Parthenogenetic Research Articles

Developmental potential and kinetics of pig embryos with different cytoplasmic volume

The effects of cytoplasmic volumes on development and developmental kinetics of in vitro produced porcine embryos were investigated. During hand-made cloning (HMC), selected cytoplasts were separated into two groups according to their size in relation to the initial oocyte: ~75% or ~50%. Following two fusion steps and activation (day 0), reconstructed embryos were cultured in vitro for 6 days. Cleavage rates on day 2 as well as blastocyst rates and cell numbers on day 6 were recorded. Results showed that embryo development was no different for ~50% versus ~75% cytoplasm at first fusion. This result was used in the following experiments, where the effect of varying cytoplasm volume in second fusion to obtain a final cytoplasm volume of ~75% to ~200% was tested. The results showed that the lowest quality was obtained when the final cytoplasm volume was ~75% and the highest quality at ~200% of the original oocyte. Similar results were observed in parthenogenetic (PA) embryos activated with different cytoplasmic volumes. A common pattern for the developmental kinetics of HMC and PA embryos was observed: the smaller group tended to have a longer time for the first two cell cycles, but subsequently a shorter time to form morula and blastocyst. In conclusion, the developmental kinetics of in vitro produced embryos was affected by the cytoplasm volume of the initial oocyte, and this further accounted for the developmental ability of the reconstructed embryos.

Meiosis in rare males in parthenogenetic Cacopsylla myrtilli (Wagner, 1947) (Hemiptera, Psyllidae) populations from northern Europe

For studying meiosis in males, large samples of Cacopsylla myrtilli (Wagner, 1947) (Hemiptera, Psyllidae) were collected in Norway, Sweden, Finland and northwest Russia. In addition to all-female populations, males were present in 10 out of 47 populations; still, all populations were highly female-biased, the proportion of males varying from 0.1% to 9.1%. These males are thus rare or so-called spanandric males. Males in northern Norway, Finland and northwest Russia showed normal chiasmate meiosis, while complete absence of chiasmata due to asynapsis was found in males collected in Norway and northern Sweden. In asynaptic meiosis, all univalent chromosomes divided during the first meiotic division resulting in incomplete second meiotic division and formation of diploid sperms. Hence, males in these populations are nonfunctional and do not contribute to the genetic constitution of the population, but appear in every generation as reversals from apomictic parthenogenesis and the mode of parthenogenesis is of obligatory type.

Differential response of IVP, parthenogenetic and nuclear transfer derived bovine embryos upon environmental heat stress-implications for expression of autosomal and X-linked genes

The present study was conducted to examine the metabolic activities and expression differences of X-linked and autosomal genes upon exposure to heat stress using different types of bovine embryos. Embryos from in vitro fertilized (IVF), parthenogenetic (PA) and nuclear transfer (NT) were given heat treatment at morula stages at 40°C for 6 hrs. Treated embryos were studied for metabolic activities by quantification of G6PD-activity through Brilliant Cresyl Blue (BCB) staining and were used for X-linked and autosomal gene expression studies. Quantification of G6PD, PGK1, XIST as sex linked and SOX, BAX, OCT4, HSP70.1 as autosomal genes and GAPDH (endogenous control) mRNAs in each cDNA sample from heat treated and untreated embryos were assessed by quantitative PCR. Results of the present study indicate sexually dimorphic expression of selected X-linked and autosomal genes upon exposure to heat stress at morula stage in IVF, PA and NT embryos. Moreover, our study showed that female IVP derived embryos show highly decreased levels of XIST after heat shock. That could be one of the reasons for reduced in vitro developmental competence of female bovine embryos since XIST is the responsible gene for X-chromosome inactivation. Any disturbance would result in dysregulation of all X-Chromosomal encoded genes. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15759 Bang. J. Anim. Sci. 2013. 42 (1): 1-10

THE EGG OF THE STICK-INSECT BACILLUS ATTICUS CAPRAI NASCETTI & BULLINI: A SCANNING ELECTRON MICROSCOPE STUDY (CHELEUTOPTERA BACILLIDAE)

SUMMARY The morphology of the egg of the stick-insect Bacillus atticus caprai Nascetti & Bullini (Insecta Cheleutoptera Bacillidae), the Italian member of the parthenogenetic B. atticus complex, was observed with the scanning electron microscope (SEM). A structural organization common to all the Cheleutoptera so far investigated at the SEM level is described. Peculiar and diagnostic characters, concerning mainly the sculpturing of the capsule and the morphology of the operculum, were found, which differentiate B. atticus caprai from the other Bacillinae so far studied, B. rossius (Rossi) and Clonopsis gallica (Charp.). A summary of the main differences between the eggs of these three species clearly shows that B. atticus caprai is more related to B. rossius than to C. gallica. A comparison of the egg of B. atticus caprai with those of the other parthenogenetic members of this complex, as well as with the more closely related bisexual species, is expected to clarify both the taxonomic status and the phylog...

