Mohammad Miryounesi1, Karim Nayernia2, Maryam Beigom Mobasheri3, Mahdi Dianatpour4, Richard Oko5, Shahram Savad6, Mohammad Hossein Modarressi7. 1. 1)Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2)Department of Medical Genetics, School of Medicine, Tehran university of Medical Sciences, Tehran, Iran.modaresi@tums.ac.ir. 2. GENEOCELL, Advanced Molecular and Cellular Technologies, Montreal, Canada. 3. 2)Department of Medical Genetics, School of Medicine, Tehran university of Medical Sciences, Tehran, Iran. 4)Cancer Research Center, Tehran University of Medical Sciences, Tehran, Iran. 4. Department of Medical Genetics, School of Medicine, Shiraz university of Medical Sciences, Shiraz, Iran. 5. Departments of Anatomy and Cell Biology, Queen's University, 9th Floor, Botterell Hall, Kingston, Ontario, Canada K7L 3N6. 6. Kamali Hospital, Alborz University of Medical Sciences, Karaj, Iran. 7. Department of Medical Genetics, School of Medicine, Tehran university of Medical Sciences, Tehran, Iran.
Abstract
BACKGROUND: 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 (mESC) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. METHODS: 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. Expression of Tsga10 in RNA and protein levels was then analyzed. RESULTS: Transition from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression 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 forms of the related protein was similar to those in vivo as well. CONCLUSIONS: 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.
BACKGROUND: 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 (mESC) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. METHODS: 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. Expression of Tsga10 in RNA and protein levels was then analyzed. RESULTS: Transition from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression 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 forms of the related protein was similar to those in vivo as well. CONCLUSIONS: 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.
Authors: Sicilia T Grady; Ashlee E Watts; James A Thompson; M Cecilia T Penedo; Kranti Konganti; Katrin Hinrichs Journal: J Assist Reprod Genet Date: 2018-11-23 Impact factor: 3.412