Pronuclear DNA Synthesis Research Articles

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.

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm.

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.

P53-Dependent S-Phase Damage Checkpoint and Pronuclear Cross Talk in Mouse Zygotes with X-Irradiated Sperm

One difficulty in analyzing the damage response is that the effect of damage itself and that of cellular response are hard to distinguish in irradiated cells. In mouse zygotes, damage can be introduced by irradiated sperm, while damage response can be studied in the unirradiated maternal pronucleus. We have analyzed the p53-dependent damage responses in irradiated-sperm mouse zygotes and found that a p53-responsive reporter was efficiently activated in the female pronucleus. [(3)H]thymidine labeling experiments indicated that irradiated-sperm zygotes were devoid of G(1)/S arrest, but pronuclear DNA synthesis was suppressed equally in male and female pronuclei. p53(-/-) zygotes lacked this suppression, which was corrected by microinjection of glutathione S-transferase-p53 fusion protein. In contrast, p21(-/-) zygotes exhibited the same level of suppression upon fertilization by irradiated sperm. About a half of the 6-Gy-irradiated-sperm zygotes managed to synthesize a full DNA content by prolonging S phase, while the other half failed to do so. Regardless of the DNA content, all the zygotes cleaved to become two-cell-stage embryos. These results revealed the presence of p53-dependent pronuclear cross talk and a novel function of p53 in the S-phase DNA damage checkpoint of mouse zygotes.

Time course of pronuclear deoxyribonucleic acid synthesis in parthenogenetically activated bovine oocytes.

The progress of pronuclear DNA synthesis was monitored by the radioactive precursor 3H-thymidine during the first cell cycle of parthenogenetically activated bovine oocytes. Bovine oocytes were exposed to Ca2+ ionophore A23187 at 24, 30, or 36 h after the onset of in vitro maturation. Young 24-h oocytes were subsequently cultured for 6 h in the protein synthesis inhibitor, cycloheximide (CHX), to ensure similar rates of activation (96-100%) and pronuclear formation (93-97%) among all groups of oocytes. Subsequent autoradiographic experiments revealed a slightly, but not significantly, accelerated start of DNA synthesis in aged (36 h) oocytes. Maximum levels of DNA labeling were reached within 4 h regardless of oocyte maturation age and persisted for 4 h in 30-h oocytes compared to 2 h in 36-h and 24-h oocytes. The period of DNA synthesis lasted for a total of 12-14 h in all groups of oocytes, and the duration of S-phase was less than 6 h. Since rates of pronuclear formation (58%) and labeling (58%) corresponded to each other, it is argued that only a fully developed pronucleus can synthesize DNA. Oocyte labeling performed in the presence of CHX revealed the capability of CHX to inhibit DNA synthesis up to 8 h postactivation. Removal of CHX by washing when the majority (94%) of oocytes had formed a fully developed pronucleus (at 8 h postactivation) led to the synchronous start of DNA synthesis within 1.5-2 h post-CHX culture. This concomitantly defined the time required for synthesis of vital proteins needed for the entry into S-phase and/or DNA replication. The prolonged exposure of activated oocytes to CHX (10-12 h) negatively affected the pattern of DNA synthesis. The start of DNA synthesis was postponed and reduced pronuclear labeling was observed. In addition, CHX-treated oocytes often exhibited a characteristic punctate pattern of pronuclear labeling in which silver grains were accumulated into clusters. In conclusion, the present results provide knowledge about timing and a possible synchronization of DNA synthesis in parthenogenetically activated bovine oocytes.

Tripronuclear human zygotes: the first cell cycle and subsequent development.

About 6.6% of human oocytes fertilized in vitro were tripronuclear. The first cell cycle and developmental ability of these triploid zygotes were studied by autoradiographic analysis of pronuclear [3H]thymidine incorporation and by morphological observations of living zygotes at various times after insemination. Weak labelling over the pronuclei was first detected at 10 h after insemination and in many zygotes the pronuclei were heavily saturated with radioactive grains by 11-13 h. Thymidine incorporation was not apparent in the majority of tripronuclear zygotes by 21-22 h. At this time, 96% of living triploids (65/68) had visible pronuclei while the others entered the first mitosis and their pronuclei disappeared. By 25 h, 70% of triploid zygotes (37/58) still exhibited visible pronuclei and 26% were already in mitosis. The first divided embryos were seen by 25 h. The results suggest that the length of the complete cell cycle of human 1-cell stage triploids is approximately 25 h. Pronuclear DNA synthesis starts approximately 9-10 h after insemination and seems to last a minimum of 4 h (the precise time of S phase could not be determined). The minimal duration times of G2 phase and mitosis are estimated in the range of 4-6 h and 3.5-4 h, respectively. Tripronuclear zygotes were capable of substantial in-vitro development. About half of examined triploids attained at least the 4-6-cell stage of development (14/32; 44%); however, approximately 19% of these exhibited multinuclear cells. The remaining triploids had reached early morula (8 and 13 cells; 25 and 19%) or blastocyst (6%) stage.

