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PubMed2003

Nucleolus in apoptosis-induced mouse preimplantation embryos.

Apoptosis may occur in early embryos in which the execution of essential developmental events has failed. Thus the initiation of the apoptotic mechanism may be related to activation of the embryonic genome. In this way, developmentally incompetent cells or whole embryos are eliminated. It is likely that some link exists between failed resumption of rRNA synthesis and the incidence of apoptosis in cleaving embryos. In this context, decreased developmental potential in cleaving nucleotransferred embryos is consistent with cell loss, and very likely due to programmed cell death. The effects of apoptosis inducers on cleaving embryos have not been characterised in comparable detail to that in the case of somatic cells. Early embryos provide a very good model for study of these processes because of the specificity of rRNA transcription resumption after fertilization. In our experiments three apoptosis inducers (staurosporin 10 mM, actinomycin D 0.05 mg/ml and camptothecin 0.1 mg/ml) were used in a culture medium for 15 h at the 4-cell stage (day 2) of mouse embryos, followed by further development in a pure culture medium until fixation on days 3, 4 and 5. In staurosporin-induced embryos, light microscopy immunostaining of nucleolar proteins (fibrillarin, Nopp140, protein B23) did not reveal changes in nucleolar morphology on day 3. On days 4 and 5, more compact (roundish) nucleoli (in comparison with controls) were observed. The embryos treated with camptothecin displayed a similar staining pattern to those with staurosporin at each day. In actinomycin-D-treated embryos, marked changes in nucleolar appearance were visible as early as day 3. These changes in nucleolar morphology consisted of loss of the reticulation appearance and fragmentation of nucleoli. In addition to nucleolar changes, significantly decreased cell proliferation was observed. The induced embryos did not reach the blastocyst stage. The number of blastomeres was decreased, and staining with Hoechst 33342 revealed a significant percentage of apoptotic nuclei (condensed/fragmented nuclei) from day 4.

Animals↗
PubMed2004

Serotonin localization and its functional significance during mouse preimplantation embryo development.

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.

Animals↗
PubMed2002

Improvement of development of vitrified two-cell mouse embryos by vero cell coculture.

PURPOSE: The purpose of this study was to determine the developmental potential of two-cell mouse embryos resulting from vitrification could increased using monolayer of Vero cells. METHODS: Two-cell mouse embryos were divided into vitrified and nonvitrified groups. Embryos in the vitrified group were frozen with a combination of 10% ethylene glycol, 30% ficoll, and 0.5% M sucrose (EFS10) as cryoprotectants, and thawed rapidly with 0.5 M sucrose. The survived embryos were cultured either with Vero cells monolayer or in T6 medium. Accordingly the embryos of the nonvitrified group were also cultured. The rates of the development in all the groups were daily determined and statistically compared. At the end of the cultivation period, several expanded blastocysts from each group were stained with ethidium bromide and the mean number of the blastomers were counted and statistically compared. RESULTS: After 4 days of culture, the developmental potential of vitrified-thawed embryos were significantly reduced in Vero cell-free medium, and the mean cell number of embryos reaching the expanded blastocyst stage were also lower than that of nonvitrified embryos. With exception of last day of culture, Vero cell coculture, resulted in a significant increase in the rate of development of vitrified-thawed embryos as well as improved the mean cell number of expanded blastocysts. On the other hand, the mean cell number of expanded blastocysts of nonvitrified group was significantly improved in coculture group. However, the rate of embryo development except for the first day of culture was similar to that of medium alone. CONCLUSIONS: The developmental potential of vitrified-thawed embryos appears to be retarded in conventional medium and Vero cell monolayer is capable to eliminate the postthaw deleterious effect of vitrification during the first 3 days of cultivation, but not for a longer period.

Animals↗
PubMed1997

Efficient selection of preimplantation transgenic embryos by an improved procedure using Dpn I-Bal 31 digestion and the polymerase chain reaction.

