This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic
embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the ...expanded blastocyst stage were cryopreserved by vitrification
using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly
improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell
stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of
nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved
by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine
embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction
with delipation.
An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of ...cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 micro g/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.
This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the ...expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.
MafA is a basic leucine zipper (b-Zip) type transcription factor that binds to the insulin promoter and regulates insulin transcription synergistically with Pdx-1 and NeuroD. Transforming growth ...factor-β (TGF-β) signaling has been reported to regulate activity of b-Zip transcription factor such as ATF-2 and acts as an important regulator of insulin gene transcription and pancreatic β cell maintenance. To investigate the relationship between MafA-dependent transcriptional activation and TGF-β signaling, we examined the effects of TGF-β signal on MafA-dependent transactivation of the rat insulin II gene promoter (RIPII-251) and a synthetic MafA-dependent promoter. MafA-dependent activation of the reporters was inhibited in the presence of Smad2/Smad4 or Smad3/Smad4 and a constitutively active TGF-β type I receptor and this inhibition was dependent upon the presence of MafA. Co-immunoprecipitation analyses revealed that MafA physically interacts with Smad2 or Smad3. These results suggest that MafA-dependent transcriptional activation is negatively regulated by TGF-β signaling.
GPR40 is a G protein-coupled receptor regulating free fatty acid-induced insulin secretion. We generated transgenic mice overexpressing the hGPR40 gene under control of the mouse insulin II promoter ...and used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis. Normal (C57BL/6J) and diabetic (KK) mice overexpressing the hGPR40 gene under control of the insulin II promoter were generated, and their glucose metabolism and islet function were analyzed. In comparison with nontransgenic littermates, hGPR40 transgenic mice exhibited improved oral glucose tolerance with an increase in insulin secretion. Although islet morphologic analysis showed no obvious differences between hGPR40 transgenic and nontransgenic mice, isolated islets from hGPR40 transgenic mice had enhanced insulin secretion in response to high glucose (16 mmol/l) compared with those from nontransgenic mice, and they both had similar low glucose (3 mmol/l)-stimulated insulin secretion. In addition, hGPR40 transgenic islets significantly increased insulin secretion against a naturally occurring agonist palmitate in the presence of 11 mmol/l glucose. hGPR40 transgenic mice were also found to be resistant to high-fat diet-induced glucose intolerance, and hGPR40 transgenic mice harboring KK background showed augmented insulin secretion and improved oral glucose tolerance compared with nontransgenic littermates. Our results suggest that GPR40 may have a role in regulating glucose-stimulated insulin secretion and plasma glucose levels in vivo and that pharmacological activation of GPR40 may provide a novel insulin secretagogue beneficial for the treatment of type 2 diabetes.
Background and Study Aims Capsule endoscopy (CE) does not necessarily identify positive findings in patients with overt obscure gastrointestinal bleeding (OGIB). We aimed to identify factors ...predictive of positive CE findings and those of re-bleeding after negative CE in overt OGIB. Patients and Methods We retrospectively analyzed 68 patients who underwent CE for overt OGIB. CE findings, therapeutic interventions, and clinical course after CE were reviewed. Clinical variables associated with positive CE findings and those associated with re-bleeding after negative CE findings were investigated. Results Positive CE finding was found in 36 (53%) patients. Marked decrease in hemoglobin value OR; 18.8, 95% CI; 3.4-152.0 and earlier CE examination within a week after the last episode of bleeding OR; 8.0, 95% CI; 2.2-35.9 were factors associated with positive CE findings. Nine (28%) of 32 patients with negative CE findings re-bled. Marked decrease in hemoglobin value was more frequent in patients with re-bleeding than those without (P = 0.07). Conclusion Patients with massive and overt OGIB are the best candidates for CE. Earlier CE, virtually within a week, contributes to the better diagnostic yield of the procedure. Careful follow-up seems necessary for patients with massive bleeding even in cases of negative CE findings.
Background Small-intestinal adenoma occurs in patients with familial adenomatous polyposis (FAP). Objectives The aim was to analyze the diagnostic yield of a double-balloon endoscopy (DBE) and an ...intraoperative enteroscopy (IOE) for small-intestinal involvement in FAP. Patients Forty-one patients with FAP. Interventions We examined 12 patients with FAP by using oral DBE before a colectomy and 29 patients with FAP by using IOE. The incidence and the endoscopic findings of adenoma were compared between the 2 procedures. Phenotypes of FAP and genotypes of adenomatous polyposis coli ( APC ) were then compared between patients with small-intestinal adenomas and those without. The genotype was classified into a 5' mutation (exons 1-14), a 3' mutation (exon 15), and a negative mutation of APC. Main Outcome Measurement The prevalence of adenoma. Results A DBE detected small-intestinal adenomas in 9 of 12 patients (75%), as did an IOE in 15 of 29 patients (52%, P > .05). The adenomas occurred predominantly in the jejunum, with a configuration of diminutive polyps in 22 patients. In addition, a DBE detected nonpolypoid adenoma in a patient, and nodular, broad-based protrusion (advanced lesions) in 3 patients, whereas an IOE detected advanced lesions in a patient. Patients with small-intestinal adenoma had more severe duodenal adenomatosis than those patients without small-intestinal adenoma ( P < .001). In cases in which APC was analyzed, the prevalence of small-intestinal adenoma was higher in patients with a 3' mutation (100%) than in those with a 5' mutation (44%) and with a negative mutation (42%, P < .02). Limitation Not a prospective randomized study. Conclusions A DBE is equal to an IOE for scrutiny of small-intestinal adenomas in FAP. There seems to be a genotype-jejunal phenotype correlation in FAP.
PDF
