Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos.

Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.     

Role of glucose in cloned mouse embryo development

Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early beneficial effect during the first cell cycle. This early beneficial effect of glucose is not displayed by parthenogenetic, fertilized, or tetraploid nuclear transfer control embryos, indicating that it is specific to diploid clones. Precocious localization of the glucose transporter SLC2A1 to the cell surface, as well as increased expression of glucose transporters and increased uptake of glucose at the one- and two-cell stages, is also seen in cloned embryos. To examine the role of glucose in early cloned embryo development, we examined glucose metabolism and associated metabolites, as well as mitochondrial ultrastructure, distribution, and number. Clones prepared with cumulus cell nuclei displayed significantly enhanced glucose metabolism at the two-cell stage relative to parthenogenetic controls. Despite the increase in metabolism, ATP content was reduced in clones relative to parthenotes and fertilized controls. Clones at both stages displayed elevated concentrations of glycogen compared with parthenogenetic controls. There was no difference in the number of mitochondria, but clone mitochondria displayed ultrastructural alterations. Interestingly, glucose availability positively affected mitochondrial structure and localization. We conclude that cloned embryos may be severely compromised in terms of ATP-dependent processes during the first two cell cycles and that glucose may exert its early beneficial effects via positive effects on the mitochondria.     

Heat shock induces apoptosis related gene expression and apoptosis in porcine parthenotes developing in vitro

Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. However, the molecular aspects involved in the thermoprotection of embryos, against heat and cold stress it is not clear. The aim of this study was to determine the effects of heat and cold shock on the viability and development of porcine diploid parthenotes developing in vitro. Exposure of two-cell stage embryos to 41 degrees C did not affect further cleavage. However, prolonged heat shock, greater than 12h, reduced the percentage of blastocysts that developed from two-cell stage parthenotes, as well as the total number of nuclei in the blastocysts that formed. Furthermore, the degree of apoptosis was increased (P<0.05) in these blastocyst stage parthenotes. In contrast, exposure of two-cell parthenotes to cold (30 degrees C) for 24h did not affect the cleavage rates, development to blastocyst, nor the total cell numbers per blastocyst. Real time PCR revealed that quantitative expression of the Bcl-xL gene was not different, but amounts of HSP 70.2, Bak, and Caspase 3mRNA were significantly increased in the heat shocked embryos, as compared with untreated controls. These results suggest that porcine embryos are more tolerant to cold shock than to heat shock. Heat stress seems to induce apoptosis related gene expression in porcine parthenotes developing in vitro, which results in diminished parthenote viability.     

Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos

The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (∼10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT).     

Haploidy influences Bak and Bcl-xL mRNA expression and increases incidence of apoptosis in porcine embryos

The objective of this study was to determine developmental pattern, total cell number, apoptosis and apoptosis-related gene expression in haploid and diploid embryos following parthenogenetic activation. In vitro-matured porcine oocytes were activated by electrical pulses and cultured in the absence or presence of cytochalasin B for 3 h. Zygotes with two polar bodies (haploid) and one polar body (diploid) were carefully selected and were further cultured in NCSU 23 medium containing 0.4% bovine serum albumin (BSA) for 7 days. The percentage of development to blastocyst stage was higher (p < 0.01) in the diploid than in the haploid parthenotes. In haploid blastocysts, average total cell number was significantly reduced (p < 0.05) and apoptosis was increased at day 7. The relative abundance of Bcl-xL and Bak mRNA in the diploid blastocysts was similar to that of in vivo-fertilized embryos. However, Bcl-xL was significantly decreased, and Bak mRNA was significantly increased (p < 0.05) in haploid parthenotes compared with the diploid parthenotes. These results suggest that the haploid state affects apoptosis-related gene expression which results in increased apoptosis and decreased developmental competence of haploid parthenotes.     

