Parthenogenetic Mouse Embryos Research Articles

Comparative maternal protein profiling of mouse biparental and uniparental embryos.

BackgroundMaternal proteins have important roles during early embryonic development. However, our understanding of maternal proteins is still very limited. The integrated analysis of mouse uniparental (parthenogenetic) and biparental (fertilized) embryos at the protein level creates a protein expression landscape that can be used to explore preimplantation mouse development.ResultsUsing label-free quantitative mass spectrometry (MS) analysis, we report on the maternal proteome of mouse parthenogenetic embryos at pronucleus, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages and highlight dynamic changes in protein expression. In addition, comparison of proteomic profiles of parthenogenotes and fertilized embryos highlights the different fates of maternal proteins. Enrichment analysis uncovered a set of maternal proteins that are strongly correlated with the subcortical maternal complex, and we report that in parthenogenotes, some of these maternal proteins escape the fate of protein degradation. Moreover, we identified a new maternal factor-Fbxw24, and highlight its importance in early embryonic development. We report that Fbxw24 interacts with Ddb1-Cul4b and may regulate maternal protein degradation in mouse.ConclusionsOur study provides an invaluable resource for mechanistic analysis of maternal proteins and highlights the role of the novel maternal factor Fbw24 in regulating maternal protein degradation during preimplantation embryo development.

Effects of PXD101 and Embryo Aggregation on the In Vitro Development of Mouse Parthenogenetic Embryos.

To improve the isolation efficiency of parthenogenetic embryonic stem cells (pESCs) in mice, it is necessary to optimize the method to increase in vitro developmental competence of mice parthenogenetic blastocysts. Therefore, this study aims to investigate an optimal method for the production of mouse parthenogenetic blastocysts and isolation of pESC colonies by comparing the effects of two methods: (1) the treatment of histone deacetylase inhibitor PXD101 before, during, or after parthenogenetic activation; (2) parthenogenetic embryo aggregation; and (3) their combination treatment. The results suggest that application of PXD101 treatment and embryo aggregation could both improve the development of mouse parthenogenetic blastocysts (50 nM PXD101 treated 4 hours during activation and further 4 hours after activation: 40.0% vs. 20.0%; p < 0.05; two-cell embryo aggregation: 38.3% vs. 20.0%; p < 0.05) and also enhance the isolation rate of pESC colonies (PXD101: 33.3% vs. 11.8%; p < 0.05; two-cell embryo aggregation: 36.4% vs. 11.8%; p < 0.05). The combination of their treatments had the higher rate of parthenogenetic blastocyst development (41.7%) and significantly higher rate of pESC colony isolation from parthenogenetic blastocysts (45.0%); therefore, we concluded that the combination of these two methods (50 nM PXD101 treated for 8 hours and then aggregated at two-cell stage with 0.25% pronase for 10 minutes in our self-made concave) is considered the optimal way for the in vitro development of parthenogenetic blastocysts and subsequent pESC colony isolation in mice, opening new opportunities for application of this combination method to improve the parthenogenetic embryo development in other species.

The perturbed expression of m6A in parthenogenetic mouse embryos.

Parthenogenetically activated oocytes cannot develop to term in mammals owing to abnormal epigenetic modifications. Methylation of the N6 position of adenosine (m6A) is a post-transcriptional epigenetic modification of RNA. To investigate the role of m6A methylation in parthenogenetic (PA) embryonic development, we analyzed METTL3, METTL14, FTO, ALKBH5, YTHDF2, IGF2BP1, and IGF2BP2 expression by quantitative real-time PCR. These genes were found dynamically expressed during the 2-cell, 4-cell, 8-cell, and blastocyst stages of the embryo. Compared to normally fertilized embryos, the expression of these genes was perturbed in PA embryos, especially at the 8-cell stage. Furthermore, immunofluorescence was used to detect m6A expression. The results demonstrated that m6A expression decreased in the 2-cell stage, whereas it increased in the 8-cell stage of PA embryos. Taken together, these results suggest that the expression of RNA methylation-related genes was perturbed, leading to abnormal m6A modification during early development in PA embryos.

