Diploid Parthenogenetic Embryos Research Articles

Oocyte activation is a cytoplasm-confined event so far: what about the nucleus?

The fertilizing spermatozoa induce a Ca2+ oscillatory pattern, the universal hallmark of oocyte activation, in all sexually reproducing animals. Assisted reproductive technologies (ARTs) like intracytoplasmic sperm injection (ICSI) bypass the physiological pathway; however, while a normal Ca2+ release pattern occurs in some species, particularly humans, artificial activation is compulsory for ICSI-fertilized oocytes to develop in most farm animals. Unlike the normal oscillatory pattern, most artificial activation protocols induce a single Ca2+ spike, undermining proper ICSI-derived embryo development in these species. Curiously, diploid parthenogenetic embryos activated by the same treatments develop normally at high frequencies and implant upon transfer in the uterus. We hypothesized that, at least in ruminant embryos, the oscillatory calcium waves late in the first cell cycle target preferentially the paternal pronucleus and are fundamentally important for paternal nuclear remodeling. We believe that Ca2+ signaling is central to full totipotency deployment of the paternal genome. Research in this area could highlight the asymmetry between the parental genome reprogramming timing/mechanisms in early development and impact ARTs like ICSI and cloning.

Caffeine improves the developmental competence of parthenogenetic embryos derived from aging porcine oocytes

Oocytes are committed to deterioration in quality as they aged due to a long duration manipulation which leads to the reduced success rate of somatic cell nuclear transfer (SCNT). Caffeine with an effect to maintain the maturation-promoting factor (MPF) from the metaphase of oocytes is expected to enhance the quality of the aging oocytes. To investigate the timely treatment of caffeine to rescue aging oocytes, caffeine was supplemented after in vitro maturation (IVM) or during metaphase I – metaphase II (MI – MII) transition. First, the effect of caffeine after IVM of oocytes was examined. After IVM for 42 h, oocytes were left for aging within 6 or 8 hours in supplement with various concentrations of caffeine (0, 5 and 10 mM), and then, examined the quality of embryo from aged oocyte through parthenogenesis activation. We found that 5 mM caffeine for the first 6 hours of aging process was suggested to improve the early development of parthenogenetic diploid embryos. However, the cytoplasmic homogeneity is significantly reduced in aging oocyte compared to fresh oocyte and it could not be improved by caffeine treatment. Next, the effect of caffeine during MI – MII transition of oocyte was examined. Caffeine was supplemented during MI – MII transition (27 – 42 h) of IVM. Then mature oocytes were left for aging within 6 h to examine on aging porcine oocyte quality via parthenogenesis embryos. The results indicated that 5 mM caffeine during MI-MII transition could efficiently rescue aged oocytes and improve the development of embryos derived from aging oocytes to four-cell, eight-cell and blastocyst stage as compared to fresh oocytes. Especially, these aged oocytes treated by caffeine could improve the cytoplasmic homogeneity in embryos and the quality of blastocysts by increasing cell number similar to fresh oocytes.

Production and development of porcine tetraploid parthenogenetic embryos.

The aim of this study was to produce porcine tetraploid (4N) parthenogenetic embryos using various methods and evaluate their developmental potential. In method 1 (M1), porcine 4N parthenogenetic embryos were obtained by inhibiting extrusion of both first (PB1) and second (PB2) polar bodies; in methods 2 (M2) and 3 (M3), 4N parthenogenetic embryos were obtained by electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 or PB1 extrusion, respectively. We found no differences in the rates of cleavage or blastocyst formation or the proportion of 4N embryos among M1, M2, and M3 groups. The different methods also did not influence apoptosis rates (number of TUNEL-positive cells/number of total cells) or expression levels of apoptosis-related BAX and BCL2L1 genes. However, total cell and EdU (5-ethynyl-2’-deoxyuridine)-positive cell numbers in 4N parthenogenetic blastocysts derived from M1 were higher (p < 0.05) than those for M2 and M3 groups. Our results suggest that, although porcine 4N parthenogenetic embryos could be produced by a variety of methods, inhibition of PB1 and PB2 extrusion (M1) is superior to electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 (M2) or PB1 (M3) extrusion.

