Cryopreservation of isolated fish blastomeres: effects of cell stage, cryoprotectant concentration, and cooling rate on postthawing survival

The toxicity of the cryoprotectant dimethyl sulfoxide (Me(2)SO) to isolated blastomeres was examined in three fish species representative of distinct environments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odontesthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects of embryonic stage, Me(2)SO concentration, and cooling rate on the cryopreservation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomeres from the three species tolerated Me(2)SO concentrations up to 9% relatively well for over 5 h but lost viability rapidly at 18%. Cells from later embryonic stages (512 or 1024 cells) were more tolerant of Me(2)SO than those from earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres after freezing and thawing, but the extent of the effect and the optimum conditions varied with the species. In general, the highest rates of successful cryopreservation were observed with older rather than younger blastomeres, slower rather than faster cooling, and with 9-18% rather than 0% Me(2)SO. Survival rates for blastomeres cryopreserved under the most effective combination of the three factors examined for each species were 19.9 +/- 10.1% for whiting, 34.1 +/- 8.5% for medaka, and 67.4 +/- 12.8% for pejerrey. Copyright 1999 Academic Press.

Crow-Fukase syndrome associated with pulmonary hypertension.

A 60-year-old woman suffering from heart failure was found to have Crow-Fukase syndrome. A precise cardiovascular study revealed the existence of pulmonary hypertension (PH), cardiomegaly and myocardial hypertrophy in addition to manifestations of this syndrome such as hyperpigmentation, hypertrichosis, finger clubbing, polyneuropathy, organomegaly and plasma cell dyscrasia. These findings suggest the possibility that patients with Crow-Fukase syndrome have cardiopulmonary disorders including PH and cardiomegaly which might cause some cardiovascular symptoms such as peripheral edema and finger clubbing.

Cloning and sequencing of cDNAs of the ß-globin gene family in carp.

A total blood cell cDNA library was constructed using a 3-year-old carp Cyprinus carpio. A ß-globin cDNA (CßG1) was identified from the library by the polymerase chain reaction using a ß-globin-specific primer deduced from the carp ß-globin-A amino acid sequence. Also, five additional types of ß-globin cDNAs (CßG2∼6) were isolated by colony hybridization using CßG1 as a probe. Sequence analysis revealed that these CßGs encoded 147 amino acids, and the deduced amino acid sequences showed high identity (89·1-95·2%) to previously reported carp ß-globin amino acid sequences. The nucleotide sequences of the CßGs were very similar (identity 96·0-99·6%) and the expression levels of CßG1∼6 were 28·6, 28·6, 21·4, 14·3, 3·6 and 3·6% of the total number of cloned CßGs, respectively. Although the complete amino acid sequence identities between the CßGs and the ß-globin of higher vertebrates were low, functionally important regions such as the α-ßcontact region and haem contact region were well conserved. These data showed that, as in higher vertebrates, the adult carp has a multiple ß-globin gene family (at least six members). However, transcripts encoding four types of peptides (CßG1 type, CßG2 and 3 type, CßG5 type, and CßG4 and 6 type) were expressed at relatively high levels, this being a unique character of the carp haemoglobin system.

Cloning and characterization of pejerrey mitochondrial DNA and its application for RFLP analysis

Probes were cloned, characterized, and developed for all regions of the mitochondrial DNA (mtDNA) of pejerrey Odontesthes bonariensis to provide the basis for the study of genetic diversity of South American atherinopsinii and to enable species identification from small amounts of tissue. The mtDNA was extracted from liver and cleaved with Eco RI, producing four fragments (7·4, 3·4, 3·1 and 2·9 kb) which were cloned using pUC118 plasmid vectors. Sequence analysis from both ends of the fragments showed that they encode tRNA (Asp, Phe, and Ser-TGA), 12 S rRNA, cytochrome oxidase (CO) II, NADH 4, 5, and 6, and the d-loop, and that the relative positions of these genes are identical to those in the mtDNA of other teleosts. A comparison of homology with carp mtDNA nucleotide sequences revealed that tRNA (Phe and Ser-TGA) and CO II were relatively conserved, whereas the d-loop region was highly divergent. The cloned mtDNA probes detected mtDNA fragments from about 800 ng of total DNA extracted from liver, muscle, and single embryos of O. bonariensis, and were effective for restriction length fragment polymorphism (RFLP) analysis of Patagonina hatcheri, the most distant atherinopsine relative of pejerrey. The cloned mtDNA probes may be useful for the analysis of genetic diversity and non-destructive species identification, including the examin-ation of eggs, larvae and juveniles. The mtDNA sequences reported here provide the basis for the design of primers for PCR-based RFLP analysis. 1997 The Fisheries Society of the British Isles

Development of a new high-speed measuring system to analyze the dental cast form.

