Enhanced Synergistic-Antioxidant Activity of Melatonin and Tretinoin by Co-encapsulation into Amphiphilic Chitosan Nanocarriers: During Mice In Vitro Matured Oocyte/Morula-Compact Stage Embryo Culture Model.

The use of exogenous antioxidants or the combination of them during in vitro oocyte/embryo culture media is reasonable. Co-delivery by nanocarrier has been designed to overcome the limitations of combining them traditionally. In this work, amphiphilic chitosan nanocarrier (ACN) was applied to co-encapsulate melatonin (Mel) and tretinoin (TTN) by the self-assembled method and evaluate their synergistic antioxidant efficacy in mice oocytes/embryos. The formation of single/dual-ACN was confirmed by Fourier-transformed infrared spectroscopy (FT-IR). The average particle diameter, size distribution, polydispersity index (PDI), and zeta potential of them were measured by dynamic light scattering (DLS), and the morphology was evaluated by TEM and SEM technologies. Also, the encapsulation efficiency (EE%) and drug loading content (DL%) of the nanocapsules were determined by UV-vis spectrophotometry. Studies of the in vitro release showed a continued drug release without any bursting effect of Mel+TTN-ACNs compared with single Mel/TTN-ACNs. Then, in both experiments, nuclear staining (Aceto-orcein and Hoechst 33342), fluorescent staining of H2DCFDA, chemiluminescence test, and qRT-PCR technique were performed as in vitro toxicity studies. The results of all these evaluations demonstrated that the dual delivery of Mel and TTN could accumulate a safety (without high-dose toxicity) synergistic anti-oxidative effect in oocyte/embryo by passive controlled, and inhibit intra/extracellular ROS levels by an enhanced intracellular penetration.

In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin.

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

Embryotoxicity in vitro with extract of Indigofera suffruticosa leaves.

Aqueous extract of leaves of Indigofera suffruticosa (AELIs) were studied for adverse effects in preimplantation mouse embryos. Two-cell mouse embryos were cultured for 94 h in human tubal fluid medium (HTF), and AELIs at a concentration of 5 or 10 mg/ml. On Day 4 of culture, morulae and blastocysts were collected for morphological analysis of blastomeres. We found that embryos exposed to the higher concentration of AELIs (10 mg/ml) did not develop and all embryos persisted at the two-cell stage. Those embryos exposed to the lower concentration (5 mg/ml) showed development until morula, blastocyst and hatched blastocyst stages that were similar to the controls. These results suggest that use of AELIs may be hazardous to humans who make use of it in folk medicine.

Ruta graveolens aqueous extract retards mouse preimplantation embryo development.

This work was undertaken to examine possible embryotoxicity of Ruta graveolens (rue), a plant used by indigenous communities for the purposes of therapeutic and fertility regulation. Superovulated mice were mated and isolated after copulation. They were given aqueous extract of R. graveolens (5, 10, and 20% w/v) or plain water (control) orally for 4 days. Ninety-eight hours post-human chorionic gonadotrophin (hCG), embryos were flushed from oviducts and uterine horns to assess their state of development and extent of embryo transport. Ingestion of rue at 10 and 20% resulted in a high proportion of abnormal embryos (36.7 and 63.6%, respectively, P<0.05). Cell number was diminished (P<0.01) and embryo transport was slightly delayed in the highest dose group. These findings demonstrate that oral administration of R. graveolens extract can interfere with preimplantation development and embryo transport.

Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development.

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.

Improved development of ICR mouse 2-cell embryos by the addition of amino acids to a serum-, phosphate-and glucose-free medium.

