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

Fit a MWC model C₅-O₅ to the macroscopic currents of BK channels from <i>Xenopus</i> oocyte.

<p>(A) Activation traces of BK currents were recorded from an inside-out patch from a <i>Xenopus</i> oocyte injected with cRNA encoding mSlo1 α subunits. Channels were activated by voltage steps ranging from −200 to +200 mV with 10 mV increments from a holding potential of −180 mV with a cytosolic [Ca²⁺]_i as indicated. The voltage protocol is not shown here. The red lines were coming from the globally fitting the model C₅-O₅ to BK currents by PSO-GSS algorithm. The channel count N_C is 314 for 1 µM, 365 for 10 µM and 433 for 300 µM. The different Nc in the same patch is probably coming from the smaller single-channel conductance at the higher Ca²⁺, which will not change the channel kinetics. (B) Deactivation currents were obtained from the same patch as we described in (A). Currents were elicited by voltage steps ranging from −200 to +180 mV with 10 mV increments from a 20 ms-prepulse of +180 mV with a cytosolic [Ca²⁺]_i as indicated. The red lines are fits by a PSO-GSS algorithm. The channel count N_C is 301 for 1 µM, 354 for 10 µM and 387 for 300 µM. The score σ² is 41.60. All the capacitive currents of 0.15 ms were pre-substituted with straight lines before run and not counted during run. The dash line is zero current.</p

Fit a five-parameter voltage-dependent C-O model to the target current traces.

<p>(A) A five-parameter Markov model consisting of a closed state and an open state labeled with the letter C and O, respectively. The forward and backward rate constants separately are a*exp(v/b) and c*exp(−v/d). Here v represents voltage in mV, a and c the pre-exponential factors in ms⁻¹ and b and d the exponential factors in mV. The fifth parameter is the channel number N_C. (B) The errors relative to their target values were obtained by estimation of initial values (left) or by fit (right). (C) Convergence of PSO-GSS with (solid line) or without (dotted line) direct estimation. (D) In this model, target parameters a = 1 ms⁻¹, b = 50 mV, c = 1 ms⁻¹ and d = 200 mV; the reversal potential of channels V_r = 0 mV; the single-channel conductance G = 250 pS and the channel count N_C = 1. The empty circles represent the target currents at the various voltages shown under each of current traces, and the solid lines represent fitted currents.</p

Fit a 13-parameter Na_v-like C₅-O-I-CI₅ model to the target current traces.

<p>(A) A 13-parameter Markov model consisting of five closed states (C0, C1, C2, C3 and C4), an open state (O), an inactivation state (C₁₀) and five closed-inactivation states (C₅, C₆, C₇, C₈ and C₉). Each of parameters to be fitted is similar to that we described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035208#pone-0035208-g001" target="_blank">Figure 1(A)</a>. (B–C) See the description in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035208#pone-0035208-g001" target="_blank">Figure 1B–C</a>. (D) In this model, the target parameters m = 288.655598 ms⁻¹, n = 12 mV, p = 22.144593 ms⁻¹, q = 48 mV, r = 7.5 ms⁻¹, s = 2 ms⁻¹, g = 0.001, d = 0.5 ms⁻¹, c = 4.436203, u = 0.9 ms⁻¹, w = 0.006 ms⁻¹ and j = 4 ms⁻¹; the reversal potential of channels V_r = +55 mV; the single-channel conductance G = 250 pS and the channel count N_C = 1. A dependent parameter i = 15 ms⁻¹. The empty circles denote target currents and the solid lines represent fitted currents. The voltage protocols are placed below each of current traces.</p

Schematic diagram for GSS and PSO.

<p>(A) A schematic drawing for golden section search algorithm (GSS). (B) A flow graph for the PSO-GSS algorithm.</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

Comparison of kinetic characteristics between simulation data and target data.

<p>(A) The G-V curves of BK channels were plotted for data and best-fit, in the presence of 1, 10 and 300 µM Ca²⁺, respectively. (B) Time constants of activation (Left) and deactivation (Right) of BK channels were plotted for data and best-fit, in the presence of 1, 10 and 300 µM Ca²⁺, respectively. Here data are in black and fits in red.</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

A 7-state BK model used for describing the same currents in

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035208#pone-0035208-g004" target="_blank"><b>Figure 4</b></a><b>.</b> (A) A 7-state BK model simplified from the 50-state MWC model of BK channels. (B) The parameters of the 7-state model were: a = 342.37 s⁻¹; b = 67.90 mV; c = 56821.90 s⁻¹; d = 168.47 mV; f = 10.21 s⁻¹; i = 232.72 mV; g = 71130.28 s⁻¹; j = 121.54 mV; h = 1330.129 s⁻¹ M⁻¹; c1 = 3.97; k_O = 562.23 M; f1 = 10⁵ s⁻¹; g1 = 1323.36 s⁻¹; f2 = 10.02 s⁻¹; g2 = 99575.30 s⁻¹. The score σ² is 84.99. Compared with the 10-state MWC model, we have .</p