Mouse pronuclear oocytes and 2-cell embryos derived from in vitro fertilization were cryopreserved by a novel simple vitrification procedure. Most cryopreserved oocytes/embryos were morphologically normal after warming, and 89-92% of them developed to the blastocyst stage during the culture. Moreover, the rate of morphologically normal pronuclear oocytes after being repeatedly cooled and warmed three times was as high as that of oocytes cooled and warmed only once, and 85% of them developed to the blastocyst stage. In addition, 43-57% of the cryopreserved oocytes/embryos transferred to recipients had developed normally to live fetuses observed on day 18.5 of pregnancy.

Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disorder, is an inherited disease characterized by the accumulation of intracellular unesterified cholesterol. A solubilizing agent of lipophilic compounds, 2-hydroxypropyl-β-cyclodextrin (HPBCD), is an attractive drug candidate against NPC disease. However, establishment of the optimum dosage of HPBCD remains to be determined. In this study, we evaluated the effective dosage of HPBCD in NPC model (Npc1 / ) mice, and determined serum HPBCD concentrations. Subcutaneous injection of 1000-4000 mg/kg HPBCD improved the lifespan of Npc1 / mice. In addition, liver injury and cholesterol sequestration were significantly prevented by 4000 mg/kg HPBCD in Npc1 / mice. Serum HPBCD concentrations, when treated at the effective dosages (1000-4000 mg/kg), were approximately 1200-2500 µg/mL at 0.5 h after subcutaneous injection, and blood HPBCD concentrations were immediately eliminated in Npc1 / mice. Furthermore, we examined serum HPBCD concentrations when treated at 40000 mg (approximately 2500 mg/kg) in a patient with NPC. We observed that the effective concentration in the in vivo study using Npc1 / mice was similar to that in the patient. In the patient, systemic clearance and the volume of distribution of HPBCD were in accordance with the glomerular filtration rate and extracellular fluid volume, respectively. These results could provide useful information for developing the optimal dosage regimen for HPBCD therapy when administered intravenously to NPC patients.

We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken beta-actin promoter-driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1- to 2-cell stage. RNA transcripts of the luciferase gene were first detected in the 1-cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon-count imaging, functional luciferase was identified at the 2-cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1-cell embryos, although paternally derived protein is not synthesized until the 2-cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1-cell stage.

Unfertilized mouse oocytes were cooled rapidly by directly plunging them into liquid nitrogen, immediately after exposure to a highly concentrated solution (modified VS1: 2.53 M-dimethyl sulphoxide, 2.36 M-acetamide, 1.19 M-propylene glycol, 5.4% (w/v) polyethylene glycol (Mr 8000) in PB1), and later warmed in a 37 degree C waterbath. After warming, 305 out of 348 oocytes (87.6%) were morphologically normal. After fertilization in vitro of cryopreserved oocytes, the proportions of pronuclear oocytes and 2-cell embryos 5 and 24 h after insemination were 81.6% (124/152) and 78.4% (120/153), respectively. All 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 45.8% (55/120) developed to normal young.

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Although the cellular and molecular mechanisms governing angiogenesis are only beginning to be understood, signaling through endothelial-restricted receptors, particularly receptor tyrosine kinases, has been shown to play a pivotal role in these events. Recent reports show that EphB receptor tyrosine kinases and their transmembrane-type ephrin-B2 ligands play essential roles in the embryonic vasculature. These studies suggest that cell-to-cell repellent effects due to bidirectional EphB/ephrin-B2 signaling may be crucial for vascular development, similar to the mechanism described for neuronal development. To test this hypothesis, we disrupted the precise expression pattern of EphB/ephrin-B2 in vivo by generating transgenic (CAGp-ephrin-B2 Tg) mice that express ephrin-B2 under the control of a ubiquitous and constitutive promoter, CMV enhancer-β-actin promoter-β-globin splicing acceptor (CAG). These mice displayed an abnormal segmental arrangement of intersomitic vessels, while such anomalies were not observed in Tie-2p-ephrin-B2 Tg mice in which ephrin-B2 was overexpressed in only vascular endothelial cells (ECs). This finding suggests that non-ECs expressing ephrin-B2 alter the migration of ECs expressing EphB receptors into the intersomitic region where ephrin-B2 expression is normally absent. CAGp-ephrin-B2 Tg mice show sudden death at neonatal stages from aortic dissecting aneurysms due to defective recruitment of vascular smooth muscle cells to the ascending aorta. EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells plays an essential role in vasculogenesis, angiogenesis, and vessel maturation.

Cryopreservation of mouse spermatozoa has become the foremost technique for preserving large numbers of different strains of mice with induced mutations. Recently, we have established procedures for cryopreservation of mouse spermatozoa and in vitro fertilization using cryopreserved spermatozoa to obtain a relatively high fertilization rate. This chapter attempts to show these procedures in simple terms.