Disruption of the sea bass skin-scale barrier by antidepressant fluoxetine and estradiol: in vivo and in vitro evidence

Trabajo presentado en la Joint 30th Conference of the European Society for Comparative Endocrinology and of the 9th International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.Fluoxetine (FLX) is a highly prescribed selective inhibitor of serotonin-reuptake and an emerging pollutant affecting fish behaviour, stress and reproduction, but little is known about possible actions and mechanisms in barrier tissues. We combined in vivo and in vitro approaches to demonstrate multi-level impacts of FLX on the sea bass (Dicentrarchus labrax) skin-scale barrier and on the estrogenic system. Juvenile sea bass intraperitoneally injected with FLX had significantly increased levels of FLX and its metabolite nor-FLX. In contrast to the natural estrogen E2, FLX did not increase plasma calcium, phosphorus (P) or vitellogenin, although a slight decrease in scale P content was detected. Quantitative SWATH-MS proteomics of the scales identified 134 proteins that were affected by FLX. Modified proteins were mainly related to extracellular matrix and protein turnover and energy production, 31 of which were also affected by E2. Multiple estrogen receptors and genes related to serotonin activity, transport and degradation were expressed in sea bass scales and transcript abundance of some of them was modulated by E2 and/or FLX. Using a minimally invasive in vitro bioassay with cultured sea bass scales and adhering epithelia we showed direct effects of FLX exposure on enzymatic activity associated with mineral mobilization, while the expression of estrogen receptors was not significantly affected. In in vitro receptor-reporter assays, FLX alone did not activate any of the three sea bass nuclear estrogen receptors but had antiestrogenic effects on Esr1/2b when in co-treatment with E2, and directly activated both plasma membrane Gprotein-coupled estrogen receptors. The combination of in vitro and in vivo assays substantiated the notion that FLX disrupted scale physiology through several different processes, with probable consequences for fish health, and revealed that some of the mechanisms of disruption can result from direct interaction with multiple estrogen .Projects UIDB/04326/2020, PTDC/AAG-GLO/4003/2012 and DL57/2016/CP1361/CT0015 from FCT (Pt); EU Interreg FR-UK project RedPol; grant AGL2015-67477-C2-1- R (Sp)

Molecular physiology of a teleost oocyte aquaporin: evolution, regulation and role during oocyte hydration / Fisiología molecular de una acuaporina ovocitaria de teleósteos: Evolución, regulación y papel durante la hidratación del oocito

