Rationing in practice: the case of in vitro fertilisation.

One of the few examples of explicit rationing in the National Health Service is provided by in vitro fertilisation. Of six purchasing authorities examined three have decided against buying in vitro fertilisation while three have decided in favour. The decisions reflect local factors such as the absence or presence of local providers and the views of the public and health professionals. But in vitro fertilisation also illustrates some of the wider issues involved in all decisions about purchasing: questions about what should be provided by the National Health Service, about what procedures should be compared when weighing up value for money, and whether equity demands national decisions about what to provide.     

In vitro fertilisation of mouse oocytes reconstructed by transfer of metaphase II chromosomes results in live births

The interaction between nucleus and cytoplasm can be explored through nuclear transfer. We describe here another tool to investigate this interaction: MII meiotic apparatus transfer (MAT) between mouse oocytes. In this study, the MII oocyte meiotic apparatus or spindle from C57BL/6 mice, a black strain, was transferred into an enucleated metaphase oocyte from Kunming mouse, a white strain. The results showed that the enucleation rate by treating oocytes with 3% sucrose was 100%, but the electrofusion efficiency was very low, with only 17.6% of reconstructed karyoplast-recipient cytoplasm pairs fused. When the fused oocytes were exposed to spermatozoa from C57BL/6 mice, 9 of 11 (82%) were fertilised. Eight reconstructed embryos at 1- to 4-cell stages were transferred into the oviducts of two synchronously pregnant Kunming strain fosters and one delivered two normal C57BL/6 offspring. This study indicates that MII meiotic apparatus or spindle sustains normal structure and function after micromanipulation and electrofusion. MAT provides a model for further research on the application of this technique to assisted human reproduction.     

In vitro selection of RNA aptamers against a composite small molecule-protein surface

A particularly challenging problem in chemical biology entails developing systems for modulating the activity of RNA using small molecules. One promising new approach towards this problem exploits the phenomenon of ‘surface borrowing,’ in which the small molecule is presented to the RNA in complex with a protein, thereby expanding the overall surface area available for interaction with RNA. To extend the utility of surface borrowing to include potential applications in synthetic biology, we set out to create an ‘orthogonal’ RNA-targeting system, one in which all components are foreign to the cell. Here we report the identification of small RNA modules selected in vitro to bind a surface-engineered protein, but only when the two macromolecules are bound to a synthetic bifunctional small molecule.     

In vitro translation of the genome of a small RNA virus from Trichoplusia ni

The genomic RNA of a member of the “Nudaurelia β virus” group functioned as a mRNA in vitro. The translation products included a protein, which comigrated with the single virus capsid protein, and a stable 100 × 10³ MW protein, which was synthesized by cleavage of a precursor protein. No precursor proteins were involved in synthesis of the putative capsid protein. Attempts to inhibit proteolytic cleavage did not result in the appearance of a product corresponding to the entire coding capacity of the genome.     

In vitro maturation and fertilisation of bovine oocytes in relation to GH gene polymorphism (Leu/Val)

The present study describes the analysis of the associations between the growth hormone gene polymorphism (Leu/Val) and oocyte maturation and in vitro fertilisation in cattle. Two independent experiments were carried out. In the first one, oocytes were collected from 49 single ovaries, matured in vitro, measured and cytogenetically analysed. One ovary was considered as a donor. The procedure of the donor's genotyping at the GH locus was based on DNA extracted from the granulosa cells. The GH genotype did not influence the oocyte diameter nor the number of oocytes collected, which were selected for maturation and matured. An unreduced chromosome number was found in 8.8% of the cells at the second metaphase stage and 42.9% of the donors. This anomaly was observed in all genotype groups with a higher frequency in the VV cows (P < 0.01). In the second experiment, the oocytes collected from 72 single ovaries were matured and fertilised in vitro. The GH genotype of a donor did not influence the number of zygotes cleaved on day-2. It has to be mentioned, that due to the low frequency of the VV genotype (0.03), the results of the present study should be treated as preliminary and need further analysis.     

In vitro synthesis of the unit that links teichoic acid to peptidoglycan.

The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated. Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp. 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer. In toluenized cells of Micrococcus sp. 2102, some of this product became linked to the wall. In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine. In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer. Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan. It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S. aureus H.     

In vitro evidence for the recognition of 8-oxoGTP by Ras, a small GTP-binding protein

Oxygen radicals attack guanine bases in DNA but they also attack cytoplasmic GTP forming 8-oxoGTP. The presence of 8-oxoGTP in cytoplasm is evidenced by the fact that cells contain MutT/MTH1 which hydrolyze 8-oxoGTP into 8-oxoGMP. In this study, the interaction between 8-oxoGTP and Ras, a small GTP-binding protein, was tested in vitro, and the action of 8-oxoGTP was compared to that of GTP. When purified Ras was treated with 8-oxoGTPgammaS, Ras was activated, as indicated by the enhanced binding of Ras with Raf-1. GTPgammaS also activated Ras but 8-oxoGTPgammaS had a much more potent effect. In lysates of human embryo kidney 293 cells, 8-oxoGTPgammaS activated not only Ras but also the downstream effectors of the Ras-ERK pathway, i.e., Raf-1 and ERK1/2. In contrast to Ras, other small GTP-binding proteins, Rac1 and Cdc42, were inactivated by 8-oxoGTPgammaS, whereas both of these proteins were activated by GTPgammaS, indicating that the biological natures of 8-oxoGTP and GTP differ. These results suggest the possibility that 8-oxoGTP is not a simple by-product but a functional molecule transmitting an oxidative signal to small GTP-binding proteins like Ras.     

In vitro study of the biology of small cell carcinoma of the lung

We have developed three types of experimental systems for the study of SCCL: (1) serially heterotransplanted tumors in athymic nude mice; (2) continuous, clonable cell cultures; and (3) direct clonogenic assays for tumor specimens. These systems have their own individual advantages, applications, and limitations, but these are interrelated and complementary. The study of these systems has greatly aided our understanding of the biology of SCCL, and its relationship to other lung cancers and the APUD cell system. In addition, new markers for SCCL have been identified, such as a creatine kinase and its BB isoenzyme (CK-BB). These cellular markers may have clinical applications, as serum levels of CK-BB are an indicator of tumor burden. Assays for clonogenic tumor cells may permit selection of optimal drug combinations for the treatment of individual tumors. Variant cultures having the morphology of SCCL, but lacking some or all of the other features, have been identified. While our systems have been used primarily for biological studies, they have clinical applications for both diagnostic and therapeutic purposes.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.