In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach

Development of new technology related to in vitro embryo production has allowed for the commercial use of this method of reproduction. In the present work, we evaluate the efficiency of this technology compared with conventional embryo production based on results obtained with a standard procedure, including the sexing of embryos. The donor animals were mature nonlactating dairy cows (n = 92) kept under a constant environment and feeding program in an ET center. Ultrasound guided transvaginal ovum pick-up following 48 h pre-treatment with FSH has been used for the IVF-IVC protocol. A total of 437 oocyte recovery sessions performed on 92 cows yielded 4145 oocytes, which were used in an IVF-IVC protocol. Using the conventional approach, 156 embryo collections on 49 cows yielded 1652 ova and embryos. All Quality 1 and 2 embryos were sexed by a PCR procedure, and embryos of the desired sex were transferred to synchronized recipients located at the center. The results obtained in the IVF protocol showed that 4 oocyte collections per cow performed within 60 d, yielded 38 oocytes, which resulted in 18.8 viable embryos, of which 7.05 were female. After transfer of the female embryos, an average of 3.8 recipients were pregnant at 60 d. One embryo collection under the conventional approach yielded an average of 1.2 female pregnancies, which was confirmed during the same 60-d time period. These results indicate that IVF procedures can effectively replace conventional embryo production methods when a predetermined number of pregnancies of known sex are needed within a short period of time.     

A wheat embryo cell‐free protein synthesis system not requiring an exogenous supply of GTP

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous‐flow wheat embryo cell‐free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC‐based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 μM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C‐terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B_1‐320) gave almost the same result. The wheat embryo cell‐free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48‐h period at 26°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The effect of wheat cytoplasm on the synthesis of wheat x rye hybrids

Summary Four nuclear genotypes of Triticum aestivum L., each substituted into the cytoplasm of T. timococcum Kostoff, Aegilops ventricosa Tausch and T. timopheevi Zhuk., were crossed with four homozygous Secale cereale L. inbred lines to produce 48 alloplasmic octoploid primary triticales. Crossability, embryo differentiation, amphihaploid plantlet recovery in vitro, and response to colchicine treatment were investigated. It was found that in general the effects of the wheat cytoplasm on the characteristics studied were of equal importance as those of the wheat and the rye nuclear genotypes. Cytoplasm can be regarded as an additional source of variation in the synthesis of primary triticales. Cross-compatibility in wheat x rye hybrids appeared to be determined by specific parental genotypes and their nucleo-cytoplasmic interactions rather than by any general compatibility of particular nuclear genotypes and/or cytoplasms.     

Control of ribosomal protein synthesis during wheat embryo imbibition

Dry wheat embryos contain large quantities of ribosomes, synthesized and assembled during embryogenesis. When messenger RNA isolated from dry embryos is translated, in vitro, a significant proportion of the total translation products (approx. 10%) is identifiable as ribosomal proteins, by electrophoresis in two distinct two-dimensional polyacrylamide gel electrophoretic systems. When germinating embryos are labelled with [³⁵S]methionine, during the first 24 h of imbibition, the appearance of newly synthesized ribosomal proteins in the cytosolic fraction is barely detectable. However, this low level (< 1% of total cytosolic protein synthesis) of observed ribosomal protein synthesis is not correlated with a correspondingly low level of ribosomal protein mRNA. Ribosomal proteins constitute at least 10% of the products of translation, in vitro, of mRNA isolated from germinating wheat embryos. Ribosomal proteins are also conspicuous products of translation when polyribosomes isolated from imbibing embryos are used to direct protein synthesis in a cell-free ‘run-off’ system, and newly synthesized ribosomal proteins can be detected in the nuclei isolated from germinating embryos. It is proposed that their absence from the cytosolic fraction is a consequence of post-translational regulatory events.     

An assay for albumin messenger RNA in an vitro protein synthesizing system from wheat germ.

The synthesis of serum albumin in an in vitro protein-synthesizing system from wheat germ stimulated with rat liver polysomal RNA is demonstrated by immunoprecipitation. The newly synthesized albumin has the same electrophoretic mobility as rat serum albumin. There is a linear increase in precursor incorporation into total protein and albumin with increasing RNA concentration. Potassium and magnesium optima for albumin synthesis are different from those for total protein synthesis.     

The protein synthesis inhibitors from wheat, barley, and rye have identical antigenic determinants

We have purified the protein synthesis inhibitors from wheat (tritin) and investigated their antigenic relationship to inhibitors from other grains and more distantly related plants. Three forms of tritin (tritin 1, 2, and 3) indistinguishable with regards to their molecular weight and enzymatic properties were found. Antiserum against the most abundant tritin, tritin 2, cross reacted with tritin 1, tritin 3, and with the translation inhibitors from barley and rye, but not with other plant-derived translational inhibitors. The results indicate that only closely related plants contain antigenically similar protein synthesis inhibitors.     

