ABSTRACT
Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.
Subject(s)
Cryopreservation/methods , Epididymis/drug effects , Glutathione/pharmacology , Lysophospholipids/pharmacology , Sperm Motility/drug effects , Spermatozoa/drug effects , Sphingosine/analogs & derivatives , Animals , Embryo Implantation/drug effects , Embryo Implantation/physiology , Epididymis/cytology , Epididymis/physiology , Female , Fertilization/drug effects , Fertilization/physiology , Fertilization in Vitro/methods , Freezing , Male , Mice , Mice, Transgenic , Organ Preservation Solutions/chemistry , Pregnancy , Sperm Motility/physiology , Spermatozoa/physiology , Sphingosine/pharmacologyABSTRACT
The exchange of genetically engineered mouse strains between research facilities requires transporting fresh mouse sperm under refrigerated temperatures. Although sperm generally maintains fertility for 48 h at cold temperatures, in vitro fertilization rates of C57BL/6 mouse sperm are low after 48-h cold storage. Furthermore, 48 h is often not sufficient for the specimens to reach their destinations. To increase the availability of this technology, we aimed to extend the cold storage period while maintaining sperm fertility. In this study, we determined the optimal medium for sperm preservation and evaluated the effect of reduced glutathione in the fertilization medium on sperm fertility after cold storage. We found that higher fertility levels were maintained after 72-h cold storage in the preservation medium Lifor compared with storage in paraffin oil, M2 medium, or CPS-1 medium. In addition, 1.0 mM glutathione enhanced sperm fertility. After transporting sperm from Asahikawa Medical University to our laboratory, embryos were efficiently produced from the cold-stored sperm. After transfer, these embryos developed normally into live pups. Finally, we tested the transport system using genetically engineered mouse strains and obtained similar high fertilization rates with all specimens. In summary, we demonstrated that cold storage of sperm in Lifor maintains fertility, and glutathione supplementation increased the in vitro fertilization rates of sperm after up to 96 h of cold storage. This improved protocol provides a simple alternative to transporting live animals or cryopreserved samples for the exchange of genetically engineered mouse strains among research facilities.
Subject(s)
Cryopreservation/methods , Epididymis/cytology , Mice/physiology , Semen Preservation/methods , Spermatozoa/cytology , Animals , Cryopreservation/veterinary , Cryoprotective Agents/metabolism , Embryo Transfer/methods , Embryo Transfer/veterinary , Female , Fertilization , Fertilization in Vitro/methods , Fertilization in Vitro/veterinary , Glutathione/metabolism , Male , Mice/embryology , Mice, Inbred C57BL , Semen Preservation/veterinary , Sperm Motility , Spermatozoa/metabolismABSTRACT
OBJECTIVE: Chronic hypoxia has been recognized as a key regulator in renal tubulointerstitial fibrosis, as seen in diabetic nephropathy, which is associated with the activation of hypoxia-inducible factor (HIF)-1α. We assess here the effects of the biguanide, metformin, on the expression of HIF-1α in diabetic nephropathy using renal proximal tubular cells and type 2 diabetic rats. RESEARCH DESIGN AND METHODS: We explored the effects of metformin on the expression of HIF-1α using human renal proximal tubular epithelial cells (HRPTECs). Male Zucker diabetic fatty (ZDF; Gmi-fa/fa) rats were treated from 9 to 39 weeks with metformin (250 mg â kg(-1) â day(-1)) or insulin. RESULTS: Metformin inhibited hypoxia-induced HIF-1α accumulation and the expression of HIF-1-targeted genes in HRPTECs. Although metformin activated the downstream pathways of AMP-activated protein kinase (AMPK), neither the AMPK activator, AICAR, nor the mTOR inhibitor, rapamycin, suppressed hypoxia-induced HIF-1α expression. In addition, knockdown of AMPK-α did not abolish the inhibitory effects of metformin on HIF-1α expression. The proteasome inhibitor, MG-132, completely eradicated the suppression of hypoxia-induced HIF-1α accumulation by metformin. The inhibitors of mitochondrial respiration similarly suppressed hypoxia-induced HIF-1α expression. Metformin significantly decreased ATP production and oxygen consumption rates, which subsequently led to increased cellular oxygen tension. Finally, metformin, but not insulin, attenuated tubular HIF-1α expression and pimonidazole staining and ameliorated tubular injury in ZDF rats. CONCLUSIONS: Our data suggest that hypoxia-induced HIF-1α accumulation in diabetic nephropathy could be suppressed by the antidiabetes drug, metformin, through the repression of oxygen consumption.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Diabetic Nephropathies/prevention & control , Hypoglycemic Agents/therapeutic use , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kidney Tubules, Proximal/metabolism , Metformin/therapeutic use , Oxygen Consumption/physiology , Adenylate Kinase/genetics , Adenylate Kinase/metabolism , Analysis of Variance , Animals , Cell Line , Cysteine Proteinase Inhibitors/pharmacology , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/genetics , Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Hypoglycemic Agents/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immunohistochemistry , Insulin/pharmacology , Insulin/therapeutic use , Kidney Tubules, Proximal/drug effects , Leupeptins/pharmacology , Male , Metformin/pharmacology , Oxygen Consumption/drug effects , Rats , Rats, Zucker , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
At refrigerated temperatures, mouse embryos can maintain developmental ability for short periods. Previously, we succeeded in transporting vitrified and warmed 2-cell mouse embryos while maintaining developmental ability at refrigerated temperatures for 50 h. Transport of nonfrozen embryos is an easier and more useful means of exchanging genetically engineered mice between laboratories than is transport of cryopreserved embryos. Here we examined the developmental ability of transported 2-cell embryos that were produced through in vitro fertilization using cryopreserved sperm. Results show that 2-cell embryos produced by cryopreserved sperm can develop into blastocysts after cold storage for 24, 48, and 72 h. Transported 2-cell embryos produced by cryopreserved sperm yielded a favorable number of pups in all of the receiving laboratories after transport lasting 48 to 52 h. In summary, cold storage and transport of 2-cell embryos derived from cryopreserved sperm at refrigerated temperatures provides a novel means of transporting genetically engineered mice as an alternative to the transport of cryopreserved embryos and sperm.
