Mutagen-induced fetal anomalies and death following treatment of females within hours after mating

In an earlier study (Generoso et al., 1987), it was observed that the mutagen, ethylene oxide (EtO), produced remarkable increases in the incidence of developmental abnormalities and death of fetuses when early zygotic stages were exposed. This is a major finding in experimental induction of embryopathy, implicating genetic damage to the zygotes as the likely cause. In the subsequent study reported here, 3 other mutagens--ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), and triethylene melamine (TEM), were studied for embryopathic effects following exposure of dictyate oocytes, prefertilization oviducal eggs and sperm, early pronuclear zygotes, zygotes undergoing pronuclear DNA synthesis, and two-cell embryos. All 4 mutagens produced developmental abnormalities among living fetuses following exposure of early pronuclear zygotes (the only stage studied for this endpoint in this report). With respect to stage specificity and gestational timing of death of conceptuses, EMS and EtO on one hand and ENU and TEM on the other, are very similar to one another. EMS, like EtO, produced a high incidence of midgestation and late fetal deaths only in prefertilization oviducal eggs and sperm and in early pronuclear eggs. In contrast, ENU and TEM produced high losses of conceptuses in all postmating stages studied but death occurred primarily prior to or around the time of implantation. Thus, the frequency of induction and the expression of embryopathy, which ranged from early embryonic preimplantation and late fetal deaths to subtle fetal anomalies, are dependent upon the stage exposed and the mutagen used.     

Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.     

Studies in vitro on mouse-egg radiosensitivity from fertilization up to the first cleavage

Super-ovulated eggs from the Balb/c strain were incubated, at various times after injection of HCG, in Whitten's medium containing tritiated thymidine. They were fixed on the following day at the 2-cell stage and prepared for autoradiography. On the basis of the results, pregnant mice were irradiated with various doses of X-rays at 15 h post HCG (fertilization), 19 h (phonuclear stage before DNA synthesis), 24 h (maximal DNA synthesis) and 27.5 h (DNA synthesis completed). On the day following irradiation, embryos were collected and classified into incleaved or 2-cell embryos, and development of the 2-cell embryos was followed in culture.

Irradiation was most effective when administered at 19 h after injection of HCG. Such a treatment increased the mortality before the first cleavage and, thereafter, from the 8-cell (100 rad) or morula stage (25, 50 rad). Blastocyst hatching and implantation were also impaired. Irradiation at other times was much less harmful for the embryos, which died mainly from the blastocyst stage. Finally, radiosensitivities of the mouse zygote at the various times studied can be estimated as follows: fertilization, + + +; pronuclear stage before DNA synthesis, + + + + +; maximal DNA synthesis, +; DNA synthesis terminated, + +.     

DEPENDENCE OF PERIODIC DNA SYNTHESIS ON PROTEIN SYNTHESIS: DELAY OF DNA SYNTHESIS IN THE EARLY CLEAVAGE STAGE OF SEA URCHIN EMBRYOS TREATED WITH CYCLOHEXIMIDE AND COLCHICINE

The effects of cycloheximide and colchicine on cleavage and syntheses of DNA and proteins in cleaving embryos of the sea urchin, Hemicentrotus pulcherrimus , were examined. Cycloheximide caused delay of cell division with prolongation of the streak stage. Both inhibitors also caused delay in initiation of DNA synthesis. The decrease in the rate and prolongation of the period of DNA synthesis caused by these inhibitors varied with their concentrations and the time of administration. Initiation of DNA synthesis was delayed when cycloheximide was added to suspensions of embryos between the time after preceding DNA synthesis terminated and a definite time before the predicted time of initiation of the next synthesis of DNA, except at the stage of pronuclear fusion. However, when the inhibitor was added after initiation of the synthesis, the latter proceeded normally. Addition of 10 m m cycloheximide immediately after fertilization or 2 m m cycloheximide 60 min before fertilization also delayed DNA synthesis at the stage of pronuclear fusion, indicating that synthesis at this stage also required prior protein synthesis. Colchicine had less inhibitory effect on protein synthesis, but greatly delayed initiation of DNA synthesis and prolonged its duration. These facts suggest that a definite amount of a particular protein must be synthesized and accumulated in each synthetic cycle before initiation of DNA synthesis.     

