TYPE Original Research PUBLISHED 11 August 2023 DOI 10.3389/fendo.2023.1222724 Neurohypophysial and paracrine OPEN ACCESS vasopressinergic signaling EDITED BY Julang Li, University of Guelph, Canada regulates aquaporin trafficking to REVIEWED BY Yoko Yamaguchi, hydrate marine teleost oocytes Shimane University, Japan Maho Ogoshi, Okayama University, Japan Alba Ferré 1, François Chauvigné 2, Magdalena Gozdowska 3, *CORRESPONDENCE Ewa Kulczykowska 3, Roderick Nigel Finn 1,4 and Joan Cerdà 1* Joan Cerdà joan.cerda@irta.cat 1 Institute of Agrifood Research and Technology (IRTA)-Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Institute of Marine Sciences, Spanish RECEIVED 15 May 2023 National Research Council (CSIC), Barcelona, Spain, 3 Department of Genetics and Marine ACCEPTED 18 July 2023 Biotechnology, Institute of Oceanology, Polish Academy of Sciences, Sopot, Poland, 4 Department of PUBLISHED 11 August 2023 Biological Sciences, University of Bergen, Bergen, Norway CITATION Ferré A, Chauvigné F, Gozdowska M, Kulczykowska E, Finn RN and Cerdà J (2023) Neurohypophysial and paracrine The dual aquaporin (Aqp1ab1/Aqp1ab2)-mediated hydration of marine teleost vasopressinergic signaling regulates aquaporin trafficking to hydrate marine eggs, which occurs during oocyte meiosis resumption (maturation), is teleost oocytes. considered a key adaptation underpinning their evolutionary success in the Front. Endocrinol. 14:1222724. oceans. However, the endocrine signals controlling this mechanism are almost doi: 10.3389/fendo.2023.1222724 unknown. Here, we investigated whether the nonapeptides arginine vasopressin COPYRIGHT © 2023 Ferré, Chauvigné, Gozdowska, (Avp, formerly vasotocin) and oxytocin (Oxt, formerly isotocin) are involved in Kulczykowska, Finn and Cerdà. This is an marine teleost oocyte hydration using the gilthead seabream (Sparus aurata) as a open-access article distributed under the model. We show that concomitant with an increased systemic production of Avp terms of the Creative Commons Attribution License (CC BY). The use, distribution or and Oxt, the nonapeptides are also produced and accumulated locally in the reproduction in other forums is permitted, ovarian follicles during oocyte maturation and hydration. Functional provided the original author(s) and the copyright owner(s) are credited and that characterization of representative Avp and Oxt receptor subtypes indicates the original publication in this journal is that Avpr1aa and Oxtrb, expressed in the postvitellogenic oocyte, activate cited, in accordance with accepted phospholipase C and protein kinase C pathways, while Avpr2aa, which is highly academic practice. No use, distribution or reproduction is permitted which does not expressed in the oocyte and in the follicular theca and granulosa cells, activates comply with these terms. 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. KEYWORDS aquaporin, trafficking, vasopressin, oxytocin, progestin, oocyte hydration, teleost Frontiers in Endocrinology 01 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 1 Introduction to form Avpr1, Avpr2 and oxytocin (Oxt, formerly isotocin) receptor (Oxtr) subtypes (25–29). In mammals, the AVPR1 and Water transport during vertebrate oogenesis determines the OXTR subtypes activate phospholipase C (PLC) for calcium phenotypic nature of antral follicles in mammals and the pelagic signaling and protein kinase (PKC) activation, while the AVPR2 (floating) eggs of marine teleosts (1, 2). In both cases, aquaporins subtype activates the cAMP-PKA-mediated transduction are recognized as the primary channels of transmembrane water mechanism (30, 31). The orthologous teleost Avpr and Oxtr are flux resulting in the fluid-driven expansion of the follicles (2, 3). more numerous than the four paralogs present in mammals due in However, little is known of the endocrine signals regulating their part to a specific whole genome duplication at the root of the crown follicular trafficking. Aquaporins belong to a large family of integral clade (28). Despite only a few studies being conducted to membrane proteins that evolved to facilitate the flux of water and characterize which receptor-mediated pathways are induced, the other small molecules through their central pores (4). They function available evidence indicates that the teleost Avpr1, Avpr2 and Oxtr in tetrameric assemblages, with each aquaporin monomer that subtypes regulate the same signal transduction cascades as in forms a water pore comprised of six transmembrane domains, mammals (32–35). More recently, two studies using the Xenopus two hemi-helices bearing conserved Asn-Pro-Asp (NPA) motifs, laevis oocyte expression system have shown that the Avp-Avpr2aa three extracellular loops (A, C, E), two intracellular loops (B, D), (formerly Avp2r)-cAMP-PKA axis can regulate the intracellular and intracellular N- and C-terminal domains of varying lengths (5). trafficking of teleost Aqp1ab1 and Aqp14 channels, while Oxt acting Up to 15 subfamilies (AQP0-14) are recognized in egg-laying through the Oxtrb (formerly Itr) and a PKC-mediated mechanism mammals, but due to gene loss, only 13 subfamilies (AQP0-12) can regulate the trafficking of Aqp1aa (36, 37). remain in placental mammals (6). Conversely, although a similar In the stinging catfish, in addition to the neuroendocrine/ number of orthologous subfamilies have been identified in teleost reproductive centers of the brain (28), an intraovarian fishes, the gene copy numbers are higher (19-26 in diploids, 38-48 vasopressinergic system has been described that could also be in paleotetraploids) due to extra rounds of whole genome involved in the paracrine regulation of oocyte hydration (22–24, duplications combined with cladal- and lineage-level tandem 38). However, although the Aqp1ab2 channel has been implicated duplications and gene losses (6–12). in this mechanism (23), no study has yet demonstrated its In mammalian follicles AQP1, -5, -7, -8 and -9 are thought to subcellular localization or indeed that of the Aqp1aa and mediate water transport via the granulosa cells to form the Aqp1ab1 channels, which are also encoded in the TSA1C of extracellular antrum (3, 13, 14). In contrast, in marine teleosts catfishes (12). In contrast to freshwater fishes in which only ~1% water flows intracellularly into the oocyte across the plasma produce semi-buoyant eggs, the great majority of modern membrane via tandemly duplicated orthologs of mammalian euacanthomorph marine teleosts, such as the gilthead seabream AQP1 (Aqp1ab1 and/or Aqp1ab2) (12, 15–18). The Aqp1ab-type (Sparus aurata), produce highly hydrated pelagic eggs (2, 12, 39), paralogs are encoded in an ancient teleost-specific aquaporin-1 yet the ovarian production of Avp or Oxt has not been reported. cluster (TSA1C: aqp1aa-aqp1ab2-aqp1ab1) that evolved ~300 Ma Since we have recently observed that PKA- and PKC-mediated with the emergence of the clade (12). The aqp1ab-type genes of the signal transduction pathways stimulate relocation of each of the TSA1C are highly selectively retained in marine species that spawn TSA1C channels (Aqp1aa, Aqp1ab1 and Aqp1ab2) in surrogate pelagic eggs, and their loop D and C-terminal subdomains co- amphibian oocytes (12), here we investigated the potential roles of evolved together with protein kinase (PKA) pathways and 14-3-3z- the Avp-Avpr and Oxt-Oxtr axes in the regulation of aquaporin like (YwhazL) binding proteins to establish a two-step regulated trafficking that facilitates oocyte hydration in the seabream. trafficking mechanism that avoids competitive occupancy of the same oocyte membrane space (12). This preovulatory process occurs during the maturational break down of the germinal 2 Results vesicle (GVBD) and meiosis resumption to form highly hydrated pelagic eggs (>90% H2O) that are subsequently dispersed in the 2.1 A paracrine vasopressinergic and oceanic currents (12, 19). oxytocinergic system is present in the With the exception of a study showing that teleost aqp1ab1 seabream ovary transcription is activated by the nuclear progestin receptor (nPgr) (20), very little is known of the endocrine regulation of water To investigate whether teleost Avp and Oxt are produced locally transport in marine teleost oocytes during meiosis resumption. in the seabream ovary, we initially determined the changes in the Studies in the freshwater stinging catfish (Heteropneusteus fossilis), concentration of the nonapeptides both in the plasma and in the which spawns partially hydrated benthic eggs (21), have, however, ovary