Paternally and maternally imprinted gene perturbed expression in parthenogenetic diploid embryos in Capra hircus

The present research work was undertaken to compare the expression of paternally imprinted (P57KIP2, Grb10 and Xist) and maternally imprinted gene (Dlk1) among diploid parthenogenetic (DIP), female sexed in vivo and female sexed in vitro produced embryonic cell colony. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and activated by ethanol (7%) for 5 min followed by incubation with 2 mM 6-dimethyl amino purine (DMAP) for 4 h for DIP embryos production. In vivo embryo was collected by surgical method from 10 super ovulated goats using Pluset®. In vitro embryos were produced by maturation of good quality oocyte followed by fertilization with superior male germplasm from the institute flock. Embryonic cell colony were developed from 8 to 16 and morula stage DIP, IVF and in vivo derived embryos. These colonies were used for studying the paternal and maternal imprinted genes expression. Karyotyping and sexing of embryonic cell colony was done to get only female sexed colony for this purpose. Paternally imprinted genes (P57KIP2, Grb10 and Xist) showed more expression in DIP compared to in vivo and IVF derived embryonic cell colony. The expression of maternally imprinted genes (Dlk1) was low in DIP compared to in vivo and IVF derived embryonic cell colony. The present study demonstrated that the expression profile of both paternal and maternally imprinted genes were perturbed, which may be responsible for developmental failure of parthenogenetic embryos to full term following parthenogenetic embryo transfer in goat.

ID: 1066 Effects of biotin supplementation on the in vitro maturation of oocytes and the development of parthenogenetic diploid porcine embryos

In mammals, Biotin serves a coenzyme in the metabolism of glucose, amino acids, and fatty acids. Biotin deficiency causes decreased rates of cell proliferation, disfunction in germ cells and fetal development. This study was carried out to determine the influence of Biotin supplementation to invitro maturation medium on the development of porcine oocyte and embryos. Biotin (0.0, 1.0, 10.0, 100.0 mg/l, respectively) was added into the oocyte maturation medium, the quality of mature oocytes was evaluated after 42h culturing. The parthenogenetic diploid embryos were produced by using electro-activation system, the quality of embryos was noted at 1-4 cells stage. The results showed that, Biotin can enhance the formation of the first polar body at the concentration of 10 mg/l, it can also improve the activation efficiency of parthenogenetic diploid embryos at the preimplantation stage from 2-4 cells. Therefore, the supplementation of 10mg/l Biotin to the in-vitro maturation medium has a beneficial effect on the parthenogenetic diploid embryos development in the pig.

ID: 1069 Effect of Diutyryl-CAMP and follicle stimulating hormone on in vitro maturation of porcine oocyte

In this study, we examine the effects of dibutyryl cyclic adenosine monophosphate (dbcAMP) and follicle stimulating hormone (FSH) on the maturation of nucleus and cytoplasm of porcine oocytes after in vitro maturation. The oocytes-granulosa cell complexes (OCGs) were cultured in three different maturation media: basic, basic+dbcAMP, and basic+dbcAMP+FSH to investigate the cumulus expansion and maturation of the fully-grown oocytes. Treated mature oocytes underwent parthenogenetic activation to examine the quality of the mature oocytes via the development of embryos. The results showed that there was a higher rate of cumulus expansion and maturation between the combination of dbcAMP and FSH group (95.1% and 85.3%) compare with the none treatment or only dbcAMP treated group (40.8% and 47.7%; 44.9% and 42.9% respectively). The results in embryos development showed that mature oocyte cultured in dbcAMP and FSH group had a higher rate of the embryos developed to the 8-cell stage (49.2%) than in the none treated or only dbcAMP treated group (26.5% and 27.6%). These results confirmed that the combination of dbcAMP 1mM and FSH 0.01 IU/ml could increase the quality of the mature oocytes and improved the preimplantation development of parthenogenetic diploid embryos

