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Biology of Reproduction 65, 1050-1056 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Genetic and Epigenetic Factors Affecting Blastomere Fragmentation in Two-Cell Stage Mouse Embryos1

Susan M. Hawesa, Young Gie Chunga, and Keith E. Latham2,a,b

a The Fels Institute for Cancer Research and Molecular Biology and b Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT

We report here that mouse embryos can exhibit a significant incidence of blastomere fragmentation at the two-cell stage. The incidence of this is influenced by both the maternal and paternal genotype. Embryos from C57BL/6 mothers exhibit a very low incidence of fragmentation at the two-cell stage in crosses involving males of C57BL/6, DBA/2, AKR/J, or SJL strains but exhibit a significantly increased incidence of fragmentation in crosses involving C3H/HeJ males. Increased fragmentation is seen in embryos from C3H/HeJ females crossed with C57BL/6 males but not with C3H/HeJ males. Embryos obtained from reciprocal (C57BL/6 x C3H/HeJ) F1 hybrid females also exhibit an increased incidence of fragmentation at the two-cell stage when the hybrid females are mated to either C57BL/6 or C3H/HeJ males. Interestingly, the results differ significantly between reciprocal F1 hybrid females, indicating a parental origin effect, possibly a result of either genomic imprinting or differences in mitochondrial origin. We conclude that the incidence of blastomere fragmentation at the two-cell stage in the mouse is under the control of more than one genetic locus. We also conclude that blastomere fragmentation is affected by both parental genotypes. These results are relevant to understanding the genetic control blastomere fragmentation, which may contribute to evolutionary processes, affect the success of procedures such as cloning, and affect the outcome of assisted reproduction techniques.

apoptosis, blastomere fragmentation, embryo

INTRODUCTION

Life begins as a cooperative venture between two gametes, each of which is separately fated to die but which, when united, become capable of initiating the developmental program that leads to the creation of a new individual. One requirement along the path to normal development is to suppress or override the molecular and cellular predisposition toward death. Maternally encoded factors deposited in the developing oocyte must interact with the incoming paternal genome, direct the formation and proper programming of the embryonic genome, and subsequently activate and control the expression of the embryonic genome with the correct temporal and spatial specificity, leading to the appropriate differentiative and morphologic events.

One can hypothesize that both gametes may contribute information, in the form of various macromolecules as well as developmental programming of the parental genomes, and that such information plays a vital role in diverting the zygote from a pathway leading to death to one leading to the creation of a new life. In some organisms, sperm-supplied factors are essential for early development [1–3], and early-acting maternal effect mutations may prevent the interaction of maternal factors in the oocyte with factors introduced via the sperm [1]. The correct interaction of maternally and paternally supplied factors is essential for basic processes such as nuclear decondensation, pronuclear formation, progression through mitosis, and maintenance of genome stability [1]. Available data also indicate that genetic differences may exert profound effects on nuclear function and embryo viability in other species, including humans. For example, genetic differences in ooplasm-nuclear interactions have been observed among different mouse strains (reviewed, [14]). Of the many factors that may affect the outcome of in vitro fertilization of human oocytes, heterogeneity among the oocyte donors can account for approximately 90% of the observed variability in outcome (R. Gosden, personal communication). In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL, to detect DNA fragmentation correlated with apoptosis) in the human oocyte and cleavage-stage embryo exhibits patient-specific effects [5, 6]. Such patient-specific effects are likely the result largely of genetic differences that affect oocyte function or oocyte quality and, subsequently, the integrity of early embryogenesis.

How genetic differences in gametes affect early mammalian development remains largely unknown. One potential pathway through which such effects may be mediated is the control of blastomere fragmentation and, possibly, apoptosis. Blastomere fragmentation is common during early human embryo development, when approximately 40% of embryos exhibit fragmented cells before developmental arrest [6–8]. Recent studies have suggested that blastomere fragmentation in mouse and human preimplantation embryos may be indicative of apoptosis [9–11], although it has also been suggested that apoptosis may occur as a secondary consequence of fragmentation [6]. The precise relationship between blastomere fragmentation and apoptosis remains to be clarified, but available data indicate that a significant proportion of human and mouse embryos may undergo blastomere fragmentation during early cleavage stages.

