The cryoprotective effect of Guar gum co-supplemented with ethylene glycol and glycerol in Simmental bull semen.

Sperm cryopreservation often reduces sperm quality by forming of intra- and extracellular ice crystals. Various compounds widely used to counteract this effect. The guar gum was considered as an extracellular cryoprotective substance. The present study evaluated the impact of the co-supplementation of guar gum with ethylene glycol or glycerol in the cryopreservation of bull sperm. Four ejaculates from 4 bulls were pooled and divided into ten groups consisting of 4 controls (glycerol 6%, ethylene glycol 6%, glycerol 3.5%, and ethylene glycol 3.5%, and six treatment groups including guar gum in 0.001% and 0.002% alone and or co-supplemented either with 3.5% glycerol or 3.5% ethylene glycol and frozen in liquid nitrogen. The sperm motility, viability, plasma membrane and DNA integrity, apoptotic-like changes, antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities evaluated. The groups contained 3.5% glycerol + 0.001% guar gum, 3.5% ethylene glycol + 0.001% guar gum, and 0.001% guar gum alone showed higher values for live sperm, antioxidant enzymes, membrane integrity, mitochondrial membrane potential (MMP), fertilization, cleavage, and blastocyst rates; and lower values for apoptotic-like changes, H2O2 level, and DNA damage than the control groups. In conclusion, adding guar gum to the bull sperm diluent either alone or combined with glycerol or ethylene glycol ameliorated sperm viability and kinematic parameters and antioxidant capacity while reducing DNA damage and apoptotic-like changes. Guar gum also has improved embryo development. Due to its cost-effectiveness and physicochemical properties, guar gum is a promising supplement for bull sperm cryopreservation.

Feline ovarian tissue vitrification: The effect of fragment size and base medium on follicular viability and morphology.

To achieve optimal vitrification, tissue structure and fragment size represent a challenge for obtaining sufficient cooling velocity. Theoretically, thin ovarian tissue fragments lead to higher surface contact, hence higher solute penetration. Another critical factor is the concentration of cryoprotectants (CPA): CPA toxicity may occur with high concentrations, and as such, this may induce local apoptosis. Therefore two experiments were conducted: In experiment I, we compared the effect of sucrose supplementation in vitrification solution along with ovarian fragments of different sizes on post-warming tissue viability and follicle architecture. Fragments of two different sizes, with a thickness and radius of 1.5 × 0.75 mm and 3 × 1.5 mm respectively were vitrified in vitrification solution without sucrose and with 0.5 M sucrose supplementation. Post-warming, fragments of ovarian tissue (fresh and vitrified) were evaluated for viability (Calcein AM/Propidium Iodide) and for morphology (hematoxylin-eosin). In experiment II, we aimed to reduce cryoprotectant toxicity by using lower CPA concentrations in combination with an optimized carrier medium (HypThermosol®; HTS). Ovarian tissue fragments were randomly allocated to five groups (A: fresh controls; B: vitrified in GLOBAL® TOTAL® LP w/HEPES with 15% ethylene glycol (EG) and 15% DMSO; C: vitrified in HTS with 5% EG and 5% DMSO; D: vitrified in HTS with 10% EG and 10% DMSO; E: vitrified in HTS with 15% EG and 15% DMSO). Fragments (fresh and vitrified) were evaluated for morphology (hematoxylin-eosin) and for apoptosis through the activity of caspase-3. Results showed that follicular morphology was affected by the size of the fragment; smaller sized fragments contained a greater proportion of intact follicles (53.8 ± 2.0%) compared to the larger fragments (40.3 ± 2.0%). Our results demonstrated that 1.5 × 0.75 mm sized pieces vitrified in a vitrification solution supplemented with 0.5 M sucrose had more intact follicles (54.8 ± 1.3%; P = 0.0002) after vitrification. In addition, HTS presented no additional protective effect as a base medium, neither for follicular morphology nor apoptotic rate.

Pyrrosia petiolosa Extract Ameliorates Ethylene Glycol-Induced Urolithiasis in Rats by Inhibiting Oxidative Stress and Inflammatory Response.