Tetraploidy-associated centrosome overduplication in mouse early embryos

ABSTRACTRecently, we observed that tetraploidization of certain types of human cancer cells resulted in upregulation of centrosome duplication cycles and chronic generation of the extra centrosome. Here, we investigated whether tetraploidy-linked upregulation of centrosome duplication also occurs in non-cancer cells using tetraploidized parthenogenetic mouse embryos. Cytokinesis blockage at early embryonic stage before de novo centriole biogenesis provided the unique opportunity in which tetraploidization can be induced without transient doubling of centrosome number. The extra numbers of the centrioles and the centrosomes were observed more frequently in tetraploidized embryos during the blastocyst stage than in their diploid counterparts, demonstrating the generality of the newly found tetraploidy-driven centrosome overduplication in mammalian non-cancer systems.

Microarray Analysis of Differentially Expressed Genes in Inner Cell Mass and Trophectoderm of Parthenogenetic Embryos

Mouse parthenogenetic (PG) embryos do not survive beyond day 9.5 of pregnancy. In this study, to understand the molecular mechanisms underlying the failure of parthenogenesis at the early developmental stage, we performed global gene expression profiling of the PG inner cell mass (ICM) and trophectoderm (TE) using microarray analysis, compared the results with those from in vitro-fertilized embryos, and identified genes whose expression levels showed more than a 4-fold change as a cutoff. Eighty probe sets were up-regulated and 59 probe sets down-regulated in the PG ICM, while 169 and 43 probe sets were respectively up-regulated and downregulated in PG TE. We selected two genes (Sfmbt2 and Gab1) that were down-regulated in both the PG ICM and TE, one gene (Nat1) that was down-regulated in the PG ICM, and one gene (Lysmd2) that was up-regulated in the PG TE, and analyzed the gene expression levels using real-time PCR. The quantitative expression levels of these four genes were confirmed by real-time PCR. In the present study, we identified differentially expressed genes in PG embryos and also identified those that were ICM- or TE-specific in PG embryos.

Microarray Analysis of Differentially Expressed Genes in Inner Cell Mass and Trophectoderm of Parthenogenetic Embryos

Abstract: Mouse parthenogenetic (PG) embryos do not survive beyond day 9.5 of pregnancy. In this study, to understand the molecular mechanisms underlying the failure of parthenogenesis at the early developmental stage, we performed global gene expression profiling of the PG inner cell mass (ICM) and trophectoderm (TE) using microarray analysis, compared the results with those from in vitro-fertilized embryos, and identified genes whose expression levels showed more than a 4-fold change as a cutoff. Eighty probe sets were up-regulated and 59 probe sets down-regulated in the PG ICM, while 169 and 43 probe sets were respectively up-regulated and downregulated in PG TE. We selected two genes (Sfmbt2 and Gab1) that were down-regulated in both the PG ICM and TE, one gene (Nat1) that was down-regulated in the PG ICM, and one gene (Lysmd2) that was up-regulated in the PG TE, and analyzed the gene expression levels using real-time PCR. The quantitative expression levels of these four genes were confirmed by real-t...

Epigenetic disruptions of histone signatures for the trophectoderm and inner cell mass in mouse parthenogenetic embryos.

