Lipid bilayer composition modulates the unfolding free energy of a knotted α-helical membrane protein.

α-Helical membrane proteins have eluded investigation of their thermodynamic stability in lipid bilayers. Reversible denaturation curves have enabled some headway in determining unfolding free energies. However, these parameters have been limited to detergent micelles or lipid bicelles, which do not possess the same mechanical properties as lipid bilayers that comprise the basis of natural membranes. We establish reversible unfolding of the membrane transporter LeuT in lipid bilayers, enabling the comparison of apparent unfolding free energies in different lipid compositions. LeuT is a bacterial ortholog of neurotransmitter transporters and contains a knot within its 12-transmembrane helical structure. Urea is used as a denaturant for LeuT in proteoliposomes, resulting in the loss of up to 30% helical structure depending upon the lipid bilayer composition. Urea unfolding of LeuT in liposomes is reversible, with refolding in the bilayer recovering the original helical structure and transport activity. A linear dependence of the unfolding free energy on urea concentration enables the free energy to be extrapolated to zero denaturant. Increasing lipid headgroup charge or chain lateral pressure increases the thermodynamic stability of LeuT. The mechanical and charge properties of the bilayer also affect the ability of urea to denature the protein. Thus, we not only gain insight to the long-sought-after thermodynamic stability of an α-helical protein in a lipid bilayer but also provide a basis for studies of the folding of knotted proteins in a membrane environment.

Application of the WFD cost proportionality principle to diffuse pollution mitigation: a case study for Scottish Lochs.

The Water Framework Directive (WFD) aims to deliver good ecological status (GES) for Europe's waters. It prescribes the use of economic principles, such as derogation from GES on grounds of disproportionate costs of mitigation. This paper proposes an application of the proportionality principle to mitigation of phosphorus (P) pollution of 544 Scottish lochs at national and local water body scales. P loading estimates were derived from a national diffuse pollution screening tool. For 293 of these lochs (31% of the loch area), GES already occurred. Mitigation cost-effectiveness was assessed using combined mitigation cost curves for managed grassland, rough grazing, arable land, sewage and septic tank sources. These provided sufficient mitigation (92% of national P load) for GES to be achieved on another 31% of loch area at annualised cost of £2.09 m/y. Mitigation of the residual P loading preventing other lochs achieving GES was considered by using a "mop-up" cost of £200/kg P (assumed cost effectiveness of removal of P directly from lochs), leading to a total cost of £189 m/y. Lochs were ranked by mitigation costs per loch area to give a national scale marginal mitigation cost curve. A published choice experiment valuation of WFD targets for Scottish lochs was used to estimate marginal benefits at national scale and combined with the marginal cost curve. This gave proportionate costs of £5.7 m/y leading to GES in 72% of loch area. Using national mean marginal benefits with a scheme to estimate changes in individual loch value with P loading gave proportionate costs of £25.6 m/y leading to GES in 77% of loch area (491 lochs).

Membrane protein folding.

Investigating the in vitro refolding of proteins that naturally reside in biological membranes is a notoriously difficult task. Biophysical studies on model systems are beginning to provide a sound physical basis for membrane protein folding that should help to alleviate this problem. Highlights of these studies include insights into the interaction of transmembrane alpha helices, as well as into the important role that membrane lipids play in folding.

Haematological and biochemical values for grey seal pups (Halichoerus grypus) during early rehabilitation.

Haematological and biochemical data were collected over a period of six years from grey seal (Halichoerus grypus) pups undergoing rehabilitation. Pups bled during the first three days were allocated retrospectively to one of seven clinical groups and statistical analyses were carried out on six of these groups (130 pups). Compared with reference ranges, all the groups had lower mean packed-cell volumes (pcv), red blood cell (rbc) counts haemoglobin and albumin levels, pups with severe trauma had higher total white blood cell (wbc) and neutrophil counts, pups with severe trauma and malnourished pups had higher total bilirubin levels, and pups in all the clinical groups except those with severe trauma had lower sodium levels. There were significant differences (P<0.05) between some clinical groups for mean pcv, rbc counts, all wbc counts except monocytes, haemoglobin, total bilirubin, creatine kinase, amylase and potassium levels. Forty-nine pups were bled more than once during the first 10 days of rehabilitation for haematology, and 11 were bled more than once for biochemistry. There were significant decreases (P<0.05) during this period in pcv, and in the activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatine kinase and lactate dehydrogenase, and significant increases in all wbc counts, total protein, globulin, amylase and calcium levels.

