The Effect of Gentamicin on the Motility of Hormonally Induced Spermatozoa of Toad Bufo bufo during Storage at 4°C.

We studied the effect of antibiotic gentamicin at concentrations of 0.05, 0.1, 0.2, 0.4, and 1 mg/ml on the maintenance of sperm motility of the common toad Bufo bufo during cold storage of spermic urine samples at 4°C. Parameters of sperm motility during storage of samples with gentamicin at concentrations of 0.05-0.4 mg/ml did not differ significantly, but were higher (p<0.0001) than in the control (storage without antibiotic). Gentamicin at a concentration of 1 mg/ml had a negative effect on sperm motility. After 2 weeks of storage of toad spermic urine samples with gentamicin, the largest number of sperm was preserved when using antibiotic at a concentration of 0.4 mg/ml.

Influence of Xenon on Survival of Sperm of Common Frog Rana temporaria during Slow Freezing.

We studied the effect of xenon on the survival rate of the spermatozoa of the common frog Rana temporaria during slow freezing with saturation of the suspension with xenon at a pressure of up to 1.2 bar. The cryoprotective properties of xenon were analyzed in comparison with nitrogen. No specific cryoprotective effect of xenon was revealed. Viability of spermatozoa pretreated with xenon at atmospheric pressure (0 bar) or under excess pressure of 0.6 bar and frozen in a cryoprotective medium with dimethylformamide, sucrose, and BSA did not differ significantly. The use of overpressure of xenon of 1.0 or 1.2 bar in the pretreatment and freezing process significantly impaired viability of the biomaterial.

[Strategies for Conservation of Endangered Amphibian and Reptile Species].

Strategies for conservation of endangered amphibian and reptile species are discussed. One-fifth of all vertebrates belongs to the category of "endangered species," and amphibians are first on the list (41%). Every fifth reptile species is in danger of extinction, and insufficient information is characteristic of every other fifth. As has been demonstrated, efficient development of a network of nature conservation areas, cryopreservation, and methods for laboratory breeding and reintroduction play.the key roles in adequate strategies for preservation of amphibians and reptiles.

Sperm motility of externally fertilizing fish and amphibians.

We review the phylogeny, sperm competition, morphology, physiology, and fertilization environments of the sperm of externally fertilizing fish and amphibians. Increased sperm competition in both fish and anurans generally increases sperm numbers, sperm length, and energy reserves. The difference between the internal osmolarity and iconicity of sperm cells and those of the aquatic medium control the activation, longevity, and velocity of sperm motility. Hypo-osmolarity of the aquatic medium activates the motility of freshwater fish and amphibian sperm and hyperosmolarity activates the motility of marine fish sperm. The average longevity of the motility of marine fish sperm (~550 seconds) was significantly (P < 0.05) greater than that of freshwater fish sperm (~150 seconds), with the longevities of both marine and freshwater fish being significantly (P < 0.05) lower than that of anuran sperm (~4100 seconds). The average velocity of anuran sperm (25 µm/s) was significantly (P < 0.05) lower than that of marine fish (140 µm/s) or freshwater fish (135 µm/s) sperm. The longevity of the sperm of giant salamanders (Cryptobranchoidea) of approximately 600 seconds was greater than that of freshwater fish sperm but much lower than anuran sperm. Our research and information from the literature showed that higher osmolarities promote greater longevity in anuran sperm, and some freshwater fish sperm, and that anuran and cryptobranchid sperm maintained membrane integrity long after the cessation of motility, demonstrating a preferential sharing of energy reserves toward the maintenance of membrane integrity. The maintenance of the membrane integrity of anuran sperm in fresh water for up to 6 hours showed an extremely high osmotic tolerance relative to fish sperm. The very high longevity and osmotic tolerance of anuran sperm and high longevity of cryptobranchid sperm, relative to those of freshwater fish, may reflect the complex fertilization history of amphibian sperm in general and anurans reversion from internal to external fertilization. Our findings provide a greater understanding of the reproductive biology of externally fertilizing fish and amphibians, and a biological foundation for the further development of reproduction technologies for their sustainable management.

[Intensive protein synthesis in neurons and phosphorylation of beta-amyloid precursor protein and tau-protein are triggering factors of neuronal amyloidosis and Alzheimer's disease].