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.

Paternal influence on timing of pronuclear DNA synthesis in naturally ovulated and fertilized mouse eggs.

Fertilized oocytes of the inbred genotypes AKR (AK), C57BL/6 (B6), DBA/2 (D2), and CBA (CB) and the hybrid genotypes F1 (female AK X male B6) and F1 (female B6 X male AK) were collected by flushing the oviducts of female mice every 2 h from 2 until 26 h post coitum. Developmental stages of the embryos and DNA content of the pronuclei were estimated by morphological criteria and cytofluorometric measurement of the pronuclei (ethidium bromide-stained DNA), respectively. In all genotypes, S-phase started about 4 h post conception (h.p.c.). The duration of S-phase amounted to 5.9 h (F1 [female B6 X male AK]), 6.4 h (AK), 8.5 h (B6), 9.4 h (F1 [female AK X male B6]), 9.8 h (D2), and 11.4 h (CB). In each of the reciprocal F1 hybrids, the length of S-phase differed from the maternal genotype (p less than 0.01) and resembled closely the paternal genotype (p greater than 0.25). Cleavage from one-cell stage to two-cell stage occurred between 16 and 21 h.p.c.

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.

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.

Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death

When previously mated female mice were exposed to inhaled ethylene oxide at the time of fertilization of their eggs or during early pronuclear stage of the zygote (before DNA synthesis), a high incidence of mortality among conceptuses and of congenital abnormalities among both the dead and the surviving fetuses was observed. The developmental stage at which death occured ranged from near the time of implantation to day 17 of gestation when examination of the uterine contents was performed. In comparison, midgestation and late fetal deaths were absent or minimal when the females were exposed either before mating or when conceptuses were in later zygotic stages (pronuclear DNA synthesis) or had reached the early two-cell stage. The random types of congenital abnormality observed and the remarkable stage-dependent sensitivity suggest a genetic basis for the response. The effects differ, both from genetic damages induced in premating germ cells, which lead only to death near the time of implantation, and from teratogenic damage, which leads to malformation only when exposure of embryos occurs during the period of major organogenesis.

Repair in fertilized eggs of mice and its role in the production of chromosomal aberrations.

The fertilized egg may influence the yield of dominant-lethal mutations produced from chemical treatment of male postmeiotic germ cells to a small or large extent depending upon the mutagen used and the competence of the egg to repair the premutational lesions induced. The strain of females has little influence on the yield of dominant-lethal mutations induced by triethylenemelamine or ethyl methane-sulfonate in spermatids and spermatozoa, but it has a large influence in the case of isopropyl methanesulfonate. In addition to this difference, triethylenemelamine and ethyl methanesulfonate induce high levels of heritable translocations at these germ cell stages whereas isopropyl methanesulfonate is practically ineffective, even though doses of these chemicals produced comparable levels of dominant-lethal mutations. These differences between ethyl methanesulfonate and triethylenemelamine on one hand and isopropyl methanesulfonate on the other were hypothesized to be a function of the types of chromosomal lesions present at the time of repair activity and whether or not chromosomal aberrations were already fixed at the time of postfertilization pronuclear DNA synthesis.

RELATIVE RATES AT WHICH DOMINANT-LETHAL MUTATIONS AND HERITABLE TRANSLOCATIONS ARE INDUCED BY ALKYLATING CHEMICALS IN POSTMEIOTIC MALE GERM CELLS OF MICE

There is a close relationship between the rates at which dominant lethal mutations and heritable translocations are induced by ethyl methanesulfonate (EMS) or triethylenemelamine (TEM) in male postmeiotic germ cells. This relationship does not hold for isopropyl methanesulfonate (IMS), which induced only negligible frequencies of heritable translocations at doses that induced high levels of dominant lethal mutations. Nor does IMS behave like EMS and TEM in the degree to which eggs of different stocks of females repair premutational lesions that are carried in the sperm-large differences between stocks for IMS treatment and small differences for EMS or TEM treatment. These dissimilarities between IMS and the other two alkylating chemicals are postulated to be attributable to differences in the types of lesions present at the time of repair activity and to whether or not chromosomal aberrations are already fixed prior to postfertilization pronuclear DNA synthesis.

