Consumption of amino acids by bovine preimplantation embryos

1996 ◽  
Vol 8 (6) ◽  
pp. 945 ◽  
Author(s):  
RJ Partridge ◽  
HJ Leese

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.

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental 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 ◽  
...  

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.


2012 ◽  
Vol 56 (2) ◽  
pp. 211-216 ◽  
Author(s):  
Ján Bystriansky ◽  
Ján Burkuš ◽  
Štefan Juhás ◽  
Dušan Fabian ◽  
Juraj Koppel

Abstract High plasma urea nitrogen concentration has been proposed as an important factor contributing to the decline in reproductive parameters of domestic animals. The aim of this study was to evaluate the effect of urea on the development of preimplantation embryos in a mouse model. During in vivo tests, acute renal failure (ARF) accompanied by hyper-uraemia was induced by intramuscular administration of glycerol (50%) into hind limbs of fertilised dams. During in vitro tests, embryos collected from healthy dams were cultured in a medium with the addition of various concentrations of urea from the 4-cell stage to the blastocyst stage. Stereomicroscopic evaluation and fluorescence staining of embryos obtained from dams with ARF showed that high blood urea is connected with an increase in the number blastocysts containing at least one apoptotic cell and in the incidences of dead cells per blastocyst, but it did not affect their ability to reach the blastocyst stage. In vitro tests showed that culture of embryos with urea at concentration of 10 mM negatively affected the quality of obtained blastocysts. Blastocysts showed significantly lower numbers of cells and increased incidence of dead cells. An increase in apoptosis incidence was observed even in blastocysts obtained from cultures with 5 mM urea. Urea at concentrations 50 mM and higher negatively affected the ability of embryos to reach the blastocyst stage and the highest used concentrations (from 500 mM) caused overall developmental arrest of embryos at the 4- or 5- cell stage. These results show that elevated levels of urea may cause changes in the microenvironment of developing preimplantation embryos, which can negatively affect their quality. Embryo growth remains un-affected up to very high concentrations of urea.


2010 ◽  
Vol 22 (1) ◽  
pp. 343
Author(s):  
R. G. Sturmey ◽  
P. Bermejo-Alvarez ◽  
A. Gutierrez-Adan ◽  
D. Rizos ◽  
H. J. Leese ◽  
...  

Sex-dependent differences in mammalian embryo phenotype are apparent at the preimplantation stage of development, before the appearance of sex-specific cells. The ratio of male:female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling (AAP), a defined, non-invasive metabolic marker of developmental potential to compare the effect of sex on the metabolism of bovine preimplantation blastocysts and expanded blastocysts conceived in vivo (n = 35) or produced in vitro (n = 172). Blastocysts were incubated individually for 24 h in synthetic oviduct fluid medium plus a close-to-physiological mixture of amino acids. The depletion or appearance of 18 amino acids was measured using high-performance liquid chromatography. Blastocysts were then sexed by PCR and the outcome related to AAP. Amino acid depletion by in vitro-produced blastocysts was higher than in embryos conceived in vivo (P = 0.02). Net appearance of amino acids was higher in the medium from early blastocysts produced in vitro (P = 0.018) although this rise was lost at the expanded stage. There were marked differences in the amino acid profiles of male and female embryos produced in vitro: female embryos exhibited significantly increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited gender-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced blastocysts exhibited differences in 2 out of 18 amino acids. These differences had disappeared by the expanded blastocyst stage. Our experiments reveal striking differences in the metabolism of preimplantation embryos conceived in vivo and in vitro, some of which, particularly in the case of the in vitro-produced embryos, are dependent on embryo sex. Moreover, in vivo-derived embryos tend to have a reduced metabolism consistent with the Quiet Embryo Hypothesis, which proposes that higher quality embryos have less molecular and cellular damage than those of a lower quality and thus have a reduced need to take up nutrients for repair processes. Supported by a Wellcome-VIP/University of York Fellowship to RGS.


Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 485-493 ◽  
Author(s):  
A.F. Pereira ◽  
L.M. Melo ◽  
V.J.F. Freitas ◽  
D.F. Salamone

SummaryIn vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.


2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
D. Tesfaye ◽  
K. Wimmers ◽  
M. Gilles ◽  
S. Ponsuksili ◽  
K. Schellander

A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P&lt;0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P&lt;0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P&lt;0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P&lt;0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.