From oocyte to embryo: a model, deduced from in vitro fertilization, for natural selection against chromosome abnormalities

A cytogenetical analysis was performed on 151 unfertilized oocytes, 22 fertilized eggs at the pronuclear stage, and 108 cleaved embryos obtained in the course of in vitro fertilization (IVF). Thirty-two per cent of unfertilized oocytes were abnormal, carrying nullisomies or disomies, mainly of D and G chromosomes, and a structural anomaly (Gq-) in one case. Fertilized eggs showed frequent asynchronism in the development of pronuclei and only 2 out of 8 karyotyped pronuclei were normal. Cleaved embryos were classified according to the number of pronuclei observed 17 hours after insemination. One per cent displayed a single pronucleus, and haploid chromosome complements were found in the corresponding cleaved embryos which were considered to be parthenotes. The rate of chromosome abnormalities of diploid eggs depended on their morphological aspect. Healthy cleaved embryos carried 12.5% of anomalies while this rate reached 37% in fragmented embryos (p less than 0.05). Lastly, 6% of fertilized eggs displayed three pronuclei or more. Only 41% of the corresponding embryos were triploid. Diploidy or diploidtriploid mosaicism were often encountered. This leads to a 21% rate of abnormalities in the preimplantation embryos. Parental karyotyping and HLA typing were carried out in a series of eight couples with in vitro idiopathic infertility or recurrent embryo degeneration in vitro. No abnormality was noted. According to these results, a model of natural selection of normal conceptuses is proposed.     

The functioning of mammalian mitochondria injected into fish embryos

The possibility of mammalian mitochondria functioning in fish embryos has been studied. Suspension of mitochondria isolated from the mouse fibroblast B-82/cap (chloramphenicol-resistant) and B-82 (chloramphenicol sensitive) cell cultures, were injected into the fertilized loach eggs. These embryos with an artificially increased number of mouse mitochondria developed and lived till the larval stages. Activity of cytochrome oxidase in these embryos was 1.5-2 times that in the control several hours after the injection, decreased during development and reached the control level by the gastrula stage. If these embryos with artificially increased number of mouse mitochondria were incubated in presence of chloramphenicol, only embryos that contained mitochondria from chloramphenicol-resistant cells survived, thus suggesting that the injected mitochondria do not degrade but are preserved and function in the cytoplasm of developing loach embryos.     

The histochemical identification of primordial germ cells in diploid parthenogenetic mouse embryos

An attempt has been made to improve the early post-implantation development potential of diploid parthenogenetic mouse embryos by transferring parthenogenetic blastocysts to one uterine horn of a pseudopregnant recipient and a similar number of fertilized embryos to the contralateral horn. In control studies, diploid parthenogenetic embryos were transferred to both uterine horns of appropriate recipients. Unfortunately no obvious advantage appeared to be gained by carrying out the former manoeuvre. A significant improvement in the development potential of the parthenogenones could have indicated that their poor post-implantation survival might have been associated with a deficiency, possibly of hormonal origin, in the functioning of their decidual reaction. However, sufficient somite-containing parthenogenetic embryos were obtained in this study to allow a comparison to be made between them and fertilized embryos that were morphologically at a comparable stage of development. The parthenogenones were found to have a markedly smaller crown-rump length than their fertilized counterparts. A high proportion of both the parthenogenetic and fertilized embryos were subsequently fixed and appropriately stained in order to localize alkaline phosphatase activity. The analysis of this material clearly demonstrated that parthenogenetic mouse embryos are in fact capable of producing primordial germ cells. The latter were recognized by their morphology, histochemical staining appearance, and characteristic location, being found in the early 'turned' embryos within the dorsal mesentery in close proximity to the developing gut tube, and in the more advanced limb-bud stage embryos within the gonadal ridges.     

Blastomeres from somatic cell nuclear transfer embryos are not allocated randomly in chimeric blastocysts

A marker has been developed to allow detection of blastomeres that originate from embryos produced by nuclear transfer (NT) of genetically engineered fetal fibroblasts. A plasmid (phEFnGFP) was constructed with a G418 resistance cassette for selection in fibroblasts and a nuclear localized green fluorescent protein (nGFP) expression cassette that expresses in every cell of day-6, -7, and -8 bovine embryos. This construct was utilized to follow the blastomere distribution in aggregation chimeras produced from fertilized embryos (in vitro produced, IVP) or parthenotes and NT embryos. Fluorescent and nonfluorescent NT embryos were aggregated early on day 4 and evaluated on day 8. Nuclei of blastomeres that carried the transgene were fluorescent under both UV epifluorescence (Hoechst 33342) and blue epifluorescence (nGFP). There was no bias in the distribution of green fluorescent blastomeres in the inner cell mass (ICM) or trophectoderm in NT<>NT chimeras. However, there was a strong bias for NT blastomeres to populate the ICM when aggregated with IVP embryos or parthenotes. There was also a strong bias against NT blastomeres in the trophectoderm when aggregated to IVP embryos. However, the bias against NT blastomeres in the trophectoderm was significantly less (p < 0.05) when aggregated with parthenotes as compared to aggregation with IVP embryos. In NT<>NT aggregates, no chimeric embryos were produced that had an ICM composed of blastomeres from a single origin. However, in NT<>Parthenote aggregates, 67% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 0% to 83% of the ICM that expressed nGFP. Similarly, in NT<>IVP aggregates 50% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 19% to 71% of the ICM being of NT origin. We conclude that production of divaricated chimeras from NT origin is feasible. Other applications of this technology are discussed.     

