Effect of amino acids and α-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor α-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O₂:5% CO₂:90% N₂. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's IX NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8–16-cell stage embryos past the morula stage. In experiment 3, the addition of IX NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P<0.05) and 86, 77, 77, 78, and 69% on Day 5 (P<0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when α-amanitin (20 μM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the α-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of α-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development. © 1996 Wiley-Liss, Inc.     

Modifications made to culture medium by bovine oviduct epithelial cells: Changes to carbohydrates stimulate bovine embryo development

Co-culture remains a common method to support the development of bovine embryos, derived from IVM/IVF procedures. However, the mechanism by which somatic cells confer their benefit to the developing embryo remains undetermined. This study therefore analysed the changes made to the culture medium TCM-199, used in bovine embryo co-culture systems, by somatic cells and determined the effects of specific changes in medium composition on bovine embryo development in culture. Bovine oviduct epithelial (BOE), Buffalo rat liver (BRL) and fibroblast (3T3) cells were compared. The concentrations of glucose, L-lactate, pyruvate, amino acids, NH₄⁺, H⁺ and the gas tensions of O₂ and CO₂ were measured in TCM-199 supplemented with 10% fetal calf serum (FCS) prior to and directly following 48 h incubation periods with each cell type. All three somatic cell types modified the carbohydrate composition of the media in a similar manner with the greatest changes made by the BOE cells. Notable alterations were an increase in the levels of L-lactate and pyruvate and a reduction in glucose concentration, which in the case of the BOE cells, fell from 5.55 mM to 2.67 mM. In order to determine the relevance of such changes in carbohydrate concentrations on bovine embryo development, modifications were made to carbohydrate levels in synthetic oviduct fluid (SOF) medium and their effect on blastocyst development in vitro assessed. In SOF medium supplemented with amino acids and BSA (SOFaa), significantly more zygotes developed to the blastocyst stage (64%; P < 0.01) than in SOFaa medium with the concentrations of glucose, D/L-lactate and pyruvate equivalent to those in TCM-199 (11%). Interestingly, when the levels of carbohydrates in SOFaa mimicked those present in TCM-199 following a 48 h incubation with BOE cells, 57% of zygotes reached the blastocyst stage. This improvement was ascribed to the reduction in glucose and increases in D/L-lactate and pyruvate concentrations in the culture system. Results from this study demonstrate that BOE cells create an environment favourable to embryonic development. The analysis of media samples by enzymatic methods meant that only the biologically active L-isomer of lactate was quantified. However, in SOFaa, both the L-isomer and inactive D-isomer are present in equimolar amounts. As such, culture media in which D/L-lactate syrup is used actually contain only 50% biologically active lactate meaning that all D/L-lactate concentrations are reported at twice the effective concentration. Therefore the effect of D/L-lactate concentration on blastocyst development was subsequently determined in this study. Blastocyst development was poor (24–36%) until the total D/L-lactate was present in the culture system at concentrations equal to or greater than 0.82 mM. However, blastocyst cell numbers remained low (60.1 ± 6.9 – 78.5 ± 6.6) until a total D/L-lactate concentration of 3.3 mM. This data reinforces that embryo morphological appearance is not sensitive enough to be used as the sole criterion for assessing embryo development. Mol Reprod Dev 46:146–154, 1997. © 1997 Wiley-Liss, Inc.     

The structure of α-amanitin in dimethylsulfoxide solution

The backbone and side-chain conformations of the bicyclic octapeptide α-amanitin indimethylsulfoxide (DMSO) solution have ben deduced from analysis of the nmr spectrl parameters and conformational energy calculations. Several ambiguities in the nmr spectral assignments were resolved following a comparison with the recently published conformation of β-amanitin in the crystalline state. The peptide proton exchange and temperature coefficient data demonstrate strong intramolecular hyfrogen bonds for the GLY⁵ and Cys⁸ peptide protons. The vicinal proton coupling constants are consistent with the cyclic octapeptide udergoing chain reversl at the Ile⁶-Gly⁷ abd the Hyp²-Hyi³ dipeptide segments. The upfield shifts of the glycine and isoleucine protons demonstrate the folding of the indole ring of the Trp⁴-Cys⁸ brifge towards the Gly⁵-Ile⁶-Gly⁷ half of the Ile-amanitin molecule. The structure af α-amanitin in DMSO is defined by the (?ψ) backbone rotation angles Trp⁴(?90, ?60), Gly⁵ (+120, ?120), Ile⁶(?6, +120), Gly⁷ (+45, +60), Cys⁸(?120, ?60), Asn¹ (+175, ?175), Hyp² (?160, ?45), and Hyi³ (?90, ?60). The study demonstrates that the structure of α-amanitin in solution is similar to the structure f β-amanitin in the crystalline state.     