[spa] La hidratación de los oocitos de teleósteos marinos que producen huevos pelágicos es clave para la supervivencia de los embriones en el océano. Estudios previos han descu-bierto el papel de una acuaporina específica de teleósteos (Aqp1b) durante este proceso, pero se carece todavía de evidencias experimentales directas, así como información sobre la regulación molecular de la Aqp1ab. En la presente tesis, estudios iniciales con el fletan Atlántico (Hippoglossus hippoglossus) confirman que los parálogos aqp1aa y -1ab de teleósteos han surgido probablemente por duplicación génica en tándem, y han demostrado el papel esencial de la Aqp1ab durante la hidratación del oocito. Para investigar el control transcripcional del gen aqp1ab en los oocitos de la dorada (Sparus aurata) se ha aislado su región promotora, la cual contiene elementos cis-reguladores de unión al receptor nuclear de progestinas (Pgr), como la 17α,20β-dihidroxi-4-pregnen-3-ona (17,20β-P), y factores de transcripción Sox. Estudios de localización subcelular indican que el Pgr aparece en el citoplasma de las oogonias, así como en el núcleo de oocitos en crecimiento primario (pre-vitelogénicos) coincidiendo con la activación de la expresión de aqp1ab. En este estadio también se expresan cuatro isoformas diferentes del Pgr, dos de las cuales pueden inhibir la transcripción mediada por el Pgr de forma dominante negativa. Las oogonias también expresan sox3 y -8b, mientras que el sox9b aparece en el estadio de alveolo cortical, cuando se reduce la expresión de aqp1ab. Ex-perimentos de transactivación indican que el Pgr activa la transcripción de aqp1ab, con Sox3 y -8b actuando de forma sinérgica, mientras que el Sox9b reprime este mecanismo. El papel del Pgr se ha investigado sobre explantes ováricos pre-vitelogénicos incubados in vitro, lo cual ha demostrado que la 17,20β-P, producida en las células de la granulosa en respuesta a la hormona folículo estimulante, activa el promotor de aqp1ab en el oocito induciendo la síntesis de Aqp1ab. Los resultados de esta tesis revelan por primera vez una estricta regulación transcripcional del gen aqp1ab durante la oogénesis de teleósteos marinos, lo cual remarca la función fisiológica esencial de este canal de agua durante la formación de los huevos pelágicos[eng] In marine teleosts that spawn pelagic eggs (pelagophils), the process of oocyte hydration that occurs during meiosis resumption is a key physiological process for the survival of the eggs in the ocean. Previous studies have discovered the role of a teleost-specific aquaporin water channel (Aqp1b) during fish oocyte hydration, but direct experimental evidence for the function of Aqp1b in oocytes is still lacking. In addition, the molecular regulation of the Aqp1b-mediated mechanism remains poorly understood. In this context, the main objectives of the present thesis were to investigate the evolutionary origin of aqp1ab in teleosts, to provide functional evidence of the role of Aqp1b during oocyte hydration, and to begin to dissect the molecular mechanisms involved in the transcriptional regulation of aqp1b in the oocyte of marine teleosts. By integrating the molecular phylogeny with synteny and structural analyses we show that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication, and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut (Hippoglossus hippoglossus), a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. Using immunological inhibition of Aqp1ab in halibut oocytes and artificial expression of the halibut paralog Aqp1aa, we demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. To investigate the aqp1ab transcriptional regulation in oocytes, we isolated the 5’-flanking region of the gilthead seabream (Sparus aurata) aqp1ab gene which contains regulatory cis-elements for the nuclear progestin receptor (Pgr) and SOX transcription factors. The Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oo-gonia, whereas in primary growth oocytes, when aqp1ab mRNA and protein are synthesized, the Pgr is translocated into the nucleus. In contrast, sox9b is highly expressed in more advanced oocytes showing the depletion of aqp1ab transcripts. In the seabream, four different pgr transcript variants are expressed in primary growth ovaries which are generated by alternative pre-mRNA splicing. Seabream wild-type Pgr shows the highest transactivation response to progestins such as 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), whereas two of the N-terminally truncated Pgr isoforms regulate novel nuclear and cytosolic mechanisms of dominant-negative repression of Pgr-mediated transcription. Transactivation assays on the aqp1ab promoter demonstrated that aqp1ab transcription is dependent on wild-type Pgr, with Sox3 and -8b acting synergistically, while Sox9b acts as a repressor. Incubation of primary ovarian explants in vitro with 17,20β-P, followed by chromatin immunoprecipitation, confirmed that 17,20β-P-activated Pgr enhanced aqp1ab promoter activation. The production of 17,20β-P in seabream primary growth ovaries in vivo was consistent with the expression of P450c17-II (Cyp17a2) and 20β-hydroxysteroid dehydrogenase (Cbr1), enzymes needed for progestins synthesis, in granulosa cells associated with primary growth oocytes, and with a high concentration of 17,20β-P. Incubation of primary ovarian explants with recombinant piscine follicle-stimulating hormone (rFsh) in vitro stimulated 17,20β-P synthesis, which was reduced in the presence of Cbr1 inhibitors. The rFsh-mediated production of 17,20β-P correlated with the up-regulation of cyp17a2 and cbr1 transcription, as well as of wild-type pgr mRNA and protein levels. Altogether, these data suggest that aqp1ab transcription in seabream primary growth oocytes is under Fsh regulation through the synthesis of progestins. The results of this thesis show that the Aqp1ab mediated mechanism for oocyte hy-dration is likely conserved in marine teleosts. In addition, the tight transcriptional reg-ulation of Aqp1ab during oogenesis highlights the essential physiological role of this water channel and opens new research avenues for understanding the molecular basis of egg formation in marine fish

