Androgenetic Embryos Research Articles

Effect of increased copper ion content in the medium on the regeneration of androgenetic embryos of carrot (Daucus carota L.)

The study was conducted to determine the effect of elevated concentrations of copper in the medium on the regeneration of androgenetic embryos of the carrot cultivar ‘Kazan F<sub>1</sub>’ obtained in anther cultures and to determine the level of soluble phenols produced in the regenerates under copper stress. Green embryos were laid out on 4 regeneration media based on B5 medium (G a m b o r g et al. 1968) without hormones, containing 0.1 – control, 1, 10, and 100 μM CuSO<sub>4</sub>×5H<sub>2</sub>O. The plant material was passaged 3 times, after 4, 9 and 15 weeks. During these passages the emerging structures were examined; they were classified in terms of growth and development <i>in vitro</i>, weighed and counted. The levels of soluble phenols in the freeze-dried regenerates were determined. The elevated concentrations of copper in the regeneration media affected positively the formation of complete plants (rooted rosettes) and secondary embryos during the first 4 weeks of culture. After a longer regeneration time (9, 15 weeks), the elevated concentrations of copper caused negative effects: deformation of rosettes. After 15 weeks, the number of rooted rosettes decreased. The 9-week culture subjected to copper stress brought about an increase in the amounts of soluble phenols. The highest values were recorded in the rosettes treated with 10 μM CuSO<sub>4</sub>. Prolonged exposure to media containing elevated concentrations of CuSO<sub>4</sub> caused a reduction in the accumulation of phenolic compounds in the rosettes.

Efficient Production and Cellular Characterization of Sheep Androgenetic Embryos

The production of androgenetic embryos in large animals is a complex procedure. Androgenetic embryos have been produced so far only in cattle and sheep using pronuclear transfer (PT) between zygotes derived from in vitro fertilization (IVF) of previously enucleated oocytes. PT is required due to the poor developmental potential of androgenotes derived from IVF of enucleated oocytes. Here we compare the developemt to blastocyst of androgenetic embryos produced by the standard pronuclear transfer and by fertilization of oocytes enucleated in Ca2+/Mg2+-free medium, without pronuclear transfer. The enucleation in Ca2+/Mg2+-free medium abolished almost completely the manipulation-induced activation, significantly improving the development to blastocyst of the androgenetic embryos (IVF followed by PT; 18.6%: IVF only; 17.7%, respectively). Karyotype analysis of IVF revealed a similar proportion of diploid embryos in androgenetic and control blastocysts (35% and 36%, respectively), although mixoploid blastocysts were frequently observed in both groups (64%). Androgenotes had lower total cell numbers than control and parthenogenetic embryos, but more cells in ICM cells comparing to parthenogenotes (30.42 vs. 17.15%). Higher expression of the pluripotency-associated gene NANOG, and trophoblastic-specific gene CDX2, were also observed in androgenotes compared to parthenogenotes and controls. The global methytion profile of androgenetic embryos was comparable to controls, but was lower than parthenogenetic embryos. The cell composition and methylation pattern we have detected in monoparental sheep monoparental embryos are unprecedented, and differ considerably from the standard reference mouse embryos. Altogether, these finding indicate significant differences across species in the molecular mechanisms regulating early development of monoparental embryos, and highlights the need to study postimplantation development of androgenetic embryos in sheep.

Essential role of paternal chromatin in the regulation of transcriptional activity during mouse preimplantation development