Because developmental arrest and blastomere fragmentation occur predominately at or just before the time of global embryonic genome activation at the two- and four-cell stages in mouse and human embryos, respectively, it has been suggested that the incidence of fragmentation and apoptosis may relate to the success of temporally and functionally correct embryonic genome activation [9]. Increased cellular fragmentation in rat embryos fertilized with sperm from 5-aza-cytidine-treated males has also been suggested to result from aberrant gene transcription at the time of genome activation [12]. Interestingly, doxorubicin treatment enhances apoptosis in unfertilized oocytes but not in fertilized embryos [13], indicating important differences between fertilized embryos and oocytes, possibly related to a developmental acquisition of antiapoptotic mechanisms within the early embryo. In addition to possible effects related to embryonic gene transcription, other effects related to mitochondrial activity [14, 15] and responses to reactive oxygen species, to which the embryo may be especially sensitive [16], might also affect the incidence of blastomere fragmentation and apoptosis. Finally, aneuploidy (observed most commonly in human embryos of poor morphology [17, 18]), DNA damage, or incomplete sperm DNA decondensation may lead to cell cycle-dependent activation of apoptotic pathways [19]. Thus, multiple biochemical pathways may affect the incidence of blastomere fragmentation or apoptosis. To our knowledge, however, the factors that govern the probability of an embryo succumbing to these processes have not been defined.

Understanding the molecular mechanisms of early embryo blastomere fragmentation and the genetic factors that control its incidence would provide important new insight regarding the underlying biochemistry and physiology of early preimplantation development and embryo survival, and it would also be of great practical value in the clinic. Because the underlying molecular and cellular basis of blastomere fragmentation is likely complex, we reasoned that a genetic model, if available, could provide an important new tool with which to discover the identities of specific gene products involved in the process. We therefore conducted a genetic study employing inbred mouse strains to identify genetic differences that affect the ability of early mouse embryos to remain intact or to undergo blastomere fragmentation. We report here the discovery of striking genetic differences between inbred mouse strains that affect the incidence of blastomere fragmentation at the two-cell stage. These results thus establish a valuable new genetic system that will be useful for investigating the molecular and cellular basis of blastomere fragmentation and for determining the genetic basis for the effects of maternal and paternal genotypes on blastomere fragmentation.

MATERIALS AND METHODS

Embryo Isolation and Culture

Female C57BL/6 (Harlan Sprague Dawley, Indianapolis, IN) mice (~8 wk of age) were superovulated and mated to C57BL/6 (Harlan Sprague Dawley), AKR/J (Jackson Laboratory, Bar Harbor, ME), DBA/2 (Taconic, Germantown, NY), CD-1 (Jackson Laboratory), C3H/HeJ (Jackson Laboratory), or SJ/L (Jackson Laboratory) males (2.5–6 mo old). Superovulated C3H/HeJ (Jackson Laboratory) females (~8 wk of age) were mated to C57BL/6 or C3H/HeJ males. Other crosses employing F1 hybrid mice were performed with superovulated females as indicated below. Superovulation was achieved by i.p. injection of 5 IU of eCG, followed 48 h later by 5 IU of hCG. Embryos were isolated at 17 h after injection of hCG (17 hphCG) as described by Latham and Westhusin [20] and cultured in CZB medium [21] at 37°C in 5% CO2, 5% O2, and 90% N2. Fertile embryos were identified at 17 hphCG on the basis of possessing second polar bodies and lacking the prominent protrusion typically located above the metaphase spindle in unfertilized oocytes. Embryos thus identified were examined again at 22 hphCG for pronuclei to confirm fertilization. In this way, only fertile embryos were analyzed, and potential effects of different efficiencies of fertilization were avoided. The embryos were examined periodically and the number of fragmented embryos recorded at 29 hphCG (late one-cell stage) and 39 hphCG (mid two-cell stage). All studies adhered to procedures consistent with the National Research Council Guide for the Care and Use of Laboratory Animals.