In vitro synthesis of lectins in cell-free extracts from dry wheat and rye embryos

Cell-free extracts from dry wheat (Triticum vulgare L.) and rye (Secale cereale L.) embryos do not synthesize their corresponding lectins when incubated under conditions optimalized for translation of either exogenous or endogenous mRNA. Only when the extracts are prepared and incubated in the complete absence of sulphydryl containing compounds lectins are synthesized in vitro. Since immunoprecipitation techniques could not demonstrate unequivocally the presence of lectin among the cell-free translation products a specific affinity purification procedure has been developed in order to proof the presence of stored lectin mRNAs in dry wheat and rye embryos.Abbreviations DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCA trichloracetic acid     

The contribution of the male to ovine embryogenesis in an in vitro embryo production system

The differences in the embryo production potential of four rams used in a commercial embryo transfer program were examined in both in vivo and in vitro embryo production systems. Processing frozen-thawed spermatozoa through Percoll density gradients prior to in vitro insemination eliminated differences in the estimates of sperm viability between the four rams, and yet, differences in embryo production persisted throughout the in vitro culture period. However, there was no effect of ejaculate within ram on embryo production rates. In addition, the timing of the onset of the differences between the rams at each stage of in vitro embryo development were revealed. Ram 2 differed from ram 4 in the proportion of fertilized oocytes at 17 h post-insemination (pi) and by 52 h, ram 3 differed from ram 4 in the proportion of cleaved embryos, and the observed differences between ram 1 and ram 2 in their blastocyst production were initiated prior to activation of the embryonic genome. Once differences in embryo development rates were detected among the four rams, they persisted throughout the in vitro culture period. The reduced in vitro fertilization (IVF) rates from ram 2 compared with the other rams was paralleled in vivo by the significantly lower proportion of embryos recovered from ewes mated to ram 2, and this was further exacerbated by a significantly lower embryo survival rate after transfer. However, the subtle differences observed in the timing of the contribution of each sire to embryo development during in vitro culture were not able to be detected in vivo. However, the higher proportions of transferable quality blastocysts obtained from ewes mated to ram 4 did not result in increased embryo survival throughout the remainder of gestation. Therefore, in this study, the blastocyst production potential for a particular sire, either in vitro or in vivo, does not necessarily reflect the potential for the production of live offspring.     

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.     

Genetic control of embryo lethality in crosses between common wheat and rye

The phenotypic manifestation and genetic control of embryo lethality observed in crosses between common wheat and rye were studied. It was found that crosses between common wheat and inbred self-fertile rye lines L2 and 535 gave rise to ungerminating grains, in which the development and differentiation of the hybrid embryo are arrested. Study of the degree of embryo development in the hybrid grains obtained by crossing common wheat varieties with inbred rye lines L2 and 535 showed that genotypes of the parents affected the ratio between undifferentiated embryos of various sizes. Analysis of this trait was performed by test crosses according to a novel pedigree program with the use of interlinear hybrids and a set of fourth-generation hybrid recombinant inbred lines. Rye line L2 was shown to bear the Eml (Embryo lethality) gene, which terminates the development of the hybrid embryo in amphihaploids. The suggestion of complementary interaction between wheat and rye genes during formation of a "n ew" character in wheat-rye F1 hybrids is discussed. A method of detecting an allele not complementary to the rye Eml allele in wheat is proposed. The proposed test program allows appropriate study of the system of wheat and rye genes involved in complementary interaction in the genotype of a distant hybrid.     

Lectin synthesis in developing and germinating wheat and rye embryos

Wheat (Triticum aestivum L.) and rye (Secale cereale L.) lectins are specifically synthesized during seed formation. They accumulate exponentially in the primary axes in a period coinciding with the development of this complex organ. Since the specific lectin content also increases dramatically, there is apparently an outburst of lectin synthesis during the development of the primary axes. Germinating embryos also synthesize some lectin. The fortunate availability of a highly specific procedure for the isolation of cereal lectins enabled us to follow the kinetics of their synthesis during early germination. Stored mRNAs appear to be involved in this residual lectin synthesis.     

The occurrence and role of DNA synthesis during meiosis in wheat and rye

The incorporation of ³H-thymidine into the DNA of rye meiocytes at zygotene, pachytene-diplotene and metaphase I to telophase II stages has been studied. Low levels of ³H were found in highly purified DNA from meiocytes at all these stages, though there was more in the DNA from pachytene-diplotene meiocytes, and it is highly likely that the zygotene groups of anthers contained a proportion at pachytene. The buoyant density distributions of the labelled DNA from zygotene and pachytene-diplotene cells were indistinguishable, in contrast to the situation in Lilium, the only other example studied so far.The DNA synthesis inhibitor 2′-deoxyadenosine halted meiotic development of anthers in culture only at late zygotene and pachytene. It did not inhibit development at early zygotene, prevent chromosome pairing as judged by light microscopy or cause extensive chromosome fragmentation during zygotene as in Lilium. These results indicate that extensive synthesis of DNA does not occur at zygotene in cereals and does not suggest that zygotene DNA synthesis is a prerequisite for chromosome pairing as in Lilium.