Isolation of informoferes from rat liver : Effects of α-amanitin and actinomycin D

RNP particles carrying rapidly labelled RNA (informoferes) were isolated from rat liver nuclei by extraction with isotonic and 0.3 M salt buffer at pH 8 either with or without ultrasonic treatment. The importance of preliminary extraction of the nuclei with the isotonic salt buffer at a lower pH of 7 or in the presence of 50 mM EDTA is demonstrated. Administration of α-amanitin or of actinomycin D, in doses inhibiting the labelling of the heterogeneous RNA with RNA precursors in the range of 60–85%, reduces the amount of informoferes recovered. The informoferes isolated from treated animals are highly depleted in HnRNA. They still contain, however, low molecular RNA species with a slower turnover than the HnRNA. The polypeptide pattern observed after acrylamide gel electrophoresis of the informofere proteins is qualitatively changed in the treated preparations, whereas the ratio of protein to RNA decreases. In the presence of RNase inhibitor the informoferes are recovered in form of polymer structures. α-Amanitin and actinomycin D significantly reduce the amount of polymers recovered.     

Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality

When embryos are cultured individually or in small groups, blastocyst yield efficiency and quality are usually reduced. The aim of this work was to investigate the effect of supplementation of the embryo culture medium (CM) with several growth factors (GFs) on embryo development and apoptosis rate when a reduced number of embryos were in vitro cultured. Two experimental studies (ES) were carried out. In ES 1, five treatments were tested to study the effect of GF on embryo development: Control (∼30 to 50 embryos cultured in 500 μl of CM); Control 5 (Five embryos cultured in 50 μl microdrops of CM), without addition of GF in either of the two control groups; epidermal GF (EGF); IGF-I; and transforming GF-α (TGF-α) (Five embryos were cultured in 50 μl microdrops of CM with 10 ng/ml EGF, 10 ng/ml IGF-I or 10 ng/ml TGF-α, respectively). In ES 2, following the results obtained in ES 1, four different treatments were tested to study their effect on embryo development and quality (number of cells per blastocyst and apoptotic rate): Control; Control 5; EGF, all three similar to ES 1; EGF + IGF-I group (five embryos cultured in 50 μl microdrops of CM with 10 ng/ml EGF and 10 ng/ml IGF-I). In both ESs, it was observed that a higher proportion of embryos cultured in larger groups achieved blastocyst stage than embryos cultured in reduced groups (22.6% v. 14.0%, 12.6% and 5.3% for Control v. Control 5, IGF-I, TGF-α groups in ES 1, and 24.9% v. 17.1% and 19.0% for Control v. Control 5 and EGF in ES 2, respectively; P < 0.05), with the exception of embryos cultured in medium supplemented with EGF (18.5%) or with EGF + IGF-I (23.5%), in ES 1 and ES 2, respectively. With regard to blastocyst quality, embryos cultured in reduced groups and supplemented with EGF, alone or combined with IGF-I, presented lower apoptosis rates than embryos cultured in reduced groups without GF supplementation (11.6% and 10.5% v. 21.9% for EGF, EGF + IGF-I and Control 5 groups, respectively; P < 0.05). The experimental group did not affect the total number of cells per blastocyst. In conclusion, this study showed that supplementation of the CM with EGF and IGF could partially avoid the deleterious effect of in vitro culture of small groups of bovine embryos, increasing blastocyst rates and decreasing apoptosis rates of these blastocysts.     

Temporal and differential effects of amino acids on bovine embryo development in culture.

The aim of the study was to determine the amino acid requirements of the in vitro-produced bovine embryo as it develops from the zygote to the blastocyst, using a two-step culture system. When added to synthetic oviduct fluid (SOF) for the first 72-h culture, Eagle's nonessential amino acids and glutamine (NeGln) significantly increased development to the 8- to 16-cell stage (Day 4 postinsemination [pi]) and subsequent blastocyst development (Day 7 pi). Glutamine alone during the first 72-h culture did not stimulate development to the 8- to 16-cell stage (p > 0.05); however, the removal of glutamine from NeGln reduced the stimulatory effects of the nonessential amino acids. Replacing glutamine with betaine (an organic osmolyte) in NeGln did not stimulate development to the 8- to 16-cell stage compared to culture in SOF, but it did improve subsequent blastocyst development, indicating an osmolytic function of glutamine during the first 72-h culture. The addition of Eagle's essential amino acids and glutamine to SOF, or to medium already containing nonessential amino acids and glutamine for the first 72-h culture, did not affect cleavage to the 8- to 16-cell stage or subsequent blastocyst development (p > 0.05). Beyond Day 4 pi, culture with 20aa (nonessential and essential amino acids and glutamine) increased blastocyst development, total cell number, and the number of cells in both the trophectoderm and inner cell mass, compared to culture with other groups of amino acids (p < 0.05). Substituting betaine for glutamine in 20aa reduced blastocyst formation, indicating a non-osmolytic function of glutamine during the second 72-h culture. Further, there was a significant negative correlation between the concentration of essential amino acids (quarter, half, or single strength) and embryo development during both the first 72-h and second 72-h culture (p < 0.01), indicating that the concentration of essential amino acids was too high during culture of the bovine embryo. This study identified the temporal and differential effects of amino acids during development of the bovine embryo from the zygote to the blastocyst.     