Fisiología molecular de una acuaporina ovocitaria de teleósteos: Evolución, regulación y papel durante la hidratación del oocito

Memoria de tesis doctoral presentada por Cinta Zapater Cardona para obtener el título de Doctora por la Universitat de Barcelona (UB), realizada bajo la dirección del Dr. Joan Cerdà Luque del Institut de Ciències del Mar (ICM-CSIC) y el Institut de Recerca i Tecnologia Agroalimentaria (IRTA).-- 357 pagesPeer Reviewe

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

10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageIntroduction 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 teleostsPeer Reviewe

Dual Neofunctionalization of a Rapidly Evolving Aquaporin-1 Paralog Resulted in Constrained and Relaxed Traits Controlling Channel Function during Meiosis Resumption in Teleosts

19 pages, 7 figures, 1 tableThe preovulatory hydration of teleost oocytes is a unique process among vertebrates. The hydration mechanism is most pronounced in marine acanthomorph teleosts that spawn pelagic (floating) eggs; however, the molecular pathway for water influx remains poorly understood. Recently, we revealed that whole-genome duplication (WGD) resulted in teleosts harboring the largest repertoire of molecular water channels in the vertebrate lineage and that a duplicated aquaporin-1 paralog is implicated in the oocyte hydration process. However, the origin and function of the aquaporin-1 paralogs remain equivocal. By integrating the molecular phylogeny with synteny and structural analyses, we show here that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication rather than WGD and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut, a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. We demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. We further show that the rapid structural divergence of the C-terminal regulatory domain causes ex vivo loss of function of halibut Aqp1ab when expressed in amphibian oocytes but not in zebrafish or native oocytes. However, by using chimeric constructs of halibut Aqp1aa and -1ab and antisera specifically raised against the C-terminus of Aqp1ab, we found that this cytoplasmic domain regulates in vivo trafficking to the microvillar portion of the oocyte plasma membrane when intraoocytic osmotic pressure is at a maximum. Interestingly, by coinjecting polyA+ mRNA from postvitellogenic halibut follicles, ex vivo intracellular trafficking of Aqp1ab is rescued in amphibian oocytes. These data reveal that the physiological role of Aqp1ab during meiosis resumption is conserved in teleosts, but the remarkable degeneracy of the cytoplasmic domain has resulted in alternative regulation of the trafficking mechanismThe Spanish Ministry of Science and Innovation (MICINN; AGL2007-60261/ACU) and European Commission (MRTN-CT-2006-035995-Aquaglyceroporins) to J.C.; Research Council of Norway (204813) to R.N.F. C.Z. and F.C. were supported by predoctoral (FPI) and postdoctoral (Juan de la Cierva Programme) fellowships, respectively, from MICINNPeer reviewe

Adaptive plasticity of killifish (Fundulus heteroclitus) embryos: dehydration-stimulated development and differential aquaporin-3 expression

