Genetic variants in the aqp1ab gene are associated with egg quality in the gilthead seabream (Sparus aurata)

Abstract

Introduction In the aquaculture of marine pelagic teleosts, egg buoyancy is the most widely used phenotypic marker of egg quality. The buoyancy of pelagic eggs and early embryos is acquired by the accumulation of lipids and water, the latter resulting from a massive hydration of the oocyte occurring during meiotic maturation. This process is mediated by the aquaporin-1ab (aqp1ab) gene which is tightly regulated both at the transcriptional and post-translational level during oogenesis. Given the essential requirement for the timely expression, trafficking and function of the Aqp1ab water channel during oocyte hydration, we hypothesized that dysfunctions in the Aqp1ab pathway might underlie poor egg quality in cultured marine teleosts. To test this hypothesis, in the present study we investigated whether allelic variants of aqp1ab may be associated with egg buoyancy in gilthead seabream (Sparus aurata) reared in captivity. Methods During the natural spawning season, eggs were collected during two consecutive days from one tank containing 8 functional (spermiating) males and 8 females raised in captivity. Buoyant and sinking eggs were recovered separately, and samples from each group containing 90-95% embryos at the 4-18-cell stage and 5-10% embryos at later stages (blastula, gastrula and neurula) were used for mRNA and protein extraction. For each sample, the full-length Aqp1ab cDNA was amplified by PCR and sequenced, and Western blot was carried out using a seabream Aqp1ab specific antibody. Wild-type aqp1ab and allelic variants identified in buoyant and sinking eggs were functionally characterized by ectopic expression in Xenopus laevis oocytes. Results and Discussion The aqp1ab mRNA levels in buoyant and sinking eggs were similar, but non-phosphorylated and phosphorylated Aqp1ab polypeptides appeared to be reduced in sinking eggs. Sequence analysis of 115 different clones indicated the expression of 14% and 43% of aqp1ab allelic variants, showing one or two non-synonymous single-nucleotide polymorphisms (SNPs), in buoyant and sinking eggs, respectively. The mutations were more abundant in the transmembrane helices and connecting loops of the encoded protein, whereas a few mutations were detected in the C terminus. Expression of the 28 different alleles identified in X. laevis oocytes showed that 43% and 100% of the variants from buoyant and sinking eggs, respectively, encoded channels with lower permeability than the wild-type due to partial or complete retention and/or degradation of the channel in the oocyte cytoplasm. Conclusion These data demonstrate that broodstock seabream raised in captivity can carry aberrant alleles in the aqp1ab gene that can modify channel trafficking. Our results suggest that aqp1ab-based polymorphisms may be employed as novel molecular markers of egg quality in marine teleosts