Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells.

Late Embryogenesis Abundant (LEA) proteins are commonly found in plants and other organisms capable of undergoing severe and reversible dehydration, a phenomenon termed "anhydrobiosis". Here, we have produced a tagged version for three different LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, binds to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein group 3 that targets the mitochondria. Sheep fibroblasts transfected with single or all three LEA proteins were subjected to air drying under controlled conditions. After rehydration, cell viability and functionality of the membrane/mitochondria were assessed. After 4 h of air drying, cells from the un-transfected control group were almost completely nonviable (1% cell alive), while cells expressing LEA proteins showed high viability (more than 30%), with the highest viability (58%) observed in fibroblasts expressing all three LEA proteins. Growth rate was markedly compromised in control cells, while LEA-expressing cells proliferated at a rate comparable to non-air-dried cells. Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming the protection conferred by LEA proteins on these organelles during dehydration stress. This is likely to be an effective strategy when aiming to confer desiccation tolerance to mammalian cells.

High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation.

BACKGROUND: The process of implantation is mediated by various molecules, one of which is anandamide (AEA), a lipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by binding to the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whether the endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s) AEA had on the development of sheep blastocysts in vitro. METHODS: We analysed the expression and activity of the endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were then exposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and 5-bromo-deoxyuridine incorporation. RESULTS: We show that the AEA signalling system is present in sheep and that high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism. Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonist SR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normal growth rate upon AEA removal from the medium. CONCLUSIONS: Our results suggest that disturbed regulation of AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.

Selective deafferentation of hand cutaneous territory is followed by changes in fibre type distribution of a forearm muscle in the horse.

Based on previous observations that capsaicin can selectively damage group III and IV afferents and induce muscle fibre transformation, we hypothesized that eliminating, by means of capsaicin, the group III and IV afferents of a peripheral territory it could lead to a fibre transformation in a muscle involved in the flexor reflexes of the same peripheral territory. Therefore, capsaicin was injected into the palmar nerves of the forelimb of the horse to investigate if eliminating group III and IV afferents from the hand of the horse a muscle fibre transition would occur in the flexor carpi radialis (FCR) muscle, which is involved in the flexor reflexes of the finger itself. 120 days after capsaicin injection, type I slow fibres increased and type IIA fast fibres decreased. We presume that the long lasting deafferentation of the ergo-nociceptive fibres causes a plastic remodelling in the central nervous system and indirectly influences the motoneuron excitability via short or long loop-pathways enhancing their tonic discharge.

Follicle activation involves vascular endothelial growth factor production and increased blood vessel extension.

The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4-5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% +/- 0.58% vs. 1.29% +/- 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150-300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.

Nerve fiber composition of the intracranial portion of the oculomotor, trochlear, and abducens nerves in the sheep.

In the present investigation, the fiber content and the diameter spectra of the intracranial portion of the three oculomotor nerves (oculomotor, trochlear, and abducens nerves) were analysed in sheep by light and electron microscopy. It was determined that up to 14.98% of fibers in the oculomotor nerve, 17.01% in the trochlear nerve, and 11.87% in the abducens nerve were unmyelinated. The myelinated fibers showed a bimodal distribution in their size spectrum in all three nerves, with a majority of large myelinated axons, but a considerable proportion of small myelinated fibers, as well. The sensory function of the unmyelinated fibers present in the three oculomotor nerves is discussed also on the basis of our previous morphofunctional investigations.

Mesencephalic trigeminal nucleus neurons supplying the jaw closing muscles have no spinal projection: a fluorescent double-labeling study in birds and mammals.