Recently the studies of Alzheimer's disease have become particularly actual and have attracted scientists from all over the world to this problem as a result of dissemination of this dangerous disorder. The reason for such pathogenesis is not known, but the final image, for the first time obtained on microscopic brain sections from patients with this disease more than a hundred years ago, is well known to clinicists. This is the deposition of Abeta amyloid in the brain tissue of senile plaques and fibrils. Many authors suppose that the deposition of beta-amyloid provokes secondary neuronal changes which are the reason of neuron death. Other authors associate the death of neurons with hyperphosphorylation oftau-proteins which form neurofibrillar coils inside nerve cells and lead to their death. For creation of methods of preclinical diagnostics and effective treatment of Alzheimer's disease novel knowledge is required on the nature of triggering factors of sporadic isoforms of Alzheimer's disease, on cause-effect relationships of phosphorylation of amyloid precursor protein with formation of pathogenic beta-amyloids, on the relationship with these factors of hyperphosphorylation of tau-protein and neuron death. In this review we analyze the papers describing the increasing of intensity of biosynthesis in neurons in normal conditions and under the stress, the possibility of development of energetic unbalanced neurons and activation of their protective systems. Phosphorylation and hyperphosphorylation of tau-proteins is also tightly connected with protective mechanisms of cells and with processes of evacuation of phosphates, adenosine mono-phosphates and pyrophosphates from the region of protein synthesis. Upon long and high intensity of protein synthesis the protective mechanisms are overloaded and the complementarity of metabolitic processes is disturbed. This results in dysfunction of neurons, transport collapse, and neuron death.

Hormonal induction of spermatozoa from amphibians with Rana temporaria and Bufo bufo as anuran models.

The use of hormonally induced spermatozoa expressed in urine (HISu) is a valuable component of reproduction technologies for amphibians. Five protocols for sampling HISu from the European common frog (Rana temporaria) were compared: (1) pituitary extracts, (2) 0.12 µg g⁻¹ luteinising hormone-releasing hormone analogue (LHRHa), (3) 1.20 µg g⁻¹ LHRHa, (4) 11.7 IU g⁻¹ human chorionic gonadotrophin (hCG) and (5) 23.4 IU g⁻¹ hCG (g⁻¹ = per gram bodyweight). From 1 to 24h after administration we assessed the number and concentration of spermatozoa in spermic urine and in holding water, and in urine the percentage of motile spermatozoa and their progressive motility. The protocol using 1.20 µg g⁻¹ LHRHa gave the highest total sperm numbers (650 × 106) and the highest percentage (40%) of samples with sperm concentrations above 200 × 106 mL⁻¹. The percentage motility and progressive motility was similar from all protocols. Considerable amounts of spermatozoa were expressed by R. temporaria into their holding water. We tested hormonal priming and spermiation in the common toad (Bufo bufo) using 0.13 µg g⁻¹ LHRHa administered 24h before a final spermiating dose of 12.8 IU g⁻¹ hCG. No spermatozoa were expressed in holding water. Priming resulted in 35% more spermatozoa than without; however, there were no differences in sperm concentrations. Primed B. bufo produced spermatozoa with significantly higher percentage motility, but not progressive motility, membrane integrity, or abnormal spermatozoa than unprimed males.

Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species.

The survival of hundreds of threatened amphibian species is increasingly dependent on conservation breeding programs (CBPs). However, there is an ongoing loss of genetic variation in CBPs for most amphibians, reptiles, birds, and mammals. Low genetic variation results in the failure of CBPs to provide genetically competent individuals for release in supplementation or rehabitation programs. In contrast, in the aquaculture of fish the perpetuation of genetic variation and the production of large numbers of genetically competent individuals for release is accomplished through the cryopreservation of sperm. Successful protocols for the cryopreservation of amphibian sperm from excised testes, and the use of motile frozen then thawed sperm for fertilisation, have been adapted from those used with fish. However, there have been no protocols published for the cryopreservation of amphibian hormonally induced sperm (HIS) that have achieved fertility. We investigated protocols for the cryopreservation of amphibian HIS with the European common frog (Rana temporaria) as a model research species. We induced spermiation in R. temporaria through the intraperitoneal administration of 50 µg LHRHa and sampled HIS through expression in spermic urine. Highly motile HIS at a concentration of 200 × 10(6)/mL was then mixed 1:1 with cryodiluents to form cryosuspensions. Initial studies showed that; 1) concentrations of ∼15 × 10(6)/mL of HIS achieve maximum fertilisation, 2) TRIS buffer in cryodiluents did not improve the recovery of sperm after cryopreservation, and 3) high concentrations of DMSO (dimethylsulphoxide) cryoprotectant reduce egg and larval survival. We then compared four optimised cryopreservation protocols for HIS with the final concentrations of cryodiluents in cryosuspensions of; 1) DMSO, (½ Ringer Solution (RS), 10% sucrose, 12% DMSO); 2) DMSO/egg yolk, (½ RS, 10% sucrose, 12% DMSO, 10% egg yolk), 3) DMFA, (½ RS, 10% sucrose, 12% dimethylformamide (DMFA)), and 4) MIS/glycerol, (Motility Inhibiting Saline (MIS), 5% glycerol, 2.5% sucrose, 5% egg yolk). Cryosuspensions were frozen in LN(2) vapour, stored in LN(2), thawed in 40° C water bath, and activated by slow equilibration with 1:3 dilutions of cryosuspensions with 20 mM/L NaCl. Protocol efficacies were assessed through the post-thaw percentage of; 1) sperm motility, 2) sperm membrane integrity, 3) fertilisation, 4) fertilised eggs hatching, and 5) larval survival from fertilised eggs to 7 d. The DMFA cryodiluent proved superior to the DMSO based cryodiluents in recovery of sperm motility and fertility after cryopreservation. MIS/glycerol cryodiluent provided low sperm viability and no fertility. Considering the ease of obtaining HIS from many Rana species, the success of our protocols offer the potential for the perpetuation of the genetic variation of the 42 threatened Rana species and the 193 threatened Ranid species in total.