Unravelling the folding of bacteriorhodopsin.

The folding mechanism of integral membrane proteins has eluded detailed study, largely as a result of the inherent difficulties in folding these proteins in vitro. The seven-transmembrane helical protein bacteriorhodopsin has, however, allowed major advances to be made, not just on the folding of this particular protein, but also on the factors governing folding of transmembrane alpha-helical proteins in general. This review focusses on kinetic and equilibrium studies of bacteriorhodopsin folding in vitro. It covers what is currently known about secondary and tertiary structure formation as well as the events accompanying retinal binding, for protein in detergent and lipid systems, including native membrane samples.

Location of the calcium binding site in Photosystem II: A Mn2+ substitution study.

The whereabouts of the Ca2+ site in Photosystem II (PSII) was investigated by experiments in which Mn2+ was substituted for Ca2+. When stoichiometric amounts of Mn2+ ions were added to Ca2+-depleted PSII, the Mn2+ was not detected by EPR. The titration of Ca2+ back into Ca2+-depleted/Mn2+-containing PSII resulted in the simultaneous release of the Mn2+ and the loss of the two EPR signals which are characteristic of the Ca2+-depleted enzyme (i.e., the stable, modified S2 multiline signal arising from the intrinsic Mn cluster and the split S3 signal from an organic radical interacting with the Mn cluster). These results indicate that the Mn2+ occupies the functional Ca2+ site. The S2 and S3 EPR signal characteristic of this kind of Ca2+-depleted preparation were unaffected by the binding of the Mn2+ Since, from earlier results, it seems likely that the modification and stability of S2 multiline signal in these PSII preparations is due to binding of chelator to or close to the Mn cluster, the present results indicate that the Ca2+ site (at least when occupied by Mn2+) does not overlap with the chelator binding site. Since Mn2+ binding does not effect the S2 EPR signal from the Mn cluster, it can be concluded that the Mn2+ is not involved in detectable magnetic interactions with the cluster. This result indicates that the Mn2+-occupied Ca2+ binding site is outside the first co-ordination sphere of the Mn cluster. The relaxation properties of TyrD. were enhanced by the presence of the Mn2+ when the Mn cluster was in the S1 state.

Estimations of lipid bilayer geometry in fluid lamellar phases.

The excess water bilayer thickness, d(l,0), and molecular area, A(0), of lipid amphiphiles in the fluid lamellar phases of dioleoylphosphatidylcholine (DOPC) and dipalmitoleoylphosphatidylcholine (DPolPC) have been estimated between 15 and 50 degrees C and for dimyristoylphosphatidylcholine (DMPC) between 25 and 50 degrees C. These determinations have been made from X-ray measurements on samples of known water composition. With respect to temperature, T, d(l,0) and A(0) are well fitted to a linear equation. We find d(l,0) (A)=(35.68+/-0.02)-(0.0333+/-0.0006)T (degrees C) and A(0) (A(2))=(70.97+/-0.05)+(0.136+/-0.001)T (degrees C) for DOPC, d(l,0) (A)=(35.2+/-0.1)-(0.068+/-0.003)T (degrees C) and A(0) (A(2))=(59.7+/-0.2)+(0.210+/-0.006)T (degrees C) for DMPC, and d(l,0) (A)=(34.54+/-0.03)-(0.0531+/-0.0009)T (degrees C) and A(0) (A(2))=(67.12+/-0.09)+(0.173+/-0.003)T (degrees C) for DPolPC. The accuracy of these estimates depends largely on how accurately the excess water point is determined. Ideally, reliable X-ray and compositional data will be available around the excess water and it may be found by simple inspection, but this is the exception rather than the rule, since samples close to water excess normally sequester sizeable amounts of water in defects, which lead to an underestimate of d(l,0). and overestimate of A(0). In this paper, we report a methodology for identifying and removing such data points and fitting the remaining data in order to determine the excess water point.

The final stages of folding of the membrane protein bacteriorhodopsin occur by kinetically indistinguishable parallel folding paths that are mediated by pH.

