Karyotypes of water frogs from the Pelophylax esculentus complex: results of cross-species chromosomal painting.

Amphibian species have the largest genome size enriched with repetitive sequences and relatively similar karyotypes. Moreover, many amphibian species frequently hybridize causing nuclear and mitochondrial genome introgressions. In addition, hybridization in some amphibian species may lead to clonality and polyploidization. All such events were found in water frogs from the genus Pelophylax. Among the species within the genus Pelophylax, P. esculentus complex is the most widely distributed and well-studied. This complex includes two parental species, P. ridibundus and P. lessonae, and their hybrids, P. esculentus, reproducing hemiclonally. Parental species and their hybrids have similar but slightly polymorphic karyotypes, so their precise identification is still required. Here, we have developed a complete set of 13 chromosome painting probes for two parental species allowing the precise identification of all chromosomes. Applying chromosomal painting, we identified homologous chromosomes in both parental species and orthologous chromosomes in their diploid hemiclonal hybrids. Comparative painting did not reveal interchromosomal exchanges between the studied water frog species and their hybrids. Using cross-specific chromosome painting, we detected unequal distribution of the signals along chromosomes suggesting the presence of species-specific tandem repeats. Application of chromosomal paints to the karyotypes of hybrids revealed differences in the intensity of staining for P. ridibundus and P. lessonae chromosomes. Thus, both parental genomes have a divergence in unique sequences. Obtained chromosome probes may serve as a powerful tool to unravel chromosomal evolution in phylogenetically related species, identify individual chromosomes in different cell types, and investigate the elimination of chromosomes in hybrid water frogs.

Nuclear gene introgressions in hybrid populations of water frog Pelophylax esculentus complex: geographical analysis of the phenomenon and its interpretation.

Reproduction of water frog hybrids Pelophylax esculentus (Pelophylax ridibundus x Pelophylax lessonae) is associated with hemiclonal reproduction and backcrossing. The hemiclonal mode of reproduction occurs within P. esculentus allodiploids. In this case, the unrecombined genome of one parental species is transmitted to the offspring after premeiotic elimination of the chromosome set of the second parental species. Usually, the chromosome set of P. lessonae is eliminated, and the altered genome of P. ridibundus is passed on to the progeny. The hemiclonal inheritance within diploid Pelophylax esculentus hybrids may be accompanied by certain aberrations of premeiotic elimination. As a result, the formation of P. ridibundus specimens with introgressions of the P. lessonae genetic material, or the formation of recombinant hybrids occurs, depending on which of the parental species backcrossing takes place. The aim of our study is to describe the aberration of premeiotic elimination within the water frog P. esculentus complex detected by the nuclear gene Ldh-B inheritance, with an attempt to find out the causes of this phenomenon. It has been established that aberrations of premeiotic elimination are widespread, but only within populations of water frog from the river system of Upper Dnieper within Ukraine. The highest level of introgression takes place in the water frog populations within Kiev metropolis under conditions of expressed anthropogenization, while the maximum frequency of recombinants was detected within populations from the basin of Desna River, that has preserved native ecosystems. It was demonstrated that the frequency of premeiotic aberrations does not correlate with the intensity of interspecific water frog hybridization. Populations with introgressions are more common than populations with recombinants, however, within the latter, the frequency of recombination events is higher. The primary factor of gametogenesis aberrations, most likely, is the genetic characteristics of the local populations of parental species, since unambiguous explanations of this phenomenon based on the action of environmental stress (pollution of water systems) are not obvious.

A Variety of Fungal Species on the Green Frogs' Skin (Pelophylax esculentus complex) in South Banat.

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin.

Environmental, anthropogenic, and spatial factors affecting species composition and species associations in helminth communities of water frogs (Pelophylax esculentus complex) in Latvia.