MicroRNA‐29b regulates DNA methylation by targeting Dnmt3a/3b and Tet1/2/3 in porcine early embryo development

MicroRNA‐29b (miR‐29b) is a member of the miR‐29 family, which targets DNA methyltransferases (DNMTs) and ten eleven translocation enzymes (TETs), thereby regulating DNA methylation. However, the role of miR‐29b in porcine early embryo development has not been reported. In this study, we examined the effects of miR‐29b in porcine in vitro fertilization (IVF) embryos to investigate the mechanism by which miR‐29b regulated DNA methylation. The interference of miR‐29b by its special miRNA inhibitor significantly up‐regulated Dnmt3a/b and Tet1 but downregulated Tet2/3; meanwhile it increased DNA methylation levels of the global genome and Nanog promoter region but decreased global DNA demethylation levels. The inhibition of miR‐29b also resulted in a decrease in the development rate and quality of blastocysts. In addition, the pluripotency genes Nanog and Sox2 were significantly downregulated, and the apoptosis genes Bax and Casp3 were upregulated, but anti‐apoptosis gene Bcl‐2 was downregulated in blastocysts. Our study indicated that miR‐29b could regulate DNA methylation mediated by miR29b‐ Dnmt3a/b – Tet1/2/3 signaling during porcine early embryo development.     

Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos

This study examined the effects of incorporating an ovine oviducal oestrus-associated glycoprotein (oEGP) and amino acids, at the concentrations present in the ovine oviduct around the time of oestrus, on in vitro production and subsequent viability of bovine embryos. The first experiment compared the influence of ovine oviducal concentrations of amino acids with MEM and BME amino acids. There was no treatment effect on cleavage rate (74.9% vs. 75.5%), but there was a higher (P < 0.05) blastocyst yield (30.4 vs. 25.2) and a shorter time (P < 0.05) to blastocyst formation (7.16 ± 0.64 vs. 7.27 ± 0.56 days) following use of oviducal concentrations of amino acids. Experiment 2 examined the influence of oEGP in combination with each of the amino acid treatments. oEGP had no effect on cleavage or blastocyst yield within amino acid treatments. Day of blastocyst formation significantly influenced nuclei numbers (P < 0.001) with higher numbers being obtained on day 7 than on either day 6 or day 8. There was also a significant (P < 0.01) interaction between day of blastocyst formation and amino acid treatment on blastocyst nuclei numbers. The third experiment studied the effects of the amino acid treatments on embryo viability. There was no effect of amino acid treatment of embryos on pregnancy rates (34.5 vs. 44.4%) following transfer of days 6 and 7 blastocysts to synchronized recipients. oEGP did not influence any of the parameters of bovine embryo development that were measured, suggesting that effects of this protein observed on ovine embryos are species specific. It is concluded that ovine oviducal amino acid concentrations are beneficial to blastocyst development in vitro but do not have any further beneficial effect following transfer of blastocysts to recipients. Mol. Reprod. Dev. 47:164–169, 1997. © 1997 Wiley-Liss, Inc.     

The effects of aphidicolin and α-amanitin on DNA synthesis in preimplantation mouse embryos

The effects of aphidicolin and α-amanitin on DNA synthesis by preimplantation mouse embryos were studied. It was found that both blastocyst and 8-cell embryos showed marked inhibition of ³H-thymidine incorporation into DNA by aphidicolin at concentrations of 20–50 μg/ml. However, aphidicolin did not inhibit the conversion of morula embryos to blastocyst embryos, although aphidicolin-treated blastocysts lost their blastocoel and collapsed into a compact form after prolonged exposure to the drug. Both 8-cell and blastocyst embryos were found to be susceptible to inhibition of DNA synthesis by α-amanitin.     