The objective of this study was to determine whether genetic factors influence the ability of mouse two-cell stage embryos to remain morphologically intact. In previous studies of human embryos, fragmented embryos were graded according to severity of fragmentation [6]. Because no evidence indicates that fundamentally different mechanisms lead to more or less severe phenotypes, such distinctions were not employed for this study. Thus, embryos were classified as either intact two-cell stage or as possessing fragmented blastomeres (e.g., compare Fig. 1, A and B with G). This approach avoided any possible subjectivity in morphological classification or ambiguity in ascertaining the blastomere origin of fragments. Thus, the data provided a straightforward evaluation regarding the effects of parental genotypes on the ability of two-cell stage embryos to remain morphologically intact.



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  		FIG. 1. Fragmentation of mouse two-cell stage embryos. A and B) Examples of embryos exhibiting fragmented blastomeres. C–E) Negative TUNEL reaction with fragmented embryos. Note positive staining of polar bodies in D and E. F) Negative TUNEL reaction in unfragmented, negative-control embryo not treated with DNase. G and H) Differential interference contrast and fluorescence images of DNase-treated positive control for TUNEL labeling

TUNEL Labeling of Individual Embryos

Fragmented embryos (39 hphCG) were analyzed for evidence of DNA degradation characteristic of apoptosis using a TUNEL-labeling kit (Boehringer Mannheim, Roche Molecular Diagnostics, Indianapolis, IN). Embryos were fixed in 3.7% (w/v) formaldehyde in PBS for 30 min at room temperature, washed three times for 15 min in PBS, and then permeabilized for 30 min in 0.1% (w/v) Triton X-100. Permeabilized embryos were washed three times in PBS for 15 min each wash and then transferred to the TUNEL-labeling solution containing terminal deoxynucleotidyl transferase and fluorescein-dUTP, dNTP (TUNEL label) in reaction buffer for 1 h at 37°C. The embryos were then washed three times in PBS for 15 min each. Negative controls were unfragmented two-cell stage embryos. Positive controls were unfragmented embryos (39 hphCG) incubated in DNase I (1000 IU) for 30 min at 37°C. Stained embryos were mounted in Citiflour (Molecular Probes, Eugene, OR) and examined by confocal microscopy using an Olympus laser scanning confocal microscope (Olympus, Nanuet, NY) equipped with a krypton/argon mixed-gas laser at 488 nm to visualize the incorporated label (excitation filter, 490 nm; emission filter, 525 nm).

Statistical Analysis

Significance of differences was evaluated using a chi-square test of independence to determine whether the proportion of embryos with fragmented blastomeres was affected by genotype.

RESULTS

Genetic Factors Affecting the Incidence of Embryo Fragmentation at the Two-Cell Stage

A previous study reported that the paternal genotype could affect the incidence of mouse embryo fragmentation at the one-cell stage, and it was suggested that this might relate to a need for early embryonic gene expression [9]. To determine whether parental genotypes might also affect the incidence of embryo fragmentation during the two-cell stage, when the embryonic genome becomes transcriptionally more highly activated, we isolated embryos at 17 hphCG and removed unfertilized oocytes and embryos that had already undergone fragmentation. We then determined the fraction of fertilized embryos (fertilization confirmed by the presence of pronuclei) exhibiting blastomere fragmentation at times corresponding to the late one-cell and mid two-cell stages. This permitted specific determination of the incidence of fragmentation in fertilized embryos at the two-cell stage (Table 1).