We investigated factors affecting species composition and patterns of species associations in parasite communities of water frogs (Pelophylax esculentus complex), applying the distance-based redundancy analysis for component communities (assemblages harboured by host populations) and Markov random fields modelling for infracommunities (assemblages harboured by individual hosts), respectively. We asked (a) What are the relative effects of variation in environmental, land use (i.e., anthropogenic), and spatial factors on the variation in the species composition of component communities (i.e., in a locality)? and (b) What is the dominant pattern of species associations in infracommunities (in a host individual), and how do these associations vary along environmental and/or anthropogenic gradients? In component communities, the greatest portion of variation in helminth species composition was explained by the combined effects of space, anthropogenic pressure, and NDVI, with the pure effect of the spatial predictor being much stronger than the effects of the remaining predictors. In infracommunities, the probability of occurrence of some, but not all, helminth species depended on the occurrence of another species, with the numbers of negative and positive co-occurrences being equal. The strength and/or sign of associations of some species pairs were spatially stable, whereas interactions between other species pairs varied along the gradient of the amount of green vegetation, from negative to positive and vice versa. We conclude that the processes in parasite infracommunities and component communities in frogs are intertwined, with both bottom-up and top-down effects acting at different hierarchical scales.

Distribution of gonadotropin-inhibitory hormone (GnIH)-like immunoreactivity in the brain and pituitary of the frog (Pelophylax esculentus) during development.

Gonadotropin-inhibitory hormone (GnIH) is a newly discovered hypothalamic RFamide peptide that influences reproduction by regulating brain and pituitary neuroendocrine functions in vertebrates. We report here for the first time, the ontogenetic description of GnIH-like immunoreactivity in the brain, olfactory system, and pituitary of the frog, Pelophylax esculentus. GnIH-like immunoreactive (GnIH-ir) elements were first observed in larvae at stage 24 in the olfactory mucosa, ventral telencephalon, and diencephalon. GnIH-ir-positive staining progressively increased in frequency and intensity during larval growth and other ir perikarya appeared in the medial septum, anterior commissure, dorsal hypothalamus, and posterior tuberculum. A decline in GnIH-ir neurons was seen along the olfactory/vomeronasal/terminal nerve complex in the stages following the pre- and prometamorphosis, while other GnIH-ir neurons showed positivity in the ventromedial surface of the olfactory bulbs and into the habenular nuclei, but the latter are no longer observed in the following stages of development. The anterior-posterior axis in several brain areas, along with the median eminence and pars intermedia of the hypophysis had the appearance of GnIH-ir fibers from early stages, with a progressive increase in the number till metamorphosis in all major subdivisions of the brain. After premetamorphosis, GnIH-ir fibers arising from labeled neurons in the suprachiasmatic nucleus could be seen contacting the ventricular lumen. The transient appearance of GnIH-ir elements in the olfactory system may hint at an olfactory placode origin in the extracranial region. The distribution of GnIH in several brain regions throughout development suggests important involvement of GnIH in multiple brain functions during development.

Oxidative stress in Pelophylax esculentus complex frogs in the wild during transition from aquatic to terrestrial life.

During life, anuran individuals undergo drastic changes in the course of transition from aquatic to terrestrial habitat, when they are faced with metabolically demanding processes (growth, responses to developmental pressures), which result in increased production of reactive oxygen species (ROS), signaling molecules involved in development that can induce oxidative damage and stress. This situation can be further complicated by environmental influences. The aim of this study was to investigate oxidative stress parameters in naturally developing Pelophylax esculentus complex frogs during four developmental periods: premetamorphosis, prometamorphosis, metamorphic climax and juvenile stage, in order to examine changes in the response of the antioxidative system (AOS) and oxidative damage during the transition from aquatic to terrestrial life. Results show that ontogenetic shifts in anurans are accompanied by different levels of damage and AOS responses, which vary from the increased first-line enzymatic activities during the early period of development (premetamorphosis), through increased changes in the non-enzymatic complement during the metamorphic climax, to changes in both the enzymatic and non-enzymatic components observed in juvenile individuals. Premetamorphic individuals and individuals in metamorphosis displayed higher levels of lipid peroxidation, indicating that direct exposure to the environment for the first time and the modulation of organs are the most susceptible stages for oxidative damage. On the other hand, lower oxidative damage in juveniles points to the ability of their AOS to efficiently respond to challenges of the terrestrial environment. This study highlights the importance of ROS and the AOS of anurans in response to different developmental and/or environmental pressures that individuals face.

