ARF6, a component of intercellular bridges, is essential for spermatogenesis in mice.

Male germ cells are connected by intercellular bridges (ICBs) in a syncytium due to incomplete cytokinesis. Syncytium is thought to be important for synchronized germ cell development by interchange of cytoplasmic factors via ICBs. Mammalian ADP-ribosylation factor 6 (ARF6) is a small GTPase that is involved in many cellular mechanisms including but not limited to regulating cellular structure, motility, vesicle trafficking and cytokinesis. ARF6 localizes to ICBs in spermatogonia and spermatocytes in mice. Here we report that mice with global depletion of ARF6 in adulthood using Ubc-CreERT2 display no observable phenotypes but are male sterile. ARF6-deficient males display a progressive loss of germ cells, including LIN28A-expressing spermatogonia, and ultimately develop Sertoli-cell-only syndrome. Specifically, intercellular bridges are lost in ARF6-deficient testis. Furthermore, germ cell-specific inactivation using the Ddx4-CreERT2 results in the same testicular morphological phenotype, showing the germ cell-intrinsic requirement of ARF6. Therefore, ARF6 is essential for spermatogenesis in mice and this function is conserved from Drosophila to mammals.

Przewaquinone A inhibits Angiotensin II-induced endothelial diastolic dysfunction activation of AMPK.

BACKGROUND: Endothelial dysfunction (ED), characterized by markedly reduced nitric oxide (NO) bioavailability, vasoconstriction, and a shift toward a proinflammatory and prothrombotic state, is an important contributor to hypertension, atherosclerosis, and other cardiovascular diseases. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is widely involved in cardiovascular development. Przewaquinone A (PA), a lipophilic diterpene quinone extracted from Salvia przewalskii Maxim, inhibits vascular contraction. PURPOSE: Herein, the goal was to explore the protective effect of PA on ED in vivo and in vitro, as well as the underlying mechanisms. METHODS: A human umbilical vein endothelial cell (HUVEC) model of ED induced by angiotensin II (AngII) was used for in vitro observations. Levels of AMPK, endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule-1 (VCAM-1), nitric oxide (NO), and endothelin-1 (ET-1) were detected by western blotting and ELISA. A mouse model of hypertension was established by continuous infusion of AngII (1000 ng/kg/min) for 4 weeks using osmotic pumps. Following PA and/or valsartan administration, NO and ET-1 levels were measured. The levels of AMPK signaling-related proteins in the thoracic aorta were evaluated by immunohistochemistry. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were measured using the tail cuff method. Isolated aortic vascular tone measurements were used to evaluate the vasodilatory function in mice. Molecular docking, molecular dynamics, and surface plasmon resonance imaging (SPRi) were used to confirm AMPK and PA interactions. RESULTS: PA inhibited AngII-induced vasoconstriction and vascular adhesion as well as activated AMPK signaling in a dose-dependent manner. Moreover, PA markedly suppressed blood pressure, activated vasodilation in mice following AngII stimulation, and promoted the activation of AMPK signaling. Furthermore, molecular simulations and SPRi revealed that PA directly targeted AMPK. AMPK inhibition partly abolished the protective effects of PA against endothelial dysfunction. CONCLUSION: PA activates AMPK and ameliorates endothelial dysfunction during hypertension.