Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification.

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140-170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

Hydroxypropyl cellulose as an option for supplementation of cryoprotectant solutions for embryo vitrification in human assisted reproductive technologies.

Hydroxypropyl cellulose (HPC) was investigated as a replacement for serum substitute supplement (SSS) for use in cryoprotectant solutions for embryo vitrification. Mouse blastocysts from inbred (n = 1056), hybrid (n = 128) strains, and 121 vitrified blastocysts donated by infertile patients (n = 102) were used. Mouse and human blastocysts, with or without zona pellucida, were vitrified and warmed in either 1% or 5% HPC or in 5% or 20% SSS-supplemented media using the Cryotop (Kitazato BioPharma Co. Ltd, Fuji, Japan) method, and the survival and oxygen consumption rates were assessed. Viscosity of each vitrification solution was compared. Survival rates of mouse hybrid blastocysts and human zona pellucida-intact blastocysts were comparable among the groups. Mouse and human zona pellucida-free blastocysts, which normally exhibit poor cryoresistance, showed significantly higher survival rates in 5% HPC than 5% SSS (P < 0.05). The 5% HPC-supplemented vitrification solution showed a significantly higher viscosity (P < 0.05). The blastocysts were easily detached from the Cryotop strip during warming when HPC-supplemented vitrification solution was used. The oxygen consumption rates were similar between non-vitrified and 5% HPC groups. The results suggest possible use of HPC for supplementation of cryoprotectant solutions and provide useful information to improve vitrification protocols.

Aqueous extracts of Rhizopus oryzae induced nitric oxide production in rat hepatocyte cell line RLN-10.

Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22(phox) and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1-carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.

Nitric oxide induces vascular endothelial growth factor expression in the rat placenta in vivo and in vitro.

We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.

Prevention of mitochondrial disease inheritance by assisted reproductive technologies: prospects and challenges.

BACKGROUND: Mitochondrial diseases are caused by the mutations in both nuclear and mitochondrial DNA (mtDNA) and the treatment options for patients who have mitochondrial disease are rather limited. Mitochondrial DNA is transmitted maternally and does not follow a Mendelian pattern of inheritance. Since reliable and predictable detection of mitochondrial disorders in embryos and oocytes is unattainable at present, an alternative approach to this problem has emerged as partial or complete replacement of mutated mtDNA with the wild-type mtDNA through embryo manipulations. Currently available methods to achieve this goal are germinal vesicle transfer (GVT), metaphase chromosome transfer (CT), pronuclear transfer (PNT) and ooplasmic transfer (OT). SCOPE OF REVIEW: We summarize the state of the art regarding these technologies and discuss the implications of recent advances in the field for clinical practice. MAJOR CONCLUSIONS: CT, PNT and GVT techniques hold promise to prevent transmission of mutant mtDNA through ARTs. However, it is clear that mtDNA heteroplasmy in oocytes, embryos and offspring produced by these methods remains as a legitimate concern. GENERAL SIGNIFICANCE: New approaches to eliminate transmission of mutant mtDNA certainly need to be explored in order to bring the promise of clinical application for the treatment of mitochondrial disorders. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.

Changes in nitric oxide production levels and expression of nitric oxide synthase isoforms in the rat uterus during pregnancy.

We clarified nitric oxide (NO) production in the rat uterus by electron paramagnetic resonance spectroscopy and with Fe-N-(dithiocarboxy) sarcosine complex (an NO-trapping reagent). We examined changes in NO production in the whole uterus, decidua, and myometrium (gestational days 13.5-21.5). The expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative reverse transcription-polymerase chain reaction. The uterine NO levels were low on day 13.5, peaked on day 17.5, and thereafter decreased significantly. The NO production levels in the decidua and myometrium were the same on day 13.5, but the levels in the decidua were 2- to 4-fold higher than those in the myometrium from day 15.5 onwards. The NOS-2 mRNA expression pattern correlated well with changes in the NO levels in the decidua, whereas the NOS-3 mRNA was expressed constantly during gestation. Thus NOS-2-generated NO in the decidua contributed significantly to uterine NO levels.