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  		TABLE 1. Fragmentation of mouse embryos from different crosses.a

For embryos isolated from C57BL/6 mothers, we observed a striking effect of paternal genotype on the incidence of blastomere fragmentation (Table 1). Very little fragmentation was observed in crosses of C57BL/6 females to C57BL/6, AKR/J, DBA/2, or SJL males (range, 0%–2.3%). Fragmentation appeared increased in crosses of C57BL/6 females to CD1 males, but this difference was not statistically different from C57BL/6 x C57BL/6 matings. For crosses of C57BL/6 females to C3H/HeJ males, however, a significantly increased incidence of fragmentation (12%, P < 0.001) was observed at 39 hphCG (two-cell stage). In the reciprocal cross of C3H/HeJ females to C57BL/6 males, the incidence of fragmentation was again elevated at 39 hphCG (15%, P < 0.01), whereas fragmentation was not significantly increased for C3H/HeJ x C3H/HeJ matings (4.5%) as compared with the crosses employing C57BL/6 females and males other than CD1 or C3H/HeJ (P > 0.052; Table 1). Fragmented embryos were generally not observed at the late one-cell stage (29 hphCG), with the exception of a few embryos in the C57BL/6 x CD1 and C57BL/6 x C3H/HeJ crosses (Table 1), and all the remaining embryos progressed to the two-cell stage, indicating that the fragmentation observed at 39 hphCG indeed occurred during the two-cell stage.

To obtain additional information about the genetic basis of the increased incidence of fragmentation observed for crosses involving the two inbred strains C57BL/6 and C3H/HeJ, we produced reciprocal F1 hybrid males and females and evaluated the incidence of embryo fragmentation at the two-cell stage in backcrosses to mice of either inbred parental strain (Table 2). Crosses involving F1 hybrid females to either type of inbred male produced a significantly higher incidence of fragmentation than observed for crosses between C57BL/6 females and C57BL/6, AKR/J, SJL, and DBA/2 males (compare with Table 1; P < 0.001). The incidence of fragmentation differed significantly (P < 0.001) between the reciprocal F1 hybrid females, so that embryos from (C57BL/6 x C3H/HeJ) F1 females mated to either strain of male exhibited approximately twice the incidence of fragmentation compared with embryos from (C3H/HeJ x C57BL/6) F1 females. Fertilization of oocytes from C57BL/6 females by F1 hybrid males produced a significantly increased incidence of blastomere fragmentation (Table 2) compared with fertilization by C57BL/6 males (Table 1; P < 0.001). Crosses of C3H/HeJ females to F1 hybrid males (Table 2) did not produce a significant increase in fragmentation compared with fertilization by C3H/HeJ males (Table 1). Less fragmentation was observed following fertilization of C57BL/6 oocytes by either genotype of F1 hybrid male (Table 2) compared with crosses to C3H/HeJ males (Table 1; P < 0.05). Fertilization of oocytes from C3H/HeJ females by either F1 hybrid male genotype (Table 2) resulted in less fragmentation than fertilization by C57BL/6 males (Table 1; P < 0.05). Interestingly, matings between F1 mice of the same type or matings between reciprocal F1 mice resulted in very low fragmentation frequencies (Table 2). Among these crosses, the only significant difference (P < 0.05) observed was between BC x BC and CB x CB matings (Table 2).


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  		TABLE 2. Fragmentation in crosses employing F1 hybrid mice.a

Contribution of Gene Transcription to Embryo Fragmentation

In some experiments, [C57BL/6 x (C57BL/6XC3H/HeJ)F1] and C57BL/6 x C3H/HeJ embryos were treated with {alpha}-amanitin, an inhibitor of RNA polymerase II, to determine whether blastomere fragmentation and, specifically, the effect of paternal genotype required embryonic gene transcription (Table 3). Treatment with {alpha}-amanitin had no significant effect on the incidence of fragmentation in two-cell stage embryos.


