Language:
English
In:
Biology of the cell, 2012-11, Vol.104 (11), p.677-692
Description:
Background information
Recently, it became apparent that microRNAs (miRNAs) can regulate gene expression post‐transcriptionally. Despite the advances in identifying the testis‐expressed miRNAs and their role in spermatogenesis, only few data are available showing the spatiotemporal expression of miRNAs during this process.
Results
To understand how different miRNAs can regulate germ cells differentiation, we generated a transgenic mouse model and purified pure populations of premeiotic (PrM) cells and primary spermatocytes (meiotic cells). We also established spermatogonial stem cell (SSC) culture using relatively simple and robust culture conditions. Comparison of global miRNA expression in these germ cell populations revealed 17 SSC‐, 11 PrM‐ and 13 meiotic‐specific miRNAs. We identified nine miRNAs as specific for both SSC and PrM cells and another nine miRNAs as specific for PrM and meiotic cells. Additionally, 45 miRNAs were identified as commonly expressed in all three cell types. Several of PrM‐ and meiotic‐specific miRNAs were identified as exclusively/preferentially expressed in testis. We were able to identify the relevant target genes for many of these miRNAs. The luciferase reporter assays with SSC (miR‐221)‐, PrM (miR‐203)‐ and meiotic (miR‐34b‐5p)‐specific miRNAs and 3′‐untranslated region constructs of their targets, c‐Kit, Rbm44 and Cdk6, respectively, showed an approximately 30%–40% decrease in reporter activity. Moreover, we observed a reduced expression of endogenous proteins, c‐Kit and Cdk6, when the testis‐derived cell lines, GC‐1 and GC‐4, were transfected with miRNA mimics for miR‐221 and miR‐34b‐5p, respectively.
Conclusions
Taken together, we established the miRNA signature of SSC, PrM and meiotic cells and show evidence for their functional relevance during the process of spermatogenesis by target prediction and validation. Through our observations, we propose a working model in which the stage‐specific miRNAs such as miR‐221, ‐203 and ‐34b‐5p coordinate the regulation of spermatogenesis.
The spatiotemporal expression of miRNAs and their post‐transcriptional regulatory mechanisms during spermatogenesis are largely unknown. In the current study, we demonstrate a miRNA signature of spermatogonial stem cells, pre‐meiotic cells and meiotic cells using a pure population of cells obtained from reporter mouse lines (Stra8/EGFP and Sycp3/DsRed). Through our observations, we propose that miRNA‐221, ‐203 and ‐34b‐5p coordinate the spermatogenesis process either by inhibition or activation of germ cell differentiation.
Subject(s):
Animals ; Cell Differentiation - genetics ; Cell Differentiation - physiology ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Gene Expression - genetics ; Gene Expression Profiling ; Male ; Meiosis ; Mice ; Mice, Transgenic ; MicroRNAs - genetics ; MicroRNAs - metabolism ; miRNA ; Spermatogenesis ; Spermatogenesis - genetics ; SSC ; Sycp3 transgenic mice ; Testis - cytology ; Testis - metabolism
ISSN:
0248-4900
E-ISSN:
1768-322X
DOI:
10.1111/boc.201200014
Source:
Wiley Online Library All Backfiles
Source:
Get It Now
URL:
https://www.ncbi.nlm.nih.gov/pubmed/22909339$$D View this record in MEDLINE/PubMed
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