ABSTRACT
Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a ß-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.
Subject(s)
Amylases , Cathepsin B , Animals , Rats , Adrenergic beta-Agonists/pharmacology , Amylases/metabolism , Cathepsin B/metabolism , Cytoplasmic Granules/metabolism , Isoproterenol/pharmacology , Parotid Gland/metabolism , Secretory Vesicles/metabolismABSTRACT
Objective: To identify factors that affect salivary gland recovery, we investigated the expression and function of bone morphogenetic protein 2 (BMP2) in mice. Materials and Methods: Using a micro clip, mice parotid glands were removed 7 days after the ligation of the unilateral parotid excretory duct. Thereafter, they were weighed and stained with hematoxylin and eosin, and BMP2 expression was examined via real-time reverse transcription-polymerase chain reaction. Primary cultures of parotid glands were prepared, and BMP2 protein was added to the culture medium for 48 hr to examine its effect on cell proliferation. E-cadherin and vimentin expression was examined using western blotting. Finally, immunohistochemical staining using an anti-Ki67 antibody was performed. Results: Duct-ligated parotid glands weighed less than those that were collected after sham surgery and showed acinar cell atrophy. They also showed higher BMP2 expression than control glands. Primary-cultured parotid acinar cells supplemented with BMP2 showed higher proliferative potential than control cells. Furthermore, they showed E-cadherin, but not vimentin, expression, and their percentage of Ki67-positive cells were higher than that corresponding to the controls. Conclusions: Injury to salivary glands by excretory duct ligation increased BMP2 expression, which may be involved in maintaining salivary gland function by inducing acinar cell proliferation.
ABSTRACT
Waste management for radioiodine is a key issue for the sustainable nuclear fuel cycle. The iodine adsorption behavior on a bed column of a silver-impregnated alumina sorbent (AgA) under conditions designed to match those of the Rokkasho reprocessing facility dissolver off-gas (DOG) system was investigated using different volatilized iodine concentrations. Cross-sectional observations of iodine-bearing AgA grains revealed that iodine was adsorbed as silver iodide and silver iodate, and gradually distributed from the surface to the inside of the AgA. The iodine distribution throughout the AgA beds allowed us to estimate the length of the mass-transfer zone. This suggests that the iodine load fraction in AgA (adsorbed iodine/total impregnated silver) will be averaged to 50% in the expected facility equipment design. This study also describes the waste form durability after disposal. To reproduce the average iodine loading in the waste form, 100%-loaded AgA grains were mixed with an equal amount of commercially available alumina reagents and consolidated through hot isostatic pressing at 175 MPa and 1,325°C for 3 h. The resultant 50%-loaded solid was used for the static leaching test over 4.5 years, where the leached iodine was less than 0.2% under simple reducing conditions. This suggested that the HIPed solid of AgA from Rokkasho DOG showed preferable water resistance for after disposal safety.
ABSTRACT
The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the ß-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.
Subject(s)
Acinar Cells/metabolism , Cell Movement/physiology , Protein Transport/physiology , Secretory Pathway/physiology , Amylases/metabolism , Animals , Cells, Cultured , Exocytosis/physiology , Parotid Gland/metabolismABSTRACT
Salivary gland atrophy and consequent hyposalivation are serious problems in clinical dentistry, as saliva regulates the environment of the oral cavity. To clarify the mechanisms underlying salivary gland dysfunction, a system for primary culture of parotid acinar cells has been established. It has been reported previously that the process of cell isolation from parotid glands triggers stress signaling mediated by Src and p38 mitogen-activated protein (MAP) kinase (p38), leading to dedifferentiation of acinar cells, and that an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor suppresses this activation of Src and p38, suggesting that reactive oxygen species initiate the dedifferentiation signal. The present study examined the effect of a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (also termed MCI-186 or edaravone), on activation of the stress signal and the secretory function of parotid acinar cells. Activation of p38 during cell isolation was suppressed by addition of MCI-186. The retention of the activity of amylase, a major salivary protein, and the number of amylase-containing secretory granules were improved by isolation and culture in the presence of MCI-186. In addition, calcium elevation upon stimulation with a muscarinic agonist was higher in MCI-186-treated cells than in untreated cells. These results suggest that MCI-186 (edaravone) is a promising agent for prevention of salivary gland dysfunction.