13 pages, 7 figures, 3 tablesEmbryos of the marine killifish Fundulus heteroclitus are adapted to survive aerially. However, it is unknown if they are able to control development under dehydration conditions. Here, we show that air-exposed blastula embryos under saturated relative humidity were able to stimulate development, and hence the time of hatching was advanced with respect to embryos continuously immersed in seawater. Embryos exposed to air at later developmental stages did not hatch until water was added, while development was not arrested. Air-exposed embryos avoided dehydration probably because of their thickened egg envelope, although it suffered significant evaporative water loss. The potential role of aquaporins as part of the embryo response to dehydration was investigated by cloning the aquaporin-0 (FhAqp0), -1a (FhAqp1a), and -3 (FhAqp3) cDNAs. Functional expression in Xenopus laevis oocytes showed that FhaAqp1a was a water-selective channel, whereas FhAqp3 was permeable to water, glycerol, and urea. Expression of fhaqp0 and fhaqp1a was prominent during organogenesis, and their mRNA levels were similar between water- and air-incubated embryos. However, fhaqp3 transcripts were highly and transiently accumulated during gastrulation, and the protein product was localized in the basolateral membrane of the enveloping epithelial cell layer and in the membrane of ingressing and migrating blastomers. Interestingly, both fhaqp3 transcripts and FhAqp3 polypeptides were downregulated in air-exposed embryos. These data demonstrate that killifish embryos respond adaptively to environmental desiccation by accelerating development and that embryos are able to transduce dehydration conditions into molecular responses. The reduced synthesis of FhAqp3 may be one of these mechanisms to regulate water and/or solute transport in the embryo.This study was supported by the European Commission New and Emerging Science and Technologies (NEST) program (contract no. 012674-2 Sleeping Beauty) and by a grant from the Spanish Ministry of Education and Science (MEC; AGL2004-00316/ACU) to J. Cerda`. Participation of C. Zapater and F. Chauvigne´ was financed by a predoctoral fellowship from MEC (Spain) and by the European Commission [Marie Curie Research Training Network Aqua (glycero)porins, MRTN-CT-2006-035995], respectively.Peer reviewe

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

Gonadotropin sialylation in European sea bass (Dicentrarchus labrax), another way of activity regulation?

Trabajo presentado en el 6th International Workshop on the Biology of Fish Gametes, celebrado en Vodňany (República Checa), del 4 al 7 de septiembre de 2017The pituitary gland synthesizes and secrets differently glycosylated forms of gonadotropins that differ in their oligosaccharide structure. In the case of FSH, several enzymes are involved in its post-translational glycosylation processing; particularly the sialyltransferases (STs) that transfer sialic acid residues into the FSH carbohydrate chain. Previous studies with recombinant sea bass Fsh showed that, as occurs in mammals, less sialylated isoforms exhibit higher in-vitro biological activity but shorter plasma half-life than their more sialylated counterparts. These facts suggest that gonadotropin heterogeneity represents an alternative mechanism of the pituitary gland to regulate the intensity and duration of gonadotropin stimuli in the gonads. In addition, the composition of the FSH carbohydrate moiety is likely regulated by the endocrine milieu, and the GnRH and gonadal sex steroids have been proposed as the main regulators of FSH microheterogeneity. Our aim in this study was to know the expression profile of several sea bass ST genes in male pituitary during a whole reproductive cycle and to elucidate whether treatments with gonadal steroids (Estradiol, Testosterone, Dihydroxyprogesterone) and LHRHa can affect the expression of these genes in a pituitary primary cell culture, and therefore to the potential sialylation of sea bass Fsh.Founded by MINECO (AGL 2015-67477-C2-1-R), CSIC (201640E073) and GV (PROMETEO II-2014/051)Peer reviewe

Gonadotropin-Activated Androgen-Dependent and Independent Pathways Regulate Aquaporin Expression during Teleost (Sparus aurata) Spermatogenesis.