Efficient selection of preimplantation transgenic embryos by an improved method after pronuclear injection of exogenous DNA is described. The method is based on subjecting DNA extracted from the embryos to restriction enzymes as well as the polymerase chain reaction (PCR). The incorporated procedure included recovery of the digested DNA with glassmilk before PCR, which markedly enhanced the rate of accurate detection of transgenic embryos. When exogenous DNA sequences in the mouse embryos were not integrated into the genome they were digested with both Dpn I and Bal 31, and subsequent PCR analysis generated DNA fragments of the injected DNA sequence in only 1.5% of cases examined. However, DNA extracted from mouse embryos containing the transgene sequences integrated into the genome evaded digestion by both enzymes and yielded transgene-specific PCR products in 68.6% of the embryos tested. When bovine embryos were used, sequences of the endogenous haemoglobin gene used as a control genomic DNA sequence were protected from enzyme digestion (PCR products in 70.5% of the embryos examined); by contrast, the non-integrated injected sequences were almost completely eliminated by the same treatment (PCR products in 1.4% of the embryos examined). It is suggested that this method might be useful for the selection of transgenic embryos before embryo transfer, thereby reducing the number of recipient females required.

Animals↗
PubMed2004

Genetics and imaging to assess oocyte and preimplantation embryo health.

Two major criteria are currently used in human assisted reproductive technologies (ART) to evaluate oocyte and preimplantation embryo health: (1) rate of preimplantation embryonic development; and (2) overall morphology. A major gene that regulates the rate of preimplantation development is the preimplantation embryo development (Ped) gene, discovered in our laboratory. In mice, presence of the Ped gene product, Qa-2 protein, results in a fast rate of preimplantation embryonic development, compared with a slow rate of preimplantation embryonic development for embryos that are lacking Qa-2 protein. Moreover, mice that express Qa-2 protein have an overall reproductive advantage that extends beyond the preimplantation period, including higher survival to birth, higher birthweight, and higher survival to weaning. Data are presented that suggest that Qa-2 increases the rate of development of early embryos by acting as a cell-signalling molecule and that phosphatidylinositol-32 kinase is involved in the cell-signalling pathway. The most likely human homologue of Qa-2 has recently been identified as human leukocyte antigen (HLA)-G. Data are presented which show that HLA-G, like Qa-2, is located in lipid rafts, implying that HLA-G also acts as a signalling molecule. In order to better evaluate the second criterion used in ART (i.e. overall morphology), a unique and innovative imaging microscope has been constructed, the Keck 3-D fusion microscope (Keck 3DFM). The Keck 3DFM combines five different microscopic modes into a single platform, allowing multi-modal imaging of the specimen. One of the modes, the quadrature tomographic microscope (QTM), creates digital images of non-stained transparent cells by measuring changes in the index of refraction. Quadrature tomographic microscope images of oocytes and preimplantation mouse embryos are presented for the first time. The digital information from the QTM images should allow the number of cells in a preimplantation embryo to be counted non-invasively. The Keck 3DFM is also being used to assess mitochondrial distribution in mouse oocytes and embryos by using the k-means clustering algorithm. Both the number of cells in preimplantation embryos and mitochondrial distribution are related to oocyte and embryo health. New imaging data obtained from the Keck 3DFM, combined with genetic and biochemical approaches, have the promise of being able to distinguish healthy from unhealthy oocytes and embryos in a non-invasive manner. The goal is to apply the information from our mouse model system to the clinic in order to identify one and only one healthy embryo for transfer back to the mother undergoing an ART procedure. This approach has the potential to increase the success rate of ART and to decrease the high, and undesirable, multiple birth rate presently associated with ART.

Animals↗
PubMed1989

In vivo cleavage rates and viability obtained for early cleavage mouse embryos in co-culture with oviduct cells.