BACKGROUND: The present study deals with the possibility that the mesencephalic trigeminal nucleus (MeV) neurons that innervate the muscle spindles of the jaw closing muscles may also have collaterals projecting to the cervical spinal cord. At the same time, we reexamine the morphology of these cells and their location within the MeV. METHODS: The fluorescent retrograde tracers Fast Blue (FB) and Diamidino Yellow dihydrochloride (DY) were injected into the jaw closing muscles and C2-C3 spinal cord segments, respectively, of duck, rat, and rabbit in one series of experiments. In a second series of animals, the targets of the tracers were reversed. RESULTS: Retrogradely double-labeled cells (FB+DY) were not found in the MeV. On the contrary, the tracer injected into the muscles retrogradely labeled only large unipolar MeV cells, whereas the tracer injected into C2-C3 spinal cord segments labeled only small multipolar cells which were intermingled with the MeV somata of muscle spindle afferents. CONCLUSIONS: These findings exclude the possibility of spinal projections via collaterals of MeV cells supplying muscle spindles of jaw closing muscles in duck, rat, and rabbit. Moreover, the retrograde double-labeling technique evidences two cellular populations within the MeV of the duck, rat, and rabbit: large unipolar neurons which are the cell bodies of primary afferents from jaw closing muscles and small multipolar cells projecting to the upper cervical spinal cord.

Differentiation and growth of muscle in the fish Sparus aurata (L): I. Myosin expression and organization of fibre types in lateral muscle from hatching to adult.

Post-hatching development of lateral muscle in a teleost fish, Sparus aurata (L) was examined. At hatching only two fibre types were present, several layers of mitochondria-poor, myofibril-rich deep muscle fibres surrounded the notochord and were covered by a superficial monolayer of mitochondria-rich, myofibril-poor A third ultrastructurally distinct fibre type first appeared as one or two fibres located just under the lateral line at 6 days post-hatching. This type, which gradually increased in number during larval life, contained a slow isoform of myosin, identified by mATPase staining and immunostaining with myosin isoform-specific antibodies. Deep muscle fibres--the presumptive fast-white type--contained a fast myosin, and superficial monolayer fibres an isoform similar but not identical to that in adult pink muscle fibres. The only fibres present during larval life which showed a clear change in myosin expression were the superficial monolayer fibres, which gradually transformed into the slow type post-larvally. Pink muscle fibres first appeared near the end of larval life. Both slow and pink muscle fibres remained concentrated around the horizontal septum under the lateral line during larval life, expanding outwards towards the apices of the myotomes only after metamorphosis. Between 60 and 90 days very small diameter fibres with a distinct mATPase profile appeared scattered throughout the deep, fast-white muscle layer, giving it a 'mosaic' appearance, which persisted into adult life. A marked expansion in the slow muscle layer began at the same time, partly by transformation of superficial monolayer fibres, but mainly by addition of new fibres both on the deep surface of the superficial monolayer and close to the lateral line. The order of appearance of these fibre types, their myosin composition, and the significance of the superficial monolayer layer are discussed and compared to muscle fibre type development in higher vertebrates.

Motoneuron organisation of the muscles of the spinal accessory complex of the sheep investigated with the fluorescent retrograde tracer technique.

Retrograde transport of the fluorescent tracers Diamidino Yellow dihydrochloride and Fast Blue was used to determine the location of the spinal nucleus of the accessory nerve in the sheep. We also considered whether in this species the sternocephalic, brachiocephalic, omotransversarius and trapezius muscles, i.e. the muscles of the spinal accessory complex, are supplied by more than one population of motoneurons. The spinal accessory nucleus extends as a single column of neurons from C1 to C7 spinal cord segments and occupies a lateral position within the ventral horn. The most rostral portion of this column is located dorsolaterally, whereas the remaining portion from C2 to C7 occupies a ventrolateral position. At C1 and C4 levels the nucleus also possesses some cells with a medial location. All the muscles of the spinal accessory complex receive their motor innervation both from the spinal accessory nucleus and from motoneurons forming the cervical spinal nerves. A double motor innervation of these muscles is thus present in the sheep.

Muscle growth and myosin isoform transitions during development of a small teleost fish, Poecilia reticulata (Peters) (Atheriniformes, Poeciliidae): a histochemical, immunohistochemical, ultrastructural and morphometric study.

