Zygotic Transition Research Articles

Chromatin signature of embryonic pluripotency is established during genome activation

After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing lysine trimethylation modifications before and after the maternal-zygotic transition in zebrafish. Histone H3 lysine 27 trimethylation (H3K27me3), which is repressive, and H3K4me3, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by H3K4me3, including many inactive developmental regulatory genes that were also marked by H3K27me3. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed. Furthermore, we found many inactive genes that were uniquely marked by H3K4me3. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published data sets revealed similar monovalent domains in embryonic stem cells. Moreover, H3K4me3 marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA polymerase II, as indicated by the analysis of an inducible transgene. These results indicate that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal-zygotic transition.

Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes

BackgroundThe maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system.ResultsTwo novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start codon shows promoter activity at least as early as 3 hours in the developing Ae. aegypti embryo. The AaKLC2.1 promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition.ConclusionsTranscriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

Imprinting: RNA expression for homocysteine recycling in the human oocyte

To investigate whether homocysteine, a well known inhibitor of methylation, which is produced after imprinting and other methylation processes, can be recycled to methionine in the oocyte, at least until the stage of maternal to zygotic transition (i.e., four- to eight-cell stage); before this stage, most of the biochemical processes are carried out with the use of maternal stores of protein and mRNA. A first approach using microarrays and then reverse-transcription polymerase chain reaction (RT-PCR) for methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase [MTR]), betaine-homocysteine methyltransferase (BHMT), and cystathionine-beta synthase (CBS). Two private hospitals. Patients involved in IVF/ICSI procedures. Germinal vesicle oocytes collected at the time of oocyte retrieval, RNA extraction amplification, RT-PCR, microarrays. mRNA expression of all the enzymes involved in the chain of methylation and recycling of homocysteine to methionine. All of the enzymes required for methylation are present in the oocyte. Homocysteine can be recycled with BHMT and MTR. The human oocyte is able to regulate its Hcy level via remethylation using MTR and BHMT but not CBS. This aspect is important, because recent studies have shown that controlled ovarian hyperstimulation affects the homocysteine concentration in follicular fluid. This may regulate, at least in part, the risk of imprinting problems during IVF procedures.

Temporal Reciprocity of miRNAs and Their Targets during the Maternal-to-Zygotic Transition in Drosophila

During oogenesis, female animals load their eggs with messenger RNAs (mRNAs) that will be translated to produce new proteins in the developing embryo. Some of these maternally provided mRNAs are stable and continue to contribute to development long after the onset of transcription of the embryonic (zygotic) genome. However, a subset of maternal mRNAs are degraded during the transition from purely maternal to mixed maternal-zygotic gene expression. In Drosophila, two independent RNA degradation pathways are used to promote turnover of maternal transcripts during the maternal-to-zygotic transition [1]. The first is driven by maternally encoded factors, including SMAUG [2], whereas the second is activated about 2 hr after fertilization, coinciding with the onset of zygotic transcription. Here, we report that a cluster of zygotically expressed microRNAs (miRNAs) targets maternal mRNAs for turnover, as part of the zygotic degradation pathway. miRNAs are small noncoding RNAs that silence gene expression by repressing translation of their target mRNAs and by promoting mRNA turnover. Intriguingly, use of miRNAs to promote mRNA turnover during the maternal-to-zygotic transition appears to be a conserved phenomenon because a comparable role was reported for miR-430 in zebrafish [3]. The finding that unrelated miRNAs regulate the maternal to zygotic transition in different animals suggests convergent evolution.

The Maternal to Zygotic Transition in Animals and Plants

In the animal kingdom, maternal control of early development is a common feature. The onset of zygotic control over early development, defined as the maternal to zygotic transition (MZT), follows fertilization with a delay of a variable number of cell divisions, depending on the species. The MZT has been well defined in animals, but investigations remain in their infancy in plants. Recent evidence suggests, however, that in plants as in animals, the MZT also occurs several division cycles after fertilization. The likely convergent evolution of the MZT in the animal and plant kingdoms is fascinating and raises major questions regarding its biological significance, particularly with regard to its importance in genome reprogramming and the acquisition of totipotency by the embryo.

Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.

The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5–3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.

