Gene expression in bovine nuclear transfer embryos in relation to donor cell efficiency in producing live offspring.

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this regard, is the analysis of gene expression patterns in nuclear transfer (NT) embryos to dissect the processes that failed and develop means to overcome the limitations imposed by these factors. In this study, we investigated expression patterns of histone deacetylase-1, -2, -3 (HDAC-1, -2, -3), DNA methyltransferase-3a (DNMT3A), and octamer binding protein-4 gene (OCT4) in donor cells with different cloning efficiencies and NT embryos derived from these cells employing a real-time RT-PCR assay. All genes investigated followed altered expression patterns in NT embryos when compared to IVF-derived embryos. In general, expression of HDAC genes was elevated especially at the compact morula stage and comparable to in vitro fertilized (IVF) embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than IVF embryos at all stages. Oct-4 transcript levels were also reduced in cloned compared to IVF embryos at the compact morula and blastocyst stages. This difference disappeared at the hatched blastocyst stage. There was a donor cell effect on the expression patterns of all genes investigated. These results demonstrate altered gene expression patterns for certain genes, in cloned cattle embryos from our donor cells of different efficiency in producing live offspring. Therefore we suggest that differences in expression of developmentally important genes during early embryo development may characterize the efficiency of donor cells in producing live offspring.

Dynamics of global transcriptome in bovine matured oocytes and preimplantation embryos.

Global activation of the embryonic genome is the most critical event in early mammalian development. After fertilization, a rich supply of maternal proteins and RNAs support development whereas a number of zygotic and embryonic genes are expressed in a stage-specific manner leading to embryonic genome activation (EGA). However, the identities of embryonic genes expressed and the mechanism(s) of EGA are poorly defined in the bovine. Using the Affymetrix bovine-specific DNA microarray as the biggest available array at present, we analyzed gene expression at two key stages of bovine development, matured oocytes (MII) and 8-cell-stage embryos, constituting the ultimate reservoir for life and a stage during which EGA takes place, respectively. Key genes in regulation of transcription, chromatin-structure cell adhesion, and signal transduction were up-regulated at the 8-cell stage as compared with 8-cell embryos treated with alpha-amanitin and MII. Genes controlling DNA methylation and metabolism were up-regulated in MII. These changes in gene expression, related to transcriptional machinery, chromatin structure, and the other cellular functions occurring during several cleavage stages, are expected to result in a unique chromatin structure capable of maintaining totipotency during embryogenesis and leading to differentiation during postimplantation development. Dramatic reprogramming of gene expression at the onset of development also has implications for cell plasticity in somatic cell nuclear transfer, genomic imprinting, and cancer.

Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro.

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.

Expression patterns of histone deacetylases in bovine oocytes and early embryos, and the effect of their inhibition on embryo development.

Gene expression at the onset of bovine embryogenesis is developmentally regulated and histone deacetylases (HDACs) have been shown to play a key role in the control of gene expression during this period of development in other species. We determined expression pattern(s) of powerful repressors, namely histone deacetylase-1, -2 and -3, that may in part regulate gene expression during bovine oogenesis and early embryogenesis at the mRNA and protein levels. Detected fragments of the hdac genes were sequenced and comparison of the sequences showed very high homologies between DNA and amino acid sequences of bovine HDACs and those of human and mouse. RPD3, a yeast global regulator of transcription, was also detected in bovine oocytes and embryos. Results suggest that HDACs may be operative in regulation of zygotic/embryonic gene expression in cattle.

Pluripotency of bovine embryonic cell line derived from precompacting embryos.

We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.

Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species.