of females showing different stages of ovarian follicle suggested that vasotocinergic mechanisms acting through distinct development (Figure 1A) using high performance liquid arginine vasotocin receptors (Avtr1 and Avtr2) could differentially chromatography (HPLC). Plasma and ovarian levels of Avp regulate GVBD and ovulation, and aquaporin-mediated oocyte progressively increased in females with ovaries at the vitellogenic hydration, respectively (22–24). Such receptors are now termed stage and undergoing meiotic maturation and hydration arginine vasopressin receptors (Avpr) that bind vasopressin (Figure 1B). However, in females with ovaries containing fully (formerly vasotocin), which diverged early in vertebrate evolution matured and hydrated follicle-enclosed oocytes and ovulated eggs, Frontiers in Endocrinology 02 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 A B C FIGURE 1 In vivo levels of arginine vasopressin (Avp) and oxytocin (Oxt) in seabream plasma and ovary during the natural reproductive cycle. (A) Representative photomicrographs of histological sections from ovaries containing ovarian follicle-enclosing oocytes at different developmental stages: previtellogenesis or primary growth stage (PVT), vitellogenesis (VT), oocytes undergoing meiosis resumption and hydration (HYD), and fully matured and hydrated oocytes and ovulated eggs (MAT). Scale bars, 100 µm (PVT), 400 µm (VT-MAT). (B, C) Levels of Avp (A) and Oxt (B) in plasma (upper panels) and ovary (lower panels) from females at the different reproductive stages as shown in (A) In each panel, values (mean ± SEM) with different superscript are significantly (ANOVA, P < 0.05) different. The number of fish analyzed at each stage are indicated in each plot. the ovarian Avp levels markedly dropped, while the plasma levels pro-avp and pro-oxt staining was also detected (Figure 2B). For both remained elevated (Figure 1B). By contrast, the plasma Oxt levels in transcripts, no extrafollicular expression (i.e., interstitial tissue) was females were approximately three times higher than those of Avp noted in the ovarian stages examined, and control sections and showed a cyclical pattern (Figure 1C). Thus, circulating Oxt incubated with sense probes were negative (Figure 2B). concentrations were higher during vitellogenesis and the fully The above observations were further confirmed by mature and hydrated stages compared to other stages (Figure 1C). immunofluorescence microscopy using anti-Avp and anti-Oxt However, the intraovarian changes of Oxt during the reproductive rabbit antisera, which were previously validated by immunostaining cycle reflected those observed for Avp, with a precipitous drop of the seabream posterior pituitary gland (Supplementary Figure S1). during the maturation and hydration stage (Figure 1C). These experiments showed that primordial follicle cells associated to To rule out the possibility that intraovarian Avp and Oxt primary growth oocytes were negative for Avp or Oxt detected in our analysis were the result of their accumulation immunostaining, and confirmed that granulosa cells surrounding from circulating products, we investigated whether these peptides vitellogenic oocytes, as well as those surrounding oocytes undergoing can also be produced within the seabream ovary, as reported for the meiotic maturation and hydration, synthesize Avp (Figure 2C). In stinging catfish (38, 40). For this, we first assessed the expression of contrast, Oxt immunoreaction was observed in theca and granulosa pro-avp and pro-oxt genes in ovarian follicles by reverse cells in both follicular stages, in agreement with the ISH data transcription-polymerase chain reaction (RT-PCR) and in situ (Figure 2C). Preadsorption of the antisera with the corresponding hybridization (ISH) using gene-specific oligonucleotide primers Avp and Oxt peptides led to a complete absence of staining in all the and riboprobes, respectively. The RT-PCR experiments indicated follicular stages analyzed, except a cross-reaction of the Avp that pro-avp and pro-oxt mRNAs were expressed in the pituitary (as antiserum with the vitelline envelope in vitellogenic follicles a positive control) and the ovary, whereas low but detectable (Figure 2C), indicating the specificity of the signals in the transcripts from both genes were amplified in isolated vitellogenic follicular cells. ovarian follicles (Figure 2A). The ISH performed with histological To assess whether Avprs and Oxtrs are also expressed in ovarian sections of ovaries at the primary growth and vitellogenic stages follicles we carried out RT-PCR and ISH using specific primers and showed that primordial follicle cells adjacent to previtellogenic riboprobes for two seabream Avpr subtypes, Avpr1aa and Avpr2aa oocytes were strongly positive for pro-avp, whereas these cells (previously termed Avt1a2r and Avt2r, respectively), and one were devoid of pro-oxt mRNAs (Figure 2B). In vitellogenic previously identified Oxtrb (formerly Itr) (41). These receptors ovarian follicles, in which follicle cells are fully differentiated into were selected as representative Avprs and Oxtrs with a potential cuboidal granulosa cells and enlarged and elongated theca cells, pro- for different ligand specificities and the activation of different signal avp transcripts were detected only in the granulosa cells, while both transduction pathways. The results of the RT-PCR assays, using granulosa and theca cells appear to express pro-oxt mRNAs kidney total RNA as a positive control, showed that both avpr1aa (Figure 2B). In the cytoplasm of vitellogenic oocytes, some weak and avpr2aa transcripts could be amplified from the ovary and Frontiers in Endocrinology 03 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 A B C FIGURE 2 Identification of the cellular sites of Avp and Oxt production in seabream ovarian follicles. (A) Representative RT-PCR analysis of the expression of mRNAs encoding the Avp and Oxt pro-peptides (pro-avp and pro-oxt, respectively) in the seabream ovary and isolated vitellogenic ovarian folliclesand in the pituitary as positive control. The ribosomal protein S18 (rps18) was used as the reference. The minus indicates absence of RT during cDNA synthesis. The sizes (bp) of the molecular markers are indicated on the left. (B) Localization of pro-avp and pro-oxt transcripts in previtellogenic (PVT) and vitellogenic (VT) ovarian follicles. Paraffin sections were hybridized with antisense DIG-labeled riboprobes specific for pro- avp and pro-oxt or sense probes (rigth panels, negative controls). Scale bars: 10 µm (PVT), 20 µm and 8 µm insets (VT). (C) Immunostaining of Avp and Oxt peptides (red, right panels) in PVT and VT follicles, and in follicles undergoing meiosis resumption and hydration (HYD). The corresponding brightfield (BF) images are also shown (left panels). The reactions were visualized with Cy3-conjugated sheep anti-rabbit IgG and the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Control sections were incubated with preabsorbed antiserum. In B and C, theca and granulosa cells are indicated by an arrowhead and arrow, respectively. Scale bars, 20 µm (PVT), 10 µm (VT and HYD). Abbreviations: GV, germinal vesicle; O, oocyte; VE, vitelline envelope. isolated follicles, while oxtrb expression was also noted in the ovary 2.2 Pharmacological characterization of but not detected in follicles (Figure 3A). The ISH data indicated that seabream Avpr1aa, Avpr2aa and Oxtrb previtellogenic oocytes express avpr2aa and oxtrb, but not avpr1aa, receptor subtypes whereas oxtrb mRNAs were also observed in the primordial follicle cells (Figure 3B). During vitellogenesis, follicles start to express Since the different Avprs and the Oxtrs are considered to trigger avpr1aa, but only in the oocyte and not in the follicle cells, whereas distinct PKA and PKC signaling cascades, and it has been shown avpr2aa expression in oocytes is maintained and activated in the that these kinases can regulate seabream Aqp1aa, -1ab1 and -1ab2 differentiated theca and granulosa cells (Figure 3B). At this stage, trafficking (12, 36), we next investigated the signaling pathways the oxtrb transcripts in oocytes seemed to be slightly reduced and activated by seabream Avpr1aa, Avpr2aa and Oxtrb. For this, were no longer detected in the follicle cells (Figure 3B). For the three human embryonic kidney cells 293T (HEK293T) were transiently transcripts sense probes gave negative results (Figure 3B). transfected with avpr1aa, avpr2aa or oxtrb full-length cDNAs, Altogether, the above data