COMPARATIVE RESEARCH ON THE SEXUALIZATION OF FISSIPAROUS PLANARIANS TREATED WITH SUBSTANCES CONTAINED IN SEXUAL PLANARIANS

SUMMARY Previous research had shown that asexual specimens of fissiparous Dugesia gonocephala s.l. stopped fissioning and developed a hermaphroditic genital apparatus if fed for several weeks on the crushed tissues of sexually mature Polycelis nigra Ehr., a non-fissiparous planarian. The present research shows that the sexualizing substance, probably a neurosecretion, is also present in the tissues of sexually mature Dugesia lugubris s.l., Dugesia dorolocephala (Woodworth), Dugesia anceps Kenk and Dendrocoelum lacteum (O. F. Muller), in concentrations which may differ according to the species but without any zoological specificity. There is a clear different in the capacity of each fissiparous species or strain to react to the sexualizing substances: while many asexual strains of Dugesia gonocephala s.l., Dugesia tigrina (Girard) and Dugesia Pdorotocephala (race from Sabino) reacted more-or-less positively to the treatment, agamic Dugesia dorotocephala, Dugesia mediterranea Benazzi et al. (fissiparous rac...

Pig oocytes with a large perivitelline space matured in vitro show greater developmental competence after parthenogenesis and somatic cell nuclear transfer

The objective of this study was to examine the developmental competence of pig oocytes in relation to the size of the perivitelline space (PVS) of oocytes matured in vitro. Immature oocytes were matured in medium 199 or porcine zygote medium (PZM)-3 containing 108 or 61.6 mM NaCl. In vitro-matured (IVM) oocytes were examined for intracellular glutathione (GSH) level; cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNA levels; and developmental competence after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). IVM oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.00 pixels/oocyte vs. 0.57 pixels/oocyte) and blastocyst formation (54.3% vs. 37.3%) after PA than oocytes with a smaller PVS. Culturing oocytes for maturation in PZM-3 with reduced (61.6 mM) NaCl increased (P < 0.05) the size of the PVS (6.4 µm vs. 2.8 µm) compared to control oocytes that were matured in normal PZM-3 containing 108 mM NaCl. Moreover, oocytes with a larger PVS showed higher CDK1, PCNA, and ERK2 mRNA and intracellular GSH levels (1.6 pixels/oocyte vs. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52.1% vs. 40.6%) and SCNT (31.8% vs. 18.2%) than control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation based on the enhanced GSH level and transcription factor expression. Further, enlargement of the PVS by culturing in low-NaCl medium improves the developmental competence of pig oocytes.

Research with parthenogenetic stem cells will help decide whether a safer clinical use is possible

The derivation and use of parthenogenetic stem cells (pESCs) are envisaged as a reliable alternative to conventional embryonic stem cells. Similar to embryonic stem cells in their proliferation, expression of pluripotency markers and capacity to multilineage differentiation, pESCs are at a lower risk of immune rejection within stem cell-based therapeutics. Moreover, pESCs represent an important model system to study the effect of paternally imprinted genes on cell differentiation. However, currently available information about the genetic and epigenetic behaviour of pESCs is limited. Thus, a detailed look at the biology of parthenogenetic (PG) embryos and PG-derived cell lines would allow gaining insight into the full potential of pESC in biotechnology. In this commentary article we review some features related to the biology of PG embryos and pESCs. In addition, novel traits on bovine pESCs (bpESCs) are discussed.