Several lines of evidence indicate that the formation of a transcriptionally repressive state during the two-cell stage in the preimplantation mouse embryo is superimposed on the activation of the embryonic genome. However, it is difficult to determine the profile of newly synthesized (nascent) RNA during this phase because large amounts of maternal RNA accumulate in maturing oocytes to support early development. Using 5-bromouridine-5'-triphosphate labeling of RNA, we have verified that nascent RNA synthesis was repressed between the two-cell and four-cell transition in normally fertilized but not in parthenogenetic embryos. Moreover, this repression was contributed by sperm (male) chromatin, which we confirmed by studying androgenetic embryos. The source of factors responsible for repressing nascent RNA production was investigated using different stages of sperm development. Fertilization with immature round spermatids resulted in a lower level of transcriptional activity than with ICSI at the two-cell stage, and this was consistent with further repression at the four-cell stage in the ICSI group. Finally, study on DNA replication and chromatin remodeling was performed using labeled histones H3 and H4 to differentiate between male and female pronuclei. The combination of male and female chromatin appeared to decrease nascent RNA production in the fertilized embryo. This study indicates that paternal chromatin is important in the regulation of transcriptional activity during mouse preimplantation development and that this capacity is acquired during spermiogenesis.

Chromosome rearrangements and survival of androgenetic rainbow trout (Oncorhynchus mykiss)

The purpose of this work was to quantify the impact of spontaneous and X-radiation-induced chromosome rearrangements on survival rate of androgenetic rainbow trout (Oncorhynchus mykiss). Various doses of X irradiation (50, 150, 250, 350 Gy) were used for inactivation of nuclear DNA in oocytes. After the irradiation, eggs were inseminated with normal sperm from 4 males derived from a strain characterized by Robertsonian rearrangements and length polymorphism of the Y chromosome. The haploid zygotes were exposed to a high hydrostatic pressure (7000 psi) to duplicate the paternal DNA. Neither Robertsonian chromosome polymorphism nor the Y chromosome morphology impaired the viability of the androgenetic embryos and alevins. Moreover, survival of eyed embryos of the androgenetic rainbow trout increased significantly with increasing doses of oocyte X irradiation. After 6 months of rearing, only specimens from the 250 and 350 Gy variants survived. The number of fingerlings with remnants of the maternal genome in the forms of chromosome fragments was higher in the 250 Gy group. Intraindividual variation of chromosome fragment number was observed, and some individuals exhibited haploid/diploid mosaicism and body malformations. Individuals irradiated with less than 250 Gy died, presumably because of the conflict between intact paternally derived chromosomes and the residues of maternal genome in the form of chromosome fragments.

Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures

Microspores were cultured on the modified B5 liquid medium containing 2.4D (0.1 mg L(-1)), NAA (0.1 mg L(-1)), L-glutamine (500 mg L(-1), L-serine (100 mg L(-1)), and sucrose (100 g L(-1)). The developmental stages of microspores and divisions were observed. Initially, the formation of binuclear and multicellular structures was noticed. Plants regenerated in the cultures in which the tetrad stage of microsporogenesis had predominated. Embryoids were still forming 24 weeks after the cultures were set up. Six weeks after the transfer of androgenetic embryos onto the B5 regeneration medium, they were converted into complete plants. Out of 90 androgenetic plants planted in a growth chamber, 42 plants adapted to the new conditions. All of those plants proved to be diploids in cytometric analysis.

6 EFFICIENT TRANSGENESIS IN BOVINE EMBRYOS BY FERTILIZATION WITH ANDROGENETIC TRANSGENIC BLASTOMERES

Pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression is observed in embryos and offspring produced by these techniques. A possible explanation is that the transgene integrates in the embryo genome after the first cell division. Our main objective was to develop a new technique to generate transgenic bovine embryos without mosaic expression and with high efficiency. We hypothesize that fertilizing metaphase II (MII) oocytes with transgenic androgenetic haploidblastomeres (AHB) (from mosaic embryos) would result in non-mosaic transgenic embryos. To this aim, in the first experiment we generated AHB by enucleating IVM MII oocytes, before or after injecting with a single spermatozoon incubated with pCX-EGFP (enhanced green fluorescent protein) plasmid. These treatments were analyzed by Fisher test (P < 0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated before and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116), respectively (P < 0.05). These embryos showed expression of EGFPof 11.8% (4/34) and 42.3% (30/71) (P < 0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analysis. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4 to 16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out by fusing a single AHB to a zona-free MII oocyte, followed by chemical activation. Presumptive zygotes were cultured in SOF medium in the well of the well (WOW) system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them were checked by PCR using Y- and X-specific sequence primers. PCR analysis confirmed Y-specific sequences in all the AHB and XY-specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the expression pattern of Oct-4 (pluripotent marker gene) was examined in the blastocysts by inmunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to that of IVF embryos, indicating efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n = 84). One hundred percent of the embryos showed EGFP expression, with 90.1% non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB is a highly efficient technique for the generation of transgenic non-mosaic bovine embryos.