Objective: To study the therapeutic effect and mechanism of Pyrrosia petiolosa (P. petiolosa) extract on ethylene glycol- (EG-) induced urolithiasis in rats. Methods: Thirty SD male rats were randomly divided into five groups (n = 6): control group, EG group, and P. petiolosa group (25 mg/kg, 50 mg/kg group, and 100 mg/kg). Biochemical testing was adopted for measuring the blood and urine parameters, as well as the level of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde acid (MDA) in kidney tissues. HE staining and ELISA were utilized to observe the histopathological changes and detect the level of IL-1ß, IL-6, MCP-1, and TNF-α in the kidney tissue, respectively. And western blot was performed for checking NOX2, NOX4, TGF-ß1, p-Smad3, Smad3, p-Smad2, and Smad2 protein expression level in kidney tissues. Results: EG could significantly increase the level of blood urea nitrogen, creatinine, and Na in serum and 24-hour urinary protein, oxalate, uric acid, creatinine, calcium, and phosphorus in urine and decreased the urine volume in rats. Whereas P. petiolosa extract was able to greatly decrease the level of related parameters in serum and urine in a dose-dependent manner, but did not affect the urine pH. In addition, P. petiolosa extract notably ameliorated EG-induced renal tissue injury. Compared with the EG group, P. petiolosa extract markedly raised the level of SOD and GSH and decreased the MDA level and the expression of NOX2 and NOX4 in the kidney tissue. Moreover, P. petiolosa extract also lowered the level of IL-1ß, IL-6, MCP-1, and TNF-α in EG-stimulated kidney tissue and inhibited the protein level of EG-induced TGF-ß1, p-Smad3, and p-Smad2 in a concentration-dependent manner. Conclusion: P. petiolosa extract can improve EG-induced urolithiasis in rats by inhibiting oxidative stress, inflammatory response, and the activation of TGF-ß pathway.

HydroZitLa inhibits calcium oxalate stone formation in nephrolithic rats and promotes longevity in nematode Caenorhabditis elegans.

Low fluid intake, low urinary citrate excretion, and high oxidative stress are main causative factors of calcium oxalate (CaOx) nephrolithiasis. HydroZitLa contains citrate and natural antioxidants and is developed to correct these three factors simultaneously. Antioxidants theoretically can prolong the lifespan of organisms. In this study, we preclinically investigated the antilithogenic, lifespan-extending and anti-aging effects of HydroZitLa in HK-2 cells, male Wistar rats, and Caenorhabditis elegans. HydroZitLa significantly inhibited CaOx crystal aggregation in vitro and reduced oxidative stress in HK-2 cells challenged with lithogenic factors. For experimental nephrolithiasis, rats were divided into four groups: ethylene glycol (EG), EG + HydroZitLa, EG + Uralyt-U, and untreated control. CaOx deposits in kidneys of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. Intrarenal expression of 4-hydroxynonenal in EG + HydroZitLa rats was significantly lower than that of EG rats. The urinary oxalate levels of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. The urinary citrate levels of EG + HydroZitLa and EG + Uralyt-U rats were restored to the level in normal control rats. In C. elegans, HydroZitLa supplementation significantly extended the median lifespan of nematodes up to 34% without altering feeding ability. Lipofuscin accumulation in HydroZitLa-supplemented nematodes was significantly lower than that of non-supplemented control. Additionally, HydroZitLa inhibited telomere shortening, p16 upregulation, and premature senescence in HK-2 cells exposed to lithogenic stressors. Conclusions, HydroZitLa inhibited oxidative stress and CaOx formation both in vitro and in vivo. HydroZitLa extended the lifespan and delayed the onset of aging in C. elegans and human kidney cells. This preclinical evidence suggests that HydroZitLa is beneficial for inhibiting CaOx stone formation, promoting longevity, and slowing down aging.

Supplementary cryoprotective effect of carboxylated ε-poly-l-lysine during vitrification of rat pancreatic islets.

