Sperm aster in rabbit zygotes: its structure and function

Experimental neurosis was achieved in fifteen rabbits (both fixed on a stand and unrestrained) by sudden, drastic enhancement of a pain electrocutaneous stimulation used as reinforcement, at the stage of a stable elaborated avoidance reflex. The experimental neurosis was characterized by a prolonged (one to two months) disturbance of conditioned activity, greater motor restlessness, predominance of the fear type reactions, the animals' reduced weight, higher pulse and respiratory rate. Dynamics of the respiratory rate and brain bioelectrical activity became chaotic. Simultaneously, bioelectrical activity revealed greater similarity of the processes in the cortex and subcortical structures at one and the same moment. Analysis of the electrogram frequency spectra of different brain formations, pointed to an enhancement in all frequency ranges (with the exception of gamma), with a clear predominance of the 6 to 10 c/s band which was most distinctly expressed in the amygdaloid structures.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.     

F-actin marks the rhizoid pole in living Pelvetia compressa zygotes

Most chicken strains are highly susceptible to avian leukosis virus (ALV) induction of bursal lymphoma, involving proviral integration within the c-myc proto-oncogene, while certain strains are genetically resistant to lymphomagenesis. A nested PCR assay was developed to analyse the appearance of proviral c-myc integrations after ALV infection of lymphoma-susceptible birds, and to determine whether these integrations arise in lymphoma-resistant birds. Proviral c-myc integrations are detected in bursa and other tissues from 6 day-old lymphoma-susceptible birds infected as embryos. The abundance of bursal cells carrying these integrations increases roughly 40-fold by 35 days of age, indicating that these cells hyperproliferate within the bursal environment. Bursal cells with proviral c-myc integrations also arise soon after infection of lymphoma-resistant embryos. However, these cells expand much more slowly than cells from lymphoma-susceptible birds. Both strains show the same rate of viral infection, so that resistance to lymphomagenesis occurs at a step subsequent to proviral c-myc integration. Proviral c-erbB gene integrations arise at the same frequency in bursa and other tissues of both strains, and they do not increase in abundance during development. These findings indicate that the mechanism of resistance to lymphomagenesis involves specific inhibition of cells with proviral c-myc integrations within the bursa.     

Formation and localization of cytoplasmic domains in leech and ascidian zygotes

Leech and ascidian embryos are well suited for the study of certain developmental processes. Although leeches and ascidians belong to different bilateralia groups (protostomes and deuterostomes, respectively) they share important developmental features and, in particular, the determinate character of their embryogenesis. In both types of embryos this property is related to the presence of specific cytoplasmic domains that are selectively allocated to different blastomeres during cleavage. In this review leech and ascidian eggs and zygotes are compared in terms of the structure of these cytoplasmic domains and of the cellular mechanisms involved in their formation and localization. During meiosis the zygote of leeches and ascidians undergo stereotypic actin-dependent contraction movements related to both the emission of the polar bodies and the formation and relocalization of cytoplasmic domains. After completion of meiosis, during first interphase, monaster microtubules nucleated from the sperm-derived centrosome play a key role in pronuclear migration. In addition, these astral microtubules direct the relocalization of cytoplasmic domains and the translocation and accumulation of organelles in the interior of the zygote. Microtubules and microfilaments, on the other hand, are involved in cortical reorganizations and organelle translocations in both zygote species during interphase and cleavage divisions. In the case of leech zygotes, this process leads to formation of characteristic polar cytoplasmic domains called teloplasms. These domains are selectively inherited by teloblasts, precursor stem cells of ectodermal and mesodermal tissues in the leech embryo. In the ascidian zygote, the cytoplasmic movements observed during interphase and mitosis lead to relocalization of the bulk of a mitochondria-rich domain, called the myoplasm, along with an endoplasmic reticulum-rich domain towards the future posterior pole of the embryo. The myoplasm is inherited by a subset of posterior blastomeres committed to become the primary muscle cells of the ascidian tadpole.     

Low energy charge and high adenosine content in smooth muscle of human bladder in comparison with striated muscle

This study determined the energy charge, adenosine and inosine content of human bladder smooth muscle in comparison with striated muscle of the same individual. Biopsies were obtained from 21 women who were subjected to urethrocystopexy because of urinary stress incontinence. We found that the ATP content of bladder smooth muscle was only about one-eighth of that of striated muscle. The energy charge of bladder smooth muscle was 0.78 +/- 0.13, which is low compared with striated muscle (0.92 +/- 0.02). The adenosine content of bladder smooth muscle was 6.7 times higher than striated muscle and the adenosine/ATP ratio was 1:9 compared with 1:450 for striated muscle. These findings were in accordance with our previous studies on uterine smooth muscle.     

