Information on the dynamics of gamete interaction in marsupials is very limited and not available for any species from the major Australian Order Diprotodontia which includes most of the more familiar animals such as kangaroos, possums and the koala. This study addressed this deficiency by examining the ultrastructure of in vivo fertilised eggs from common brushtail possums (Trichosurus vulpecula). Females were superovulated by treatment with 15 IU PMSG and then 4 mg porcine LH 3 days later, and inseminations were performed 910-13 h after LH) using epididymal spermatozoa. Between 33 and 39 h after LH injection females were killed, reproductive tracts excised and the oviduct ampulla segment flushed for eggs. Three of the six eggs examined were fertilised as judged by the presence of sperm remnants in the cytoplasm. On the basis of these eggs it was found that sperm penetration left a large hole in the zona pellucida (ZP), suggesting that sperm zona penetration occurs primarily by the enzymatic action of acrosomal enzymes. Sperm lying within the perivitelline space were lacking both an outer acrosomal membrane and the associated acrosomal contents, while both these structures were found on sperm embedded within the mucoid layer, which is consistent with induction of the acrosome reaction by binding to the ZP. Once inside the egg cytoplasm, the sperm head travelled only a short distance before chromatin decondensation occurred. Fertilised eggs showed signs of cytoplasmic activation including cytoskeleton association with apparently dividing mitochondria and prominent rough endoplasmic reticulum. Unfertilised eggs appeared to be undergoing degenerative changes and lacked any evidence of activation. This study has demonstrated that superovulation and laparoscopic intravaginal artificial insemination provide a system through which perifertilisation events in the possum and other monovular Australian marsupials can be examined experimentally.

The low involvement of glucose metabolism in early preimplantation embryos has suggested the presence of metabolic blocks in the glycolytic pathway. Genetic expression of hexokinase (HK), glucose-6-phosphate isomerase (GPI) or phosphofructokinase (PFK) were qualitatively analysed using the reverse transcription nested polymerase chain reaction (RT-nested PCR) in individual human immature oocytes at the germinal vesicle (GV) stage and in single non-fertilised human metaphase II (MII) oocytes, after IVF or ICSI failures. Transcripts encoding for HK, GPI and PFK were detectable in the majority of the oocytes analysed, and with different expression patterns. GPI transcripts were consistently present and appear to be constitutively expressed during GV and MII stages. In contrast, low quantities of transcripts encoding for HK and PFK were observed in all oocytes analysed. Our data revealed that the metabolic block previously reported at GPI or PFK levels underwent post-transcriptional regulation. The expression profiles of HK and PFK transcripts in GV and MII reflect a low level of transcription or active translation of maternal transcripts during oocyte maturation. Nevertheless, enzymatic activities of HK and PFK have previously been determined in human oocytes. This suggests that HK and PFK may be accumulated in protein (enzyme) form instead of maternal mRNA during human oocyte maturation. The expression pathway of glycolytic metabolism reflects the presence of different mechanisms involved in gene expression/regulation at the transcriptional and translational level and their accumulation during human oocyte maturation.

This study examined the event of protein phosphorylation in bovine oocytes in response to sperm penetration and parthenogenetic activation. In vitro matured oocytes were labelled with [32P]orthophosphate at 3 h intervals from 3 h to 18 h or from 0 h to 12 h following in vitro fertilisation and parthenogenetic activation, respectively. The level of protein dephosphorylation, at approximately 43 kDa, was similar in fertilised and parthenogenetically activated bovine oocytes. However, the level of protein phosphorylation at 40 kDa, 23 kDa and 18 kDa was different between these two samples. There were no such changes of protein phosphorylation and dephosphorylation in the control oocytes. Further, by two-dimensional gel electrophoresis there is a difference in the level of protein phosphorylation at 18 kDa between the fertilised and activated oocytes. These results suggest that this protein phosphorylation may be related to the formation of the male pronucleus in bovine oocytes.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24–26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18–20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.