This study was conducted to evaluate how exogenous amino acids could affect preimplantation development of ICR mouse embryos. Two-cell embryos collected from naturally mated mice were cultured in amino acid-, glucose- and phosphate-free preimplantation (P)-1 medium. In Experiments 1, 19 amino acids (aa; 1% and 0.5% of MEM essential and nonessential amino acid solutions, respectively) were added to P-1 medium supplemented with either fatty acid-free bovine serum albumin (BSA; 3 mg/mL) or human follicular fluid (hFF; 10%). Regardless of BSA or hFF addition, embryo development to the morula (84 to 86% vs. 97 to 100%) and the blastocyst (54% vs. 93 to 94%) stages was significantly (P<0.05) enhanced by the addition of aa compared with no addition. In Experiment 2, the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts and the ratio of ICM cell to trophectodermal cell (TE) were evaluated after aa addition. In both BSA- and hFF-containing P-1 medium, a significant increase in total blastomere number were found after aa addition (47 to 52 vs. 62 to 63 cells) compared with no addition. However, the ICM/TE ratio was not significantly affected by aa supplementation in both media, while ICM cell number was greatly increased after aa addition in hFF-containing medium (12 vs. 17 cells). When blastocysts were further cultured up to 162 hr post-hCG injection, development to the hatched blastocyst stage was significantly promoted by aa addition (0% vs. 11 to 20%) in both BSA- and hFF-containing media. In conclusion, aa significantly promote the preimplantation development to the hatched blastocyst stage and such effect mainly exerted on supporting blastomere proliferation.

Effect of linoleic acid-albumin in the culture medium on freezing sensitivity of in vitro-produced bovine morulae.

The objective of this study was to improve the survival of in vitro-produced bovine morulae after cry opreservation. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) were cultured in a mixture of modified synthetic oviduct fluid (m-SOF)/0.3% BSA and m-SOF/0.3% linoleic acid-albumin from bovine serum (LAA) at 39.0 degrees C in 5% O2, 5% CO2 and 90% N2 (final LAA concentration: 0, 0.01, 0.03, 0.1 or 0.3%). Morulae harvested at 138 hpi were frozen and thawed in m-PBS/0.3% BSA containing 1.5 M ethylene glycol and were cultured for 96 h in m-SOF/10% FBS to assess further development. The post-thaw survival of morulae derived from culture in 0.1% LAA (60%, P < 0.01) and in 0.03% LAA (55%, P < 0.05) was higher than that in 0% LAA (32%). Lowering the LAA concentration below 0.1% resulted in similar rates of morula development as in m-SOF/0.3% BSA. In Experiment 2, zygotes were cultured in m-SOF/0.1% LAA from 20 to 90 hpi and/or from 90 to 138 hpi. Post-thaw survival of morulae that had been exposed to LAA from 20 to 90 hpi (39%) or from 90 to 138 hpi (56%) was higher than that of morulae cultured without LAA from 20 to 138 hpi (12%, P < 0.02). These survival rates were lower than that of morulae cultured with LAA over a period of 20 to 138 hpi (76%, P < 0.001). The results indicate that cell-free culture of IVM/IVF bovine zygotes in m-SOF supplemented with LAA produces morula-stage embryos relatively tolerant to the process of freezing and thawing.

Uptake and salvage of hypoxanthine mediates developmental arrest in preimplantation mouse embryos.

Preimplantation mouse embryos become arrested after first or second cleavage when cultured in hypoxanthine-supplemented Whitten's medium. We present evidence that the hypoxanthine-induced arrest is dependent on uptake and salvage of hypoxanthine and depletion of phosphoribosylpyrophosphate (PRPP) levels. Hypoxanthine uptake increased during the 2-cell stage and was augmented by glucose. HPLC analysis of [14C]hypoxanthine metabolism revealed that hypoxanthine was salvaged and converted to ATP and guanosine triphosphate (GTP), with a shift to more guanyl nucleotide production at the 3- to 4-cell stage. In embryos from mice with a null mutation for the salvage enzyme hypoxanthine-guanine phosphoribosyltransferase, hypoxanthine did not block development nor was it taken up by the embryos. Glucose, which is required for the hypoxanthine-induced arrest, produced a 5.3-fold increase in PRPP levels at the 2-cell stage, which was eliminated by hypoxanthine. We conclude that metabolism of hypoxanthine to nucleotides mediates its inhibitory action on preimplantation mouse embryos via negative feedback on PRPP synthetase, ultimately resulting in decreased PRPP availability and arrest of other PRPP-dependent pathways. Finally, reversal of the block by EDTA and cAMP-elevating agents may be mediated by alterations in hypoxanthine or glucose uptake, or by changes in the relative metabolism of hypoxanthine.

Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by vitro-produced bovine morulae and blastocysts.