Predictive variables of in vitro fertilization and pre-implantation embryo development in the mouse

The present study aims to analyze the cause-effect relationships among several in-vitro fertilization and pre-implantation embryo development variables in the mouse. Superovulation of hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of pregnant mare's serum gonadotropin at the estrus stage of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Ovulated cumulus-enclosed oocytes were inseminated with sperm from hybrid males of 12-16 weeks of age. The multiple linear regression analyses performed indicated that (a) total number of ovulated oocytes is a good predictor of both fertilization frequency and total number of cells in day-5 blastocysts; (b) fertilization frequency predicts percentage of day-5 blastocysts; (c) total number of cells in day-5 blastocysts is predicted by percentage of day-5 blastocysts; and (d) total number of cells in day-5 blastocysts predicts percentage of apoptotic cells, number of inner cell mass (ICM) and trophectoderm (TE) cells, and ICM/TE ratio in day-5 blastocysts. Mitotic index in day-5 blastocysts was positively correlated with total number of ovulated oocytes, percentage of ovulated cumulus-enclosed oocytes, fertilization frequency, percentage of day-5 blastocysts and total number of cells in day-5 blastocysts. On the contrary, it was negatively correlated with percentage of apoptotic cells in day-5 blastocysts.     

Proteolytically stable peptides by incorporation of α-Tfm amino acids

A series of model peptides containing α-trifluoromethyl-substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease α-chymotrypsin was synthesized by solution methods to investigate the influence of α-Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted in the P₁ position and still considerable proteolytic stability for peptides substituted at the P₂ and P′₂ positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine-free disubstituted amino acid α-aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the α-Tfm-substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P′₁ substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)-diasteromer the steric constraints exhibited by the α-Tfm group can be outweighed by an advantageous interaction of the fluorine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)-diastereomer. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

MUC1 is a substrate for γ‐secretase

Understanding the underlying mechanisms by which a normal cell avoids the oncogenic potential of MUC1 signaling requires further definition of the pathways by which the MUC1 cytoplasmic tail is processed in both normal and tumor‐derived cells. In the present study we describe the processing pathway initiated by TACE/ADAM17 cleavage of MUC1. Utilizing the human uterine epithelial cell line, HES, derived from normal endometrium, we show that endogenous full length MUC1 undergoes regulated intramembranous proteolysis mediated by presenillin‐dependent γ‐secretase. Cytokine‐stimulated HES cells exposed to γ‐secretase inhibitors accumulated a membrane‐associated 15 kDa fragment of the MUC1 C‐terminal subunit (CTF15). Inhibitors of TACE/ADAM17‐mediated shedding inhibited accumulation of MUC1‐CTF15 and MUC1 ectodomain release to a similar extent consistent with MUC1‐CTF15 being a product of TACE/ADAM17 action. Reduction of catalytically active γ‐secretase complex by nicastrin siRNA treatment also resulted in CTF15 accumulation. Furthermore, mature nicastrin, the substrate receptor for γ‐secretase, co‐immunoprecipitated with CTF15 in the presence of γ‐secretase inhibitors indicating the formation of CTF15: nicastrin complexes. MUC1‐CTF15 accumulation in response to γ‐secretase inhibition was demonstrated in both normal and tumor‐derived cells from humans and mice indicating that this processing pathway exists in many cell contexts. We did not detect products of MUC1 cleavage by γ‐secretase in the presence of various proteasomal inhibitors indicating that subsequent degradation is either non‐proteasomal or extremely efficient. We suggest that this efficient pathway attenuates potential signaling mediated by cytoplasmic tail fragments. J. Cell. Biochem. 108: 802–815, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.     

Preferred conformations of peptides containing α,α-disubstituted α-amino acids

The conformational preferences of linear peptides containing α,α-disubstituted α-amino acids, derived from the crystal structures of 28 compounds, are reviewed. In particular, the sensitivity of peptide conformation to the geometry of these unusual amino acids is underlined. We also consider possible future directions of research, which, we hope, will result in a complete understanding of the structures adopted by peptaibol antibiotics.     

Effects of acetoacetate and D-beta-hydroxybutyrate on bovine in vitro embryo development in serum-free medium

It is known that the ketone bodies acetoacetate and D-beta-hydroxybutyrate can be metabolized by the early bovine embryo for in vitro development. In the present work, we report experiments leading to the culture of bovine embryos in the absence of serum. In vitro-produced bovine zygotes were cultured in modified synthetic oviduct fluid medium supplemented with acetoacetate derivatives, acetoacetate and D-beta-hydroxybutyrate. Acetoacetate and its derivatives prevented blastocysts from forming in the absence of serum during the whole culture period. However, from Days 6 to 8 of culture in the absence of serum, acetoacetate did not affect development as compared to controls containing lactate and pyruvate or no substrate. Interestingly, D-beta-hydroxybutyrate stimulated blastocyst and expansion development, and allowed lipid mobilization. In feeder cells coculture, embryos produced with D-beta-hydroxybutyrate showed improved hatching. Embryos cultured in D-beta-hydroxybutyrate were viable upon transfer to recipients, although no pregnancies were confirmed later by ultrasonic scanning. The protective effect of serum upon embryos cultured in medium containing acetoacetate is apparently not required in the presence of D-beta-hydroxybutyrate.