Epigenetic asymmetry has been shown to be associated with the first lineage allocation event in preimplantation development, that is, the formation of the trophectoderm (TE) and inner cell mass (ICM) lineages in the blastocyst. Since parthenogenesis causes aberrant segregation between the TE and ICM lineages, we examined several development-associated histone modifications in parthenotes, including those involved in (i) transcriptional activation [acetylated histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac), trimethylated histone H3 lysine 4 (H3K4Me3), and dimethylated histone H3 arginine 26 (H3R26Me2)] and (ii) transcriptional repression [trimethylated histone H3 lysine 9 (H3K9Me3) and lysine 27 (H3K27Me3), and mono-ubiquitinated histone H2A lysine 119 (H2AK119u1)]. Here, we report that in parthenotes, H3R26Me2 expression decreased from the morula stage, while expression patterns and levels of H3K9Ac, H3K27Me3, and H2AK119u1 were unchanged until the blastocyst stage; whereas H3K14Ac, H3K4Me3, and H3K9Me3 showed normal patterns and levels of expressions. Relative to the decrease of H3K9Ac in the ICM and increase in the TE of parthenotes, we detected reduced expression of TAT-interactive protein 60 acetyltransferase and histone deacetylase 1 deacetylase in the ICM and TE of parthenotes, respectively. Relative to the decrease of H3R26Me2, we also observed decreased expression of coactivator-associated arginine methyltransferase 1 methyltransferase and increased expression of the Wnt effector transcription factor 7L2 and miR-181c microRNA in parthenotes. Furthermore, relative to the decrease in H3K27Me3 and H2AK119u1, we found increased phosphorylation of Akt1 and enhancer of zeste homolog 2 in parthenogenetic TE. Therefore, our findings that histone signatures are impaired in parthenotes provide a mechanistic explanation for aberrant lineage segregation and TE defects.

Genome wide functional genetics in haploid cells.

Some organisms such as yeast or males of social insects are haploid, i.e. they carry a single set of chromosomes, while haploidy in mammals is exclusively restricted to mature germ cells. A single copy of the genome provides the basis for genetic analyses where any recessive mutation of essential genes will show a clear phenotype due to the absence of a second gene copy. Most prominently, haploidy in yeast has been utilized for recessive genetic screens that have markedly contributed to our understanding of development, basic physiology, and disease. Somatic mammalian cells carry two copies of chromosomes (diploidy) that obscure genetic analysis. Near haploid human leukemic cells however have been developed as a high throughput screening tool. Although deemed impossible, we and others have generated mammalian haploid embryonic stem cells from parthenogenetic mouse embryos. Haploid stem cells open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale. Haploid genetics has thus become a powerful alternative to RNAi or CRISPR based screens.

Gene dosage in mammals: characterization of haploid embryonic stem cells

Most animals are diploid but in several cases haploid genomes can support development to some extent. In contrast to fish, haploidy is not compatible with embryonic development in mammals. Albeit, haploid cells have been observed in egg cylinder stage mouse embryos the majority of surviving embryos becomes diploid. Differences between the development of mammals and fish include genomic imprinting and dosage compensation. Both processes are thought to contribute to the loss of haploid embryos. Haploid embryonic stem cells (ESCs) have recently been derived from mouse parthenogenetic embryos. These cells maintain key properties of mouse ESCs and might overcome existing limitations in developmental genetic approaches in mice. Haploid ESCs have been established through the activation of unfertilized oocytes from a variety of mouse strains and maintain a wide developmental potential in culture and in chimeric mice. Notably, parthenogenetic haploid ESCs possess robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. Contribution to the embryo correlates with an efficient gain of a diploid karyotype. We have also observed that differentiation in culture results in diploidization, which likely is the result of endoreduplication and not cell fusion. In contrast to differentiation into embryonic cell types a haploid karyotype is maintained under certain conditions during forced differentiation to extraembryonic cell fates. Exploring the differentiation potential of a haploid karyotype highlights developmental constraints imposed by evolutionary adaptations specific for the mammalian genome namely genomic imprinting and X chromosome inactivation. Our data would suggest that early embryos possess a remarkable ability to buffer disturbances of both epigenetic processes.

Germline potential of parthenogenetic haploid mouse embryonic stem cells.

Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.

Derivation of haploid embryonic stem cells from mouse embryos

Most animals are diploid but haploid-only and male-haplo species have been described 1. Diploid genomes of complex organisms limit genetic approaches in biomedical model species such as in mice. To overcome this problem experimental induction of haploidy has been used in fish 2,3. In contrast, haploidy appears less compatible with development in mammals 4,5. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.

Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation

Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to find the best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In a first set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were artificially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents (electrical pulses followed by ionomycin or SrCl(2)) was applied in an aim to improve developmental competence. All embryos were evaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P<0.05). For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro matured oocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed that for both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation compared with chemical activation. Combining activating agents had no further positive effect on developmental potential.

Effect of 5-azadeoxycytidine and retinoic acid on expression of genomic imprinting in parthenogenetic mouse embryos

The action of two types of substances has been studied: 5-azadeoxycytidine and retinoic acid, which have a demethylation effect on DNA in the development process of diploid parthenogenetic mouse embryos. The effect of 5-azadeoxycytidine on hybrid mice (CBAxC57BL/6)F1 in vitro for 6 h, in the presence of single cell parthenogenetic embryos during the S-phase of the cell cycle has been studied. After developing to the blastocyst stage in vitro, parthenogenetic embryos were transplanted into the uterus of false pregnant females. It has been determined that a concentration of 0.1 microM 5-azadeoxycytidine activates embryonic development in the preimplantation period until the blastocyst stage (69% in experiment; 61% in the control) and during postimplantation, it increases the number of available space in the uterus for implantation (76% in experiment; 63% in the control). The effect of retinoic acid on parthenogenetic embryos from inbred C57BL/6 or CBA mice lines was studied by adding it to single cell embryos in a medium in vitro for 96 h. Treating parthenogenetic embryos C57BL/6 with retinoic acid concentrations 0.1 microM or 0.5 microM significantly increased the number of spaces for embryo implantation, 76% and 78% respectively, as against 57% for untreated embryos. Addition of similar doses of retinoic acid to the nutrient medium containing CBA parthenogenetic mouse embryos does not improve implantation (as with embryos C57BL/6), and a concentration of 2.0 microM is toxic to the embryos. During the period of postimplantation, parthenogenetic embryos of mouse lines C57BL/6 treated with retinoic acid just as the controls, did not develop to the somite stage. Mouse lines CBA had 45% of their embryos which were used as controls, developing to the advanced somite stages. However, the number of embryos treated with retinoic acid does not increase. Thus the treatment of two parthenogenetic embryos from inbred mice lines and their hybrids with compounds which demethylate DNA (5-azadeoxycytidine and retinoic acid) creates an opportunity for partial modulation of genomic imprinting and an increase in the survival rate of such embryos.

Modulating the Expression of Imprinted Genes in Mouse Parthenogenetic Embryos

ABSTRACTThe effect of transforming growth factor α (TGFα) on the expression of imprinted genes Igf2, H19 and Pegl/Mest in diploid parthenogenetic mouse embryos (PE) from hybrids (CBA x C57BL/6)F1 in posimplantational period of embryonal development has been studied. PE were treated with TGFα in vitro at morula stage and after development to blastocyst stage were transplanted in the uterus of pseudopregnant females. At day 10 from the begining of pregnancy PE were explanted for culturing in vitro for 48 hours. The expression of imprinted genes Igf2, H19 and Peg1/Mest was studied by the methods RT-PCR u whole mount in situ RNA-RNA hybridization by labelled with digoxygenin antisens RNA probes. It has been demonstrated, that before beginning of culturing in 60% from treated PE at day 10 (stage of 21–25 somites) expression of Igf2 has been established only in forebrain, expression of Peg1/Mest has not been established and expression of H19 was significantly diminished. In the control non-treated with TGFα PE at the same stages (21–25 somites) expression of the genes Igf2 and Peg1/Mest has not been detected. After 48 hours culture in vitro in all PE treated with TGFα and developed to stage of 40–45 somites, expression of Igf2 has been established in the cerebrum, heart, forming maxillae, liver and somites, expression of Peg1/Mest was found in the cerebrum, heart and liver. In the control non-treated with TGFα PE at similar stages of development expression of Igf2 and Peg1/Mest was not detected. The data received shows, that exogenic TGFα is able to reactivate expression of two imprinted genes (Igf2 u Peg1/Mest) and to repress third imprinted gene (H19). In this way TGFα modulates the effects of genomic imprinting and improve significantly viability and development of PE. The possibility that gene coding for TGFα is imprinted is discussed‥

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.