demonstrate the local production of isolated from the seabream ovary based on publicly available data. Avp and Oxt nonapeptides in the seabream ovarian follicle, as well Cells were transfected at the same time with b-Galactosidase (b- as the expression of their cognate receptors in the follicle cells and/ Gal) together with a cAMP-responsive reporter gene plasmid or oocyte, which would be consistent with a role of a (pCRE-luc), or a plasmid for activation of the phospholipase C vasopressinergic and oxytocinergic regulatory system controlling (PLC)/PKC pathway (pSRE-luc), and exposed to increasing doses of aquaporin intracellular trafficking and oocyte hydration. the nonapeptides ranging from 10-12 to 10-5 M (Figure 4). The Frontiers in Endocrinology 04 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 A B FIGURE 3 Localization of Avp and Oxt receptor mRNA expression in seabream ovarian follicles. (A) Representative RT-PCR analysis of the expression of avpr1aa, avpr2aa and oxtrb mRNAs in the seabream ovary and isolated ovarian follicles, and in kidney as positive control. The ribosomal protein S18 (rps18) was used as the reference. The minus indicates absence of RT during cDNA synthesis. The sizes (bp) of the molecular markers are indicated on the left. (B) Localization of avpr1aa, avpr2aa and oxtrb transcripts in previtellogenic (PVT) and vitellogenic (VT) ovarian follicles. Paraffin sections were hybridized with specific antisense DIG-labeled riboprobes or sense probes (rigth panels, negative controls). Theca and granulosa cells are indicated by an arrow and arrowhead, respectively. Scale bars, 10 µm (PVT), 20 µm (VT). GV, germinal vesicle; O, oocyte; VE, vitelline envelope. receptor-induced increase in intracellular Ca2+ concentration increasing doses (from 10-12 to 10-5 M) of the different receptor ([Ca2+]i), as a second messenger in the PLC signal transduction antagonists, and then subsequently treated with the approximate pathway, was determined using Fluo-4 AM, a cell-permeable, EC50 of Avp or Oxt on each cognate receptor (10-8 M). The results fluorescent Ca2+ indicator. showed that activation of the Avpr1aa was equally inhibited by In cells expressing the Avpr1aa or Oxtrb receptors, incubation SR49059 and TLV, which showed half maximal inhibitory with Avp or Oxt, respectively, resulted in a concentration- concentration (IC50) of ~10-9 M, although the SR49059 was dependent increase of pSRE-induced luciferase activity and of slightly more effective (Figure 4G). Both SR49059 and TLV also [Ca2+]i, while it did not induce measurable increases in pCRE- inhibited the Avpr2aa, although in this case TLV was ~10 times mediated luciferase activity (Figures 4A–C). However, the Avpr1aa more potent than SR49059 at blocking the receptor (Figure 4H). In was less affective at activating PKC or increasing the [Ca2+]i than contrast, the L-371,257 antagonist had no effect on Avpr1aa or the Oxtrb, since the half-maximal effective concentrations (EC50) of Avpr2aa activation (Figure 4G, H). Finally, the Oxtrb was well Avp on its cognate receptor were ~10 times higher compared with blocked by L-371,257, showing an IC50 of ~10-9 M, while it was not those of Oxt on the Oxtrb (Figures 4B, C). In contrast, in Avpr2aa affected by SR49059 or TLV (Figure 4I). These data therefore expressing cells Avp induced a potent dose-dependent response in indicate that TLV and L-371,257 are, respectively, potent and pCRE-induced luciferase activity, while PKC was not activated and selective inhibitors of seabream Avpr and Oxtr receptor subtypes. the [Ca2+]i was not affected (Figures 4A–C). Receptor cross- activation experiments showed that both Avpr1aa and Avpr2aa could be activated by Avp and Oxt, although the EC50 for Oxt on 2.3 Avp and Oxt differentially regulate the these receptors was ~10 times higher with respect to that of Avp intracellular trafficking of the seabream (Figures 4D, E). On the contrary, the Oxtrb was more specific for TSA1C channels Oxt, since only a slight increase of the [Ca2+]i in response to Avp was noted in cells expressing this receptor (Figure 4F). To investigate whether Avprs and Oxtrb can regulate the To search for effective and selective inhibitors of seabream intracellular trafficking of Aqp1aa, -1ab1 or -1ab2, we initially Avprs and Oxtrb, which could be used in further physiological employed oocytes from X. laevis as a surrogate experimental experiments, we tested the effect of different non-peptide system (Figure 5). Oocytes were injected with avpr1aa, avpr2aa antagonists selective for mammalian AVPR1A (SR49059) (42), or oxtrb cRNAs, synthesized from human influenza hemagglutinin AVPR2 (tolvaptan, TLV) (43) and OXTR (L-371,257) (44). In (HA)-tagged cDNAs, which drives the constitutive expression of these experiments, cells were transfected with Avpr1aa, Avpr2aa the receptors in the oocyte plasma membrane (Supplementary or Oxtrb and reporter plasmids as above, and preincubated with Figure S2). To test the effect of receptor activation on the Frontiers in Endocrinology 05 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 A D G B E H C F I FIGURE 4 Functional characterization of seabream Avp and Oxt receptors in HEK293T cells. (A–F) Activation of the Avpr1aa, Avpr2aa and Oxtrb by graded concentrations of the corresponding ligands (A–C), and ligand selectivity (D–F) of each receptor. (G–I) Inhibition of the activation of the Avpr1aa, Avpr2aa and Oxtrb by 10-8 M of the corresponding ligands in the presence of increasing concentrations of vasopressin (SR49049 and Tolvaptan) and oxytocin (L-371 257) receptor antagonists. In all experiments, hormone-induced cAMP production and PKC activation were indirectly quantified by measuring the luciferase activity from the CRE-Luc and SRE-Luc reporter vectors, respectively, which were cotransfected with the receptor cDNA containing plasmids. Intracellular Ca2+ was determined by loading the cells with the Fluo-4 Ca2+-sensitive dye before activation of the receptors. The data (mean ± SEM) from each plot are derived from three to six independent transfections. trafficking of the TSA1C channels, each receptor was coinjected the activation of the PLC/PKC signaling pathway by the with cRNAs encoding Aqp1aa, Aqp1ab1 or Aqp1ab2, or water as a Avpr1aa previously observed in HK293T cells. Immunoblotting negative control. Oocytes were exposed to Avp or Oxt, depending of total and plasma membrane extracts of oocytes and on the type of receptor expressed, or to the hormone vehicle immunofluorescence microscopy using Aqp1aa-specific antibodies dimethyl sulfoxide (DMSO) as control, and subsequently revealed that the hormone-induced reduction in the oocyte Pf was submitted to swelling assays to determine the osmotic water due to the internalization of the channel in response to Avpr1aa permeability (Pf). Since Aqp1ab2 can only traffic to the frog activation, which was blocked in the presence of Bim-II (Figures 5C, oocyte plasma membrane when it is bound to the teleost-specific D). The same results were obtained in oocytes expressing the Oxtrb YwhazLb protein in the presence of the intracellular cAMP and Aqp1aa, Aqp1ab1 or Aqp1ab2 plus YwhazLb (Figures 5I–L), activator forskolin (FSK) (12), Aqp1ab2-injected oocytes were indicating that activation of Avpr1aa or Oxtrb by their cognate also coinjected with seabream YwhazLb cRNA and, in some ligands inhibit Aqp1aa trafficking to the oocyte surface through experiments, exposed to FSK prior to nonapeptide treatment. PKC signaling, while Aqp1ab1 and -1ab2 are not regulated by Oocytes expressing the Avpr1aa and Aqp1aa or Aqp1ab1 these receptors. showed a 3-4-fold increase in Pf with respect to the control In contrast, the Pf of oocytes expressing Avpr2aa and Aqp1ab1 oocytes after a hypoosmotic challenge. However, Avp treatment or Aqp1ab2+YwhazLb (in the latter case without FSK pretreatment) reduced the Pf by 80% in oocytes expressing Aqp1aa, but not in was increased by ~2- and ~4-fold, respectively, after Avp exposure, those injected with Aqp1ab1 (Figure 5A). In the case of oocytes whereas that of the oocytes co-expressing Avpr2aa and Aqp1aa did expressing Avpr1aa plus Aqp1ab2, treatment with Avp did not not change (Figure 5E). The Avpr2aa-mediated increase in the Pf of affect the oocyte Pf triggered by FSK exposure (Figure 5A). oocytes expressing Aqp1ab1 or Aqp1ab2+YwhazLb occurred Preincubation of Avpr1aa+Aqp1aa oocytes with the PKC through the Avp-induced insertion of the channels into the inhibitor bisindolylmaleimide II (Bim-II) prevented the Avp- oocyte plasma