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Theriogenology Feb 2024In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture...
Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation.
In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.
Topics: Pregnancy; Female; Swine; Animals; Oocytes; Embryonic Development; In Vitro Oocyte Maturation Techniques; Parthenogenesis; Blastocyst; Proto-Oncogene Proteins c-bcl-2; Cumulus Cells
PubMed: 38008049
DOI: 10.1016/j.theriogenology.2023.11.011 -
Studies in Health Technology and... Nov 2023The growth and development of early mammalian embryos mainly take place in the fallopian tube, which not only provides nutrients for embryonic growth and development but...
The growth and development of early mammalian embryos mainly take place in the fallopian tube, which not only provides nutrients for embryonic growth and development but also offers suitable mechanical conditions. The embryo culture system established in assisted reproductive technology mainly simulates the environment in which oocytes and embryos grow and develop in vivo. However, current in vitro embryo culture is mainly static and cannot completely mimic the mechanical environment in which embryos grow and develop in vivo. Therefore, to more accurately simulate the mechanical environment of embryos in the fallopian tube, we have developed a dynamic culture device to investigate the effects of mechanical stimulation on the in vitro maturation of immature oocytes and their parthenogenetic developmental potential. Immature mice oocytes were subjected to in vitro maturation by static culture and vibration (3 Hz, 6 Hz) with tilting for 15∼16 hours. The maturation of oocytes was observed after the culture period. The mature oocytes were activated by parthenogenesis and the rate of embryo compaction and formation of parthenogenetic blastocysts was analyzed. The results showed that using 3 Hz vibration and tilting can significantly improve the parthenogenetic development potential of immature mice oocytes.
Topics: Female; Animals; Mice; Vibration; Embryonic Development; Parthenogenesis; Oocytes; Blastocyst; Mammals
PubMed: 38007729
DOI: 10.3233/SHTI230828 -
Methods in Molecular Biology (Clifton,... 2024The maternally transmitted reproductive manipulator Wolbachia can impact sex ratios of its arthropod host by different mechanisms, ultimately promoting the spread of...
The maternally transmitted reproductive manipulator Wolbachia can impact sex ratios of its arthropod host by different mechanisms, ultimately promoting the spread of infection across a population. One of these reproductive phenotypes, parthenogenesis induction (PI), is characterized by the asexual production of female offspring, which in many cases results in an entirely female population. Cases of Wolbachia-mediated PI are most common in the order Hymenoptera, specifically in parasitoid wasps. The complex sex determination pathways of hymenopterans, their diverse life histories, the multiple cytogenetic mechanisms of PI, and the lack of males make functional studies of parthenogenesis induction challenging. Here, we describe the mechanisms of PI, outline methods to recognize and cure PI-Wolbachia infection, and note possible complications when working with PI-Wolbachia strains and their parthenogenetic hosts.
Topics: Animals; Male; Female; Wolbachia; Parthenogenesis; Wasps; Reproduction
PubMed: 38006545
DOI: 10.1007/978-1-0716-3553-7_4 -
Molecular Plant Jan 2024The current apomixis system used in fixing heterozygosity suffers from the problems of low fertility and limited apomixis induction rate. This study implies that...
The current apomixis system used in fixing heterozygosity suffers from the problems of low fertility and limited apomixis induction rate. This study implies that egg-cell-specific expression of dandelion's PAR combined with MiMe in hybrid rice can efficiently trigger highly fertile synthetic apomixis for effective clonal propagation of hybrids.
Topics: Oryza; Apomixis; Fertility; Phenotype; Seeds
PubMed: 37990497
DOI: 10.1016/j.molp.2023.11.007 -
Annual Review of Animal Biosciences Feb 2024Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of... (Review)
Review
Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of renewed attention as a tool for rescuing endangered or extinct species, it seems timely to dissect the role of the numerous reproductive techniques encompassed by this term in animal species conservation. Although cloning is typically associated with somatic cell nuclear transfer, the recent advent of additional techniques that allow genome replication without genetic recombination demands that the use of induced pluripotent stem cells to generate gametes or embryos, as well as older methods such as embryo splitting, all be included in this discussion. Additionally, the phenomenon of natural cloning (e.g., a subset of fish, birds, invertebrates, and reptilian species that reproduce via parthenogenesis) must also be pointed out. Beyond the biology of these techniques are practical considerations and the ethics of using cloning and associated procedures in endangered or extinct species. All of these must be examined in concert to determine whether cloning has a place in species conservation. Therefore, we synthesize progress in cloning and associated techniques and dissect the practical and ethical aspects of these methods as they pertain to endangered species conservation.
Topics: Animals; Endangered Species; Cloning, Organism; Nuclear Transfer Techniques; Fishes; Cloning, Molecular
PubMed: 37988633
DOI: 10.1146/annurev-animal-071423-093523 -
International Journal of Molecular... Oct 2023Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we...
Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1, and p-CDK1, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations ( < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments ( < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of did not show differences. In addition, the ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment ( < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.
Topics: Cattle; Animals; Parthenogenesis; Maturation-Promoting Factor; Mitogen-Activated Protein Kinases; Adenine; Oocytes; Cycloheximide; Blastocyst; Anisomycin; Mammals
PubMed: 37958778
DOI: 10.3390/ijms242115794 -
Nature Communications Nov 2023The formation and consequences of polyploidization in animals with clonal reproduction remain largely unknown. Clade I root-knot nematodes (RKNs), characterized by...