Plant regeneration from carrot (Daucus carota L.) anther culture derived embryos

The research concerned of the regeneration of plants from embryos obtained from anther cultures of seven carrot (Daucus carota L.) cultivars. The aim was to determine the influence of the regeneration medium on the efficiency of the regeneration process. The optimization of the adaptation of the obtained plants was also carried out. Embryogenesis occurred on four of the tested media: B5 and MS without hormones, MS with charcoal, and MS with 1 mg dm−3 BA and 0.001 mg dm−3 NAA. Embryos obtained from the anther cultures produced secondary embryos, from which the regenerations of plants was observed. Secondary embryos were formed most extensively on the B5 medium without hormones. The efficiency of the regeneration process depended on the cultivar. Most of the secondary embryos were formed by androgenetic embryos of the cultivar ‘Feria F1’. The highest number of plants (102) regenerated from one embryo during 12 weeks of culture was also obtained in case of the cultivar ‘Feria F1’. Secondary embryogenesis and plant regeneration from embryos allow to omit the difficult stage of root induction applied when plants are regenerated form shoots' explants. This makes the plant regeneration process quicker and easier. The plants regenerated by the conversion of embryos are better adapted to the ex vitro conditions than those obtained in the two-stage organogenesis involving the regeneration of shoots and in second stage roots induction.

Developmental Ability of Trophoblast Stem Cells in Uniparental Mouse Embryos

Neither parthenogenetic (PG) nor androgenetic (AG) mouse embryos survive after day 9.5 of pregnancy, owing to the inadequate growth of extraembryonic tissues, including the placenta. At day 9.5 of pregnancy, the placental structures are poorly developed in PG embryos, while trophoblast giant cells are abundant at the implantation site in AG embryos. These findings suggest that both parental genomes are required for placental development. To gain further insight into the trophoblast lineage in PG and AG embryos, we attempted to derive trophoblast stem (TS)-like cell lines from uniparental embryos. Furthermore, we sought to assess their ability to differentiate into cells of the trophoblast lineage by using gene expression analysis. Three cell lines that expressed marker genes for undifferentiated TS cells ( Cdx2 and Errβ) were derived from AG embryos. Under differentiation conditions, these cells expressed the trophoblast giant cell-specific genes, but did not express the spongiotrophoblast-specific genes. In contrast, none of the four cell lines from PG embryos expressed marker genes for undifferentiated TS cells, but they expressed Oct3/4, a marker gene for embryonic stem cells. Immunohistochemical analysis indicated that PG blastocysts expressed Oct3/4 and Cdx2 specifically in inner cell mass and the trophectoderm respectively. These results suggest that PG embryos do not possess TS cells, because of the lack of the developmental ability of trophoblast cells.

DNA methylation in the IGF2 intragenic DMR is re-established in a sex-specific manner in bovine blastocysts after somatic cloning

The recent identification of an intragenic differentially methylated region (DMR) within the last exon of the bovine Insulin-like growth factor 2 ( IGF2) gene provides a diagnostic tool for in-depth investigation of bovine imprinting and regulatory mechanisms which are active during embryo development. Here, we used bisulfite sequencing to compare sex-specific DNA methylation patterns within this DMR in bovine blastocysts produced in vivo, by in vitro fertilization and culture, SCNT, androgenesis or parthenogenesis. In in vivo derived embryos, DNA methylation was removed from this intragenic DMR after fertilization, but partially replaced by the time the embryo reached the blastocyst stage. Among embryos developing in vivo, the level of DNA methylation was significantly lower in female than in male blastocysts. This sexual dimorphism was also found between parthenogenetic and androgenetic embryos, and followed the donor cell sex in SCNT derived blastocysts and is evidence for correct methylation reprogramming in SCNT embryos.