Identification and target-modifications of temporin-PE: A novel antimicrobial peptide in the defensive skin secretions of the edible frog, Pelophylax kl. esculentus.

A potent natural antimicrobial peptide named temporin-PE was identified and encoded from the skin secretions of Pelophylax kl. esculentus via "shotgun" cloning and LC-MS/MS fragmentation analysis. Target-modifications were carried out to further enhance the antimicrobial and anti-proliferative bioactivities, whilst decreasing the hemolytic effect. A range of bioassays demonstrated that replacing a proline with a tyrosine residue resulted in a loss of the bioactivity against Gram-negative bacteria, but dramatically improved the hemolytic and anti-proliferative activity, indicating the FLP- motif influences the hemolytic activity of temporins. Moreover, the coupling of TAT to the peptide dramatically improved its antimicrobial activity, indicating coupling TAT to these peptides could be considered as a potential tool to improve their antimicrobial activity. Overall, we have shown that targeted modifications of this natural antimicrobial peptide can adjust its bioactivities to help its development as an antibiotic or anti-proliferative agent.

Mutual maintenance of di- and triploid Pelophylax esculentus hybrids in R-E systems: results from artificial crossings experiments.

BACKGROUND: Interspecies animal hybrids can employ clonal or hemiclonal reproduction modes where one or all parental genomes are transmitted to the progeny without recombination. Nevertheless, some interspecies hybrids retain strong connection with the parental species needed for successful reproduction. Appearance of polyploid hybrid animals may play an important role in the substitution of parental species and in the speciation process. RESULTS: To establish the mechanisms that enable parental species, diploid and polyploid hybrids coexist we have performed artificial crossing experiments of water frogs of Pelophylax esculentus complex. We identified tadpole karyotypes and oocyte genome composition in all females involved in the crossings. The majority of diploid and triploid hybrid frogs produced oocytes with 13 bivalents leading to haploid gametes with the same genome as parental species hybrids usually coexist with. After fertilization of such gametes only diploid animals appeared. Oocytes with 26 bivalents produced by some diploid hybrid frogs lead to diploid gametes, which give rise to triploid hybrids after fertilization. In gonads of all diploid and triploid hybrid tadpoles we found DAPI-positive micronuclei (nucleus-like bodies) involved in selective genome elimination. Hybrid male and female individuals produced tadpoles with variable karyotype and ploidy even in one crossing owing to gametes with various genome composition. CONCLUSIONS: We propose a model of diploid and triploid hybrid frog reproduction in R-E population systems. Triploid Pelophylax esculentus hybrids can transmit genome of parental species they coexist with by producing haploid gametes with the same genome composition. Triploid hybrids cannot produce triploid individuals after crossings with each other and depend on diploid hybrid females producing diploid eggs. In contrast to other population systems, the majority of diploid and triploid hybrid females unexpectedly produced gametes with the same genome as parental species hybrids coexist with.

Kisspeptin regulates steroidogenesis and spermiation in anuran amphibian.