The zona pellucida (ZP) is an extracellular glycoprotein coat that is deposited around the oocyte during folliculogenesis and performs several functions that relate to fertilisation and preimplantion development. In eutherian mammals it consists of three major glycoproteins – ZPA, ZPB, and ZPC – but little is known about its molecular constitution in marsupials. We have isolated the cDNA encoding the ZPA homologue in two distantly related marsupial series: the possum, Trichosurus vulpecula (a phalangerid) and the dunnart Sminthopsis crassicaudata (a dasyurid). The two cDNA sequences were 86% identical and showed extensive regions of homology to eutherian ZPA proteins, particularly in the central region of the molecule. Many other features of the ZPA protein, except the positioning of the N-linked glycosylation sites, were also conserved between marsupials and eutherians. ZPA expression was shown to occur maximally in the cytoplasm of the oocyte primary follicles with a little, but significant, expression in oocytes of both primordial follicles and in the cytoplasm of the oocyte in follicles with an antral cavity. No expression was seen in surrounding follicle or granulosa cells

Summary

The present study was undertaken to investigate whether a time lag in sperm nuclear decondensation and male pronuclear formation in the course of development of eggs is associated with any occurrence of structural chromosome aberrations in male genomes of hybrid zygotes between Chinese hamster spermatozoa and zona-free Syrian hamster oocytes. Shortly after insemination, hybrid zygotes were treated with dithiothreitol (DTT) at different concentrations (0.1–10.0 mM) for 30 min to reduce protamine disulphide (S–S) bonds and thereby accelerate sperm nuclear decondensation and male pronuclear formation. The incidence of sperm nuclear decondensation and male pronuclear formation increased with increasing DTT concentrations, indicating that a reduction in S–S bonds effectively induces these cytological events. Chromosomes of male genomes in hybrid zygotes generated by treatment with 1.0 mM, 2.5 mM and 10.0 mM DTT were analysed at the first cleavage metaphase. Incidence of structural chromosome aberrations in each treatment was 34.5%, 27.1% and 24.7%, respectively. There was a significant difference between the incidences with 1.0 mM and 10.0 mM DTT treatment. As the time lag in nuclear decondensation and male pronuclear formation was greatest in the 1.0 mM treatment condition, followed in order by 2.5 mM and 10.0 mM, it is suggested that the lag in sperm nuclear development behind egg development is responsible for structural chromosome aberrations in male genomes of hybrid zygotes.

Acrosin, an acrosomal serine protease, has been associated with binding of spermatozoa and their penetration through the zona pellucida. This study was aimed at determining whether the remaining proacrosin/acrosin system on rabbit perivitelline spermatozoa still has proteolytic activity and whether this activity is involved in further penetration of unfertilised rabbit eggs. Eight hundred and sixty-five rabbit perivitelline spermatozoa were evaluated by the gelatin-substrate film technique for the detection of acrosin on individual spermatozoan. Fifteen per cent of the studied spermatozoa showed small digestion halos on the gelatin film. The proteolytic activity of rabbit perivitelline spermatozoa was inhibited in the presence of 1 mg/ml of soybean trypsin inhibitor (SBTI) or with 20 μg/ml of a mixture of the monoclonal anti-proacrosin/acrosin antibody. In vitro fertilisation occurred in 21.8% of rabbit oocytes co-incubated with perivitelline spermatozoa and was completely inhibited when oocytes were incubated with 600 μg/ml of a mixture of three anti-acrosin monoclonal antibodies (ACRO-A8C10, ACRO-C2B10 and ACRO-C5F10). Inseminations in the presence of anti-cholera monoclonal antibody (irrelevant to spermatozoa) resulted in 17.6% fertilisation. These results support the idea that the residual proacrosin/acrosin system in perivitelline spermatozoa might be involved in spermatozoal binding and/or second penetration through the zona pellucida.

Accumulation of total RNA and poly(A+)RNA was determined in the oocytes of Japanese quail (Coturnix coturnix japonica) during oogenesis, by a standard spectrophotometric method, after RNA extraction. Intensive RNA accumulation was observed in the oocytes 0.25–2.0 mm in diameter (correlated with the presence of lampbrush chromosomes), followed by a plateau in 2.0–3.0 mm oocytes (correlated with the condensation of lampbrush chromosomes). Resumption of the RNA accumulation in the 3.0–5.0 mm oocytes is interpreted as a result of external RNA delivery by the granulosa cells. Most of the RNA (~3/4, 3–4 μg) contained in the ovulated oocyte was found under the vitelline membrane surrounding the yolk. This RNA has been designated as ‘extraembryonic RNA’, as it is located outside the germinal disc region where the embryo is formed. The extraembryonic RNA is rapidly degraded within 24 h, from ovulation until oviposition, during egg passage through the oviduct, while the RNA present in a germinal disc (~1.1 μg) is stable. In bird oocytes the presence of two, territorially separated pools of RNA has been postulated: one contained in a germinal disc and needed for early embryo development, and the second present in the cytoplasmic layer around the yolk, supporting oocyte growth and development during oogenesis.