Histochemistry of some acid hydrolases in striated muscles of the rat

Endoscopic sphincterotomy of the ampulla of Vater was performed in seven patients suffering from gallstones in the common bile duct and/or bile flow stasis. After this procedure, spontaneous delivery of gallstones occurred in two cases and a gallstone was extracted by our basket-tipped catheter in two cases. The electromyogram of the sphincter of Oddi after endoscopic sphincterotomy showed the similar patterns and rhythms to those before this procedure. Thus, the function (bile flow mechanism) of the sphincter was not destroyed by this procedure. Morphologic and physiologic data, therefore, lead to the conclusion that endoscopic sphincterotomy is a safe and useful procedure.     

Interstitial photodynamic therapy with tetra(m-hydroxyphenyl)chlorin: tumor versus striated muscle damage

In this study, we have investigated the changes in the number of individual presynaptic boutons in the neocortex of rats and correlated them with cognitive performance. Brown Norway x Fischer 344 F1 hybrid rats, aged from one to 24 months, were used. Using synaptophysin as a marker for presynaptic boutons, we found that in the parietal II region of the neocortex an age-related decrease in the density of immunostained punctae representing presynaptic boutons occurred. Regression analysis showed that this decline in the number of presynaptic boutons correlates with ageing (r=0.495, P<0.05). Interestingly, we found that this age-related depletion of presynaptic boutons was more intense in the deeper cortical lamina, such as laminae V and VI (mean decrease of 18%), than in the superficial laminae (mean decrease of 8% in laminae I-IV). Using the Morris water maze test, we observed that young rats acquired the task at twice the speed of aged animals (48.9 +/- 9.0 s and 91.0 +/- 4.9 s for young and aged animals, respectively). Furthermore, at the end of the training period, the aged cohort still showed significantly higher escape latencies in the Morris water maze. The present findings support the concept that the decline in cognitive performances in ageing is related to the loss of synapses in the cerebral cortex.     

Post-traumatic reconnection of the cervical spinal cord with skeletal striated muscles. Study in adult rats and marmosets

In an attempt at repairing the injured spinal cord of adult mammals (rat, dog and marmoset) and its damaged muscular connections, we are currently using: 1) peripheral nerve autografts (PNG), containing Schwann cells, to trigger and direct axonal regrowth from host and/or transplanted motoneurons towards denervated muscular targets; 2) foetal spinal cord transplants to replace lost neurons. In adult rats and marmosets, a PNG bridge was used to joint the injured cervical spinal cord to a denervated skeletal muscle (longissimus atlantis [rat] or biceps brachii [rat and marmoset]). The spinal lesion was obtained by the implantation procedure of the PNG. After a post-operative delay ranging from 2 to 22 months, the animals were checked electrophysiologically for functional muscular reconnection and processed for a morphological study including retrograde axonal tracing (HRP, Fast Blue, True Blue), histochemistry (AChE, ATPase), immunocytochemistry (ChAT) and EM. It was thus demonstrated that host motoneurons of the cervical enlargement could extend axons all the way through the PNG bridge as: a) in anaesthetized animals, contraction of the reconnected muscle could be obtained by electrical stimulation of the grafted nerve; b) the retrograde axonal tracing studies indicated that a great number of host cervical neurons extended axons into the PNG bridge up to the muscle; c) many of them were assumed to be motoneurons (double labelling with True Blue and an antibody against ChAT); and even alpha-motoneurons (type C axosomatic synapses in HRP labelled neurons seen in EM in the rat); d) numerous ectopic endplates were seen around the intramuscular tip of the PNG. In larger (cavitation) spinal lesions (rat), foetal motoneurons contained in E14 spinal cord transplants could similarly grow axons through PNG bridges up to the reconnected muscle. Taking all these data into account, it can be concluded that neural transplants are interesting tools for evaluating both the plasticity and the repair capacities of the mammalian spinal cord and of its muscular connections.     

Facial alopecia in the rabbit associated with Cheyletiella parasitivorax

A facial dermatitis characterized by alopecia over the frontal area with varying degrees of erythema and scale formation was observed in 46 newly arrived New Zealand White rabbits within a 5-month period. The clinical history, nature of the lesion, and laboratory findings indicated that the mite Cheyletiella parasitivorax was the most likely cause of the dermatitis.     