Kisspeptin (Kp) system has a recognized role in the control of gonadotropic axis, at multiple levels. Recently, a major focus of research has been to assess any direct activity of this system on testis physiology. Using the amphibian anuran, Pelophylax esculentus, as animal model, we demonstrate - for the first time in non-mammalian vertebrate - that testis expresses both Kiss-1 and Gpr54 proteins during the annual sexual cycle and that ex vivo 17B-estradiol (E2, 10-6 M) increases both proteins over control group. Since the interstitium is the main site of localization of both ligand and receptor, its possible involvement in the regulation of steroidogenesis has been evaluated by ex vivo treatment of testis pieces with increasing doses of Kp-10 (10-9-10-6 M). Treatments have been carried out in February - when a new wave of spermatogenesis occurs - and affect the expression of key enzymes of steroidogenesis inducing opposite effects on testosterone and estradiol intratesticular levels. Morphological analysis of Kp-treated testes reveals higher number of tubules with spermatozoa detached from Sertoli cells than control group and the expression of connexin 43, the main junctional protein in testis, is deeply affected by the treatment. In spite of the effects on spermatozoa observed ex vivo, in vivo administration of Kp-10 has been unable to induce sperm release in cloacal fluid. In conclusion, we demonstrate Kp-10 effects on steroidogenesis with possible involvement in the balance between testosterone and estradiol levels, and report new Kp-10 activities on spermatozoa-Sertoli cell interaction.

Pharmacological Effects of Two Novel Bombesin-Like Peptides from the Skin Secretions of Chinese Piebald Odorous Frog (Odorrana schmackeri) and European Edible Frog (Pelophylax kl. esculentus) on Smooth Muscle.

Bombesin-like peptides, which were identified from a diversity of amphibian skin secretions, have been demonstrated to possess several biological functions such as stimulation of smooth muscle contraction and regulation of food intake. Here, we report two novel bombesin-like peptides, bombesin-OS and bombesin-PE, which were isolated from Odorrana schmackeri and Pelophylax kl. esculentus, respectively. The mature peptides were identified and structurally confirmed by high performance Scliquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the effects of these purified chemically-synthetic peptides on smooth muscle were determined in bladder, uterus, and ileum. The synthetic replications were revealed to have significant pharmacological effects on these tissues. The EC50 values of bombesin-OS for bladder, uterus and ileum, were 10.8 nM, 33.64 nM, and 12.29 nM, respectively. Furthermore, compared with bombesin-OS, bombesin-PE showed similar contractile activity on ileum smooth muscle and uterus smooth muscle, but had a higher potency on bladder smooth muscle. The EC50 value of bombesin-OS for bladder was around 1000-fold less than that of bombesin-PE. This suggests that bombesin-OS and bombesin-PE have unique binding properties to their receptors. The precursor of bombesin-OS was homologous with that of a bombesin-like peptide, odorranain-BLP-5, and bombesin-PE belongs to the ranatensin subfamily. We identified the structure of bombesin-OS and bombesin-PE, two homologues peptides whose actions may provide a further clue in the classification of ranid frogs, also in the provision of new drugs for human health.

Bioaccumulation and effects of metals on oxidative stress and neurotoxicity parameters in the frogs from the Pelophylax esculentus complex.

Metals are involved in the formation of reactive oxygen species and can induce oxidative stress. The aim of this study was to assess the effects of several metals on oxidative stress in the skin and muscle of the Pelophylax esculentus "complex" frogs (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax esculentus) that inhabit the wetland Obedska Bara in Serbia, and the potential use of these species as bioindicator organisms in biomonitoring studies. The biomarkers of oxidative stress (SOD, CAT, GSH-Px, GR, GST activities and GSH, SH concentrations) and cholinesterase activity were investigated. The concentrations of nine metals (Fe, Cu, Zn, As, Cd, Cr, Hg, Ni, and Pb) were measured in the water and tissues. Correlations were established between metals and biomarkers in the tissues. The results of metal accumulation distinguished the skin of P. lessonae and muscle of P. ridibundus from other P. esculentus complex species. The oxidative stress biomarkers observed in P. ridibundus and P. esculentus had greater similarity than in P. lessonae. The P. lessonae displayed the highest number of correlations between biomarkers and metals. The results of tissue responses revealed that skin was more susceptible to metal-induced oxidative stress, with only exception of As. In the light of these findings, we can suggest the use of P. esculentus complex species as a biomonitoring species in studies of metal accumulation and metal-induced oxidative stress, but with special emphasis on P. lessonae.