Efficient production of androgenetic embryos by round spermatid injection

Mammalian androgenetic embryos can be produced by pronuclear exchange of fertilized oocytes or by dispermic in vitro fertilization of enucleated oocytes. Here, we report a new technique for producing mouse androgenetic embryos by injection of two round spermatid nuclei into oocytes, followed by female chromosome removal. We found that injection of round spermatids resulted in high rates of oocyte survival (88%). Androgenetic embryos thus produced developed into mid-gestation fetuses at various rates, depending on the mouse strain used. All the fetuses examined maintained paternally specific genomic imprinting memories. This technique also enabled us to produce complete heterozygous F1 embryos by injecting two spermatids from different strains. The best rate of fetal survival (12% per embryos transferred) was obtained with C57BL/6 x DBA/2 androgenetic embryos. We also generated embryonic stem cell lines efficiently with the genotype of Mus musculus domesticus x M. m. molossinus. Thus, injection of two round spermatid nuclei followed by maternal enucleation is an effective alternative method of producing androgenetic embryos that consistently develop into blastocysts and mid-gestation fetuses.

Genome-wide and gene specific paternal demethylation in androgenetic embryos

In the present study, a genome-wide measure of demethylation employing the 5 methylcytidine antibody as well as a gene specific approach by bisulphite sequencing of a house keeping and imprinted genes was investigated to confirm if the active paternal demethylation was occurred correctly in mouse haploid, diploid androgenetic and triploid polyspermic embryos. The results indicated that the active demethylation of paternal genome in haploid, diploid, and triploid embryos occurred as similar as the normal ICSI embryos and completed the full demethylation within 6, 8, and 10 h after fertilization, respectively. The methylated CpG dinucleotides sites of alpha-actin, paternally expressed Igf2, paternal methylated Gtl2 and H19 gene loci were hypermethylated in mature sperm. After fertilization, methylated sites of two paternal methylated genes retained their methylation status whereas the paternal alleles of alpha-actin and Igf2 rapidly underwent active demethylation in the diploid androgenetic embryos produced by two sperm injection into enucleated oocytes or pronuclear transplantation. These results indicated that no specificity of paternal specific active demethylation was found in androgenetic or triploid polyspermic embryos.

Effect of Increased Copper Concentrations on Deformations of the Regenerates of Carrot Obtained from Androgenetic Embryos

Effect of Increased Copper Concentrations on Deformations of the Regenerates of Carrot Obtained from Androgenetic Embryos The experiment aimed at determining the effect of high copper concentrations on the process of regeneration of carrot plants cv. Feria F1 from androgenetic embryos. The starting material consisted of green embryos obtained in anther cultures on a medium for the induction of androgenesis. Five B5 regeneration media of Gamborg et al. (1968) containing 0.1 (control), 1, 10, 100, and 1000 μM·L-1 CuSO4·5H2O were used. The plant material was passaged onto fresh media 3 times after 4, 9, and 15 weeks from culture set-up date. During the passages, the regenerated structures were examined and categorized according to their growth, development and the course of regeneration in the in vitro culture. The B5 regeneration medium that contained 1000 μM·L-1 CuSO4 was highly toxic for the growth and development of androgenetic embryos of carrot. The other concentrations of CuSO4 that were higher than the level of the control medium, i.e. 1, 10, and 100 μM·L-1, had a negative effect on the regeneration from androgenetic embryos of carrot in the second and third passages (after 9 and 15 weeks). They caused deformations of the leaves and the formation of an abnormal callus heel between the above-ground part and the root.

Development of Sheep Androgenetic Embryos Is Boosted following Transfer of Male Pronuclei into Androgenetic Hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.