Early embryonic development is largely dependent on maternal RNAs and proteins synthesised during oogenesis. Zygotic transcription is an essential event that occurs at a species-specific time after fertilization. In the absence of zygotic transcription the embryo dies since it can no longer support requirements for successful embryo development. Molecular genetics of gene expression during early embryogenesis, especially in the bovine species, remain one of the unsolved questions in modern biology. Earlier studies suggested that embryonic transcription in cattle begins at the late 4-cell or 8-cell stage. However, more recent studies suggest that bovine zygotes and 2-cell embryos are both transcriptionally and translationally active. Moreover, changes in chromatin structure due to acetylation of core histones and DNA replication play important roles in the regulation of zygotic/embryonic gene expression. This review will summarise results of recent studies about the timing and mechanisms of zygotic/embryonic gene expression in cattle. In addition, terminology in the literature regarding gene expression during early embryogenesis will be clarified. These terminologies include: 'zygotic/embryonic gene expression', 'maternal to embryonic transition in control of development (MET)' and 'zygotic/embryonic genome activation (ZEGA)'.

A combination of EGF and IGF-I accelerates the progression of meiosis in bovine follicular oocytes in vitro and fetal calf serum neutralizes the acceleration effect.

The effects of a combination of EGF and IGF-I (GFs) on the progress of meiosis and on their developmental competence were examined in cumulus-enclosed bovine oocytes. Exposure to GFs in serum-free, 0.3% PVP-containing maturation medium significantly (P<0.05) increased the frequency of oocytes with the first polar body (PB) at 16 h of culture and decreased those with PB at 20 h. The cleavage rates of PB-extruded oocytes after fertilization were not affected by treatment of GFs during maturation culture, and blastocyst yield was not improved by GFs treatment. Although replacement of PVP from GFs-containing medium with fatty acid-free BSA did not affect the timing of PB extrusion, replacement with 10% FCS neutralized the acceleration effects of GFs. Replacement for macromolecule in maturation medium did not improve blastocyst yield of PB-extruded oocytes after fertilization. These results indicate that the progression of meiosis in bovine oocytes with cumulus cells is accelerated by exposure to GFs in serum-free maturation medium but their developmental competence is not improved, and that the acceleration effects on the progress of meiosis is neutralized by the presence of FCS in maturation medium with no improvement of developmental competence after in vitro fertilization.

Control of gene expression at the onset of bovine embryonic development.

The objective of this study was to examine the timing and mechanisms involved in transcription initiation in bovine embryos. Transcriptional activity and its regulation were explored by labeling 1-cell zygotes and 2-cell embryos with [(3)H]uridine in the presence or absence of alpha-amanitin, aphidicolin, and tricostatin A (TSA) (inhibitors of mRNA synthesis, DNA replication, and histone deacetylases, respectively) followed by a total RNA isolation and determination of [(3)H]uridine incorporation. We also analyzed translation of zygotic/embryonic mRNAs by labeling zygotes and 2-cell embryos with [(35)S]methionine in the presence or absence of alpha-amanitin, aphidicolin, and TSA followed by two-dimensional PAGE and autoradiography. We show that bovine 1-cell zygotes and 2-cell embryos are transcriptionally and translationally active. The first and second rounds of DNA replication are important regulators of early gene expression as the inhibition of DNA replication resulted in a dramatic decrease in both transcriptional and translational activity. Moreover, acetylation of histones plays an important role in this early gene activation at the onset of embryonic development in the cow.

Checkpoint control of the G2/M phase transition during the first mitotic cycle in mammalian eggs.

The high incidence of chromosomally abnormal human embryos is frequently assumed to be due to a lack of checkpoint controls operating during early embryogenesis. In our study we have analysed when these mechanisms first become functional. Mouse oocytes treated in late metaphase I with either of two different cyclin-dependent kinase inhibitors [butyrolactone 1 (BL1) or 6-dimethylaminopurine (6-DMAP)] form nuclei in the cytoplasm. BL1-treated eggs enter S-phase at 16-18 h post-treatment and, after completion of DNA synthesis, cleave to 2-cell stage embryos. 6-DMAP treatment results in the rapid initiation of DNA synthesis, its completion by 12 h and then arrest in the G2 phase. Thus, two different cell cycle stages can be obtained at the same time point after the initiation of treatment: G1- after BL1 and G2-staged nuclei after 6-DMAP treatment. That this approach greatly facilitates cell cycle studies has been shown by analysing checkpoint function during the first division. Whilst G2-staged eggs enter M phase within 2-3 h when 6-DMAP is washed out, the onset of M phase is delayed after their fusion to G1 (BL1) cells. Here M phase occurs only after the less advanced nucleus completes DNA replication. Our results indicate that checkpoints in mammalian eggs are functional during the first mitotic cycle.

Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species.

The transfer of nuclei from one cell to another provides a powerful tool for studying the interactions between the cytoplasm of one cell and the nucleus of another. This study was designed to examine the ability of the bovine metaphase oocyte cytoplasm to support mitotic cell cycles under the direction of differentiated somatic cell nuclei of various mammalian species. Skin fibroblast cells from cows, sheep, pigs, monkeys, and rats were used as sources of donor nuclei. Nuclear transfer units produced by fusion of enucleated bovine oocytes and individual fibroblasts from all species examined underwent transition to interphase accompanied by nuclear swelling, further progression through the cell cycle, and completion of the first mitosis. Regardless of the species of donor fibroblasts used, some cleaving units progressed further and developed to advanced stages, as evidenced by continuation of cell proliferation and formation of a blastocoele cavity at the time appropriate for the donor fibroblast species. Although no pregnancies have been carried to term after transfer of embryos into surrogate animals, these observations suggest that mechanisms regulating early embryonic development may be conserved among mammalian species and that bovine oocyte cytoplasm can support the introduced differentiated nucleus regardless of chromosome number, species, or age of the donor fibroblast.

Sexual dimorphism in IVM-IVF bovine embryos produced from X and Y chromosome-bearing spermatozoa sorted by high speed flow cytometry.

The objective of this study was to examine preimplantation development and sperm aster characteristics of bovine male and female embryos produced by using spermatozoa sorted for the X or Y chromosome. In vitro matured oocytes were inseminated at 24 h of maturation with sorted X or Y chromosome-bearing spermatozoa, using either fresh or frozen-thawed semen. Samples were taken from each sperm group 12 h post insemination (hpi), fixed, and immunostained for the microtubule cytoskeleton. Confocal microscopy enabled visualization of sperm aster formation and microtubule characteristics of each zygote during early fertilization. Cultured embryos were checked for cleavage at 30, 35, 40 and 45 hpi, embryo development was examined daily until Day 8 of culture. Blastocyst cell numbers were determined at the end of the experiments. Reanalysis of the sorted sperm cells for DNA content showed purity rates of 90.1 and 92.1% for X and Y chromosome-bearing spermatozoa, respectively. Reduced fertilization and development rates were observed when sorted spermatozoa were used compared with fresh and frozen-thawed spermatozoa. Penetration rates at 12 hpi were 39.5, 44.7, 55.9 and 79.0%, while blastocyst formation rates at Day 8 were 26.7, 26.5, 31.7 and 40.7% for X and Y chromosome-bearing spermatozoa, using fresh and frozen-thawed semen groups, respectively. Sperm aster size was larger in males than females, while the size of pronuclei and subjective grade of sperm aster quality showed no differences between sexes. In this study, a greater cleavage rate and sperm aster size in male embryos indicated a dimorphic pattern of development in male and female embryos during fertilization and first cleavage.

Cloning by somatic cell nuclear transfer.

The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development. Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process?

Developmental changes in RNA polymerase II in bovine oocytes, early embryos, and effect of alpha-amanitin on embryo development.

Development of mammalian early embryos relies on stored maternal messenger RNAs (mRNAs) that have been synthesized during oogenesis until embryonic genome activation. Although embryonic genome acti vation in bovine embryos has been proposed to start at the late 4-cell stage, recent evidences suggest that embryonic genome activation starts earlier than the 4-cell stage, and molecular details of this event are not known. RNA polymerase II in eukaryotes is responsible for transcription of mRNA and most of the small nuclear RNAs. The unphosphorylated form of RNA polymerase II (IIA) has been shown to function in transcriptional initiation, and the hyperphosphorylated form (IIO) functions in translational elongation and mRNA splicing. In this study, we examined the changes in the amount of RNA polymerase IIA by immunoblotting in immature oocytes; mature oocytes; and 2-, 4- and 8-cell bovine embryos. We also examined the levels of IIO and the multiple intermediately phosphorylated form in the same oocytes and embryos. The IIA reached the highest level at the 2-cell stage and decreased gradually at the 4- and 8-cell stages, and IIO was at very low levels in mature oocytes and 2-cell stage embryos and was not detectable at later stages. The multiple intermediately phosphorylated form was present at the highest level in mature oocytes and was detectable at the other stages. We demonstrate that RNA polymerase IIA, which is responsible for initiation of transcription, is present in oocytes and preimplantation embryos and reaches the highest levels in the 2-cell stage embryos. Inhibition of RNA polymerase II-dependent transcription during any of the first four embryonic cell cycles has detrimental effects on progression of embryonic development beyond the 16-cell stage, indicating the importance of early transcripts for continuation of development. The results indicate that expression of all the genes whose transcription is inhibited by alpha-amanitin is essential for embryo development.

