Parthenogenetic Embryos Research Articles

Procyanidin B1 promotes in vitro maturation of pig oocytes by reducing oxidative stress.

Oxidative stress negatively affects the in vitro maturation (IVM) of oocytes. Procyanidin B1 (PB1) is a natural polyphenolic compound that has antioxidant properties. In this study, we investigated the effect of PB1 supplementation during IVM of porcine oocytes. Treatment with 100 μM PB1 significantly increased the MII oocytes rate (p <0.05), the parthenogenetic (PA) blastocyst rate (p <0.01) and the total cell number in the PA blastocyst (p < 0.01) which were cultured in regular in vitro culture (IVC) medium. The PA blastocyst rate of regular MII oocytes activated and cultured in IVC medium supplemented with 100 and 150 μM PB1 significantly increased compared with control (p < 0.01 and p < 0.05). We also evaluated the reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δψm) levels, glutathione (GSH) levels, and apoptotic levels in MII oocytes and cumulus cells following 100 μM PB1 treatment. The results showed that the PB1 supplementation decreased ROS production and apoptotic levels. In addition, PB1 was found to increase Δψm levels and GSH levels. In conclusion, PB1 inhibited apoptosis of oocytes and cumulus cells by reducing oxidative stress. Moreover, PB1 improved the quality of oocytes and promoted PA embryo development. Taken together, our results suggest that PB1 is a promising antioxidant additive for IVM of oocytes.

Pluripotent pig embryonic stem cell lines originating from in vitro-fertilized and parthenogenetic embryos.

Here, we report in vivo developmental competent pig embryonic stem cells (ESCs) originating from in vitro-fertilized and parthenogenetic embryos cultured in chemically defined culture media. The pig ESC lines expressed pluripotency markers and were capable of forming teratomas following subcutaneous injection into nude mice. These cell lines would be a useful resource for comparative developmental biology and agricultural biotechnology.

Proteomic analysis demonstrates that parthenogenetically activated swamp buffalo embryos have dysregulated energy metabolism.

The comprehensive understanding of early embryo development is essential to optimize in vitro culture conditions. Protein expression landscape of parthenogenetically produced embryo remains unexplored. This study aimed to investigate the protein expression dynamics with a particular focus on energy metabolism throughout the early developmental stages of parthenogenetic buffalo embryos. For this purpose, we performed iTRAQ-based quantitative mass spectrometry and identified 280 proteins common in all stages. A total of 933 proteins were identified during the proteomics analysis. The data depicted that morula and blastocyst had distinct protein expression dynamics as compared to 2- to 16-cell-stage embryo. KEGG pathway analysis showed 23 proteins belonging to energy metabolism appeared in the data. Study of energy metabolism-related protein's expression pattern demonstrated that there was asynchrony in proteins related to glycolysis throughout the examined developmental stages. The expression pattern of pyruvate kinase mutase (PKM), an essential protein of glycolysis, indicated a slightly decreasing trend from 2-cell-stage embryo to blastocyst, and it was supported by expression of proteins involved in lactate production (LDHA and LDHB) suggesting the decreasing rate of aerobic glycolysis (Warburg Effect) at morula and blastocyst stage. The increased Warburg Effect is considered as the hallmark of proliferating cells or embryo at the blastocyst stage. Furthermore, the proteins involved in the citric acid cycle also showed down-regulation at the blastocyst stage, indicating a lesser role of oxidative phosphorylation at this stage. Therefore, it could be divulged from the study that there may be an irregular pattern of energy metabolism in early parthenogenetic embryos. Further studies are recommended to understand this phenomenon.

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

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

The Landscape of Genomic Imprinting at the Porcine SGCE/PEG10 Locus from Methylome and Transcriptome of Parthenogenetic Embryos.