The folding of the transmembrane protein bacteriorhodopsin that occurs during the binding of its retinal cofactor is investigated in a membrane-like environment. Changes in the retinal absorption band reveal two transient retinal-protein intermediate states, with apparent absorption maxima at 380 nm and 440 nm, respectively. Studies on a bacteriorhodopsin mutant of Lys216, which cannot bind retinal covalently, add to evidence that retinal is non-covalently bound in these intermediate states. The two retinal-protein intermediates are genuine intermediate states that form in parallel, each with an observed rate constant of 1.1 s-1. Meanwhile no formation of the folded state is detected. Folded bacteriorhodopsin, with all trans retinal covalently bound, forms from both retinal-bound intermediates with the same apparent rate constant of 0.0070 s-1 that is independent of retinal concentration. Retinal isomerisation then occurs with a rate constant of 0.00033 s-1 to give bacteriorhodopsin containing all trans and 13 cis-retinal. These results provide experimental evidence for multiple folding routes for a membrane protein that are pH dependent, with pH conditions determining the apparent folding route. These observed parallel folding paths are kinetically indistinguishable, which contrasts with most other observations of parallel folding pathways where only pathways with different kinetics have been reported. Furthermore, together with previous work, this study shows that bacteriorhodopsin has to populate at least two folding intermediates, during folding in the mixed lipid micelles investigated here, before the final fold is attained.

Proline residues in transmembrane alpha helices affect the folding of bacteriorhodopsin.

Proline residues occur frequently in transmembrane alpha helices, which contrasts with their behaviour as helix-breakers in water-soluble proteins. The three membrane-embedded proline residues of bacteriorhodopsin have been replaced individually by alanine and glycine to give P50A, or P50G on helix B, P91A, or P91G on helix C, and P186A or P186G on helix F, and the effect on the protein folding kinetics has been investigated. The rate-limiting apoprotein folding step, which results in formation of a seven transmembrane, alpha helical state, was slower than wild-type protein for the Pro50 and Pro91 mutants, regardless of whether they were mutated to Ala or Gly. These proline residues give rise to several inter-helix contacts, which are therefore important in folding to the seven transmembrane helix state. No evidence for cis-trans isomerisations of the peptidyl prolyl bonds was found during this rate-limiting apoprotein folding step. Mutations of all three membrane-embedded proline residues affected the subsequent retinal binding and final folding to bacteriorhodopsin, suggesting that these proline residues contribute to formation of the retinal binding pocket within the helix bundle, again via helix/helix interactions. These results point to proline residues in transmembrane alpha helices being important in the folding of integral membrane proteins. The helix/helix interactions and hydrogen bonds that arise from the presence of proline residues in transmembrane alpha helices can affect the formation of transmembrane alpha helix bundles as well as cofactor binding pockets.

Effects of chlorophyll a, chlorophyll b, and xanthophylls on the in vitro assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb.

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio < or = 1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin.

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics.

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.

Fit to fly: practical challenges in neonatal transfers by air.

Air transport of infants in incubators should be undertaken in a manner that is safe for both staff and infant and satisfies all directives by appropriate regulatory bodies. In Scotland during the last two years, certification of an infant incubator system for use in both rotary and fixed wing aircraft has been accomplished. This is a report of the issues addressed during this project, which will be common to all neonatal transport services intending to develop air transport capability.

A comparison of the glycosaminoglycans of weight-bearing and non-weight-bearing human dermis.

Twenty-six samples of human skin from three non-weight-bearing and 27 samples from three weight-bearing sites were obtained at autopsy from 11 subjects varying in age from 4 to 91 years. The dermis was idssected free of other tissues and after proteolytic digestion, the glycosaminoglycans were isolated quantitatively by ion-exchange chromatography. Analysis of the preparations showed that there was significantly more glycosaminoglycan in the dermis from weight-bearing sites (p less than 0.001). The major glycosaminoglycans were hyaluronic acid and dermatan sulfate with chondroitin sulfate as a minor component, but the relative proportions of these components were not significantly different in the two sets of samples (p greater than 0.1). It is suggested that the quantity of glycosaminoglycan in the dermis is influenced by the functional requirements of the dermis at that site.

Assembly and cloning of coding sequences for neurotrophic factors directly from genomic DNA using polymerase chain reaction and uracil DNA glycosylase.

The polymerase chain reaction (PCR) was used to amplify individual exons of the gene (CNTF) coding for human ciliary neurotrophic factor (CNTF) directly from genomic DNA. Inclusion of deoxyuracil in place of thymine in the PCR primers permits removal of dU residues in the primer after amplification using uracil DNA glycosylase, generating single-stranded 3' overhangs. Thus, the individual exons were assembled to generate the full-length CNTF sequence. A similar strategy was also used to generate a chimeric gene (BDNF) encoding brain-derived neurotrophic factor (BDNF) with the pre-pro sequence of nerve growth factor (NGF). The method described allows direct amplification of coding sequence from genomic DNA and ordered assembly of amplified exons to generate a clone containing the complete coding sequence of the gene without the need for splicing; a clone which is equivalent to a cDNA clone.