Immunolocalization of the TASK2 Potassium Channel in Frog Kidney.

TASK2 (K2P5. 1, KCNK5) is a two-pore domain K⁺ channel belonging to the TALK subgroup of the K2P family of proteins. TASK2 expression has been reported in a variety of cells and tissues ranging from kidney to immune cells and including specific neurons, its proposed functions spanning from involvement in the regulation of cell volume to control of excitability. The purpose of this study was to determine the tubule location ofthe TASK2 K⁺ channel protein in frog kidney applying polyclonal antibody against the carboxyl terminus of human TASK2 (KCNK5) protein. Immunohistochemical analysis revealed that TASK2 is expressed on distal tubules and proximal epithelial cells. TASK2 is strongly expressed predominantly on the luminal part ofthe proximal epithelial cells and slightly cytoplasmatic staining is expressed. Distal tubules showed diffuse cytoplasmatic staining as well as slight staining on the apical parts ofthe cells. These findings suggest that the TASK2 K⁺ channel has cell-specific roles in renal potassium ion transport.

Post-zygotic selection against parental genotypes during larval development maintains all-hybrid populations of the frog Pelophylax esculentus.

BACKGROUND: Hybridization between two species usually leads to inviable or infertile offspring, due to endogenous or exogenous selection pressures. Yet, hybrid taxa are found in several plant and animal genera, and some of these hybrid taxa are ecologically and evolutionarily very successful. One example of such a successful hybrid is the water frog, Pelophylax esculentus which originated from matings between the two species P. ridibundus (genotype RR) and P. lessonae (LL). At the northern border of the distribution all-hybrid populations consisting of diploid (LR) and one or two triploid (LLR, LRR) frog types have been established. Here, the hybrid has achieved reproductive independence from its sexual ancestors and forms a self-sustaining evolutionary unit. Based on the gamete production of these hybrids, certain mating combinations should lead to LL and RR offspring, but these parental forms are absent among the adults. RESULTS: In order to investigate the mechanisms that maintain such an all-hybrid system, we performed a field study and a crossing experiment. In the field we sampled several ponds for water frog larvae at different developmental stages. Genotype compositions were then analysed and life-history differences between the genotypes examined. In the experiment we crossed diploid and triploid males and females from different ponds and determined fertilization success as well as development speed and survival rates of the offspring under high, medium and low food availability. In both parts of the study, we found numerous LL and RR offspring during the egg and early larval stages; but the frequency of these parental genotypes decreased drastically during later stages. In natural ponds almost all of them had disappeared already before metamorphosis; under the more benign experimental conditions the last ones died as juveniles during the following year. CONCLUSIONS: From the combined results we conclude that the absence of parental genotypes in all-hybrid populations is due to post-zygotic selection against them, rather than to pre-zygotic mechanisms that might prevent their formation in the first place. For this post-zygotic selection, genetic mechanisms resulting from low genetic diversity and fixation of deleterious mutations seem to be a more likely explanation than ecological factors.

Neuroanatomical organization of the brain gonadotropin-inhibitory hormone and gonadotropin-releasing hormone systems in the frog Pelophylax esculentus.

