Abstract
Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3'UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.
Highlights
In all sexually reproducing organisms, germ cells (GCs)1, in contrast to somatic cells, are the only cells that can give rise
In addition to this “classic” mechanism, a novel system of post-transcriptional control mediated by microRNAs is lately emerging with an important role during spermatogenesis ((8 –10), and reviewed in [11]). miRNAs are endogenous, single-stranded, noncoding RNAs of ϳ22 nucleotides that act as post-transcriptional regulators of gene expression
We were able to detect already by postnatal day 5 (P5), a delay in Sertoli cells (SCs) maturation and an initial increase in SC proliferation followed by highly elevated levels of SC and GC apoptosis, events that led to a dramatic testicular degeneration during adulthood (Fig. 1A)
Summary
Affymetrix Microarray Analysis—Microarray analysis is described in [10]. All microarray data are available through ArrayExpress (http:// www.ebi.ac.uk/arrayexpress/, accession number: E-TABM-426). Relative protein quantification was obtained using the WarpLC 1.1 software (Bruker Daltonik, GmbH) This automatically calculates the heavy-to-light (H/L) ratios by comparing the relative intensities of the extracted ion chromatograms (EIC) that are reconstituted by extraction of the intensities of m/z ratios corresponding to the labeled peptides observed on MS spectra. Reproducibility and accuracy of ICPL experiments performed by ESI-ITMS were evaluated in five independent technical replicates using a standard mixture of ICPL labeled proteins containing bovine serum albumin with a heavy-to-light ratio of 1:1 (ICPL-kit, Serva Electrophoresis). For each AQUA peptide, the corresponding EIC area was automatically determined by the QuantAnalysis 1.8 software (Bruker Daltonik, GmbH) and plotted against the injected amount to obtain the calibration curves. Protein extracts used for the ICPL experiment, that is, one sample of 20 pairs of control and one sample of 20 pairs of mutant testes, were used for AQUA peptide analysis.
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