A new non-contact measurement system employing of a line laser and CCD camera was developed to rapidly measure maloccluded dental casts with large depth and high accuracy. In this system, the measuring interval along the line laser and the model scanning direction perpendicular to the line laser were both 0.2 mm. By averaging the raw data through repetitious input and smoothing by local filtering, the noise was effectively decreased. Sub-pixel processing, which calculates the pixel position with the largest brightness within one pixel, improved the accuracy in the measurement to 22 microns compared with 65 microns before sub-pixel processing. Dental casts with malocclusion were measured with this system. The time and the depth for the measurement were about 10 minutes and 60 mm, respectively. This was a vast improvement compared with our previous system employing a point laser. In the CG of the measured dental casts, smooth and detailed surface morphology were sufficiently reproduced, and recognition of the characteristic points in the cast could be accomplished with the accuracy of 30 microns. This measurement system will be helpful to analyze the dental cast form with malocclusion in progress.

Development of the computerized dental cast form analyzing system--three dimensional diagnosis of dental arch form and the investigation of measuring condition.

To analyze the functional and morphological harmonies of the tooth and dental arch, a computerized system to measure a dental cast and to detect the apex of dental cusps and angle points of incisors was developed. Detailed morphology in the measured dental cast could be displayed distinctively by computer graphics (CG) with a surface model. Accuracy in determining the position of the apex of the dental cusp was examined by increasing the measuring pitch from 50 microns to 400 microns, the error increased gradually with increased measuring pitch. Taking the measuring error, the time for measurement and the memory size for analysis into account, a measuring pitch of 200 microns was determined to be reasonable. Three-dimensional data measurements from the apex of the dental cusp in molar, and the mesial and distal angles in canine and incisor were fitted to a polynomial formula. In the present measured model, the 4th order polynomial formula was used for the dental arch, and the 2nd order polynomial formula was used for the anteroposterior and lateral occlusal curves. These formulae could be simultaneously superimposed on the surface model of the dental cast, and displayed using CG.

[Stress analysis of titanium-hydroxyapatite implant. Effect of titanium diameter on reinforcement and calculation of implant diameter].

In order to evaluate the effect of titanium diameter on reinforcement of titanium-hydroxyapatite implant and calculate the diameter of the implant under loading, stress analysis was performed using the finite element method. In finite element model, 100% or 70% of root length was supported by PMMA in place of bone and the implant was loaded 15 kgf at 45 degrees of the implant axis. The results were obtained as follows, 1. In the case of 100% support, the titanium diameter had little effect on the reinforcement of the implant, whereas in the case of 70% support, it had much effect. 2. The diameter of this implant was calculated to be more than 3.7 mm under loading. From these results and the view point of manufacturing techniques, the diameter of this implant was supposed to need 4.0 mm and that of titanium was to be 2.0 mm.

[Dynamic study of titanium-hydroxyapatite implant by finite element method].

In order to evaluate the strength of titanium-hydroxyapatite implant, adhesive tests and loading tests were performed. The results of the loading tests were compared with the computed results by finite element method. Axisymmetric 8-node quadrilateral element for arbitrary loading was used for the analysis. Implants of 3.2 mm diameter were loaded at 45 degrees of the implant axis by using an Instron testing machine at crosshead speed of 0.5 mm/min. The results were obtained as follows: 1. The mean value of the adhesive test of titanium-resin (Panavia)-hydroxyapatite was 2.3 kgf/mm2. The fracture was caused at Panavia layer. 2. The mean value of the fracture loads of implants was 10.9 kgf. The fracture was caused at Panavia layer, similarly to the result described in 1. 3. The principal stress caused at Panavia layer of the implant was approximately 2.3 kgf/mm2, which was calculated under the load of 10.9 kgf by the finite element method. This result corresponds to that of the loading test described in 1 and 2. It was ascertained by these findings that finite element method is effective for the evaluation of the strength of this implant.

