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.

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.