Systematic identification and characterization of long non-coding RNAs in mouse mature sperm

<div><p>Increasing studies have shown that mature spermatozoa contain many transcripts including mRNAs and miRNAs. However, the expression profile of long non-coding RNAs (lncRNAs) in mammalian sperm has not been systematically investigated. Here, we used highly purified RNA to investigate lncRNA expression profiles in mouse mature sperm by stranded-specific RNA-seq. We identified 20,907 known and 4,088 novel lncRNAs transcripts, and the existence of intact lncRNAs was confirmed by RT-PCR and fluorescence in situ hybridization on two representative lncRNAs. Compared to round spermatids, 1,794 upregulated and 165 downregulated lncRNAs and 4,435 upregulated and 3,920 downregulated mRNAs were identified in sperm. Based on the “Cis and Trans” RNA-RNA interaction principle, we found 14,259 targeted coding genes of differently expressed lncRNAs. In terms of Gene ontology (GO) analysis, differentially expressed lncRNAs targeted genes mainly related to nucleic acid metabolic, protein modification, chromatin and histone modification, heterocycle compound metabolic, sperm function, spermatogenesis and other processes. In contrast, differentially expressed transcripts of mRNAs were highly enriched for protein metabolic process and RNA metabolic, spermatogenesis, sperm motility, cell cycle, chromatin organization, heterocycle and aromatic compound metabolic processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the differentially expressed lncRNAs were involved in RNA transport, mRNA surveillance pathway, PI3K-Akt signaling pathway, AMPK signaling pathway, protein processing in endoplasmic reticulum. Metabolic pathways, mRNA surveillance pathway, AMPK signaling pathway, cell cycle, RNA transport splicesome and endocytosis incorporated with the differentially expressed mRNA. Furthermore, many lncRNAs were specifically expressed in testis/sperm, and 880 lncRNAs were conserved between human and mouse. In summary, this study provides a preliminary database valuable for identifying lncRNAs critical in the late stage of spermatogenesis or important for sperm function regulation, fertilization and early embryo development.</p></div

Quality control of sperm RNA.

<p><b>Confirmation of sperm purity and electrophoretic size distribution of extracted total sperm RNA.</b> (A) Morphology of collected sperm under phase contrast microscopy. (B) Biomarkers of leukocytes (<i>CD4</i>), testicular germ cells (<i>c-kit</i>) and epithelial cells (<i>E-cadherin</i>) could be amplified easily from the RNA extracted from unpurified sperm sample. (C) After sperm was purified, non-sperm cell markers (<i>CD4</i>, <i>c-kit</i>, <i>E-cadherin</i>) were unable to be amplified from the RNA extracted from our purified sperm sample, while positive markers of sperm (<i>Prm1</i> and <i>Prm2</i>) were easily detected. (D, E) The 442 bp PCR product of <i>Prm2</i> indicated DNA contamination (D), which disappeared after DNase I digestion (E). (F) The DNA PCR product of <i>Prm1 and Prm2</i> could be amplified from no reverse transcription (RT) sperm RNA. (G, H) Electrophoretic size distribution of RNAs in mouse testis (G) and mature sperm (H) analyzed by Agilent Bioanalyzer.</p

The putative functional lncRNAs in mature sperm.

<p>LncRNA target genes were predicted based on the “Cis and Trans” RNA-RNA interaction principle. One-to-one pairs of lncRNA and mRNA were deemed to putative functional lncRNAs modulating the spermatogenesis or sperm function.</p

RT-PCR and qPCR primers of examined coding genes.

<p>RT-PCR and qPCR primers of examined coding genes.</p

qPCR validation of the upregulated RNAs.

<p>After total sperm RNA was isolated, qPCR was performed to detect the RNA expression in sperm and testes. <i>Rplp1</i> and <i>β-actin</i> genes were used as loading controls to normalize RNA expression levels. Data are expressed as the mean ± standard deviation (n = 3).</p

Integrity evaluation and existing forms of sperm lncRNAs.

