Androgenetic Embryos Research Articles

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.

In vitro culture of isolated living sunflower (Helianthus annuus L.) embryo sacs

Ovules of sunflower tubular florets were observed histologically by serial sectioning and clearing to study the correlation between flower morphology and developmental stage. On the basis of these data, florets with visible stigma and receptive "curling" surfaces were chosen for embryo sac (ES) isolation. In such florets, ~20% of the ovules were unfertilized; fertilized zygote-stage ES (~75% of ovules) or ES containing two-celled proembryo occurred in the remaining ovules. ES were dissected manually using needles under a stereomicroscope or were treated with enzymes for 4–5 h after manual isolation. The viability of ES isolated without enzymes was more than 90% as assessed by fluorescein diacetate staining, and decreased to ~3% after 72 h of culture. The use of enzymes during isolation resulted in diminished ES viability, suggesting that removal of the protective layers of ovular cells may be part of the problem. Another factor affecting ES viability is medium osmolality. Living ES were then cultured in vitro. On liquid medium containing 9% sucrose, globular embryos developed in ~10% of zygote-stage ES, but further growth of the embryos was abnormal and callus was produced. Transfer of embryo-derived callus to solid Murashige-Skoog medium supplemented with 1-naphtaleneacetic acid and kinetin resulted in organogenesis. When ES were co-cultured with androgenetic microspores and microspore-derived embryos of Brassica napus, ~11% of the ES showed growth and development. Embryological study of the cultured ES revealed outgrowth of endothelium.

Imprinting.

Over 3000 years ago, mule breeders had already observed parent-specific effects in the offspring of crossbred horses and donkeys. A mare crossed with a donkey yields a mule, whereas a stallion crossed with a donkey yields a hinny, which has stronger legs and shorter ears than a mule. The parent-of-origin effects point to different functions for maternal and paternal genomes. This is due to a process now generally known as genomic imprinting. Despite these early clues, it was not until the early 1980’s before the phenomenon of genomic imprinting was discovered. Pronuclear transplantation studies in mice showed that gynogenetic embryos with two maternal genomes and androgenetic embryos with two paternal genomes were arrested early in development. The gynogenetic embryos showed a relatively normal embryo but poor extraembryonic growth whereas there were mainly extraembryonic tissues and poor embryonic development in the androgenetic embryos. These experiments demonstrated that certain genes are preferentially expressed from only one parental genome and that both paternal and maternal genomes are required for normal embryonic development (McGrath and Solter, 1984; Barton et al 1984).KeywordsImprint GenePaternal AlleleMaternal AlleleCTCF BindingPaternal GenomeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Comparison of Gene Expression During Preimplantation Development Between Diploid and Haploid Mouse Embryos1

Haploid development is a normal part of the life cycle for some animals, but it has not been observed in mammals. Studies in mice have revealed that the preimplantation developmental potential of haploid embryos is significantly impaired relative to diploid embryos. The reasons for the severely limited developmental potential of haploid embryos in mammals have not been discerned. To examine the effects of haploid development on gene expression, and in particular on X-linked gene expression, and to evaluate to what degree newer techniques of producing and culturing such embryos might affect developmental potential, haploid and diploid parthenogenetic and androgenetic embryos were produced and reevaluated for developmental potential, genomic integrity, and relative expression levels of specific autosomal and X-linked gene transcripts. Our data confirm the previously observed restriction in haploid developmental potential, eliminate chromosomal abnormalities as a major factor in this restriction, and reveal subtle alterations in gene expression. Haploid parthenogenones display only very subtle alterations in the expression of most mRNAs but a consistent elevation in X-linked Bex1 mRNA expression. Haploid androgenones seem to lack repression of the Pgk1 gene that is seen in diploid androgenones, but this may reflect ongoing loss of those haploid androgenones that experience X chromosome inactivation. The significance and possible explanations for these differences are discussed.