Subject(s)
Acinar Cells , Parotid Gland , Edaravone , Free Radical Scavengers , Signal TransductionABSTRACT
PURPOSE: The aim of this study was to introduce a new method of producing three-dimensional (3D) images of vertebral venous plexuses (VVPs) by 3D-MRI with and without contrast media, to identify pathoanatomical features that might accelerate or modify spinal canal stenosis. METHODS: We used a 1.5-T MRI unit with two different 3D sequences with and without contrast media. Multi planar reconstruction (MPR) images of VVPs could be obtained by volume image subtraction methods with a workstation for dural sac from whole 3D volume MPR without contrast media, using images before and after gadoteridol injection. Three patients with degenerative lumbar spine disease and one with cervical ossification of the posterior longitudinal ligament (OPLL) were studied with and without contrast media. As three patients underwent operations, we investigated intraoperative microscopic findings, and compared VVP images. RESULTS: Abundant components of internal VVPs were identified on MRI in correlation with neural tissues such as dura and nerve roots. CONCLUSIONS: Using 3D MRI without and with gadoteridol, we can evaluate morphological changes in VVP under degenerative spinal conditions. The MR anatomy of VVPs of the spine is important, as it has been implicated in many pathophysiological mechanisms and may also cause pitfalls in MRI.
Subject(s)
Dura Mater/diagnostic imaging , Imaging, Three-Dimensional/methods , Magnetic Resonance Imaging/methods , Spinal Canal/blood supply , Spinal Nerve Roots/diagnostic imaging , Spinal Stenosis/diagnostic imaging , Aged , Contrast Media , Female , Gadolinium , Heterocyclic Compounds , Humans , Image Enhancement/methods , Image Processing, Computer-Assisted/methods , Male , Middle Aged , Organometallic Compounds , Spinal Canal/diagnostic imagingABSTRACT
OBJECTIVES: Salivary acinar and duct cells show different expression patterns of claudins, which may reflect their different functions. To study the role of claudins in saliva secretion, we examined alterations in the expression patterns of cell adhesion molecules in parotid glands of γ-irradiated rats and analyzed the influence of those changes on intercellular barrier function using primary cultures of parotid acinar cells. DESIGN: Rats were γ-irradiated with doses of 5, 15 or 20Gy, and expression levels of cell adhesion molecules were examined by immunoblotting analysis. Acinar cells were isolated from parotid glands and were cultured in the absence or presence of the Src kinase inhibitor PP1. Changes in protein and mRNA expression patterns were determined by immunoblotting and by RT-PCR analyses, respectively. Intercellular barrier function was examined by measuring transepithelial electrical resistance and the paracellular flux of FITC-dextran. RESULTS: In irradiated parotid glands, the expression of claudin-4 was enhanced at 15Gy or higher, levels that induce the hyposecretion of saliva, although that increase was transient. At 30days after irradiation, expression levels of cell adhesion molecules were decreased. In primary cultures, the expression of claudin-4 was also increased transiently but the expression of claudin-3 and E-cadherin was decreased. The barrier function of tight junctions was disrupted although the localization of occludin was maintained. The Src kinase inhibitor PP1 suppressed those changes in gene expression and retained the intercellular barrier function. CONCLUSIONS: These results suggest that the inhibition of Src signaling maintains the barrier functions of intercellular junctions in salivary glands, which can be lost due to tissue injury.
Subject(s)
Acinar Cells/metabolism , Claudin-3/metabolism , Intercellular Junctions/metabolism , Parotid Gland/metabolism , Parotid Gland/radiation effects , src-Family Kinases/metabolism , Animals , Cadherins/metabolism , Cells, Cultured , Immunoblotting , Male , Occludin/metabolism , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Signal TransductionABSTRACT
Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKC-δ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKC-δ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane to the cytosol. When acini were lysed by a detergent, Triton X-100, CCK partially induced displacement of the MARCKS from the GM1a-rich detergent-resistant membrane fractions (DRMs) in which Syntaxin2 is distributed. A MARCKS-related peptide inhibited the CCK-induced amylase release. These findings suggest that MARCKS phosphorylation by PKC-δ and then MARCKS translocation from the GM1a-rich DRMs to the cytosol are involved in the CCK-induced amylase release in pancreatic acinar cells.
