Deep freezing of mouse one-cell embryos and oocytes using different cryoprotectants

The objective of this study was to compare iso-osmolar concentrations (1.5 M) of 1,2-propanediol, glycerol, dimethylsulphoxide and a combination of 1 M propanediol + 0.5M glycerol (PDGLY) as cryoprotectants for murine ovulated oocytes and one-cell embryos. A higher (P < 0.01) percentage of one-cell embryos developed to the two-cell stage when frozen-thawed with 1,2-propanediol (83%) as compared with glycerol (43%), dimethylsulfoxide (51%) or PDGLY (7%). Data recalculated on the basis of two-cell embryos/number of normal one-cell embryos after thawing indicated no differences among single cryoprotectant groups. More (P < 0.01) frozen-thawed, in-vitro fertilized oocytes developed to the two-cell stage when 1,2-propanediol (35%) was used as cryoprotectant as compared with glycerol (15%). Freezing-thawing resulted in a reduced number of two-cell embryos after oocytes were fertilized in-vitro as compared with fresh oocytes. 1,2-propanediol was a better cryoprotectant than glycerol, dimethylsulphoxide or PDGLY for deep freezing of murine oocytes or one-cell embryos.     

Minimal nutrient requirements for culture of one-cell rabbit embryos

The minimal nutrient requirements of one-cell rabbit embryos for cleavage during in vitro culture were investigated. One-cell rabbit embryos were cultivated in a simple salt solution supplemented with the macromolecule polyvinylalcohol (PVA) either alone or with bovine serum albumin (BSA), amino acids, or one of a number of potential energy sources. At the end of 48 h culture, the embryos were stained with aceto-orcein HCl and the number of nucleated cells per embryo counted. One-cell embryos in medium with PVA but without an exogenous, fixed nitrogen source or energy substrate cleaved to a mean of 10.4 cells per embryo. Addition of the putative energy substrates--phosphoenolpyruvate, malate, acetate, and lactate--resulted in nonsignificant increases in cleavage rate. Glucose, pyruvate, a group of 20 amino acids from Ham's F-10 medium, and BSA gave a statistically significant doubling of the cleavage rate. These results indicate that the one-cell rabbit embryo, unlike the mouse embryo, has significant endogenous energy sources and that an exogenous, fixed nitrogen source is not essential for cleavage.     

Long-term and transgenerational effects of cryopreservation on rabbit embryos

The short-term effects of cryopreservation and embryo transfer are well documented (reduced embryo viability, changes in pattern expression), but little is known about their long-term effects. We examined the possibility that embryo vitrification and transfer in rabbit could have an impact on the long-term reproductive physiology of the offspring and whether these phenotypes could be transferred to the progeny. Vitrified rabbit embryos were warmed and transferred to recipient females (F0). The offspring of the F0 generation were the F1 generation (cryopreserved animals). Females from F1 generation offspring were bred to F1 males to generate an F2 generation. In addition, two counterpart groups of noncryopreserved animals were bred and housed simultaneously to F1 and F2 generations (CF1 and CF2, respectively). The reproductive traits studied in all studied groups were litter size (LS), number born alive at birth (BA), and postnatal survival at Day 28 (number of weaned/number born alive expressed as percentage). The reproductive traits were analyzed using Bayesian methodology. Features of the estimated marginal posterior distributions of the differences between F1 and their counterparts (F1 − CF1) and between F2 and their counterparts (F2 − CF2) in reproductive characters found that vitrification and transfer procedures cause a consistent increase in LS and BA between F1 and CF1 females (more than 1.4 kits in LS and more than 1.3 BA) and also between F2 and CF2 females (0.96 kits in LS and 0.94 BA). We concluded that embryo cryopreservation and transfer procedures have long-term effects on derived female reproduction (F1 females) and transgenerational effects on female F1 offspring (F2 females).     