Olfactory responses of several species of teleost to F-prostaglandins.

Olfactory responses (EOG: electro-olfactogram) of 11 species of teleost (five Cypriniformes and six non-Cypriniformes) to F-prostaglandins (PGF2 alpha, 15-keto-PGF2 alpha, 13,14-dihydro-15-keto-PGF2 alpha) were examined. All the Cypriniformes showed distinct EOG responses to F-prostaglandins at 10(-7) M. By contrast, non-Cypriniformes with the exception of ayu, Pleccoglossus altivelis, showed responses to 10(-7) M F-prostaglandins of < 22% of that to 10(-5) M L-serine, and they were anosmic to 10(-8) M F-prostaglandins. These results indicate that F-prostaglandins are potent chemical stimulants in Cypriniformes, and suggest that F-prostaglandins may be commonly used as sex pheromones in Cypriniformes, whereas they seem not to have pheromonal functions in most non-Cypriniformes examined.

Molecular cloning and hormonal control in the ovary of connexin 31.5 mRNA and correlation with the appearance of oocyte maturational competence in red seabream.

Gap junctions are aggregates of intercellular channels, composed of the protein connexin (Cx), between adjacent cells. This study examined whether, in the ovary of the red seabream Pagrus major, the connexin gene essential for the production of RNA and protein during the acquisition of oocyte maturational competence is active. Mixed primers for this reaction were designed on the basis of the high sequence homology of selected regions of known connexin genes. Polymerase-chain-reaction-amplified cDNA fragments generated by 3' and 5' rapid amplication of cDNA ends were combined to generate full-length cDNA sequences. The resulting 2400 base pair cDNA had an open reading frame encoding a polypeptide containing 275 amino acid residues (31493 Da; Cx31.5). Hydropathicity analysis of the predicted amino acid sequence indicated that red seabream Cx31.5 has four major hydrophobic regions and four major hydrophilic regions indicative of a topology similar to that of known connexins. Typical connexin consensus sequences were also observed in the first and second extracellular loops. During the acquisition of oocyte maturational competence, red seabream Cx31.5 mRNA transcription levels increased after treatment with gonadotropin-II. It is therefore proposed that expression of Cx31.5 contributes to the acquisition of oocyte maturational competence in this species.

Heat-induced germ cell deficiency in the teleosts Odontesthes bonariensis and Patagonina hatcheri.

Pathological or experimental elevation of testicular temperature is known to trigger degeneration and disappearance of germ cells in scrotal mammals. In contrast, there are no reports of heat-induced germ cell deficiency in males of non-scrotal mammals and other vertebrate taxa, nor in females of any species. This study describes the induction of germ cell deficiency up to complete sterility in the teleosts Patagonina hatcheri and Odontesthes bonariensis by rearing larvae and juveniles for prolonged periods at incipient lethal, high temperatures (27-28.5 and 29 degrees C, respectively). It was shown that female germ cells are also heat-sensitive and disappear under high temperatures. The potential implications of these findings for physiological, ecological, and environmental studies and the usefulness of heat-induced germ cell-deficient fish in research and animal production are discussed.

Histological study on bone response to resin-hydroxyapatite.

Resin-Hydroxyapatite has been developed to take the advantages of bioactivity of hydroxyapatite (HAP) and plasticity of resin (Bis-GMA). It was composed of 75% HAP, 15% Bis-GMA and 10% quartz reinforced filler. Tested pellets of Resin-HAP as well as its constituents were implanted into subcutaneous connective tissue of femurs of adult dogs. Histological observation at 12 weeks postoperatively showed that each pellet was surrounded by the fibrous connective tissue almost free of the inflammatory cells. The thickness of the fibrous layer around HAP was relatively thin, while those around Bis-GMA and Resin-HAP showed some variance. Taking our previous data into consideration, it was suggested that Resin-HAP had bio-compatibility both in soft tissue and bone, although its long-term stability as well as its stability under function should further be examined.