Degeneration and regeneration of striated skeletal muscle fibers in Duchenne muscular dystrophy

Like all other muscular dystrophies, Duchenne muscular dystrophy is characterized by the coexistence of degenerative lesions of the muscle fibers and of regenerative changes. The present study has been carried out in order to precise the degree of regeneration at different stages of the disease, by analyzing the expression of several markers of cell proliferation and of muscular differentiation. In the two affected foetuses of our series, the m. quadriceps is histologically normal, except for the absent expression of immunoreactive dystrophin. The quadriceps from the eight children of our series (20 months-16 years) all present clear dystrophic changes. Muscle regeneration is characterized by activation of the satellite cells, by their multiplication followed by their fusion giving birth to regenerative fibers. By studying the expression of muscular markers (vimentin, desmin, isoforms of the myosin heavy chains), it has been possible to define more precisely the degree of maturation and of differentiation of these regenerative fibers. Our results suggest that an abortive regeneration of the muscle fibers in Duchenne muscular dystrophy can explain, at least partly, the progressive evolution of this disease.     

Rapid chromosome detection in human gametes, zygotes, and preimplantation embryos using the PRINS technique

PURPOSE: The analysis of the chromosomal constitution of human gametes and embryos is of particular importance for investigation of aneuploidy occurrence and diagnostic purposes. The PRINS method constitutes an alternative to FISH for in situ chromosomal identification. We have adapted this method to human gametes, zygotes, and preimplantation embryos. RESULTS: Chromosome-specific labeling was obtained in gametes, zygotes, and isolated blastomeres. Simultaneous detection of two or three chromosomes can be completed in less than 3 hr using fluorochrome-labeled nucleotides. CONCLUSIONS: The PRINS technique appears to be more efficient than FISH for detection and discrimination of alpha-satellite DNA sequences. The present study demonstrates the usefulness of PRINS for chromosomal screening and preimplantation diagnosis.     

Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro

Due to the complicated media used for culturing bovine embryos, most of the nutrient requirements are unknown. Recently, we developed a simple, serum-free medium (CR1) that allows bovine embryos to develop in vitro. Therefore, our objective was to determine whether development of bovine embryos would be improved by the addition of free amino acids and vitamins to CR1. Oocytes were recovered from slaughterhouse ovaries and matured 22 +/- 2 h, following which the oocytes were randomly allotted to treatment. The experiment was a randomized block design with a 2 x 5 factorial treatment structure. The oocytes were fertilized with or without cumulus cells intact. The five fertilization media were 1) Control (CR1 +/- 10 micrograms/mL of phenol red); 2) control + basal medium Eagle (BME) essential amino acids (EAA) + minimum essential medium (MEM) nonessential amino acids (NEA) + MEM vitamins (VIT); 3) control + EAA + NEA; 4) control + EAA + VIT; and 5) control + NEA + VIT. Cleavage rate was greater (P < .001) when cumulus cells remained on the oocytes during fertilization (51.7 vs 73.2% without and with cumulus cells, respectively). The frequency of blastocysts was increased (P < .001) when EAA or NEA were added to CR1; however, adding VIT had no effect or tended (P = .12) to decrease the frequency of embryos attaining the blastocyst stage. This experiment demonstrates that development of bovine embryos in vitro can be improved by the addition of free amino acids to a simple medium. Contrary to work in rodents, the mixture of vitamins in MEM was not beneficial for bovine embryos.     

Genotoxicity of trophosphamide in mouse germ cells: assessment of micronuclei in spermatids and chromosome aberrations in one-cell zygotes

We have used the muscle history dependence of the sensitivity of muscle spindles to stretch, to provide evidence for their contribution to kinaesthesia, the sense of position and movement. Stretch sensitivity is altered depending on whether or not slack has been introduced in intrafusal fibres [13]. At the human elbow joint detection threshold was measured to passive movements applied at different speeds to the forearm after a conditioning co-contraction of muscles of the upper arm, with the arm held either flexed ('hold short') or extended ('hold long'). Test measurements were made with the elbow joint at 90 degrees. For the three speeds of movement, 2 degrees s-1, 0.2 degree s-1 and 0.02 degree s-1, after 'hold short' conditioning thresholds were lower for movements into extension, after 'hold long' conditioning they were lower for movements into flexion. It is concluded that when muscle conditioning introduces slack in the intrafusal fibres of muscle spindles, this must be taken up by the test movements before they can be detected by the subject. It means that whenever detection thresholds to passive movements are measured at a joint, the contraction history of the muscles acting at that joint must be taken into account.     