The time- and age-dependent effects of the juvenile hormone analog pesticide, pyriproxyfen on Daphnia magna reproduction

Pyriproxyfen is an insecticidal juvenile hormone analog that perturbs insect and tick development. Pyriproxyfen also alters parthenogenic reproduction in non-target cladoceran species as it induces male production that can lead to a decrease in fecundity, a reduction in population density, and subsequent ecological effects. In this study, we investigate the impacts of pyriproxyfen on Daphnia magna reproduction using a series of male production screening assays. These assays demonstrate that pyriproxyfen increases male production in a concentration-dependent fashion with an EC50 of 156pM (50.24ngL−1); a concentration considered environmentally relevant. Furthermore, pyriproxyfen decreases overall fecundity at all ages tested (7, 14, 21-d old female parthenogenic daphnids). Juvenile (3-d old) and reproductively mature (10-d old) female daphnids were also exposed to 155pM pyriproxyfen for 2–12d and reproduction measured for 16d to compare the effects of short-term and prolonged exposures, and determine the potential for recovery. Results indicate that longer pyriproxyfen exposures (8–12d) extend male production and decrease reproduction; however, daphnids exposed for only 2–4d recover and produce a relatively normal abundance of neonates. In addition, juvenile daphnids are also very sensitive to pyriproxyfen, but the primary effect on juvenile daphnids is reduced reproduction and protracted development not male production. Taken together, continued use of pyriproxyfen around water bodies needs due caution because of its potential adverse effects with significant developmental delays and male production compounded by prolonged exposure.

SPERMATOGENESIS AND SPERM STRUCTURE OF THE NORMALLY PARTHENOGENETIC FRESHWATER SNAIL MELANOIDES TUBERCOLATA

ABSTRACT The freshwater snail Melanoides tuberculata ranges throughout Asia and Africa, and throughout much of its range it reproduces parthenogenetically. In Israel, however, males have been found. These males produce two types of spermatozoa, euspermatozoa and paraspermatozoa. The euspermatozoa are filiform (about 230 μ m in length) with a head comprising a nucleus and conical acrosome, a mid-piece (98 μ m long) of four equal-sized mitochondria, and a tail. Unlike in other cerithiaceans, the nucleus is unusually elongate (31 μ m long × 1 μ m diameter) and the axoneme is housed within an intranuclear canal. The paraspermatozoa consists of a head of electron-dense blocks (up to 25 μ m long) which surround up to 15 axonemes, a mid-piece of elongate mitochondria and a tail tuft, as in other cerithiaceans. Structural changes during spermatogenesis resemble those described for other gastropods with dimorphic spermatozoa.

Assessment of possible hybridization between Bt cotton (Gossypium hirsutum L.) and other Malvaceae species (Abelmoschus spp. and Hibiscus spp.) cultivated in Burkina Faso

The cultivation of transgenic cotton in Burkina Faso should consider the possible hybridizations with cultivated cotton relatives like okra (Abelmoschus spp.) and roselle (Hibiscus spp.), when these crops are often sympatric and co-flowering. The possibility for Bt cotton and these crops to outcross was investigated in Farako-Bâ research centre, in the south sudanian zone. The study was carried out in 2007 using both open-pollinated and artificial pollination experimental designs. Results show that the studied species are effectively reproductively isolated because of low overlapping of their optimum flowering periods. In the case of the artificial crossings (direct and reciprocal) between the studied species and the Bt cotton, the results show that fruits were produced at different rates depending on the species being crossed and the crossing direction. Immunological tests done on progeny seeds resulting from the crosses with transgenic cotton as the male parent confirmed that there was a transfer of transgenes from Bt cotton to the conventional cotton (99.2%). The absence of the transgenes in the seeds of okra and roselle means that these seeds are not the result of intergeneric hybridization but could be attributed to self-pollination or parthenogenetic development of un-fertilized ovules. Key words: Transgenic cotton, relatives’ species, intergenera hybridizations, immunological test, Burkina Faso.

Optimizing of Activation Protocols and Production of Transgenic Pigs Expressing Human Lysozyme by Somatic Cell Nuclear Transfer