Dynamics of actin filaments during tension-dependent formation of actin bundles

The actin cytoskeleton stress fiber is an actomyosin-based contractile structure seen as a bundle of actin filaments. Although tension development in a cell is believed to regulate stress fiber formation, little is known for the underlying biophysical mechanisms. To address this question, we examined the effects of tension on the behaviors of individual actin filaments during stress fiber (actin bundle) formation using cytosol-free semi-intact fibroblast cells that were pre-treated with the Rho kinase inhibitor Y-27632 to disassemble stress fibers into a meshwork of actin filaments. These filaments were sparsely labeled with quantum dots for live tracking of their motions. When ATP and Ca(2+) were applied to the semi-intact cells to generate actomyosin-based forces, actin meshwork in the protruded lamellae was dragged toward the cell body, while the periphery of the meshwork remained in the original region, indicating that centripetally directed tension developed in the meshwork. Then the individual actin filaments in the meshwork moved towards the cell body accompanied with sudden changes in the direction of their movements, finally forming actin bundles along the direction of tension. Dragging the meshwork by externally applied mechanical forces also exerted essentially the same effects. These results suggest the existence of tension-dependent remodeling of cross-links within the meshwork during the rearrangement of actin filaments, thus demonstrating that tension is a key player to regulate the dynamics of individual actin filaments that leads to actin bundle formation.     

Bending flexibility of actin filaments during motor-induced sliding

Muscle contraction and other forms of cell motility occur as a result of cyclic interactions between myosin molecules and actin filaments. Force generation is generally attributed to ATP-driven structural changes in myosin, whereas a passive role is ascribed to actin. However, some results challenge this view, predicting structural changes in actin during motor activity, e.g., when the actin filaments slide on a myosin-coated surface in vitro. Here, we analyzed statistical properties of the sliding filament paths, allowing us to detect changes of this type. It is interesting to note that evidence for substantial structural changes that led to increased bending flexibility of the filaments was found in phalloidin-stabilized, but not in phalloidin-free, actin filaments. The results are in accordance with the idea that a high-flexibility structural state of actin is a prerequisite for force production, but not the idea that a low-to-high flexibility transition of the actin filament should be an important component of the force-generating step per se. Finally, our data challenge the general view that phalloidin-stabilized filaments behave as native actin filaments in their interaction with myosin. This has important implications, since phalloidin stabilization is a routine procedure in most studies of actomyosin function.     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

Effects of low molecular weight oviductal factors on the development of mouse one-cell embryos in vitro.

The relationship between the oviduct and embryo development in the mouse was investigated and the period at which the influence of oviduct can be concerned in the development of mouse embryos in vitro was identified. In addition, the relative molecular weight of oviductal factors that promote embryo development was demonstrated. Mouse zygotes developed to the blastocyst stage when co-cultured with ampulla. The period of embryo co-culture significantly affected the further development of the embryos. Fewer one-cell embryos co-cultured with dissected ampullae for less than 24 h developed to blastocysts than those co-cultured for more than 28 h (P < 0.001). A high percentage of embryos co-cultured with ampullae after 24 h of culture in vitro developed to the blastocyst stage, which suggests that the influences of ampulla on the development of mouse embryos are restricted to a specific period at the two-cell stage (about 55-56 h after hCG injection) in vitro. Mouse ova that were cultured in media conditioned by ampullae could also develop to the blastocyst stage. The fractionated medium that contained low molecular weight fractions was more effective (P < 0.001) on the development of embryos to the blastocyst stage than that containing high molecular weight fractions. These results suggest that the low molecular weight oviductal factors play an important role in the development of mouse embryos at a certain critical age in vitro.     

Factors influencing renal cryopreservation. I. Effects of three vehicle solutions and the permeation kinetics of three cryoprotectants assessed with rabbit cortical slices

A renal cortical slice model was used to assess the effects on viability of three vehicle solutions-Krebs-Henseleit (K-H), solution A, and RPS-2--at 25 degrees C. After 120 min incubation no differences in [K+]/[Na+] ratios were found. Tracer techniques were used to study the osmotic effects and permeation kinetics at 25 degrees C of three cryoprotectants (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) and the effect of the vehicle solution (K-H or RPS-2) on Me2SO kinetics. It was found that Me2SO was most permeable and ethylene glycol least, and that ethylene glycol had unusual effects which suggest that it may not act as a simple solute. Differences were found when Me2SO was introduced in K-H and RPS-2 that are believed to be related to the binding properties of Me2SO to cell constituents.     