Subject(s)
Amylases/metabolism , Cholecystokinin/pharmacology , Intracellular Signaling Peptides and Proteins/biosynthesis , Membrane Proteins/biosynthesis , Pancreas/metabolism , Protein Kinase C/metabolism , Acetophenones/pharmacology , Animals , Benzopyrans/pharmacology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cholecystokinin/antagonists & inhibitors , Cytosol/drug effects , Cytosol/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Male , Membrane Microdomains/metabolism , Membrane Proteins/genetics , Myristoylated Alanine-Rich C Kinase Substrate , Pancreas/drug effects , Pancreas/enzymology , Phosphorylation , Protein Kinase C-delta/drug effects , Protein Kinase C-delta/metabolism , Qa-SNARE Proteins/metabolism , Rats , Rats, Sprague-Dawley , Translocation, GeneticABSTRACT
Aneurysmal degeneration of a saphenous vein graft (SVG) is a rare, but potentially fatal complication of coronary artery bypass graft (CABG) surgery. In this case report, a patient that had undergone prior CABG surgery and bare metal stent (BMS) implantation at the site of a stenotic SVG lesion presented at our hospital with chest pain, and an SVG aneurysm was detected at the previous BMS implantation site. In addition, the implanted BMS was fractured and floating in the SVG aneurysm. The SVG aneurysm was successfully occluded by percutaneous intervention, using a combination of distal covered stent deployment at the site of the anastomosis between the native coronary artery and the SVG and proximal coil embolization of the aneurysm.
Subject(s)
Aneurysm/therapy , Coronary Artery Bypass/adverse effects , Embolization, Therapeutic/instrumentation , Metals , Percutaneous Coronary Intervention/instrumentation , Prosthesis Failure , Saphenous Vein/transplantation , Stents , Aged, 80 and over , Aneurysm/diagnostic imaging , Aneurysm/etiology , Computed Tomography Angiography , Coronary Angiography/methods , Female , Humans , Percutaneous Coronary Intervention/adverse effects , Saphenous Vein/diagnostic imaging , Treatment OutcomeABSTRACT
BACKGROUND: There have been several imaging studies of cervical radiculopathy, but no three-dimensional (3D) images have shown the path, position, and pathological changes of the cervical nerve roots and spinal root ganglion relative to the cervical bony structure. The objective of this study was to introduce a technique that enables the virtual pathology of the nerve root to be assessed using 3D magnetic resonance (MR)/computed tomography (CT) fusion images that show the compression of the proximal portion of the cervical nerve root by both the herniated disc and the preforaminal or foraminal bony spur in patients with cervical radiculopathy. FINDINGS: MR and CT images were obtained from three patients with cervical radiculopathy. 3D MR images were placed onto 3D CT images using a computer workstation. The entire nerve root could be visualized in 3D with or without the vertebrae. The most important characteristic evident on the images was flattening of the nerve root by a bony spur. The affected root was constricted at a pre-ganglion site. In cases of severe deformity, the flattened portion of the root seemed to change the angle of its path, resulting in twisted condition. CONCLUSIONS: The 3D MR/CT fusion imaging technique enhances visualization of pathoanatomy in cervical hidden area that is composed of the root and intervertebral foramen. This technique provides two distinct advantages for diagnosis of cervical radiculopathy. First, the isolation of individual vertebra clarifies the deformities of the whole root groove, including both the uncinate process and superior articular process in the cervical spine. Second, the tortuous or twisted condition of a compressed root can be visualized. The surgeon can identify the narrowest face of the root if they view the MR/CT fusion image from the posterolateral-inferior direction. Surgeons use MR/CT fusion images as a pre-operative map and for intraoperative navigation. The MR/CT fusion images can also be used as educational materials for all hospital staff and for patients and patients' families who provide informed consent for treatments.
ABSTRACT
OBJECTIVE: The acinar cells of the parotid gland are filled with numerous secretory granules (SGs), which accumulate the digestion enzyme amylase. SGs mature accompanied with membrane remodelling such as fusion and budding of small vesicles. However, little is understood about the mechanism of the condensation of SG contents during maturation. In this study, we examined whether secretory proteins need a specific signal to be retained in SGs. DESIGN: To induce internalization of the luminal membrane after exocytosis, we injected the ß-adrenergic agonist isoproterenol into rats. Acinar cells were then incubated with Lucifer Yellow (LY) dye as a tracer for 3h for uptake into immature secretory granules (ISGs). To observe whether LY was retained in SGs after maturation, we continued incubating the cultured acinar cells for 2 days. RESULTS: The localization of LY into ISGs was confirmed by the following four methods: (1) co-localization of the fluorescence of LY and amylase by confocal laser microscopy, (2) detection of the fluorescence from purified ISGs, (3) secretion of the fluorescence together with amylase upon stimulation, and (4) observation of the intracellular localization of LY by electron microscopy. Moreover, we observed co-localization of some of the SGs with the fluorescence of LY after cell culture. CONCLUSIONS: Although the fusion and budding of small vesicles may contribute to the process of granule maturation, LY remained in the SGs even after maturation. These results suggest that secretory proteins that have no transport signal are not excluded from SGs, and they are retained in SGs during granule maturation in exocrine parotid glands.
