Production of Functional Gametes from Cryopreserved Primordial Germ Cells of the Japanese Quail

The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at –196 C for 77–185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (85.5% vs. 95.1%). Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into recipient embryos migrated toward and were incorporated into recipient gonads, although the number of PGCs settled in the gonads was 48.5% lower in the frozen group than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that one female and two male recipients produced live progeny derived from the frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than that of unfrozen controls, but the difference was not significant (4.0 vs. 14.0%). These results revealed that freeze-thaw treatment causes a decrease in viability, migration ability and germline transmission ability of PGCs in quail.     

Occurrence of Spontaneous Tumors in the Central Nervous System (CNS) of F344 and SD Rats

In order to accurately assess the carcinogenicity of chemicals with regard to rare tumors such as rat CNS tumors, sufficient information about spontaneous tumors are very important. This paper presents the data on the type, incidence and detected age of CNS tumors in F344/DuCrlCrlj (a total of 1363 males and 1363 females) and Crl:CD(SD) rats (a total of 1650 males and 1705 females) collected from in-house background data-collection studies and control groups of carcinogenicity studies at our laboratory, together with those previously reported in F344 and SD rats. The present data on F344/DuCrlCrlj rats (F344 rats) and Crl:CD(SD) rats (SD rats) clarified the following. (1) The incidences of all CNS tumors observed in F344 rats were less than 1%. (2) The incidences of malignant astrocytoma and granular cell tumor were higher in male SD rats than in female SD rats. (3) The incidences of astrocytoma and granular cell tumor were higher in SD rats than in F344 rats. (4) Among astrocytoma, oligodendroglioma and granular cell tumor, oligodendroglioma was detected at the youngest age, followed by astrocytoma, and ultimately, granular cell tumor developed in both strains. The incidences observed in our study were almost consistent with those previously reported in F344 and SD rats.     

Availability of a Microglia and Macrophage Marker,Iba-1, for Differential Diagnosis of Spontaneous Malignant Reticuloses from Astrocytomas in Rats

In rats, it is sometimes difficult to distinguish malignant reticuloses from astrocytomas in routine histopathological assessment. In the present study, four spontaneous brain neoplasms developing in the cerebrum of one Wistar Hannover rat and three Sprague-Dawley rats were immunohistochemically examined using microglia and macrophage markers. Histopathologically, these neoplasms were localized mainly in the cerebral cortex, hypothalamus or piriform lobe, and the portions showing solid growth did not show characteristic cellular arrangement but had an indistinct boundary with the surrounding brain parenchyma. Neoplastic cells had oval or pleomorphic small nuclei with abundant eosinophilic cytoplasm. Two cases showed neoplastic cell infiltration into the meninges and perivascular spaces. Silver staining showed lack of reticulin fiber production in the stroma of the neoplasms. Immunohistochemically, the neoplastic cells were strongly positive for Iba-1 and sporadically positive for CD68 in all four cases. On the basis of these results, all the neoplasms examined here could be distinguished from astrocytomas and diagnosed as malignant reticuloses. Thus, immunohistochemical demonstration of microglia/macrophage characters, such as using Iba-1, is considered to be helpful for differential diagnosis of malignant reticuloses from astrocytomas among spontaneously occurring primary brain neoplasms in rats.     

Highly invasive intracranial malignant schwannoma in a rat

A highly invasive intracranial malignant schwannoma containing several masses was detected in a 28-week-old male Crl:CD(SD) rat. Macroscopically, 3 masses were noted in the cranial cavity; one was present at the bottom of the cranial cavity and involved the trigeminal nerve, and the other two were in the parietal bone. Histologically, each mass consisted of fusiform cells with interlacing fascicular, wavy and nuclear pseudopalisading arrangements and round cells with cystic lesions. The tumor cells invaded not only the brain but also the parietal bone. In the brain, the tumor cells infiltrated diffusely into the leptomeningeal and perivascular spaces and parenchyma, in which the tumor cell morphology and invasive pattern closely resembled those of malignant astrocytoma and malignant reticulosis. Immunohistochemically, the tumor cells in the masses showed positive reactions for both S-100 protein and GFAP, while those in the cerebral invasion sites were negative for GFAP and less positive for S-100 protein. Electron microscopically, a single basal lamina layer and short intricate cell processes were confirmed in the tumor cells. From these results, the present tumor was diagnosed as a malignant schwannoma arising in the cranial cavity, probably originating from the trigeminal nerve. The present tumor is considered to be a relatively unique malignant schwannoma based on its growth and invasion patterns.     

Influence of delayed timing of owners' actions on the behaviors of their dogs,Canis familiaris

This study examined the influence of delayed actions from the owner, including commands, reinforcement, and punishment, on already-learned behaviors in 10 dogs. The delay times were set to 2.0, 1.0, 0.5, 0.27, and 0.13 seconds (s). Responses to commands with a delay (Delayed) were compared with those that were not delayed (Nondelayed). The results indicated that appropriate responses to commands decreased in 2.0, 1.0, and 0.5 s delayed conditions. As delay time increased, response to commands decreased. The numbers of commands used by handlers to make their dogs obey was significantly increased with a 2.0 and 1.0 second delay compared to the nondelayed trials. The time required for dogs to obey the commands was significantly increased in 2.0, 1.0, and 0.5 s delayed conditions compared to those of the nondelayed trials. There were no significant differences between the 0.27 s, 0.13 s, and the nondelayed condition. These results suggest that timing is an important factor affecting a dog's behavior not only while learning new things, but also in the production of learned desirable behaviors that could occur during everyday interactions.     

Evaluation of a d-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.