ANALYSIS OF THE ALBINO-LOCUS REGION OF THE MOUSE. II. MOSAIC MUTANTS

Among 119 mutations involving the c locus that were recovered in the course of mouse specific-locus experiments with external radiations, 16 were found in mosaic, or fractional, mutants. The number of additional c-locus fractionals that could have occurred in these experiments and, for a variety of reasons, might not have been clearly identified, probably does not exceed the present number.-There was no evidence for radiation induction of the fractionals, and even those occurring in the irradiated groups may thus be assumed to be of spontaneous origin. Since only two mutations in the control groups were found in whole-body mutants, it appears that the bulk of spontaneous c-locus mutations are fractionals.-None of the mutations recovered in fractional mutants was homozygous lethal; 25% were viable intermediate alleles, and the remainder were albino-like mutants, all viable except for one subvital and one not tested.-Genetic tests of the fractionals indicated no major selection against the new mutations, either gametically or in the progeny.-For the group of fractionals as a whole, about one-half of the germinal tissue carried the mutation, indicating that the fractionals came from an overall blastomere population that was one-half mutant. Such a population could result from mutation in one strand of the gamete DNA, in a daughter chromosome derived from pronuclear DNA synthesis of the zygote, or in one of the first two blastomeres prior to replication. Since the mouse embryo does not stem from all of the cleavage products of the zygote, the frequency of fractionals observed underestimates the frequency of mutational events that result in two types of blastomeres.

Adenylation and ADP-ribosylation in the mouse 1-cell embryo

The incorporation of [3H]AAadenosine into cold trichloroacetic acid (TCA) insoluble material by the mouse 1-cell embryo has been studied. Incorporation of label was high immediately after fertilization, then decreased over the next 7 h with the sharpest decline occurring 3-5 h after fertilization. A small maximum was observed at the time of pronuclear DNA synthesis. Actinomycin D at a concentration which inhibited the cleavage of 1-cell embryos by 50% had little effect on this incorporation, which in the period 1-6 h post-fertilization was shown by autoradiography to be confined to the ooplasm of the newly fertilized ovum. [3H]Adenosine and poly ([3H]A) were released from embryo RNA labelled 1-3 h after fertilization with [3H]adenosine by digestion with a mixture of ribonucleases A and T1. The poly ([3H]A) segments were hydrolysed by alkali to 3'-[3H]AMP and [3H]adenosine ([3H]AMP/[3H]adenosine = 5/1), and by snake venom phosphodiesterase to 5'-[3H]AMP but very little [3H]adenosine. These results suggest that adenylation of RNA occurs soon after fertilization, that this is a cytoplasmic event, and that most of the newly synthesized poly ([3H]A) segments are joined to pre-existing poly (A) tracts. The unusual polynucleotide, poly (ADP-ribose), identified by its resistance to alkali and the release of 2'-(5''-phosphoribosyl)-5'[3H]AMP on incubation with snake venom phosphodiesterase, was also found in the ribonuclease digest.

Mammalian ova and one-cell embryos do not incorporate phosphate into nucleic acids.

The incorporation of [32P]Phosphate by unfertilized mouse ova and 1-cell embryos has been studied. [32P]Phosphate was incorporated into material insoluble in cold trichloroacetic acid by unfertilized ova throughout the period 1–6 h after ovulation with the lowest level of incorporation occurring 2–4 h after ovulation. Incorporation of label by 1-cell embryos was highest soon after fertilization but rapidly fell to a minimum within 2 h before rising to a second maximum at about the time of commencement of pronuclear DNA synthesis. A variety of extraction procedures failed to liberate labeled RNA from ova or embryo lysates and less than 10% of the incorporated label was soluble in chloroform/methanol. The 32P-labeled material precipitated from embryo lysates by ethanol/sodium acetate, pH 5 was resistant to snake venom phosphodiesterase before or after digestion with pronase. However, 32P-labeled material insoluble in cold trichloroacetic acid was hydrolysed by hot alkali to 32Pi and by 6 M HCl to 32P-labeled serine and threonine phosphates and 32Pi thus showing that [32]phosphate was incorporated solely into protein. Paper electrophoresis and thin-layer chromatography showed that 32P-labeled diphosphates and triphosphates but not monophosphates were present in the 32P-labeled soluble fraction from embryo lysates. 32P-labeled nucleoside monophosphates were also absent from the snake venom phosphodiesterase digest of the labeled soluble fraction but 32Pi and 32PPi were products of hydrolysis. Those results show that the mouse ovum does not contain a store of nucleosides, that pathways for mononucleotide synthesis are not functional in the 1-cell embryo, and suggest that the ovum contains a large endogenous store of nucleotides.