Production of Haploids and Doubled Haploids of the Amphiploids Aegilops Variabilis × Secale Cereale

Anther culture was used to produce haploid plants from Aegilops variabilis ssp. euvariabilis × Secale cereale (inbred line) amphiploids (2n=42) and their parental forms. The anthers were cultured on modified induction media - C 1 7 and PII. Androgenetic embryos were obtained only from amphiploid plants and the frequency depended on genotypes. On C 1 7 medium 20.0 - 65.2 and on PII medium 1.4 - 15.7 androgenetic embryos /100 anthers were produced. The embryos were transferred to a regeneration medium 190-2 and 0.1 - 13.4 of green plants /100 androgenetic embryos were obtained. The haploid (n=21) chromosome number was found in all plants. Doubled haploids were produced by haploid colchicine treatment.

Asb4, Ata3, and Dcn Are Novel Imprinted Genes Identified by High-Throughput Screening Using RIKEN cDNA Microarray

Genes differentially expressed between parthenogenetic and androgenetic embryos are candidates for the identification of imprinted genes, which are expressed specifically from the maternal or paternal allele. To search for genes differentially expressed between parthenogenetic and androgenetic embryos, we used the RIKEN full-length enriched mouse cDNA microarray. The 25 candidates obtained included 8 known imprinted genes (such as IgfII, Snrpn, and Neuronatin) and 3 new ones—Asb4 (ankyrin repeat and SOCS box-containing protein 4), Ata3 (amino acid transport system A3), and Decorin—which were confirmed by using normal diploid embryos from the reciprocal F1 crosses of B6 and JF1 mice. The 25 candidates also included genes that showed no imprinting-associated expression in normal diploid embryos. We describe a feasible high-throughput method of screening for novel imprinted genes by using the RIKEN cDNA microarray.

Subtraction-hybridization method for the identification of imprinted genes.

Imprinted genes show monoallelic expression from either the paternal or maternal genome (1,2), and their regulated expression is usually associated with the existence of parentally differentially methylated regions on genomic DNAs (3,4). Because of this, essentially two different approaches, using either cDNA or genomic DNA as starting material (5) have been developed for systematic isolation of imprinted genes. In this chapter, we describe a subtraction-hybridization method (6-8) as an example of the former approach. Both parthenogenetic embryos and androgenetic embryos (9,10) are the most suitable biological materials for the subtraction or detection of imprinted genes. However, it is difficult to obtain a large amount of such special materials because only a small number of these embryos develop to the d 10 stage (9,10). Thus, polymerase chain reaction (PCR)-based techniques, such as the differential display (11-13) and subtraction-hybridization methods, are necessary to accomplish this experiment. The subtraction-hybridization method has been successfully applied for isolation of both paternally expressed genes (Pegs) (6,14,15) and maternally expressed genes (Megs) (7), and it allows cDNA libraries to be made from a very small amount of biological material. We are convinced that this method can be applied in many fields of biological science.

Segregation distortion at marker loci: variation during microspore embryogenesis in maize

In the present study, we analyzed the segregation distortions of markers during in vitro androgenesis in maize. This was based on four segregating populations derived from the A188×DH7 one-way-cross. These populations consisted of very young androgenetic embryos, well-developed calluses, haploid regenerated plantlets and spontaneous diploid plantlets. These structures all represented different developmental stages, from that of microspores to the regenerated plantlets. This study complemented a previous one by Murigneux et al. 1994, where distorted segregations of RFLP markers were detected in a single-seed-descent population and in a doubled-haploid population derived from the same cross. The weakly biased SSD maize genetic map was used as a reference to locate 145 AFLP loci whose allelic segregations were also analyzed in the androgenetic segregating populations. Segregation distortions were determined based on chi-square analysis (P<0.01 and P<0.001). Regions on chromosomes 2 and 8 showed distortions from the beginning of embryo formation, with large effects throughout the process. Regions on chromosomes 3, 4, 6 and 10 could control callus formation from microspores. Other deviations of marker genotypes on chromosomes 1, 4, 6 and 10 could be associated with the regeneration phase. Moreover, the statistical method of Cheng et al. for mapping a lethal factor locus inside segments of linked distorted markers was used to estimate the position of seven partial lethal androgenetic factors on chromosomes 1, 2, 8 and 10. These factors could represent selective genes actively involved in maize androgenesis.

