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Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca2+ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon.

E2F4 was initially described as a transcription factor with a key function in the regulation of cell quiescence. Nevertheless, a number of recent studies have established that E2F4 can also play a relevant role in cell and tissue homeostasis, as well as tissue regeneration. For these non-canonical functions, E2F4 can also act in the cytoplasm, where it is able to interact with many homeostatic and synaptic regulators. Since E2F4 is expressed in the nervous system, it may fulfill a crucial role in brain function and homeostasis, being a promising multifactorial target for neurodegenerative diseases and brain aging. The regulation of E2F4 is complex, as it can be chemically modified through acetylation, from which we present evidence in the brain, as well as methylation, and phosphorylation. The phosphorylation of E2F4 within a conserved threonine motif induces cell cycle re-entry in neurons, while a dominant negative form of E2F4 (E2F4DN), in which the conserved threonines have been substituted by alanines, has been shown to act as a multifactorial therapeutic agent for Alzheimer’s disease (AD). We generated transgenic mice neuronally expressing E2F4DN. We have recently shown using this mouse strain that expression of E2F4DN in 5xFAD mice, a known murine model of AD, improved cognitive function, reduced neuronal tetraploidization, and induced a transcriptional program consistent with modulation of amyloid-β (Aβ) peptide proteostasis and brain homeostasis recovery. 5xFAD/E2F4DN mice also showed reduced microgliosis and astrogliosis in both the cerebral cortex and hippocampus at 3-6 months of age. Here, we analyzed the immune response in 1 year-old 5xFAD/E2F4DN mice, concluding that reduced microgliosis and astrogliosis is maintained at this late stage. In addition, the expression of E2F4DN also reduced age-associated microgliosis in wild-type mice, thus stressing its role as a brain homeostatic agent. We conclude that E2F4DN transgenic mice represent a promising tool for the evaluation of E2F4 as a therapeutic target in neuropathology and brain aging.