The mediation of fluid homeostasis by multiple classes of aquaporins has been suggested to be essential during spermatogenesis and spermiation. In the marine teleost gilthead seabream (Sparus aurata), seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the spermiating testis, but the endocrine regulation of these channels during germ cell development is unknown. In this study, we investigated the in vivo developmental expression of aquaporins in the seabream testis together with plasma androgen concentrations. We then examined the in vitro regulatory effects of recombinant piscine gonadotropins, follicle-stimulating (rFsh) and luteinizing (rLh) hormones, and sex steroids on aquaporin mRNA levels during the spermatogenic cycle. During the resting phase, when plasma levels of androgens were low, the testis exclusively contained proliferating spermatogonia expressing Aqp1ab, whereas Aqp10b and -9b were localized in Sertoli and Leydig cells, respectively. At the onset of spermatogenesis and during spermiation, the increase of androgen plasma levels correlated with the additional appearance of Aqp0a and -7 in Sertoli cells, Aqp0a in spermatogonia and spermatocytes, Aqp1ab, -7 and -10b from spermatogonia to spermatozoa, and Aqp1aa and -8b in spermatids and spermatozoa. Short-term in vitro incubation of testis explants indicated that most aquaporins in Sertoli cells and early germ cells were upregulated by rFsh and/or rLh through androgen-dependent pathways, although Aqp1ab in proliferating spermatogonia was also activated by estrogens. However, expression of Aqp9b in Leydig cells, and of Aqp1aa and -7 in spermatocytes and spermatids, was also directly stimulated by rLh. These results reveal a complex gonadotropic control of aquaporin expression during seabream germ cell development, apparently involving both androgen-dependent and independent pathways, which may assure the fine tuning of aquaporin-mediated fluid secretion and absorption mechanisms in the seabream testis

Endocrine control of aquaporin expression during gilthead seabream (Sparus aurata) spermatogenesis

10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageIntroduction Recent studies in mammals have inferred that multiple classes of aquaporins may play different roles for water and solute transport during spermatogenesis and spermiogenesis. In teleosts, immunolocalization experiments in gilthead seabream have demonstrated that seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the mature (spermiating) testis. In this work, we used recombinant piscine gonadotropins and steroid hormones to investigate the in vivo and in vitro endocrine regulation of aquaporin expression during gilthead seabream spermatogenesis. Methods Males were sampled during the resting phase, at the onset of spermatogenesis and during the spermiating period. Plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) were measured by EIA. Testicular subsamples were used for immunofluorescence microscopy using paralog-specific antibodies, or incubated in vitro in the presence of European seabass recombinant follicle-stimulating hormone (rFsh) or luteinizing hormone (rLh), T, estradiol (E2), 11-KT or 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Changes in aquaporin gene expression were determined by real-time qPCR. Results and Discussion During the resting phase, when plasma levels of T and 11-KT were low and the testis exclusively contained spermatogonia, only Aqp1ab was detected in the cytoplasm of spermatogonia, whereas Aqp9b and -10b were expressed in Leydig and Sertoli cells, respectively. At the onset of spermatogenesis and in the spermiating testis, androgen plasma levels progressively increased which correlated with the appearance of spermatocytes and spermatids, and few spermatozoa, in the seminiferous tubules. At both stages, Aqp0a and -9b were localized in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and spermatozoa. In vitro incubations of testis explants with rFsh and rLh indicated that both gonadotropins induced elevated expression levels of aqp0a, - 9b and - 10b at the onset of spermatogenesis, whereas aqp1aa and - 7 only increased in the presence of rLh. At the spermiating stage, gonadotropins only promoted the expression of aqp7 and - 8b. The response to the steroids was also different depending on the spermatogenic stage. Thus, at the onset of spermatogenesis aqp0a, - 1aa, - 7, - 9b and - 10b mRNA levels increased in the presence of 11-KT, and in the case of aqp9b also with E2 and 17,20β-P. In contrast, during spermiogenesis the mRNA levels of aqp0a, -8b, - 9b and - 10b were only stimulated with T, and 17,20β-P for aqp9b, whereas those of aqp7 were still responsive to 11-KT. Interestingly, aqp1ab expression was not regulated by gonadotropins or steroids at the onset of spermatogenesis or during spermiation, suggesting an earlier control of this channel. Conclusion These observations suggest a differential and complex gonadotropic and steroid regulation of aquaporin expression during gilthead seabream germ cell developmentPeer Reviewe