The effect of glycine and alanine on the development of 2- to 4-cell bovine embryos, and amino acid uptake by bovine morulae and blastocysts, were examined through the use of a chemically defined medium. Bovine embryos at 2- to 4-cell stages were prepared by in vitro maturation and fertilization and cultured in a synthetic oviduct fluid medium (SOFM) containing polyvinyl alcohol (PVA) instead of BSA in order to examine the effect of amino acids. Morulae or blastocysts obtained from culture in SOFM containing BSA were cultured for 10 h in SOFM containing PVA to determine amino acid uptake. The combination of essential and nonessential amino acids with or without glutamine improved development to the blastocyst stage over that observed in the control (34% or 32% vs. 19%, respectively; p < 0.05). The optimal supplemental concentrations were 5 mM for alanine and 10 mM for glycine. At these concentrations, development to blastocysts was enhanced by the addition of alanine or glycine independently (35% or 36% vs. 26%, respectively; p < 0.05); the combined addition of alanine and glycine greatly (p < 0.01) improved proportions of blastocysts (45% vs. 26%) and hatched blastocysts (10% vs. 2%) compared to those obtained in the control. Addition of glycine with or without alanine to culture medium significantly increased cell number per blastocyst over that in the control (137 +/- 5 or 131 +/- 5 vs. 106 +/- 4, respectively; p < 0.01). Bovine morulae and blastocysts depleted aspartate, serine, and glutamate at a highly significant rate (p < 0.001) and arginine at a significant rate (p < 0.05), and produced alanine at a highly significant rate (p < 0.001) when cultured in medium containing 20 essential and nonessential amino acids. Serine, asparagine, glycine, alanine, and glutamine were highly (p < 0.001) produced by bovine morulae and blastocysts cultured in a medium containing essential amino acids without glutamine. These results indicate that alanine and glycine in a defined medium synergistically improve development of in vitro-produced bovine embryos, and also that bovine morulae and blastocysts prefer aspartate, glutamate, serine, and arginine and produce glutamine and several nonessential amino acids (serine, asparagine, alanine, and glycine).

Nicotinamide, a component of complex culture media, inhibits mouse embryo development in vitro and reduces subsequent developmental potential after transfer.

OBJECTIVE: To determine the effects of B-group vitamins present in culture media on mouse embryo development in vitro and subsequent viability. DESIGN: Mouse zygotes were cultured in the presence of B-group vitamins. Embryo morphology and cell numbers were determined at 96 and 120 hours after hCG. Viability was assessed by transfer of embryos after 3 days of culture to pseudopregnant recipients. Resultant pregnancy rates (PRs) and fetal weights were determined. RESULTS: Supplementation of an amino acid-free medium with minimal essential medium (MEM) B-group vitamins significantly decreased embryo cleavage rates, whereas the inclusion of Ham's F-10 medium B-group vitamins significantly reduced both cleavage rates and morphological development. Subsequent experiments determined that nicotinamide (5 microM) significantly reduced blastocyst cell number, implantation rate, viable PR, and fetal weight. CONCLUSION: The data indicate that nicotinamide inhibits mouse embryo development in culture and reduces viability. Nicotinamide is present at high levels in Ham's F-10 and MEM media that are used routinely in human embryo culture. The role of vitamins in human embryo development in vitro warrants investigation.

Design of vitrification solutions for the cryopreservation of embryos.

A series of experiments was performed to determine the concentrations at which ten cryoprotectants singly and in pairs would vitrify on plunging into liquid nitrogen and remain vitreous when warmed by plunging into a water bath at 25 degrees C. From these tests eight solutions (VS) were selected for testing of toxicity to mouse morulae in vitro. One of these (VS1) was modified as a further five VS of which one (VS11) was tested for toxicity to all stages of mouse embryos and to sheep compacted morulae. The concentrations at which the cryoprotectants vitrified on cooling were: butylene glycol, 3.0 mol l-1; propylene glycol, 4.0 mol l-1; dimethyl sulfoxide (DMSO) and glycerol 5.0 mol l-1; ethylene glycol, 6.5 mol l-1. None of these, at the highest concentration tested, remained vitreous during warming. Methanol and the high molecular weight polymers, dextran, Ficoll, polyethylene glycol and polyvinylpyrrolidone, did not vitrify at the concentrations tested. Toxicity studies showed the order of increasing toxicity to be ethylene glycol, methanol, DMSO, glycerol, propylene glycol and butylene glycol. Of the mixtures composed of two cryoprotectants, those containing ethylene glycol and glycerol were the least toxic at vitrifying concentrations. VS11 (6.0 mol ethylene glycol l-1 and 1.8 mol glycerol l-1) was well tolerated by mouse morulae, less well by eight- and one-cell embryos and poorly by two-cell embryos. Dilution of the VS11 from mouse embryos by exposure to 1.0 mol sucrose l-1 for 10 min did not enhance their survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of phosphate, energy substrates, and amino acids on development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium.

