A mutation in alpha-tropomyosin(slow) affects muscle strength, maturation and hypertrophy in a mouse model for nemaline myopathy.

Nemaline myopathy is a hereditary disease of skeletal muscle defined by a distinct pathology of electron-dense accumulations within the sarcomeric units called rods, muscle weakness and, in most cases, a slow oxidative (type 1) fiber predominance. We generated a transgenic mouse model to study this disorder by expressing an autosomal dominant mutant of alpha-tropomyosin(slow) previously identified in a human cohort. Rods were found in all muscles, but to varying extents which did not correlate with the amount of mutant protein present. In addition, a pathological feature not commonly associated with this disorder, cytoplasmic bodies, was found in the mouse and subsequently identified in human samples. Muscle weakness is a major feature of this disease and was examined with respect to fiber composition, degree of rod-containing fibers, fiber mechanics and fiber diameter. Hypertrophy of fast, glycolytic (type 2B) fibers was apparent at 2 months of age. Muscle weakness was apparent in mice at 5-6 months of age, mimicking the late onset observed in humans with this mutation. The late onset did not correlate with observed changes in fiber type and rod pathology. Rather, the onset of muscle weakness correlates with an age-related decrease in fiber diameter and suggests that early onset is prevented by hypertrophy of fast, glycolytic fibers. We suggest that the clinical phenotype is precipitated by a failure of the hypertrophy to persist and therefore compensate for muscle weakness.

Rat embryonic myoblasts are restricted to forming primary fibres while later myogenic populations are pluripotent.

Three populations of myoblasts, embryonic, foetal and adult, appear sequentially during myogenesis. The present study uses retroviruses to mark myoblasts clones in vivo from these populations. Myoblasts labelled at E15 (embryonic) contributed to primary fibres only. The majority of marked primary fibres were slow but a small number of clones contained marked primaries which were no longer slow at E19. Myoblasts labelled at E17 (foetal) fused with both primary and secondary fibres and most clones contained both fast and slow fibres. Similarly, adult myoblasts marked at P0 fused with all fibre types. These results indicate that embryonic myoblasts are restricted to producing only primary fibres which are initially slow but which can convert to being fast. Clones of foetal and adult myoblasts fuse with both primary and secondary fibres which may be either fast or slow.

The generation of fiber diversity during myogenesis.

Adult muscle is composed of different fiber types distinguished by their speed of contraction and metabolism. The generation of these differences is related both to the sequence in which muscle fibers form and to differences between the myogenic cells involved. Fibers form in two successive waves (primary and secondary) whose time of appearance can be correlated with the existence of successive populations of myogenic cells (embryonic and fetal). The differences between fibers arise through an interplay between heritable cellular commitment, where cells are preprogrammed to produce particular types of fiber and influences from the limb environment. The techniques of genetically marking cells and clonal analysis in vivo and in vitro are starting to reveal the relationship between these different influences. Although the process of myogenesis is similar in birds and mammals it is likely that cell autonomous behaviour plays a more important role during avian development as compared to mammals. The identification of muscle specific transcription factors has provided some clues to the mechanisms by which development is controlled but the expression of relatively few of these has been correlated with the sequence of events seen in myogenesis.

Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium.

The aim of this study was to investigate the effect of leukaemia inhibitory factor (LIF) on human blastocyst formation rates in vitro. To do this, it was first necessary to devise a complex serum-free medium (CSFM) in which to test its activity. Blastocyst formation rates in CSFM microdrops (18.4%) showed no difference to those obtained previously for embryos cultured in 1 ml T6 medium containing 10% serum (25%). The majority of blastocysts were of optimal blastocyst grade (BG1/BG2) as compared to the poor grade (BG3). The percentage of BG1/BG2 blastocysts was decreased in CSFM microdrops (10.2%) compared to that observed in T6 medium (24.8%). Addition of 1000 IU/ml LIF to CSFM microdrops increased the blastocyst formation rate from 18.4 to 43.6% (P = 0.025) and increased the percentage of BG1/BG2 blastocysts (33%; P = 0.025) to levels comparable with those observed in T6 medium. Thus LIF significantly increased the quality and number of human blastocysts formed in CSFM medium, increasing the potential for blastocyst transfer in human in-vitro fertilization (IVF).

Stimulation of endocytosis in mouse blastocysts by insulin: a quantitative morphological analysis.