membrane as revealed by immunoblotting and induced inhibition of P f (Figure 5B), in agreement with immunolocalization experiments using paralog-specific antibodies Frontiers in Endocrinology 06 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 A B C D G H E F I J K L FIGURE 5 Avp and Oxt regulation of seabream Aqp1aa, Aqp1ab1 and Aqp1ab2 intracellullar trafficking in X. laevis oocytes. (A, B, E, F, I, J) Osmotic water permeability (Pf) of oocytes expressing the seabream Avpr1aa (A, B), Avpr2aa (E, F) or Oxtrb (I, J), and injected with either water, wild-type Aqp1aa, Aqp1ab1 or Aqp1ab2 plus YwhazLb. Oocytes were treated with DMSO (controls) or 10 mM Avp or Oxt (A, E, I), in the presence or absence of 10 mM of PKC and PKA inhibitors (Bim-II and H89, respectively) (B, F, J). Oocytes injected with Aqp1ab2+ YwhazLb were also treated with or without 10 µM forskolin (FSK). Data are the mean ± SEM with the number of oocytes indicated in each bar. **P < 0.01; ***P < 0.001 (unpaired Student’s t-test), with respect to water-injected oocytes, or as indicated in brackets. (C, D, G, H, K, L) Representative immunoblot (C, G, K) of Aqp1aa, Aqp1ab1 and Aqp1ab2 in total (TM) and plasma (PM) membrane extracts from oocyes treated as above, and corresponding immunostaining (D, H, L), using paralog-specific antibodies. The arrowheads indicate the PM, whereas the arrows indicate aquaporin-containing intracellular vesicles. Scale bars, 10 µm. (Figures 5F–H). However, this trafficking was prevented by the binding motif, while that of Aqp1ab2, as mentioned above, PKA inhibitor H89 (Figures 5F–H). These data thus show that strictly relies on PKA-mediated Thr262 phosphorylation and the Avpr2aa can positively regulate Aqp1ab1 and Aqp1ab2 trafficking YwhazLb interaction (12) (Figures 6A, D, I). Therefore, in order to to the plasma membrane through the PKA signaling cascade, which investigate whether Avp and Oxt control Aqp1aa, Aqp1ab1 and is activated in both oocytes and cultured cells. Aqp1ab2 trafficking through the same mechanisms, X. laevis oocytes were injected with cRNAs for avpr1aa, avpr2aa or oxtrb and wild-type TSA1C channels or mutant forms in which the 2.4 Molecular basis of the nonapeptide PKA and PKC phosphorylation sites were replaced by non- regulation of aquaporin trafficking phosphorylatable Ala residues (12), together with HA-tagged YwhazLa or -zLb. The Pf of oocytes, the trafficking of the The above data are consistent with our recent study using channels to the oocyte plasma membrane, and the interaction direct pharmacological activation of PKC and PKA in X. laevis of Aqp1ab1 and Aqp1ab2 with YwhazLa or -zLb carrier oocytes, which showed that in euacanthomorph teleosts, such as the proteins, respectively, in response to the nonapeptides, were seabream, PKC negatively regulates Aqp1aa trafficking to the then examined by using swelling assays, immunoblotting and plasma membrane, while PKA positively regulates this co-immunoprecipitation using Aqp1ab1- and Aqp1ab2- mechanism in Aqp1ab1 and Aqp1ab2 (12). This latter study also specific antibodies. identified the kinase phosphorylation sites involved in trafficking The results of these experiments showed that the reduction of regulation of the different channels: Thr145 (loop D) in Aqp1aa, oocyte permeability and channel accumulation in the plasma Ser156 and Ser253 (intracellular loop D and C-terminus, respectively) membrane in response to Avp or Oxt, in oocytes expressing wild- in Aqp1ab1, and Thr262 (C-terminus) in Aqp1ab2 (Figures 6A, D, type Aqp1aa plus Avpr1aa or Oxtrb, was completely reversed when I). However, plasma membrane trafficking of Aqp1ab1 partially oocytes expressed the Aqp1aa-V145T mutant instead of the wild- depends on channel binding to the teleost-specific YwhazLa, type (Figures 6B, C), as observed when PKC is directly activated in through PKA phosphorylation of Ser 253 within the YWHA Aqp1aa expressing oocytes (12). In oocytes expressing wild-type Frontiers in Endocrinology 07 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 A B C E F D G H I J K L FIGURE 6 Molecular basis of the vasopressinergic and oxytocinergic regulation of Aqp1aa, Aqp1ab1 and Aqp1ab2 trafficking. (A, D, I) Schematic representation of seabream Aqp1aa, Aqp1ab1 and Aqp1ab2 monomers showing the six transmembrane domains, the five connecting loops (A–E), and the PKC and PKA phosphorylation sites involved in channel trafficking (12). The predicted YWHA-binding regions in the Aqp1ab1 and Aqp1ab2 C-termini are underlined. (B, C) Osmotic water permeability (Pf) (B) and immunoblots of total (TM) and plasma (PM) membrane extracts (C) of oocytes expressing the Oxtrb or Avpr1aa and wild-type (WT) Aqp1aa or the Aqp1aa-V145T mutant, and exposed to DMSO (controls) or 10 mM Avp or Oxt. (E–G) Pf of oocytes expressing the Avpr2aa and WT Aqp1ab1 or Aqp1ab1 mutants (S156A and S253A), together with HA-tagged YwhazLa (YwhazLa-HA), and treated with Avp in the presence or absence of 10 µm of H89. The corresponding immunoblots are shown in (F, G). (H) Co-immunoprecipitation of WT and mutant Aqp1ab1 channels and YwhazLa-HA in the same oocytes using Aqp1ab1 and HA specific antibodies. (J, K) Pf (J) and immunoblot (K) of oocytes expressing the Avpr2aa and WT Aqp1ab2 or Aqp1ab2-T262A, together with YwhazLb-HA, and treated with Avp as above. (L) Co- immunoprecipitation of both channels in the same oocytes carried out as in (H). Data in B, E and J are the mean ± SEM with the number of oocytes indicated in each bar. **P < 0.01; ***P < 0.001 (unpaired Student’s t-test), with respect to water-injected oocytes, or as indicated in brackets. In C, F- H, K and L, molecular mass markers (kDa) are on the right. Aqp1ab1, YwhazLa-HA and Avpr2aa, Avp triggered a more robust mutant, which did not traffic to the oocyte surface after Avp increment in the oocyte Pf and of the relative amount of the channel stimulation, was not able to interact with YwhazLa regardless of in the plasma membrane (Figures 6E, F) than in oocytes without H89 treatment (Figure 6H). These data agree with our previous YwhazLa (Figure 5E), which was partially or completely prevented, study (12) and suggest that Avp-mediated targeting of Aqp1ab1 to respectively, in oocytes expressing the Aqp1ab1-S156A or -S253A the plasma membrane depends on PKA phosphorylation of C- mutants (Figures 6E, F). However, the positive effect of Avp on terminal Ser253, which drives YwhazLa binding, and of loop D plasma membrane trafficking of both wild-type Aqp1ab1 and Ser156 once the complex Aqp1ab1-YwhazLa is formed. In contrast, Aqp1ab1-S156A channels was equally abolished in the presence oocytes expressing Aqp1ab2 and Avpr2aa, together with YwhazLb- of the H89 inhibitor (Figures 6E, G). Co-immunoprecipitation HA, only showed a higher Pf, together with an increased channel experiments using the Aqp1ab1 antibody confirmed that Avp expression in the oocyte plasma membrane and channel binding to promoted the interaction of wild-type Aqp1ab1 and Aqp1ab1- YwhazLb, after Avp treatment, which were all blocked in oocytes S156A with YwhazLa, which was inhibited in both cases in the expressing the Aqp1ab2-T262A mutant (Figures 6J–L). These presence of H89 (Figure 6H). However, the Aqp1ab1-S253A findings therefore indicate that the Avp-mediated regulation of Frontiers in Endocrinology 08 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 Aqp1ab2 trafficking is specifically me diate d by PKA response to piscine single-chain recombinant Lh (rLh) was phosphorylation of Thr262 in the C-terminus, allowing YwhazLb evaluated by a custom made Avp enzyme-linked immunosorbent binding and further insertion of the channel into the assay (ELISA) (Supplementary Figure S3). To investigate the plasma membrane. specificity of the rLh potential regulation, in these experiments we also tested the effect of single-chain recombinant follicle- stimulating hormone (rFsh). The results showed that rLh 2.5 Intraovarian Avp production increased the concentration of Avp in the explants by ~4-fold is regulated by gonadotropin- with respect to the water-exposed explants (controls), while the induced progestin Avp levels in the rFsh-treated explants did not differ from the controls (Figure 7A). The previous experiments show that seabream ovarian follicles Since the progestin 17a,20b-dihydroxypregn-4-en-3-one can synthesize Avp, and that the ovarian levels of the nonapeptide (17,20b-P) is known to be the maturation-inducing steroid in vivo increase during the stage of oocyte maturation and secreted by granulosa cells in response