The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozenthawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.fish genetic resources | testes cryopreservation | germ cell transplantation | stem cell | triploid trout B ecause of the environmental effects of human activities, a growing number of fish species have become threatened or are already extinct (1). Salmonid species are particularly vulnerable to habitat destruction and the effects of climate change as they occupy a unique ecological niche resulting from their longrange migration between freshwater and marine habitats (2, 3). Conservation strategies for these threatened fish must therefore be developed and implemented with all possible haste. Complicating the conservation of threatened salmonid populations are the risks associated with captive breeding and translocation of live fish, such as facility accidents, pathogen infections, genetic drift, and reduced fitness of individuals within natural habitats (4, 5).Cryobanking of fish gametes to semipermanently (6) store genetic resources could serve as an alternative approach to traditional conservation methods (7); however, past attempts at cryopreservation of fish embryos and mature oocytes have been unsuccessful (8), as a result of their large size, high yolk content, and low membrane permeability (9, 10). Although androgenesis performed by using frozen sperm and γ-ray-inactivated xenogenic eggs can regenerate live fish, their survival rate is extremely low, and resulting offspring become nuclear-cytoplasmic hybrids (11). The loss of maternally inherited materials including mitochondrial DNA makes this method impractical, as it also does for the transfer of nuclei from cryopreserved somatic cells into xenogenic oocytes (12, 13). In zebrafish, in vitro maturation of immature oocytes subsequent to cryopreservation is also impossible. Although techniques for cryopreservation of stage I and II oocytes (diameter, 90-350 μm) (14) and ...

Skeletal resistance to parathyroid hormone (PTH) is well known to the phenomenon in chronic renal failure patient, but the detailed mechanism has not been elucidated. In the process of analyzing an animal model of renal failure with low bone turnover, we demonstrated decreased expression of PTH receptor (PTHR) accompanying renal dysfunction in this model. In the present study, we focused on the accumulation of uremic toxins (UTx) in blood, and examined whether indoxyl sulfate (IS), a UTx, is associated with PTH resistance. We established primary osteoblast cultures from mouse calvariae and cultured the cells in the presence of IS. The intracellular cyclic adenosine 3',5' monophosphate (cAMP) production, PTHR expression, and free radical production in the primary osteoblast culture were studied. We found that the addition of IS suppressed PTH-stimulated intracellular cAMP production and decreased PTHR expression in this culture system. Free radical production in osteoblasts increased depending on the concentration of IS added. Furthermore, expression of organic anion transporter-3 (OAT-3) that is known to mediate cellular uptake of IS was identified in the primary osteoblast culture. These results suggest that IS taken up by osteoblasts via OAT-3 present in these cells augments oxidative stress to impair osteoblast function and downregulate PTHR expression. These finding strongly suggest that IS accumulated in blood due to renal dysfunction is at least one of the factors that induce skeletal resistance to PTH.

No abstract

The sexual plasticity of fish gonads declines after the sex differentiation period; however, information about the plasticity of the germ cells themselves after sex differentiation is limited. Using rainbow trout (Oncorhynchus mykiss), we recently established a novel germ cell transplantation system that provides a unique platform with which to dissect the developmental and cellular mechanisms underlying gametogenesis. Using this technique, we show here that transplanted ovarian germ cells isolated from 6- to 9-month-old donors can colonize sexually undifferentiated embryonic gonads and resume gametogenesis. Ovarian germ cells containing oogonia and early oocytes isolated from female rainbow trout were transplanted into the peritoneal cavities of hatching-stage fry of both sexes and the behavior of the donor cells was observed. The transplanted ovarian germ cells migrated towards the recipient gonads, interacted with embryonic gonadal somatic cells, proliferated rapidly, and eventually differentiated into eggs in female recipients and sperm in male recipients. Furthermore, the donor-derived eggs and sperm obtained from the recipient fish were functional and were able to produce normal offspring. These findings indicate that mitotic germ cells, the oogonia, possess a high level of sexual plasticity.

No abstract

We describe the surface plasmon resonance (SPR) detection of an enzymatic turnover reaction and the measurement of glucose concentration using a multienzyme layer modified gold electrode. We constructed an osmium redox polymer mediated enzyme sensor on a gold thin-film electrode and monitored electrochemical reaction by SPR measurement. Unlike the usual binding assay with SPR, here we used SPR to detect the redox state of an electron mediator that was the result of the electron-transfer reaction of sequential enzymatic reactions. Therefore, the degree of refractive index change was independent of the dielectric property of the substrate and enzymatic molecular recognition was converted to refractive index change with amplification. For the quantitative evaluation of glucose with this method, we used chronopotentiometry and a linear relation was obtained between the glucose concentration and the rate of refractive index change.

The effect of thyrotrophin-releasing hormone (TRH) or luteinizing hormone-releasing hormone (LH-RH) on plasma levels of growth hormone (GH), prolactin (PRL), thyrotrophin (TSH), and luteinizing hormone (LH), were studied in patients with anorexia nervosa. The basal plasma GH levels were elevated in 6 of 11 patients studied. Intravenous injection of synthetic TRH (500 μg) significantly raised the plasma GH levels in 9 of 11 patients. The peak values of plasma GH after TRH ranged from 6.0 to 31.5 ng/ml. Plasma GH concentrations also increased following the administration of synthetic LH-RH (100μg) in 1 of 7 patients. The intravenous injection of saline solution caused no significant change in plasma GH in these patients. The plasma LH responses to LH-RH were significantly blunted in all patients, whereas the plasma PRL and TSH responses to TRH were almost normal in the patients examined. These results suggest that the hypothalamo-pituitary function regulating GH and LH secretion is altered in patients with anorexia nervosa.

Abnormalities in bone turnover, mineralization, and volume represent one of the three components of chronic kidney disease–related mineral and bone disorder (CKD-MBD). The risk of hip fracture is considerably high, while the risk of spinal compression fracture may not be more elevated among CKD patients than in general population. The relationship between bone fracture and bone mineral density in CKD patients is more complex than in those without kidney disease. An increase in the rate of falls has been reported to be a major cause of high hip fracture risk among CKD patients; however, it certainly is not the only underlying mechanism. Abnormal parathyroid function is not likely to be a major cause of hip fracture among CKD patients. In experimental CKD animals, mechanical elasticity properties of long bones showed an inverse correlation with kidney function. The deterioration of bone elasticity showed a significant correlation with bone biochemical changes. Of note, administration of the oral absorbent AST-120 was capable of preventing both changes. These findings suggest that uremic toxins cause a deterioration of bone material properties, and changes in material properties disturb bone elasticity. This disease concept cannot be considered to be a direct consequence of CKD-MBD. We therefore would like to call it ‘uremic osteoporosis'. This entity may be a major cause of increased hip fracture risk among CKD patients.