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.

Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos

Zygote ◽  
2014 ◽  
Vol 24 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Yong-Xun Jin ◽  
Zhong Zheng ◽  
Xian-Feng Yu ◽  
Jia-Bao Zhang ◽  
Suk Namgoong ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Light Chain ◽  
Positive Control ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Intracytoplasmic Injection ◽  
Early Embryos ◽  
Morula Stage ◽  
Zygote Stage

SummaryThe mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI).

25 THE EFFECTS OF OOCYTE SOURCES AND ACTIVATION PROTOCOLS ON THE DEVELOPMENT OF CLONED GOAT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 131
Author(s):  
M. Apimeteeumrong ◽  
A. Thuangsanthia ◽  
N. Leingchaloen ◽  
V. Yiengvisavakul ◽  
A. Harintharanon ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Cytochalasin B ◽  
Vero Cells ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates ◽  
Matured Oocyte

The objective of this study was to compare the development to the morula and blastocyst stages, after either cycloheximide (CHX) or ethanol (ETOH) activation, in somatic nuclear transfer (NT) goat embryos derived from 2 sources of oocytes. In vivo- and in vitro-matured oocytes were obtained from FSH-stimulated goats (Native, Saanen, and Native-Saanen crossbred goats). Gonadotropin treatment was performed with a modified program of a previous report (Reggio et al. 2001 Biol. Reprod. 64, 849-856). In vivo-matured oocytes were flushed from the oviduct of donor goats by exposing the reproductive tract via a small ventral laparotomy incision. In vitro-matured oocytes were aspirated and cultured in maturation medium (M199 + 10% FCS, 10 �g mL-1 FSH, 10 �g mL-1 LH, and 1 �g mL-1 17�-estradiol) for 22 h, at 38.5�C in 5% CO2 and air. Donor cells were prepared from ear skin fibroblasts of a female goat (Native breed). Cells, at passage 3-9, starved by culturing in 0.5% FCS for 4-8 days, were used for NT. Matured oocytes were enucleated, and cell-cytoplast couplets (n = 162 in vivo-, and n = 190 in vitro-matured oocyte groups, respectively) were fused by applying 2 DC pulses of 2.2 kV cm-1 for 30 �s. One to 2 h after fusion, fused embryos were either incubated in 10 �g mL-1 cycloheximide plus 5 �g mL-1 cytochalasin B for 5 h (CHX treatment) or in 7% ethanol for 5 min followed by a 4-h incubation in 2 mM 6-dimethylaminopurine plus 5 �g mL-1 cytochalasin B (ETOH treatment). NT embryos were then cultured in B2 medium supplemented with 5% FCS and Vero cells for 9 days. At the end of the culture period, the NT embryos were fixed and stained with Hoechst 33342 (Begin et al. 2003 Theriogenology 59, 1839-1850). The numbers of nuclei were counted under ultraviolet light. Fusion, cleavage, and development rates were compared using chi-square test or Fisher's exact test. For the in vivo-matured oocyte group, there were no significant differences in fusion rates (78.1% vs. 68.7%), cleavage rates (87.7% vs. 87.0%, based on the numbers of embryos fused) between the CHX and ETOH treatment groups, respectively (P > 0.05). However, the development rates to morula and blastocyst stages of NT embryos derived from either in vivo- or in vitro-matured oocytes were significantly higher in the ETOH group than in the CHX group (in vivo: 15.2% vs. 0%, and in vitro: 7.1% vs. 0%, for ETOH and CHX groups, respectively; P < 0.05). For the in vitro-matured oocyte group, no significant differences were found between the CHX and ETOH groups in fusion rates (78.6% vs. 83.6%; P > 0.05), cleavage rates (80.5% vs. 83.9%: P > 0.05, based on the numbers of embryos fused). NT embryos from the CHX treatment group derived from in vivo- or in vitro-matured oocytes did not develop beyond the 16-cell stage. These results demonstrate that activation with CHX plus cytochalasin B treatment affects the development to the blastocyst stage of cloned goat embryos whether derived from in vivo- or in vitro-matured oocytes. This work was supported by the RGJ PhD program, Thailand Research Fund, and the Bureau of Biotechnology in Animal Production, Department of Livestock Development.