Genomic DNA Damage in Mouse Transgenesis1

Creating transgenic mammals is currently a very inefficient process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In the present study, we demonstrate that this loss is due to chromosomal damage. We examined the integrity of chromosomes in embryos produced by microinjection of pronuclei, intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF)-mediated transgenesis, and correlated these findings with the abilities of embryos to develop in vitro and yield transgenic morulas/blastocysts. Chromosomal analysis was performed after microinjection of the pronuclei in zygotes, as well as in parthenogenetic and androgenetic embryos. In all the pronuclei injection groups, significant oocyte arrest and increased incidence of chromosome breaks were observed after both transgenic DNA injection and sham injection. This indicates that the DNA damage is a transgene-independent effect. In ICSI-mediated transgenesis, there was no significant oocyte arrest. The observed chromosomal damage was lower than that after pronuclei microinjection in zygotes and was dependent upon the presence of exogenous DNA. The occurrence of DNA breaks, as measured by comet assay performed on the sperm prior to ICSI, showed that DNA damage was present in the sperm before fertilization. Embryonic development in vitro and transgene expression at the morula/blastocyst stage were higher in ICSI-mediated transgenesis than after microinjection of pronuclei into zygotes. Sperm-mediated gene transfer via IVF did not affect chromosome integrity, allowed good embryo development, but did not yield any transgenic embryos. The present study demonstrates that DNA damage occurs after both the microinjection of pronuclei and ICSI-mediated transgenesis, albeit through different mechanisms.

Evaluation of different doses of UV irradiation to loach eggs for Genetic inactivation of the maternal genome

Genetic inactivation of the egg nucleus is an indispensable step in the production of androgenetic embryos in teleosts. However, few experimental studies have focused on determining the most effective means of achieving complete inactivation of the maternal genome. Here, we sought to identify the optimum conditions of ultraviolet (UV) irradiation for complete inactivation of the loach egg nucleus. Unfertilized eggs were UV irradiated from above with a dose in the range 0-200 mJ/cm2. Successful inactivation of the maternal genome was evaluated by the exclusive expression of a paternally inherited color phenotype. The presence or absence of putative maternal chromosome fragments was screened by flow cytometry of DNA content and by cytogenetic analysis. The majority of the larvae derived from irradiated eggs had an abnormal appearance. Haploid individuals were detected by measurement of DNA content flow cytometry and by chromosome counting in the groups that received more than 75 mJ/cm2 groups. Although the coefficient of variation of DNA content was apparently reduced in the 125-200 mJ/cm2 groups, chromosome fragments were still detected in all the groups from irradiated eggs. Inactivation of the egg nucleus was also histologically elucidated by the presence or absence of residual nuclear material in anuclear embryos that developed from UV-irradiated eggs fertilized with UV-irradiated sperm. Embryos that were completely or near-completely anuclear were found in the 150 and 200 mJ/cm2 groups. We conclude that the optimum UV dose for complete genetic inactivation of the egg nucleus is more than 150 mJ/cm2.

Regeneration and Evaluation of Androgenetic Plants of Head Cabbage (Brassica Oleracea Var. Capitata L.)

Regeneration and Evaluation of Androgenetic Plants of Head Cabbage (Brassica Oleracea Var. Capitata L.) Plant regeneration from head cabbage embryos obtained in anther culture, the way of colchicine using for doubling number of chromosomes in haploid plants and estimation of the double haploid plants with regard to vigour, typicality of leaves and fertility were worked out. The best medium for plant regeneration from androgenetic embryos was B5 medium, enriched with sucrose 20 g.L-1 and kinetin 20 mg.L-1. On this medium, the highest number of embryos formed shoots, usually several from 1 embryo, particularly in subsequent passages. The most effective method of doubling the number of chromosomes was soaking the roots of young plants, when they had 5-6 leaves in a 0.1% hydrous solution of colchicine with 4.0% DMSO and 25 ppm GA3, for 20 hrs. The tested cultivars of head cabbage differed considerably with regard to the vigour of the plants obtained from anther culture. The highest number of plants characterized by strong vigour was observed in the cultivar Langendijker. About 90% of head cabbage plants obtained from androgenetic embryos had typical leaves and the differences between the cultivars in this aspect were insignificant. The highest number of androgenetic androfertile plants was found in the cultivar Sława z Enkhuizen. The genotype was the factor which had an impact in the processes examined: ability to shoot proliferation, rooting, the sensitivity to colchicine treatment, vigour of produced doubled haploid plants and their fertility.