The cleavage rate and development of two-cell mouse embryos to the morulae stage in co-culture with mouse oviduct cells was studied in vitro and compared with those achieved in vivo. Embryos were cultured in Whittingham's T6 (T6), T6 supplemented with fetal calf serum (FCS) and in co-culture with either Dulbecco's Modified Eagles Medium supplemented with sodium lactate (DMEM + 1a) or a modification of T6 medium containing vitamins and amino acids (T6 + v + aa). Co-culture of oviductal cells with DMEM + la medium supported two-cell mouse embryo development to eight cells at a rate significantly better (P less than 0.001) than T6, but the rate of embryo development was not equivalent to that in vivo. DMEM + la alone was inadequate as an embryo culture medium. Co-cultures using T6 + v + aa with mouse oviductal cells were prepared from mice at days 1, 2 or 3 of pseudopregnancy. Day 2 and 3 co-cultures allowed two-cell embryos to develop at a rate comparable to that in vivo up to the mid eight-cell stage (68 h after hCG), but by 76 h after hCG embryos were retarded. Transfer to pseudopregnant recipients of embryos co-cultured with day 2 oviductal cells until 68 h after hCG resulted in a rate of fetal development equivalent to that of embryos grown in vivo. Our results show that co-culture of early cleavage-stage embryos with mouse oviductal cells allows embryos to retain cleavage rates and viability comparable to in vivo development.

Animals↗
PubMed1992

Antisense c-myc effects on preimplantation mouse embryo development.

Antisense DNA inhibition of gene expression was explored as an approach toward elucidating mechanisms regulating development of preimplantation mammalian embryos. Specifically, a role for the c-myc protooncogene was examined. Detection of c-myc mRNA and immunoreactive nuclear c-myc protein in preimplantation mouse embryos at the eight-cell/morula and blastocyst stages suggested that this DNA-binding protein could be important during early embryo-genesis. The effects of c-myc oligodeoxyribonucleotides (oligos) on the in vitro development of two-cell mouse embryos were examined. Embryos cultured in medium containing an unmodified (phosphodiester) antisense c-myc oligo complementary to the translation initiation codon and spanning the first seven codons exhibited a dose-dependent arrest at the eight-cell/morula stage. At lower concentrations (7.5 microM) this inhibitory effect was specific to the antisense oligo and did not occur with the sense-strand complement or with duplexes of the antisense and sense oligos. However, at 4-fold higher concentrations of DNA (30 microM), all unmodified c-myc oligos were embryotoxic, causing embryos to arrest at the two-cell to four-cell stages. In contrast, almost all (98%) two-cell embryos cultured with a modified (chimeric phosphorothioate/phosphodiester) antisense c-myc oligo (7.5 microM) exhibited developmental arrest at the eight-cell/morula stage, whereas no developmental arrest occurred following incubation with high concentrations of the modified sense complement (30 microM). Culture of freshly recovered eight-cell embryos with antisense c-myc led to the absence of c-myc protein but no change in epidermal growth factor receptor in those embryos that developed a blastocoel. These effects on c-myc were specific for the antisense oligo. These results suggest that c-myc function becomes particularly critical for preimplantation mouse embryos at the eight-cell/morula stage of development and establish that antisense DNA can be successfully applied as an approach toward elucidating the roles of specific genes in preimplantation mammalian embryo development.

Animals↗
PubMed1995

Morphological effects of caffeine, okadaic acid and genistein in one-cell mouse embryos blocked in G2 by X-irradiation.

