The effect of ovine oocyte vitrification on expression of subset of genes involved in epigenetic modifications during oocyte maturation and early embryo development.

Apart from ultrastructural damages in oocytes subjected to cryopreservation procedures, little is known about the status of epigenetic modification and chromatin remodeling in vitrified oocytes. In present study, the expression patterns of eight genes involved in epigenetic modification (HAT1, HDAC1, SUV39H1, DNMT1, and DNMT3b), chromatin remodeling (HMGN3a and SMARCAL1), and transcription (STAT3), were investigated in fresh and vitrified germinal vesicle and metaphase II oocytes and their resulting embryos at 2 to 7 cells, 8 to 16 cells, morula, and blastocyst stages. The mRNA relative abundance was quantified by reverse transcriptase real-time polymerase chain reaction, as fold change relative to the value obtained for fresh germinal vesicle oocytes. Vitrified oocytes showed lower cleavage (38.1% vs. 95.5%, P < 0.001) and development to blastocyst (8.2% vs. 50.8%, P < 0.001) compared with controls. In both fresh and vitrified groups, the genes expressions in oocytes were lower than cleaving embryos, with an exception of HMGN3a. Compared with fresh derived embryos, in vitrified groups, the overall expressions of HMGN3a and HDAC1 were decreased, whereas the expressions of STAT3, SMARCAL1, and DNMT3B were increased. Altogether, despite some differences in expression pattern of some genes, the overall pattern of increase and/or decrease in gene expression was almost the same in most of the genes studied between vitrified and fresh groups. Thus, apart from some mismatch in pattern of genes expression in a number of cases, the difference in magnitude and/or primacy and recency in reaching to the maximum expression, in association to embryonic genome activation, between fresh and vitrified groups, might be the reason for the lower developmental competence of vitrified-warmed oocytes compared with fresh ones.

Comparison of proliferative and multilineage differentiation potential of sheep mesenchymal stem cells derived from bone marrow, liver, and adipose tissue.

BACKGROUND: Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow Mesenchymal Stem Cells (MSCs) has remained to be challenged. In this study successful isolation, multilineage differentiation, and proliferation potentials of sheep MSCs derived from bone marrow, adipose tissue, and liver were widely investigated. METHODS: The primary cell cultures were prepared form tissue samples obtained from sheep 30-35 day fetus. Passage-3 cells were plated either at varying cell densities or different serum concentrations for a week. The Population Doubling Time (PDT), growth curves, and Colony Forming Unit (CFU) of MSCs was determined. The stemness and trilineage differentiation potential of MSCs were analyzed by using molecullar and cytochemical staining approaches. The data was analyzed through one way ANOVA using SigmaStat (ver. 2). RESULTS: The highest PDT and lowest CFU were observed in adipose tissue group compared with other groups (p<0.001). Comparing different serum concentrations (5, 10, 15, and 20%), irrespective of cell sources, the highest proliferation rate was achieved in the presence of 20% serum (p<0.001). Additionally, there was an inverse relation between cell seeding density at culture initiation and proliferation rate, except for L-MSC at 300 cell seeding density. CONCLUSION: All three sources of fetal sheep MSCs had the identical trilineage differentiation potential. The proliferative capacity of liver and bone marrow derived MSCs were similar at different cell seeding densities except for the higher fold increase in B-MSCs at 2700 cells/cm (2) density. Moreover, the adipose tissue derived MSCs had the lowest proliferative indices.

The effect of biopsy during precompacted morula stage on post vitrification development of blastocyst derived bovine embryos.

Improvements on embryo micromanipulation techniques led to the use of embryo biopsy in commercial embryo transfer programs for genetic analysis of preimplantation bovine embryos. The aim of this study was to evaluate the quality of bovine blastocyst derived from embryos biopsied at different pre-compacted morulae stages by assessment of cryosurvivability of the resulting blastocysts. The in vitro produced bovine embryos were subjected to biopsy at days 2, 3, and 4 post-insemination with different cell numbers (4 to 16-cells). Embryo cell biopsy was carried out in a 100 µl drop of H-SOF following pronase drilling by aspiration of one blastomere. The biopsied embryos were then cultured in SOFaaBSA co-cultured with oviduct cells-monolayer until blastocyst formation. The blastocysts were cryopreserved at room temperature after exposure of equilibration (glycerol 1.4 M for 5 min and then glycerol 1.4 M and ethylene glycol 3.6 M for 5 min) and vitrification solutions (3.4 M glycerol and 4.6 M ethylene glycol). The blastocysts were loaded into the center of 0.25 ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. There was no significant difference in cryosurvivability of vitrified-warmed blastocysts derived form biopsied embryos at different pre-compacted morula stages. The quality of biopsy derived blastocysts was identical to that of non-biopsy derived ones in terms of post vitrifcation survival and hatching rates. In conclusion there was no preference between different times of embryo biopsy at precompacted morula stages in term of cryosurvivability of biopsy derived bovine blastocysts.

The Effect of the Duration of In Vitro Maturation (IVM) on Parthenogenetic Development of Ovine Oocytes.

The aim of this study was to compare the effect of time of parthenogenetic activation (22 hr versus 27 hr after In Vitro Maturation-IVM) on in vitro development of ovine oocytes using either single (Ionomycin 5 µM for 5 min or Ethanol 7% for 7 min) or combined (ionomycin and ethanol with 6-DMAP 2 mM for 3 hr) activation treatments. The abattoir-derived in vitro matured activated oocytes were cultured in modified synthetic oviductal fluid and assessed for the cleavage, blastocyst, and hatching rates. The single-activated oocytes had a reduction in cleavage, blastocyst and hatching rates compared to the combined-activated oocytes (except for the cleavage at 27 hr). In single-treated groups the rates of cleavage and blastocyst were increased as the maturation time was extended from 22 hr to 27 hr. The numbers of total cells and Inner Cell Mass (ICM), though insignificant, were greater in combined-treated groups compared to the single treatment. The number of ICM in Eth + 6-DMAP group activated at 27 hr was lower than 22 hr. Nonetheless, irrespective of the activation protocol, development to the blastocyst stage, the numbers of total cell, ICM, and cell allocation (ICM/total cells) were significantly lower in parthenogenetic than fertilized embryos. In conclusion, though the cleavage and blastocyst rates in single-treated groups were positively influenced by the extension of duration of IVM (27 hr), there was a trend of decreased numbers of total cells and ICM in slightly aged oocytes. Moreover, developmental potential of ovine parthenotes, especially in young oocytes, was improved by the addition of 6-DMAP to the activation regimen.