Over the past two decades advances in reproductive biology and medicine have made important strides in alleviating many conditions of sub-sterility in the human. It is the general consensus that gametes and embryos from other mammalian species are not good models for the study of human embryology and therefore assisted reproductive technologies have also been fundamental and opportune in providing material for the systematic study of human embryology. A global increase in human reproductive incompetence due to environmental stress make such studies essential.

Sperm motility in amphibians is thought to be initiated by a decrease in environmental osmolarity. However, fertilisation in the newt, Cynops pyrrhogaster, is achieved in an environment without osmotic change. We show here that sperm motility initiating activity is present in jelly layer extract (JE). JE was gel-filtrated and a single peak with sperm motility initiating activity was detected in the fraction corresponding to about 50 kDa. The activity was strengthened by heat treatment of JE at 100 °C for 30 min. This suggests that JE includes the inactive form of sperm motility inducing substance (SMIS) in addition to active substance. Thus JE was fractionated before and after the heat treatment. When JE was fractionated first and then each fraction was heated, the activity was detected in the fraction both above 500 kDa and below 500 kDa. When heat-treated JE was fractionated, the activity was detected only in the fraction below 500 kDa. These results suggest that JE includes the inactive form of SMIS of more than 500 kDa in molecular weight. A regulatory mechanism for the initiation of sperm motility in C. pyrrhogaster is proposed according to the results of the present study.

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 × DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4–5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 ± 0.16) and testicular spermatozoa (4.03 ± 0.34), and then decreased sharply (p < 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 ± 0.03). However, the ratio was not different among corpus (0.69 ± 0.01), cauda epididymis (0.68 ± 0.11) and ejaculated spermatozoa (0.63 ± 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p < 0.01) during passage of spermatozoa from testis (4.74 ± 0.16) through epididymis (caput, 2.72 ± 0.08; corpus, 1.07 ± 0.03; cauda, −0.05 ± 0.05; ejaculated, 0.08 ± 0.03). The acridine orange red/green fluorescence ratio increased (p < 0.01) during zona penetration (binding sperm, 0.52 ± 0.09; perivitelline sperm, 0.64 ± 0.16) and sperm decondensation (decondensed sperm, 0.69 ± 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a ‘sum of parts’ is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.

Controlled ovarian stimulation was induced in 19 women for in vitro fertilisation/embryo transfer. After ovum pick-up, haptoglobin titres were determined by ELISA in sera and homologous follicular fluids. The haptoglobin phenotype of each subject was assessed and the penetration of the protein forms through the blood-follicle barrier was predicted on the basis of their molecular weight. The penetration threshold compatible with the barrier integrity was calculated as 92%, 73% and 57% of the blood level of phenotypes Hpt 1-1, Hpt 1-2 and Hpt 2-2 respectively. Penetration values comparable/lower or higher than threshold were found associated with 46 of 49 and 3 of 49 fertilised oocytes, respectively. Complexes of haptoglobin with apolipoprotein A-1 were isolated from follicular fluids by affinity chromatography with haemoglobin. The haptoglobin β chain, after Western blotting and incubation with apolipoprotein A-1, was found to be involved in the protein–protein interaction as detected by anti-apolipoprotein A-1 antibodies. Complexes from separate fluids were analysed by electrophoresis and densitometry: the plain β chain/apolipoprotein A-1 stoichiometric ratio was 0.75 and 1.40 in fluids associated with fertilised and unfertilised oocytes respectively. The results suggest that haptoglobin transport in the follicle depends on the integrity of the blood–follicle barrier and might be associated with oocyte quality, possibly by interfering with the role of apoliprotein A-1 in cholesterol or vitamin E exchange between high-density lipoproteins and granulosa cells.

Fertilisation in ascidian oocytes triggers a plasma membrane current, the release of intracellular calcium and the degradation of Maturation Promoting Factor (MPF) activity leading to the completion of meiosis and the initiation of embryo development. We have previously shown that the fertilisation current in ascidians is produced through the metabolism of nicotinamide nucleotide (NN) metabolites to ADP ribose. In this study we have used nicotinamide to test whether NN metabolism plays additional roles in fertilisation in ascidians. Nicotinamide treatment blocked calcium-induced calcium release (CICR) and arrested the cell cycle prior to the completion of meiosis I. Nicotinamide further prevented the abolition of MPF activity after fertilisation. Interestingly, nicotinamide treatment caused ascidian oocytes to form interphase-like pronuclei after fertilisation, despite the high MPF activity. The data demonstrate that NN metabolism is involved in calcium signalling through CICR and further suggest that a NN metabolite acts as a messenger connecting MPF activity to the formation of the meiotic apparatus.

