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.

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.

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.

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).

Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: work in progress.

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.

We describe a bovine embryo culture system that supports repeatable high development in the presence of serum or BSA as well as under defined conditions in the absence of those components. In the first experiment, embryo development in SOF with amino acids (SOFaa), sodium citrate (SOFaac) and myo-inositol (SOFaaci) and with BSA or polyvinyl alcohol (PVA) was compared with that in a M199 granulosa cell co-culture (M199 co-culture). Subsequently, development and cell numbers of blastocysts cultured under defined conditions in SOFaaci with PVA (SOFaaci-PVA), or under undefined conditions in SOFaaci with 5% cow serum (SOFaaci-CS) or M199 co-culture were compared. The repeatability of culture results in SOFaaci-CS was checked by weekly replicates (n = 30) spread over 11 months. The viability of embryos developed in SOFaaci-PVA was estimated by transfer of morphologically good blastocysts (n = 10) to synchronized recipients. In the second experiment, the effect of omitting CS or BSA from IVM and IVM-IVF on subsequent embryo development in SOFaaci-PVA or in SOFaaci-CS was investigated. Blastocyst development in SOFaa-PVA, SOFaac-PVA, SOFaa-BSA and M199 was 16 +/- 3b, 23 +/- 2ab, 30 +/- 8a and 36 +/- 7a%, respectively (Pab < 0.05). Additional inclusion of myoinositol resulted in 42 +/- 1a% blastocysts in SOFaaci-PVA vs 19 +/- 3b% in SOFaac-PVA, 47 +/- 7a% in SOFaac-BSA, and 36 +/- 7a% in M199 co-culture, respectively (Pab < 0.01). In 30 replicates, the average cleavage and blastocyst rates of oocytes in SOFaaci-CS were 87 +/- 4 and 49 +/- 5%, respectively. Five normal calves were produced after transfer of 10 blastocysts developed in defined culture medium (i.e., SOFaaci-PVA). Defined IVM or IVM-IVF (i.e., in absence of CS and BSA) reduced cleavage rates (83 +/- 3 and 55 +/- 3% vs 90 +/- 1% in presence of CS; P < 0.01). Subsequent embryo development in SOFaaci-CS was not affected in either of these defined conditions. However, cleavage and blastocyst rates under completely defined IVP conditions were 54 +/- 7 and 19 +/- 4%, respectively. It was concluded that under defined culture conditions, addition of citrate and myo-inositol improved blastocyst development to rates comparable to those obtained with serum, BSA or co-culture and that the quality of blastocysts was not affected by the absence of serum or BSA. However, serum was essential during IVM/IVF for normal fertilization and subsequent high blastocyst development.

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos.

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.

Nutritional manipulation of growth and metabolic and reproductive status in prepubertal gilts.

Reproductive status, metabolic state, and carcass composition were compared in prepubertal littermate gilts of 85 kg BW and identical ages after allocation to one of two feeding regimens at 75 kg, either twice-daily feeding to appetite up to 85 kg and then maintenance feeding until slaughter (Group 85AR) or an initial period of maintenance feeding at 75 kg and then feeding to appetite to reach 85 kg (Group 85RA). Other littermates were studied at the start of treatment (Group 75C) and after the initial period of feeding to appetite (Group 85A) or maintenance feeding (Group 75R). In the absence of differences in backfat depth or longissimus muscle area, Group 85RA gilts showed increased follicular development (P < .001) compared with their feed-restricted Group 85AR littermates. Plasma LH (P < .001) and FSH (P < .02) responses to an LHRH challenge differed between these same groups. Feed restriction reduced preprandial glucose, postprandial cortisol, and insulin (P < .05), total plasma IGF-I, and basal and postprandial free triiodothyronine (T3; all P < .001) at all stages of the experiment. Postprandial insulin, pre- (P < .001) and postprandial free T3, and plasma IGF-I (P < .001) increased in response to refeeding (Group 75R vs 85RA). These data indicate that changes in metabolic status can mediate short-term nutritional effects on reproductive function, in the absence of changes in age, weight, and widely used indices of body composition, and also establish the short-term, feed-restricted, prepubertal gilt as an effective model for studying the mechanisms mediating nutrition-reproduction interactions.

Endocrine and metabolic responses to realimentation in feed-restricted prepubertal gilts: associations among gonadotropins, metabolic hormones, glucose, and uteroovarian development.

Temporal relationships among plasma LH, FSH, metabolic hormones, and glucose concentrations were studied in nutritionally manipulated prepubertal gilts. Twenty-four growth-matched littermate gilts (73.6 +/- 1.2 kg) were fed at maintenance for 7 d (d 1 to 7). From d 8 to 14, littermates were divided between two treatments and were either fed twice daily to appetite or continued on feed restriction. Feeding to appetite increased episodic LH secretion during the first 6 h of sampling on d 8 (P < .05). Plasma FSH concentrations were not affected by treatment between d 8 and 14. Nocturnal increases in plasma LH (P < .01) and FSH (P < .002) concentrations were detected in feed-restricted gilts but not realimented gilts. Mean total plasma IGF-I concentrations gradually increased in gilts fed to appetite (d 12, P < .02; d 13 to 14, P < .001). Mean postprandial insulin concentrations were also greater in gilts fed to appetite (P < .01). Treatment differences in mean postprandial glucose concentrations on d 9 and 10 indicate that normoglycemia was established by d 9 in gilts fed to appetite. At slaughter on d 15, numbers of ovarian follicles (P < .01), follicular volume (P < .001), and uterine weight (P < .05) were greater in gilts fed to appetite. In conclusion, uteroovarian responses to realimentation after short-term feed restriction in prepubertal gilts are mediated by enhanced LH secretion. This rapid enhancement of LH secretion in response to dietary repletion might have been mediated by changes in glucose and insulin status. Ovarian responses to gonadotropins may also be potentiated by observed increases in plasma glucose, insulin, and IGF-I. During feed restriction, a distinct diurnal rhythm in gonadotropin secretion was evident.