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

2020 ◽  
Vol 87 (4) ◽  
pp. 493-502
Author(s):  
Chen Liao ◽  
Xinghui Shen ◽  
Yuwei Zhang ◽  
Lei Lei
Keyword(s):  
Paternal Genome ◽  
Androgenetic Embryos

scholarly journals Amount of maternal factor within the male pronucleus affects the reprogramming and developmental efficiency of androgenetic embryos

2019 ◽  
Author(s):  
Chen Liao ◽  
Xinghui Shen ◽  
Yuwei Zhang ◽  
Lei Lei
Keyword(s):  
Embryonic Development ◽  
Biological Significance ◽  
Specific Gene ◽  
Paternal Genome ◽  
Developmental Potential ◽  
Reprogramming Factors ◽  
Androgenetic Embryos ◽  
Early Developmental ◽  
Parthenogenetic Embryos ◽  
Lower Expression

Abstract Background Uniparental embryos have uniparental genomes and are very useful models for studying parental specific gene expression 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 the nuclei of sperm after fertilization to guarantee embryonic development by maternal derived reprogramming factors, which accumulate during oogenesis. However, importance of maternal materials in the efficiency of reprogramming the pronucleus of androgenetic embryos has not been ascertained.Results Androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS) in our experiments. Compared with the androgenetic embryos constructed artificially by DS, those constructed by PT, which derived from two zygotes, contained more maternal material (like Tet3 and H3.3). This study confirmed the better developmental potential of PT embryos, with higher blastocyst rates, the stronger expression of pluripotent genes, the lower expression of apoptotic genes, and superior blastocyst quality.Conclusions The aggregation of more maternal materials in the paternal pronucleus facilitated the reprogramming of the paternal genome, improving embryonic development in pronucleus transfer androgenesis.

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

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 4195-4202 ◽  
Author(s):  
H. Sasaki ◽  
A.C. Ferguson-Smith ◽  
A.S. Shum ◽  
S.C. Barton ◽  
M.A. Surani
Keyword(s):  
Preimplantation Embryos ◽  
Upstream Region ◽  
Transcription Start ◽  
Paternal Genome ◽  
Spatial Regulation ◽  
Inactivation Mechanism ◽  
H19 Gene ◽  
Temporal And Spatial ◽  
Androgenetic Embryos ◽  
Parthenogenetic Embryos

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

203 PRODUCTION OF ANDROGENETIC SHEEP BLASTOCYSTS

2007 ◽  
Vol 19 (1) ◽  
pp. 218
Author(s):  
K. Matsukawa ◽  
M. Y. Turco ◽  
G. Ptak ◽  
P. Loi
Keyword(s):  
Uv Light ◽  
Sperm Concentration ◽  
Control Group ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Paternal Genome ◽  
Significant Difference ◽  
Pronuclear Transfer ◽  
Androgenetic Embryos

