This study investigated cold plasmas for multiple biological applications.  Our previous work has found dielectric barrier discharge plasma improves chicken sperm quality.  The number of Sertoli cells (SCs) decides spermatogenesis.  However, whether cold plasma can regulate SC proliferation remains unclear.  This study explored the effects of cold plasma on immature chicken SC proliferation and the regulation mechanism.  Results showed that cold plasma exposure at 2.4 W for 30 s twice with an interval of 6 h produced (P<0.05) the maximum SC viability, cell growth, and cell cycle progression.  SC proliferation-promoting effect of cold plasma treatment was regulated by increasing (P<0.05) the adenosine triphosphate production and the respiratory enzyme activity in the mitochondria.  This process was potentially mediated by the adenosine monophosphate-activated protein kinase (AMPK)–mammalian target of rapamycin (mTOR) signaling pathway, which was regulated by the microRNA (miRNA) targeting regulation directly and by the intracellular reactive oxygen species homeostasis indirectly.  The cold plasma treatment increased (P<0.01) the miR-7450-5p expression and led to a decreased (P<0.01) AMPKα1 level.  On the other hand, miR-100-5p expression was reduced (P<0.05) and led to an increased (P<0.05) mTOR level in SCs.  A single-stranded synthetic miR-7450-5p antagomir and a double-stranded synthetic miR-100-5p agomir reduced (P<0.05) the SC proliferation.  However, this could be ameliorated (P<0.05) by the cold plasma treatment.  Our findings suggest that appropriate cold plasma treatment provides a safe strategy to improve SC proliferation, which is beneficial to elevating male chicken reproductive capacity.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small non-coding RNAs (ncRNAs) that trigger RNA interference (RNAi) in eukaryotic organisms.  The biogenesis pathways for these ncRNAs are well established in Drosophila melanogaster, Aedes aegypti, Bombyx mori and other insects, but lacking in hymenopteran species, particularly in parasitoid wasps.  Pteromalus puparum is a parasitoid of pupal butterflies.  This study identified and analyzed two pathways by interrogating the P. puparum genome.  All core genes of the two pathways are present in the genome as a single copy, except for two genes in the siRNA pathway, R2D2 (two copies) and Argonaute-2 (three).  Conserved domain analyses showed the protein structures in P. puparum were similar to cognate proteins in other insect species.  Phylogenetic analyses of hymenopteran Dicer and Argonaute genes suggested that the siRNA pathway-related genes evolved faster than those in the miRNA pathway.  The study found a decelerated evolution rate of P. puparum Dicer-2 with respect to Dicer-1, which was contrary to other hymenopterans.  Expression analyses revealed high mRNA levels for all miRNA pathway genes in P. puparum adults and the siRNA related genes were expressed in different patterns.  The findings add valuable new knowledge of the miRNA and siRNA pathways and their regulatory actions in parasitoid wasps.

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase (N6AMT) is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens (Nlu-N6AMT) and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenineMlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding dsRNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

Estrogen plays an important role in regulating testicular Sertoli cell number. Furthermore, S-phase kinase-associated protein 2 (SKP2) plays a central role in mammalian cell cycle progression. The objective of this study was to determine whether 17β-estradiol can regulate the expression of SKP2, and the Sertoli cell cycle, via estrogen receptor β (ERβ), the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and extracellular signal-regulated kinase (ERK1/2) pathway. When cultured immature boar Sertoli cells were treated with 17β-estradiol, a time-dependent increase in SKP2 mRNA and protein level was observed by real-time PCR and Western blot, and 17β-estradiol activity peaked at 30 min. Treatment with ICI182780 and ERβ antagonist reduced 17β-estradiol-induced expression of SKP2 and proliferating cell nuclear antigen (PCNA), while increasing the protein concentration of p27kip1. However, the effect of ERa antagonist on these parameters was lower than that of ICI182780 and ERβ. Forskolin had a similar effect as 17β-estradiol on the expression of SKP2, PCNA and p27kip1. Rp-cAMP, H-89 and U0126 treatment reduced 17β-estradiol-induced changes, while H-89 also inhibited ERK1/2 activation. Therefore, 17β-estradiol mainly regulates SKP2 mRNA and protein expression via ERβ-cAMP-PKA and ERK1/2 activation. SKP2 and PCNA expression were positively correlated, while increased SKP2 expression likely resulted in p27kip1 degradation.

A cDNA clone encoding a putative EBF-like protein (DCEBF1) was obtained from total RNA isolated from senescing carnation (Dianthus caryophyllus L.) petals using reverse transcription PCR and rapid-amplification of cDNA ends techniques. The cDNA contained an open reading frame of 1 878 bp corresponding to 625 amino acids. Results of Northern blot indicated DCEBF1 expression was enhanced by endogenous and exogenous ethylene, and was inhibited by STS in petals and ovaries. Upon wounding treatment, DCEBF1 showed a quick increase in mRNA accumulation which was positively correlated with the increase in ethylene production. The levels of DCEBF1 mRNA increased in both petals and ovaries by sucrose treatment compared with the control.