One-cell mouse embryos of the Balb/c strain normally divide at 18.5 h p.c. (post conception), but they suffer an extremely long G2 arrest when irradiated with 2 Gy X-rays 8 h p.c. at the early pronuclear stage. This could be an indirect effect of radiation on tyrosine dephosphorylation of the p34cdc2 subunit of a maturation or mitosis promoting factor (MPF), which normally occurs at the end of G2. This, in turn, would maintain MPF in an inactivated form and block entry into mitosis. Preliminary studies were undertaken at the morphological level to assess indirectly the validity of this hypothesis. For this purpose, irradiated and control embryos were exposed to different compounds, which are known to interfere, directly or indirectly, with the state of phosphorylation/dephosphorylation of p34cdc2. Caffeine (CAF; 2 mM) did not affect the time of first division of control embryos, but it completely suppressed the radiation-induced G2 arrest of embryos exposed to this compound from 17 h p.c., i.e. 1.5 h before the normal time of first cleavage. Under the same conditions, okadaic acid (OA; 3 microM), a specific inhibitor of phosphatases I and IIA, induced a rapid pronuclear membrane breakdown and a block of all control and irradiated embryos at metaphase. Genistein (GEN; 92 or 185 microM). A potent inhibitor of tyrosine kinases, increased the radiation-induced G2 arrest and even induced a dose-dependent G2 arrest in the control embryos. Embryos were exposed at different times following irradiation to a mixture of either CAF (2 or 5 mM) or OA (3 or 10 microM), and cycloheximide (CH; 5 micrograms/ml), a potent protein synthesis inhibitor. Reversion of G2-arrest by CAF was still seen in embryos exposed to CAF+CH from 17 h p.c. However, the proportion of irradiated embryos eventually able to cleave was lower than that obtained under the conditions of exposure to CAF alone. Embryos exposed to CAF+CH before 17 h p.c. were not able to cleave, regardless of the concentration of CAF used. Nuclear envelope breakdown still occurred in 100% control and irradiated embryos, following exposure to 3 microM OA+CH from 10 h p.c., or to 10 microM OA+CH from 8.5 p.c.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Proliferation, maturation and germination of Castanea sativa Mill. Somatic embryos originated from leaf explants.

Experiments were performed to determine the influence of proliferation medium on the maintenance of embryogenic competence and on repetitive embryogenesis in Castanea sativa Mill. somatic embryos derived from leaf explants. Somatic embryo proliferation was carried out by both direct secondary embryogenesis and by the culture of nodular callus tissue originated from cotyledons of somatic embryos. Both systems led to the production of cotyledonary somatic embryos on Murashige and Skoog proliferation medium supplemented with 0.1 mg l-1 benzyladenine and 0.1 mg l-1 naphthaleneacetic acid. Carbon source and concentration had a marked influence on maturation and subsequent germination ability of chestnut somatic embryos. Plantlet conversion was achieved in embryos matured on media with 6 % sucrose, and on 3 or 6 % maltose, whereas mean shoot length, root length and leaf number of produced plants were not significantly affected by these maturation media. Overall, the best results were obtained with 3 % maltose-matured somatic embryos, giving rise to 6 % plant recovery in addition to 33 % of embryos exhibiting only shoot development. The application of a 2-month cold treatment at 4 degrees C to somatic embryos matured on medium with 3 % maltose was necessary for achieving plant conversion, while partial desiccation did not appear to influence this response. A total of 39 % of embryos eventually produced plants either through conversion to plantlets or indirectly through rooting of shoots. Shoots formed by somatic embryos could be excised, multiplied and rooted following the micropropagation procedures previously developed for chestnut.

Culture Media↗
PubMed1996

Programmed cell death and human embryo fragmentation.

The quality of embryos produced by in-vitro fertilization (IVF) is variable. Many embryos contain unequal sized blastomeres and multiple cellular fragments. Embryos with excessive fragmentation have limited developmental potential both in vitro and in vivo. Histologically, some blastomeres of fragmented embryos resemble cells undergoing apoptosis as a result of programmed cell death (PCD). The objective of the present study was to determine if the morphological features of apoptosis are observed in fragmented human preimplantation embryos, supporting the possible involvement of PCD in early human embryo arrest and demise. Using combined nuclear and terminal transferase-mediated DNA end labelling (TUNEL) on arrested, fragmented human embryos, we were able to detect extensive condensation and degradation of chromatin, compatible with apoptosis. Electron microscopy confirmed the typical morphological features of apoptosis. No such abnormalities were observed in spare embryos with regular sized blastomeres without fragmentation. The high incidence of condensed chromatin, TUNEL detection of degraded DNA, cell corpses and apoptotic bodies in fragmented human embryos strongly suggest that PCD is triggered in human embryos at a stage prior to blastocyst formation. At such early stages, occurrence of apoptosis seemed to be detrimental, leading to preimplantation embryo death.