Growing evidence suggests that gonadotropin-inhibitory hormone (GnIH) may play a key role in mediating vertebrate reproduction. GnIH inhibits gonadotropin synthesis and release by decreasing the activity of gonadotropin-releasing hormone (GnRH) neurons as well as by directly regulating gonadotropin secretion from the pituitary. Whereas the presence of GnIH has been widely investigated in various classes of vertebrates, there are very few immunohistochemical reports focusing on GnIH in amphibians. The aim of this study was to assess the presence and neuroanatomical distribution of GnIH-like immunoreactivity in the brain of the anuran amphibian Pelophylax (Rana) esculentus (esculenta) and to explore any potential anatomical relationship with mammalian GnRH-immunoreactive (mGnRH-ir) elements. The GnIH-like immunoreactive (GnIH-ir) system constitutes two distinct subpopulations in the telencephalon and diencephalon, with the highest number of immunoreactive cells located in the preoptic and suprachiasmatic areas. GnIH-ir neurons were also observed in the medial septum, the anterior commissure, the dorsal hypothalamus, the periventricular nucleus of the hypothalamus, and the posterior tuberculum. Scattered GnIH-ir fibers were present in all major subdivisions of the brain but only occasionally in the median eminence. mGnRH-ir neurons were distributed in the mediobasal telencephalon, the medial septal area, and the anterior preoptic area. Double-label immunohistochemistry revealed that the GnRH and GnIH systems coexist and have overlapping distributions at the level of the anterior preoptic area. Some GnIH-ir fibers were in close proximity to mGnRH-ir cell bodies. Our results suggest that both the neuroanatomy and the functional regulation of GnRH release are conserved properties of the hypothalamic GnIH-ir system among vertebrate species.

Kisspeptin drives germ cell progression in the anuran amphibian Pelophylax esculentus: a study carried out in ex vivo testes.

Kisspeptin, via Gpr54 receptor, regulates puberty onset in most vertebrates. Thus, the direct involvement of kisspeptin activity in testis physiology was investigated in the anuran amphibian, Pelophylax esculentus. In this vertebrate gpr54 mRNA has been localized in both interstitial compartment and spermatogonia (SPG), whereas SPG proliferation requires the cooperation between estradiol and testicular Gonadotropin releasing hormone (Gnrh). In the pre-reproductive period, dose response curve to assess the effects of Kisspeptin-10 (Kp-10) was carried out in vitro (dose range: 10(-9)-10(-6)M; incubation times: 1 and 4h); proliferative activity and germ cell progression were evaluated by expression analysis of proliferating cell nuclear antigen (pcna), estrogen receptor beta (erß), Gnrh system (gnrh1, gnrh2, gnrhr1, r2, r3) and by the count of empty, mitotic and meiotic tubules. All selected markers were up regulated at 4h Kp-10 incubation. Histological analysis also proved the increase of mitotic activity and the progression of spermatogenesis. Besides Kp-10 modulation of testicular Gnrh system, in vitro treatment with 17ß-estradiol (10(-6)M) ± the antagonist ICI182-780 (10(-5)M) revealed gnrh2 and gnrhr3 estrogen dependent expression. In the reproductive period, testes were incubated for 1 and 4h with Kp-10 (10(-7)M) or Kp-10 (10(-7)M)+kisspeptin antagonist [Kp-234 (10(-6)M)]. Results obtained in the pre-reproductive period were confirmed and Kp-234 completely counteracted Kp-10 effects. In conclusion, Kp-10 modulated the expression of pcna, erß, gnrhs and gnrhrs, inducing the progression of the spermatogenesis.

Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog.

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 µm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 µm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.

The non-linear elasticity of the muscle sarcomere and the compliance of myosin motors.

Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre-microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4 °C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa(-1) (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model.

The contributions of filaments and cross-bridges to sarcomere compliance in skeletal muscle.

Force generation in the muscle sarcomere is driven by the head domain of the myosin molecule extending from the thick filament to form cross-bridges with the actin-containing thin filament. Following attachment, a structural working stroke in the head pulls the thin filament towards the centre of the sarcomere, producing, under unloaded conditions, a filament sliding of ∼ 11 nm. The mechanism of force generation by the myosin head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (C(cb)) and filaments. By measuring the force dependence of the spacing of the high-order myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined filament compliance (Cf) of 13.1 ± 1.2 nm MPa(-1), close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa(-1)). C(cb) calculated using these estimates is 0.37 ± 0.12 nm pN(-1), a value fully accounted for by the compliance of the myosin head domain, 0.38 ± 0.06 nm pN(-1), obtained from the intensity changes of the 14.5 nm myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to C(cb) from the myosin tail that joins the head to the thick filament is excluded. The low C(cb) value indicates that the myosin head generates isometric force by a small sub-step of the 11 nm stroke that drives filament sliding at low load. The implications of these results for the mechanism of force generation by myosins have general relevance for cardiac and non-muscle myosins as well as for skeletal muscle.