to Lh in sparids (47), we hydration. These observations would be compatible with a role of further investigated whether 17,20b-P could mediate the Lh Avp regulating the intracellular trafficking of Aqp1ab1 and induction of Avp synthesis in postvitellogenic ovarian explants. In Aqp1ab2 in maturing oocytes, as observed in surrogate addition, to examine the type of progestin receptor potentially amphibian oocytes. However, our data also suggest that the local involved in this mechanism, treatment with 17,20b-P was applied production of Avp may be under gonadotropic regulation, since a in the presence or absence of the nuclear progesterone receptor surge of the circulating luteinizing hormone (Lh) typically occurs in inhibitor RU486 (20), while other explants were treated with the seabream females during the maturation stage (45, 46). To test this membrane progestin receptor agonist OD 02-0 (10- ethenyl-19- hypothesis, we carried out in vitro experiments in which the norprogesterone) (48) in the absence of 17,20b-P. The results of production of Avp by postvitellogenic ovarian explants in these trials confirmed that 17,20b-P induced a 3-fold increase in A B FIGURE 7 Gonadotropin and progestin induction of ovarian Avp production in vitro. (A) Amounts of Avp measured in ovarian extracts after ~20 h of exposure to 100 ng/ml of single-chain recombinant seabass Fsh or Lh (rFsh and rLh, respectively), or to the hormone vehicle (controls). Data are the mean ± SEM (n = 10 replicates compiled from two separated experiments on two females). (B) Avp content in ovarian explants incubated with 0.1 µg/ml 17,20b-P, 100 µM of the nuclear progesterone receptor inhibitor RU486, 17,20b-P+RU486, or to 10 µM of the membrane progestin receptor agonist OD 02-0. Controls were treated with 0.5% ethanol. Values are the mean ± SEM (n = 4 independent experiments on different females). *P < 0.05; ***P < 0.001 (unpaired Student’s t-test), with respect to vehicle exposed explants, or as indicated in brackets. Frontiers in Endocrinology 09 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 Avp synthesis in ovarian explants, comparable to that observed in we determined the dynamics of the subcellular localization of response to rLh, which could be completely abolished by RU486, endogenous Aqp1aa, Aqp1ab1, and Aqp1ab2, and their and not reproduced by OD 02-0 treatment (Figure 7B). These data interactions with YwhazLa and -zLb proteins, after Avp treatment therefore suggest that the specific Lh induction of Avp synthesis in in vitro, using the Aqp1ab1- and Aqp1ab2-specific antisera and the granulosa cells of seabream postvitellogenic follicles is mediated commercial mammalian YWHAZ antibodies specific for seabream by a genomic mechanism through the nuclear progestin receptor. YwhazLa/b or -zLb (12). For this, we incubated isolated seabream vitellogenic ovarian follicles with Avp, in the presence or absence of the seabream Avpr1aa and Avpr2aa antagonist TLV or the PKA 2.6 The Avp-Avpr2aa-PKA signaling inhibitor H89, while control follicles were exposed to DMSO vehicle pathway differentially regulates Aqp1ab1 alone (Figure 8). and Aqp1ab2 trafficking in postvitellogenic The experiments showed that Avp did not induce meiotic seabream oocytes maturation or oocyte hydration, in the latter case because the osmotic drive generated by yolk hydrolysis was not activated by To investigate the physiological significance of the Avp the nonapeptide. Immunofluorescence microscopy indicated that regulation of the TSA1C channel trafficking in seabream oocytes, Aqp1aa was localized to the epithelium surrounding the ovarian A E B C F D G FIGURE 8 Avp regulation of aquaporin trafficking in seabream ovarian follicles in vitro. (A–D) Representative brightfield (BF, left panels) and immunostaining of Aqp1aa, Aqp1ab1 and Aqp1ab2 (red color, right panels) in follicles exposed to 10 µM Avp, in the presence or absence of 10 µM of the Avpr1aa and Avpr2aa antagonist Tolvaptan (TLV) or the PKA inhibitor H89. The immunostaining of follicles incubated with TLV or H89 alone are shown in Supplementary Figure S4. The nuclei were counterstained with DAPI (blue color). The theca and granulosa cells are indicated by an arrowhead and arrow, respectively. ve, vitelline envelope; o, oocyte; yg, yolk globules. Scale bars, 10 µm. (E) Percentage (mean ± SEM; n = 5 follicles) of Aqp1ab1 and Aqp1ab2 in the oocyte plasma membrane (PM) after each treatment determined by image analysis. (F) Uptake (mean ± SEM; n = 3 pools of 10 oocytes) of 3H2O by follicle-enclosed oocytes treated with Avp as in E. ***P < 0.001 (unpaired Student’s t-test) as indicated in brackets. (G) Co- immunoprecipitation of Aqp1ab1 and Aqp1ab2 with YwhazL proteins in ovarian follicles (n = 10) treated as in A-D. Left panel shows the immunoblotting of Aqp1ab1, Aqp1ab2, YwhazLa and YwhazLb in the input extracts before immunoprecipitation. The middle and right panels show co-immunoprecipitated YwhazLa and/or YwhazLb with Aqp1ab1 and Aqp1ab2. Controls were immunoprecipitated with IgG or IgY for the Aqp1ab1 and Aqp1ab2 antibodies, respectively. The arrowheads indicate phosphorylated forms of Aqp1ab1 and Aqp1ab2. Molecular mass markers (kDa) are on the left. Frontiers in Endocrinology 10 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 follicles, and not in the theca cell as previously reported (12), and In the present context, we isolated paralogs from each of the that this localization did not appear to change with Avp treatment major Avp and Oxt receptor subtypes (Avpr1aa, Avpr2aa and (Figures 8A, B). In contrast, both Aqp1ab1 and Aqp1ab2 were more Oxtrb) and functionally characterized the signal transduction accumulated in the most cortical cytoplasmic region of the oocytes, pathways they induce in order to determine how the systemic just below the plasma membrane, while Aqp1ab2 was also localized and paracrine systems might integrate to regulate aquaporin- in the granulosa and theca cells (Figures 8A, B). After Avp exposure, mediated oocyte hydration. The data show that the Avpr1aa and Aqp1ab1 immunoreactivity appeared more concentrated in the Oxtrb subtypes activate PLC-PKC cascades while the Avpr2aa most distal part of the microvilli crossing the vitelline envelope, subtype activates the cAMP-PKA pathway. These results are whereas the Aqp1ab2 channel became localized in a more basal consistent with the notion that the functional pathway dichotomy region of the microvilli (Figures 8A, B). The differential spatial between the Avpr1/Oxtr and Avpr2 subtypes may have emerged in localization of Aqp1ab1 and Aqp1ab2 in the oocyte microvilli in the common ancestor of jawed vertebrates (29). However, since response to Avp mimics that observed in FSK-treated teleosts encode up to five copies of the Avpr2 subtype (see postvitellogenic follicles (12). However, it was prevented in Supplementary Figure S5), it will be important to validate the follicle-enclosed oocytes preincubated with TLV or H89 prior to pathway specificity of each paralog to avoid expectation bias. For Avp treatment (Figures 8C, D). These observations were example, in related experiments, our ligand specificity analyses corroborated by Aqp 1ab1 and Aqp1ab2 flu o r es ce n ce show that seabream Avpr1aa and Avpr2aa display some quantification in the oocyte plasma membrane (Figure 8E), as promiscuity for Avp and Oxt. By contrast, the Avpr1ab ortholog well as by radiolabeled water uptake assays, which showed that of the white sucker (Catostomus commersoni) is only activated by water permeability of Avp-treated follicles was higher than that of Avp (32). Similarly, our data for the seabream Oxtrb show a high follicles treated with DMSO alone and reduced in the presence of specificity for Oxt, while the white sucker Oxtra can also be Avp plus the TLV or H89 inhibitors (Figure 8F). activated by Avp, albeit with lower potency (33). The potential changes in the phosphorylation state of Aqp1ab1 Since we had previously established that TSA1C channel and Aqp1ab2 channels, and of their interactions with YwhazLa and trafficking is differentially regulated by the PKC-mediated -zLb carrier proteins, during Avp treatment were subsequently phosphorylation of loop D sites in Aqp1aa and the PKA- investigated by co-immunoprecipitation and immunoblotting. mediated phosphorylation of loop D and C-terminal sites in These trials confirmed previous observations (12, 16) indicating Aqp1ab1 and the C-terminus of Aqp1ab2 (12), we confirmed that in control postvitellogenic follicles, when Aqp1ab1 is held in