Apoptosis↗
PubMed1996

Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium.

The formulation of chemically defined culture media that support primate embryo development would facilitate studies on primate preimplantation embryogenesis. The specific aims of this study were (i) to evaluate the development of the macaque embryos in a simple, chemically defined, protein-free medium developed for a rodent embryo model, and (ii) to determine if a two-step progressive culture system could enhance blastocyst development and zona escape. In experiment 1, in-vitro-fertilized pronucleate-stage embryos (n = 81) from nine monkeys were randomly allocated to one of three treatments: (a) hamster embryo culture medium-6 (HECM-6; chemically defined, protein-free medium), (b) CMRL-BCS medium (modified CMRL-1066 medium containing 20% bovine calf serum; BCS), and (c) a two-step culture procedure (HECM-6 through to the 8- to 12-cell stage, and CMRL-BCS medium beyond that stage). Optimal development was attained equally (P > or = 0.05) with embryos cultured in CMRL-BCS medium or the two-step procedure (48 and 61%), but not to the blastocysts respectively). HECM-6 alone supported development to the morula stage (72%) equally as well as CMRL-BCS medium (80%) or the two-step procedure (69%), but not to the blastocyst stage (22 versus 48 and 61% respectively). Hatching of the blastocysts was essentially limited to the serum-containing media (CMRL-BCS medium, 31%; two-step procedure, 44%). In experiment 2, in-vitro-fertilized pronucleate-stage embryos (n = 87) from nine monkeys were randomly placed in each of four two-step treatments: (a) HECM-6 through to the 8- to 12-cell stage and CMRL-BCS medium beyond that stage, (b) HECM-6 through the 8- to 12-cell stage and HECM-6-BCS beyond that stage, (c) HECM-6 through to the morula stage and CMRL-BCS medium beyond that stage, and (d) HECM-6 through to the morula stage and HECM-6-BCS beyond that stage. Greater (P < or = 0.05) percentages of embryos developed into blastocysts, expanded blastocysts and hatched blastocysts when switched at the 8- to 12-cell versus the morula stage in the second step medium. When transferred into BCS-containing medium at either the 8- to 12-cell or morula stage, embryos underwent blastulation and expansion equally well in CMRL-BCS medium versus HECM-6-BCS. However, when embryos were switched to the second step medium at the 8- to 12-cell stage, hatched blastocysts were obtained more (P < or = 0.05) frequently in CMRL-BCS medium (50.9%) than in HECM-6-BCS (37%). This work is the first to produce in-vitro-fertilized primate blastocysts cultured from the pronucleate stage in chemically defined, protein-free medium, and demonstrates that while primate embryos can form morulae in such a medium, their requirements for blastocoel formation and zona escape appear to be more demanding, and may be acquired as early as the 8-cell stage.

Animals↗
PubMed1993

Microsurgical correction of partially degenerate mouse embryos promotes hatching and restores their viability.

We have evaluated the effects of degeneration of blastomeres on the developmental fate of mouse embryos. Micromanipulation techniques were used first to destroy one or two blastomeres of a 4-cell embryo (thereby creating three-quarter and half embryos), and later to repair the anomaly by removing the degenerate material. The embryos were either cultured in protein-free or protein-supplemented medium. When cultured in protein-supplemented medium, three-quarter embryos hatched at the same rate as intact embryos (84 and 91%, respectively), but this rate was reduced (54%; 67/125) when the embryos were cultured in a protein-free environment. Destruction of two blastomeres of a 4-cell embryo and culture in protein-free medium was detrimental, as only 3.2% (4/124) hatched. By supplementing the culture medium with protein, some of these half embryos were rescued, as shown by a 34% (32/95) hatching rate. A more dramatic increase in hatching was achieved, however, after repair of the half embryos by microsurgical removal of the degenerate material. In this case, 72% (78/109) of the repaired embryos were able to hatch. These findings may have implications for human in-vitro fertilization where partial embryonic degeneration or fragmentation often leads to embryonic demise and reduced implantation. Moreover, these observations may provide important clues to mechanisms of mammalian embryonic hatching.