Nuclear reprogramming: the zygotic transcription program is established through an “erase-and-rebuild” strategy

Oocytes display a maternal-specific gene expression profile, which is switched to a zygotic profile when a haploid set of chromatin is passed on to the fertilized egg that develops into an embryo. The mechanism underlying this transcription reprogramming is currently unknown. Here we demonstrate that by the time when transcription is shut down in germinal vesicle oocytes, a range of general transcription factors and transcriptional regulators are dissociated from the chromatin. The global dissociation of chromatin factors (CFs) disrupts physical contacts between the chromatin and CFs and leads to erasure of the maternal transcription program at the functional level. Critical transcription factors and regulators remain separated from chromatin for a prolonged period, and become re-associated with chromatin shortly after pronuclear formation. This is followed temporally by the re-establishment of nuclear functions such as DNA replication and transcription. We propose that the maternal transcription program is erased during oogenesis to generate a relatively naïve chromatin and the zygotic transcription program is rebuilt de novo after fertilization. This process is termed as the "erase-and-rebuild" process, which is used to reset the transcription program, and most likely other nuclear processes as well, from a maternal one to that of the embryo. We further show in the accompanying paper (Gao T, et al., Cell Res 2007; 17: 135-150.) that the same strategy is also employed to reprogram transcriptional profiles in somatic cell nuclear transfer and parthenogenesis, suggesting that this model is universally applicable to all forms of transcriptional reprogramming during early embryogenesis. Displacement of CFs from chromatin also offers an explanation for the phenomenon of transcription silence during the maternal to zygotic transition.

Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro

BackgroundReal-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data.ResultsQuantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly.ConclusionCompared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies.

The effect of glucosamine concentration on the development and sex ratio of bovine embryos

Glucosamine is a component of hyaluronic acid and an alternative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP- N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5 mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal–zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-α- d-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT.

Interspecies nuclear transfer of Tibetan antelope using caprine oocyte as recipient

Interspecies nuclear transfer is an invalulable tool for studying nucleus-cytoplasm interactions; and at the same time, it provides a possible alternative to clone endangered animals whose oocytes are difficult to obtain. In the present study, we investigated the possibility of cloning Tibetan antelope embryos using abattoir-derived caprine oocytes as recipients. Effects of culture conditions, enucleation timing, and donor cell passages on the in vitro development of Tibetan antelope-goat cloned embryos were studied. Maternal to zygotic transition timing of interspecies Tibetan antelope embryos was also investigated using two types of cloned embryos, Tibetan antelope-rabbit and Tibetan antelope-goat embryos. Our results indicate that: (1) goat oocyte is able to reprogram somatic cells of different genus and supports development to blastocyst in vitro. (2) Coculture system supported the development of Tibetan antelope-goat embryos to blastocyst rate stage (4.0%), while CR1aa alone did not. (3) When MII phase enucleated caprine cytoplast and TII phase enucleated caprine cytoplast were used as recipients, the fusion rate and blastocyst rate of hybrid embryos were not statistically different (73.9% vs. 67.4%; 4.0% vs. 1.1%). (4) When donor cells at 3-8 passages were used, 2.9% hybrid embryos developed to blastocysts, while none developed to blastocysts when cells at 10-17 passages were used. (5) There may be a morula-to-blastocyst block for Tibetan antelope-goat, while there may be an 8- to 16-cell block for Tibetan antelope-rabbit embryos.

Role of oocyte-specific genes in the development of mammalian embryos.

Studies on oocyte-specific genes are important in understanding the genetic pathways essential for folliculogenesis, oogenesis and early embryogenesis. Although the molecular mechanisms regulating oocyte growth and embryo development in mammals have partially been unraveled by gene knockout studies, many aspects concerning reproduction remain to be determined. Development of mammalian embryos starts with the fusion of sperm and egg. After fertilization, the first major developmental transition, maternal to zygotic transition, occurs at the specific stages of preimplantation development in each mammal. The transition is called zygotic gene activation (ZGA) or embryonic genome activation. The ZGA is one of the most important events that occur during preimplantation development; however, the mechanism of the event remains unknown. Because the development until the transition is maintained by maternally inherited proteins and transcripts stored in the oocytes, it is highly likely that these products play an important role in the initiation of ZGA. Several maternal-effects genes that are specifically expressed in oocytes have been identified and their involvement in preimplantation development has been revealed. Therefore, to study oocyte-specific gene regulation would help not only to understand the precise mechanisms of mammalian development, but also to show the mechanisms of reproductive disorders, such as premature ovarian failure and infertility. (Reprod Med Biol 2006; 5: 175-182).

Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment

Paternal effect on embryonic development occurs as early as fertilization. Incorrect formation of the spermatozoon due to centrosome defects and abnormal concentrations of any components involved in the activation process lead to failure immediately or in the subsequent cell cycles. Sperm chromosomal abnormalities result in early embryo developmental arrests. Generally poor spermatozoa lead to poor blastocyst formation. Sperm DNA fragmentation may impair even late post-implantation development. The DNA repair capacity of the oocytes is of major importance. Early preimplantation development, i.e. until maternal to zygotic transition, is maternally driven. Maternal mRNAs and proteins are of major importance, as there is an unavoidable turnover of these reserves. Polyadenylation of these mRNAs is precisely controlled, in order to avoid too early or too late transcription and translation of the housekeeping genes. An important set of maternal regulations, such as DNA stability, transcriptional regulation and protection against oxidative stress, are impaired by age. The embryo biochemical endogenous pool is very important and may depend upon the environment, i.e. the culture medium. Paternal, maternal and environmental factors are unavoidable parameters; they become evident when age impairs oocyte quality.

Transcription profile during maternal to zygotic transition in the mouse embryo

To gain insight into early embryo development, we used microarray technology to compare gene expression profiles in metaphase II oocytes and one- (1C), two- (2C) and four-cell (4C) embryos. Differences in spot intensities were normalised and grouped using Avadis Prophetic software platform (version 3.3; Strand Genomics, Bangalore, India) and categories were based on the PANTHER and Gene Ontology classification systems. We examined 6927 genes and identified those preferentially expressed in 1C or 2C embryos. We found 1261 genes that were more highly expressed (P < 0.05) in 1C compared with 2C embryos and 1480 genes showing enhanced expression in 2C embryos. Similarly, we investigated 6581 genes present in 2C and 4C embryos and identified 841 that are expressed to a greater extent at the 2C stage and 905 that are more highly expressed at the 4C stage. Using PANTHER classification, genes that were upregulated and downregulated in 2C embryos compared with 1C and 4C embryos were grouped according to their protein functions, which included developmental processes, electron transport, lipid, fatty acid and steroid metabolism, nucleoside, nucleotide and nucleic acid metabolism, protein metabolism and modification, signal transduction and transport, among others. Real time reverse transcription-polymerase chain reaction was performed to confirm differential expression of 14 selected genes. The identification of the genes being expressed will provide insight into the complex gene regulatory networks affecting zygotic genome activation and further development.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &amp;lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &amp;lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

Mechanism of Translation in the Period of Oocyte to Zygote Transition in Mammals

In the mouse, maturing oocytes and zygotes until the late 1-cell stage are transcriptionally inert. The development of early preimplantation embryos, including reprogramming of differentiated germ cells into totipotent embryos, is regulated by the translation of mRNAs in oocytes preliminarily stored during oocytogenesis (maternal mRNAs). In the period of oocyte to zygote transition in mammals, the translational promotion or repression of maternal mRNAs does not start in unison. For example, a set of maternal mRNAs that are translationally quiescent in the growing stage become translated after the start of maturation and/or fertilization, but another group of maternal mRNAs that are actively translated in the growing stage become inactivated during maturation. This selective and temporal translational profile of maternal mRNAs seems to be regulated by RNA-binding proteins that bind to maternal mRNAs and decide the timing of their entry into the ribosome and by special short sequences in maternal mRNAs that recognize specific RNA-binding proteins. In this review, we focus on the mechanisms that modulate the profile of post-transcriptional regulation in the period of oocyte to zygote transition in mammals.

Comparison of gene expression in individual preimplantation bovine embryos produced by in vitro fertilisation or somatic cell nuclear transfer

In vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) have been implicated in a variety of developmental abnormalities. Aberrant gene expression is likely to account for much of the diminished viability and developmental abnormalities observed. In the present study, the expression of multiple genes in IVF and SCNT bovine blastocyst-stage embryos were evaluated and compared with in vivo-produced embryos. Eleven genes expressed at and following maternal-zygotic transcription transition were evaluated in individual blastocysts by real-time polymerase chain reaction following RNA amplification. A subset of those genes was also evaluated in individual IVF and SCNT eight-cell embryos. A fibroblast-specific gene, expressed by nuclear donor cells, was also evaluated in IVF and SCNT embryos. The observed gene expression pattern at the eight-cell stage was not different between IVF and SCNT embryos (P > 0.05). In vitro fertilisation and SCNT blastocyst expression was lower (P < 0.01) for all genes compared with their in vivo-produced counterparts, except for lactate dehydrogenase isoenzyme A (P < 0.001). The patterns of gene expression of the IVF and SCNT blastocysts were indistinguishable. Neither SCNT eight-cell nor blastocyst-stage embryos expressed the gene used as a fibroblast marker (collagen VIalpha1). For the genes evaluated, the level of expression was influenced more by the environment than by the method used to produce the embryos. These results support the notion that if developmental differences observed in IVF- and SCNT-produced fetuses and neonates are the result of aberrant gene expression during the preimplantation stage, those differences in expression are subtle.