Chemical induction of presumed dominant-lethal mutations in postcopulation germ cells and zygotes of mice. II. Sensitivity of different postcopulation-precleavage stages to three alkylating chemicals.

The relative sensitivities of various postcopulation-precleabage and pronuclear stages to dominant-lethal effects of isopropyl methanesulfonate (IMS), ethyl methanesulfonate (EMS), and triethylenemelamine (TEM) were investigated. The pattern of sensitivity differed with the chemical. IMS was most effective when pronuclear formation was already completed and the majority of the zygotes were presumably undergoing DNA synthesis. EMS, on the other hand, induced its most pronounced effects when eggs in the course of second meiotic division and zygotes in early pronuclear stages were treated. The greatest effect of TEM was observed when zygotes were treated at the early pronuclear stage. EMS and TEM, in contrast to IMS, are similar to radiations in that zygotes undergoing DNA synthesis are more resistant to them than are the early pronuclear stages. In the case of IMS, effects induced in the most sensitive postcopulation-precleavage stage were 6 to 9 times greater than in the most sensitive precopulatory dictyate oocytes or male germ cells. On the other hand, in the case of EMS and TEM, the most sensitive precopulatory male germ cells, but not the dictyate oocytes, were more sensitive than the most sensitive postcopulation stages.     

Fetal anomalies produced subsequent to treatment of zygotes with ethylene oxide or ethyl methanesulfonate are not likely due to the usual genetic causes

Earlier studies in this laboratory revealed that ethylene oxide (EtO) or ethyl methanesulfonate (EMS) induced high frequencies of midgestation and late fetal deaths,and of malformations among some of the surviving fetuses, when female mice were exposed at the time of fertilization of their eggs or during the early pronuclear stage of the zygote. Effects of the two mutagens are virtually identical. Thus, in investigating the mechanisms responsible for the dramatic effects in the early pronuclear zygotes, the two compounds were used interchangeably in the experiments. First, a reciprocal zygote-transfer study was conducted in order to determine whether the effect is directly on the zygotes or indirectly through maternal toxicity. And second, cytogenetic analyses of pronuclear metaphases, early cleavage embryos, and midgestation fetuses were carried out.

The zygote transplantation experiment rules out maternal toxicity as a factor in the fetal maldevelopment. Together with the strict stage specificity observed in the earlier studies, this result points to a genetic cause for the abnormalities. However, the cytogenetic studies failed to show structural or numerical chromosome aberrations. Since intragenic base changes and deletions may also be ruled out, it appears that the lesions in question induced in zygotes by the two mutagens are different from conventional ones and, therefore, could be a novel one in experimental mammalian mutagenesis. Alternatively, the mechanism could invlve a non-mutational ‘imprinting’ process that caused changes in gene expression.     

Nucleic acid synthesis and development of human male pronucleus

Polyspermically penetrated human zona-free eggs prepared from oocytes that had failed to be fertilized in an in-vitro fertilization programme were used. The pronuclear synthetic activity was evaluated by high-resolution autoradiography and correlated with the development of pronuclear structure. Incorporation of [3H]-thymidine, signalling the occurrence of a DNA synthetic phase, was only detected in structurally fully developed pronuclei previously shown to appear no sooner than 12 h after gamete union. However, [3H]adenosine was incorporated into very early pronuclei which had not yet completed the development of their nuclear envelopes and which first appeared about 4 h after sperm-egg fusion. In the absence of DNA synthesis (shown by the lack of thymidine incorporation), this early adenosine incorporation apparently reflects an early pronuclear RNA synthesis. Taken together, these results indicate that nucleic acid synthesis in human male pronuclei is tightly bound to the development of a corresponding pronuclear structure and that DNA synthesis, beginning about 12 h after fertilization, is preceded by a slight but evident RNA synthesis taking place during an early stage of human male pronuclear formation.     