In order to improve the cloning efficiency and obtain human lysozyme(hLY) gene transgenic pigs,the present study was carried out to investigate the effects of different electric activation parameters and chemicals on in vitro development of embryos derived from parthenogenesis(PA) and somatic cell nuclear transfer(SCNT).The results showed that the electric strength could influence the fusion rate and developmental ability of SCNT embryos(P 0.05),and number of electric pulses had no significant effect on SCNT embryos development(P 0.05),yet different variation tendency was found in developmental ability between PA and SCNT embryos under same activation parameters.The blastocyst rate of SCNT embryos was improved when treated with CHX+CB for 4 h after electric activation(P 0.05),whereas 6-DMAP did not(P 0.05).On the contrary,either CHX+CB or 6-DMAP treatment after electric pulses could improve the blastocyst rate of PA embryos,indicating that the best activation method for PA was not necessarily the best for SCNT.The best activation protocol of SCNT embryos in our study is two pulses of 100 μs,direct current of 1.6 kV/cm electric strength with 100 μs interval,and followed by CHX+CB treatment for 4 h.With the activation protocol,the mammary gland expressed hLY transgenic pigs were generated.It could help for improving piglets survival rate in transgenic breeding.

Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx platform. The average depth of sequencing coverage was 14,611 for IVV and 17,068 for PA. Quantitative analysis of the methylation profiles of both input samples for each genomic locus showed distinct differences in methylation profiles between IVV and PA samples for six of the target loci, and subtle differences in four loci. It was concluded that high throughput sequencing technologies can be effectively applied to provide a powerful, cost-effective approach to targeted DNA methylation analysis of embryonic and other reproductive tissues.

Effect of Trans-ε-Viniferin on &lt;i&gt;In Vitro&lt;/i&gt; Porcine Oocyte Maturation and Subsequent Developmental Competence in Preimplantation Embryos

ABSTRACTTrans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoid family that has been isolated from Vitis amurensis, one of the most common wild grapes in Asia. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after in vitro fertilization (IVF) or parthenogenesis (PA). We observed that trans-ε-viniferin treatment during IVM did not improve nuclear maturation rates of oocytes in any group, but significantly increased (P<0.05) intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS) levels in the 0.5 µM treatment group. Trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (P<0.05) expression of DNA methyltransferase-1 (DNMT1) mRNA in the 0.5 µM treatment group as compared with the control group. However, the expression of essential transcriptional and apoptosis-related genes did not significantly differ from that of the control. In cumulus cells, pro-apoptosis gene expressions were changed as apoptosis decreased. Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after PA, but total cell numbers were significantly higher (P<0.05) in the 0.5 and 5.0 µM treatment groups compared with those in the control group. IVF embryos showed similar results. In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased the total cell number of blastocysts, possibly by increasing intracellular GSH synthesis, reducing ROS levels, increasing DNMT1 gene expression of oocytes and decreasing pro-apoptosis gene expressions of cumulus cells.

Sexual reproduction in monogonont rotifers: A review

作为轮虫动物门的主体,单巢类轮虫是淡水浮游动物的重要组成部分.它们通常作为一类主要的初级消费者在淡水生态系统中起着重要作用,因而深入了解它们在自然水体中的种群动态及其决定机制对淡水生态系统理论与应用研究都有重要意义.作为一类具有典型周期性孤雌生殖生活史的动物,有性生殖的发生和进化策略对其种群动态和进化都有重要影响,因而单巢类轮虫有性生殖的机理和进化研究一直是轮虫学研究领域的一个核心内容.本文就当前单巢类轮虫有性生殖的主要研究进展,包括其发生机理、信息素、理论模型、影响因素和相关实证研究等进行了系统综述;特别就单巢类轮虫有性生殖策略的三个理论模型:bang-bang模型、中等有性生殖比率模型和非延迟的有性生殖模型进行了详细论述.此外,本文还对影响单巢类有性生殖的内外源因素进行了总结,主要的外源因素包括食物的浓度及种类、温度、盐度和特殊的细菌等,而主要的内源性因素则包括母体年龄、食物浓度诱导的跨代母系效应、延迟的有性生殖和其他一些潜在的母系效应因素等.其中,因为延迟的有性生殖现象广泛存在且可能是轮虫物种共存或克隆共存现象的一种潜在解释,所以就此现象的理论模型、机理和实证研究等方面进行了深入讨论.另外,本文还简要讨论性比率资源分配与单巢类轮虫有性生殖的关系.最后,对未来几个可能的热点方向简单评述,以期为国内轮虫学和相关方面的研究提供帮助.;This paper focuses on the recent findings of sexual reproduction in monogonont rotifers,which account for the main bulk of phylum Rotifera.Generally,they act as one of the major groups of primary consumers in freshwater ecosystem.Thus,studies on population dynamics and their mechanisms of monogonont rotifers may provide important data to both theoretical and applied research on freshwater ecosystems.As a group with parthenogenetic life history,sexual reproductive strategy in monogonont rotifers may profoundly affect their population dynamics and evolution.This review summarized several aspects of sexual reproduction in the monogonont rotifers,such as the mechanism,pheromones,models,influence factors,and particularly,empirical evidences.More specifically,we emphasized three models about their sexual reproduction patterns: bang-bangmodel,intermediate mictic ratios model and no-delaying mixis model.Then we summarized the exogenous and endogenous factors that affect sexual reproduction.The major exogenous factors include the concentration and type of food,temperature,salinity and special bacteria,while the major endogenous factors include the maternal age,a transgenerational maternal effect induced by food concentration,mixis delay and some potential maternal effects.We especially highlighted the model,mechanism and empirical evidences of mixis delay,since this phenomenon existed widely and was deem to be a potential explanation for species or clones coexistence.We also gave some personal views on the relationship between sex allocation theory and sexual reproduction in the monogonont rotifers.Finally,several potential hotspots in the future study were highlighted.