The formation and consequences of polyploidization in animals with clonal reproduction remain largely unknown. Clade I root-knot nematodes (RKNs), characterized by parthenogenesis and allopolyploidy, show a widespread geographical distribution and extensive agricultural destruction. Here, we generated 4 unzipped polyploid RKN genomes and identified a putative novel alternative telomeric element. Then we reconstructed 4 chromosome-level assemblies and resolved their genome structures as AAB for triploid and AABB for tetraploid. The phylogeny of subgenomes revealed polyploid RKN origin patterns as hybridization between haploid and unreduced gametes. We also observed extensive chromosomal fusions and homologous gene expression decrease after polyploidization, which might offset the disadvantages of clonal reproduction and increase fitness in polyploid RKNs. Our results reveal a rare pathway of polyploidization in parthenogenic polyploid animals and provide a large number of high-precision genetic resources that could be used for RKN prevention and control.
Topics: Animals; Polyploidy; Hybridization, Genetic; Triploidy; Germ Cells; Chromosomes; Nematoda
PubMed: 37935661
DOI: 10.1038/s41467-023-42700-w -
TAG. Theoretical and Applied Genetics.... Nov 2023We present the highest-density genetic map for the hexaploid Urochloa humidicola. SNP markers expose genetic organization, reproduction, and species origin, aiding...
We present the highest-density genetic map for the hexaploid Urochloa humidicola. SNP markers expose genetic organization, reproduction, and species origin, aiding polyploid and tropical forage research. Tropical forage grasses are an important food source for animal feeding, with Urochloa humidicola, also known as Koronivia grass, being one of the main pasture grasses for poorly drained soils in the tropics. However, genetic and genomic resources for this species are lacking due to its genomic complexity, including high heterozygosity, evidence of segmental allopolyploidy, and reproduction by apomixis. These complexities hinder the application of marker-assisted selection (MAS) in breeding programs. Here, we developed the highest-density linkage map currently available for the hexaploid tropical forage grass U. humidicola. This map was constructed using a biparental F population generated from a cross between the female parent H031 (CIAT 26146), the only known sexual genotype for the species, and the apomictic male parent H016 (BRS cv. Tupi). The linkage analysis included 4873 single nucleotide polymorphism (SNP) markers with allele dosage information. It allowed mapping of the ASGR locus and apospory phenotype to linkage group 3, in a region syntenic with chromosome 3 of Urochloa ruziziensis and chromosome 1 of Setaria italica. We also identified hexaploid haplotypes for all individuals, assessed the meiotic configuration, and estimated the level of preferential pairing in parents during the meiotic process, which revealed the autopolyploid origin of sexual H031 in contrast to apomictic H016, which presented allopolyploid behavior in preferential pairing analysis. These results provide new information regarding the genetic organization, mode of reproduction, and allopolyploid origin of U. humidicola, potential SNPs markers associated with apomixis for MAS and resources for research on polyploids and tropical forage grasses.
Topics: Humans; Female; Male; Apomixis; Plant Breeding; Poaceae; Polyploidy; Genomics
PubMed: 37919432
DOI: 10.1007/s00122-023-04485-w -
FASEB Journal : Official Publication of... Dec 2023Glucose-regulated protein 78 (GRP78) binds to and stabilizes melanocortin 4 receptor (MC4R), which activates protein kinase A (PKA) by regulating G proteins. GRP78 is...
Glucose-regulated protein 78 (GRP78) binds to and stabilizes melanocortin 4 receptor (MC4R), which activates protein kinase A (PKA) by regulating G proteins. GRP78 is primarily used as a marker for endoplasmic reticulum stress; however, its other functions have not been well studied. Therefore, in this study, we aimed to investigate the function of GRP78 during porcine embryonic development. The developmental quality of porcine embryos, expression of cell cycle proteins, and function of mitochondria were evaluated by inhibiting the function of GRP78. Porcine oocytes were activated to undergo parthenogenesis, and blastocysts were obtained after 7 days of in vitro culture. GRP78 function was inhibited by adding 20 μM HA15 to the in vitro culture medium. The inhibition in GRP78 function led to a decrease in G proteins release, which subsequently downregulated the cyclic adenosine monophosphate (cAMP)/PKA pathway. Ultimately, inhibition of GRP78 function induced the inhibition of CDK1 and cyclin B expression and disruption of the cell cycle. In addition, inhibition of GRP78 function regulated DRP1 and SIRT1 expression, resulting in mitochondrial dysfunction. This study provides new insights into the role of GRP78 in porcine embryonic development, particularly its involvement in the regulation of the MC4R pathway and downstream cAMP/PKA signaling. The results suggest that the inhibition of GRP78 function in porcine embryos by HA15 treatment may have negative effects on embryo quality and development. This study also demonstrated that GRP78 plays a crucial role in the functioning of MC4R, which releases the G protein during porcine embryonic development.
Topics: Female; Pregnancy; Swine; Animals; Receptor, Melanocortin, Type 4; Endoplasmic Reticulum Chaperone BiP; Embryonic Development; Parthenogenesis; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; GTP-Binding Proteins
PubMed: 37917004
DOI: 10.1096/fj.202301356R -
Zygote (Cambridge, England) Dec 2023Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during maturation improves...
Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy . However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.
Topics: Pregnancy; Animals; Female; Swine; RNA, Guide, CRISPR-Cas Systems; Oocytes; Blastocyst; Embryonic Development; Parthenogenesis
PubMed: 37905414
DOI: 10.1017/S096719942300045X