The use of chimeric mice in studying the effects of genomic imprinting

This is a review of the data of clonal analysis of developing tissues in parthenogenetic and androgenetic chimeric mice. The time and causes of death of the parthenogenetic and androgenetic cell clones in chimeras are considered. The data obtained suggest that the development of cell clones, derivatives of the mesoderm and endoderm, is determined by the expression of alleles of the imprinted loci of paternal chromosomes, while the formation of cell clones, derivatives of the ectoderm, depends on the expression of other imprinted loci of maternal chromosomes. The death of androgenetic and parthenogenetic (gynogenetic) mammalian embryos is due to the lack of the expression of certain imprinted loci of the maternal and paternal genome, respectively.

EFFECTS OF THE HORMONAL BALANCE ON CLAUSENA EXCAVATA ANDROGENESIS

Clausena excavata, a native of Southeast Asia, displays characteristics of disease tolerance and adaptability to different kinds of soils. This species contains potential parents for the improvement of Citrus fruit rootstocks. However, it is tetraploid, unlike most Citrus and related species, which are diploid. Bringing the ploidy level back to 2n=2x would allow us to consider integrating this species into the varietal creation programme by somatic hybridisation. A diplohaploidisation programme by anther culture is currently being conducted. The effects of the hormonal balance on androgenesis and on the maturation of androgenetic embryos are described. Thirty-six hormonal combinations using benzylamino purine (BAP) and dichlorophenoxyacetic acid (2,4-D) were examined. The tendency to embryogenesis varies from 0 to 9% depending on the hormonal concentration and combination. A total of 106 plants were regenerated, of which 91 were hexaploid, 11 were tetraploid, and only two were diploid after the flow cytometry evaluation. Histological study showed that the embryos were derived not from somatic embryogenesis of the mother tissues but from microspores. (Resume d'auteur)

Post-implantation development of mouse androgenetic embryos produced by in-vitro fertilization of enucleated oocytes.

We report here on the precise ability of mouse androgenetic embryos produced by in-vitro fertilization of enucleated oocytes to develop to day 9.5 of gestation when cultured with M16 and CZB media. Androgenetic embryos cultured with CZB rather than M16 medium developed to the blastocyst stage in a more significant proportion (56.6% versus 45.0%, P < 0.001). However, after cavitation, the rate of cell proliferation of androgenetic embryos cultured with CZB medium was significantly decreased (P < 0.05). Embryo transfer experiments showed that blastocysts cultured with M16 medium were superior to those cultured with CZB medium in their ability to develop to 9.5-day-old fetuses (28.1% versus 11.1%, P < 0.001). These results showed that the present procedure for producing androgenetic mouse embryos is reliable and that M16 medium is superior for culturing the embryos. Fetal sexing by polymerase chain reaction (PCR) also demonstrated that both XX and XY embryos develop to 9.5-day fetuses at theoretical rates (1:2). This is the first finding that mouse XX androgenones survive after implantation.

Genetic improvement of anther culture response in maize: relationships with molecular, Mendelian and agronomic traits

Two cycles of androgenetic in vitro doubled haploid (DH) plant production and intermating were implemented in an experimental synthetic population of maize. In vitro traits, including androgenetic embryo production, the regeneration potential and the frequency of spontaneous chromosome doubling, were studied. The success of the regenerated plants to self pollinate was also observed. Impressive genetic progress is reported for all the steps of the androgenetic process including seed set. Differential genetic progress is recorded according to the trait measured. Using a set of Mendelian and molecular markers that mapped to the different maize chromosomes, we were able to characterize the variation in the genetic variability in the population. Progress in the in vitro response was not found to be associated with any noticeable decline in global genetic variability. In addition, the QTL chromosomic regions tested, which were involved in androgenetic response, were not found to be subjected to a strong variation during the breeding experiment. Some phenotypical and morphological traits were also evaluated, and these showed that there was no depreciation effect in the agronomic value of the population. DH plant production and intermating the regenerated plants may be considered for the introduction and use of androgenesis in material which responds poorly.

Direct gene delivery into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants.

A procedure for direct gene transfer into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants is presented. By modifying the microspore culture method and adopting the firefly luciferase (Luc) gene as a non-destructive marker, we could obtain stably transformed androgenetic embryos from bombarded microspores. Luc-positive embryos were easily isolated from the large non-transformed population using a high-sensitivity bioluminescent image analyzer. PCR and Southern blot analyses confirmed that the introduced transgene was integrated stably into the genome of the selected embryos. Diploidized plants obtained from the haploid embryos were self-pollinated, and all of the offspring tested were Luc-positive, indicating rapid fixation of the transgene which is characteristic of doubled haploids.