Subject(s)
Acinar Cells/metabolism , Cytoplasmic Granules/metabolism , Isoproterenol/pharmacology , Parotid Gland/metabolism , Secretory Vesicles/metabolism , Amylases/metabolism , Animals , Cells, Cultured , Endocytosis , Microscopy, Confocal , Microscopy, Electron , Rats , Signal TransductionABSTRACT
The adverse effects of the anti-cancer agent cyclophosphamide (CTX) on follicular growth and ovarian angiogenesis were investigated in mice. CTX treatment irreversibly induced a loss of follicles through apoptosis and decreased microvascularization of the corpora lutea and follicles in a dose-dependent manner. Our findings demonstrated that CTX adversely affected the ovaries indicating the need to support an awareness of fertility preservation before chemotherapy is initiated.
Subject(s)
Antineoplastic Agents, Alkylating/adverse effects , Cyclophosphamide/adverse effects , Neovascularization, Physiologic/drug effects , Oogenesis/drug effects , Ovarian Follicle/drug effects , Ovary/drug effects , Animals , Antineoplastic Agents, Alkylating/administration & dosage , Apoptosis/drug effects , Corpus Luteum/blood supply , Corpus Luteum/cytology , Corpus Luteum/drug effects , Corpus Luteum/metabolism , Cyclophosphamide/administration & dosage , DNA Damage , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Developmental/drug effects , Immunohistochemistry , Injections, Intraperitoneal , Mice, Inbred C57BL , Microvessels/drug effects , Microvessels/growth & development , Ovarian Follicle/cytology , Ovarian Follicle/growth & development , Ovarian Follicle/metabolism , Ovary/blood supply , Ovary/growth & development , Ovary/metabolism , Random Allocation , Time FactorsABSTRACT
Some of the low level radioactive wastes from reprocessing of spent nuclear fuels contain nitrates. Nitrates can be present in the form of soluble salts and can be reduced by various reactions. Among them, reduction by metal compounds and microorganisms seems to be important in the underground repository. Reduction by microorganism is more important in near field area than inside the repository because high pH and extremely high salt concentration would prevent microorganism activities. In the near field, pH is more moderate (pH is around 8) and salt concentration is lower. However, the electron donor may be limited there and it might be the control factor for microorganism's denitrification activities. In this study, in-vitro experiments of the nitrate reduction reaction were conducted using model organic materials purported to exist in underground conditions relevant to geological disposal. Two kinds of organic materials were selected. A super plasticizer was selected as being representative of the geological disposal system and humic acid was selected as being representative of pre-existing organic materials in the bedrock. Nitrates were reduced almost to N2 gas in the existence of super plasticizer. In the case of humic acids, although nitrates were reduced, the rate was much lower and, in this case, dead organism was used as an electron donor instead of humic acids. A reaction model was developed based on the in-vitro experiments and verified by running simulations against data obtained from in-situ experiments using actual groundwaters and microorganisms. The simulation showed a good correlation with the experimental data and contributes to the understanding of microbially mediated denitrification in geological disposal systems.
ABSTRACT
The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a ß-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.