In mammals, imprinted genes often exist in the form of clusters in specific chromosome regions. However, in pigs, genomic imprinting of a relatively few genes and clusters has been identified, and genes within or adjacent to putative imprinted clusters need to be investigated including those at the SGCE/PEG10 locus. The objective of this study was to, using porcine parthenogenetic embryos, investigate imprinting status of genes within the genomic region spans between the COL1A2 and ASB4 genes in chromosome 9. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were conducted with normal and parthenogenetic embryos, and methylome and transcriptome were analyzed. As a result, differentially methylated regions (DMRs) between the embryos were identified, and parental allele-specific expressions of the SGCE and PEG10 genes were verified. The pig imprinted interval was limited between SGCE and PEG10, since both the COL1A2 and CASD1 genes at the centromere-proximal region and the genes between PPP1R9A and ASB4 toward the telomere were non-imprinted and biallelically expressed. Consequently, our combining analyses of methylome, transcriptome, and informative polymorphisms revealed the boundary of imprinting cluster at the SGCE/PEG10 locus in pig chromosome 9 and consolidated the landscape of genomic imprinting in pigs.

Elevation of MPF and MAPK gene expression, GSH content and mitochondrial distribution quality induced by melatonin promotes porcine oocyte maturation and development in vitro.

The MPF and MAPK genes play crucial roles during oocyte maturation processes. However, the pattern of MPF and MAPK gene expression induced by melatonin (MT) and its correlation to oocyte maturation quality during the process of porcine oocyte maturation in vitro remains unexplored. To unravel it, in this study, we cultured the porcine oocytes in maturation medium supplemented with 0, 10−6, 10−9, and 10−12 mol/L melatonin. Later, we analyzed the MPF and MAPK gene expression levels by RT-PCR and determined the maturation index (survival and maturation rate of oocytes). The GSH content in the single oocyte, and cytoplasmic mitochondrial maturation distribution after porcine oocyte maturation in vitro was also evaluated. We also assessed the effects of these changes on parthenogenetic embryonic developmental potential. The oocytes cultured with 10−9mol/L melatonin concentration showed higher oocyte maturation rate, and MPF and MAPK genes expression levels along with better mitochondrial distribution than the 0, 10−6, and 10−12 mol/L melatonin concentrations (p < 0.05). No significant difference was observed in the survival rates when the oocytes were cultured with different melatonin concentrations. The expression of the MPF gene in the oocytes cultured with 10−6 mol/L melatonin was higher than with 10−12 and 0 mol/L melatonin, and the expression of the MAPK gene in 10−6 and 10−12 group was higher than the control (p < 0.05). As far as the embryonic developmental potential is concerned, the cleavage and blastocyst rate of oocytes cultured with 10−6 and 10−9 mol/L melatonin was significantly higher than the 10−12 mol/L melatonin and control. In conclusion, 10−9–10−6 mol/L melatonin significantly induced the MPF and MAPK gene expression; besides, it could also be correlated with GSH content of single oocyte, mitochondrial maturation distribution, and the first polar body expulsion. These changes were also found to be associated with parthenogenetic embryo developmental potential in vitro.

Opportunities and Challenges in Doubled Haploids and Haploid Inducer-Mediated Genome-Editing Systems in Cucurbits

Doubled haploids have played a major role in cucurbit breeding for the past four decades. In situ parthenogenesis via irradiated pollen is the preferred technique to obtain haploid plantlets whose chromosomes are then doubled in Cucurbitaceae, such as melon, cucumber, pumpkin, squash and winter squash. In contrast to doubled haploid procedures in other species, in situ parthenogenesis in cucurbits presents many limiting factors which impede efficient production of haploids. In addition, it is very time-consuming and labor-intensive. However, the haploid inducer-mediated genome-editing system is a breakthrough technology for producing doubled haploids. Several reports have described using the CRISPR/Cas9 system in cucurbit species, and although its application has many bottlenecks, the targeted knock-out of the CENH3 gene will allow breeders to obtain haploid inducer lines that can be used to obtain parthenogenetic embryos. In this review, we discuss the progress made towards the development of doubled haploids and haploid inducer genotypes using CRISPR/Cas9 technologies in cucurbit species. The present review provides insights for the application of haploid inducer-mediated genome-editing system in cucurbit species

Haploidy in Tobacco Induced by PsASGR-BBML Transgenes via Parthenogenesis.

