Mohammad Miryounesi , Zeinab Jamali , Masoumeh Razipour , Elahe Alavinejad , Mohammad Hossein Modarressi ,
Volume 72, Issue 11 (February 2015)
Abstract
Background: About 15% of couples have fertility problems and male factor in fertility accounts for half of the cases. In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (mESCs) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Testis specific 10 (Tsga10) during this process.
Methods: This is an in vitro study that was performed in department of Medical Genetics in Tehran University of Medical Sciences from February 2012 to March 2013. Mouse embryonic stem cells were cultured on mouse embryonic fibroblast as feeder layer. Then mESCs were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th and 25th days of the culture and were subjected to expression analysis of a panel of germ cell specific genes (Stra8 as pre-meiotic, Dazl and Sycp3 as meiotic and Protamin1 and Spata19 as Post-meiotic). Expression of Testis Specific Gene 10 (Tsga10) at RNA and protein levels was then analyzed.
Results: It was shown that transition of embryonic stem cells from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until 25th day of the culture. Results showed low level of Tsga10 expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This finding is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed form of the related protein was similar to those in vivo as well.
Conclusion: Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.