This study was designed to investigate whether cryosurvival of rat pancreatic islets can be improved by carboxylated ε-poly-l-lysine (CPLL). Islets isolated from Wistar × Brown-Norway F1 rats (101-200 µm in diameter) were cryopreserved in three vitrification solutions containing ethylene glycol (EG; 30%, v/v) and CPLL (0%, 10%, or 20%, v/v) by Cryotop® protocol (10 islets per device). The post-warm survival rate of the islets vitrified in the presence of 20% CPLL (74%), assessed by FDA/PI double staining, was higher than those in 0% and 10% CPLL (65% and 66%, respectively). Decreased EG concentrations (10% and 20%) in the presence of 20% CPLL resulted in impaired post-warm islet survival rates (50% and 64%, respectively). Value of stimulus index (SI) for 20 mM/3 mM glucose-stimulated insulin secretion was 4.1 in islets vitrified-warmed in the presence of 30% EG and 20% CPLL, which was comparable with those in fresh control islets and vitrified islets in 30% EG alone (4.1 and 4.4, respectively). A large number of islets (50 islets per device) could be cryopreserved in the presence of 30% EG and 20% CPLL by using nylon mesh as the device, without considerable loss of post-warm survival (68%) and SI value (3.7). In conclusion, supplementation of antifreeze 20% CPLL was effective in improving the post-warm survival of isolated rat pancreatic islets when vitrification solution containing 30% EG was used.

Cryopreservation of Orchids - A Review.

BACKGROUND: The orchids are one of the beautiful creations of nature which stand apart from any other assemblage of flowering plants. They are highly evolutionary and ecologically significant group of plants that have effectively occupied almost every habitat on the earth. Indiscriminate collections and extermination of their natural habitats have threatened many species of orchids with extinction, resulting in a severe reduction of their genetic resources in nature according to recent patents. It is necessary to adopt sound scientific protocols for the preservation of orchid species. METHOD: This cost-effective technique provides large storage time for the conservation of germplasm. Presently, efforts have been made to explore various cryopreservation techniques utilized so far and factors affecting the longevity of the propagules (in vivo and in vitro) while cryopreserving them. The sample to be cryopreserved is freeze-preserved in two ways, a) stepwise at two different subzero temperatures and b) in the rapid method, the samples are placed directly in the liquid nitrogen. RESULTS: The orchid seeds and pollen are the most suitable propagules for cryopreservation of orchids due to their minute size and less space requirement. CONCLUSION: Among the tissues (such as seeds, pollen, protocorms etc.) seeds are the most reliable. The present article reviews the cryopreservation techniques and factors effecting the cryopreservation, for in vitro conservation of orchid gene pool.

Antiurolithic effect of olive oil in a mouse model of ethylene glycol-induced urolithiasis.

PURPOSE: At present, commercially available antiurolithic drugs have more adverse effects than potential therapeutic or preventive effects with chronic use. With this in mind, the present study was designed to assess the antiurolithic effect of olive oil in a mouse model of ethylene glycol (EG)-induced urolithiasis. MATERIALS AND METHODS: Adult albino mice were divided into 6 groups. Group I was fed the vehicle only. Group II was supplemented with 0.75% EG alone in drinking water during the experimental period to initiate deposition of calcium oxalate in kidneys, which leads to urolithiasis in animals. Groups III (olive oil control group) through V were fed olive oil orally at various doses during the experimental period. Group VI received cystone (750 mg/kg). Groups IV-VI additionally received 0.75% EG in drinking water ad libitum. SPSS ver.17.0 was used for statistical analysis. RESULTS: The study results showed significantly higher levels of serum urea, uric acid, and creatinine (p<0.05) in group II than in groups III-VI and I. Administration of olive oil at different doses restored the elevated serum parameters in groups IV and V compared with group II. Urine and kidney calcium, oxalate, and phosphate levels in groups IV-VI were significantly lower (p<0.05) than in animals with EG-induced urolithiasis (group II). Group V mice showed a significant restoration effect on serum as well as urine and kidney parameters compared with group II. CONCLUSIONS: Supplementation with olive oil (1.7 mL/kg body weight) reduced and prevented the growth of urinary stones, possibly by inhibiting renal tubular membrane damage due to peroxidative stress induced by hyperoxaluria.

Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation.