The effects of insulin on the endocytic activity of mouse blastocysts in vitro were investigated using confocal laser scanning microscopy, quantitative image analysis and electron microscopy. Confocal studies showed that fluorescein isothiocyanate-labelled markers, dextran (fluid phase) and albumin (combined membrane and fluid phase), were endocytosed by blastocysts and localized within vesicles (about 2.5 microns in diameter) in the outer trophectoderm cells. No labelling was detected in the inner cell mass cells or the blastocoel cavity. Treatment with 170 nmol insulin l-1 stimulated the endocytosis of fluorescently labelled dextran in freshly collected blastocysts, increasing mean vesicle diameter per embryo by 15% (P < 0.05) after incubation with insulin for 2.5 h and mean vesicle number per embryo by 56% (P < 0.01) after 6 h. Both effects were also evident in blastocysts that had been cultured from the late eight-cell stage. Blastocysts incubated for 6 h with insulin displayed increased convolutions in the trophectoderm apical membrane compared with controls, indicating increased membrane activity and suggesting macropinosome formation. Collectively, these results suggest that insulin enhances endocytosis in the trophectoderm by stimulating uptake at the apical membrane into larger and more numerous endocytic vesicles and with some evidence of vesicle fusion. This mechanism may provide a metabolic basis for the stimulation by insulin of biosynthesis, proliferation and morphological development in early embryos.

Endocytosis in mouse blastocysts: characterization and quantification of the fluid phase component.

Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, 3H-dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy-requiring process, with only 20% accountable by diffusion as indicated by analysis at 4 degrees C. Insulin stimulated uptake of 3H-dextran by 30% (P < 0.05) over the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin-treated blastocysts contained 38% more 3H-dextran (38%; P < 0.01) than control blastocysts. Incubation of blastocysts in protein-free medium increased 3H-dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 +/- 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/l and 10 g BSA/l, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid-phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient-containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin regulates protein metabolism in mouse blastocysts.

Mouse blastocysts, in vitro, endocytosed 100 micrograms/ml 125I-labelled bovine serum albumin (BSA) at a rate equivalent to 192 +/- 27 microliters/hr/mg embryonic protein over the first 20 min. Insulin stimulated this initial uptake by 30% (P < 0.05). After this time, accumulation of 125I-labelled BSA began to plateau as the endocytosed 125I-labelled BSA was catabolized and 125I was released from the cells. Insulin caused an approximately 72% (P < 0.05) increase in the amount of uncatabolized 125I-labelled BSA remaining in insulin-treated blastocysts after 2 hr as compared to control blastocysts. Insulin partially inhibited catabolism of endocytosed 125I-labelled BSA during the first 2 hr following transfer to nonradioactive medium. After this time, degradation ceased in both control and insulin-treated blastocysts, leaving a small, uncatabolized protein pool remaining in the embryos; however, as a result of insulin's inhibitory effects on the initial catabolic rate, the uncatabolized protein pool was 30% (P < 0.05) larger in insulin-treated blastocysts after the 4 hr chase. Insulin inhibited endogenous protein degradation in blastocysts by 37% (P < 0.05). Combined with previous studies showing a 90% increase in endogenous protein synthesis in blastocysts following short-term stimulation with insulin (Harvey and Kaye, 1988), these results suggest that insulin acts to increase the endogenous protein reserves in the embryo. Dose-response studies indicated an EC50 of 0.5 pM for insulin's stimulation of 125I-labelled BSA accumulation, consistent with action via its own receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies to early pregnancy factor retard embryonic development in mice in vivo.

Previous work in this laboratory has shown that passive immunization of mice against early pregnancy factor (EPF) leads to failure to maintain pregnancy. The findings presented in this paper demonstrate that this treatment affects the development of the embryos very early in gestation. By Day 3, 54 and 25% of embryos in the 2 groups treated with anti-EPF immunoglobulin (Ig)G and IgM, respectively, had not developed to the 4-cell stage, compared with 12 and 1% in the control groups. None of the embryos in the mice treated with anti-EPF had developed beyond the 8-cell stage. A similar delay in development after treatment was observed on Day 4. The effect apparent during the early stages of cleavage is an indirect rather than a direct one, as 2-cell embryos (32-36 h post coitum), cultured in vitro in the presence of anti-EPF antibodies, developed uninterrupted to the morula and blastocyst stage. The delay in development did not appear to be caused by a disruption of the normal pattern of circulating progesterone, as progesterone concentrations on Day 4 were within the normal range for Quackenbush mice.

Autoradiographic studies on the effect of ethanol on muscarinic receptors in rat brain.

The effect of ethanol vapour inhalation on the binding characteristics of muscarinic receptors in rat brain has been studied using quantitative autoradiography. After 14 days in ethanol vapour there was increased binding of [(3)H]QNB to the cortex of 4- and 6-week-old animals, but not 11-week-old animals, and to the hippocampus of the 6-week-old animals. Displacement studies indicated that the effect of ethanol was due to an increase in the density of low-affinity carbachol binding sites. There was no effect on the binding of [(3)H]QNB in the presence of pirenzipine. The data strongly suggest that the increase in binding after ethanol exposure is due to an increase in M(1) sites in the younger animals possibly linked to age-related plasticity of the nervous system.