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.

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 125-130 ◽  
Author(s):  
Lei Lei ◽  
Na Guan ◽  
Yan-Ning Xu ◽  
Qing-Hua Zhang ◽  
Jing-Ling Shen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Karyotype Analysis ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lower Number ◽  
Mouse Embryos ◽  
Developmental Pattern ◽  
Cell Stage ◽  
Positive Expression

SummaryPolyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 ± 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 ± 7.2; 18.4 ± 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. β-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

Effect of culture from the zygote stage on the metabolism of glucose and glutamine by 2-cell embryos and blastocysts recovered from outbred or F1 hybrid female mice

1993 ◽  
Vol 5 (5) ◽  
pp. 555 ◽  
Author(s):  
ZF Du ◽  
RG Wales
Keyword(s):  
Reproductive Tract ◽  
Soluble Fraction ◽  
Insoluble Fraction ◽  
Blastocyst Stage ◽  
Hybrid Female ◽  
Cell Stage ◽  
F1 Hybrid ◽  
Maternal Genotype ◽  
Oxidation Of Glucose ◽  
Zygote Stage

The oxidation and incorporation of glucose and glutamine by embryos derived from cultured zygotes was compared with the utilization of these substrates by embryos recovered directly from the reproductive tract of pregnant females. The oxidation of glutamine was greater at the blastocyst stage than at the 2-cell stage. Embryos derived from outbred females (Qs) were less active in the oxidation of glutamine than those from hybrid (B10D2F1) females and development in culture was detrimental to this oxidation, especially in blastocysts from the outbred stock. The oxidation of glutamine was stimulated by the presence of glucose at the 2-cell stage but reduced by its presence at the blastocyst stage. Maternal genotype had no effect on the oxidation of glucose at either the 2-cell or blastocyst stage, and only at the blastocyst stage was there evidence of a detrimental effect of culture. The oxidation of glucose was stimulated by the presence of glutamine at the 2-cell stage but depressed by its addition at the blastocyst stage. Incorporation of glutamine increased with development, but this was reduced at the blastocyst stage by development in culture, especially if the blastocysts were derived from outbred females. Incorporation of glucose also increased with development. At the 2-cell stage, culture reduced incorporation of this substrate, especially into the acid-soluble fraction of embryos from outbred females. In blastocysts, incorporation of glucose into the acid-insoluble fraction was depressed by culture and in embryos from outbred females. In contrast to glucose oxidation, incorporation of glucose into the acid-soluble fraction was reduced by the addition of glutamine at the 2-cell stage but increased by its addition at the blastocyst stage.

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

Zygote ◽  
2008 ◽  
Vol 16 (1) ◽  
pp. 87-91 ◽  
Author(s):  
P. Chrenek ◽  
A.V. Makarevich ◽  
M. Bauer ◽  
R. Jurcik
Keyword(s):  
Developmental Rate ◽  
Blastocyst Stage ◽  
Egfp Expression ◽  
Control Group ◽  
Cell Stage ◽  
Embryonic Shield ◽  
Developmental Capacity ◽  
Transgenic Cells ◽  
Average Total Number ◽  
Rabbit Embryos

SummaryThe objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR<>N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage.All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR<>N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175 ± 13.10, of which 58 ± 2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24 ± 5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage.Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.

scholarly journals Changes in responsiveness of preimplantation mouse embryos to serum

Development ◽  
1978 ◽  
Vol 45 (1) ◽  
pp. 295-301
Author(s):  
Simon B. Fishel ◽  
M. Azim H. Surani
Keyword(s):  
Bovine Serum ◽  
Rna Synthesis ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
The Other ◽  
Cell Stage ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Serum Albumen

Changes in uptake of radioactive uridine and its incorporation into RNA were determined in preimplantation mouse embryos, from the 2-cell to the blastocyst stage, as a measure of their responsiveness to extracellular conditions. Two media were tested, one contained serum and the other contained bovine serum albumen as a control. An increase in the acid-soluble pool occurred at the 8-cell stage and a marked increase in RNA synthesis occurred at the early blastocyst stage when the embryos were incubated with serum.