Insulin like growth factor 2 (Igf2) and its receptor gene (Igf2r) showed opposite expression in diploid parthenogenetic embryos in Capra hircus

The present research work was proposed to compare the expression of development related genes (Igf2 and Igf2r) among diploid parthenogenetic (DIP), female sexed in vivo and female sexed in vitro produced embryonic cell colony. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and activated by ethanol (7%) for 5 min followed by incubation with 2 mM 6-dimethyl amino purine (DMAP) for 4 h for DIP embryos production and embryo development was recorded. We have explored comparative expression profile of paternally (Igf2r) and maternally (Igf2) imprinted genes among DIP, female sexed IVF and female sexed in vivo derived embryos. Embryonic cell colony were developed from 8–16 and morula stage DIP, IVF and in vivo derived embryos and these colonies were used for studying the paternal and maternal imprinted genes. Overall, we observed higher expression of maternally expressed genes (Igf2r) in DIP compared to in vivo and IVF derived embryonic cell colony. The expression of paternal genes (Igf2) was low in DIP compared to in vivo and IVF derived embryonic cell colony.

Microarray analysis of gene expression in parthenotes and in vitro–derived goat embryos

The present work was carried out to investigate the global gene expression profile to search differentially expressed candidate transcripts between parthenogenetic and in vitro–fertilized (IVF) caprine morula. For this study, total RNA was isolated from diploid parthenogenetic and IVF embryos, and complementary DNA was synthesized. Microarray and relative real-time polymerase chain reaction analysis were performed to check global gene expression profile and validation, respectively. According to the microarray analysis, the total number of upregulated (UR) and downregulated (DR) genes was 613 and 220, respectively in diploid parthenogenetic morula as compared with IVF morula. The number of genes showing about two-, two- to five-, five- to 10-, 10- to 20-, and above 20-fold UR and DR genes was 147, 229, 122, 59, and 56 and 94, 73, 18, 13, and 22, respectively. Five UR genes validated (PTEN, PHF3, CTNNB1, SELK, and NPDC1) and all of them were significantly higher in parthenotes, which was in accordance with microarray results, whereas the expression of DR (AURKC and KLF15) genes were downregulated in parthenotes as observed in microarray results but the difference was not significant (P < 0.05). In conclusion, our findings demonstrate differential expression of a large number of genes in parthenotes compared with IVF embryos, which may be the reason for aberrant parthenogenetic embryo development in caprine species.

Effect of Actin Polymerization Inhibitor During Oocyte Maturation on Parthenogenetic Embryo Development and Ploidy in Capra hircus

This study was designed to observe the effect of cytochalasin B (CCB) concentrations on ploidy and early development of parthenogenetic embryos in a caprine species. Caprine oocytes were matured in the presence of different concentrations of CCB (5, 10, 15, and 20 μg/ml) and activated by 7% ethanol followed by incubation with 2 mM DMAP. For embryos fertilized in vitro, oocytes were matured in maturation medium without CCB. The cleavage rate and further embryo development were significantly higher (P < 0.05) when oocytes were treated in this way. The percentage of embryos showed higher diploid values in 15 μg/ml CCB (83.66 ± 1.13), followed by 20 (72.22 ± 1.22), 10 (68.57 ± 1.17), and 5 μg/ml (62.00 ± 2.48). These results indicate that CCB with a concentration of 15 μg/ml in maturation medium can be used for the production of diploid parthenogenetic embryos in the caprine species.

Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.

Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Expression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α- was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α- throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

Ploidy Assessment of Porcine Haploid and Diploid Parthenogenetic Embryos by Fluorescent In Situ Hybridization Detecting a Chromosome 1-Specific Sequence, Sus scrofa Mc1 Satellite DNA

The aim of the present study was to examine the feasibility of fluorescent in situ hybridization (FISH) for detecting a chromosome 1-specific sequence as a means of assessing the ploidy of porcine parthenotes. In vitro-matured oocytes with the first polar body (PB) were electrically activated; some were treated with cytochalasin B to prevent second PB extrusion (1PB embryos), and the others extruded the second PB (2PB embryos). At the 2-cell stage, one and two FISH signals were detected in each nucleus of 2PB and 1PB embryos, respectively. Almost all cells of blastocysts derived from 1PB embryos retained two signals. In contrast, cells of blastocysts derived from 2PB embryos had two signals. These data demonstrate that FISH analysis allows precise ploidy assessment of porcine parthenogenetic embryos, hence providing a practical means of detecting ploidy transition during parthenogenetic embryogenesis.

63 OOCYTE GENOME CLONING USED IN TRANSGENIC BOVINE EMBRYO PRODUCTION

Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogenetic haploid blastomeres (PHB) from these embryos should be considered as a clone of the original gamete. Our main objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Moreover, we generated biparental homogeneous transgene-expressing embryos using PHB that express a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation in 5 μM ionomycin for 4 min followed by culture in SOF for 3 h to permit second polar body extrusion and subsequently treated with 1.9 mM DMAP for 3 h. To generate transgene-expressing PHB, parthenogenetic embryos were injected 3 h post-activation with pCX-EGFP–liposome complexes. These treatments were analysed by Fisher test (P &lt; 0.05). The cleavage rate of the haploid and diploid parthenogenetic embryos control was 87.3% (103/118) and 88.5% (108/122) respectively; (P &gt; 0.05). When the haploid activated oocytes were injected with pCX-EGFP–liposome complexes, the cleavage rate was 84.4% (54/64). These embryos showed 54.7% (35/54) of EGFP expression. In the second experiment, haploid parthenogenetic embryos (4 to 16 cells) were disaggregated and the PHB obtained were fused with a zona-free haploid male hemizygotes in order to reconstruct biparental embryos. Haploid male hemizygotes were generated by fertilization of enucleated oocytes. The haploid condition of parthenogenetic embryos and male hemizygotes was confirmed by karyotype analyzes. Reconstructed embryos were cultured in SOF medium in well of well system in 5% O2, 5% CO2, and 90% N2 at 39°C. The development of the reconstructed embryos expressing or not expressing EGFP reached 78.4% (29/37) and 61.1% (80/131) of cleavage; and 10.8% (4/37) and 8.4% (11/131) of blastocysts, respectively; (P &gt; 0.05). EGFP expression was observed in 100% of the reconstructed embryos, with 96.6% (28/29) of homogenic expression. The biparental IVF control showed 64.4% of cleavage and 22.5% of blastocysts (n = 98). The mean cell number of blastocysts were significantly different among the reconstructed biparental embryos (mean = 72.8) and the biparental IVF-embryos control (mean = 97.0). Additionally, the Oct-4 pattern expression was examined in the blastocysts, by inmunocytochemistry. Blastocysts displayed a pattern of Oct-4 expression similar to IVF-blastocyst control. Finally, biparental reconstructed blastocysts (n = 2) were transferred in recipient cows obtaining 1 pregnancy detected at 2 months after embryo transfer. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that it is an efficient technique to generate homogeneous transgene-expressing embryos. Haploid oocyte genome replication would have high potential in livestock production, as it increases the number of embryos produced from a single oocyte, and allows the selection of favourable genetic characteristics before biparental embryo reconstruction.

Expression of imprinted Igf2 and Peg1/Mest genes in postimplantation parthenogenetic mouse embryos treated with transforming growth factor α in vitro

The effect of transforming growth factor alpha (TGFt) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA x C57BL/6)F1 mice during the postimplantation period of embryogenesis. The PEs were treated with TGFalpha in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf2 and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf2 before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFalpha) PEs, neither gene was expressed at the same 21- to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30- to 35-somite stages, while the Peg1/Mest expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30- to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFalpha-treated PEs at the 40- to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40-45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFalpha can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.

Diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes

Epigenetic modifications are closely associated with embryo developmental potential. One of the epigenetic modifications thought to be involved in genomic imprinting is DNA methylation. Here we show that the maternally imprinted genes Snrpn and Peg1/ Mest were nearly unmethylated or heavily methylated, respectively, in their differentially methylated regions (DMRs) at the two-cell stage in parthenogenetic embryos. However, both genes were gradually de novo methylated, with almost complete methylation of all CpG sites by the morula stage in parthenogenetic embryos. Unexpectedly, another maternally imprinted gene, Peg3, showed distinct dynamics of methylation during preimplantation development of diploid parthenogenetic embryos. Peg3 showed seemingly normal methylation patterns at the two-cell and morula stages, but was also strongly de novo methylated in parthenogenetic blastocysts. In contrast, the paternally imprinted genes H19 and Rasgrf1 showed complete unmethylation of their DMRs at the morula stage in parthenogenetic embryos. These results indicate that diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes and that the aberrantly homogeneous status of methylation imprints may partially account for developmental failure.

Genomic Imprinting and Problem of Parthenogenesis in Mammals

Genomic imprinting belongs by its nature to problems of epigenetics, which studies hereditary changes in gene expression not related to defective sequences of DNA nucleotides. Epigenetic mechanisms of control, including genomic imprinting, are involved in many processes of normal and pathological development of humans and animals. Disturbances of genomic imprinting may lead to various consequences, such as formation of developmental anomalies and syndromes in humans, appearance of the large offspring syndrome and increased mortality upon cloning of mammals, and death of parthenogenetic embryos soon after implantation and beginning of organogenesis. The death of diploid parthenogenetic or androgenetic mammalian embryos is determined by the absence of expression of the genes of imprinted loci of the maternal or paternal genome, which leads to significant defects in development of tissues and organs. A review is provided of the studies aimed at search of possible normalization of misbalanced gene activity and modulation of genomic imprinting effects during parthenogenetic development in mammals.

Effects of growth factors FGF4, TGFα, and TGFβ1 on development of parthenogenetic embryos of C57BL/6 mice

We studied the effects of three growth factors, fibroblast growth factor (FGF4), transforming growth factor α (TGFα), and transforming growth factor β1 (TGFβ1), on development of diploid parthenogenetic embryos of C57BL/6 mice, which are not capable of developing to somatic stages. Parthenogenetic embryos were treated with growth factors at optimal doses in vitro at the morula-blastocyst stages and transplanted in the uterus of pseudopregnant females. FGF4 and TGFα improved the development of parthenogenetic embryos at the preimplantation stages and the number of blastocysts increased under the influence of TGFα. All three growth factors improved the implantation of embryos in the uterus. When FGF4 or TGFβ1 2.4 were added to the nutrient medium, 2.4 or 1.6%, respectively, of parthenogenetic embryos reached the somatic stages in utero. No somitic embryos were observed in the control. The treatment of parthenogenetic embryos with two growth factors, FGF4 and TGFβ1 , simultaneously increased the amount of somatic embryos to 7.5%, while combination of three growth factors in creased the amount of such embryos to 16.7%. In the latter case, some parthenogenetic embryos reached the stage of 25–27 pairs of somites and were 2.0–2.5 mm long. The data we obtained suggest that, when combined, the growth factors FGF4, TGFα, and TGFβ1 possessed a synergistic effect leading to a significant improvement of the development of parthenogenetic C57BL/6 embryos.

