Developmental potential of bovine androgenetic and parthenogenetic embryos: a comparative study

In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24-37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.     

Genetic inactivation of Leuciscus idus L. (ide) oocytes using UV irradiation

Oocytes of Leuciscus idus were genetically inactivated using ultraviolet (UV) irradiation. Eggs for the experiment were obtained from dark-coloured females, whereas milt was taken from yellow-coloured (recessive marker) males. The survival at the eleutheroembryo stage (free embryo) in all experimental groups fertilized with genetically inactivated spermatozoa was much lower than in control groups. All haploid embryos showed morphological abnormalities, such as a stunted body and a poorly formed retina, and the condition was referred to as the haploid syndrome. The androgenetic origin (haploid or diploid embryos) was checked using a recessive colour marker ('blond'). The optimal doses of UV irradiation were 3,456-4,608 Jm(-2) at which almost 100% haploid embryos were produced at a hatching rate of >15%. Lower UV-ray doses influenced abnormal embryo development. Ploidy level recognition showed a typical value of mean active nucleoli per cell in haploid and diploid (control fish and spontaneous androgenotes) specimens. Abnormal dark embryos were classified as aneuploids.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

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

Autophagy and apoptosis: parent-of-origin genome-dependent mechanisms of cellular self-destruction

Functional genomic imprinting is necessary for the transfer of maternal resources to mammalian embryos. Imprint-free embryos are unable to establish a viable placental vascular network necessary for the transfer of resources such as nutrients and oxygen. How the parental origin of inherited genes influences cellular response to resource limitation is currently not well understood. Because such limitations are initially realized by the placenta, we studied how maternal and paternal genomes influence the cellular self-destruction responses of this organ specifically. Here, we show that cellular autophagy is prevalent in androgenetic (i.e. having only a paternal genome) placentae, while apoptosis is prevalent in parthenogenetic (i.e. having only a maternal genome) placentae. Our findings indicate that the parental origin of inherited genes determines the placenta''s cellular death pathway: autophagy for androgenotes and apoptosis for parthenogenotes. The difference in time of arrest between androgenotes and parthenogenotes can be attributed, at least in part, to their placentae''s selective use of these two cell death pathways. We anticipate our findings to be a starting point for general studies on the parent-of-origin regulation of autophagy. Furthermore, our work opens the door to new studies on the involvement of autophagy in pathologies of pregnancy in which the restricted transfer of maternal resources is diagnosed.     

Cadaveric sperm induces intergeneric androgenesis in the fish, Hemigrammus caudovittatus

Intergeneric androgenetic golden Buenos Aires tetra (BT), Hemigrammus caudovittatus was generated using sperm drawn from post-mortem males preserved at -20 degrees C for 10, 20, 30 and 40 days or fresh sperm to activate the UV-irradiated oocytes of black widow tetra (WT), Gymnocorymbus ternetzi. UV-irradiation (4.2 W/m(2)) of the oocytes for 3 min inactivated their nuclear genome. Fry hatched out from these activated oocytes were haploids; suffering haploid syndrome, they died before or within 48 h after hatching. Fresh BT sperm activated 95% oocytes; however, the sperm drawn from post-mortem males preserved at -20 degrees C for 60 (within glycerol packing) and 30 days (without glycerol packing) activated only 24 and 19% oocytes, respectively. Following activation, diploidy was restored by shocking the 25-min-old embryos at 41 degrees C for 2 min. Nuclear genomic inactivation of the oocytes was confirmed by (i) production of 100% haploids, (ii) karyotype and erythrocyte measurements, (iii) phenotypic markers, (iv) progeny testing and (v) species-specific marker. At hatching, survival of androgenotes decreased from 11% for those induced with fresh sperm to 4% for those generated using sperm from 30-day-old post-mortem males. Reproductive performance of the 'fresh' and 'cadaveric' F(0) and F(1) androgenetic males (Y(2)Y(2)) was superior to the control (X(1)Y(2)). Crosses involving homozygous (Y(2)Y(2)) 'fresh' F(0) androgenetic males with heterozygous females (X(1)X(2)) and F(0) homozygous males (Y(2)Y(2)) with females (X(2)X(2)) produced 2-4% unexpected female progenies. Paternal autosomes, inherited by the homozygous androgenetic female (X(2)X(2)), induced the production of female progenies in significantly less number of crosses than the crosses with heterozygous females (X(1)X(2)), which carried equal number of paternal and maternal autosomes. PCR analyses of the genomic DNA of normal male and unexpected F(1) and F(2) female progenies amplified by DMRT 1 specific primer produced bands of 237 and 300 bp length, and thereby confirmed that these unexpected females were genetic males. RAPD analyses of the androgenetic progenies showed that their genome was not contaminated with maternal genome.     