Timing of sperm penetration, pronuclear formation, pronuclear DNA synthesis, and first cleavage in naturally ovulated mouse eggs.

Timing of development of naturally ovulated mouse eggs from sperm penetration to first cleavage, including that of DNA synthesis, was established. In an attempt to limit variability, partial synchronization of ovulation was accomplished by shortening the length of the dark period to five hours, and partial synchronization of sperm entry was attempted by mating the females soon after the ovulated eggs reached the ampulla and by limiting the period at which mating could occur to only 20 minutes. Evidence of sperm penetration (presence of one or more sperm in perivitelline space or inside the vitellus) was found beginning 1.75 hours after the end of the mating period. Pronuclei were formed three to four hours after sperm entry. Pronuclear DNA, synthesis began about eight hours postmating, 3.25 to 4.5 hours after pronuclear formation, or about 6.25 to 8.5 hours after sperm entry; it was completed in almost all zygotes by 16 hours postmating. The first completed cleavage division was found 17 to 18 hours postmating, and almost all eggs had cleaved by 20 hours.

DELAYED FORMATION OF CHROMOSOME ABERRATIONS IN MOUSE PACHYTENE SPERMATOCYTES TREATED WITH TRIETHYLENEMELAMINE (TEM)

Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis-metaphase I stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis-metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed--a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis.

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.

Duplication of DNA during the first cell cycle in the mouse embryo.

Institutes of Histology and General Embryology of the University of Rome and of the School of Medicine of L'Aquila, Italy (Received 5th August 1974) Microdensitometric studies of DNA content in Feulgen-stained mouse zygotes (Alfert, 1950) have shown that the pronuclei duplicate their DNA before the prophase of the first cleavage division. The earliest incorporation of labelled adenine into DNA was autoradiographically detected by Sirlin & Edwards (1959) 13 hr after ovulation; according to Mintz (1965), DNA synthesis takes place by the end of the first third of the cycle. In the present study, we have investigated the timing of DNA duplication in the mouse zygote. While we were analysing our data, Luthardt & Donahue (1973) published an extensive work on pronuclear DNA synthesis in the mouse. Their findings will be discussed. Virgin Swiss female mice (CD-I COBS) purchased from Charles River Italia were kept in a room with controlled temperature

Pronuclear DNA synthesis in mouse eggs: An autoradiographic study

A total of 4738 eggs, collected at 2 h intervals, were obtained from mated CF1 (Carworth) female mice between 15 and 31 h following human chorionic gonadotropin (HCG) administration. Eggs pooled from several females were exposed to 3H-thymidine in vitro for 1 h. Pronuclear DNA synthesis began in some eggs as early as 21 h after HCG, approx. 5 h after pronuclear formation, and was completed in all zygotes by 31 h. The maximum number of eggs with labeled pronuclei occurred at 25 h. Some cumulus cells and second polar bodies were also labeled. Pronuclear incorporation of 3H-thymidine indicates DNA synthesis since no labeled pronuclei occurred following exposure to deoxyribonuclease, and labeled first cleavage metaphase chromosomes were obtained when pronuclear eggs were pulsed with 3H-thymidine during the S period. In each zygote the S period is about 4 h with DNA synthesis beginning and terminating slightly earlier in the male pronucleus. Cytoplasmic factors and not pronuclear volume per se are implicated in controlling the onset of pronuclear DNA synthesis. Pronuclear labeling was not affected by removal of cumulus cells or zona pellucida. When 3H-thymidine was injected into the Fallopian tube, the timing and frequency of labeled pronuclei corroborated the findings obtained in vitro.