Viable offspring derived from cryopreserved haploid rabbit parthenotes

The female parthenogenetic haploid embryos can be stored long-term by cryopreservation. Briefly, rabbit haploid parthenotes at the 32-cell stage were produced by electroactivation and in vitro culture. At this embryonic stage, parthenotes were individually cryopreserved by a slow-freezing procedure. After thawing, every embryo was disaggregated and blastomeres used as haploid maternal donors of nuclei. These nuclei were transferred to androgenetic haploid hemizygotes, obtained by female pronuclear removal offertilizedova. In the firstexperiment, 38 out of 87 reconstructedheteroparental diploid zygotes reachedthe hatched blastocyst stage invitro. In the second experiment, ourpurpose was toobtain live pups from each frozen-thawed parthenote. Viable offspring (at least one live pup at delivery) were obtained from five out of seven frozen-thawed haploid morula used as donors, with three live hemiclones being the highest number of pups produced from a single thawed parthenote. These results indicate that the rabbit female gametic endowment can be successfully stored by cryopreservation of parthenogenetic haploid embryos.     

In vitro and in vivo developmental competence of ovulated and in vitro matured porcine oocytes activated by electrical activation

The objective of this study was to evaluate the in vitro and in vivo developmental competence of parthenogenetic (parthenote) pig embryos derived from ovulated and in vitro matured (IVM) oocytes. A total of four experiments were carried out. These demonstrated that the mean blastocyst rates from stimulated ovulated and IVM pig oocytes were not significantly different (61% vs. 46%, p > 0.05) following in vitro culture. Both ovulated and IVM pig parthenotes were able to develop in vivo for 30 days. Parthenote fetuses collected 21 and 30 days post estrus were morphologically normal but significantly smaller and lighter than fertilized controls (p < 0.01). IVM pig parthenotes stopped development around 31 days post estrus.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Developmental rate and ploidy of embryos produced by nuclear transfer with different activation treatments in cattle

Bovine oocyte activation is one of the essential elements that determine the success of nuclear transfer and the subsequent development of cloned embryos. Three methods for oocyte activation, including 5 microM ionomycin (5 min, Group 1) alone, ionomycin+1.9 mM 6-dimethylaminopurine (DMAP, 3h, Group 2), and ionomycin+10 microg/ml cycloheximide (CHX, 3h, Group 3) were compared for the development of embryos produced by somatic nuclear transfer (SCNT) to parthenotes and IVF counterparts. At 19-h post-activation/insemination (hpa/hpi), 27.5% of oocytes in Group 2 cleaved and this rate was greater (P<0.05) than other groups (Group 1, 2.1%; Group 3, 3.0%). None of the oocytes in the IVF control group cleaved at 19-22 hpi. At 24 hpa, the rates of cleavage of oocytes in Group 2 (52.1%) were greater (P<0.05) than those in Groups 1 and 3 (7 and 38.3%, respectively). Only six oocytes (3.3%) in the IVF control group cleaved at 24 hpi. The overall cleavage rates of oocytes in Group 2 (85.5%) at 48 hpa were greater (P<0.05) than other treatments, but it did not show any difference when compared with the IVF control group (75.0%). The development rate to two-cell stage embryos of Group 2 was consistently greater at all observation points followed by Groups 3 and 1. Similar results were obtained in SCNT embryos, but the rates of cleavage at 48 hpi and blastocyst development in Group 2 (68.4 and 16.3%, respectively) did not differ from Group 3 (63.0 and 13.1%, respectively). The chromosomal composition in the parthenotes and SCNT embryos differed (P<0.05) among treatments. In Groups 1 and 3, greater percentages of haploid parthenotes (86 and 71%, respectively) were observed. In contrast, 84% of parthenotes in Group 2 had abnormal ploidy (44% polyploid and 40% mixoploid). In the case of SCNT embryos, Groups 1 and 3 had greater percentages of diploid chromosomal sets (77 and 70%, respectively), whereas 54% in Group 2 were polyploid or mixoploid. These results indicate that DMAP treatment after ionomycin greatly increases the developmental rates of parthenotes, but did not differ in blastocyst development compare with CHX treatment. However, DMAP treatment increased the time-dependent cleavage rate to two-cell stage embryos. Further, it greatly enhanced the incidence of chromosomal abnormalities in parthenotes and SCNT embryos. Hence, it is concluded that CHX combined with ionomycin is more desirable than DMAP for oocyte activation during nuclear transfer in cattle.     

Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos

Parthenote embryos offer multiple possibilities in biotechnological investigation, such as stem cell research. However, there is still a dearth of knowledge of this kind of embryo. In this study, development and ploidy were analysed in parthenotes under in vitro and in vivo culture conditions. Subsequently, using real-time PCR, the expressions of factor OCT-4, Vascular Endothelial Growth Factor, Epidermal Growth Factor Receptor 3 and Transforming Growth Factor β2 genes were analysed to compare the embryo types at the blastocyst stage. Development and implantation of parthenote embryos were described after transfer at day 10 of pregnancy. Parthenotes showed similar blastocyst development for both culture conditions and most of the parthenotes produced were diploid. However, parthenotes developed under in vivo conditions showed similar mRNA expression of OCT-4, VEGF and TGF-β2 to 5 and 6 day old blastocysts. In contrast, parthenotes developed under in vitro conditions had altered the expression pattern of these genes, except for erbB3 mRNA. Finally, transferred parthenotes had the ability to implant but showed severe growth retardation and lesser size. This is the first demonstration of the influence of culture conditions on parthenote mRNA expression. Our study highlights the importance of culture conditions in subsequent uses of parthenotes, such as the production of stem cell lines.     

Injection of mitochondria into oocytes and fertilized eggs]

The suspension of mitochondria isolated from the loach embryos or the frog heart were injected in the oocytes or fertilized eggs of the loach, newt, toad and frog in the amount roughly equivalent to the content of mitochondria in the egg. After the injection the oocytes did not differ during several days from the normal ones and the fertilized eggs of the loach, newt and South Afican clawed toad developed normally. The activity of cytochrome oxidase in the injected oocytes was kept at a somewhat higher level (1.4 to 1.9 vs 1.0 in the control) during several days. In the developing eggs the activity of cytochrome oxidase began to decrease from the blastula stage and attained rapidly the control level. The decrease of the enzyme activity is due to non-specific degradation of excessive mitochondria or to compensatory inactivation of the enzyme ensuring the maintenance of its normal activity during the development.     

The effect of energy substrate concentration and amino acids on the in vitro development of preimplantation porcine embryos

As the pig becomes increasingly used for biomedical research, an effective and efficient in vitro culture system is essential. This study aimed to improve the commonly used porcine embryo culture medium, NCSU23, by altering the energy substrates and adding amino acids, using electrically activated diploid parthenotes from oocytes obtained from the ovaries of prepubertal and adult animals. Morphological development to day 6 and blastocyst cell number were examined. Glucose (5.56 mM) was replaced by pyruvate and lactate (0.2 mM and 5.7 mM, respectively) for either the entire culture period or for the first 48 h only. Blastocyst rates were not different between any of the treatments, and were similar for prepubertal and adult oocytes. When the embryos were cultured with pyruvate and lactate for the first 48 h and then glucose, there was a significant increase in blastocyst cell number compared to glucose only. Blastocysts produced using pyruvate and lactate for the entire time tended to have more cells than those exposed to glucose only and less than those who were cultured in pyruvate and lactate for the first 48 h and then glucose. Nonessential amino acids added for the first 48 h and nonessential and essential amino acids added for the remaining time significantly increased blastocyst cell number only when the embryos were grown in pyruvate and lactate followed by glucose. Blastocyst rates were not different between any of the treatments, and this result was the same when using sow or gilt oocytes. The modified medium was then tested using in vitro matured and fertilized embryos from sow oocytes. Blastocyst rates and cell number were significantly increased in the modified medium compared to those grown in unmodified NCSU23. This shows that altering energy substrates and adding amino acids can increase the quantity and cell number of IVP blastocysts compared with NCSU23.