Background: Engineering apomixis in sexually reproducing plants has been long desired because of the potential to fix hybrid vigor. Validating the functionality of genes originated from apomictic species that contribute to apomixis upon transfer to sexually reproducing species is an important step. The PsASGR-BABYBOOM-like (PsASGR-BBML) gene from Pennisetum squamulatum confers parthenogenesis in this apomict, and its functionality was demonstrated in several sexually reproducing monocots but not in any dicots. Methods: We introduced the PsASGR-BBML gene regulated by egg cell-specific promoters, either AtDD45 or AtRKD2, into tobacco, and analyzed progeny of the transgenic lines resulting from self-pollination and crossing by flow cytometry. Results: We identified haploid progeny at a frequency lower than 1% in the AtDD45pro lines, while at a frequency of 9.3% for an octoploid (2n = 8x) AtRKD2pro line. Haploid production in the T2 generation, derived from the tetraploid T1 offspring of this original octoploid AtRKD2pro line, was also observed. Pollinated by homozygous transgenic tobacco carrying a DsRed marker gene, 4x progeny of the AtRKD2pro line yielded parthenogenetic embryos identified as DsRed negative. We verified that the DsRed negative seedlings recovered were haploid (2x). Conclusion: The PsASGR-BBML gene regulated by egg cell-specific promoters could enable parthenogenesis in tobacco, a dicotyledon species.

Assessment of different stages of parthenogenetic embryos for production of embryonic stem cell like colonies

Parthenogenetic activation of oocytes has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. In this study, we described the production of goat parthenogenetic ES like cells from different stages of parthenogenetic embryos. We compared the source material on the pESCs outgrowth and culture. 8-16 cell stage embryos, morula, blastocysts and ICM (inner cell mass) from hatched blastocysts produced from chemical activation were cultured on goat fetal fibroblast monolayer in stem cell culture media. Colonies were passaged when signs of differentiation were visible. ESC like colonies were cultured on feeder cells in the presence of hLIF however, some ESC like colonies were also cultured in absence of hLIF for random differentiation. In our study, ESC like colony formation with ICM of hatched blastocyst was comparatively higher as compared to blastocysts while ESC like colony formation with blastocysts and ICM of hatched blastocyst was significantly higher as compared 8-16 cells and morula. It is observed that inner cell mass source is an important criterion for the ES like cells derivation in goats. Also, in comparison to the expanded blastocysts (80.10%), hatched blastocysts showed higher (86.06%) attachment rate and primary colony formation rate. ESC like colonies in absence of hLIF differentiated in vitro into epithelial like and neuronal like cells. Undifferentiated ESC like colonies stained positive for alkaline phosphatase, SSEA-3 and OCT-4.

Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq.

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

CHIR99021 and rpIL6 promote porcine parthenogenetic embryo development and blastocyst quality

Signaling pathways and transcription factors are involved in porcine embryonic development. Here, we demonstrate that glycogen synthase kinase-3 (GSK3) inhibitor, CHIR99021 and recombinant porcine interleukin-6 (rpIL6) significantly promote porcine parthenogenetic blastocyst formation (49.23 ± 8.40% vs 32.34 ± 4.15%), with increased inner cell mass (ICM) cell numbers (7.72 ± 2.30 vs 4.28 ± 1.60) and higher expression of pluripotent genes, such as OCT4, SOX2 and NANOG. Furthermore, CHIR99021 and rpIL6 improve blastocyst quality with increased blastocyst hatching percentage (16.19 ± 1.96% vs 10.25 ± 1.12%) and subsequently porcine pluripotent stem cells (pPSCs) derivation efficiency. These results advance the understanding of porcine pre-implantation development and provide evidences in improving the blastocyst quality.

Effect of Triclosan Exposure on Developmental Competence in Parthenogenetic Porcine Embryo during Preimplantation.