We have previously reported the occurrence and partial characterisation of an α-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25–30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 °C in 5% CO2 in air. The sperm–zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These results suggest that mannose-binding molecule(s) such as α-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.

Our previous study indicated that thimerosal is one of the most effective artificial activators to mimic sperm-induced increases in the intracellular free calcium concentration ([Ca2+]i) and other activation events in pig oocytes (Macháty et al., 1997). The present study was conducted to examine the temporal relationship between intracellular calcium transients, cortical granule (CG) exocytosis and the zona reaction induced by thimerosal. When pig oocytes matured in vitro were exposed to 200 μM thimerosal the first intracellular calcium transient, with a mean peak ratio of 4.97 ± 1.14, was observed 509.64 ± 122.03 s after addition of thimerosal. The density of CGs fell significantly from 63.3 ± 11.7 CGs/100 μm2 of cortex in control oocytes to 25.7 ± 19.2 CGs/100 μm2 of cortex (59.4% release) at 2 min after the first intracellular calcium transient. At 5 min after the calcium transient the residual CG density had been reduced to 10.7 ± 10.4 CGs/100 μm2 of cortex (83.1% release). This degree of CG exocytosis was the same as that in oocytes penetrated by sperm (9.5 ± 5.1 CGs/100 μm2 of cortex). No further decrease in residual CG density was observed at 10 min (10.3 ± 14.8 CGs/100 μm2 of cortex). Whereas 77.4% (120/155) of control oocytes were penetrated by spermatozoa only 1.4% (2/144) of thimerosal-treated oocytes were penetrated. Further experimental results obtained by in vitro fertilisation of oocytes with preincubated (capacitated) spermatozoa suggested that the zona block to sperm penetration in thimerosal-treated oocytes occurred within 35 min after CG exocytosis and 40 min after the first calcium transient. These results indicate that polyspermic penetration of pig oocytes inseminated in vitro is not due to delayed or incomplete CG exocytosis but more likely to a delayed zona reaction and/or simultaneous sperm penetration.

The objective of this study was to determine whether Calomys laucha and Calomys musculinus super-ovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6–8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.

During progesterone-induced nuclear maturation the oocytes of Bufo arenarum undergo a series of nuclear and cytoplasmic changes. The breakdown of heterocellular communications between the follicular cell projections and the oocyte microvilli, and the consequent enlargement of the perivitelline space, were observed at the animal pole. The more evident cytoplasmic feature during nuclear maturation comprised the gathering of glycogen granules in clusters, some phagocytosed by empty vesicles. With respect to the location of these vesicles, some were observed in close proximity to the oolemma and others were freely suspended in the perivitelline space, extruded from the oocyte. Other visible events were the disruption of the annulate lamellae, the formation of an elaborate cortical endoplasmic reticulum and the rearrangement of the cortical granules in a monolayer immediately beneath the oolemma together with aggregates of endoplasmic reticulum cisternae. Our results show that during nuclear maturation the nuclear oocyte changes include a flattening of the spherical oocyte nucleus, its migration towards the surface of the animal pole, the disappearance of the nucleoli and the dissolution of the nuclear envelope.

Temporal and spatial distribution of the sites of DNA replication were examined in 1-cell mouse embryos. Embryos were labelled with bromodeoxyuridine (BrdU) at hourly intervals after fertilisation, and the incorporation of BrdU was examined by laser-scanning confocal microscopy following immunostaining with an anti-BrdU antibody. DNA replication first started uniformly in both the male and female pronuclei in the intranuclear region and then was observed in the peripheral regions of nucleus and nucleolus. These changes, however, occurred asynchronously in that the female pronucleus required a longer time to complete replication in the intranuclear region but not in the peripheral regions. Inhibiting transcription with α-amanitin had no effect on the temporal and spatial patterns of DNA replication. Treatment of the embryos with trapoxin, a specific inhibitor of histone deacetylase, accelerated the completion of replication in the peripheral regions but not in the intranuclear region. These results suggest that DNA replication is temporally and spatially regulated in the 1-cell embryos and that acetylation of histones, but not transcription, is involved in the regulation of DNA replication.