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  		TABLE 3. Fragmentation in embryos treated with {{alpha}}-amanitin

TUNEL Labeling of Fragmented Embryos

To determine whether the blastomere fragmentation observed in two-cell stage embryos was associated with DNA degradation typically associated with apoptosis, some of the fragmented embryos were subjected to TUNEL labeling. The TUNEL labeling of nuclear DNA was observed only in 2 of 59 fragmented embryos analyzed, so the vast majority were unlabeled. The two TUNEL-positive fragmented embryos exhibited an intermediate degree of fluorescence (i.e., less intense than shown in Fig. 1H). Diffuse background staining over the cytoplasm was observed in some instances (Fig. 1, C and D), but similar diffuse signals were observed in unfragmented negative control embryos (Fig. 1F). No intense staining of nuclei or nuclear fragments typical of apoptotic embryos was seen in fragmented embryos. Intense staining in the majority of fragmented embryos was thus limited to polar bodies (Fig. 1, D and E). Positive-control, DNase-treated embryos exhibited the expected intense TUNEL labeling over blastomere nuclei as well as the polar body (Fig. 1H).

DISCUSSION

The results presented here reveal a striking and, to our knowledge, previously unobserved effect of both parental genotypes on blastomere fragmentation in two-cell stage mouse embryos. Specifically, we have demonstrated that two-cell stage mouse embryos can undergo cellular fragmentation at substantial frequencies, and that the incidence of fragmentation is dependent on both maternal and paternal genotypes, with specific genetic combinations leading to a much greater proportion of embryos with fragmented blastomeres than others. Embryos from certain genotypes of females exhibited an increased incidence of blastomere fragmentation at the two-cell stage when fertilized by certain genotypes of males, but not when fertilized by other genotypes of males. Conversely, specific genotypes of males produced elevated incidences of fragmentation only in progeny embryos from specific genotypes of females. For example, neither the C57BL/6 oocyte nor the C3H/HeJ oocyte were inherently destined to produce embryos with an elevated likelihood of fragmentation, and neither were C57BL/6 nor C3H/HeJ sperm. Accordingly, the different incidences of fragmentation observed cannot be ascribed merely to differences in inherent gamete quality, because for both sperm and oocytes, the incidence of fragmentation was dependent on the genotype of the opposite gamete. Moreover, because the effect of parental genotype is not an intrinsic feature of the gametes or the parental genotypes, and because our experimental design eliminated unfertilized oocytes from the analysis, the effects of maternal and paternal genotypes on fragmentation cannot be attributed merely to strain-dependent differences related to other events (e.g., superovulation or efficiency of fertilization) or to maternal effects related simply to oocyte quality. Rather, our data indicate that specific genetic combinations of oocyte and sperm resulted in enhanced fragmentation. These observations establish a new and valuable genetic system with which to examine the genetic factors that may control oocyte and embryo fragmentation.

The crosses employing F1 hybrids between C57BL/6 and C3H/HeJ strains reveal additional interesting results. First, the results with reciprocal F1 females indicate that the incidence of fragmentation exhibits a significant parental origin effect. Specifically, a greater incidence of fragmentation was observed with crosses involving (C57BL/6 x C3H/HeJ) F1 hybrid females compared with (C3H/HeJ x C57BL/6) F1 hybrid females. Second, this difference was unaffected by whether C57BL/6 or C3H/HeJ males were used. This indicates that a combination of allelic variants of gene products expressed either in the oocyte or from the maternal genome of the embryo may interact with products encoded by either paternal genotype to enhance fragmentation, and that the parental origin effect is superimposed on this interaction. Third, the incidence of fragmentation for F1 intercrosses was very low, further indicating that the parental origin effect is itself sensitive to the combinations of specific genotypes at one or more other loci.