Triclosan (TCS) is included in various healthcare products because of its antimicrobial activity; therefore, many humans are exposed to TCS daily. While detrimental effects of TCS exposure have been reported in various species and cell types, the effects of TCS exposure on early embryonic development are largely unknown. The aim of this study was to determine if TCS exerts toxic effects during early embryonic development using porcine parthenogenetic embryos in vitro. Porcine parthenogenetic embryos were cultured in in vitro culture medium with 50 or 100 µM TCS for 6 days. Developmental parameters including cleavage and blastocyst formation rates, developmental kinetics, and the number of blastomeres were assessed. To determine the toxic effects of TCS, apoptosis, oxidative stress, and mitochondrial dysfunction were assessed. TCS exposure resulted in a significant decrease in 2-cell rate and blastocyst formation rate, as well as number of blastomeres, but not in the cleavage rate. TCS also increased the number of apoptotic blastomeres and the production of reactive oxygen species. Finally, TCS treatment resulted in a diffuse distribution of mitochondria and decreased the mitochondrial membrane potential. Our results showed that TCS exposure impaired porcine early embryonic development by inducing DNA damage, oxidative stress, and mitochondrial dysfunction.

Housekeeping genes for RT-qPCR in ovine preimplantation embryos.

Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20-105.96%), with correlation coefficients ranging from -0.922 to -0.998 and slopes from -3.22 to -3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.

Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

Effects of cobalamin on meiotic resumption and developmental competence of growing porcine oocytes

This study was performed to explore the effects of cobalamin treatment during in vitro maturation (IVM) of porcine oocytes. Specifically, the effects of cobalamin exposure on nuclear and cytoplasmic maturation of oocytes, diameter, glutathione (GSH) and reactive oxygen species (ROS) levels of matured oocytes, as well as development and gene expression of porcine parthenogenetic and cloned embryos were assessed. Cumulus–oocyte complexes were exposed to 200 pM cobalamin for 22 h or incubated for 22 h without cobalamin (controls). The mean diameter of cobalamin-treated oocytes was greater than that of control oocytes (160.0 vs. 154.5 μm; p < 0.05). GSH level increased but ROS level decreased in the cobalamin-treated oocyte group. Parthenogenetic embryos derived from cobalamin-treated oocytes showed improved oocyte maturation (91.3% vs. 83.8%), cleavage (88.9% vs. 82.1%), and blastocyst formation (38.7% vs. 31.9%) rates compared with control embryos (p < 0.05). Similarly, cloned embryos derived from cobalamin-treated oocytes showed higher oocyte maturation (89.2% vs. 82.2%), cleavage (87.5% vs. 80.3%), and blastocyst formation (30.0% vs. 23.8%) rates than control embryos (p < 0.05). Furthermore, in parthenogenetic and cloned embryos, total cell number, inner cell mass (ICM), trophectoderm (TE) expression, and ICM: TE ratio were higher in the cobalamin-treated group compared to that in the control group (p < 0.05). Cloned embryos in the cobalamin-treated group showed higher mRNA expression levels of POU5F1, DPPA2, and NDP52IL than control group embryos. Together, these results demonstrate that cobalamin treatment during IVM improves the developmental competence of porcine oocytes by neutralizing the free radicals produced in the IVM medium.

Fatty acid induced lipolysis influences embryo development, gene expression and lipid droplet formation in the porcine cumulus cells†.

The pig oocyte maturation protocol differs from other mammalian species due to dependence on follicular fluid (FF) supplementation. One of the most abundant components of the porcine follicular fluid are fatty acids (FAs). Although evidence from other mammalian models revealed a negative impact of saturated fatty acids (SFA) on developmental competence of oocytes, pig has not yet been widely analyzed. Therefore, we aimed to investigate whether supplementation of IVM medium with 150 μM of stearic acid (SA) and oleic acid (OA) affects lipid content and expression of genes related to fatty acid metabolism in porcine cumulus–oocyte complexes and parthenogenetic embryo development. We found significant influence of fatty acids on lipid metabolism in cumulus cells without affecting the oocyte proper. The expression of ACACA, SCD, PLIN2, FADS1, and FADS2 genes was upregulated (P < 0.01) in cumulus cells, while their expression in oocytes did not change. The increase in gene expression was more pronounced in the case of OA (e.g., up to 30-fold increase in PLIN2 transcript level compared to the control). The number of lipid droplets and occupied area increased significantly in the cumulus cells and did not change in oocytes after SA treatment. Oleic acid improved the blastocyst rate (48 vs 32% in control), whereas stearic acid did not affect this parameter (27%). Additionally, we have discovered a phenotypic diversity of LD in cumulus cells in response to FA supplementation, suggesting extensive lipolysis in response to SA. Stearic acid excess in maturation media led to the formation of multiple micro lipid droplets in cumulus cells.