Reproduction ◽  
2002 ◽  
pp. 667-673 ◽  
Author(s):  
DG Morris ◽  
PG Humpherson ◽  
HJ Leese ◽  
JM Sreenan

Blastocyst elongation from day 14 to day 16 after insemination coincides with a major phase of embryo loss in cattle. Protein synthesis, reflected in protein content, increases markedly over this period but little is known about the amino acid requirement of elongating blastocysts at this time. Cattle blastocysts produced in vivo were recovered on days 14-16 after insemination and cultured individually for up to 8 h in synthetic oviduct fluid containing a physiological mixture of amino acids plus 1 mmol glutamine l(-1) and 0.1% (w/v) polyvinyl alcohol (SOFaaPVA). After 1, 4 and 8 h in culture, an aliquot of culture medium was removed and the rate of amino acid depletion or production was calculated per unit of protein and per hour of culture. Amino acids were depleted or produced at different rates. Arginine was depleted from the medium at a significant rate (P < 0.05) during all culture periods. Alanine and glutamate were produced at a significant rate (P < 0.05) during all culture periods. The rate of alanine production was significantly greater (P < 0.05) in blastocysts recovered on day 14 compared with days 15 or 16 after insemination. Alanine production and arginine depletion tended to be greater in smaller embryos recovered on day 14 compared with larger and later stage embryos, indicating that earlier stage embryos may have higher metabolic activity than later stage embryos. Qualitatively, the pattern of amino acid consumption and production during elongation was similar to that shown from the zygote to early blastocyst stage.


1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.


2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...


2005 ◽  
Vol 17 (2) ◽  
pp. 216
Author(s):  
P. Booth ◽  
T. Watson ◽  
H. Leese

Pre-implantation embryos can produce and consume amino acids in a manner dependent upon stage of embryonic development (Partridge and Leese 1996 Reprod. Fert. Dev. 8, 945) that may also be predictive of subsequent viability (Houghton et al. 2002 Hum. Reprod. 17, 999). To examine these relationships in the pig, the appearance or depletion of 18 amino acids from a presumptive near-physiological mixture was determined by HPLC in porcine in vitro-produced embryos from the zygote to the blastocyst stage. Cumulus oocyte complexes derived from slaughterhouse prepubertal pig ovaries were matured for 40 h in modified TCM-199 before being fertilized (Day 0) with frozen thawed semen in tris-based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20 in NCSU medium modified to contain a physiological mixture of 18 amino acids including 0.1 mM glutamine (NCSUaa). Groups of 2–10 embryos (dependent on stage) were removed on Day 0 (1 cell), Day 1 (2- and 4-cell), Day 4 (compact morula), and Day 6 (blastocyst) and placed in 4 μL NCSUaa for 24 h. After incubation, the embryos were removed and the medium analyzed by HPLC. Each stage was replicated 3–9 times. Since amino acid profiles of 2- and 4-cell embryos were not different, data were combined. Overall, arginine (1.19 ± 0.33), glutamine (0.78 ± 0.34) and threonine (0.05 ± 0.04) were significantly (P < 0.01) depleted from the medium whereas alanine (0.21 ± 0.1), glycine (0.20 ± 0.06), asparagine (0.13 ± 0.5), lysine (0.1 ± 0.03), isoleucine (0.08 ± 0.01), valine (0.05 ± 0.01), leucine (0.04 ± 0.02), phenylalanine (0.03 ± 0.01), and histidine (0.02 ± 0.04) significantly (P < 0.05) accumulated (mean of the 4 sampling timepoints; all values pmol/embryo/h ± SEM). The difference between amino acid accumulation and depletion (balance) was approximately equivalent between Day 0 and the morula stage although turnover (sum of depletion and accumulation) steadily decreased during this period from 3.1 on Day 0 to 1.35 pmol/embryo/h at the morula stage. However, at the blastocyst stage, turnover and balance increased to 6.32 and 2.42 pmol/embryo/h, respectively, i.e. net appearance occurred. Notable changes in amino acid profile during development included decreases in accumulation of asparagine, glutamate, and glycine in the medium and the depletion of glutamine over Days 0, 1, and 4, followed by reversal of these trends by Day 6. These data suggest that pig embryos can alter the accumulation and depletion rates of amino acids in a manner that is dependent on the specific amino acid and the stage of embryonic development. This work was supported by BBSRC.


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