Some perspectives on the viscosity of actin filaments

Measurements of the dynamic viscosity of various actin filament preparations under conditions of low and controlled shear: (a) confirm the shear rate dependence of F-actin viscosities and show that this dependence obeys the power law relationship observed for entangled synthetic polymers; (b) permit estimation of the extent to which shear artifact amplifies changes in the apparent viscosity of F-actin measured in a falling ball viscometer; (c) show that gel-filtration chromatography of actin and the addition of cytochalasin B to F-actin bring about small (20-40%) changes in the viscosity of the F-actin solutions. These variations are consistent with alterations in the actin-binding protein concentrations required for incipient gelation, a parameter inversely related to average filament length. Therefore: (a) changes in the viscosity of F-actin can be magnified by use of the falling ball viscometer, and may exaggerate their biological importance; (b) chromatography of actin may not be required to obtain meaningful information about the rheology of actin filaments; (c) changes in actin filament length can satisfactorily explain alterations in F-actin viscosity exerted by cytochalasin B and by chromatography, obviating the need to postulate specific interfilament interactions.     

ADP-ribosylated actin caps the barbed ends of actin filaments

The mode of action on actin polymerization of skeletal muscle actin ADP-ribosylated on arginine 177 by perfringens iota toxin was investigated. ADP-ribosylated actin decreased the rate of nucleated actin polymerization at substoichiometric ratios of ADP-ribosylated actin to monomeric actin. ADP-ribosylated actin did not tend to copolymerize with actin. Actin filaments were depolymerized by the addition of ADP-ribosylated actin. The maximal monomer concentration reached by addition of ADP-ribosylated actin was similar to the critical concentration of the pointed ends of actin filaments. ADP-ribosylated actin had no effect on the rate of polymerization of gelsolin-capped actin filaments which polymerize at the pointed ends. The results suggest that ADP-ribosylated actin acts as a capping protein which binds to the barbed ends of actin filaments to inhibit polymerization. Based on an analysis of the depolymerizing effect of ADP-ribosylated actin, the equilibrium constant for binding of ADP-ribosylated actin to the barbed ends of actin filaments was determined to be about 10(8) M-1. As actin is ADP-ribosylated by perfringens iota toxin and by botulinum C2 toxin, it appears that conversion of actin into a capping protein by ADP-ribosylation is a pathophysiological reaction catalyzed by bacterial toxins which ultimately leads to inhibition of actin assembly.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Preliminary characterization of pyruvate uptake by one-cell ovine embryos

Pyruvate uptake was measured by a noninvasive method on a total of 59 individual one-cell ovine embryos. Uptake was measured over a 4-h period in Whitten's medium containing 1.0 mM glucose and either 0.0 mM, 0.33 mM, 0.67 mM or 1.0 mM sodium pyruvate. Glucose uptake was also assessed to determine the effect of altering pyruvate concentration on glucose uptake. Embryos incubated in medium containing 1.0 mM or 0.67 mM pyruvate showed average uptake values of 20.36 and 5.00 pmoles per embryo per 4 h, respectively. In contrast, when pyruvate concentrations were 0.0 mM and 0.33 mM, the embryos released an average of 24.60 and 10.40 pmoles pyruvate per embryo per 4 h. Uptake or release of pyruvate was found to vary linearly with concentration level. Increasing pyruvate concentration resulted in a significant decrease in glucose uptake.     

Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments

We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1 microM CB and 0.02 microM CD for ATP-actin and at 0.1 microM CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2 microM) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem. 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Understanding the cooperative interaction between myosin II and actin cross-linkers mediated by actin filaments during mechanosensation

Myosin II is a central mechanoenzyme in a wide range of cellular morphogenic processes. Its cellular localization is dependent not only on signal transduction pathways, but also on mechanical stress. We suggest that this stress-dependent distribution is the result of both the force-dependent binding to actin filaments and cooperative interactions between bound myosin heads. By assuming that the binding of myosin heads induces and/or stabilizes local conformational changes in the actin filaments that enhances myosin II binding locally, we successfully simulate the cooperative binding of myosin to actin observed experimentally. In addition, we can interpret the cooperative interactions between myosin and actin cross-linking proteins observed in cellular mechanosensation, provided that a similar mechanism operates among different proteins. Finally, we present a model that couples cooperative interactions to the assembly dynamics of myosin bipolar thick filaments and that accounts for the transient behaviors of the myosin II accumulation during mechanosensation. This mechanism is likely to be general for a range of myosin II-dependent cellular mechanosensory processes.