Transport of cationic and zwitterionic amino acids in preimplantation rat conceptuses

The ability of preimplantation rat conceptuses to take up several amino acids was examined under a variety of conditions, and the characteristics of uptake were compared to those determined previously for mouse conceptuses. Mediated leucine transport in two-cell rat conceptuses is Na(+)-independent and inhibited almost completely by 2-amino-endobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), so it resembles system L which predominates in two-cell mouse conceptuses. System L becomes less conspicuous than homoarginine-sensitive, Na(+)-independent leucine transport (provisionally designated system bo,+) by the time rat conceptuses develop into blastocysts, as is also the case for mouse conceptuses. In contrast to leucine transport, system bo,+ appears to be the most conspicuous transporter of cationic amino acids throughout preimplantation development of both species. A Na(+)-independent cation-preferring amino acid transport process also appears to be present in rat as well as in mouse conceptuses. Moreover, rat conceptuses resemble mouse conceptuses because Na(+)-dependent transport system Gly activity virtually disappears from them by the time they form blastocysts. Unlike mouse conceptuses, however, Na(+)-dependent system Bo,+ activity appears to be present throughout preimplantation development of rat conceptuses, whereas it has not been detected until at least the two-cell stage in the mouse. Although system Bo,+ becomes more conspicuous in mouse than in rat conceptuses by the time they form blastocysts, system Bo,+ activity appears to increase when blastocysts of both species are removed from the uterus just prior to implantation. The latter observation is consistent with the possibility that system Bo,+ activity is controlled, in part, by the uterus near the time of implantation, although further studies are needed to verify this possibility. Similarities as well as differences in the amino acid transport processes present in conceptuses of rats and mice may eventually be understood best in relation to the environments in which they develop in vitro and in situ.     

The onset of DNA synthesis and its relation to morphogenetic events of the pronuclei in activated eggs of the sea urchin, Arbacia punctulata

Experiments were carried out in order to investigate the onset of DNA synthesis and its possible relation to pronuclear morphogenesis prior to, during, and following pronuclear fusion in the sea urchin, Arbacia punctulata. Analysis of the acid-soluble radioactivity of zygotes continuously incubated in tritiated thymidine (³H-TdR) at 22°C demonstrated the production of thymidine triphosphate prior to and during pronuclear migration (5–10 min post-insemination) which continued to increase until prophase (about 45 min post-insemination). DNA synthesis was initiated subsequent to pronuclear fusion, i.e., 15–20 min post-insemination. Little or no cytoplasmic label was detected during fertilization. Silver grains were first detected over the zygote nucleus in sections prepared for light and electron microscopic radioautography from zygotes fixed at 18 min post-insemination. Incubation of Arbacia zygotes at 12°C dramatically slowed development but did not alter the temporal relation between pronuclear fusion and DNA synthesis. Radioautographs of polyspermic zygotes demonstrated that unfused male pronuclei were able to synthesize DNA and substantiate the claim that pronuclear fusion is not required for the initiation of DNA synthesis. Analysis of autoradiographs of artificially activated eggs also lead to the same conclusion. Uptake of ³H-TdR or ³H-bromodeoxyuridine was not found in spermatozoa incorporated into polyspermic oocytes regardless of whether or not they had initiated or developed into male pronuclei. It is concluded that the relation between DNA synthesis and pronuclear fusion is not close or a direct one, i.e., pronuclear fusion does not appear to be a prerequisite for the onset of DNA synthesis in Arbacia.     

Experiments Pertaining to the Suppression of Accessory Sperm in Fertilized Newt Eggs*

In the physiologically polyspermic eggs of the newt, Cynops pyrrhogaster, a number of accessory sperm undergo pronuclear formation along with a concomitant DNA synthesis, but degenerate after zygote nucleus formation. When denuded eggs were divided into two halves at various post-fertilization stages, the andromerogons produced before zygote nucleus formation but not after that stage cleaved at a high frequency. The accessory sperm were unable to participate in the cleavage when they were located in the half of the egg which was connected with the diploid merogon by a cytoplasmic bridge higher than 100 μm in height. The removal of the egg nucleus or the retardation of early post-fertilization nuclear events by treatment with cycloheximide resulted in the induction of multipolar cleavage. Continuous exposure of the fertilized eggs to aphidicolin showed that in the appreciable absence of the DNA synthesis many eggs underwent a first cleavage cytokinesis of a mostly abortive type, but failed to initiate the following cytokinesis at all. Cytological examinations in association with these experiments suggest that the observed suppression of accessory sperm includes the inhibition of centriolar replication under the influence of the zygote nucleus, resulting in the failure of cytasters corporating with nuclear-independent activity of cortical cytoplasm.     