Subject(s)
Acinar Cells/physiology , Pancreas/cytology , Parotid Gland/cytology , Protein Sorting Signals/physiology , Secretory Vesicles/metabolism , Acinar Cells/cytology , Animals , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation , Male , Parotid Gland/metabolism , Protein Transport , Rats , Rats, Sprague-Dawley , Staining and LabelingABSTRACT
We experienced an outbreak of extensively drug-resistant pulmonary tuberculosis (XDR-TB) in a hemodialysis facility. The primary case involved a 51-year-old male hemodialysis patient, with a history of treatment for Mycobacterium tuberculosis infection seven years previously. There was no drug resistance, and the patient completely recovered after undergoing treatment with isoniazid (INH), rifampicin (RFP) and ethambutol (EB). He was admitted to another hospital due to a recurrence of pulmonary tuberculosis in June 2006. At first, he was treated with HRS [INH, RFP and streptomycin (SM)]; however, the drug regimen was changed to INH, EB, levofloxacin (LVFX) and kanamycin (KM) in August following the results of a drug susceptibility test. Although the patient was receiving outpatient tuberculous therapy, he was readmitted in June 2007 due to relapse and conversion of a sputum culture to positive status. Additionally, the XDR-TB organism was identified. Following these events, five staff members of the hemodialysis facility and a member of the patient's family were diagnosed with XDR-TB infection. The staffs who were infected with XDR-TB had worked in the same dialysis room, drug resistance was found in all cases and drug resistant gene mutations were found in three cases; therefore, we considered this to be an outbreak. As XDR-TB infection was suspected in all cases, no patients took drugs to treat latent tuberculosis infection (LTBI). Regarding the causes of the outbreak, the first is the delay of four months in making a diagnosis of re-exacerbation of tuberculosis. Second, in Case 2, the patient developed laryngeal and tracheobronchial tuberculosis after first being diagnosed with asthma, and the tuberculosis diagnosis was delayed. Third, the sputum smear of Case 2 was strongly positive. There is only one previously reported outbreak of XDR-TB in Japan; therefore, we consider this outbreak to be educational.
Subject(s)
Disease Outbreaks , Extensively Drug-Resistant Tuberculosis/transmission , Hemodialysis Units, Hospital , Tuberculosis, Pulmonary/transmission , Adult , Extensively Drug-Resistant Tuberculosis/diagnosis , Female , Humans , Male , Middle Aged , Tuberculosis, Pulmonary/diagnosisABSTRACT
BACKGROUND: For fertility preservation of women patients scheduled to undergo chemotherapy or radiotherapy, unilateral oophorectomy was performed, and the ovary was cryopreserved. METHODS: Two-port surgery was conducted in 3 patients, and single-port surgery using a single-incision laparoscopic surgery port in 3. An 18-G Cathelin needle equipped with a syringe was directly inserted transabdominally to reach the small follicle on the ovarian surface; then, follicular fluid was recovered by aspiration through the syringe as with in vitro fertilization procedures, and immature oocytes were collected from the resulting culture medium under microscopy and cryopreserved. Vitrification of the ovarian tissue was performed using the cryotissue method. RESULTS: The operative time and estimated blood loss were 39.7 minutes (17-57) and 8.6 mL (2-20), and the numbers of ovarian cortical tissues and immature oocytes collected were 10.1 (5.5-15) and 16.3 (0-36), respectively. CONCLUSIONS: It is suggested that fertility preservation operations before chemotherapy or radiotherapy can be safely done using reduced-port surgery.
Subject(s)
Fertility Preservation/methods , Laparoscopy/methods , Minimally Invasive Surgical Procedures/methods , Ovariectomy/methods , Adult , Cryopreservation , Female , Humans , Oocytes/cytology , Oocytes/physiology , VitrificationABSTRACT
The clinical application of human adipose-derived mesenchymal stem cells (MSCs) as treatment for intractable diseases or traumatic tissue damage has attracted attention. To address the ability of reactivating injured ovaries, we prepared a rat model with damaged ovaries by using an anticancer agent, cyclophosphamide (CTX). We then investigated the restorative effects on ovarian function and the safety of adipose-derived MSCs (A-MSCs). MSCs were shown to be capable of inducing angiogenesis and restoring the number of ovarian follicles and corpus lutea in ovaries. No deformities, tumor formation or deaths were observed in F1 and F2 rats, indicating that the local injection of MSCs into the ovary did not have any obvious side effects. In addition, the localization of the Y chromosome was investigated using the fluorescent in situ hybridization method by injecting male A-MSCs into the ovaries; as a result, the Y chromosomes were localized not in the follicles, but in the thecal layers. ELISA revealed that A-MSCs secreted higher levels of vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) than tail fibroblast cells. Quantitative real-time PCR and immunohistochemistry showed that higher expression levels of VEGF, IGF-1 and HGF were observed in CTX-treated ovaries after A-MSC transplantation. These findings suggest that MSCs may have a role in restoring damaged ovarian function and could be useful for regenerative medicine.