Duplication of the sperm genome by human androgenetic embryo production: towards testing the paternal genome prior to fertilization

There is currently no technique for evaluating the sperm genome before fertilization. However, sperm genome duplication could offer a way forward, whereby one of the sister blastomeres of a 2-cell haploid androgenetic embryo could be analysed. A method was developed for production of human androgenotes by enucleation of oocytes at telophase II (TII) after intracellular sperm injection (ICSI). The results were compared with those obtained via the more usual procedure of oocyte enucleation at metaphase II (MII) prior to ICSI. TII enucleation led to an improvement in the rate of embryo survival, increased the production rate of 1PN-embryos, and also the production of 2- to 8-cell-stage embryos (85.0, 74.9 and 65.8% in TII enucleation, versus 73.8, 48.9 and 33.3% in MII enucleation). Fluorescence in-situ hybridization (FISH) analysis of 30 2- to 5-cell androgenic embryos for two to seven chromosomes revealed the correct chromosome distribution in 76.7% of haploid human androgenotes.

203 PRODUCTION OF ANDROGENETIC SHEEP BLASTOCYSTS

The androgenetic embryo is a useful model for investigating the contribution of the paternal genome (e.g. genomic imprinting) to embryonic development. Few works on androgenetic embryo production in domestic animals exist. In this study, we compared the developmental ability of diploid, haploid, and triploid androgenetic sheep embryos. In vitro-matured metaphase II oocytes were enucleated in HEPES-buffered TCM-199 with cytochalasin B (7.5 µg mL−1) and Hoechst 33342 (5 µg mL−1) under UV light using a Narishighe Micromanipulator fitted to an inverted Nikon microscope. Enucleated oocytes were fertilized in vitro with a high sperm concentration (2.5 × 107 sperm mL−1). Fifteen hours after in vitro fertilization (IVF), embryos were centrifuged (12 000g for 10 min) to visualize the pronuclei; the number of pronuclei were scored under the inverted microscope. In Experiment 1, IVF (control), haploid (1 pronucleus), diploid (2 pronuclei), and triploid (3 pronuclei) embryos were cultured in SOFaa medium with BSA, according to the protocol in our laboratory (Ptak et al. 2002 Biol. Reprod. 67, 1719–1725). In Experiment 2, we performed pronuclear transfer to produce diploid embryos. A single pronucleus was aspirated with a bevelled pipette from haploid (haploid + haploid) or diploid (diploid + haploid) embryos and transferred into the perivitelline space of another haploid embryo. The reconstructed zygotes were electrofused in 0.27 M mannitol solution with 50 µM CaCl2 and 100 µM MgCl2 by a single DC pulse (0.8 kV cm−1 for 80 µs). As a control group for Experiment 2, IVF embryos (pronuclear stage) were centrifuged, followed by the aspiration of a small volume of cytoplasm, and fused under the same condition of diploidization. In Experiment 1, there was no significant difference in cleavage rate (91% to 98%), but there was a significant difference on blastocyst development between IVF and androgenetic embryos (IVF: 43% (26/60); haploid: 0% (0/37); diploid: 1% (1/73); and triploid: 2% (1/48)). In Experiment 2, there was no significant difference in cleavage rate (94% to 100%). However, there was a significant difference on blastocyst development (Control: 42%; haploid + diploid: 19%; and haploid + haploid: 3%; Table 1). Our results suggest that sheep androgenetic embryos show poor developmental ability compared with IVF embryos. Interestingly, diploid androgenetic embryos produced by IVF displayed very poor development; however, such poor development was rescued, for unknown reasons, by pronuclear transfer. Ongoing experiments will provide new insight into this previously uncharacterized phenomenon. In conclusion, pronuclear transfer was an effective method for producing sheep androgenetic blastocysts in vitro. Table 1.Development of androgenetic embryos after pronuclear transfer