Consumption of amino acids by bovine preimplantation embryos

1996 ◽  
Vol 8 (6) ◽  
pp. 945 ◽  
Author(s):  
RJ Partridge ◽  
HJ Leese
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Amino Acid Requirement ◽  
Zygote Stage

Bovine embryos produced in vitro from the putative zygote stage to the blastocyst stage, and blastocysts freshly flushed from the uterus, were cultured in a physiological mixture of amino acids. Depletion of amino acids from the medium and, in a few cases, their appearance, was measured by high performance liquid chromatography. Amino acids were depleted at widely differing rates. The depletion of amino acids was higher when embryos at later developmental stages were cultured, implying an increase in amino acid requirement with development. Threonine was the only amino acid to be depleted at all stages of development; depletion increased from 0.18 +/- 0.07 pmol embryo-1 h-1 at the putative zygote stage to 1.96 +/- 0.49 pmol embryo-1 h-1 at the blastocyst stage. Glutamine was depleted at the putative zygote stage and the 4-cell stage (0.76 +/- 0.05 and 0.94 +/- 0.10 pmol embryo-1 h-1 respectively), but was not significantly depleted at the later stages. Alanine was the only amino acid that appeared consistently in the medium and its production increased progressively throughout development. Aspartate, glutamate, threonine and lysine were depleted significantly by blastocysts derived both in vitro and in vivo; the embryos in vivo also depleted arginine, phenylalanine, isoleucine and tyrosine. These results indicate that individual amino acids are depleted at different rates by bovine preimplantation embryos and suggest that amino acid requirements change during development.

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.

scholarly journals The differential staining pattern of the X chromosome in the embryonic and extraembryonic tissues of postimplantation homozygous tetraploid mouse embryos

1992 ◽  
Vol 59 (3) ◽  
pp. 205-214 ◽  
Author(s):  
S. Webb ◽  
T. J. de Vries ◽  
M. H. Kaufman
Keyword(s):  
X Chromosome ◽  
High Efficiency ◽  
Indirect Evidence ◽  
X Inactivation ◽  
Differential Staining ◽  
Hybrid Female ◽  
Cell Stage ◽  
X Chromosomes ◽  
F1 Hybrid ◽  
Inactivation Pattern

Summary(C57BL × CBA)F1 hybrid female mice were mated with hemizygous Rb(X.2)2Ad males to distinguish the paternal X chromosome. Homozygous tetraploids were produced by blastomere fusion at the 2-cell stage, and 161 of these were transferred to recipients and analysed on the 10th day of gestation. 59 implants contained resorptions and 76 contained either an embryo and/or extraembryonic membranes. 38 (20, XXXX and 18, XXYY) were analysed to investigate their X-inactivation pattern. Embryonic and yolk sac endodermally- and mesodermally-derived samples were analysed by G-banding and by Kanda analysis. In the XX and XY controls, the predicted pattern of X-inactivation was observed, though 12·2% of metaphases in the XX series displayed no X-inactivation. In the XY series the Y chromosome was seen in a high proportion of metaphases.In the XXXX tetraploids, 8 cell lineages were recognized with regard to their X-inactivation pattern, though most belonged to the following 3 categories: (XmXm)XpXp, Xm(XmXp)Xp and XmXm(XpXp). The other categories were only rarely encountered. In the embryonic and mesodermally-derived tissue the ratio of these groups was close to 1:2:1, whereas in the endodermally-derived tissue it was 1:4·11:4·88, due to preferential paternal X-inactivation. A significant but small proportion of all 3 tissues analysed displayed no evidence of X-inactivation. Indirect evidence suggests that this represents a genuine group because of the high efficiency of the Kanda staining. The presence of the Xm(XmXp)Xp category is consistent with the expectation that X-inactivation occurs randomly in 2 of the 4 X chromosomes present. The presence of small numbers of preparations with no evidence of X-inactivation and other unexpected categories suggests that these are probably selected against during development.

Cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 600-601
Author(s):  
A.E. Sutherland ◽  
P.G. Calarco ◽  
C.H. Damsky
Keyword(s):  
Mouse Embryo ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Cell Stage ◽  
Early Mouse Embryo ◽  
Migratory Activity ◽  
Early Mouse ◽  
Cell Matrix Interactions