Comparison of the D1/D2/cytochrome b559 reaction centre complex of photosystem two isolated by two different methods.

Photosystem 2 reaction centre complexes prepared either by solubilisation with Triton X-100 and subsequent exchange into dodecyl maltoside or by a procedure involving a combination of dodecyl maltoside and LiClO4, were characterised in terms of chlorophyll a, pheophytin a, beta-carotene and cytochrome b559 content. Time-resolved chlorophyll fluorescence decay kinetics were measured using both types of complexes. Our data show that the isolated photosystem two reaction centre complex contain, for two pheophytin a molecules, close to six chlorophyll a, two beta-carotene and one cytochrome b559. No major differences were observed in the composition or the kinetic characteristics measured in the samples prepared by the different procedures. Time-resolved fluorescence measurements indicate that more than 94% of the chlorophyll a in both preparations is coupled to the reaction centre complex.

Manipulating the folding of membrane proteins: using the bilayer to our advantage.

The folding mechanisms of integral membrane proteins have largely eluded detailed study. This is owing to the inherent difficulties in folding these hydrophobic proteins in vitro, which, in turn, reflects the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is, however, a large body of information on lipid properties and, in particular, on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to develop efficient in vitro lipid-bilayer folding systems for the membrane protein, bacteriorhodopsin. Furthermore, we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency appear to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer, and the lateral pressure that the lipid chains exert on the their neighbouring folding proteins. These are generic properties of the bilayer that can be achieved with simple mixtures of biological lipids, and are not specific to the lipids studied here. These bilayer properties also seem to be important in modulating the function of several membrane proteins, as well as the function of membranes in vivo. Thus, it seems likely that careful manipulations of lipid properties will shed light on the forces that drive membrane protein folding, and will aid the development of bilayer folding systems for other membrane proteins.

Developmental kinetics of bovine nuclear transfer and parthenogenetic embryos.

Early developmental kinetics of nuclear transfer (NT) embryos reconstituted with blastomeres and parthenogenones produced by ionophore activation followed by either dimethylaminopurine (DMAP) or cycloheximide (CHX) treatment was studied. In vitro produced (IVP) embryos served as controls. Embryos were cultured to the hatched blastocyst stage, and images were recorded every 0.5 h throughout the culture period. The longest cell cycle shifted from 4th to 5th cycle (26 +/- 4 and 44 +/- 5 h) in NT-embryos compared to IVP-embryos (41 +/- 2 and 20 +/- 3 h) and showed greater asynchrony between blastomeres than any other embryo category. Compared to DMAP, CHX prolonged the 1(st) (23 +/- 1 vs. 33 +/- 1 h) and shortened the 3(rd) cell cycle (17 +/- 2 vs. 13 +/- 1 h). Moreover, though cytoskeleton activity was initialised, a larger proportion of CHX embryos was unable to accomplish first cleavage. The parthegenones differed from IVP embryos with respect to the lengths of the 1st, 3rd, and 4th cell cycles and time of hatching. The findings are discussed in relation to known ultrastructural, chromosomal and genomic aberrations found in NT embryos and parthenogenones. We hypothesize that the shift of the longest cell cycle in NT embryos is associated with a shift in the time of major genomic transition.

Application of the zona-free manipulation technique to porcine somatic nuclear transfer.

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.

Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique.

Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development of the system was based on results using parthenogenetic and in vitro fertilized zygotes in order to (a) select the optimal primary activation agent that induced the lowest lysis rate but highest parthenogenetic blastocyst yield, (b) evaluate the quantity and quality of zona-free blastocysts produced in WOWs, and (c) establish any potential embryotoxic effects of PHA-P. The initial data indicated that, of calcium ionophore A23187, ionomycin, and electropulse treatments as primary activation agents, the two former were equally efficient even with reduced exposure times. WOW-culture of zona-free versus zona-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20.8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1%) blastocysts per successfully fused and surviving reconstructed embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1%) recipients pregnant at 5 months after transfer. These results suggest that the zona-free nuclear transfer technique generates blastocysts of equivalent quantity and quality compared to conventional micromanipulation methods, requires less technical expertise, is less time consuming and can double the daily output of reconstructed embryos (even after taking into consideration the rejection of the half oocytes containing the metaphase plate).