131 DYNAMICS OF HISTONE H3 METHYLATION AT POSITIONS K4 AND K9 IN MOUSE, RABBIT, AND BOVINE PRE-IMPLANTATION EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 174 ◽  
Author(s):  
K. Lepikhov ◽  
F. Yang ◽  
C. Wrenzycki ◽  
V. Zakhartchenko ◽  
H. Niemann ◽  
...  
Keyword(s):  
Histone H3 ◽  
Distribution Patterns ◽  
Blastocyst Stage ◽  
Dna Demethylation ◽  
Preimplantation Embryos ◽  
Staining Procedure ◽  
Cell Stage ◽  
Paternal Genome ◽  
Histone H3 Methylation ◽  
Rabbit Embryos

In mammals, upon the penetration of sperm into the oocyte, the paternal genome undergoes dramatic epigenetic changes. Protamin packaging of DNA is replaced by histones that acquire specific modifications. In mouse zygotes, paternal DNA gets rapidly demethylated by an active mechanism. In bovine zygotes the methylation from paternal DNA is erased only partially, and in rabbit zygotes it persists at the initial level. To understand whether these reprogramming differences are also reflected in histone modifications, we examined the dynamic changes of histone H3 methylation at positions K4 and K9 in mouse, bovine, and rabbit zygotes and in preimplantation embryos using an immunofluorescence staining procedure (Lepikhov and Walter 2004 BMC Dev. Biol. 4, 12). In zygotes, maternal chromatin contains both types of histone H3 methylation. After fertilization protamines in sperm are very quickly replaced by histones. After the formation of nucleosomes, histone H3 acquires methylation at position K4 in a stepwise manner: first as mono-methylated form and later as tri-methylated. In the late zygote, both paternal and maternal pronuclei show equal levels of histone H3 methylation at position K4. Regardless of the differences in DNA reprogramming in these 3 species, H3/K9 di-methylation is not detected on paternal genomes and is only associated with maternal genomes. During the subsequent cleavage stages, H3/K9 di-methylation decreases gradually and becomes hardly detectable in 4-cell bovine and rabbit embryos. In mouse embryos, it is detectable through all the stages. Bovine embryos reacquire this type of modification at the 8-16 cell stage, and it remains at the very low levels in rabbit, embryos until the blastocyst stage. In conclusion, mouse, rabbit and bovine zygotes show similar patterns of H3/K4triMe and H3/K9diMe distribution despite the difference in paternal DNA demethylation. The dynamics of H3/K9diMe distribution patterns in cleavage stage embryos from all embryos do not correlate with embryonic genomic activation events.

scholarly journals Maternal DNMT3A-dependent de novo methylation of the paternal genome inhibits gene expression in the early embryo

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Julien Richard Albert ◽  
Wan Kin Au Yeung ◽  
Keisuke Toriyama ◽  
Hisato Kobayashi ◽  
Ryutaro Hirasawa ◽  
...  
Keyword(s):  
De Novo ◽  
Cpg Islands ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mass Spectrometry Analysis ◽  
Paternal Allele ◽  
Sequencing Data ◽  
Cell Stage ◽  
Paternal Genome ◽  
Spectrometry Analysis

Abstract De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

2008 ◽  
Vol 56 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Chang-Liang Yan ◽  
Qi-En Yang ◽  
Guang-Bin Zhou ◽  
Yun-Peng Hou ◽  
Xue-Ming Zhao ◽  
...  
Keyword(s):  
Survival Rate ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Significant Loss ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Fresh Embryos

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3–100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8–89.5%) and hatched blastocyst rates (61.1–69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3–30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

Vitrification of biopsied mouse embryos

2005 ◽  
Vol 53 (1) ◽  
pp. 103-112 ◽  
Author(s):  
B. Baranyai ◽  
Sz. Bodó ◽  
◽  
Keyword(s):  
Solid Surface ◽  
Birth Rate ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
The Novel ◽  
Cell Stage ◽  
Cryopreservation Protocol

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vi tro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.

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