The myosin composition of lateral muscle in Poecilia reticulata from birth to adult was studied by ATPase histochemistry and immunostaining with myosin isoform-specific antibodies. At birth the muscle consists of two layers containing developmental isoforms of myosin. In deep layer fibres the developmental myosin is replaced by the adult fast-white isoform soon after birth. In the epaxial and hypaxial monolayer fibres the myosin composition present at birth (J1) is replaced within 3 days by another (J2). In some fibres, this J2 composition is retained in the adult, but in others it is slowly replaced by the adult slow-red muscle isoform. Close to the lateral line, all monolayer fibres are already in transition between the J2 myosin and the adult slow-red form at birth, and rapidly complete the transition to slow-red form. These fibres, together with others generated de novo in an underlying hyperplastic zone, form the red muscle layer of the adult. The pink muscle develops during the first month after birth, and by 31 days it consists of an outer, middle and inner layer. A few middle layer fibres are already present at birth, while the outer layer fibres first appear 3 days after birth. The thin inner layer is probably a transitional form between the middle pink and adult white types, and appears at about 31 days. A morphometric analysis showed that growth of the white muscle occurs principally by hypertrophy. Even at the magnification level of the electron microscope, no satellite cells or myoblasts which could give rise to new fibres were found in the white muscle, except in the far epaxial and hypaxial regions and only in the first 10 days. A zone of hyperplastic growth was also found lying just under the superficial monolayer close to the lateral line, and this presumably contributes fibres to the red and pink muscle layers.

Neonatal myosin in bovine and pig tensor tympani muscle fibres.

In previous studies of middle ear muscles, the classification of fibre types by histochemical methods was particularly difficult in the bovine and porcine tensor tympani muscle, suggesting the presence of immature fibres. We therefore reexamined the tensor tympani from pigs and cattle of various ages immunohistochemically, using a panel of antimyosin antibodies, including one (anti-NE) specific for neonatal and embryonic myosins. Fibres positive to anti-NE were found in tensor tympani in both species in all ages examined; only a few of these fibres reacted exclusively with this antibody; some also contained slow myosin and the majority also contained adult fast (type IIA) myosin. Furthermore, although the remaining fibres included some of the classical types I and IIA, the majority of them showed a mismatch between their histochemical and immunohistochemical profiles. The morphological appearance of the muscle, the widespread presence of neonatal myosin (often together with another myosin in the same fibre) and the persistence of this composition from birth to adulthood, could be explained by an incomplete development of the muscle fibres, resulting in a 'muscle' much better suited to the role of a ligament.

Analysis of the sternotrachealis muscle fibers in some Anseriformes: histochemistry and sex differences.

Histochemical characteristics and sizes of the fibers of the sternotrachealis (ST) muscle have been investigated in some Anseriformes (mallard, Pekin duck, Muscovy duck, and goose) of both sexes. A sexual dimorphism has been shown in the muscle of the species examined. In the mallard and Pekin duck, the male ST muscle shows type IIIA fibers in addition to the type I, IIA, and IIB fibers observed also in the female. In the Muscovy duck, the male muscle has only type I and IIA fibers, whereas the female muscle presents type I fibers and both types IIA and IIB fibers. Moreover, the mean frequencies for each fiber type were significantly different between males and females. In the goose, both male and female muscles present only type I and IIA fibers. In all the species examined, the mean areas of each fiber type are significantly different between male and female, being always larger in the male muscles. The anatomical sexual dimorphism observed in the ST muscle is discussed in relation to function.

An immunohistochemical approach to the intrafusal fibers of extraocular muscle spindles in sheep, cow, and pig.

Intrafusal muscle fibers of the extraocular muscles (EOMs) of the sheep, cow, and pig were studied histochemically and immunohistochemically. In sheep and cow spindles, three intrafusal fiber types, namely the bag1, bag2, and chain fibers, were identified by a combination of standard histochemical methods and immunohistochemical staining with antibodies selective for slow-tonic (antitonic ALD) and slow twitch (anti-I BA-D5) myosin. The bag1 and bag2 fibers appeared immunologically different on the basis of their differential reactivity with the two antisera. Anti-tonic ALD preferentially stained the bag1 fibers, whereas anti-I BA-D5 labeled the bag2 fibers. Chain fibers did not react with either antisera. In the pig EOM spindles, in general, one bag and some chain intrafusal fibers were identified. The bag fiber was labeled by anti-tonic ALD, but it did not react with the anti-I BA-D5. These findings point to the existence in pig EOM spindles of only one bag fiber antigenically similar to the bag1 fiber of the other species examined.

