Discovery of Potential piRNAs from Next Generation Sequences of the Sexually Mature Porcine Testes

Piwi- interacting RNAs (piRNAs), a new class of small RNAs discovered from mammalian testes, are involved in transcriptional silencing of retrotransposons and other genetic elements in germ line cells. In order to identify a full transcriptome set of piRNAs expressed in the sexually mature porcine testes, small RNA fractions were extracted and were subjected to a Solexa deep sequencing. We cloned 6,913,561 clean reads of Sus Scrofa small RNAs (18–30 nt) and performed functional characterization. Sus Scrofa small RNAs showed a bimodal length distribution with two peaks at 21 nt and 29 nt. Then from 938,328 deep-sequenced small RNAs (26–30 nt), 375,195 piRNAs were identified by a k-mer scheme and 326 piRNAs were identified by homology searches. All piRNAs predicted by the k-mer scheme were then mapped to swine genome by Short Oligonucleotide Analysis Package (SOAP), and 81.61% of all uniquely mapping piRNAs (197,673) were located to 1124 defined genomic regions (5.85 Mb). Within these regions, 536 and 501 piRNA clusters generally distributed across only minus or plus genomic strand, 48 piRNA clusters distributed on two strands but in a divergent manner, and 39 piRNA clusters distributed on two strands in an overlapping manner. Furthermore, expression pattern of 7 piRNAs identified by homology searches showed 5 piRNAs displayed a ubiquitous expression pattern, although 2 piRNAs were specifically expressed in the testes. Overall, our results provide new information of porcine piRNAs and their specific expression pattern in porcine testes suggests that piRNAs have a role in regulating spermatogenesis

Continuous Wavelet Analysis of Leaf Reflectance Improves Classification Accuracy of Mangrove Species

Due to continuous degradation of mangrove forests, the accurate monitoring of spatial distribution and species composition of mangroves is essential for restoration, conservation and management of coastal ecosystems. With leaf hyperspectral reflectance, this study aimed to explore the potential of continuous wavelet analysis (CWA) combined with different sample subset partition (stratified random sampling (STRAT), Kennard-Stone sampling algorithm (KS), and sample subset partition based on joint X-Y distances (SPXY)) and feature extraction methods (principal component analysis (PCA), successive projections algorithm (SPA), and vegetation index (VI)) in mangrove species classification. A total of 301 mangrove leaf samples with four species (Avicennia marina, Bruguiera gymnorrhiza, Kandelia obovate and Aegiceras corniculatum) were collected across six different regions. The smoothed reflectance (Smth) and first derivative reflectance (Der) spectra were subjected to CWA using different wavelet scales, and a total of 270 random forest classification models were established and compared. Among the 120 models with CWA of Smth, 88.3% of models increased the overall accuracy (OA) values with an improvement of 0.2–28.6% compared to the model with the Smth spectra; among the 120 models with CWA of Der, 25.8% of models increased the OA values with an improvement of 0.1–11.4% compared to the model with the Der spectra. The model with CWA of Der at the scale of 23 coupling with STRAT and SPA achieved the best classification result (OA = 98.0%), while the best model with Smth and Der alone had OA values of 86.3% and 93.0%, respectively. Moreover, the models using STRAT outperformed those using KS and SPXY, and the models using PCA and SPA had better performances than those using VIs. We have concluded that CWA with suitable scales holds great potential in improving the classification accuracy of mangrove species, and that STRAT combined with the PCA or SPA method is also recommended to improve classification performance. These results may lay the foundation for further studies with UAV-acquired or satellite hyperspectral data, and the encouraging performance of CWA for mangrove species classification can also be extended to other plant species

miR-638 Inhibits immature Sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene

<p>Numerous studies have demonstrated that microRNAs (miRNAs) play important roles in cell growth, apoptosis and spermatogenesis. Our previous study showed that miR-638 was differentially expressed in sexually immature and mature testes of Large White boars. Here we reported that sperm-associated antigen 1 (<i>SPAG1</i>) was a direct target gene of miR-638. Moreover, miR-638 inhibited cell proliferation and cell cycle, and promoted apoptosis of porcine immature Sertoli cells. Key genes including phosphorylated phosphatidylinositide 3-kinases (p-PI3K) and phosphorylated serine/ threonine kinase (p-AKT) in PI3K/AKT pathway as well as cell cycle factors including c-MYC, cyclin-D1 (CCND1), cyclin-E1 (CCNE1) and cyclin-dependent kinase 4 (CDK4) were all significantly down-regulated after overexpression of miR-638 or RNAi of <i>SPAG1</i>. Notably, mRNA levels of SRY-related HMG-box 2 (<i>SOX2)</i> and POU domain, class 5, transcription factor 1 (<i>POU5F1)</i> essential for spermatogonia proliferation were significantly suppressed in <i>SPAG1</i> siRNA- transfected ST cells. This study suggests that miR-638 regulates immature Sertoli cell growth and apoptosis by targeting <i>SPAG1</i> gene which can indirectly inactivate PI3K/AKT pathway, and plays roles in pig spermatogenesis.</p

Seven piRNAs expression profile in the porcine testes.

<p>(A) Seven piRNAs expression profile in the porcine testes from 60-day and 180-day testes of Chinese Meishan and Large White pigs using stem-loop qRT-PCR method. The X-axis represents the small RNAs and the Y-axis shows the relative expression levels of small RNAs (–ΔC_t values for qRT-PCR). The significance of differences for the expression between 60-day (sexually immature) and 180-day (sexually mature) testes of Chinese Meishan and Large White pigs was calculated using two-tailed T-test. *, p≤0.05; **, p≤0.01. (B) Seven piRNAs expressed in the porcine testes by stem-loop semi-quantitative RT-PCR. M, 25 bp DNA ladder; He, heart; Li, liver; Sp, spleen; Lu, lung; Ki, kidney; LM, <i>longissimu</i>s <i>dorsi</i> muscle; BF, backfat; Te, testis; Ov, ovary.</p

The chromosomal distribution of piRNAs and piRNA clusters in the genome.

*<p>All piRNAs with the unique perfectly mapping to pig genome were used to analyze to chromosome distribution. MT, mitochondrial genome.</p

Characterization of <i>sus scrofa</i> small RNAs.

<p>(A) Length distribution of small RNAs. <i>Sus scrofa</i> small RNAs displayed a bimodal length distribution with two peaks at 21 nt and 29 nt. (B) Bar chart summarizing the annotation of small RNA populations in total RNA from testes. (C) Chromosomal distribution of small RNAs.</p