Onset of transcription in bovine oocytes and preimplantation embryos.

The transition from the maternal to embryonic control of early embryonic development (MET) in mammals is not fully understood. The objective of this study was to determine the amount of transcriptional activity in immature oocytes containing germinal vesicle (GV), mature metaphase II arrested oocytes (MII), 2-, 4- and 8-cell bovine embryos by labeling with 35S-UTP followed by isolation of total RNA and autoradiography. Expression of counts per minute (CPM) per cell showed that incorporation of 35S-UTP in GV oocytes was significantly higher than the background (P < 0.01) and decreased sharply by the time the oocytes reached MII arrest. Incorporation significantly increased during the 2-cell stage and remained at the same level during the 4- and 8-cell stages. Uptake remained constant throughout different development stages (P > 0.05) with the highest variability observed during the 2-cell stage. When CPM were expressed per oocyte or embryo incorporation remained high at the GV stage, decreased to the background levels at the time of MII and increased again at the 2-cell stage. It remained at the same level during the 4-cell stage but increased significantly for the second time during the 8-cell stage. Uptake remained at the same level until the 8-cell stage when a significant increase was observed. The negative controls showed a significantly lower amount of incorporation compared to the positive control (P < 0.05). Similar results were observed by autoradiography. Our observations suggest that MET starts as early as the 2-cell stage in bovine embryos.

Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation.

The requisite endpoint of mammalian oocyte maturation, whether in vivo or in vitro, is a metaphase II oocyte which is able to be fertilized and which can eventually support normal embryonic development. Oocytes which have been matured in vivo basically fulfill these criteria. On the other hand, a completely different situation exists when these cells are isolated from the ovaries and cultured in vitro. If they are too small (growing oocytes), they do not undergo maturation, or, if more advanced, will mature only to the metaphase I stage. Even in fully grown oocytes which are able to mature to metaphase II, the developmental potential after fertilization is disappointingly low, for reasons which remain unknown. The complexity of certain factors (nuclear, cytoplasmic or arising from our current culture systems) undoubtedly influences both the ability of oocytes to mature fully, as well as their developmental potential after fertilization.

Timing of meiotic progression in bovine oocytes and its effect on early embryo development.

This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest.

Induction of DNA replication in germinal vesicles and in nuclei formed in maturing mouse oocytes by 6-DMAP treatment.

Immature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

Relationship between the maturational state of oocytes at the time of insemination and sex ratio of subsequent early bovine embryos.

The effect of maturational state of bovine oocytes at the time of insemination on early embryo development and the sex ratio of developing embryos was evaluated. Early maturing oocytes were inseminated either immediately after the first polar body extrusion or insemination was delayed for 8 h. Most of the zygotes completed the first embryonic cell cycle and reached the 2-cell stage by 35 h after insemination regardless of the time of insemination. Delaying insemination enhanced the proportion of cleaving zygotes and significantly improved their development to the 8-cell stage. At the same time delaying insemination produced significantly higher proportions of male embryos. Cleavage and development to 8-cell stage was significantly impaired when oocytes were inseminated immediately after polar body formation. Sex ratio in these embryos did not differ from 1. These results suggest that oocytes developmental ability as well as capability to process X and Y-bearing spermatozoa may be acquired at specific times during maturation.