Cell proliferation is reduced in parthenogenetic mouse embryos at the blastocyst stage: a quantitative study.

Parthenogenetic and androgenetic embryos fail to develop to term, possibly because of genomic imprinting, an epigenetic alteration of certain genes, depending on the parent of origin. The effect of this phenomenon has been studied mainly in mid-gestation embryos, without morphological abnormalities detected during the preim-plantation period. Nevertheless, parthenogenetic mouse embryos never develop to the blastocyst stage in the same numbers as do fertilized ones, and up to 50% fail to implant, suggesting that genomic imprinting may be responsible for this lack of viability. We have made a quantitative and morphometric analysis of the cell proliferation capacity at the end of the preimplantation period in parthenogenetic mice to study if such parameter is affected by the monoparental constitution of the embryos. We have found that, apart from the different morphology and cell number induced by culture conditions, parthenogenetic mouse blastocysts have a significantly smaller cell number than do fertilized control embryos. Our interpretation of these results is that the monoparental constitution of these embryos may be responsible for the lack of some factor required to sustain cell proliferation after the morula stage.

Analysis of parent-specific gene expression in early mouse embryos and embryonic stem cells using high-resolution two-dimensional electrophoresis of proteins.

Genomic imprinting is an important genetic mechanism in mammals whereby certain genes are epigenetically modified and their expression altered according to their parental origin. The most important consequence of this is the requirement for both a maternal and a paternal genome for normal development to proceed to term. Although there are many instances of specific phenotypes (in the mouse) and diseases (in humans) resulting from imbalances in the parental chromosomes, it is only in the past few years that some of the imprinted genes responsible have been identified. It is however unclear what proportion of the genome is imprinted, particularly in the early embryo. To address the question to what extent parent-specific gene expression occurs in the early embryo and with a possible view to identifying new imprinted genes, the protein profiles of parthenogenetic and normal blastocysts were compared using the technique of high-resolution two-dimensional electrophoresis. The protein profiles of parthenogenetic, androgenetic and normal embryonic stem cells were also compared. Hence parent-specific gene expression was examined in embryonic and extraembryonic lineages of the early embryo. Approximately 1000 polypeptides were examined in each of the analyses, however no parent-specific differences were observed for any of these polypeptides. From this result, it is concluded that expression of genes encoding these polypeptides is identical from the parental chromosomes. These findings have important implications for estimates of the number of imprinted genes in the genome and for the interpretation of phenotypes of parthenogenetic and androgenetic embryos.

Establishment of Imprinted Methylation Patterns during Development

Genomic imprinting is a classic example of epigenetic control of gene expression in mammals. It represents a process that marks the parental origin of certain genes, resulting in their allele-specific expression. Imprinting, which is implicated in the inequality of the maternal and paternal genomes in mammals, results in the developmental failure of isoparental embryos to develop properly (Surani 1993). An imbalance of specific parental chromosomes in the embryo or aberrant expression of the imprinted genes results in a number of genetic disorders in man and may also contribute to tumor development (Feinberg 1993; Nicholls 1993). Several model explanations have been proposed for the evolutionary acquisition of genomic imprinting as a developmental control mechanism in mammals (Moore and Haig 1991; Barlow 1993; Varmuza and Mann 1994). One of these models (Moore and Haig 1991) suggests that imprinting might have evolved in mammals because of the conflicting “interests” of maternal and paternal genes within the embryo. This model proposes that paternally expressed genes promote embryonic growth, whereas maternal genes act to restrain the use of maternal resources. A suitable example that corroborates this argument is the paternally expressed Igf2 gene and its counterpart, maternally expressed receptor (Igf2r). The different contribution to the embryonic phenotype of paternal and maternal genes had in fact been demonstrated in parthenogenetic and androgenetic embryos. Although the development of extraembryonic tissues in parthenogenetic embryos harboring two copies of the maternal genome is impaired, the development of the embryo proper is normal. In contrast, the embryo proper in androgenones...