292. Influence of the in vitro environment on rat gametes and pre-implantation embryos

Rat in vitro fertilization (IVF) and culture (IVC) is attempted by few because of its reputation for difficulty. Currently very few functional rat in vitro systems (IVS) exist for sperm–oocyte interaction research. Successful fertilization of rat metaphase II (MII) oocytes was achieved with two different media, Enriched Krebs Ringer Bicarbonate (EKRB) (70.2%) and M16 (57.4%). Using this IVS we have shown that the rat germinal vesicle-intact (GV-i) oocyte lacks the necessary maturity to interact with capacitated caudal epididymal spermatozoa, whether zona pellucida intact (ZP-i) or free (ZP-f). Proteomic analysis of the protein profile of the oolemma from the GV-i stage through to the MII stage in oocytes is being conducted to characterize any maturational changes that may occur. In addition we provide initial evidence to suggest that an acrosome-intact spermatozoa can fuse with the oolemma of a ZP-f MII oocyte during IVF. Although high percentages of polyspermic embryos in ZP-f IVF (64.8–100%) were observed, the possibility that the rat oolemma may undergo a post-fusion block to polyspermy was implied by a small proportion of normally fertilized embryos (3.8–17.0%) in M16 supplemented with different ratios of hyperactived spermatozoa. Despite successful culture to the blastocyst stage for in vivo fertilized zygotes (33.73%) and 2-cell stage embryos (79.3%), IVF embryos have repeatedly failed to develop in culture. Two dimensional analyses of the protein profile of oocytes/embryos immediately prior to fertilization (MII oocyte–101 spots) and the maternal to zygotic transition (MZT) (zygotes–59 spots and 2-cell embryos–84 spots) has shown a difference in patterns of protein expressed. Comparison of IVF zygotes (41 spots) obtained from EKRB displayed reduced protein expression suggesting that nuclear maturation and/or MZT is not being adequately supported. These data illustrate that rat IVF and IVC require suitable media if its problematic reputation is finally to be shed.

Embryogenomics of pre-implantation mammalian development: current status.

Pre-implantation development is marked by many critical molecular events, including the maternal to zygotic transition and the first differentiation of cells. Understanding such events is important, for both basic reproductive biology and practical applications, including regenerative medicine and livestock production. Scarcity of materials has hampered the progress of the field, but systematic genomics approaches are beginning to be applied to the study of pre-implantation development, resulting in unprecedented amounts of data about the pre-implantation process. The first step in embryogenomics is to collect and sequence cDNAs (expressed sequence tags (ESTs)) for genes that are expressed and function in these early embryos. Mouse work is the most advanced, with 140 111 ESTs derived from all stages of pre-implantation development currently available in the public sequence database. For other mammals, at present only approximately 1000 ESTs can be found in the public database, but efforts by several groups are generating cDNA libraries and ESTs. In the present review, the current status of the implementation of these investigative tools for mammalian pre-implantation embryos is discussed.

Embryogenomics of pre-implantation mammalian development: current status

Pre-implantation development is marked by many critical molecular events, including the maternal to zygotic transition and the first differentiation of cells. Understanding such events is important, for both basic reproductive biology and practical applications, including regenerative medicine and livestock production. Scarcity of materials has hampered the progress of the field, but systematic genomics approaches are beginning to be applied to the study of pre-implantation development, resulting in unprecedented amounts of data about the pre-implantation process. The first step in embryogenomics is to collect and sequence cDNAs (expressed sequence tags (ESTs)) for genes that are expressed and function in these early embryos. Mouse work is the most advanced, with 140111 ESTs derived from all stages of pre-implantation development currently available in the public sequence database. For other mammals, at present only approximately 1000 ESTs can be found in the public database, but efforts by several groups are generating cDNA libraries and ESTs. In the present review, the current status of the implementation of these investigative tools for mammalian pre-implantation embryos is discussed.