The androgenetic embryo is a useful model for investigating the contribution of the paternal genome (e.g. genomic imprinting) to embryonic development. Few works on androgenetic embryo production in domestic animals exist. In this study, we compared the developmental ability of diploid, haploid, and triploid androgenetic sheep embryos. In vitro-matured metaphase II oocytes were enucleated in HEPES-buffered TCM-199 with cytochalasin B (7.5 µg mL−1) and Hoechst 33342 (5 µg mL−1) under UV light using a Narishighe Micromanipulator fitted to an inverted Nikon microscope. Enucleated oocytes were fertilized in vitro with a high sperm concentration (2.5 × 107 sperm mL−1). Fifteen hours after in vitro fertilization (IVF), embryos were centrifuged (12 000g for 10 min) to visualize the pronuclei; the number of pronuclei were scored under the inverted microscope. In Experiment 1, IVF (control), haploid (1 pronucleus), diploid (2 pronuclei), and triploid (3 pronuclei) embryos were cultured in SOFaa medium with BSA, according to the protocol in our laboratory (Ptak et al. 2002 Biol. Reprod. 67, 1719–1725). In Experiment 2, we performed pronuclear transfer to produce diploid embryos. A single pronucleus was aspirated with a bevelled pipette from haploid (haploid + haploid) or diploid (diploid + haploid) embryos and transferred into the perivitelline space of another haploid embryo. The reconstructed zygotes were electrofused in 0.27 M mannitol solution with 50 µM CaCl2 and 100 µM MgCl2 by a single DC pulse (0.8 kV cm−1 for 80 µs). As a control group for Experiment 2, IVF embryos (pronuclear stage) were centrifuged, followed by the aspiration of a small volume of cytoplasm, and fused under the same condition of diploidization. In Experiment 1, there was no significant difference in cleavage rate (91% to 98%), but there was a significant difference on blastocyst development between IVF and androgenetic embryos (IVF: 43% (26/60); haploid: 0% (0/37); diploid: 1% (1/73); and triploid: 2% (1/48)). In Experiment 2, there was no significant difference in cleavage rate (94% to 100%). However, there was a significant difference on blastocyst development (Control: 42%; haploid + diploid: 19%; and haploid + haploid: 3%; Table 1). Our results suggest that sheep androgenetic embryos show poor developmental ability compared with IVF embryos. Interestingly, diploid androgenetic embryos produced by IVF displayed very poor development; however, such poor development was rescued, for unknown reasons, by pronuclear transfer. Ongoing experiments will provide new insight into this previously uncharacterized phenomenon. In conclusion, pronuclear transfer was an effective method for producing sheep androgenetic blastocysts in vitro. Table 1.Development of androgenetic embryos after pronuclear transfer

scholarly journals Localization of genes encoding egg modifiers of paternal genome function to mouse chromosomes one and two

Development ◽  
1998 ◽  
Vol 125 (5) ◽  
pp. 929-935
Author(s):  
K.E. Latham ◽  
C. Sapienza
Keyword(s):  
Inbred Mouse ◽  
Nuclear Transplantation ◽  
F1 Hybrids ◽  
Mouse Strains ◽  
Paternal Genome ◽  
Developmental Potential ◽  
Genes Encoding ◽  
The Difference ◽  
Androgenetic Embryos ◽  
Strain Dependent

It is now well established that genomic imprinting effects in mammals require a combination of epigenetic modifications imposed during gametogenesis and additional modifications imposed after fertilization. The earliest post-fertilization modifications to be imposed on the genome are those thought to be mediated by factors in the egg cytoplasm. Strain-dependent differences in the actions of these egg modifiers in mice reveal an important potential for genetic variability in the imprinting process, and also provide valuable genetic systems with which to identify some of the factors that participate in imprinting. Previous studies documented a strain-dependent difference in the modification of paternal genome function between the C57BL/6 and DBA/2 mouse strains. This difference is revealed as a difference in developmental potential of androgenetic embryos produced with eggs from females of the two strains by nuclear transplantation. The specificity of the effect for the paternal genome is consistent with an effect on imprinted genes. The egg phenotype is largely independent of the genotype of the fertilizing sperm, and the C57BL/6 phenotype is dominant in reciprocal F1 hybrids. Genetic studies demonstrated that the difference in egg phenotypes between the two strains is most likely controlled by two independently segregating loci. We now report the results of experiments in which the egg phenotypes of the available BxD recombinant inbred mouse strains have been determined. The results of the analysis are consistent with the two locus model, and we have identified candidate chromosomal locations for the two loci. These data demonstrate clearly that differences in how the egg cytoplasm modifies the incoming paternal genome are indeed genetically determined, and vary accordingly.