[Survival of amphibian embryos after continuous ultrasound treatment].

The influence of continuous ultrasound on the embryonic development of grass frog Rana temporaria has been investigated. Intact embryos at the blastula stage were treated by ultrasound of different frequency (0.88 and 2.64 MHz), intensity (0.05-1.0 W/cm2), and duration (1-15 min). The treatment with ultrasound of frequency 0.88 MHz and intensity 0.05 W/cm2 for 1-5 min tended to increase the proportion of normally developing embryos up to hatch (10-25% of control). Increasing the intensity of ultrasound (0.88 MHz) to 0.7-1.0 W/cm2 and the duration of its action to 5-15 min induced the death of almost all of treated embryos. No significant differences were found between the development of control embryos and embryos treated with ultrasound of middle intensity (0.2-0.7 W/cm2) for 1-5 min. The exposure of amphibian embryos to ultrasound of frequency 2.64 MHz and intensity 0.05-0.7 W/cm2 for 1-5 min did not change their survival. Increasing the intensity of ultrasound (2.64 MHz) to 1.0 W/cm2 and the duration of its action to 5 min decreased the number of normal developing embryos (by 35%).

[Changes in the permeability of amphibian embryo envelope for fluorochromes under the action of ultrasound].

It was determined whether high-frequency ultra sound can change the permeability of gray toad Bufo bufo and grass frog Rana temporaria under the action of high-frequency ultrasound. The changes in the permeability of embryonic envelope were assessed by using slowly penetrating fluorochromes ANS, FDA, and fluorescein. It was found that the ultrasound of 0.88 MHz and 0.4 - 0.7 W/cm2 intensity increased the permeability of amphibian embryonic envelope for ANS and FDA, whereas the ultrasound of 2.64 MHz and the same intensity increased that for fluorescein with the retention of low permeability for FDA. Embryos continued the normal development after treatment with ultrasound under these conditions.

[The role of lipid-nucleic acid interactions in the reconstruction in vitro of the nuclear shell with pores]. / Rol' vzaimodeistvii mezhdu lipidami i nukleinovymi kislotami pri rekonstruktsii in vitro iadernoi obolochki s porami.

Previous studies of triple complexes DNA-phosphatidylcholine liposomes-Me2+ showed that, upon complexing, along with aggregation, a partial reversible fusion of liposomes takes place. In this case, DNA acts as a fusogen. The addition of the extract from Xenopus laevis or Drosophila melanogaster oocytes to the triple complex leads to a complete fusion of initial liposomes 100-200 nm in size and the formation of giant liposomes of up to 100-500 microns in size. Upon the addition of liposomes to the extract from Xenopus or Drosophila eggs, either fragments of the nuclear envelope with pores or structures like annulate lamellae are formed. The diameter of pores in these formations is approximately twice as small as in the native nuclear envelope, which may be associated with a lower surface tension of the lipid bilayer. This fact is in agreement with our model of the formation of nuclear pores suggested earlier.

Mechanisms of amnestic effect of ergot alkaloid agroclavin.

Neurochemical mechanisms of the effect of agroclavin on spatial memory in the Morris water maze in mice were studied by analyzing the effect of neurotransmitter receptor ligands on the amnestic effect of this alkaloid. D1-receptor agonist SKF-38393 and calcium channel blockers verapamil and nimodipine abolished the negative effect of agroclavin on spatial memory. The role of intracellular calcium in the mechanisms of amnestic effect of agroclavin is discussed.

[Fluorescent analysis of cryopreserved totipotent cells of amphibian embryos]. / Fluorestsentnyi analiz kriokonservirovannykh totipotentnykh kletok zarodyshei amfibii.