116 ACTIVE METHYLATION AND ACETYLATION OF HISTONE H3-K9 IN MOUSE EMBRYO WITH DIFFERENT PROPORTIONS OF MATERNAL AND PATERNAL GENOME

Epigenetic modification of parental genomes plays a prominent role in regulating genome expression in the early development of embryos. In general, histone H3 of the paternal genome is demethylated at lysine 9 (H3-K9) during the first and second mitotic divisions in fertilized embryos, while the maternal genome is methylated. We investigated the effects of maternal genomes (Mgen) and paternal genomes (Pgen) on H3-K9 methylation and acetylation during the early development of murine embryos. Histone H3-K9 methylation and acetylation were detected by anti-trimethyl histone H3-K9 and anti-triacetyl histone H3-K9 antibodies. The following embryos were used in this study: (1) intracytoplasmic sperm injection (ICSI) embryos (50% Mgen, 50% Pgen); (2) parthenogenetic diploid embryos (100% Mgen, 0% Pgen); (3) somatic nuclear transfer embryos (50% Mgen, 50% Pgen from previous generation); (4) androgenetic diploid embryos (0% Mgen, 100% Pgen); and (5) haploidized somatic nucleus and sperm embryo (about 25% Mgen, about 75% Pgen). Each experiment was repeated five times to obtain more than 120 embryos per group. Our results show that: (1) in the ICSI embryo, histone H3 methylation occurs in Mgen but not in Pgen at the first and second mitotic divisions; (2) in the parthenogenetic embryo, histone H3 methylation occurs in both nuclei at the first and second mitotic divisions; (3) in the somatic nuclear transfer embryo, histone H3 is methylated in all of the nuclei at the first and second mitotic divisions; (4) in the androgenetic embryo, methylated H3-K9 is detected weakly in the heterochromatin enclosed around the nucleolus of the pronuclei of the one-cell embryo, and methylated in the entire nuclei of the two-cell embryo; and (5) in the haploidized somatic and sperm embryo, the pattern of histone H3-L9 methylation resembles that of the ICSI embryo. While histone H3-K9 acetylation occurs in both paternal and maternal genomes during interphase, even when the nuclear membrane is completely degraded and the chromosome is condensed, it disappears rapidly when the chromosome enters the real metaphase, and reappears at the early stage of pronuclear formation in all types of embryo. These results suggest that the absence of maternal genomes results in histone H3-K9 methylation in the paternal genomes during the first and second mitotic divisions of embryos in mice. In addition, histone H3-K9 acetylation is independent of the presence or absence of maternal or paternal genomes during pre-implantation development in mice. This study was supported by grants-in-aid for Creative Scientific Research (13GS0008) and a project for the realization of regenerative medicine (the research field for the technical development of stem cell manipulation) to T.W. from MEXT, Japan.

171 NORMAL REPROGRAMMING OF IMPRINTING IN PARTHENOGENETIC FEMALE GERM CELLS

Mammalian parthenotes cannot develop normally to term. Mouse parthenogenetic embryos die by Day 10 of gestation. On the other hand, viable parthenogenetic chimeras were produced by normal host embryos, although parthenogenetic cells were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. This can be explained by the aberrant expressions of imprinted genes in parthenogenetic cells. In female mice, erasure of imprints occurs around the time that primordial germ cells enter the gonad, and establishment of imprints occurs in the postnatal growth phase of oogenesis. In this study, we investigated whether aberrant imprints in parthenogenetic embryonic stem (PgES) cells can be erased through the germline. Diploid parthenogenetic embryos were produced by activation of (CBA × C57BL/6-EGFP) F1 mouse superovulated unfertilized oocytes by exposure to Sr2+ and cytochalasin B. Ten parthenogenetic blastocysts were plated and three PgES cell lines were isolated. Chimeras were made by injecting 10–15 PgES cells into ICR(CD-1) mouse blastocysts. Chimeras and chimeric tissues were detected by fluorescent microscopy. In all, 173 chimeric blastocysts were transferred to 9 recipient females, and 101 live pups containing 9 female and 21 male chimeras were born. No significant growth retardation was apparent in PgES chimeras, irrespective of their degree of chimerism. In 5 male chimeras killed at 1 day postpartum (dpp), PgES cells showed a restricted tissue contribution. The contribution to lung, liver, and intestine was considerably lower than in the other tissues such as brain, heart, spleen, and kidney. PgES derived or host embryo derived non-growing oocytes were isolated from dissociated ovaries of female chimeras at 1 dpp under fluorescent microscopy. Methylation imprints in non-growing oocytes were analyzed for maternally methylated imprinted genes Peg1, Snrpn, and Igf2r by the combined bisulfite restriction analysis (COBRA). In normal oocytes, imprints are expected to be erased and these genes are unmethylated at this stage. We observed that these genes were unmethylated in both PgES derived and host embryo derived non-growing oocytes. These results suggest that aberrant imprints in PgES cells can also be erased normally through the germline.