The Role of Parental-Specific Methylation Imprint on Mammalian Development.

Germ cells are unique totipotent cells from which individuals are generated by the fusion of male and female gametes. However, in mammals bi-parental reproduction is necessary for the production of descendants because parent-specific epigenetic modifications occur in the genome during gametogenesis. Therefore mouse parthenogenetic embryos die by E10. However, ng/fg bi-maternal embryos that contain a non-growing (ng) oocyte genome and a fully grown (fg) oocyte genome develop to E14. Imprinted genes have prominent effects on foetal and placental development. The Igf2-H19 genes, which are controlled by paternally methylated regions on chromosomes 7, contribute to the further extended development of the bi-maternal embryos. It was found that the bi-maternal embryos harboring a 13-kb deletion (Δchr7) that spanned the differentially methylated region and the transcription unit of the H19 gene developed to term. Extended development, however, was accompanied by foetal severe growth retardation and placental dysplasia. Because of these abnormalities the foetuses died after they were recovered from the uterus. It is also known that the genes, Dlk1-Gtl2, controlled by the paternally methylated region of chromosome 12, contribute to foetal development. To obtain an insight into the association between Dlk1-Gtl2 and development of bi-maternal embryos, we reconstructed bi-maternal embryos using ng oocytes harboring an IG-DMR deletion at chromosome 12 (Δchr12). Assessment of developmental ability showed that these bi-maternal embryos also developed to term. However, the phenotype is apparently differed between the ngΔchr7/fg and ngΔchr12/fg bi-maternal fetuses/placentae. In the ngΔchr12/fg bi-maternal foetuses. Extremely severe growth retardation was observed in the latter foetuses. The ngΔchr7/fg placenta is exhibited severe dysplasia, including an expanded spongiotrophoblast layer and a malformed labyrinthine zone. The placental dysplasia was restored in the ngΔchr12/fg placentae, although the mass was severely reduced. Gene expression analysis revealed that the expressions of imprinted genes regulated by IG-DMR was corrected in the bi-maternal embryos. In order to understand more the features of bi-maternal embryos, we characterized the gene expression profiles of the ngΔchr7/fg and the ngΔchr12/fg bi-maternal foetuses and placentae by using the Affymetrix Mouse Genome 430 2.0 Array. After describing a Venn diagram using the GeneSpring software, we constructed lists including genes in the ngΔchr7/fg and ngΔchr12/fg bi-maternal embryos and placentae. Based on the obtained extensive gene expression data and developmental features, we discuss complementary role of the 2 sets of imprinted genes (Igf2-H19 and Dlk1-Gtl2) on the development of bi-maternal mouse embryos.

A novel microtube culture system that enhances the in vitro development of parthenogenetic murine embryos

Oil is an indispensable material in micro-droplet culture; it prevents medium from evaporation, and its transparency facilitates monitoring. However, lipophilic factors in the medium can be absorbed into the oil overlay, and conversely, deleterious materials can diffuse into the medium. In the present study, we describe a novel oil-free microtube culture (MTC) system. Parthenogenetic mouse embryos were placed into 0.2-mL thin-wall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as a control. Embryos in MTC had a higher blastocyst formation rate (89.2%) and larger population of cells in the blastocysts (92.0+/-6.9; mean+/-S.E.M.) compared with drop culture (78.3% and 74.7+/-8.1; P<0.05 for each). The large blastocyst cell population in MTC was due to higher numbers of trophectoderm (TE) cells (70.5+/-5.9 versus 53.8+/-7.4; P<0.05) rather than inner cell mass cells. The presence of more TE cells was attributed to faster development in MTC. Embryos cultured in oil-covered MTC had fewer TE cells (61.5+/-5.6) than oil-free cultures (70.5+/-5.9; P<0.05). In conclusion, oil-free MTC was an alternative to conventional micro-drops, without the deleterious effects of oil.