Animals↗
PubMed2003

Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei.

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.

Animals↗
PubMed2004

Apoptosis in parthenogenetic preimplantation porcine embryos.

Parthenogenesis (PA) of the oocyte is essential to a number of oocyte- or embryo-related technologies such as intracytoplasmic sperm injection and cloning by nuclear transfer. This study investigated the onset and frequency of apoptosis in PA- porcine embryos and the morphological changes that conform to the general criteria of apoptotic cell death by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. PA embryos had a higher degree of apoptotic cell death during in vitro culture, a lower cleavage rate (45% vs. 71%), and a lower development rate to the blastocyst stage (16% vs. 29%), relative to in vitro fertilization (IVF). The earliest positive TUNEL signal in the PA embryos was detected on Day 6, 1 day later than that in IVF embryos. Apoptosis in PA embryos increased from 15% of the embryos on Day 6 to 29% on Day 8. The mean level of apoptosis of the PA embryos was statistically higher than that of IVF embryos, except on Day 5. In particular, apoptosis in PA embryos was twice that of IVF embryos on Day 6 (15% vs. 6.7%) and Day 8 (29% vs. 13%). The mean cell number in PA blastocysts was significantly lower than that of IVF blastocysts, whereas the percentage of apoptosis in PA blastocysts was significantly higher than that of IVF blastocysts. There was a high percentage of haploid (62.5%) PA blastocysts. The ploidy may contribute to a high level of apoptosis. These results may help to explain the mechanism of parthenogenetic developmental failure and may lead to methods that will improve parthenogenetic development.

Animals↗
PubMed2004

Methylation and acetylation characteristics of cloned bovine embryos from donor cells treated with 5-aza-2'-deoxycytidine.

Differentiated somatic cells and embryos cloned from somatic cells by nuclear transfer (NT) have higher levels of DNA methylation than gametes and early embryos produced in vivo. Reducing DNA methylation in donor cells before NT by treating them with chemicals such as the DNA methyl-transferase inhibitor (5-aza-2'-deoxycytidine; 5-aza-dC) may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Previously, high levels of this reagent were used to treat donor cells, and decreased development of cloned embryos was observed. In this study, we tested a lower range (0.005 to 0.08 microM) of this drug and used cell cycle distribution changes as an indicator of changes in the characteristics of donor cells. We found that at 0.01 microM 5-aza-dC induced changes in the cycle stage distribution of donor cells, increased the fusion rate of NT embryos, and had no deleterious effect on the percentage of blastocyst development. Levels of 5-aza-dC greater than 0.01 microM significantly decreased embryo development. Embryos cloned from donor cells treated with a low dose of 5-aza-dC had higher levels of DNA methylation than embryos produced by in vitro fertilization, but they also had higher levels of histone acetylation. Although 5-aza-dC at 0.04 microM or higher reduced DNA methylation and histone acetylation levels to those of in vitro-fertilized embryos, development to blastocyst was reduced, suggesting that this concentration of the drug was detrimental. In summary, 5-aza-dC at 0.01 microM altered donor cell characteristics while showing no deleterious effects on embryos cloned from treated cells.

Acetylation↗
PubMed1993

Effects of glucose and fructose on fertilization, cleavage, and viability of mouse embryos in vitro.

In this study we examined the role of glucose, and the use of fructose as a replacement, in embryo culture medium. Three embryo culture media were used: a routine embryo culture medium (M16), M16 without glucose (M16-G) and M16-G supplemented with fructose (M16F-G). Their effect on fertilization, rate of cleavage, and embryo viability were examined in both an outbred (OF1) and an inbred (C57Bl) mouse strain. Of the three media, only M16 was found to support fertilization. In vitro-fertilized embryos from OF1 oocytes and C57Bl oocytes and OF1 sperm were placed in the different media at the early 2-cell stage. In 65% of OF1 embryos cultured in M16, development was blocked at the 2-cell stage, whereas in M16-G and M16F-G embryos, only 22% and 32%, respectively, were blocked. M16F-G medium also produced morulae and blastocysts with higher cell numbers than M16-G. In vitro-fertilized C57Bl 2-cell embryos cultured in M16 displayed retarded cleavage to the 4-cell stage compared to embryos cultured in M16-G and M16F-G. In contrast, the morulae and blastocyst cell numbers were significantly lower in M16-G compared to M16 and M16F-G. The viability of morulae and blastocysts obtained from OF1 and C57Bl embryos cultured in M16-G and M16F-G was lower compared to control C57Bl morulae and blastocysts cultured in M16. The results show that although morphologically normal embryos could be obtained in M16-G and M16F-G, inherent anomalies existed that limited viability.