191IN VIVO AND IN VITRO DIFFERENTIATION OF EMBRYONIC STEM CELLS DERIVED FROM PARTHENOGENETIC EMBRYOS IN MICE

Availability of embryonic stem (ES) cells opens the prospect for regenerative medicine. However, ES cells genetically mismatched to diseased individuals cause immunological rejection. In this study, we established ES cells from parthenogenetic embryos in mice and examined their pluripotency. Oocytes were collected from (C57BL/6xDBA)F1 mice (BDF1) by superovulation. Parthenogenetic diploid embryos were produced by activation treatment in 5mM SrCl2 in Ca2+-free KSOM medium for 2h, followed by cultivation in 5μgmL−1 cytochalasin B for 6h. The zonae pellucidae of embryos developed to the blastocyst stage in vitro were removed by a 5-min incubation in 0.5% pronase. Inner cell masses (ICMs) isolated immunosurgically were seeded on the feeder layers (mitomycin C-treated mouse embryonic fibroblasts) in DMEM supplemented with 15% Knock-Out Serum Replacement (Invitrogen), 2mM L-glutamine, non-essential amino acids, β-mercaptoethanol and 103UmL−1 of Leukemia inhibitory factor (LIF) at 37°C in a humidified atmosphere with 5% CO2 in air. The attached ICM cells were mechanically disaggregated and seeded on the fresh feeder layers. After several passages, parthenogenetic ES (PnES) cell lines were established. The efficacy of establishing PnES cell lines was 66% (37/56). To examine the characteristics of PnES cell lines, seven lines were subjected to histochemical and immunohistochemical analysis. All showed alkaline phosphatase activity and immunoreactivity to anti-SSEA-1 and anti-Oct4 antibodies. They maintained euploid sets of choromosomes at 29; 59%. PnES cells from two of the seven lines were injected into 59 host blastocysts obtained from ICR mice, resulting in 16 chimeric offspring (27%). In another experiment, injection of ICM cells and ES cells obtained from fertilized BDF1 blastocysts and ICM cells obtained from BDF1 parthenogenetic blastocysts also produced chimeric offspring (35%, 7/20; 46%, 6/13; and 53%, 10/19, respectively). However, no chimeric mouse with germline transmission was obtained from PnES cells. Injection of 1×107 of PnES cells into SCID mice formed teratocarcinomas. Immunohistochemical analysis showed cells positive for nestin (specific to neuroepitherial stem cells), Tu-J (class III β-tublin), NF-M (neurofilament), desmin (muscle), and albumin (hepatocytes), which indicated their differentiation potency to the cells derived from all three germ layers. Simple embryoid bodies produced from these cell lines were plated on tissue culture dishes under conditions for induction of differentiation. Immunohistochemistry and RT-PCR analysis showed their differentiation into neurons (NF-M, nestin), cardiomyocytes and hepato-like cells (albumin, α-fetoprotein). Our results indicate that PnES cells are pluripotent similar to the ES cells from fertilized embryos except for germline transmission and should be tested in cell replacement animal models.