Brainstem circuits underlying the prey-catching behavior of the frog.

Prey-catching behavior (PCB) of the frog consists of a sequence of movements as a stimulus-response chain of the behavioral pattern in which each action presents a signal for the subsequent event. The transformation of visual information into appropriate spatiotemporal patterns of motor activity is carried out by the motor pattern generators located in the brainstem reticular formation. The motor pattern generators provide input to the motoneurons either directly or via the last-order premotor interneurons (LOPI). Although the feeding program is predetermined in this way, various sensory mechanisms control the motor activity. By using neuronal labeling methods, we have studied the morphological details of sensorimotor integration related to the hypoglossal motoneurons to provide further insight into the neuronal circuits underlying the PCB in ranid frogs. Our major findings are as follows. (1) Dendrodendritic and dendrosomatic contacts established by the crossing dendrites of hypoglossal (XII) motoneurons may serve as a morphological option for co-activation, synchronization and proper timing of the bilateral activity of tongue muscles. The crossing dendrites may also provide a feedforward amplification of various signals to the XII motoneurons. The overlapping dendritic territories of the motoneurons innervating protractor and retractor muscles may facilitate the coordinated activities of the agonistic and antagonistic muscles. (2) The musculotopic organization of the XII motoneurons is reflected in the distribution of LOPI for the protractor and retractor muscles of the tongue. (3) Direct sensory inputs from the trigeminal, vestibular, glossopharyngeal-vagal, hypoglossal and spinal afferent fibers to the XII motoneurons may modulate the basic motor pattern and contribute to the plasticity of neuronal circuits. (4) The electrical couplings observed in the vestibulocerebellar neuronal circuits may synchronize and amplify the afferent signals. The combination of chemical and electrical impulse transmission provides a mechanism by which motoneurons can be activated sequentially.

Hypothalamus-pituitary axis: an obligatory target for endocannabinoids to inhibit steroidogenesis in frog testis.

Endocannabinoids - primarily anandamide (AEA) and 2-arachidonoylglycerol (2-AG) - are lipophilic molecules that bind to cannabinoid receptors (CB1 and CB2). They affect neuroendocrine activity inhibiting gonadotropin releasing hormone (GnRH) secretion and testosterone production in rodents, through a molecular mechanism supposed to be hypothalamus dependent. In order to investigate such a role, we choose the seasonal breeder, the anuran amphibian Rana esculenta, an experimental model in which components of the endocannabinoid system have been characterized. In February, at the onset of a new spermatogenetic wave, we carried out in vitro incubations of frog testis with AEA, at 10(-9)M dose. Such a treatment had no effect on the expression of cytochrome P450 17alpha hydroxylase/17,20 lyase (cyp17) nor 3-ß-hydroxysteroid dehydrogenase/Δ-5-4 isomerase (3ß-HSD), key enzymes of steroidogenesis. To understand whether or not the functionality of the hypothalamus-pituitary axis could be essential to support the role of endocannabinoids in steroidogenesis, frogs were injected with AEA, at 10(-8)M dose. Differently from in vitro experiment, the in vivo administration of AEA reduced the expression of cyp17 and 3ß-HSD. Whereas the effect on 3ß-HSD was counteracted by SR141716A (Rimonabant) - a selective antagonist of CB1, thus indicating a CB1 dependent modulation - the effect on cyp17 was not, suggesting a possible involvement of receptors other than CB1, probably the type-1 vanilloid receptor (TRPV1), since AEA works as an endocannabinoid and an endovanilloid as well. In conclusion our results indicate that endocannabinoids, via CB1, inhibit the expression of 3ß-HSD in frog testis travelling along the hypothalamus-pituitary axis.