Mouse embryos with paternal duplication of an imprinted chromosome 7 region die at midgestation and lack placental spongiotrophoblast

Imprinted genomic regions have been defined by the production of mice with uniparental inheritance or duplication of homologous chromosome regions. With most of the genome investigated, paternal duplication of only distal chromosomes 7 and 12 results in the lack of offspring, and prenatal lethality is presumed. Aberrant expression of imprinted genes in these two autosomal regions is therefore strongly implicated in the periimplantation lethality of androgenetic embryos. We report that mouse embryos with paternal duplication of distal chromosome 7 (PatDup.d7) die at midgestation and lack placental spongiotrophoblast. Thus, the much earlier death of androgenones must involve paternal duplication of other autosomal regions, acting independently of or synergistically with PatDup.d7. The phenotype observed is similar, if not identical to, that resulting from mutation of the imprinted distal chromosome 7 gene, Mash2, which in normal midgestation embryos exhibits spongiotrophoblast-specific maternally active/paternally inactive (m+/p-) allelic expression. Thus, the simplest explanation for the PatDup.d7 phenotype is p-/p- expression of this gene. We also confirm that PatDup.d7 embryos lack H19 RNA and posses excess Igf2 RNA as might be expected from the parental-specific activities of these genes in normal embryos.

Temporal and spatial regulation of H19 imprinting in normal and uniparental mouse embryos.

The mouse H19 gene is imprinted so that the paternal copy is both methylated and repressed during fetal development. However, the CpG-rich promoter region encompassing the transcription start is not methylated in sperm; this region must therefore become methylated postzygotically. We first examined the timing of DNA methylation of this region and the corresponding expression of H19. Both parental copies are initially undermethylated in blastocysts and the paternal copy then becomes fully methylated in the embryo around implantation; this methylation is more protracted in the extraembryonic lineages, especially in the trophoblast. By contrast to the lineage-dependent methylation, we observed exclusive expression of the maternal copy in preimplantation embryos and in all the lineages of early postimplantation embryos although variability may exist in cultured embryos. This indicates that methylation of the CpG-rich promoter is not a prerequisite for the paternal repression. We then examined whether methylation and expression occurs appropriately in the absence of a maternal or a paternal genome. Both H19 copies in androgenetic embryos are fully methylated while they are unmethylated in parthenogenetic embryos. This correlates with the lack of expression in androgenetic embryos but expression in parthenogenetic embryos. However, the androgenetic trophoblast was exceptional as it shows reduced methylation and expresses H19. These results suggest that promoter methylation is not the primary inactivation mechanism but is a stabilizing factor. Differential methylation in the more upstream region, which is established in the gametes, is a likely candidate for the gametic signal and may directly control H19 activity.

Molecular and biochemical markers for embryogenic potential and regenerative capacity of barley ( Hordeum vulgare L.) cell cultures

The embryogenic potential and regenerative capacity of cereal cell cultures was examined using the expression of two embryo-specific genes (the dormancy-related cDNA-clone B15C, R. Aalen, Norway; the aldose reductase cDNA-clone pG22–69, D. Bartels, Germany) in barley cell cultures. By Northern blotting the expression of both clones was limited to embryogenic cultures and not to non-embryogenic cultures. In situ hybridization showed high gene expression in the shoot and root apex of embryos and in subepidermal cell layers of embryogenic barley cultures. In androgenetic embryos the expression of the embryo-specific clones was detected as early as 2 weeks after isolation of microspores, indicating their usefulness as early markers for embryogenesis. Nevertheless, cell aggregates which were embryogenic but no longer able to regenerate plants, expressed both genes. Cell cultures consisting of embryogenic aggregates unable to regenerate plantlets were used to verify that an embryo-specific marker alone is not sufficient to characterize regenerable cultures. Comparing the protein pattern of embryogenic/regenerable and embryogenic/non-regenerable suspension cultures of barley, we identified a 85-kDa polypeptide (pI 5.8) accumulating only in non-regenerable cultures. Moreover, following the electrophoretic pattern of secreted proteins, two glycoproteins were found correlating with the embryogenic capacity (46 kDa, pI 6.1) and the loss of regenerative potential (17.4 kDa), respectively. The data indicate that at least two levels of control exist in somatic embryogenesis. One is the induction of bipolar embryos and the second the germination of somatic embryos into plantlets.

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.