Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

In relation to the growing recent interest in the establishment of sperm‐mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0–500 ng) of pcDNA/his/Lac‐Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live‐immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA‐incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X‐Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live‐immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live‐immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression. Mol. Reprod. Dev. 77:868–875, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.     

Time dependent effect of post warming interval on microtubule organization, meiotic status, and parthenogenetic activation of vitrified in vitro matured sheep oocytes

It is a common practice to rest vitrified-warmed matured oocytes for 1-3 h, as a treatment to recover spindle and cytoskeleton, before commencing a further treatment. Vitrified-warmed matured oocytes, however, are very sensitive and may resume meiosis spontaneously during this recommended rest time. Therefore, the aim of this study was to assess spindle and chromosome status as well as developmental competence of vitrified in vitro matured sheep oocytes activated parthenogenetically, either 0 h (immediately) or 2 h (delayed) after warming. There was no significant effect of post-warming interval on the proportion of degenerated oocytes. Evaluation of chromosomes and meiotic spindle configuration showed that 11.11% of oocytes in the immediate group and 8.82% of oocytes in the delayed group had normal chromosomal alignment on well-structured spindles, compared to non-vitrified group (79.41%). Meanwhile, majority of the chromosomal abnormalities in the immediate and delayed groups were categorized as absent (unobservable) (77.78%) and anaphase II (70.59%), respectively. Oocytes in immediately activated group showed significantly higher blastocyst rate (28.86%) compared to delayed activated group (16.47%). In conclusion, the results suggest that post-warming interval may have important consequence on meiotic progression and parthenogenetic activation of vitrified oocytes. In sheep, it appears that chemical activation without having to await microtubule reorganization improves embryonic development.     

Specific activation requirements of in vitro-matured sheep oocytes following vitrification-warming

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase‐II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified‐warmed, and unvitrified‐activated oocytes, the present study investigated how vitrification‐warming process may affect developmental competence of in vitro‐matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified‐warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified‐activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using the maximum ionomycin concentration (5 µM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified‐warmed oocytes (28.4 ± 1.4%) was achieved with a minimal duration of ionomycin treatment (1 min), and further extending the duration dramatically reduced developmental potential of vitrified‐warmed oocytes. These results suggested that vitrified‐warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture. Mol. Reprod. Dev. 79:434–444, 2012. © Wiley Periodicals, Inc.     

Differentiation of microfluidic-encapsulated trabecular meshwork mesenchymal stem cells into insulin producing cells and their impact on diabetic rats

Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM-MSC), into insulin-producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet-specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi-differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx-1 mRNA (1.938-fold, p-value: 0.0425) and Insulin mRNA (2.841-fold, p-value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM-MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.     

Fabrication of an Electrochemical Sensor Based on Gold Nanoparticles/Carbon Nanotubes as Nanocomposite Materials: Determination of Myricetin in Some Drinks

In this paper, the electrochemical behavior of myricetin on a gold nanoparticle/ethylenediamine/multi-walled carbon-nanotube modified glassy carbon electrode (AuNPs/en/MWCNTs/GCE) has been investigated. Myricetin effectively accumulated on the AuNPs/en/MWCNTs/GCE and caused a pair of irreversible redox peaks at around 0.408 V and 0.191 V (vs. Ag/AgCl) in 0.1 mol L⁻¹ phosphate buffer solution (pH 3.5) for oxidation and reduction reactions respectively. The heights of the redox peaks were significantly higher on AuNPs/en/MWNTs/GCE compare with MWCNTs/GC and there was no peak on bare GC. The electron-transfer reaction for myricetin on the surface of electrochemical sensor was controlled by adsorption. Some parameters including pH, accumulation potential, accumulation time and scan rate have been optimized. Under the optimum conditions, anodic peak current was proportional to myricetin concentration in the dynamic range of 5.0×10⁻⁸ to 4.0×10⁻⁵ mol L⁻¹ with the detection limit of 1.2×10⁻⁸ mol L⁻¹. The proposed method was successfully used for the determination of myricetin content in tea and fruit juices.