here that the same sites control the recycling of Aqp1aa and the most cortical region of the oocyte and some inserted in the basal membrane insertion of the Aqp1ab-type channels when co- region of the microvilli, the channel is already phosphorylated and expressed with the Avpr1aa/Oxtrb and Avpr2aa subtypes, bound to both YwhazLa and -zLb (Figure 8G). However, after Avp respectively. By further identifying the pituitary and follicular cell treatment Aqp1ab1 becomes more phosphorylated, while sources of Avp and Oxt together with the ovarian loci of their maintaining the interaction with YwhazLa and -zLb (Figure 8G), respective receptors, and monitoring the plasma and ovarian levels which coincides with the secondary loop D Ser156 phosphorylation of each nonapeptide during oocyte growth and maturation, we can and trafficking of the channel to the most distal region of the oocyte propose a model that is congruent with the suggestions for catfish microvilli. Phosphorylation of Aqp1ab2 was also increased after (24, 28), to explain the neuroendocrine and paracrine regulation of Avp stimulation, but in this case the YwhazLb interaction was only aquaporin trafficking facilitating marine teleost oocyte detected in the presence of the nonapeptide (Figure 8G), when the hydration (Figure 9). channel is translocated to the basal region of the oocyte microvilli. In this model, neurohypophysial secretion of Avp throughout The Avp-mediated increase in the phosphorylation of Aqp1ab1 and oocyte growth (vitellogenesis) arrives at the capillary beds of the Aqp1ab2, as well as the binding of the latter channel to YwhazLb, thecal layer. From there it diffuses to activate the Avpr2aa receptor, was blocked by TLV and H89 (Figure 8G), suggesting that the which is expressed in the plasma membrane of the theca and differential membrane trafficking regulation of the channels is granulosa cells, to induce the PKA-mediated phosphorylation of regulated by the Avpr2aa-PKA transduction pathway. Thr262 in the C-terminus of Aqp1ab2. This results in the coupling of the YwhazLb binding protein and the trafficking of the channel to the cell surface of the theca and granulosa cells. Meanwhile, in the 3 Discussion oocyte Aqp1ab1, which is already expressed since the primary growth stage (20) and phosphorylated at the C-terminal Ser253 by In this study we show for the first time that in addition to the a PKA-mediated mechanism (12), preferentially couples with the hypophysial neuroendocrine system, a paracrine vasopressinergic YwhazLa binding protein and is trafficked close to the basal region and oxytocinergic signaling system exists in the ovary of a marine of the oocyte microvilli during vitellogenesis. Some of Aqp1ab1 also teleost. Since expression of Avp and/or Oxt precursor mRNAs or enters the basal region of the oocyte microvilli (12), potentially to proteins are also expressed in the ovaries of freshwater catfishes (24, mediate water homeostasis during the vitellogenic growth phase. 28, 38, 40), as well as in lampreys (25), sharks (49), birds (50), and Concomitant with the preovulatory surge of Lh (45, 46), our data mammals (51, 52), it seems plausible that an ovarian show that the circulating and ovarian levels of Avp are increased vasopressinergic and/or oxytocinergic paracrine system existed during meiotic maturation and hydration of the oocytes. Such an prior to the emergence of vertebrates. elevated systemic Avp further enhances the PKA-mediated Frontiers in Endocrinology 11 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 trafficking of Aqp1ab2 channels in the theca and granulosa cells. progestin 17,20ß-P in granulosa cells (54). Thus, the 17,20ß-P could However, as in the catfish (53), the combined activation of Lh on act as a master switch regulating the reciprocal genomic production Lhcgr and Avp on Avpr2aa could also multiply the action of the of Avp via the nuclear progestin receptor in granulosa cells, and the PKA-mediated Lh induction of the steroidogenic shift from non-genomic activation of GVBD and meiosis resumption via the estrogen production to the synthesis of the maturation-inducing membrane progestin receptor that is expressed in the oocyte plasma FIGURE 9 (Continued) Frontiers in Endocrinology 12 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 FIGURE 9 (Continued) Proposed model for the vasopressinergic regulation of Aqp1ab1 and Aqp1ab2 function in seabream ovarian follicles during meiosis resumption and oocyte hydration. In primary growth oocytes, Aqp1ab1 is phosphorylated at Ser253 by protein kinase A (PKA), which is activated by a yet unknown signaling pathway, to bind YwhazLa and -zLb. The complex is then slowly transported to the oocyte cortical region during the vitellogenic period, where it is retained just below the oocyte plasma membrane in postvitellogenic oocytes, while some of Aqp1ab1 enters the basal region of the oocyte microvilli. The release of circulating hypohysial Avp and luteinizing hormone (Lh) from capillaries in the theca layer at the time of oocyte maturation activate the Avpr2aa in theca and granulosa cells, as well as the Lh/choriogonadotropin receptor (Lhcgr) in the granulosa cells. In the theca and granulosa, the Avp-stimulated Avpr2aa triggers PKA activation, which mediates the phosphorylation of Thr262 in Aqp1ab2 promoting the binding to YwhazLb and the trafficking of the channel to the plasma membrane of these cells. The activation of the Lhcgr-PKA pathway in granulosa cells drives the synthesis of the progestagen 17,20b-P, through a switch in the steroidogenic (StG) pathway from estrogen to progestagen production. The 17,20b-P, in turn stimulates the local synthesis of Avp through a genomic mechanism mediated by the nuclear progestin receptor (nPgr). The resulting release of Avp and 17,20b-P from the granulosa cells respectively activates the Avpr2aa and the membrane progestin receptor (mPgr) in the oocyte surface. The mPr triggers meiosis resumption (oocyte maturation), whereas the Avpr2aa-PKA signaling regulates the trafficking of Aqp1ab2 as in follicle cells, and a secondary phosphorylation of Ser156 in the loop D of Aqp1ab1 channels already complexed with YwhazLa/b. The secondary phosphorylation of Aqp1ab1 further shuttles the channel to the most distal region of the oocyte microvilli. Finally, systemic (circulating) and/or local levels of Avp, or a possible Lh-mediated production of Oxt, may potentially activate the protein kinase C (PKC) signaling pathway in the epithelium, through a yet unidentified receptor, to induce the recycling of Aqp1aa. This model integrates both the systemic and paracrine vasopressinergic regulation of the intracellular trafficking of Aqp1ab-type paralogs in the seabream oocyte, such that competitive plasma membrane spatial occupancy by both channels is avoided and bulk water influx is augmented during oocyte hydration. membrane (55). It is the former reciprocal genomic production of ovarian levels of Avp, thus ending the paracrine regulation of the Avp that would provide the paracrine signal between the granulosa trafficking of the Aqp1ab-type channels, and their resultant cells and the Avpr2aa in the oocyte plasma membrane, which now internalization in the oocyte (12). The subsequent cyclical activates the PKA-mediated phosphorylation of Thr262 in the increase in the level of circulating Oxt may thus prepare the Aqp1ab2 C-terminus and Ser156 in loop D of Aqp1ab1. The oocytes for ovulation as in mammals (58), leaving the eggs ready Thr262 phosphorylation promotes the coupling of the YwhazLb for fertilization and their future pelagic passage in the binding protein and the trafficking of Aqp1ab2 to the basal region oceanic currents. of the oocyte microvilli, while the secondary phosphorylation of In conclusion, the present study uncovered a paracrine loop D Ser156 in Aqp1ab1 relocalizes the channel to the distal vasopressinergic and oxytocinergic system in the ovary of a portion of the oocyte microvilli. In this way, the two Aqp1ab-type modern euacanthomorph marine teleost, the gilthead seabream, paralogs are co-regulated by the same Avp-Avpr2aa-PKA pathway which supports the notion that this mechanism may have existed to cooperatively enhance water influx while avoiding competitive prior to the emergence of vertebrates. In both the seabream and occupancy of the same membrane space. catfish, the ovarian vasopressinergic system is possibly activated via Our proposed model does not yet explain the signal systemic neuroendocrine signaling to multiply the Lh induction of