Simultaneous cell death in the trigeminal ganglion and in ganglion neurons present in the oculomotor nerve of the bovine fetus.

A well-developed ganglion and scattered ganglion cells are present in the intracranial portion of the oculomotor nerve during the first half of fetal life in the ox. In the second half of fetal life a dramatic reduction of the ganglion cells associated with the oculomotor nerve occurs because of spontaneous cell death. Concomitantly, the same phenomenon of cell death is found in the trigeminal ganglion, especially in its rostromedial portion. Free degenerating perikarya can be found in the cavernous sinus.

Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.). An ultrastructural and morphometric study.

In this EM study of lateral muscle in Dicentrarchus labrax, we observed that during the larval period, growth of the presumptive red and white muscle layers occurs both by hypertrophy (as fibres already present at hatching complete their maturation) and by production of new fibres in germinal zones specific to the two muscle layers. In the first half of larval life the presumptive white muscle increases in thickness by the addition, superficially, of new fibres derived from a germinal zone of presumptive myoblasts lying beneath the red muscle layer. In the second half of larval life new fibres produced in this same zone form the intermediate (or pink) muscle layer. Dorsoventrally the myotome grows throughout larval life, largely by addition of new fibres from germinal zones at the hypo- and epi-axial extremities. Towards the end of larval life all these germinal zones are becoming exhausted, but another source of fibres arises as satellite cells, associated with large-diameter presumptive white muscle fibres, are activated to produce new fibres. The addition of small, new fibres gives the white muscle its mosaic appearance. Morphometric analysis of fibre diameters in the white muscle confirms that whereas these hyperplastic processes are important during the larval and juvenile periods, when growth is very rapid, they have ceased by the time the adult stage is attained. By contrast, fibre hypertrophy continues through into adult life. The presumptive red muscle consists initially of a monolayer of fibres present only near the lateral line, and during larval life it grows hypo- and epi-axially by addition of fibres derived from myoblasts already present in these areas at hatching. Lying superficially to the presumptive red muscle monolayer there is a near-continuous layer of external cells with a "flattened" profile. During the second half of larval life, differentiation of these external cells into myoblasts provides the source of new fibres which are added to the red muscle layer. This process, which occurs initially in the region around the lateral line and later spreads outwards, is responsible for the increase in thickness of the red muscle.

Different intrafusal fiber composition of spindles in sheep and pig extraocular muscles.

Histochemical profiles of intrafusal fibers have been examined in muscle spindles of extraocular muscles of sheep and pig. Results show that in the sheep the intrafusal content presents, in addition to chain fibers, at least one bag1 and one bag2 fiber, whereas in the pig almost all the spindles are one-bag-fiber [corrected] spindles.

The adductor mandibulae muscle in teleost fish with protrusible or non protrusible jaws: a histochemical and immunohistochemical study.

A study was made of the morphology and fibre type composition of the adductor mandibulae (AM) muscle in Teleosts with very protrusible (carp), moderately protrusible (cod) and non-protrusible (trout and cat-fish) jaws. In contrast to the trout and cat-fish, in which the AM is formed by only 2 components (mandibular and mental), in the carp and cod there is a third portion (maxillary) which is more or less developed in relation to the extent of jaw protrusion. Fibre types were identified by means of histochemical staining for succinate dehydrogenase and myosin ATPase activities, and by immunohistochemistry with anti-sera specific for fish fast and slow myosins. In all the species AM is composed principally of white (fast) fibres, with a smaller proportion of red (slow) fibres. The red fibres, which appear in the deep layers only of the muscle are not found in all of the components, and in the different species are not always present in the same parts. In those parts of the AM which are mixed, a transition zone lies between the red and white areas, and is usually composed of a third, or intermediate, type of fibre with histochemical and immunohistochemical properties similar to those of the pink zone of lateral muscle. The anatomical characteristics and different fibre type compositions of the various components forming the AM are discussed in relation to the extent of jaw protrusion and the relevant physiological data concerning other movements in which this muscle participants.