Animals↗
PubMed1994

Developmental potential of rhesus monkey embryos produced by in vitro fertilization.

This study was an examination of the developmental potential of in vitro fertilization (IVF)-produced rhesus monkey embryos that were cultured in medium alone or cocultured with various cell types. End points were the quality and yield of embryos attaining the expanded or hatched blastocyst stage. A total of 96 IVF-produced embryos were cryopreserved and thawed, and 90 embryos were considered intact and suitable for culture. These embryos were placed into one of five treatment groups consisting of four different cell supports and medium alone. Two primary cultures (bovine oviductal cells [bOVID] and bovine cumulus cells [bCUM]) and two established cell lines (Vero cells and buffalo rat liver cells [BRL]) were utilized for coculture of embryos. Embryos were cultured for up to 14 days, and growth curves were established for all embryos that expanded and/or hatched. The developmental rate for embryos classified as viable varied substantially; in number of days to reach a given stage, early morulae ranged from Days 3 to 9 post-insemination, morulae from Days 4 to 9, blastocysts from Days 6 to 11, expanded blastocysts from Days 7 to 12, and hatched blastocysts from Days 9 to 15. On the basis of developmental curves, 30% of the embryos were arrested upon thawing or shortly after. Of the remaining embryos classified as viable, developmental efficiencies to the hatched blastocyst stage for the various treatments were 1) bOVID, 33%; 2) bCUM, 15%; 3) Vero cells, 9%; 4) BRL, 45%; and 5) medium alone, 8%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Ribosomal ribonucleic acid is transcribed at the 4-cell stage in in vitro-produced bovine embryos.

Ribosomal RNA, rRNA genes, and silver-staining nucleolar proteins were visualized in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst using a sequential fluorescent in situ hybridization (FISH) and a silver-staining procedure. At FISH, the rRNA was differentiated from the signal of the rRNA genes through comparison of RNase- and non-RNase-treated embryos. Both RNase- and non-RNase-treated 2-cell embryos revealed up to 10 small clusters of fluorescein isothiocynate (FITC) labeling in interphase nuclei. The RNase-treated 4-cell embryos displayed the same FITC pattern as the 2-cell embryos. In the non-RNase-treated 4-cell embryos, in contrast, the clusters were larger and included numerous small spots. In 2-cell as well as 4-cell embryos, almost all FITC-labeled clusters colocalized with silver-stained spots. In the RNase-treated 8- to 16-cell embryos, up to 10 clusters of FITC labeling were organized as one or more large spots surrounding a central faint but homogeneously labeled area. The non-RNase-treated 8- to 16-cell embryos displayed similar complexes, but the central areas consisted of small labeled spots. In 8- to 16-cell embryos, all FITC-labeled clusters were again colocalized with silver-stained areas. In the blastocysts, 1-6 big clusters of FITC labeling colocalized with silver staining. In the RNase-treated blastocysts, the FITC labeling was typically located at the edges of the silver-stained areas, whereas in the non-RNase-treated blastocysts, the FITC labeling totally covered the silver-stained areas. In conclusion, there is a close association between the rRNA genes and silver-staining nucleolar proteins in in vitro-produced bovine embryos from the second cell cycle, i.e., the 2-cell stage; the first rRNA is apparently transcribed during the third cell cycle, and during the fourth cell cycle the molecular composition of functional nucleoli is established.

Animals↗