The parental origin effect may be explicable on the basis of either an effect of maternal origin of mitochondria in the oocyte or a role for one or more imprinted gene(s) expressed either in the oocyte or in the early embryo. A possible role for genetic differences in mitochondria is interesting, given the central role played by mitochondria in cellular apoptosis [22–26] and the overall cellular physiology. It is possible that the oocyte mitochondria differ between strains, either genetically or physiologically, and that this difference affects the predisposition of the embryo to undergo blastomere fragmentation even into the two-cell stage. Such an effect of mitochondrial genotype need not be an obligate, inherent feature of the oocyte. Mitochondria contain proteins encoded by both the mitochondrial genome and by nuclear genes. In the newly formed embryo, the nuclear encoded proteins contained in the mitochondria are initially encoded entirely by the maternal genome. On expression of the paternal genome, the mitochondria would be expected to acquire proteins encoded by paternal alleles, and these paternally encoded variants would be required to interact with mitochondrial proteins produced during oogenesis. Any reduction in the efficiency of such interactions arising through specific parental genotype combinations could affect embryo physiology, including fragmentation. A possible role for genomic imprinting could arise through the actions of one or more imprinted genes expressed in the egg. The allelic variants of the imprinted gene(s) could confer different predispositions to fragmentation that may be uncovered following fertilization with sperm of specific genotypes. Interestingly, several imprinted genes affect apoptosis in other systems [27–34]. The majority of embryos that we examined did not exhibit positive TUNEL labeling, but this does not exclude a possible role for apoptotic mechanisms in causing blastomere fragmentation. It is possible that the embryo lacks expression of the caspase-dependent DNase, and apoptosis can, in fact, occur in the absence of DNA degradation [35]. Although the precise relationship between blastomere fragmentation and apoptosis remains to be clarified, the reports concerning involvement of imprinted genes in apoptosis in other systems present the interesting possibility that such imprinted genes may affect cell fragmentation and/or apoptosis in the early embryo. Another possible basis for the parental origin effect in F1 hybrid females would be via a grandparental effect acting on one or more genes expressed from the maternal genome of the embryo after fertilization. This, however, would require the transmission of an epigenetic mark from the grandparent to the embryo, which, although possible, requires an inherently more complicated mechanism to achieve.

Our observation that both parental genotypes affect embryonic blastomere fragmentation is consistent with the hypothesis that maternal and paternal factors must interact to promote early embryo survival. The nature of such interactions and the identities of the relevant gene products remain to be determined. The maternal genotype effect may be mediated, at least in part, through factors deposited in the ooplasm. The suppression of apoptosis in human one-cell embryos relative to unfertilized oocytes treated with chemical inducers [13] indicates that the predisposition to undergo apoptosis diminishes at fertilization. This transition could vary with maternal genotype. A role for de novo expression of antiapoptotic molecules from the maternal genome after fertilization is also conceivable. Other mechanisms not involving apoptosis are possible as well. For example, effects on the organization or function of the cytoskeleton, which controls complex movements of mitochondria and polarized distribution of specific macromolecules [36–39], as well as cytokinesis could be subject to genetic variability, and this could affect blastomere integrity. In addition, the ooplasm contains factors that impose epigenetic alterations on the paternal pronucleus [4, 40, 41], including changes in DNA methylation [42, 43], and also contains transcription factors that associate preferentially with the paternal pronucleus [44]. Genetic differences in the composition, abundance, or biochemical properties of these ooplasmic components could affect paternal genome function and, subsequently, blastomere fragmentation. Such an effect on paternal genome function could account for the combined effects of both parental genotypes on blastomere fragmentation.

