclear formation (Degen. eggs in Table 2), suggesting a problem with sperm-head decondensation and pronuclear formation. Some of these eggs had no identifiable pronuclei or chromosomes, but did contain a sperm tail, which is incorporated into the mouse egg during fertilization [52]. Others contained a female complement of meiotic chromosomes associated with a sperm head in various stages of decondensation, a sperm tail, or occasionally, a group of male-derived chromosomes that did not undergo DNA synthesis (Meiotic eggs in Table 2). These cells may represent eggs that were fertilized by highly damaged sperm. Following fertilization, a series of events take place in the egg that leads to the formation of male and female pronuclei [53–58]. Both gametes must undergo these changes in a coordinated fashion to ensure normal zygotic development. If the timing of these critical events is disrupted in one pronucleus, the development of the other pronucleus is also affected [59, 60]. Finally, eggs with two well-defined pronuclei were significantly increased after paternal exposure to AA and MLP, suggesting the induction of cell-cycle delay or arrest.

There were two exceptions (out of the 14 tested) to the linear relationship between unstable aberrations in zygotes and DL. DAPI analysis of unstable aberrations in zygotes significantly underestimated the incidence of DL for controls (P < 0.0001) and 125 mg/kg AA (P < 0.01). Several reasons may account for these discrepancies. First, aneuploidy is another class of chromosomal abnormalities that can result in the death of the developing mouse embryo [61]. When the incidence of chromosomal aberrations is low, the contribution of aneuploidy to DL may be significant. This is especially true for controls, where the incidence of spontaneous numerical abnormalities is higher than that of structural abnormalities [16]. Second, our data were obtained from matings occurring at single days after exposure, while all DL (and HT) data were obtained from matings that occurred over 3–7 days (Table 1). The sensitivity of male germ cells to mutagens may change significantly within such a short time interval [33]. Third, the discrepancy of the AA finding may have a genetic cause. The strain of females used to investigate the induction of DL after paternal exposure to 125 mg/kg AA was the (SECxC57BL)F1, which is one of the two strains of females with reduced maternal capacity to repair sperm DNA lesions [62].

Our study strongly indicates that the frequencies and types of chromosomal aberrations in zygotes predict embryonic fate and that cytogenetic abnormalities at 1-Cl are critical intermediates between paternal exposure and postimplantation abnormal reproductive outcomes. A surprising conclusion derived from these results is that preimplantation development appears to proceed to implantation regardless of the presence of unstable chromosomal aberrations of paternal origin. If zygotes with unstable aberrations were eliminated before implantation, their frequencies would have been much higher than the DL frequencies. Indeed, cell death does not seem to occur during the first few cell cycles of mammalian development [63, 64] and embryos with constitutional aneuploidy and structural aberrations are known to survive to postimplantation [61]. The reason for this survival after paternal exposure may be that these initial phases of development are controlled in part by factors stored in the egg before fertilization [65]. Although, in the mouse, transition from maternal control to embryonic control begins during the 2-cell stage [66], some of the proteins required for later preimplantation embryonic development are synthesized very early in development from stored maternal mRNAs before the embryonic genome is fully activated [67]. Additionally, cell-cycle checkpoints seem to operate inefficiently during these initial stages of embryonic development [68]. Therefore, even embryos with unstable chromosomal aberrations may develop enough to elicit the characteristic implantation reaction (resorption) that would be detected as an early postimplantation death in the DL test.

Our data also provide evidence that the chance of an abnormal zygote to reach implantation may be reduced in the presence of large amounts of chromosomal damage. When we reevaluated the frequencies of DL versus the number of chromosomal aberrations in zygotes, we found that the best correlation was achieved when we assumed that zygotes with more than four chromosomal aberrations would die before implantation (ß = 0.92, R2 = 0.89, P = 0.0001).

Another implication of the good correlation between unstable aberrations in zygotes and postimplantation death is that the preimplantation loss, which is often observed in DL tests as the difference between the numbers of corpora lutea and implants, is due primarily to a reduction in the number of eggs that reach the 1-Cl metaphase stage rather than to death of embryos with chromosomal aberrations. Although a direct comparison with DL data are not possible because not all ovulated eggs are fertilized following superovulation, mutagens that significantly reduced the frequencies of zygotes at the metaphase stages in our study should have induced preimplantation loss in DL experiments. According to the data in the last column of Table 2, AA and MLP would be expected to induce high levels of preimplantation loss, MMS and IR moderate levels, while CP and ET little to no preimplantation loss. With the exception of CP, which was found to induce moderate levels of preimplantation loss, this is also what was found in the DL tests (see Table 1 for references). This data shows that preimplantation loss after paternal exposure to mutagens has three components: reduction in the number of eggs that are fertilized, reduction in the number of fertilized eggs that reach the metaphase stage, and death of zygotes with extensive chromosomal damage before they reach implantation.

In conclusion, the results of this study provide compelling evidence that the analysis of eggs recovered within the first day after fertilization may be used to predict future embryonic outcome. As shown in Figure 3, our data are suggesting that, following paternal exposure to mutagens, a fraction of eggs, composed of unfertilized eggs, fertilized eggs arrested before the 1-Cl metaphase, and zygotes with more than four unstable chromosomal aberrations, will be lost before implantation. Zygotes with less than four unstable chromosomal aberrations or aneuploidy will proceed to implant and die before birth, while chromosomally normal zygotes and those with reciprocal translocations will reach birth and produce healthy offspring and translocation carriers, respectively. Our study provides strong evidence that the nature and frequencies of paternally transmitted chromosomal aberrations in zygotes are fully predictive of subsequent abnormal embryonic development. These findings suggest that, for paternally transmitted chromosomal aberrations, the fate of the embryo is already determined by the end of G1 of the first cell cycle of development.



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  		FIG. 3. Schematic summary of the association between the cytogenetic constitution of the egg and reproductive outcomes. Unfertilized eggs, zygotes arrested before first cleavage (1-Cl) and zygotes with more that four chromosomal aberrations are predicted to die before implantation. Zygotes with four or fewer chromosomal aberrations or aneuploidy are predicted to survive preimplantation development, implant in the uterus, and die before birth. Zygotes with normal chromosomes and those with reciprocal translocations are predicted to produce viable offspring, with the latter resulting in translocation carriers


   ACKNOWLEDGMENTS
 
We thank Dorreyah Schahin-Reed for helping with the fluorescence in situ hybridizations and Dr. Eddie Sloter for helpful discussions.


   FOOTNOTES
 
1 Funding support from NIH ES 09117-03 and NIEHS IAG Y1 ES 8016-5. This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory, under Contract W-7405-Eng-48. Back

2 Correspondence: Francesco Marchetti, Biology and Biotechnology Research Program, L-448, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550. FAX: 925 424 3130; marchetti2{at}llnl.gov Back

Received: 9 September 2003.

First decision: 5 October 2003.

Accepted: 21 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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