The aim of this study was to evaluate the effects of glycerol, ethylene glycol or DMSO in a soybean lecithin extender for freezing ram semen. In this study, 20 ejaculates were collected from four Ghezel rams and diluted with soybean lecithin extender with glycerol (7%), ethylene glycol (3%, 5% and 7%) or DMSO (3%, 5% and 7%). Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality, mitochondrial activity (Rhodamine 123) and apoptotic features (Annexin V/Propidium iodide) were assessed after thawing. There was no significant difference between glycerol and ethylene glycol at different concentrations (3% and 5%) regarding sperm total and progressive motility, viability, and membrane integrity. The least percentages of mitochondrial functionality were observed in samples frozen with all different DMSO concentrations tested (P<0.05). Moreover, the percentage of post-thawed dead sperm was the greatest for all the DMSO concentrations compared with other groups (P<0.05). Thus, DMSO had an adverse effect on the post thaw ram sperm parameters. In contrast, ethylene glycol could be a desirable substitute of glycerol in the freezing extender, in view of similar results obtained in post-thaw quality of ram semen cryopreserved in a soybean lecithin extender. We propose that glycerol in a soybean lecithin based extender could be replaced by ethylene glycol at 3% or 5% concentrations.

VITRIFICATION OF THAI NATIVE CATTLE OOCYTES: EFFECTS OF ETHYLENE GLYCOL CONCENTRATIONS AND EXPOSURE TIME, LINOLEIC ACID ALBUMIN AND CHOLESTEROL-LOADED METHYL-B-CYCLODEXTRIN.

The present study aimed to improve the oocyte vitrification procedure for preservation of Thai native cattle genetic resources. In Experiment I, oocytes were exposed to various doses (2%, 4% and 6%) of ethylene glycol (EG) in vitrification solution I (VS-I) for different equilibration times (10 or 20 min) before being exposed to VS-II and then subjected to vitrification. Experiment II was divided into two parts: (a) oocytes were matured in medium supplemented with linoleic acid albumin (LAA) (1% or 2%) and then vitrified; (b) matured oocytes were preincubated with cholesterol-loaded methyl-ß-cyclodextrin (CLC) (1% or 2%) and then vitrified. Equilibration of oocytes by exposure to 6% EG in VS-I for 10 min (Experiment I), and in vitro maturation of immature oocytes in medium supplementation with 2% LAA (Experiment II) were the most effective methods; vitrified/thawed oocytes showed higher rates of survival and subsequent embryonic development compared with the other experimental groups.

Protective effect of Urtica dioica methanol extract against experimentally induced urinary calculi in rats.

Renal calculi formation is one of the most common urological disorders. Urinary stone disease is a common disease, which affects 10­12% of the population in industrialized countries. In males, the highest prevalence of the disease occurs between the age of 20 and 40 years, while in females, the highest incidence of the disease occurs later. Previous studies have shown that long­term exposure to oxalate is toxic to renal epithelial cells and results in oxidative stress. In the present study, a methanolic extract of aerial parts of Urtica dioica was screened for antiurolithiatic activity against ethylene glycol and ammonium chloride­induced calcium oxalate renal stones in male rats. In the control rats, ethylene glycol and ammonium chloride administration was observed to cause an increase in urinary calcium, oxalate and creatinine levels, as well as an increase in renal calcium and oxalate deposition. Histopathological observations revealed calcium oxalate microcrystal deposits in the kidney sections of the rats treated with ethylene glycol and ammonium chloride, indicating the induction of lithiasis. In the test rats, treatment with the methanolic extract of Urtica dioica was found to decrease the elevated levels of urinary calcium, oxalate and creatinine, and significantly decrease the renal deposition of calcium and oxalate. Furthermore, renal histological observations revealed a significant reduction in calcium oxalate crystal deposition in the test rats. Phytochemical analysis of the Urtica dioica extract was also performed using liquid chromatography­electrospray ionization tandem mass spectrometry and high-performance liquid chromatography with photodiode array detection, to determine the chemical composition of the extract. The eight chemical constituents identified in the extract were protocatechuic acid, salicylic acid, luteolin, gossypetin, rutin, kaempferol­3­O­rutinoside, kaempferol­3­O­glucoside and chlorogenic acid. In conclusion, the results of the present study suggest that Urtica dioica has strong antiurolithiatic activity and may have potential as a natural therapeutic agent for various urological disorders.

Anti-urolithiatic effect of ethanolic extract of Pedalium murex linn. fruits on ethylene glycol-induced renal calculi.

