[ comparison between the colony formation of adult mouse spermatogonial stem cells in co cultures with sertoli and STO [mouse embryonic fibroblast cell line]]

The aim of this study was to compare the colony formation of spermatogonial stem cells [SSCs] on sertoli and STO [Mouse embryonic fibroblast cell line] feeder cell layers during a two-week period. Initially, sertoli cells and SSCs were isolated from adult mouse testes using a two-step enzymatic digestion and lectin immobilization. Characteristics of the isolated cells were immunocytochemically confirmed by examining for the presence of Oct-4, CDH1, promyelocytic leukaemia zinc finger factor [PLZF], SSC C-kit, and the distribution of Sertoli cell vimentin. SSCs were then cultured above the Sertoli, STO and the control [without co-culture] separately for two weeks. In all three groups, the number and diameter of colonies were evaluated using an invert microscope on the 3rd, 7th, 10th and 14th day. beta1 and alpha6 -integrin m-RNA expressions were assessed using a reverse transcription polymerase chain reaction [RT-PCR] and realtime PCR. Furthermore, Oct-4 m RNA expression was assessed using real time PCR. Statistical analysis was performed using ANOVA; and the paired two-sample t test and Tukey's test were used as post-hoc tests for the data analysis of the three sertoli, STO and control cocultures. At the four specified time points, our results showed significant differences [p<0.05] in colony numbers and diameters among the sertoli, STO and control groups. The number and diameter of colonies increased more rapidly in the sertoli coculture than in the other two Our results at all four time points also showed significant differences [p<0.05] in the mean colony numbers and diameters between the three groups, with the Sertoli coculture having the highest mean values for colony numbers and diameters. The RT-PCR results, after two-weeks of culturing, showed that beta1 -integrin was expressed in all three groups cocultures, but alpha6 -integrin was not expressed. Additionally, based on real time PCR results, the three genes [beta1 -integrin, alpha6 -integrin, Oct-4] mentioned were also expressed in all three co cultures groups. Based on the optimal effects of sertoli feeder cells on spermatogonial stem cells in a co culture system, as also confirmed by several other studies, their use is suggested to achieve better colonization of SSCs

Assessment of Epidermal growth factor [EGF] effects on development of vitrified mouse morulae to the blastocyst stage

Many investigators are interested in finding the new cultural systems that can support the in vitro development of pre-implantation embryos better. Previous studies suggested that growth factors such as epidermal growth factor [EGF] are important in pre-implantation embryo development and implantation process. On the other hand, it is very important to support post thaw development of frozen embryos. The purpose of this study was to determine if the developmental potential of mouse morulae survived after vitrification could be increased using medium containing EGF. Mouse morulae were divided into vitrified and non-vitrified groups. Vitrification procedure was carried out using a combination of 40% ethylene glycol, 30% ficoll and 0.5 M sucrose [EFS40] as cryoprotectant. The embryos were warmed rapidly using 0.5 M sucrose. The survived embryos were cultured either on T6 or T6+EGF media. Accordingly, the embryos of the non-vitrified group were also cultured. The developmental rates in all groups were daily recorded and compared statistically using Chi-square test. The results showed that after 4 days of culture, the developmental potential of non-vitrified embryos cultured on T6+EGF was significantly increased. There was no significant difference between vitrified embryos cultured on T6 and T6+EGF media. In conclusion, the developmental potential of vitrified-warmed embryos does not increase in the medium containing EGF, even though there was significant increased developmental potential of non-vitrified embryos after culture on medium containing EGF. It is needed to do more study about the changes which will probably happen on the embryo EGF receptors following vitrification