A mating type-linked gene cluster expressed in Chlamydomonas zygotes participates in the uniparental inheritance of the chloroplast genome

A characteristic feature of early zygote development in Chlamydomonas is the selective degradation of chloroplast DNA from the mating type minus parent. The zygote-specific gene cluster ezy-1 is linked to the mating type locus and is transcribed almost immediately upon zygote formation. We show here that the acidic Ezy-1 polypeptide is rapidly transported to both the plus and minus chloroplasts, where it interacts with each chloroplast nucleoid. Expression of ezy-1 is selectively inhibited when plus, but not minus, gametes are briefly ultraviolet irradiated just prior to mating, a treatment known to disrupt the uniparental inheritance of chloroplast traits. We propose that the Ezy-1 polypeptide participates in the destruction of the minus chloroplast DNA in zygotes and thus the uniparental inheritance of chloroplast traits. The ezy-1 gene represents a valuable molecular probe for dissecting mechanisms underlying organelle inheritance.     

Oxygenation in the rabbit myocardium: assessment with susceptibility-dependent MR imaging

PURPOSE: To determine the feasibility of using hemoglobin (Hb) desaturation as an indicator of myocardial oxygenation. MATERIALS AND METHODS: High-resolution gradient-echo nuclear magnetic resonance (MR) images of isolated, blood-perfused rabbit hearts were obtained at various blood oxygenation levels. The hearts were perfused at 37 degrees C with a Langendorff apparatus modified for nuclear MR imaging. The perfusate contained bovine red blood cells in a cardioplegic solution that eliminated motion artifacts and minimized arteriovenous oxygenation differences. Hb saturation was varied (7%-100%) randomly. Perfusion pressure was continuously monitored, and blood samples were obtained. RESULTS: There was a substantial correlation between image signal intensity in the myocardium and Hb saturation in the blood, believed to be due to susceptibility effects of the paramagnetic species deoxyhemoglobin. CONCLUSION: Direct and noninvasive determination of regional Hb saturation with susceptibility-dependent MR imaging may provide information regarding regional myocardial O2 content.     

Specific chromosome changes associated with rabbit cell lines cultured in vitro

Thirty-three rabbit cell lines were established from various fetal tissues of the inbred strain III of the New Zealand rabbit (Oryctolagus cuniculus). None of these lines exhibited senescence during a growth period of more than 2 years. Karyologic studies of most cell lines at 10 to 20 cell-passage intervals revealed that the karyotype stability of the rabbit cells in vitro was correlated with the organs from which the cell lines were derived. Thus, lines derived from cornea, spleen, and kidney tissues usually contained high frequencies of polyploidy in their early passages, whereas most of those derived from lung and skin were found to retain the normal diploid karyotype for much longer periods of time. One line derived from fetal lung tissue, designated Lung 16, remained diploid up to 100 passages. In late passages of the majority of all the lines studied, the cells became pseudodiploid, hyperdiploid, or polyploid. Among the pseudodiploid and the hyperdiploid cell lines, the chromosomal changes followed three basic patterns: (1) a gain of one or more telocentric chromosomes; (2) a loss of one telocentric chromosome plus a metacentric marker chromosome (M); or (3) a gain of a long telocentric marker chromosome with or without changes in the number of telocentric D chromosomes. By the G-banding technique, the telocentric chromosome involved in these three patterns was identified as the D-group chromosome 18 and the M marker chromosome as an isochromosome of 18. These results suggest that chromosomal rearrangement in rabbit cells involving trisomy of 18 may be responsible for the longevity of these cell lines cultured in vitro.     

Pathways linking L-phenylalanine and 2-phenylethylamine with p-tyramine in rabbit brain

Concentrations of labeled p-tyramine and 2-phenylethylamine were measured in rabbit brain 10 min after the intraventricular administration of either radioactive amine. In both cases the recoveries of the newly synthesized amine and of the unchanged precursor were significantly increased in animals pretreated with the monoamine oxidase inhibitor pargyline. Significant amounts of both amines were present in rabbit brain 10 min after the intraventricular injection of labeled L-phenylalanine. Pretreatment with pargyline increased their recoveries, whereas alpha-methyldopa (an L-aromatic amino acid decarboxylase enzyme inhibitor, L-AAADI) decreased them considerably, and no significant alteration was found in L-alpha-methyldopa hydrazine (a peripheral L-AAADI) pretreated animals. These results provide evidence for a new biochemical pathway in brain for p-tyramine biosynthesis, with L-phenylalanine (bypassing the formation of L-tyrosine) or 2-phenylethylamine as precursors. The significance and implications of these metabolic routes are discussed, especially considering that p-tyramine itself can be converted to catecholamines.     

Inhibition of white blood cell adhesion at reperfusion decreases tissue damage in postischemic striated muscle

By correlating data obtained from environmental indicators and health indicators over a five-year period, a system for measuring the effects of air pollution on health has been established and tested. Rises in air pollution levels do appear to be accompanied by increases in the relevant health problems.