PURPOSE: To evaluate effect of ethanolic extract of Pedalium murex Linn. fruits on experimental model of calcium oxalate nephrolithiasis. MATERIALS AND METHODS: Thirty-six male Wistar albino rats were randomly divided in 6 groups.Normal controls received distilled water for 28 days. Other five groups received ethylene glycol(1% v/v) in distilled water for 28 days. Pedalium murex ethanolic extract was given 200 mg/kg and 400 mg/kg orally in distilled water for 28 days in prophylactic groups (III and IV) and from 15th to 28th days in treatment groups (V and VI). The urea, creatinine, random blood sugar, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bilirubin and calcium were measured on 28th day. 24 hr urinary oxalate and volume were measured on day 0 and 28. On day 28, kidneys were removed, weighed and subjected to histopathological examination. Calcium oxalate crystallization was evaluated by renal histopathology and in-vitro method of mineralization.All parameters were analyzed by Kruskal-Wallis or one-way ANOVA with post-hoc test. RESULTS: Pedalium murex showed significant improvement in renal function and kidney weight inprophylactic groups as compared to ethylene glycol controls. It did not show any effect on urinary oxalate, urine volume and any other serological parameters. Calcium oxalate crystallization was significantly reduced in all the Pedalium murex treated groups (P < .05). Calcium oxalate and phosphate mineralization were also inhibited by 33% and 57%. CONCLUSION: Ethanolic extract of Pedalium murex fruits possess significant activity for prevention of renal calculi.

Preventive effect of specific antioxidant on oxidative renal cell injury associated with renal crystal formation.

OBJECTIVE: To investigate whether calcium oxalate monohydrate (COM), a key element of hyperoxaluria, would induce renal cell injury through oxidative stress and also whether certain antioxidants could prevent chemically induced renal crystal formation in rats. MATERIALS AND METHODS: COM-exerted oxidative stress on the kidney epithelial Madin-Darby canine kidney cells was assessed using the lipid peroxidation assay. Glyoxalase I (Gly-I) activity was also determined. Two antioxidants, vitamin C and N-acetylcysteine (NAC), were then tested to determine whether they could abolish such oxidative stress in Madin-Darby canine kidney cells. Both antioxidants were also tested to determine whether they might prevent or reduce renal crystal formation induced with ethylene glycol (EG) and vitamin D3 (VD3) in Wistar rats. RESULTS: COM (200 µg/mL) demonstrated ∼1.3-fold greater oxidative stress with a significant reduction in cell viability and Gly-I activity compared with controls. However, such adverse events were almost completely prevented with NAC but not with vitamin C. In the animal study, no renal crystals were seen in the sham group. However, numerous crystals, with reduced Gly-I activity and elevated oxidative stress, were found in the EG-VD3 group. However, markedly (>70%) fewer crystals, with full Gly-I activity and diminished oxidative stress, were detected in the EG-VD3+NAC group. CONCLUSION: COM exerted oxidative stress on Madin-Darby canine kidney cells, leading to cell viability reduction and Gly-I inactivation, with NAC fully preventing such adverse consequences. Similarly, numerous crystals with Gly-I inactivation and elevated oxidative stress seen in the rats (EG-VD3) were also significantly prevented with NAC supplement. Thus, NAC might have clinical implications in preventing oxidative renal cell injury and, ultimately, kidney stone formation.

Preventive and curative effects of Achyranthes aspera Linn. extract in experimentally induced nephrolithiasis.

The present study was undertaken to evaluate the efficacy of Achyranthes aspera in preventing and reducing the growth of calcium oxalate stones in ethylene glycol induced nephrolithiatic model. Hyperoxaluria was induced in rats using ethylene glycol (EG, 0.4%) and ammonium chloride (1%) for 15 days and was then replaced with EG (0.4%) only. Upon administration of cystone (750 mg/kg body wt.), aqueous extract of A. aspera (500 and 1000 mg/kg body wt.), levels of renal injury markers (lactate dehydrogenase and alkaline phosphatase) were normalized with a decrease in serum urea and serum creatinine. Concurrent treatment reduced changes in the architecture of renal tissue and also decreased the size of crystals thereby helping in quick expulsion of the crystals. The present results indicated that Achyranthes aspera had an ability to maintain renal functioning and reduced renal injury.

Inhibition of calcium oxalate crystal deposition on kidneys of urolithiatic rats by Hibiscus sabdariffa L. extract.

