Aquaporin splice variation differentially modulates channel function during marine teleost egg hydration.

Aquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.

Neurohypophysial and paracrine vasopressinergic signaling regulates aquaporin trafficking to hydrate marine teleost oocytes.

The dual aquaporin (Aqp1ab1/Aqp1ab2)-mediated hydration of marine teleost eggs, which occurs during oocyte meiosis resumption (maturation), is considered a key adaptation underpinning their evolutionary success in the oceans. However, the endocrine signals controlling this mechanism are almost unknown. Here, we investigated whether the nonapeptides arginine vasopressin (Avp, formerly vasotocin) and oxytocin (Oxt, formerly isotocin) are involved in marine teleost oocyte hydration using the gilthead seabream (Sparus aurata) as a model. We show that concomitant with an increased systemic production of Avp and Oxt, the nonapeptides are also produced and accumulated locally in the ovarian follicles during oocyte maturation and hydration. Functional characterization of representative Avp and Oxt receptor subtypes indicates that Avpr1aa and Oxtrb, expressed in the postvitellogenic oocyte, activate phospholipase C and protein kinase C pathways, while Avpr2aa, which is highly expressed in the oocyte and in the follicular theca and granulosa cells, activates the cAMP-protein kinase A (PKA) cascade. Using ex vivo, in vitro and mutagenesis approaches, we determined that Avpr2aa plays a major role in the PKA-mediated phosphorylation of the aquaporin subdomains driving membrane insertion of Aqp1ab2 in the theca and granulosa cells, and of Aqp1ab1 and Aqp1ab2 in the distal and proximal regions of the oocyte microvilli, respectively. The data further indicate that luteinizing hormone, which surges during oocyte maturation, induces the synthesis of Avp in the granulosa cells via progestin production and the nuclear progestin receptor. Collectively, our data suggest that both the neurohypophysial and paracrine vasopressinergic systems integrate to differentially regulate the trafficking of the Aqp1ab-type paralogs via a common Avp-Avpr2aa-PKA pathway to avoid competitive occupancy of the same plasma membrane space and maximize water influx during oocyte hydration.

Functional Evolution of Clustered Aquaporin Genes Reveals Insights into the Oceanic Success of Teleost Eggs.

Aquaporin-mediated oocyte hydration is considered important for the evolution of pelagic eggs and the radiative success of marine teleosts. However, the molecular regulatory mechanisms controlling this vital process are not fully understood. Here, we analyzed >400 piscine genomes to uncover a previously unknown teleost-specific aquaporin-1 cluster (TSA1C) comprised of tandemly arranged aqp1aa-aqp1ab2-aqp1ab1 genes. Functional evolutionary analysis of the TSA1C reveals a ∼300-million-year history of downstream aqp1ab-type gene loss, neofunctionalization, and subfunctionalization, but with marine species that spawn highly hydrated pelagic eggs almost exclusively retaining at least one of the downstream paralogs. Unexpectedly, one-third of the modern marine euacanthomorph teleosts selectively retain both aqp1ab-type channels and co-evolved protein kinase-mediated phosphorylation sites in the intracellular subdomains together with teleost-specific Ywhaz-like (14-3-3ζ-like) binding proteins for co-operative membrane trafficking regulation. To understand the selective evolutionary advantages of these mechanisms, we show that a two-step regulated channel shunt avoids competitive occupancy of the same plasma membrane space in the oocyte and accelerates hydration. These data suggest that the evolution of the adaptive molecular regulatory features of the TSA1C facilitated the rise of pelagic eggs and their subsequent geodispersal in the oceanic currents.

A multiplier peroxiporin signal transduction pathway powers piscine spermatozoa.