Subject(s)
Adipose Tissue/cytology , Disease Models, Animal , Mesenchymal Stem Cell Transplantation/methods , Ovarian Diseases/physiopathology , Ovarian Diseases/therapy , Animals , Antibodies, Monoclonal , Corpus Luteum/pathology , Cyclophosphamide/toxicity , Cytokines/metabolism , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay , Female , Fibroblasts/metabolism , Flow Cytometry , Immunohistochemistry , In Situ Hybridization, Fluorescence , Intercellular Signaling Peptides and Proteins/metabolism , Litter Size , Mice , Ovarian Diseases/chemically induced , Ovarian Follicle/pathology , Rats , Rats, Wistar , Real-Time Polymerase Chain ReactionABSTRACT
In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²âº, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²âº (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 µM Hg²âº, concentrations that activate AQP6. The Hg lysis was completely blocked by ß-mercaptoethanol which disrupts Hg²âº-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Brâ» > Iâ» > Clâ» and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²âº-sensitive anion channel in rat parotid secretory granules.
Subject(s)
Aquaporin 6/metabolism , Mercury/pharmacology , Osmosis , Parotid Gland/metabolism , Secretory Vesicles/drug effects , Secretory Vesicles/metabolism , Animals , Anions/metabolism , Biological Transport , Chlorides/metabolism , Intracellular Membranes/metabolism , Male , Permeability/drug effects , RatsABSTRACT
BACKGROUND: For women with congenital uterine infertility, or for those who have undergone hysterectomy, uterine transplantation is one of the potential treatments to regain fertility. In this study, we utilized a primate model of uterine transplantation, and evaluated the patency of our microsurgical anastomoses, and the perfusion of the transplanted uterus. METHODS: Two female cynomolgus monkeys underwent surgery. We anastomosed two arteries and one vein in Case 1 and two arteries and two veins in Case 2. The arteries used were the uterine arteries and the anastomosis was done to the external iliac artery. We used one of the ovarian veins in both animals, but resected the ovary from the Fallopian tube. Uterine arterial blood flow and uterine size were determined by intraoperative indocyanine green (ICG) angiography and ultrasonography. The biopsy of the uterine cervix was performed after surgery. RESULTS: ICG angiography showed that the unilateral uterine artery perfused the bilateral uterine bodies and cervix. In Case 1, ICG angiography showed the occlusion of one of the anastomosed arteries during the operation and the uterus appeared atrophied 2 months after operation. In Case 2, the transplanted uterus survived and normal menstruation occurred. The animal achieved a natural pregnancy and was delivered by the Caeserean section due to early separation of the placenta. The newborn suffered fetal distress. CONCLUSIONS: These results show the anastomosis of at least the bilateral uterine arteries and the unilateral ovarian vein is required for uterus transplantation. This is the first report of a natural pregnancy in a primate following uterine autotransplantation.
Subject(s)
Uterus/immunology , Uterus/transplantation , Anastomosis, Surgical , Angiography/methods , Animals , Biopsy , Blood Flow Velocity , Female , Indocyanine Green/pharmacology , Macaca fascicularis , Transplantation, Autologous , Ultrasonography, Doppler/methods , Uterus/blood supply , Uterus/pathologyABSTRACT
BACKGROUND: The two main complications associated with the use of assisted reproduction techniques, ovarian hyperstimulation syndrome and multiple pregnancies, could be eliminated by milder ovarian stimulation protocols and the increased use of a single embryo transfer (SET) policy. A retrospective, cohort study was performed in private infertility centre to evaluate the embryological and clinical results of a large exclusively SET program according to patient age (lower or equal 29, 30-34, 35-39, 40-44 and equal or higher 45 years). MATERIALS: A total of 7,244 infertile patients have undergone 20,244 cycles with a clomiphene-based minimal stimulation or natural cycle IVF protocol during 2008. Following oocyte retrieval, fertilization and embryo culture a total of 10,401 fresh or frozen single embryo transfer procedures were performed involving cleavage-stage embryos or blastocysts. RESULTS: Successful oocyte retrieval rate (78.0 %) showed no age-dependent decrease until 45 years. Fertilization (80.3 %) and cleavage (91.1 %) rates were not significantly different between age groups. Blastocyst formation (70.1 % to 22.8 %) and overall live birth rates (35.9 % to 2 %) showed an age-dependent decrease. Frozen-thawed blastocyst transfer cycles gave the highest chance of live birth per embryo transfer (41.3 % to 6.1 %). CONCLUSIONS: High fertilization and cleavage rates were obtained regardless of age whereas blastocyst formation and live birth rates showed an age-dependent decrease. An elective single embryo transfer program based on a minimal ovarian stimulation protocol yields acceptable live birth rates per embryo transfer in infertile patients up until their mid-forties. However in very advanced age patients (equal or higher 45 years old) success rates fall below 1 %.