The cryotolerance of totipotent cells from dissociated embryos of amphibian (grass frog Rana temporaria and grey toad Bufo bufo) was studied. Cell integrity and preservation of the cell barrier function were evaluated by fluorescent analysis. It was shown that the best cryopreservation of the cells was achieved by using the cryoprotective agent 10% dimethyl sulfoxide and 10% saccharose. These cells were successfully used for the homotransplantation of nuclei into enucleated eggs. The development of reconstructed eggs to the blastula stage was noted.

[The reaction of endoplasmic reticulum of Mauthner cells of Xenopus laevis tadpoles in response to the partial denervation]. / Reaktsiia éndoplasmaticheskogo retikuluma na chastichnuiu denervatsiiuMautnerovskikh neironov golovastikov shportsevoi liagushki.

The structure of the Mauthner cells in Xenopus laevis tadpole was investigated by light- and electron microscopy in norm and after early unilateral enucleation. It was found that enucleation at early stages caused a delay in morphological development of the contralateral neurons during embryogenesis. We observed a decrease in size of the soma and nucleus and in the number of dendrites, a marked structural underdevelopment of the majority of cell organoids, as well as proliferation and hypertrophy of transversal cisternae in the contralateral Mauthner cells. The ipsilateral neurostructure remained normal in embryogenesis. The data obtained suggest the availability of some unknown powerful afferent contralateral input to Mauthner cells from the optical analyzer.

[The wave-like character of the defence conditioned reflex acquisition in rats]. / Volnoobraznyi kharakter dinamiki formirovaniia u krys oboronitel'nogo uslovnogo refleksa.

The dynamic peculiarities of formation of conditioned avoidance in rats were studied during 15-day sessions using different experimental paradigms (massed and spaced training). In all the experimental situations, the acquisition curves were wave-like (independently of the paradigm). Regression curves of the 6th or 7th order most perfectly fit the experimental data, which suggests that the acquisition dynamics in both paradigms is non-linear.

[Morphogenesis of Mauthner neurons of Xenopus laevis tadpoles following early unilateral eye enucleation]. / Morfogenez Mauthner neironov golovastikov shportsevoi liagushki v usloviiakh rannei odnostoronnei énukleatsii glaza.

The connection between Mauthner neuron (CMN) and visual analyzer in Xenopus laevis tadpoles was studied using these neurons capacity to change their morphogenesis during the development in case of interruption of their connections with afferent fibres. In phase 31 of development tadpoles were subjected to unilateral enucleation of eye. Morphogenesis of contralateral (experimental) and ipsilateral (control) MN with respect to the operated eye was followed up. Comparison with intact Mauthner neuron of the tadpole of the same age who were not exposed to operation was conducted. Contralateral MN were found to be delayed in development phases 47 and 50. This was associated with 1.5-3.3 fold difference in neuron body central section area and 2-3 fold differences in nuclear section area. Three-dimensional reconstruction revealed lower number of cell processes in contralateral MN as well as decline in their length and diameter. Control and intact MN showed no difference similar to experimental and control ones in phase 41. The data obtained indicate that MN of Xenopus laevis tadpole possess powerful ipsilateral connections originating from the side of visual analyzer and contralateral ones from the side of the eye. This affects MN development and in contrast to octavolateral system (including vestibular apparatus and organs of lateral line) controls the contralateral neuron.

[Efficacy of repeated training at different stages of elaboration of a defensive reflex in the mouse]. / Effektivnost' povtornogo obucheniia na raznykh stadiiakh vyrabotki oboronitel'nogo refleksa u myshei.

It has been found that the efficiency of additional learning is in no linear dependency on interval value between initial and repeated conditioning sessions. At consecutive interval extension the efficiency of additional session changes wave-like. The effect of repeated learning session directly correlates with successful reproduction of traces of initial reflex elaboration in the second session. It is suggested that fluctuations of reproduction of unstable reflexes and of efficiency of additional learning sessions depend on changes in spatial relations of bioelectrical processes in the cortex which accompany the additional learning.

[Reproduction of a conditioned defense reflex in mice in a water maze]. / Vosproizvedenie u myshei ukslovnogo oboronitel'nogo refleksa v vodnom labirinte.

The characteristics of formation of the reflex of water avoidance in a water maze and the dynamics of its reproduction during 24 hours after its elaboration were studied in mice. In case of incomplete initial learning the reflex reproduction had a phasic, oscillating nature. It is assumed that this oscillating dynamics is due to corresponding changes in the similarity of biopotentials in the cortical areas which participate in the formation of the conditioned connection.