transduction mechanism that may activate PKC-mediated progestin which generates Avp for the paracrine signaling. In the recycling of Aqp1aa in the follicular epithelium, which occurs seabream, however, by functionally characterizing the signal when this channel is co-expressed with Avpr1aa or Oxtrb in X. transduction pathways induced by each of the Avp and Oxt laevis oocytes. Nevertheless, we did observe that in vitellogenic receptor subtypes and assaying their ligand specificities, it was ovarian follicles, both the mRNA and protein of Oxt are possible to uncover the molecular basis of the nonapeptide accumulated in the theca and granulosa cells, while those of Avp regulated trafficking of the TSA1C channels. In this respect, the are only accumulated in the granulosa cells. These observations may Avpr2aa paralog plays a vital role in the transduction of the suggest that trafficking of epithelial Aqp1aa could also be under Avp paracrine signals and the co-ordinated trafficking of the Aqp1ab- or Oxt regulation through receptors not characterized here, such as type channels so that they avoid competitive occupancy of the same the Avpr1ab or the Oxtra, since in our study neither avpr1aa nor membrane space and maximize the hydration of the oocyte. It oxtrb mRNA expression could not be detected in the follicular remains to be seen if a similar neuroendocrine/paracrine regulation epithelial layer. In any event, the potential recycling of the Aqp1aa of aquaporin trafficking exists in older lineages of teleost that retain channel in the follicular epithelium may provide an important the Aqp1ab-type paralogs. mechanism for the flow of water into the hydrating oocyte. The water path originates with the seawater imbibed by the mother and is delivered to the follicles through the thecal capillary beds. By 4 Materials and methods simultaneously recycling epithelial Aqp1aa and driving Aqp1ab2 to the membranes of the theca and granulosa cells, water is only free to 4.1 Experimental animals and sampling flow in one direction toward the oocyte. The differential proteolysis of primarily vitellogenin-Aa (VtgAa) type yolk proteins (39, 56, 57) Adult gilthead seabream were obtained from the Institut de generates the osmotic driving force that converts the oocyte into the Recerca i Tecnologia Agroalimentàries (IRTA) aquaculture facilities water sink, which then enters via the vasopressinergic induction of (Tarragona, Spain) and transported to the facilities in Institut de the Aqp1ab1 and Aqp1ab2 channels to the distal and proximal Ciències del Mar (Barcelona, Spain). Fish were maintained under regions of the microvilli. The water finally becomes trapped within natural conditions of photoperiod and temperature and fed three the highly hydrated oocyte due to the precipitous drop in the times a week with dry pellets (Seabream Alterna AE, Skretting) and Frontiers in Endocrinology 13 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 squid ad libitum. Samples of blood and gonads were collected ml of glacial acetic acid and incubated in a boiling water bath during 3.5 throughout the year from sacrificed females as previously min. The samples were centrifuged (30 min, 4°C, 10,000 × g). Plasma described (20), frozen in liquid nitrogen and stored at -80°C. The samples (1.5-2 ml each) were acidified with 1 M HCl (100 ml) and stage of ovarian development in each female was determined by centrifuged at 6000·× g for 20 min at 4 °C. In both cases, the standard histological analysis as described previously (59). supernatants were loaded onto previously equilibrated SPE columns Adult X. laevis were purchased from the Centre de Ressources (Strata-X, 30 mg/ml; Phenomenex, Torrance, USA) and eluted in 80% Biologiques Xénopes (University of Rennes, France) and maintained at acetonitrile. Eluates were evaporated to dryness using TurboVap the AQUAB facilities of the Universitat Autònoma de Barcelona (UAB, LVTM (Caliper Life Sciences, PerkinElmer, Inc.), and frozen and Spain). Frogs were kept in tanks with filtrated freshwater at 18°C, under a stored at -20°C until HPLC analysis. Before quantitative analysis, the 12-h light-dark cycle, and fed two days a week with beef heart or pellets dried samples were reconstituted with 0.1 ml of 4% acetic acid and (Xenopus Sticks, AQUA Schwarz GmbH, Göttingen, Germany). Oocytes divided into two aliquots for duplicate analysis. For plasma, samples were collected by surgical laparotomy from anesthetized females. were redissolved in 0.1 ml of acetonitrile:H2O (2:1). For pre-column derivatization, 50 ml of sample and 50 ml of 0.05 M borate buffer (pH 8) were mixed, and then 3 ml of NBD-F (4-fluoro-7-nitro-2,1,3- 4.2 Ligand and receptor nomenclature benzoxadiazole; 30 mg/ml of acetonitrile) was added. The solution was heated at 40˚C for 10 min, cooled on ice, and acidified with 5 ml of The nomenclature used for the seabream vasopressin and oxytocin 1 M HCl. Chromatographic analysis was achieved using Agilent 1200 nonapeptides and corresponding cognate receptors is adopted Series Quaternary HPLC System (Agilent Technology, USA). Peptide according to the Human Genome Nomenclature Committee separations were performed on a Kinetex C18 column (4.6 x 150 mm, 5 (HGNC) (www.genenames.org), the zebrafish information network mm, Phenomenex, USA). A gradient elution system was applied for (ZFIN) (www.zfin.org) and Ensembl (v109) (www.ensembl.org), separation of derivatized peptides. The mobile phase consisted of according to that previously described (28). The specific solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in 3:1 nomenclature of the gilthead seabream Avprs and Oxtrs was acetonitrile:H2O). A linear gradient was 40-65% of eluent B in 20 established through Bayesian inference of two million MCMC min. Flow rate was set at 1 ml/min and the column temperature was set generations (aamodel = mixed; burnin = 25%) of a ClustalX amino to 20˚C. Fluorescence detection was carried out at 530 nm with acid alignment of the seabream Avprs and Oxtrs in relation to 88 excitation at 470 nm. actinopterygian orthologs downloaded from Ensembl (v109) (Supplementary Figure S5). The resultant tree topology matches that computed by maximum likelihood by Daza et al. (27) with the 4.5 Expression constructs exception that the zebrafish Avpr2ba and Avpr2bb receptors are named in accordance with the Ensembl (v109) nomenclature. Full-length cDNAs encoding seabream Aqp1aa, Aqp1ab1, Aqp1ab2, YwhazLa and -zLb have been previously isolated (GenBank accession numbers AY626939, AY626938, MW960021, 4.3 Antibodies and reagents OK572451 and OK572452, respectively). Mutated Aqp1aa, Aqp 1a b1 and Aqp1ab2 constructs in PK A and PK C Affinity-purified rabbit or chicken polyclonal antibodies specific for phosphorylation sites were previously produced (12). Full-length seabream Aqp1aa, -1ab1 and -1ab2 have been described elsewhere (12, cDNAs encoding seabream Avpr1aa, Avpr2aa and Oxtrb were 60). Antisera against Avp and Oxt were generously provided by Prof. isolated by RT-PCR using intestine or rectum total RNA as Olivier Kah (INSERM-Université de Rennes 1, France). The HA tag previously described (18) and oligonucleotide primers designed rabbit antibody was from Thermo Fisher Scientific (Invitrogen, #PA1- based on publicly available sequences (GenBank accession 985), whereas anti-YWHAZ antibodies were purchased from GeneTex numbers KC195974, KC960488 and KC195973, respectively; (#GTX101075) and Signalway Antibody LLC (#SAB40525), which Supplementary Table S1). The cDNAs were subcloned into the specificity for seabream YwhazLa and/or YwhazLb has been validated pT7Ts (61) or pcDNA3 (Invitrogen) expression vectors, and fused elsewhere (12). Synthetic Avp (CYIQNCPRG-NH2) and Oxt with an HA epitope tag (YPYDVPDYA) in the C-terminus before (CYISNCPIG-NH2) were obtained from PeptideSynthetics (Peptide the stop codon by using PCR. The final clones were Sanger Protein Research Ltd.). All other reagents were purchased from Merck sequenced to confirm that the constructs were correct. unless indicated otherwise. 