In situ Parthenogenetic Doubled Haploid Production in Melon "Piel de Sapo" for Breeding Purposes.

Doubled haploids in cucurbit species are produced through in situ parthenogenesis via pollination with irradiated pollen for further use as parental lines for hybrid F1 production. In this study, seven genotypes of melon “Piel de Sapo” were appraised for agronomic traits and pathogen resistances to evaluate its commercial value and used as donor plant material for the parthenogenetic process. Then, in situ parthenogenetic capacity of melon “Piel de Sapo” germplasm was evaluated and optimized. Several steps of the parthenogenetic process were assessed in this study such as melon fruit set after pollination with irradiated pollen, haploid embryo obtention, in vitro germination and growth of parthenogenetic embryos and plantlets, in vitro and in vivo chromosome doubling with colchicine or oryzalin and fruit set of doubled haploid lines. Parthenogenetic efficiencies of “Piel de Sapo” genotypes showed a high genotypic dependency during the whole process. Three different methods were assayed for parthenogenetic embryo detection: one-by-one, X-ray and liquid medium. X-ray radiography of seeds was four times faster than one-by-one method and jeopardized eight times less parthenogenetic embryo obtention than liquid medium. One third of melon fruits set after pollination with irradiated pollen contained at least one parthenogenetic embryo. The 50.94% of the embryos rescued did not develop into plantlets because failed to germinate or plantlet died at the first stages of development because of deleterious gene combination in haploid homozygosity. The distribution of the ploidy-level of the 26 parthenogenetic plantlets obtained was: 73.08% haploid, 23.08% spontaneous doubled haploid and 3.84% mixoploid. Two in vitro chromosome doubling methods, with colchicine or oryzalin, were compared with a third in vivo colchicine method. In vivo immersion of apical meristems in a colchicine solution for 2 h showed the highest results of plant survival, 57.33%, and chromosome doubling, 9.30% mixoploids and 20.93% doubled haploids. Fruit set and seed recovery of doubled haploids lines was achieved. In this study, doubled haploid lines were produced from seven donor genotypes of melon “Piel de Sapo,” however, further improvements are need in order to increase the parthenogenetic efficiency.

Production and Characterization of Mouse Diploid Parthenogenetic Blastocyst Developed in Phosphate-Free Medium

Parthenogenesis is an artificial oocytes activation process without paternal contribution. Blastocyst, derived from parthenogenesis, is one of potential source for pluripotent stem cell propagation. Unfortunately, previous studies reported that parthenogenetic embryo did not achieve exhilarating blastocyst rate. One of the component that predicted inhibit parthenogenetic embryo development is phosphate. Therefore, we try to modify culture medium in order to overcome that problem. The aim of this research was to produce and analyze the characteristics of parthenogenetic blastocyst developed in phosphate-free medium. Mouse oocytes obtained from adult female DDY by superovulation. The activator was strontium chloride 10 mM and diploidization with cytochalasin B 5 μg/ml. Medium for activation and culture medium were modified rat 1 cell embryo medium (MR1ECM) which is phosphate free. The results showed that parthenotes that were cultured in phosphate free medium reached higher blastocyst rate compared to the other groups. The increase of phosphate in culture medium lead to impaired parthenogenetic embryos development. Further experiment was made to analyze the differences between fertilized and parthenogenetic embryo in this medium. The experiment showed that diploid parthenogenetic could achieve high blastocyst rate (30.9±1.3%). The quality of diploid parthenogenetic blastocyst, based on cells number, viability, and ICM ratio, was lower than fertilized blastocyst.

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos.

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10-11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.