303 THE MODIFIED CULTURE SYSTEM OF PORCINE IN VITRO PRODUCTION IMPROVES COLONIZATION OF PUTATIVE EMBRYONIC STEM CELLS

The establishment of porcine embryonic stem cells (pESC) using in vitro-produced blastocysts derived from IVF, parthenogenesis (PA), and somatic cell nuclear transfer (SCNT) have a great potential. However, porcine in vitro-produced blastocysts show a lower quality than do in vivo-derived blastocysts. In this study, to improve in vitro-derived blastocyst quality, and then to establish pESC, we treated IVF embryos and PA embryos with resveratrol (RES), porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), and β-mercaptoethanol (β-ME). The control system was produced using M199 medium in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The modified group was produced using M199 with 2 µM RES in IVM and PZM5 with 10 ng mL–1 pGM-CSF, 2 µM RES, and 10 µM β-ME in IVC. Data were analysed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test. In total, 1210 PA, 612 IVF, and 5 SCNT embryos were evaluated (for SCNT, we examined only the control system). We observed that overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (P &lt; 0.05) in the modified system (54.5%) compared with the control system (43.4%) in PA and hatched blastocysts rates at Day 6 and 7 were also increased significantly. Total blastocyst cell numbers were significantly higher (P &lt; 0.05) in the modified system (55.1) compared with the control system (45.6). Hatching rate of Day 7 IVF blastocysts was also significantly increased. After seeding porcine blastocysts, the attachment rates were higher in the modified system (32.2% in PA and 36.2% in IVF; not examined in SCNT) than the control system (19.5% in PA, 26.6% in IVF, and 40.0% in SCNT). In addition, colonization rates and cell line derivation rates were higher in the modified system than in the control system. Colonization rates of modified system were 13.1% for PA and 17.75% for IVF embryos, whereas the control system generated 2.4% for PA, 10.8% for IVF, and 20.0% for SCNT. We also investigated the correlation between blastocyst state and attachment rate. The highest attachment rate is in hatched blastocyst (78.35 ± 15.74%). Therefore, the modified system increased quality of porcine blastocysts in vitro produced, and subsequently increased attachment rates. The cell line derivation rates were 2.4% (PA), 4.2% (IVF), and 20.0% (SCNT) in control group. In modified system, they were 7.2% (PA) and 10.0% (IVF). We established 3 cell lines from PA blastocysts (1 cell line in control system and 2 cell lines in modified system) and 1 cell line from SCNT-control system. All cell lines showed alkaline phosphatase activity and expressed pluripotent markers. In conclusion, the modified system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of in vitro-produced porcine blastocysts, and subsequently increased derivation rates of putative pESC. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (#2012004885).

Ascorbic Acid Improves the Developmental Competence of Porcine Oocytes After Parthenogenetic Activation and Somatic Cell Nuclear Transplantation

In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos.

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.