Bovine oocytes that had been matured and fertilized in vitro were cultured in a simple, chemically defined, protein-free medium (mTLP-PVA). When the medium was supplemented with 19 amino acids, development to the 8-cell (14-20% vs. 38-46%), morula (0-6% vs. 27-32%), and blastocyst (0-1% vs. 9-13%) stages 96, 144, and 192 h after insemination, respectively, was significantly greater in the absence than in the presence of glucose (5.56 mM) regardless of the presence of phosphate (1.05 mM). However, blastocyst development was difficult in medium with any combination of glucose and phosphate without amino acids. In mTLP-PVA with amino acids and different concentrations of phosphate, the highest proportions of embryos reaching the > or = 8-cell (56%), morula (44%), and blastocyst (24%) stages were obtained at a 0.35 mM concentration. When lactate and pyruvate were omitted from mTLP-PVA (mT-PVA) supplemented with amino acids and 0.35 mM phosphate, the first cleavage was completely inhibited. Although lactate or pyruvate alone could support blastocyst development to a limited extent (10-15%), a significantly higher proportion (22%) of blastocysts was obtained in medium with both lactate (10 mM) and pyruvate (0.5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Thermoprotection of preimplantation bovine embryos from heat shock by glutathione and taurine.

To determine if deleterious effects of heat shock on embryos could be reduced in vitro by glutathione or taurine, morulae from superovulated cows were placed in modified Hams-F10 medium supplemented with 50 nM glutathione (GSH), 50 mM taurine or neither. Morulae were incubated for 2 hours at 38.5 degrees C, then at 42.0 degrees C (heat shock) or 38.5 degrees C for 2 hours and followed by incubation at 38.5 degrees C for 20 hours. Neither GSH nor taurine enhanced viability or blastocyst development at 38.5 degrees C. At 42.0 degrees C, however, GSH and taurine increased (P less than 0.02) viability (73%, 41% and 26% live for GSH, taurine and control); GSH increased (P less than 0.05) blastocyst development (55% for GSH vs. 30% for control). In conclusion, partial thermoprotection of bovine embryos from heat shock can be achieved in vitro by administration of GSH. Taurine is only slightly effective.

Effect of RU486 on different stages of mouse preimplantation embryos in vitro.

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.

In vivo cleavage rates and viability obtained for early cleavage mouse embryos in co-culture with oviduct cells.

The cleavage rate and development of two-cell mouse embryos to the morulae stage in co-culture with mouse oviduct cells was studied in vitro and compared with those achieved in vivo. Embryos were cultured in Whittingham's T6 (T6), T6 supplemented with fetal calf serum (FCS) and in co-culture with either Dulbecco's Modified Eagles Medium supplemented with sodium lactate (DMEM + 1a) or a modification of T6 medium containing vitamins and amino acids (T6 + v + aa). Co-culture of oviductal cells with DMEM + la medium supported two-cell mouse embryo development to eight cells at a rate significantly better (P less than 0.001) than T6, but the rate of embryo development was not equivalent to that in vivo. DMEM + la alone was inadequate as an embryo culture medium. Co-cultures using T6 + v + aa with mouse oviductal cells were prepared from mice at days 1, 2 or 3 of pseudopregnancy. Day 2 and 3 co-cultures allowed two-cell embryos to develop at a rate comparable to that in vivo up to the mid eight-cell stage (68 h after hCG), but by 76 h after hCG embryos were retarded. Transfer to pseudopregnant recipients of embryos co-cultured with day 2 oviductal cells until 68 h after hCG resulted in a rate of fetal development equivalent to that of embryos grown in vivo. Our results show that co-culture of early cleavage-stage embryos with mouse oviductal cells allows embryos to retain cleavage rates and viability comparable to in vivo development.