Cell-extracellular matrix (ECM) interactions mediated by the integrin family of receptors are critical for morphogenesis and may also play a regulatory role in differentiation during early development. We have examined the onset of expression of individual integrin subunit proteins in the early mouse embryo, and their roles in early morphogenetic events. As detected by immunoprecipitation, the α6, αV, β1, and β3 subunits are detected as early as the 4-cell stage, α5 at the hatched blastocyst stage and αl and α3 following blastocyst attachment. We tested the role of these integrins in the attachment and migratory activity of two cell populations of the early mouse embryo: the trophoblast giant cells, which invade the uterine stroma and ultimately contribute to the chorio-allantoic placenta, and the parietal endoderm, which migrates over the inner surface of the trophoblast and ultimately forms Reichert's membrane and the parietal yolk sac. Experiments were done in serum-free medium on substrates coated with laminin (Ln) and fibronectin (Fn). Trophoblast outgrowth occurs on Ln and its E8 fragment (long arm), but not on the E1’ fragment (cross region) (Figs. 1, 2 ). This outgrowth is inhibited by anti-E8, anti-Ln, and by the anti-β1 family antiserum anti-ECMR, but not by anti-αV or the function-perturbing GoH3 antibody that recognizes the α6/β1 integrin, a major Ln (E8) receptor. This suggests that trophoblast outgrowth on Ln or E8 is mediated by a different β1 integrin such as α3/β1. Early stages of trophoblast outgrowth (up to 48 hours) on Fn are inhibited by anti-Fn and by function-perturbing anti-αV antibodies, whereas at later times outgrowth becomes insensitive to anti-αV but remains sensitive to the anti-β1 family antiserum anti-ECMr, indicating that trophoblast cells modulate their interaction with Fn during outgrowth. Trophoblast outgrowth on vitronectin (Vn) is sensitive to anti-αV antibodies throughout the 5-day period examined.

Glucose utilization by sheep embryos derived in vivo and in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 571 ◽  
Author(s):  
JG Thompson ◽  
AC Simpson ◽  
PA Pugh ◽  
RW Wright ◽  
HR Tervit
Keyword(s):  
Glucose Oxidation ◽  
Glucose Utilization ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stage ◽  
Glycolytic Activity ◽  
Total Utilization ◽  
Oxidation Of Glucose

Embryos were collected from superovulated donors at various intervals from onset of oestrus, ranging from Day 1.5 to Day 6. In addition, blastocysts obtained from the culture of 1-cell embryos collected in vivo or of oocytes matured and fertilized in vitro were used to assess the effects of in vitro manipulation and culture on glucose utilization. Glycolytic activity was determined by the conversion of [5-3H]glucose to 3H2O, and oxidation of glucose was determined by the conversion of [U-14C]glucose to 14CO2. Glucose utilization increases significantly from the 8-cell stage and during compaction and blastulation. Glucose oxidation was at a relatively low level (5-12% of total utilization) compared with glycolysis. No difference was observed between the glycolytic activity of blastocysts derived from in vivo or in vitro sources. However, glucose oxidation was lower (P less than 0.05) in blastocysts derived from the culture of 1-cell embryos or from oocytes matured and fertilized in vitro. Exogenous tricarboxylic acid cycle substrates (i.e. pyruvate and lactate supplied in the medium) affected the level of glucose oxidation.

scholarly journals Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mark G Larman ◽  
Courtney B Sheehan ◽  
David K Gardner
Keyword(s):  
Ethylene Glycol ◽  
Zona Pellucida ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Initial Increase ◽  
Cell Stage ◽  
Free Treatment ◽  
Zona Hardening ◽  
Free Media ◽  
Oocyte Freezing

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

Synthesis and phosphorylation of uvomorulin during mouse early development

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 313-318 ◽  
Author(s):  
M. Sefton ◽  
M.H. Johnson ◽  
L. Clayton
Keyword(s):  
Cell Adhesion ◽  
Early Development ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Blastocyst Stage ◽  
Embryonic Stage ◽  
Cell Stage ◽  
Mouse Oocytes ◽  
Maternal Mrna ◽  
Embryonic Genome

The cell adhesion molecule, uvomorulin, is synthesised in both the 135 × 10(3) M(r) precursor and 120 × 10(3) M(r) mature forms on maternal mRNA templates in unfertilized and newly fertilized mouse oocytes. Synthesis on maternal message ceases during the 2-cell stage to resume later on mRNA encoded presumptively by the embryonic genome. Uvomorulin is detectable by immunoblotting at all stages upto the blastocyst stage, but shows variations in its total amount and processing with embryonic stage. Whilst only trace levels of phosphorylated uvomorulin are detectable in early and late 4-cell embryos, uvomorulin in 8-cell embryos is phosphorylated.

A Novel System of Fertility Rescue in Drosophila Hybrids Reveals a Link Between Hybrid Lethality and Female Sterility

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 217-226 ◽  
Author(s):  
Daniel A Barbash ◽  
Michael Ashburner
Keyword(s):  
Drosophila Melanogaster ◽  
Low Temperature ◽  
Genetic Basis ◽  
Female Sterility ◽  
Hybrid Lethality ◽  
Hybrid Male ◽  
Hybrid Female ◽  
F1 Hybrid ◽  
Drosophila Gene

Abstract Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F1 female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F1 hybrid males from these crosses remained fully sterile. Hmr+ is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality.

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