The primary task of a spermatozoon is to deliver its nuclear payload to the egg to form the next-generation zygote. With polyandry repeatedly evolving in the animal kingdom, however, sperm competition has become widespread, with the highest known intensities occurring in fish. Yet, the molecular controls regulating spermatozoon swimming performance in these organisms are largely unknown. Here, we show that the kinematic properties of postactivated piscine spermatozoa are regulated through a conserved trafficking mechanism whereby a peroxiporin ortholog of mammalian aquaporin-8 (Aqp8bb) is inserted into the inner mitochondrial membrane to facilitate H2O2 efflux in order to maintain ATP production. In teleosts from more ancestral lineages, such as the zebrafish (Danio rerio) and the Atlantic salmon (Salmo salar), in which spermatozoa are activated in freshwater, an intracellular Ca2+-signaling directly regulates this mechanism through monophosphorylation of the Aqp8bb N terminus. In contrast, in more recently evolved marine teleosts, such the gilthead seabream (Sparus aurata), in which spermatozoa activation occurs in seawater, a cross-talk between Ca2+- and oxidative stress-activated pathways generate a multiplier regulation of channel trafficking via dual N-terminal phosphorylation. These findings reveal that teleost spermatozoa evolved increasingly sophisticated detoxification pathways to maintain swimming performance under a high osmotic stress, and provide insight into molecular traits that are advantageous for postcopulatory sexual selection.

The Xenopus Oocyte as an Expression System for Functional Analyses of Fish Aquaporins.

Aquaporins are membrane proteins present in all organisms that selectively transport water and small, uncharged solutes across biological membranes along an osmotic gradient. Recent gene editing technologies in zebrafish (Danio rerio) have started to uncover the physiological functions of the aquaporins in teleosts, but these approaches require methods to establish the effects of specific mutations on channel function. The oocytes of the South African frog Xenopus laevis are widely used for the expression of bacterial, plant, and animal aquaporins, and this heterologous system has contributed to numerous discoveries in aquaporin biology. This chapter focuses on techniques used for oocyte preparation and aquaporin expression and gives an overview of specific methods to determine water and solute permeability of the channels and their intracellular trafficking in oocytes.

Unravelling the Complex Duplication History of Deuterostome Glycerol Transporters.

Transmembrane glycerol transport is an ancient biophysical property that evolved in selected subfamilies of water channel (aquaporin) proteins. Here, we conducted broad level genome (>550) and transcriptome (>300) analyses to unravel the duplication history of the glycerol-transporting channels (glps) in Deuterostomia. We found that tandem duplication (TD) was the major mechanism of gene expansion in echinoderms and hemichordates, which, together with whole genome duplications (WGD) in the chordate lineage, continued to shape the genomic repertoires in craniates. Molecular phylogenies indicated that aqp3-like and aqp13-like channels were the probable stem subfamilies in craniates, with WGD generating aqp9 and aqp10 in gnathostomes but aqp7 arising through TD in Osteichthyes. We uncovered separate examples of gene translocations, gene conversion, and concerted evolution in humans, teleosts, and starfishes, with DNA transposons the likely drivers of gene rearrangements in paleotetraploid salmonids. Currently, gene copy numbers and BLAST are poor predictors of orthologous relationships due to asymmetric glp gene evolution in the different lineages. Such asymmetries can impact estimations of divergence times by millions of years. Experimental investigations of the salmonid channels demonstrated that approximately half of the 20 ancestral paralogs are functional, with neofunctionalization occurring at the transcriptional level rather than the protein transport properties. The combined findings resolve the origins and diversification of glps over >800 million years old and thus form the novel basis for proposing a pandeuterostome glp gene nomenclature.

The vertebrate Aqp14 water channel is a neuropeptide-regulated polytransporter.

Water channels (aquaporins) were originally discovered in mammals with fourteen subfamilies now identified (AQP0-13). Here we show that a functional Aqp14 subfamily phylogenetically related to AQP4-type channels exists in all vertebrate lineages except hagfishes and eutherian mammals. In contrast to the water-selective classical aquaporins, which have four aromatic-arginine constriction residues, Aqp14 proteins present five non-aromatic constriction residues and facilitate the permeation of water, urea, ammonia, H2O2 and glycerol. Immunocytochemical assays suggest that Aqp14 channels play important osmoregulatory roles in piscine seawater adaptation. Our data indicate that Aqp14 intracellular trafficking is tightly regulated by the vasotocinergic/isotocinergic neuropeptide and receptor systems, whereby protein kinase C and A transduction pathways phosphorylate highly conserved C-terminal residues to control channel plasma membrane insertion. The neuropeptide regulation of Aqp14 channels thus predates the vasotocin/vasopressin regulation of AQP2-5-6 orthologs observed in tetrapods. These findings demonstrate that vertebrate Aqp14 channels represent an ancient subfamily of neuropeptide-regulated polytransporters.