人工诱导泥鳅与大鳞副泥鳅雄核发育单倍体子代的RAPD分析

用３０个经过筛选的随机引物对３组泥鳅雄核发育单倍体、２组大鳞副尼鳅雄核发育单倍体及其相应亲本进行了ＲＡＰＤ分析。结果表明,雄核发育倍体子代与其父本的ＲＡＰＤ谱带相似率为９７．０％－９７．８％,与母本相似率为３０．３％－５９．５％,子代中的非亲本谱眩为０－０．０２９,极少母本特异谱带。这一结果说明雄性发育鱼类单倍体的遗传信息主要来自父本,且存在着个体差异,雄核发育子代存在ＤＮＡ变异和母本ＤＮＡ非特异带,但并非其必要条件。     

Production and progeny testing of androgenetic rosy barb Puntius conchonius

Protocol for androgenetic cloning of the rosy barb, Puntius conchonius, with contrasting gray and golden strains is described. At the intensity of 4.2 W/m2, UV irradiation for 3.0 min inactivates the maternal genome in eggs of the gray barb. Following activation by the golden barb sperm, 24-min old eggs are shocked at 41 degrees C for 2 min to restore diploidy. Maternal genomic inactivation is confirmed by the (i) golden body color, (ii) karyotyping, and (iii) progeny testing of F1-F3 progenies. Estimates of stage-specific mortality of haploid and diploid androgenotes indicate no change in the time scale or developmental sequence, when sperm of related strain is used for activation, and when haploid genome regulates the development. Survival of androgenetic clones remains constant for the F1, F2, and F3 progenies and is about 15% and 7% at hatching and sexual maturity, respectively. Homozygosity of the androgenotes is shown to inflict greater mortality. Between F1 and F3 generations, the heterozygosity of the androgenetic clone is decreased, as evidenced by reduction in size hierarchy. Though the reproductive performance of the F1, F2, and F3 supermales is superior to the normal ones, the realized fecundity remains equal around 80 progenies per brood. The 92 crosses involving 16 supermales and 10 normal dams yield 75-100% male progenies, confirming the possible operation of XXfemale symbol:XYmale symbol sex determination system. The frequency of unexpected occurrence of female progenies is about 8%, the causes for which are discussed.     

Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting

The inviability of diploid androgenetic and parthenogenetic embryos suggests imprinting of paternal and maternal genes during germ cell development, and differential expression of loci depending on parental inheritance appears to be involved. To facilitate identification of imprinted genes, we have derived diploid androgenetic embryonic stem (ES) cell lines. In contrast to normal ES cells, they form tumors composed almost entirely of striated muscle when injected subcutaneously into adult mice. They also form chimeras following blastocyst injection, although many chimeras die at early postnatal stages. Surviving chimeras develop skeletal abnormalities, particularly in the rib cartilage. These results demonstrate that androgenetic ES cells are pluripotent and point to stage- and cell-specific expression of developmentally important imprinted genes.     

Inheritance and mapping of 2n-egg production in diploid alfalfa.

The production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cytokinesis after restitutional meiosis. The formation of 2n eggs through diplosporic apomeiosis has also been documented in a diploid mutant of M. sativa subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular tagging of 2n-egg formation appears to be an essential step towards marker-assisted breeding and map-based cloning strategies aimed at investigating and manipulating reproductive mutants of the M. sativa complex. We made controlled crosses between PG-F9 and three wild type plants of M. sativa subsp. coerulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F1 progenies with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa because of endosperm imbalance, and owing to the negligible selfing rate, seed set in 2x-4x crosses was used to discriminate the genetic capacity for 2n-egg production. F1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified fragment length polymorphism) markers was employed to identify a genetic linkage group related to the 2n-egg trait using one of the three F1 progenies. This approach enabled us to detect a paternal ISSR marker of 610 bp, generated by primer (CA)8-GC, located 9.8 cM from a putative gene (termed Tne1, two-n-eggs) that in its recessive form determines 2n eggs and a 30% recombination genomic window surrounding the target locus. Eight additional RAPD and AFLP markers, seven of maternal, and one of paternal origin, significantly co-segregated with the trait under investigation. The minimum number of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses was estimated by ANOVA and regression analysis. Four maternal and three paternal independent molecular markers significantly affected the trait. A paternal RAPD marker allele, mapped in the same linkage group of Tne1, explained 43% of the variation for seed set in 2x-4x crosses indicating the presence of a major QTL. A map of the PG-F9 chromosome regions carrying the minor genes that determine the expression level of 2n eggs was constructed using selected RAPD and AFLP markers. Two of these genes were linked to previously mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evidence support the involvement of at least five genes.     

Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis

The random amplified polymorphic DNA (RAPD) technique was employed in the haplo-diploid dioecious species Gracilaria gracilis to identify sex-linked PCR markers. Sixty-nine decamer oligonucleotide primers were tested on two bulks of DNA, one from five haploid males and the other from five haploid females. One of these primers (OPD13) generated a 430-bp fragment specific to males and a 620-bp fragment specific to females. The diploid individuals (tetrasporophytes) showed the co-occurrence of these two fragments. In order to verify the linkage between the sexual phenotypes and these markers, a progeny array of 59 haploid individuals (male and female) born on a diploid individual was analysed, in all of which the two markers produced by the OPD13 primer segregated perfectly with sex.     

Maternal and paternal genomes function independently in mouse ova in establishing expression of the imprinted genes Snrpn and Igf2r: no evidence for allelic trans-sensing and counting mechanisms.

It has often been suggested that the parental-specific expression of mammalian imprinted genes might be dependent on maternal-paternal intergenomic or interallelic interactions. Using quantitative allele-specific RT-PCR single nucleotide primer extension assays developed for two imprinted genes, Snrpn and Igf2r, we demonstrate: (i) No role for maternal-paternal allelic interactions: the modes of parental-specific expression of Snrpn and Igf2r in normal ova were unchanged in gynogenetic and androgenetic ova; the latter contain two maternal and two paternal genomes respectively, and cannot undergo maternal-paternal interactions. (ii) No role for allelic counting or exclusion mechanisms: in individual blastomeres of androgenetic ova, both paternal Snrpn alleles were active (Snrpn was not expressed in gynogenetic ova), and in individual gynogenetic and androgenetic blastomeres, both maternal and paternal Igf2r alleles, respectively, were active. (iii) No role for ploidy: the mode of parental-specific expression of Snrpn and Igf2r in normal diploid ova was unchanged in individual blastomeres of triploid and tetraploid ova. Thus, the maternal and paternal genomes function independently in establishing the pre-implantation mode of parental-specific expression of Snrpn and Igf2r, with no role for trans-allelic/genomic interaction phenomena. In addition, the results show that inactive and biallelic modes of expression of imprinted genes are potential mechanisms for the death of gynogenones and androgenones at the peri-implantation stage.     

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.     

Second polar body inclusion results in diploid/triploid mixoploidy

We report three cases with a typical diploid/triploid mixoploidy. Cytogenetic analysis showed a normal diploid karyotype in peripheral blood lymphocytes and a mixture of diploid and triploid cells in skin fibroblasts. We analysed microsatellite markers in patients blood lymphocytes and skin fibroblasts and compared the results with the microsatellite markers in the parents. The extra haploid set was in all three cases of maternal origin. In one case the markers were not very informative but in two cases pericentromeric markers showed a single dose of one paternal allele and a double dose of one maternal allele, more telomeric markers showed one paternal allele and two different maternal alleles. These observations can only be explained by the inclusion of the second polar body in one of the blastomeres at the cleavage stage.     

Somatic cells derived from haploid larvae are feasible as donors for nuclear transplant in zebrafish. Preliminary results

Summary Somatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.     

Functional neuronal cells generated by human parthenogenetic stem cells

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.     

Triploidy and haploid-triploid mosaicism among chick embryos (Gallus domesticus).

Homomorphic, chromosomally abnormal roosters were mated to normal hens. The 23 hens produced 67 embryos, including two triploids and a haploid-triploid mosaic at about 26 hours of incubation. Both of the triploid embryos were conceived within a 5-day period. The presence of a single genome of paternal origin with marker chromosomes in each triploid led to the conclusion that these embryos were derived from diploid, ZW-type ova fertilized by haploid, Z-type spermatozoa. The inheritance pattern of the mosaic embryo was clearly due to a spermatozoal origin for the haploid cell line; and one genome of the three in the triploid cell line was paternal. The sec chromosomes were Z/ZZZ, with one Z of each cell line being a translocation product of paternal derivation.     

Diploid mouse embryos constructed at the late 2-cell stage from haploid parthenotes and androgenotes can develop to term

Male and female gamete nuclei are required to ensure the full-term development of the mouse embryo. Differential expression of the two genomes has been proposed as the basis for this requirement. In order to investigate whether some interactions between the paternal and the maternal genomes are essential before or at the time of the activation of the embryonic genome, we have constructed diploid embryos from haploid parthenotes and androgenotes at the late 2-cell stage. These embryos developed to term into normal offsprings. This shows that the male and the female genomes can be activated separately and are still able to ensure complete development when put together in cytoplasm synchronized with the nuclei. These experiments also show that the egg cytoplasm does not need any male contribution before the late 2-cell stage.