Our data support a possible role for paternal genome expression in controlling blastomere fragmentation. The incidence of fragmentation was reduced when C57BL/6 or C3H/HeJ females were mated to F1 hybrid males, as opposed to when they were mated to inbred males of the opposite genotypes. Because parental alleles would be segregated among different sperm, this reduction is consistent with the possibility that the paternal genotype effect is exerted via postfertilization gene expression. Our studies with {alpha}-amanitin revealed neither a reduction nor an increase in blastomere fragmentation, indicating that the increased incidence of blastomere fragmentation we observed probably is not attributable to inappropriate de novo gene expression from the paternal genome or expression of gene products that promote fragmentation or, perhaps, apoptosis. This result, however, does not exclude the possibility that increased blastomere fragmentation results from the lack of expression of gene products that normally inhibit fragmentation in transcriptionally active embryos, or that the expression of such products from paternally inherited genes in the embryo plays an important part in suppressing blastomere fragmentation.

The paternal genotype effect could also be exerted, at least in part, via sperm-derived factors that may interact with ooplasmic factors. The introduction of proapoptotic regulatory proteins via the sperm, introduction of gene regulatory factors via the sperm, introduction of cellular organelles that may affect cellular physiology, or differences in signaling events mediated at the time of fertilization that subsequently regulate apoptosis or other processes leading to blastomere fragmentation provide possible means by which sperm-derived factors could affect blastomere fragmentation. The recent demonstration that sperm-derived factors can affect the timing of blastomere division [45] is especially interesting, because such an effect on cell division might also affect other processes that control fragmentation.

Because both parental genotypes affect the incidence of blastomere fragmentation, the magnitude of the effects exerted by both the maternal and paternal genotype each depend on the genotype of the opposite parent, and the effects of genotype are quantitative, it is reasonable to conclude that multiple genes, expressed either in the sperm, the oocyte, or embryonically, likely affect the process. Our studies thus indicate that the mechanism leading to blastomere fragmentation is likely complex and involves interactions between sperm and oocyte factors expressed in the gametes before fertilization, interactions between factors produced from the two parental genomes after fertilization, or interactions between gamete-derived factors and genes expressed after fertilization.

The finding that both parental genotypes affect the incidence of blastomere fragmentation has implications for human reproductive medicine, because it indicates that parameters of gamete fitness (e.g., oocyte quality) or genetic factors affecting early development cannot necessarily be evaluated for one parent independently of the genotype of the opposite parent. Our data, together with data reported earlier [9], indicate that the combined effects of maternal and paternal genotype on fragmentation at the one- and two-cell stages in mice can lead to blastomere fragmentation in 25% or more of the fertilized embryos. Thus, the data presented here reveal a previously unknown, but highly significant, effect of parental genotypes on blastomere fragmentation. Our data also raise the possibility that procedures such as cloning by somatic cell nuclear transfer [46–49] or therapeutic ooplasm transfer to enhance embryo viability [50] may, with certain genetic combinations of donor nuclei and recipient oocyte, result in the production of embryos with an enhanced predisposition to blastomere fragmentation, which may thus affect the overall efficiency and efficacy of such procedures in a way opposite to the desired outcome. Finally, the finding that particular genetic combinations of sperm and oocyte can enhance the incidence of blastomere fragmentation and, thus, may affect the viability of heterozygotes raises the possibility that such genetic effects may lead to changes in allele frequencies in populations and, in turn, may participate in the evolutionary process. The genetic system that we have established provides a useful tool with which to address these possibilities and to investigate the underlying biology of blastomere fragmentation and its effects on embryo survival.

ACKNOWLEDGMENTS

We thank Dr. Fernando Pardo-Manuel de Villena and Dr. Carmen Sapienza for their assistance with statistical analysis and comments on the manuscript and Dr. Sue Varmuza for comments on the manuscript.

FOOTNOTES

First decision: 20 March 2001.

1 Supported by grants from the National Institutes of Health (GM-56682, 5 T32 CA09214-20). Back

2 Correspondence: Keith E. Latham, Temple University School of Medicine, The Fels Institute, 3307 North Broad Street, Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}unix.temple.edu Back

Accepted: May 21, 2001.

Received: February 20, 2001.

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