Hybrid vigor and transgenerational epigenetic effects on early mouse embryo phenotype

Abstract

Mouse embryos display a strain-dependent propensity for blastomere cytofragmentation at the two-cell stage. The maternal pronucleus exerts a predominant, transcription-dependent effect on this phenotype, with lesser effects of the ooplasm and the paternal pronucleus. A parental origin effect has been observed as an inequality in the cytofragmentation rate of embryos produced through genetic crosses of reciprocal F(1) hybrid females. To understand the basis for this, we conducted an extensive series of pronuclear transfer studies employing different combinations of inbred and F(1) hybrid maternal and paternal genotypes. We find that the parental origin effect is the result of a transgenerational epigenetic modification, whereby the inherited maternal grandpaternal contribution interacts with the fertilizing paternal genome and the ooplasm. This result indicates that some epigenetic information related to grandparental origins of chromosomes (i.e., imprinting of chromosomes in the mother) is retained through oogenesis and transmitted to progeny, where it affects gene expression from the maternal pronucleus and subsequent embryo phenotype. These results reveal for the first time that mammalian embryonic development can be affected by the epigenotype of at least three individuals. Additionally, we observe a significant suppression of fragmentation by F(1) hybrid ooplasm when it is separated from the F(1) hybrid maternal pronucleus. This latter effect is a striking example of heterosis in the early mammalian embryo, and it provides a new opportunity for examining the molecular mechanisms of heterosis. These results are relevant to our understanding of the mechanisms of epigenetic effects on development and the possible fertility effects of genetic and epigenetic interactions in reproductive medicine.

FIG. 1.. Schematic illustration of maternal PN transfers performed using inbred strains of mice. Black ooplasm and black PN represent B6 (denoted B), and white ooplasm and white PN represent C3H (denoted C). The combinations of ooplasm, maternal PN (Mat. PN), and paternal PN (Pat. PN) are shown below the embryos, and are those used in Table 1.

FIG. 5.. Schematic illustration of maternal PN transfers performed using inbred and F1 hybrid strains of mice. Black ooplasm and black PN represent B6 (denoted B), and white ooplasm and white PN represent C3H (denoted C). Light stippled ooplasm are C3H×B6 (denoted CB), heavy stippled ooplasm are B6×C3H (denoted BC). Maternal PNs with left half white and right half black are from C3H×B6 (denoted CB) donors. Maternal PNs with left half black and right half white are from B6×C3H (denoted BC) donors. The combinations of ooplasm, maternal PN, and paternal PN produced are shown below the embryos, and are those used in Tables 6 and 7.

FIG. 2.. Schematic illustration of ooplasm transfers performed using inbred strains of mice. Black ooplasm and black PN represent B6 (denoted B), and white ooplasm and white PN represent C3H (denoted C). Cytoplasts were approximately the size of a polar body. The combinations of recipient ooplasm, donor ooplasm, maternal PN, and paternal PN are shown below the embryos, and are those used in Table 2.

FIG. 3.. Schematic illustration of paternal PN transfers performed using inbred strains of mice. Black ooplasm and black PN represent B6 (denoted B), and white ooplasm and white PN repersent C3H (denoted C). The combinations of ooplasm, maternal PN, paternal PN, and paternal PN source ooplasm are shown below the embryos, and are those used in Table 3.

FIG. 4.. Schematic illustration of the embryo types with different mitochondria origin and maternal F1 hybrid genotype. Black ooplasm and black PN represent C57BL/6 (B6, denoted B), and white ooplasm and white PN represent C3H (denoted C). Light stippled ooplasm is from C3H×B6 (denoted CB), heavy stippled ooplasm is from B6×C3H (denoted BC), and medium stippled ooplasm is from B6×C3H ooplasm with C3H mitochondria origin (denoted BC^MitoC). Maternal PNs with left half white and right half black are from C3H×B6 (denoted CB) donors. Maternal PNs with left half black and right half white are from B6×C3H (denoted BC) donors. The combinations of mitochondria, ooplasm, maternal PN, and paternal PN are shown below the embryos, and are those used in Table 5.

FIG. 6.. Quantitative effects of pronucleus and ooplasm strain of origin on cytofragmentation in maternal pronucleus transfer studies. The parental rates of cytofragmentation for B6 and C3H strains are indicated (6.7% and 19.6%). The effect of each manipulation is represented by a vector, where the origin and direction of each vector is the observed departure from the cytofragmentation rate expected and the length of each vector is directly proportional to the strength of the effect of each manipulation. In general, addition of C3H alleles into the pronuclei enhances the rate of fragmentation above the B6 parental level, whereas addition of B6 alleles suppresses fragmentation below the C3H parental level. Note the synergistic effect of a BC maternal pronucleus and C3H paternal pronucleus (vector no. 13), which is greater than the sum of individual effects of BC maternal PN (vector no. 11) and C3H paternal PN (vector no. 1). This degree of synergy is not seen with a CB maternal pronucleus (vector no. 12), revealing the novel transgenerational effect of the maternal grandpaternal genome. Note also the hybrid vigor effect observed with F₁ ooplasm when separated from F₁ maternal PN (vector nos. 16–21), which suppresses fragmentation.

FIG. 7.. Schematic diagram summarizing interactions between maternal and paternal pronuclei and ooplasm affecting cytofragmentation in two-cell stage mouse embryos. The initial predisposition toward cytofragmentation is established in the ooplasm. This can occur either before or after fertilization, but is dependent upon the oocyte strain of origin. The predisposition toward fragmentation can be attenuated by heterozygosity during oogenesis, accounting for the observed hybrid vigor effect. This hybrid vigor effect, however, is seen only when the ooplasm is separated from its normal F₁ maternal PN partner. This is most likely because C3H alleles inherited via the maternal PN promote fragmentation, or fail to suppress it fully, when combined with ooplasmic C3H products. The predominant effect of the maternal PN on cytofragmentation, and the absence of this effect when F₁ ooplasm is separated from the F₁ maternal PN, indicate that the primary function of the maternal PN may be to suppress the overall rate of cytofragmentation directed by the ooplasm. This effect is transcription dependent, and it may involve direct effects of maternal PN-encoded gene products on the pathway leading to cytofragmentation. A B6 maternal PN suppresses cytofragmentation much more than a C3H maternal PN, and the latter may actually promote cytofragmentation. The maternal PN effect, however, must also require interactions either between products encoded by the zygotic maternal and paternal PN, or between maternal PN-encoded products and the paternal PN, the overall effect of which is to promote cytofragmentation. This requirement arises from the finding that the parental origin effect seen with CB and BC maternal PN is sensitive to the paternal PN genotype and the ooplasm strain of origin. For example, we observe a strong synergistic interaction between a BC maternal PN and a C3H paternal PN, which clearly indicates interactions between products expressed from these two genomes in the early embryo. Additionally, the ooplasm itself appears to modify the paternal PN to promote cytofragmentation, a subtle effect that is seen in the severity of fragmentation following one combination of paternal PN transfer (Table 3, compare rows 7 and 8). Additional effects of ooplasm strain of origin are seen in that the ooplasm of one strain tends to increase cytofragmentation when combined with maternal plus paternal PN of the opposite strains (compare lines 1 and 4, and lines 7 and 8, Table 1), and also in the effect of transient exposure of the paternal PN to one kind of ooplasm followed by a need to interface with ooplasm of the opposite strain (compare grades 2 and 3 fragmentation, lines 5 and 6, and lines 7 and 8, Table 3). GP, grandpaternal.