142. microRNA AND EARLY MALE GERM CELLS
S. C. McIver A B C , S. D. Roman A B C , B. Nixon A B C and E. A. McLaughlin A B CA ARC Centre of Excelence in Biotechnology and Development, Australia.
B Reproductive Science Group, Australia.
C Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia.
Reproduction, Fertility and Development 22(9) 60-60 https://doi.org/10.1071/SRB10Abs142
Published: 6 September 2010
Abstract
MicroRNAs are short regulatory noncoding RNA molecules that bind to the 3′ untranslated region of mRNA targets to control translation therefore influencing the abundance of many different protein molecules. Aberrant expression of miRNA is linked to many diseases and developmental abnormalities. Testicular Germ Cell Tumours (TGCT) develop from Carcinoma in situ cells which have been identified as dysfunctional gonocytes. Due to the continual rise in the rate of testicular cancer in the developed world, the molecular mechanisms underlying the failure of gonocytes to differentiate into spermatogonia is of great interest. Gonocytes from post natal day 1 testes and spermatogonia from day 7–9 mice were enriched by 2–4% BSA gradient sedimentation. Total RNA including microRNA was extracted and analysed in by Illumina miRNA microarray. Total RNA was also reverse transcribed using specific primers and analysed by qPCR. Three biological replicates were performed in both the microarray and qPCR experiments. Bioinformatic analysis with SAM (Significance Analysis of Microarrays) identified seven significantly different miRNA molecules between spermatogonia and gonocytes. qPCR analysis confirmed two miRNAs were significantly upregulated in spermatogonia (743a, 463*) and three miRNAs were significantly down regulated in spermatogonia (293, 290-5p, 291a-5p). Several miRNA molecules were selected for further study (293, 290-5p, 136, and 146a) and overexpression assays first in the P19 cell line, then in isolated spermatogonia will help determine their function. In the future the role of these molecules in human seminoma will be analysed using overexpression within a seminoma cell line. It is hypothesised that these miRNA molecules control genes involved in male development and differentiation, such as stella, nanog and oct3/4, and may also play a role in tumour development. In conclusion miRNA expression is significantly different between gonocytes and spermatogonia and we propose that this results in the initiation of differentiation and commencement of spermatogenesis.