<p>(A) Integrity evaluation of sperm lncRNAs using a computational approach. To determine the uniformity of coverage for each, RNA transcripts were divided into 100 bins, and a 5-bin moving average was used to calculate localized variations in sequencing coverage. The squared deviation from expected coverage for each bin was summed and used as an intactness score to rank the 1000 lncRNAs according to their stability. (B) Intact RNAs are present in sperm; the testes served as positive control to amplify the proposed full-length mRNAs and lncRNAs. (C) The fragmented RNAs degraded or cleaved from the intact transcripts were amplified in sperm. S′ and T′: Representative fragments were amplified from sperm and testes cDNA, respectively. (D) The localization of two representatively intact lncRNAs with FISH staining.</p

LncRNAs expression profiles in mouse mature sperm.

<p>(A) Correlation analysis of all sequencing samples. (B) The numbers of lncRNAs according to length in mice testes and mature sperm. (C) The numbers of lncRNAs and mRNAs in mature sperm. (D) The average expression levels of known and novel lncRNAs and mRNAs in mice mature sperm. (E) The average expression levels of known and novel lncRNAs in mice testes and mature sperm. (F) The violin graph of lncRNAs and mRNAs in in mice testes and mature sperm. **<i>P</i> < 0.01 compared with mRNA, ##<i>P</i> < 0.01 compared with testes.</p

Data_Sheet_1_Discovery of the Consistently Well-Performed Analysis Chain for SWATH-MS Based Pharmacoproteomic Quantification.PDF

<p>Sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) has emerged as one of the most popular techniques for label-free proteome quantification in current pharmacoproteomic research. It provides more comprehensive detection and more accurate quantitation of proteins comparing with the traditional techniques. The performance of SWATH-MS is highly susceptible to the selection of processing method. Till now, ≥27 methods (transformation, normalization, and missing-value imputation) are sequentially applied to construct numerous analysis chains for SWATH-MS, but it is still not clear which analysis chain gives the optimal quantification performance. Herein, the performances of 560 analysis chains for quantifying pharmacoproteomic data were comprehensively assessed. Firstly, the most complete set of the publicly available SWATH-MS based pharmacoproteomic data were collected by comprehensive literature review. Secondly, substantial variations among the performances of various analysis chains were observed, and the consistently well-performed analysis chains (CWPACs) across various datasets were for the first time generalized. Finally, the log and power transformations sequentially followed by the total ion current normalization were discovered as one of the best performed analysis chains for the quantification of SWATH-MS based pharmacoproteomic data. In sum, the CWPACs identified here provided important guidance to the quantification of proteomic data and could therefore facilitate the cutting-edge research in any pharmacoproteomic studies requiring SWATH-MS technique.</p

Enhanced Performance of InGaN Light-Emitting Diodes via High-Quality GaN and Embedded Air Voids Grown on Hexagonal 3D Serpentine Mask Sapphire Substrates

This work demonstrates high-efficiency InGaN-based light-emitting diodes (HSM-LEDs) prepared on hexagonal 3D serpentine sapphire substrates. The 3D serpentine mask has a modulating effect on epitaxial lateral growth (ELOG), which can not only reduce the dislocation density (TDD) to 1.7 × 107 cm–2 without any high dislocation density (HDD) region but also induce the formation of a hexagonal pyramidal air-void array with an inclination angle of 65°. Compared to conventional LEDs, HSM-LEDs exhibit a 117% enhancement in EL output power at a current injection of 600 mA. This can be attributed to the improvement of crystal quality by modulated ELOG, the relief of in-plane stresses to mitigate the quantum-confined Stark effect (QCSE) through the weak connection of the epitaxial layer to the substrate, and the enhanced light extraction efficiency by an embedded air-void array. We confirmed the reduction of compressive stress from 0.94 GPa to 0.51 GPa in HSM-LEDs by Raman spectroscopy and investigated the effect of air voids on light extraction efficiency (LEE) experimentally and theoretically. Ray-tracing simulations show that the embedded pyramidal air voids can effectively re-extract the downward emitted light, and the pyramidal air voids with a 65° slant sidewall can improve the LEE by 71%