Effect of egg composition on the developmental capacity of androgenetic mouse embryos

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 561-568 ◽  
Author(s):  
K.E. Latham ◽  
D. Solter
Keyword(s):  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Paternal Genome ◽  
Maternal Genotype ◽  
Developmental Capacity ◽  
Egg Composition ◽  
Androgenetic Embryos ◽  
Dependent Modification ◽  
Strain Dependent

Analysis of the developmental capacities of androgenetic and gynogenetic mouse embryos (bearing two paternal or two maternal pronuclei, respectively) revealed a defect in blastocyst formation of androgenetic, but not gynogenetic, embryos that was a function of the maternal genotype. Androgenetic embryos constructed using fertilized eggs from C57BL/6 or (B6D2)F1 mice developed to the blastocyst stage at frequencies similar to those previously reported, whereas androgenetic embryos constructed with fertilized eggs from DBA/2 mice developed poorly, the majority failing to progress beyond the 16-cell stage and unable to form a blastocoel-like cavity, regardless of whether the male pronuclei were of C57BL6 or DBA/2 origin. This impaired development was observed even in androgenetic embryos constructed by transplanting two male pronuclei from fertilized DBA/2 eggs to enucleated C57BL/6 eggs, indicating that the defect cannot be explained as the lack of some essential component in the DBA/2 cytoplasm that might otherwise compensate for androgeny. Rather, the DBA/2 egg cytoplasm apparently modifies the incoming male pronuclei differently than does C57BL/6 egg cytoplasm. Several specific alterations in the protein synthesis pattern of DBA/2 androgenones were observed that reflect a defect in the regulatory mechanisms that normally modulate the synthesis of these proteins between the 8-cell and blastocyst stages. These results are consistent with a model in which cytoplasmic factors present in the egg direct a strain-dependent modification of paternal genome function in response to epigenetic modifications (genomic imprinting) established during gametogenesis and indicate that preimplantation development can be affected by these modifications at both the morphological and biochemical levels.

Sperm capacitation combined with removal of the sperm acrosome and plasma membrane enhances paternal nucleus remodelling and early development of bovine androgenetic embryos

2013 ◽  
Vol 25 (4) ◽  
pp. 624 ◽  
Author(s):  
Yao Xiao ◽  
Hualin Zhang ◽  
Sibtain Ahmad ◽  
Liya Bai ◽  
Xiaomin Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Early Development ◽  
Developmental Competence ◽  
Sperm Capacitation ◽  
Paternal Genome ◽  
Morula Stage ◽  
Chromatin Integrity ◽  
Androgenetic Embryos ◽  
Sperm Acrosome ◽  
Sperm Decondensation

The androgenetic embryo is a useful model for functional analysis of the paternal genome during embryogenesis. However, few studies have focused on the factors involved in the suppressed developmental competence of such embryos or why sperm cloning-derived androgenetic embryos fail to develop beyond the morula stage in large domestic animals. To overcome this developmental failure, we tried to improve sperm decondensation, as well as to enhance embryonic development by sperm capacitation and removal of the acrosome and plasma membrane before injection of the spermatozoa. Before injection of the spermatozoa, we quantified the effects of sperm capacitation combined with sperm pretreatment on the acrosome and plasma membrane status. We also evaluated sperm decondensation potential, sperm viability and chromatin integrity. Immunostaining data showed that the sperm acrosome and plasma membrane could be more efficiently removed after capacitation. Dithiothreitol-induced sperm decondensation potential was improved with capacitation and removal of the acrosome and plasma membrane. Although most spermatozoa lost viability after pretreatment, their chromatin remained integrated. The patterns of paternal chromatin remodelling within uncleaved androgenetic embryos and the nucleus morphology of cleaved embryos indicated that capacitation combined with membrane disruption could make injected spermatozoa decondense synchronously not only with each other, but also with the developmental pace of the ooplasm. We successfully produced androgenetic blastocysts, and efficiency increased with sperm pretreatment. In conclusion, sperm decondensation and the early development of androgenetic embryos were enhanced with sperm capacitation and removal of the acrosome and plasma membrane prior to sperm injection.

Evidence for expression of the paternal genome in the two-cell mouse embryo

Nature ◽  
1981 ◽  
Vol 294 (5840) ◽  
pp. 450-451 ◽  
Author(s):  
Janet A. Sawicki ◽  
Terry Magnuson ◽  
Charles J. Epstein
Keyword(s):  
Mouse Embryo ◽  
Paternal Genome ◽  
Cell Mouse Embryo

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

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