Subject(s)
Cold Temperature , Cryopreservation , Embryo Culture Techniques , Spermatozoa , Animals , Culture Media , Embryo Transfer , Embryonic Development , Female , Fertilization in Vitro , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , TransportationABSTRACT
Blood vessels deliver oxygen and nutrients to tissues, and vascular networks are spatially organized to meet the metabolic needs for maintaining homeostasis. In contrast, the vasculature of tumors is immature and leaky, resulting in insufficient delivery of nutrients and oxygen. Vasculogenic processes occur normally in adult tissues to repair "injured" blood vessels, leading us to hypothesize that bone marrow mononuclear cells (BMMNC) may be able to restore appropriate vessel function in the tumor vasculature. Culturing BMMNCs in endothelial growth medium resulted in the early outgrowth of spindle-shaped attached cells expressing CD11b/Flt1/Tie2/c-Kit/CXCR4 with proangiogenic activity. Intravenous administration of these cultured vascular proangiogenic cells (VPC) into nude mice bearing pancreatic cancer xenografts and Pdx1-Cre;LSL-Kras(G12D);p53(lox/+) genetically engineered mice that develop pancreatic ductal adenocarcinoma significantly reduced areas of hypoxia without enhancing tumor growth. The resulting vasculature structurally mimicked normal vessels with intensive pericyte coverage. Increases in vascularized areas within VPC-injected xenografts were visualized with an ultrasound diagnostic system during injection of a microbubble-based contrast agent (Sonazoid), indicating a functional "normalization" of the tumor vasculature. In addition, gene expression profiles in the VPC-transplanted xenografts revealed a marked reduction in major factors involved in drug resistance and "stemness" of cancer cells. Together, our findings identify a novel alternate approach to regulate abnormal tumor vessels, offering the potential to improve the delivery and efficacy of anticancer drugs to hypoxic tumors.
Subject(s)
Adenocarcinoma/blood supply , Adenocarcinoma/surgery , Bone Marrow Transplantation/methods , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/surgery , Adenocarcinoma/metabolism , Angiogenic Proteins/biosynthesis , Animals , CD11b Antigen/biosynthesis , Cell Growth Processes/physiology , Cell Hypoxia/physiology , Cell Line, Tumor , Cytokines/biosynthesis , Drug Resistance, Neoplasm , Female , Humans , Mice , Mice, Nude , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Oxygen/blood , Oxygen/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Phenotype , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. CONCLUSIONS/SIGNIFICANCE: We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.
Subject(s)
Angiopoietin-1/genetics , Bone Marrow Cells/metabolism , Carcinoma, Pancreatic Ductal/blood supply , Hedgehog Proteins/physiology , Insulin-Like Growth Factor I/genetics , Neovascularization, Pathologic/physiopathology , Pancreatic Neoplasms/blood supply , Animals , Bone Marrow Transplantation , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Hedgehog Proteins/antagonists & inhibitors , Immunohistochemistry , Mice , Mice, Nude , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Transplantation, Heterologous , Veratrum Alkaloids/pharmacologyABSTRACT
Cryopreservation of 2-cell embryos is an effective technology for storage of genetically engineered mouse strains. Transport of genetically engineered mice between laboratories has frequently been performed using such cryopreserved 2-cell embryos. However, the receiving laboratory requires proficient skills and special instruments to obtain live young from cryopreserved and transported embryos. Therefore, in this study, we tried to address the storage and transport of vitrified/warmed 2-cell embryos at a cold temperature. In cold storage experiments, the development rates of 2-cell embryos stored in M2 medium for 24, 48 and 72 h into blastocysts were relatively high (83%, 63% and 43%, respectively). Although, 2-cell embryos stored in PB1 and mWM maintained the developmental potency for 24h, the rates were markedly decreased to low levels after 48 h (PB1: 0%; mWM: 5%). In transport experiments, many pups were obtained from vitrified/warmed 2-cell embryos transported at a cold temperature in all receiving laboratories (incidence of successful development: 49%; 249/511). In summary, short-term storage and transport of vitrified/warmed 2-cell embryos in M2 medium at a cold temperature can maintain their ability to develop into live young.