Expression profiling of uniparental mouse embryos is inefficient in identifying novel imprinted genes

Imprinted genes are expressed from only one allele in a parent-of-origin-specific manner. We here describe a systematic approach to identify novel imprinted genes using quantification of allele-specific expression by Pyrosequencing, a highly accurate method to detect allele-specific expression differences. Sixty-eight candidate imprinted transcripts mapping to known imprinted chromosomal regions were selected from a recent expression profiling study of uniparental mouse embryos and analyzed. Three novel imprinted transcripts encoding putative non-protein-coding RNAs were identified on the basis of parent-of-origin-specific monoallelic expression in E11.5 (C57BL/6 × Cast/Ei)F1 and informative (C57BL/6 × Cast/Ei) × C57BL/6 backcross embryos. In addition, four transcripts with preferential expression of a strain-specific allele were found. Intriguingly, a vast majority of the analyzed transcripts showed no imprinting-associated expression in F1 embryos. These data strengthen the view that a large fraction of nonimprinted genes is differentially expressed between parthenogenetic and androgenetic embryos and question the efficiency of expression profiling of uniparental embryos to identify novel imprinted genes.

168 ESTABLISHMENT OF TROPHOBLAST STEM CELLS FROM MOUSE ANDROGENETIC AND PARTHENOGENETIC EMBRYOS

Mouse androgenetic and parthenogenetic embryos, which have two sets of paternal and maternal genomes, respectively, are lethal until Day 9.5 of pregnancy, because the growth of the extraembryonic tissues including placenta is inadequate. These results suggest that both parental genomes are involved in placental development. However, the reason for placental deficiency in androgenetic and parthenogenetic embryos has been unclear. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage, have been used to elucidate the mechanism of differentiation and gene function in the development of the placenta. In this study, to characterize trophoblast lineage in androgenetic and parthenogenetic embryos, we examined TS cells from these embryos, and assessed their ability to differentiate into trophoblast lineage. The ovulated MII oocytes from B6D2F1 (C57BL/6 � DBA2) female mice were used for the uniparental embryo production. In order to produce parthenogenetic embryos, the oocytes were activated in SrCl2 and cytochalasin B. The androgenetic embryos were produced by in vitro fertilization using enucleated oocytes. These uniparental embryos were cultured for 3.5–4.5 days to develop into blastocysts. TS-like cell lines were established from these blastocysts, as described previously (Tanaka et al. 1998 Science 282, 2072–2075). TS-like cells were cultured without feeder cells in conditioned medium containing FGF4 and heparin to maintain the stem cell conditions. The expression of 5 TS cell maker genes (Eomes, CdX2, Fgfr2, Ap2a, and Errb) in TS-like cells was analyzed by RT-PCR and northern blotting. In addition, the localization of CDX2 was detected using immunostaining. To cause their differentiation into giant cells, TS-like cells were cultured for 6 days in TS medium without FGF4 and heparin. The giant cells were detected by the expression of 2 giant cell marker genes, Hand1 and Pl-1. TS cells from fertilized embryos between B6D2F1 male and female mice were also established as controls. Five TS cell marker genes were expressed both in androgenetic TS-like cells (ATS) and in parthenogenetic ones (PTS). The CDX2 gene was localized in the nucleus in both ATS and PTS; however, the number of CDX2-positive cells decreased in PTS. After 6 days of differentiation, Hand1 and Pl-1 were expressed and giant cells were detected in differentiated derivatives in ATS. On the other hand, giant cells were not detected in differentiated derivatives in PTS. These results suggest that parental genomes may regulate the gene expression independently to differentiate into trophoblast lineage. In conclusion, TS-like cells from uniparental embryos are useful tools to aid the understanding of the function of the parental genomes during placental development.