The present study aims at systematic evaluation of the calyces of Hibiscus sabdariffa to establish its scientific validity for anti-urolithiatic property using ethylene glycol-induced hyperoxaluria model in male albino rats. Administration of a mixture of 0.75% ethylene glycol and 2% ammonium chloride resulted in hyperoxaluria as well as increased renal excretion of calcium and phosphate. The decrease in the serum calcium concentration indicates an increased calcium oxalate formation. Supplementation of aqueous extract of H. sabdariffa at different doses (250, 500 and 750 mg/kg body weight) significantly lowered the deposition of stone-forming constituents in the kidneys and serum of urolithiatic rats. These findings have been confirmed through histological investigations. Results of in vivo genotoxicity testing showed no significant chromosomal aberrations in the bone marrow cells of ethylene glycol-induced rats. The plant extracts at the doses investigated induced neither toxic nor lethal effects and are safe. It can be concluded that the calyces of H. sabdariffa are endowed with anti-urolithiatic activity and do not have genotoxic effects. Thus, it can be introduced in clinical practices and medicine in the form of orally administered syrup after further investigations and clinical trials.

Phytotherapy in a rat model of hyperoxaluria: the antioxidant effects of quercetin involve serum paraoxonase 1 activation.

Serum paraoxonase 1 (PON1) has been reported to be an important contributor to the antioxidant and anti-inflammatory activities of HDL, avoiding LDL oxidation. The activity of this enzyme is reduced in patients with renal insufficiency, caused by elevated oxidative stress and disturbances of apolipoprotein metabolism. Therapeutic utilization of antioxidants to control renal oxidative stress may be an effective therapy in renal protection. The aim was to investigate the protective effects of several antioxidant compounds against the oxidative stress associated to renal failure induced by ethylene glycol (EG), focusing on the possible role of serum PON1 activity. Fifty-four male Wistar rats were randomly assigned to six groups (n = 9): an untreated control (C) group, an EG-treated group, a catechin (CAT)-treated group, an epicatechin (EPI)-treated group, a quercetin (QUE)-treated group and a folk herbal extract (FHE)-treated group. After 16 d of treatment, calcium oxalate lithiasis was induced in the rats using EG. After eight days (treatment + EG), the animals were sacrificed. EG treatment impaired kidney composition, increased oxidative damage, and decreased serum paraoxonase and arylesterase activities. CAT, QUE and the FHE Fagolitos improved oxidative status by enhancing antioxidant defenses - superoxide dismutase and PON1 activities - and reducing oxidative damage, thus reinforcing the idea of a possible role of PON1 in the protective effects of QUE against the deleterious consequences of oxidative stress in kidney.

Ethylene glycol-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.

Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified/warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after in vitro fertilization, and cortical granule exocytosis (CGE) was observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.

Development of alternative plant vitrification solutions in droplet-vitrification procedures.

This study aimed at developing alternative vitrification solutions, modified either from the original PVS2 vitrification solution by increasing glycerol and sucrose and/or decreasing dimethylsulfoxide and ethylene glycol concentration, or from the original PVS3 vitrification solution by decreasing glycerol and sucrose concentration. The application of these vitrification solutions to two model species, i.e. garlic and chrysanthemum in a droplet-vitrification procedure, revealed that PVS3 and variants were superior to PVS2 and variants and that most PVS2 variants were comparable to the original PVS2. Both species were sensitive to chemical toxicity of permeating cryoprotectants and chrysanthemum was also sensitive to osmotic stress. The lower recovery of cryopreserved garlic shoot apices dehydrated with PVS2 and variants compared with those dehydrated with PVS3 and variants seemed attributed to cytotoxicity of the vitrification solutions tested as well as to insufficient protection against freezing injury. Chrysanthemum shoot tips were very sensitive to both chemical toxicity and osmotic stress and therefore, induction of cytotoxity tolerance during preconditioning was required for successful cryopreservation. The present study revealed that some of the PVS2 variants tested which have increased glycerol and sucrose and/or decreased dimethylsulfoxide and ethylene glycol concentration can be applied when explants are of medium size, tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.

Development of an in vitro reproductive screening assay for novel pharmaceutical compounds.