Two maternally derived X chromosomes contribute to parthenogenetic inviability

In certain extraembryonic tissues of normal female mouse conceptuses, X-chromosome-dosage compensation is achieved by preferential inactivation of the paternally derived X. Diploid parthenogenones have two maternally derived X chromosomes, hence this mechanism cannot operate. To examine whether this contributes to the inviability of parthenogenones, XO and XX parthenogenetic eggs were constructed by pronuclear transplantation and their development assessed after transfer to pseudopregnant recipients. In one series of experiments, the frequency of postimplantation development of XO parthenogenones was much higher than that of their XX counterparts. This result is consistent with the possibility that two maternally derived X chromosomes can contribute to parthenogenetic inviability at or very soon after implantation. However, both XO and XX parthenogenones showed similar developmental abnormalities at the postimplantation stage, demonstrating that parthenogenetic inviability is ultimately determined by the possession of two sets of maternally derived autosomes.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. II. Chromosome aberrations induced in mature oocytes and fertilized eggs at the pronuclear stage following X-irradiation

Cytological analysis of the first-cleavage metaphase of eggs exposed to X-rays at the mature oocyte stage or the pronuclear stage 4 h after fertilization was performed using the in vitro fertilization technique. The frequency of chromosome aberrations in irradiated mature oocytes increased exponentially with dose, the dose-response relationship being best fitted to the linear-quadratic model. On the other hand, in eggs irradiated at the early pronuclear stage, the frequency increased linearly with dose and the dose-response relationship was best fitted to the linear model. The aberrations were mainly chromosome-type (mature oocytes: 86.0% and pronuclear stage: 88.5%) and the majority were fragments in both cases. Eggs in the early pronuclear stage were markedly more radiation-sensitive than mature oocytes. A comparison of the present results with the previous ones (Matsuda et al., 1985b) showed that the sensitivities to induction of chromosome aberrations were in the order: egg at early pronuclear stage (highest) greater than mature oocyte greater than mature sperm.     

Potential role of somatomedin inhibitors in the production of diabetic embryopathies

Mouse conceptuses at the 18-21-somite stage were grown for 2-24 h in vitro in the presence of a serum fraction (Mr = 800-1,080 daltons) possessing somatomedin-inhibitory activity (SI) isolated from diabetic rats. Following an 8-h exposure to the SI, DNA and incorporation of 3H-thymidine were reduced in the embryos while 12 h was required to observe a reduction in total protein and RNA. At the 24-h time point, the neurectoderm was thinner than in controls, and autoradiograms of this region showed a substantial decrease in grain density with 3H-thymidine, but not 3H-leucine or -uridine. Effects of the visceral yolk sac (VYS) preceded those on the embryo. The cytoplasm of the VYS endoderm cells from conceptuses exposed to the SI contained many vacuoles by 4 h, which were larger by 24 h. Total protein was greater than in controls from 4 h onward, although 3H-leucine incorporation, which had increased after 2 h of SI exposure, returned to control levels by 8 h. As seen by SDS-polyacrylamide gel electrophoresis, VYSs from conceptuses exposed to the SI for 4 or 24 h were enriched (compared to control VYSs) in four protein bands also present in the culture medium (primarily rat serum), suggesting that protein degradation and/or transfer of amino acids and peptides to the embryo was inhibited in these VYSs. Such a conclusion was supported by a quantitative decrease in proteins and amino acids in the exocoelomic fluid of conceptuses exposed to the SI for 24 h. The altered processing of proteins may therefore represent a primary cause of the SI-induced embryonic abnormalities.     