Female parthenogenetic apomixis and androsporogenetic parthenogenesis in embryonal cells of Araucaria angustifolia: interpolation of progenesis and asexual heterospory in an artificial sporangium

Cell fate, development timing and occurrence of reproductive versus apomictic development in gymnosperms are shown to be influenced by culture conditions in vitro. In this study, female parthenogenetic apomixis (fPA), androsporogenetic parthenogenesis (mAP) and progenesis were demonstrated using embryonal initials of Araucaria angustifolia in scaled-up cell suspensions passing through a single-cell bottleneck in darkness and in an artificial sporangium (AS). Expression was based on defined nutrition, hormones and feedforward-adaptive feedback process controls at 23-25°C and in darkness. In fPA, the nucleus of an embryonal initial undergoes endomitosis and amitosis, forming a diploid egg-equivalent and an apoptotic ventral canal nucleus in a transdifferentiated archegonial tube. Discharge of egg-equivalent cells as parthenospores and their dispersal into the aqueous culture medium were followed by free-nuclear conifer-type proembryogenesis. This replaced the plesiomorphic and central features of proembryogenesis in Araucariaceae. Protoplasmic fusions of embryonal initials were used to reconstruct heterokaryotic expressions of fPA in multiwell plates. In mAP, restitutional meiosis (automixis) was responsible for androsporogenesis and the discharge of monads, dyads, tetrads and polyads. In a display of progenesis, reproductive development was brought to an earlier ontogenetic stage and expressed by embryonal initials. Colchicine increased polyploidy, but androspore formation became aberrant and fragmented. Aberrant automixis led to the formation of chromosomal bouquets, which contributed to genomic silencing in embryonal initials, cytomixis and the formation of pycnotic micronucleated cells. Dispersal of female and male parthenospores displayed heteromorphic asexual heterospory in an aqueous environment.

Continuous occurrence of intra-individual chromosome rearrangements in the peach potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

Analysis of the holocentric mitotic chromosomes of the peach-potato aphid, Myzus persicae (Sulzer), from clones labelled 50, 51 and 70 revealed different chromosome numbers, ranging from 12 to 14, even within each embryo, in contrast to the standard karyotype of this species (2n=12). Chromosome length measurements, combined with fluorescent in situ hybridization experiments, showed that the observed chromosomal mosaicisms are due to recurrent fragmentations of chromosomes X, 1 and 3. Contrary to what has generally been reported in the literature, X chromosomes were frequently involved in recurrent fragmentations, in particular at their telomeric ends opposite to the nucleolar organizer region. Supernumerary B chromosomes have been also observed in clones 50 and 51. The three aphid clones showed recurrent fissions of the same chromosomes in the same regions, thereby suggesting that the M. persicae genome has fragile sites that are at the basis of the observed changes in chromosome number. Experiments to induce males also revealed that M. persicae clones 50, 51 and 70 are obligately parthenogenetic, arguing that the reproduction by apomictic parthenogenesis favoured the stabilization and inheritance of the observed chromosomal fragments.

Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcine embryos derived from in vitro fertilization, somatic cell nuclear transfer and parthenogenesis

Evidence in many species has suggested that those embryos that cleave earliest after fertilization are more developmentally competent than those that cleave relatively later after fertilization. Herein we document this phenomenon in porcine in vitro-fertilized (IVF), somatic cell nuclear transfer (SCNT), and parthenogenetic (PA) embryos. In vitro-matured pig oocytes were used to generate IVF, SCNT, and PA embryos. At 24 hr post-activation (or insemination; hpa/hpi), embryos were visually assessed, and cleaved embryos were moved into a new culture well. This process was repeated at 30 and 48 hpa/hpi. All embryos were allowed to develop 7 days in culture. For IVF embryos, 39.9%, 24.6%, and 10.5% of fast-, intermediate-, or slow-cleaving embryos, respectively, developed into blastocysts by day 7. For SCNT embryos, 31.8% of fast-, 5.7% of intermediate-, and 2.9% of late-cleaving embryos achieved the blastocyst stage of development. For PA embryos, the percentages of those cleaved embryos that developed to blastocyst were 59.3%, 36.7%, and 7.5% for early-, intermediate-, and late-cleaving embryos, respectively. Using RNA collected from early-, intermediate-, and late-cleaving embryos, real-time PCR was performed to assess the transcript levels of 14 different genes of widely varied function. The qPCR results suggest that maternal mRNA degradation may not proceed in an appropriate pattern in slow-cleaving embryos. These findings (1) confirm that, as observed in other species, earlier-cleaving porcine embryos are more successful at developing in culture than are slower-cleaving embryos, and (2) implicate mechanisms of maternal transcript destruction as potential determinants of oocyte/embryo quality.