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Abstract. The cryopreservation of ovarian tissues is a technology with significant potential for the preservation of the genetic resource materials of working dogs, including guide dogs for the blind. However, no attempt has been reported on cryopreservation of the canine ovary. Thus, we evaluated a vitrification method for cryopreservation of canine ovaries and determined the potential functionality of vitrified-warmed canine ovaries by means of transplantation into non-obese diabeticsevere combined immunodeficiency (NOD-SCID) mice. All ovarian tissues cryopreserved by vitrification were morphologically normal in terms of histology. Cryopreserved ovaries were transplanted into the ovarian bursa of the NOD-SCID mice, and the xenografts were recovered from 23 of 23 mice (100%) 4 weeks after the operation. The transplanted canine tissue was tightly adhered to the mouse ovary. Although antral follicle formation did not occur after grafting, proliferating cell nuclear antigen immunoreactivity was detectable in many of the granulosa cells in the primary follicles of the grafts. These results indicate that cryopreservation of the canine ovary by vitrification appears to have the potential to restore endocrine function and ovulation potential.

SummaryRecent studies have revealed that 10-20% of CD4+8 -or CD4-8-thymocyte populations contain NKI.1 + T cell receptor (TCR)-cff~ + cells. This subpopulation shows characteristics that are different from NKI.1-CD4 § or NKI.1-CD8 + T calls and seems to have developed in a manner different from NKI.1-T cells. Although extensive studies have been performed on the NKI.1 + TCR-od~8 + thymocytes, the physiological role of the NKI.1 + TCR-c~/~/+ thymocytes has been totally unclear. In the present study, we found that fleshly isolated NKI.1 + TCR-odB + thymocytes, but neither whole thymocytes nor lymph node T cells, directly killed CD4 +8 § thymocytes from normal syngeneic or allogeneic mice by using a long-term cytotoxic assay in which flow cytometry was used to detect the cytotoxidty. However, only weak cytotoxicity was detected against thymocytes from lpr mice on which the Fas antigen that transduces signals for apoptosis into the cells is not expressed. Furthermore, the NKI.1 + TCR-ot/B + thymocytes exhibited high cytotoxicity against T lymphoma targets transfected withj~s genes as compared with the parental T lymphoma targets or target cells transfected with mutatedJas genes, which lack the function of transducing signals. On the other hand, NKI.1 § effector thymocytes from gld mice that carry a point mutation in Fas ligand did not kill thymocyte targets from normal mice. The present findings, thus, consistently suggest that the NKI.1 + TCR-ot/~8 + thymocytes kill a subpopulation among CD4+8 + thymocytes via Fas antigen and in this way regulate generation of T lineage cells in the thymus.

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The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.

A possible association between intrauterine inflammation and impairments of lung development has been suggested. The purpose of this study is to determine the influence of a potent proinflammatory agent, intra-amniotic lipopolysaccharide (LPS), on lung development. At 21 d gestation, an intra-amniotic injection of 1 microg LPS was administered to two subgroups of WKAH rats. One subgroup received only LPS and the other received LPS plus a fetal intraperitoneal dose of 0.25 microg granulocyte-colony stimulating factor (hrG-CSF) to produce peripheral blood neutrophilia. A third subgroup received hrG-CSF only, and a control group received maternal intraamniotic and fetal intraperitoneal normal saline. All pups were delivered by cesarean section at 22 d (term, 22.5 d) and maintained under identical conditions. Left upper lungs were obtained for morphometric analysis at 1, 3, 7, 14, 21, 45, and 60 d of age. Morphometric analysis indicated that changes in alveolar surface density (Sv), average alveolar radius (r), and numerical density of alveoli (nv) all showed that there were fewer and larger alveoli in rat lungs that had been exposed to LPS, but not to hrG-CSF alone or saline. LPS-exposed alveoli showed fewer secondary septa, suggesting an arrest of alveolarization. No destructive changes were observed in any alveoli. We concluded that these changes could be caused purely by intra-amniotic LPS. These abnormalities closely mimic those of new bronchopulmonary dysplasia. The LPS damage model may be applicable to further studies of the pathophysiology of new BPD.

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