An in vitro reproductive cell-based toxicity assay was developed using MLTC-1 (murine Leydig tumour cell line) in order to examine the reproductive toxicity of two novel nanopharmaceutical compounds, namely ethylene glycol mono allyl ether and poly(ethylene glycol) octa-functionalized polyhedral oligomeric silsesquioxane. Three commonly used cytotoxicity assays, namely the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and Crystal Violet assays, were compared, and the MTT assay proved to be the most accurate and reproducible for the MLTC-1 cell line. The doubling rate of the MLTC-1 cells was 30+/-3.5 h and the optimal seeding density for the MTT assay was 20000 cells per well, and the optimized MTT assay utilized a 4 h cell adherence followed by incubation with 0.5 mg/ml MTT for 1 h. The intra- and inter-assay CV (coefficient of variation) values were 12.3 and 11% respectively. MLTC-1 cells only produce the reproductive hormone progesterone in response to hCG (human chorionic gonadotropin), which stimulated progesterone production dose-dependently from 0 to 100 m.i.u. (milliinternational units)/ml (2706+/-1118 ng/ml). H(2)O(2) as a negative control killed 100% of cells at 1000 microg/ml. The two nanopharmaceutical compounds were cytotoxic at concentrations > or =0.1 microg/ml, but hCG decreased cytotoxicity to > or =1000 microg/ml (P<0.001). hCG-stimulated progesterone synthesis afforded some protection against the cytotoxic effects of the two novel nanotechnology compounds; therefore doses < or =100 microg/ml and an exposure period of 1 h would be recommended for testing in in vivo animal reproductive assays.

The effect of osmolytes and small molecule on Quadruplex-WC duplex equilibrium: a fluorescence resonance energy transfer study.

The structural competition between the G-quadruplex and Watson-Crick duplex has been implicated for the repetitive DNA sequences, but the factors influencing this competitive equilibrium in the natural and pharmacological context need to be elucidated. Using a 21mer 5'-Fluorescein-d[(G3TTA)3G3]-TAMRA-3' as a model system, extensive fluorescence resonance energy transfer analysis was carried out to investigate sensitivity of this equilibrium to osmotic stress and quadruplex selective small molecule. The binding affinities and kinetics involved in the hybridization of quadruplex to its complementary strand in the absence and presence of different concentrations of osmolytes (ethylene glycol and glycerol) and a quadruplex selective ligand (cationic porphyrin-TMPyP4) were determined. The presence of osmolytes and cationic porphyrin decreased the binding affinity of quadruplex to its complementary strand and slowed the kinetics of the reaction by delaying the hybridization process. Our binding data analysis indicates that the presence of either osmolytes or porphyrin increase the amount of quadruplex in the equilibrium. In 100 mM KCl solution, when 30 nM of each of the components, i.e. quadruplex and the complementary strand, were mixed together, the amount of quadruplex present in the system under equilibrium were 17.6, 23.4, 23.1 and 19.6 nM in the absence and presence of 10% ethylene glycol, 10% glycerol and 150 nM TMPyP4, respectively. Fluorescence melting profile of quadruplex in the absence and presence of these perturbants confirm the findings that osmolytes and cationic porphyrin stabilize quadruplex, and thus, shift the equilibrium to quadruplex formation.

A novel bipolar mode of attachment to aluminium-containing adjuvants by BBG2Na, a recombinant subunit hRSV vaccine.

Human respiratory syncytial virus (hRSV) is a major pathogen responsible for bronchiolitis and severe pulmonary disease in very young children, immunodeficient patients and the elderly. BBG2Na, a recombinant chimeric protein produced in Escherichia coli, is a promising subunit vaccine candidate against this respiratory pathogen, composed of G2Na, the central domain of RSV G glycoprotein, and BB, an albumin binding domain of streptococcal protein G. BBG2Na has a basic isoelectric point (pI 9.3) and as expected, is strongly adsorbed by aluminium phosphate (AP). Surprisingly, BBG2Na is also strongly adsorbed by aluminium hydroxide (AH), which normally binds molecules with acidic isoelectric points. This behaviour was unexpected according to the well established adsorption model of Hem and co-workers. Our observations may be explained by the bipolar two-domain structure of the BBG2Na chimera which is not reflected by the global basic isoelectric point of the whole protein: the BB domain has an acidic isoelectric point (pI 5.5) and the G2Na domain a highly basic one (pI 10.0). Importantly, formulation in either aluminium salt resulted in equally high immunogenicity and protective efficacy against RSV in mice. From a physicochemical point of view, this unique property of BBG2Na makes it eminently suitable for combination to either paediatric or elderly multivalent AH- or AP-containing vaccines already in the market or in development.