Abstract
In marine teleosts, such as the gilthead seabream, several aquaporin paralogs are known to be expressed during the hyperosmotic induction of spermatozoon motility in seawater. Here, we used immunological inhibition of channel function to investigate the physiological roles of Aqp1aa, Aqp1ab, and Aqp7 during seabream sperm activation. Double immunofluorescence microscopy of SW-activated sperm showed that Aqp1aa and Aqp7 were respectively distributed along the flagellum and the head, whereas Aqp1ab accumulated in the head and in discrete areas toward the anterior tail. Inhibition of Aqp1aa reduced the rise of intracellular Ca(2+), which is independent of external Ca(2+) and normally occurs upon activation, and strongly inhibited sperm motility. Impaired Aqp1aa function also prevented the intracellular trafficking of Aqp8b to the mitochondrion, where it acts as a peroxiporin allowing H2O2 efflux and ATP production during activation. However, restoring the Ca(2+) levels with a Ca(2+) ionophore in spermatozoa with immunosuppressed Aqp1aa function fully rescued mitochondrial Aqp8b accumulation and sperm motility. In contrast, exposure of sperm to Aqp1ab and Aqp7 antibodies did not affect motility during the initial phase of activation, but latently compromised the trajectory and the pattern of movement. These data reveal the coordinated action of spatially segregated aquaporins during sperm motility activation in a marine teleost, where flagellar-localized Aqp1aa plays a dual Ca(2+)-dependent role controlling the initiation of sperm motility and the activation of mitochondrial detoxification mechanisms, while Aqp1ab and Aqp7 in the head and anterior tail direct the motion pattern.
Highlights
The activation of the spermatozoon motility is crucial for successful fertilization
To investigate the possible colocalization of some of these channels, double immunofluorescence microscopy was carried out using specific affinity-purified rabbit antisera for Aqp1aa (Aqp1aa-Ab), Aqp1ab (Aqp1abAb), Aqp7 (Aqp7-Ab), and Aqp8b (Aqp8b-Ab) (Fig. 1). These experiments confirmed that in activated spermatozoa for 1–2 min strong Aqp1aa immunostaining was detected along the flagellum, while Aqp8b staining was restricted to the mitochondrion (Fig. 1, A, B, and D)
Aqp1ab and Aqp7 were immunolabeled in the cytoplasm and/or the plasma membrane surrounding the nucleus in the head (Fig. 1, B and D), whereas Aqp1ab was detected in discrete areas of the flagellum, which were more abundant toward the anterior region (Fig. 1B)
Summary
The activation of the spermatozoon motility is crucial for successful fertilization. Similar to mammals, where physiological hypotonicity is required for the activation of spermatozoon motility once released from the epididymis [1], in oviparous fishes, the initiation of motility is triggered by the hypo- or hyperosmotic aquatic environment into which the sperm are ejaculated [2,3,4,5]. Spermatozoa experience a strong hyperosmotic shock (D ;700–900 mOsm) upon release. Ó 2015 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363 into seawater (SW), which in most cases is the signal for the activation of motility [6,7,8]. It is believed that the external osmotic pressure drives an efflux of water from the spermatozoon, causing a rise of the internal concentrations of calcium and potassium, that is, [Ca2þ]i and [Kþ]i, respectively, and a change in the pH, which together activate the axonemal machinery and flagellar movement [7, 8]
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