Construction of the nuclear matrix at the transition from maternal to zygotic control of development in the mouse: an immunocytochemical study.

The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, Pl1, Pl2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two-cell-stage embryos, and eight-cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two- and eight-cell-stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two-cell stage in the presence of alpha-amanitin, the matrix morphology was altered for Pl1 and Pl2. alpha-Amanitin had no affect on the distribution of P1 or lamin B antigens. If alpha-amanitin was added 2 hr after cleavage to the two-cell stage, the normal staining pattern of Pl2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.     

Embryonic development in the pig up to the 64-cell stage, with reference to DNA replication and cell cycle times from the third cleavage division

Preimplantation cleaving-stage embryos were recovered from Dutch Landrace (DL) and F1 Dutch Landrace x Great Yorkshire (DL x GY) gilts for which the time of insemination and ovulation were known. Embryonic cell counts were performed, usually after brief in vitro culture to estimate DNA synthesis. Special attention was given to the 4-cell stage. Beyond this stage, the mean cell cycle time was 14 hours for DL and 17 hours for F1 gilts. Generally, high indices of DNA synthesis were obtained (more than 60% of nuclei). There was prominent within and between gilt variability with regard to embryonic cell numbers. Gilts are especially heterogeneous with respect to the length of the 4-cell stage. The G 2 M phase of the 4-cell stage takes approximately 3.5 hours. Especially for F1 gilts, the age of the spermatozoa at the moment of ovulation was not related to the rate of cleavage and/or embryonic death. It is postulated that variability in the length of the 4-cell stage is reflected in genetic activation of the embryos at this stage and subsequently influences embryonic survival.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. II. Alkylation of DNA and inhibition of DNA replication.

Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

An in situ transgenic enzyme marker for the midgestation mouse embryo and the visualization of inner cell mass clones during early organogenesis

In order to study the deployment of cells during gastrulation and early organogenesis, it is necessary to have an in situ cell marker which can be used to follow cell fate. To create such a marker a transgenic mouse strain, designated Tg(Act-lac Z)-1, which carries 6 copies of the Escherichia coli lac Z gene under the control of the rat beta-actin promoter, was made by pronuclear injection of DNA. Staining early postimplantation hemizygous mouse conceptuses, during gastrulation and early organogenesis, for beta-galactosidase activity shows that lac Z expression is ubiquitous and constitutive in all epiblast derivatives of the 10th day conceptus. No activity is seen in trophectoderm and primitive endoderm derivatives. Postimplantation grafts of [3H]thymidine-labelled transgenic cells establish the cell autonomy of this transgenic marker. Preliminary observations on the distribution of inner cell mass (ICM) descendant clones, identified in situ in midgestation conceptuses, confirm the pluripotency of individual ICM cells. The implications regarding patterns of cell growth in nascent fetal primordia are discussed.     

The sperm nuclear matrix is required for paternal DNA replication

The mammalian sperm nucleus provides an excellent model for studying the relationship between the formation of nuclear structure and the initiation of DNA replication. We previously demonstrated that mammalian sperm nuclei contain a nuclear matrix that organizes the DNA into loop domains in a manner similar to that of somatic cells. In this study, we tested the minimal components of the sperm nucleus that are necessary for the formation of the male pronucleus and for the initiation of DNA synthesis. We extracted mouse sperm nuclei with high salt and dithiothreitol to remove the protamines in order to form nuclear halos. These were then treated with either restriction endonucleases to release the DNA not directly associated with the nuclear matrix or with DNAse I to digest all the DNA. The treated sperm nuclei were injected into oocytes, and the paternal pronuclear formation and DNA synthesis was monitored. We found that restriction digested sperm nuclear halos were capable of forming paternal pronuclei and initiating DNA synthesis. However, when isolated mouse sperm DNA or sperm DNA reconstituted with the nuclear matrices were injected into oocytes, no paternal pronuclear formation or DNA synthesis was observed. These data suggest that the in situ nuclear matrix attachment organization of sperm DNA is required for mouse paternal pronuclear DNA synthesis.