Marked improvement of fertility of cryopreserved C57BL/6J mouse sperm by depletion of Ca2+ in medium.

Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca(2+)), phosphate (PO(4)(3-)) and lactate. In all media containing no Ca(2+), including medium lacking Ca(2+), lacking Ca(2+) and PO(4)(3-), lacking Ca(2+) and lactate and lacking Ca(2+), PO(4)(3-) and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca(2+) were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca(2+). In conclusion, preincubation of thawed sperm in medium containing no Ca(2+) markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca(2+) is practical for use in cryopreserved C57BL/6J sperm.

Development of immunocompetent lymphocytes in vivo from murine umbilical cord blood cells.

BACKGROUND: Reports concerning the immunological functions of lymphocytes derived from umbilical cord blood cell (UCBC) have been limited. METHODS: In murine syngeneic transplantation system using green fluorescent protein transgenic donors, UCBC-derived lymphocytes were studied for their immunological competence. RESULTS: Hematopoietic stem cells (HSC) among UCBC differentiated in the recipients into phenotypically mature T and B lymphocytes. The T lymphocytes were capable of specific recognition of major histocompatibility complex/peptide complex and of the subsequent activation. The antigen-induced CD4(+) T cells produced lymphokine upon in vitro antigen stimulation. CD8(+) T cells simulated in the mixed lymphocyte culture could lyze specific target cells. Furthermore, RAG2(-/-) mice reconstituted with UCBC mounted specific antibody responses to T-dependent antigen comparable to those by bone marrow chimeras and also rejected allogeneic skin grafts. CONCLUSION: Collectively the data indicated that T and B lymphocytes derived from UCBC-HSC are fully competent in immunological terms.

Successful pregnancies after transplantation of frozen-thawed mouse ovaries into chimeric mice that received lethal-dose radiation.

OBJECTIVE: To study whether fecundity was recovered in mice into which umbilical cord blood cells (UCBCs) were transfused after lethal-dose radiation, followed by transplantation of frozen-thawed ovaries. DESIGN: Prospective basic research study. SETTING: Academic research laboratory. ANIMAL(S): Female C57BL/6 mice as recipients of UCBCs and ovaries, male B6C3F1 mice for mating, and green fluorescent protein (GFP)-transgenic mice: 18.5-day-old fetuses (-/+) for UCBCs and adult GFP mice (+/+) for ovarian tissues. INTERVENTION(S): The UCBCs were transfused into each irradiated mouse, with GFP+ ovaries transplanted 4 weeks later. The chimeric mice were mated 3 weeks after ovarian transplantation and were examined 14 to 16 weeks after the transfusion of UCBCs. MAIN OUTCOME MEASURE(S): Percentage of chimerism, number of GFP+ pups. RESULT(S): The percentage of chimerism in these mice tends to increase with the radiation dose. The recovery of fecundity was observed in the chimeric mice that were transplanted with fresh and previously vitrified ovaries after exposure to radiation. CONCLUSION(S): Even when the exposure dose of radiation administered as pretreatment is lethal, the fecundity of recipients can be maintained if their ovaries are cryopreserved before they are exposed to radiation.

Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation.

Mesenchymal stem cells (MSCs) are thought to be multipotential, capable of differentiating into multiple lineages. We attempted to characterize rat cells derived from fetal circulating blood (FCBCs) that displayed a fibroblastic morphology and differentiated into osteoblastic and chondrocytic lineages. Notably, they differentiated into a chondrocyte-specific phenotype on plastic culture dishes in medium supplemented only with 10% fetal bovine serum (FBS) without the use of a three-dimensional culture substrate. Bone marrow-derived cells did not convey such phenotypic expression under the same conditions. The characteristic features of these cells were analyzed by reverse transcription polymerase chain reaction, immunohistological and von Kossa staining, and by immuno-dot blotting. In one population, expression of collagen types II and X was detected in differentiated cells at the same levels as observed in chondrocytes derived from rat rib cartilage. In another population, parathyroid hormone receptor, alkaline phosphatase, and osteocalcin were also expressed at levels almost equal to those observed in long bone-derived osteoblasts. After 3 weeks in culture, extensively condensed cell masses, stained with anti-type II collagen antibody, could be distinguished histologically from small, multilayered, von Kossa-positive nodules, which stained with anti-osteocalcin, but not with anti-type II collagen antibody. In addition, the FCBCs differentiated into adipogenic cells in the presence of methyl-isobutyl xanthine, dexamethasone, insulin, and indomethacin. These cells expressed PPARgamma2 mRNA and accumulated lipid vesicles detectable by Oil red-O staining. Our findings suggest that FCBCs have the potential to readily differentiate into multiple lineages and that they are distinct from mesenchymal stem cells derived from bone marrow or circulating blood from more mature and adults in their spontaneous differentiation in the absence of specific factors such as transforming growth factor-beta (TGF-beta) or dexamethasone, or a three-dimensional culture environment.

Full reconstitution of hematopoietic system by murine umbilical cord blood.

BACKGROUND: Murine umbilical cord blood cells (UCBCs) were studied for their ability to reconstitute the hematopoietic system. METHODS: On average, 150 microL of cord blood per fetus containing 1.2 to 2 x 10(4) nucleated cells were collected from day 18.5 fetal umbilical cord, and 3 to 6 x 10(3) cells per fetus were obtained after separation by gradient centrifugation. RESULTS: Although lineage marker-, c-Kit+, and Sca-1+ cells were detectable among UCBCs, cells designated to be in the side population (SP) by Hoechst 33342 staining were hardly detectable within this population; the frequency of cells of this phenotype was less than 1 of 105. Instead, the lineage marker-, c-Kit+, and Sca-1- population contained a considerable number of SP cells. Nevertheless, UCBCs obtained from fetuses of green fluorescent protein-transgenic mice successfully reconstituted the blood cells of lethally irradiated recipients. Fluorescent cells could be readily detected in every blood cell lineage and among immature cell populations. Furthermore, fluorescent SP cells sorted from the recipient bone marrow cells could also reconstitute the blood cells in the secondary recipients, indicating that UCBCs also replenished bone marrow stem cells. CONCLUSION: Murine UCBC could fully reconstitute the hematopoietic system of lethally irradiated recipients including hematopoietic stem cells in bone marrow.

Successful cryopreservation of mouse ovaries by vitrification.

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.