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.

Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 µg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, p<0.05), compact morulae formation (60.83 vs. 51.30%, p<0.05), and the blastomere apoptosis index (3.70 ± 1.41 vs. 4.43% ± 1.65, p<0.05) of bovine SCNT embryos. However, vitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, p<0.05) on day 7 and the hatching blastocysts formation rate on day 9 (26.51 vs. 50.65%, p<0.05) compared with that of the untreated group. Vitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

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.

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).

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.