Differential effects of oxygen on hatching of fertilized and parthenogenetic embryos

Embryos exposed to in vitro culture are prone to oxidative damage. Low oxygen tension during early preimplantation development appears beneficial, but is less studied during later development. Moreover, the effects of oxygen on development of parthenogenetic embryos remain unclear. Laboratory studies of fertilized and parthenogenetic mouse embryos, cultured in low (5%) and high (20%, atmospheric) O2, respectively, their rates of development to blastocyst, hatching, and expression profiles of apoptosis, imprinted, oxidation and protease genes. Parthenogenetic embryos (PEs) were achieved by activation of oocytes with strontiuim and cyctochalasin D. Embryos were cultured in KSOM at 37°C under 6% CO2 and 5% or 20% O2. Some embryos were processed for Oct4 expression and apoptosis by TUNEL analysis. Real-time PCR was used for gene expression analysis. Blastocysts developed from zygotes under conditions of low or high O2 were transferred into psuedopregnant recipients for term development. Both PEs and zygotes cultured under 5% or 20% O2 exhibited similar high rates for development to blastocysts (>96%). Apoptosis did not differ between parthenogenetic and fertilized blastocysts, regardless of O2 tension. Notably, blastocysts (n = 77) cultured under 5% O2 showed higher rates of implantation (71% vs. 47%) and live pups (44% vs. 20%), compared to those (n = 89) cultured under 20% O2. Total cell number of PE blastocysts was not affected by O2 tension, but the number of ICM was increased under 5% O2. Zygotes cultured under 5% O2 had markably lower rates of hatching (21%) vs. those under 20% O2 (57%). By contrast, the hatching rate (91%) of PEs cultured under 5% O2 was higher than under 20% O2 (80%). Reduced expression of ERK/MAPK but increased Sod1 was observed for PE blastocysts, with no effects of oxygen tension. High O2 reduced expression of Sod2 in both types of embryos. As expected, expression of the imprinted genes H19 was increased but Snrpn decreased in PE blastocysts, compared to fertilized blastocysts, but neither was affected by oxygen tension. Oxygen tension does not affect development to blastocyst of either fertilized or PEs, but low oxygen tension promotes embryo survival following postimplantation development. Low oxygen inhibits hatching of fertilized blastocysts, but promotes hatching of parthenogenetic blastocysts. The mechanism of oxygen's differential effects on fertilized vs. parthenogenic blastocysts remains to be determined.

Epigenetics in Male Germ Cells

Epigenetics in Male Germ Cells

Genomic imprinting in epigenetic of mammals

Genomic imprinting is one of the most remarkable and important epigenetic phenomena. A biological ban on parthenogenetic and androgenetic development of mammals is an obvious consequence of genomic imprinting. Genomic imprinting defects may cause malformations, clinical syndromes, and tumor growth in humans and to the large offspring syndrome and an increased mortality after in vitro manipulations with early embryos in mammals. Differential expression of parental alleles during ontogeny implies a mechanism of reversible, selective marking of gene alleles. These relatively stable epigenetic modifications, which do not affect the primary nucleotide sequence of DNA, may be transmitted in somatic cell lines and reproduced in the germ line. The genomic imprinting mechanism may be involved in other epigenetic processes, such as epigenetic inheritance, nonrandom allele segregation, meiotic drive, etc. Artificial modulation of genomic imprinting effects with the use of growth factors and demethylating agents permits partial “normocoping” during the development of parthenogenetic mouse embryos. Targeted changes in the transcriptional activity of imprinted genes provide prerequisites for epigenetic correction of syndromes and diseases caused by genomic impriting defects.