4.6 Pharmacological Avp and Oxt receptor 4.4 HPLC determination of Avp and Oxt characterization content in plasma and ovary Seabream Avp and Oxt receptor cDNA constructs in pcDNA3 Ovarian and plasma levels of Avp and Oxt in vivo were determined were transiently expressed in HEK293T cells (ATCC # CRL-11268). using HPLC with fluorescence detection preceded by solid-phase The cells were grown in 24 wells plates in DMEM supplemented extraction (SPE). Pieces of ovary (150-350 mg) previously frozen and with penicillin/streptomycin, 2 mM glutamine, and 10% fetal stored at -80°C were sonicated in 1 ml of Milli-Q water, acidified with 3 bovine serum (FBS) (Life Technologies) and were incubated at Frontiers in Endocrinology 14 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 37°C in 5% CO2. Receptor activation was measured using luciferase using 0.5 mg oligo(dT)17 primer, 1 mM deoxynucleotide triphosphates reporter gene assays and intracellular Ca2+ levels. At approximately (dNTPs), 40 IU RNAse out (Life Technologies Corp.), and 10 IU 50-60% confluence, cells were transfected using Lipofectamine 3000 SuperScript II Reverse Transcriptase enzyme (Life Technologies Corp.) (Invitrogen) with empty pcDNA3 or Avpr1aa (5 ng), Avpr2aa (5 for 1.5 h at 42°C. The PCR was carried out with 1 ml of the RT reaction ng), or Oxtrb (50 ng) cDNAs, 50 ng of pCRE-luc (Agilent in a final volume of 50 ml containing PCR buffer with Mg2+, 0.2 mM Technologies) or 200 ng of pSRE-luc (BD Biosciences Clontech), dNTPs, 1 IU of Taq polymerase (Roche), and 0.2 mM of forward and and 5 ng or 50 ng of b-Gal plasmid (Promega Corp.) to normalize reverse oligonucleotide primers specific for seabream pro-avp, pro-oxt, the transfection efficiency. The transfected cells were grown for 36 h avpr1aa, avpr2aa and oxtrb (Supplementary Table S1). Reactions were at 37°C in an air/CO2 [95:5 (v/v)] atmosphere before starvation for amplified using one cycle at 95°C for 2 min; 35 cycles at 95°C for 30 sec, 16 h by replacing the DMEM by serum-free DMEM medium. The 60-62°C (depending on primer Tm) for 30 sec, and 1 min at 72°C; and cells were then incubated in triplicate with different concentrations 7 min for final elongation at 72°C. The 18S ribosomal RNA (rps18) was of Avp or Oxt (from 10-5 M to 10-12 M), or sterile dimethyl used as reference gene. The PCR products were run on 1% agarose gels sulfoxide (DMSO) vehicle (0.01% final concentration), for 3 h. In and photographed. some experiments, the cells were incubated with increasing Cellular localization of pro-avp, pro-oxt, avpr1aa, avpr2aa and concentrations (from 10-12 to 10-5 M) of different AVP (SR49059, oxtrb expression in ovarian follicles was carried out by ISH using Merck #S5701; TLV, Merck #T7455) and OXT (L-371,257, Tocris digoxigenin-labelled gene-specific riboprobes as described #2410) receptor antagonists for 1 h prior to stimulation with 10-8 M previously (62). The sense and antisense riboprobes were Avp or Oxt. After incubation, the cells were harvested in lysis buffer synthesized using T7- or SP6-RNA polymerases from a PCR- (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 10% glycerol, 1% amplified template using specific oligonucleotide primers triton X-100), frozen at -80°C for 16-24 h and centrifuged at 14,000 (Supplementary Table S1). The pro-avp and pro-oxt probes × g for 1 min. To determine the luciferase activity, 20 µl of the corresponded to the C-terminal end and part of the 3´UTR, or supernatant was mixed with 100 µl of reconstituted Luciferin the proximal 3´UTR, respectively. For the receptors, the probes (BioThema) in a 96-well plate (Nunc F96 MicroWell Black and spanned ~300 bp from the middle of the avpr2aa cDNA sequence, White Polystyrene Plate, Thermo Fisher Scientific), and the and ~200 bp of the avpr1aa and oxtrb cDNAs including the C- luminescence measured in an Orion II microplate luminometer terminus and a portion of the 3´UTR. (Titertek-Berthold). Luciferase activity was normalized to b-Gal activity measured by colorimetric detection using an Infinite M200 microplate reader (Tecan Group Ltd.). The data was finally 4.8 Functional expression in X. laevis expressed as fold-change of luciferase activity with respect to oocytes and swelling assays control cells exposed to DMSO only. To determine the intracellular Ca2+ levels after receptor activation The cRNAs corresponding to the different constructs were we used the cell permeant Fluo-4-AM vital dye (Life technologies synthesized in vitro with T7 RNA polymerase from XbaI-digested #F14201). For this, HEK293T cells were transfected as above with 2.5 pT7Ts vector containing the cDNAs. The isolation and microinjection µg of Avpr1aa, Avpr2aa or Oxtrb cDNAs in 6-well plates (Thermo of oocytes, and subsequent swelling assays, were carried out as Scientific). After 16 h, the cells were transferred to a black 96-well previously described (63). Oocytes were injected with 50 nl of water microplate and incubated 24 h at 37°C in an air/CO2 [95:5 (v/v)] alone (controls) or containing 15 ng of Avpr1aa, Avpr2aa or Oxtrb atmosphere. Fluo-4 AM was diluted in Hank’s Balanced Salt Solution cRNAs. Oocytes expressing the receptors were also injected with wild- (HBSS) supplemented with glucose, containing 8% NP-40 and 20% type or mutant Aqp1aa (0.1 ng), Aqp1ab1 (0.5 ng) or Aqp1ab2 (25 ng) DMSO, and added to the cells at 5 µM final concentration. Cells were plus YwhazLb (25 ng). In some experiments, oocytes expressing incubated for 1 h at 37°C in an air/CO2 [95:5 (v/v)] atmosphere, and Avpr2aa and Aqp1ab1 constructs were also injected with YwhazLa subsequently exposed in triplicate to the nonapeptides, in the presence (25 ng). The oocyte Pf was determined after 3 h incubation with 10 µM of absence of inhibitors, as described above. After washing twice with Avp or Oxt (depending on the type of receptor expressed in oocytes). fresh DMEM, fluorescence intensity was measured at excitation and The role of PKA or PKC on the oocyte Pf was tested by preincubating emission wavelengths of 494 and 506 nm, respectively, using an Infinite the oocytes with 10 µM of H89 or Bim-II for 1 h prior to the addition of M200 microplate reader (Tecan Group Ltd.). The background signal of the hormones. Oocytes expressing Avpr1aa or Oxtrb together with cells incubated with HBSS without Fluo-4 AM was subtracted from Aqp1ab2 plus YwhazLb were treated with 10 µM FSK as previously each value. described (12). Control oocytes were treated with 0.1% DMSO. 4.7 Gene expression analyses 4.9 In vitro incubation of ovarian explants and isolated ovarian follicles For RT-PCR, total RNA was extracted from different adult tissues, including pituitary, kidney, ovary and isolated ovarian follicles, using Pieces of the ovary at the vitellogenic stage (400-500 mg) were the RNeasy Minikit (Qiagen) and DNAse I treatment, following the dissected from naturally spawning seabream females and placed in manufacturer’s instructions. Total RNA (5 mg) was reverse transcribed Petri dishes containing 75% Leivovitz L-15 culture medium with Frontiers in Endocrinology 15 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 L-glutamine and 100 µg/ml gentamicin at pH 7.5. The explants were were finally washed 3 times in PBST and each well was incubated treated with 100 ng/ml of sea bass (Dicentrarchus labrax) rFsh or with 100 µl/well of EIA grade affinity purified goat anti-rabbit IgG rLh (64), which activate the seabream Fsh and Lh receptors, (H + L) HRP conjugated (Bio-rad Laboratories, Inc.) diluted respectively (59), 0.1 µg/ml 17,20bP, in the presence or absence of 1:3000 in PBST plus 0.5% NGS for 2 h at room temperature. 100 µM of RU486 (Merck M8046), or with 10 µM of OD 02-0 After washing, enzymatic color development was carried out by (Axon Medchem, 13258-85-0), for ~20 h at 18°C in a temperature- the addition of 100 µl/well of tetramethylbenzidine (TMB) controlled incubator. Control groups were treated with an peroxidase EIA substrate kit (Bio-rad Laboratories, Inc.) in equivalent volume of the cell culture medium used for rLh and darkness during 30 min, and the reaction was stopped with 100 rFsh production, or with 0.5% ethanol. After incubation, explants µl/well of 1 N sulfuric acid. Absorbance was read at 450 nm with a were frozen and stored at -80°C until Avp extraction and VICTOR3™ Multilabel Plate Reader (PerkinElmer, Inc.). The determination by ELISA. amount of Avp was normalized to the wet weight of the ovarian In other experiments, groups of fully-grown postvitellogenic explants. The intra-assay coefficient of variation was 4.64% ovarian follicles (n = 10), manually isolated from the ovary, were (calculated by assaying eight replicates of an ovarian sample at incubated with 10 µM Avp in the same culture medium as above, in 50% of maximum binding on the same plate), whereas the inter- the presence or absence of 10 µM of the receptor antagonist TLV, or assay coefficient of variation was 8.35%. The sensitivity of the with 0.1% DMSO (controls) for 6 h at 18°C. After incubation, assay for Avp was of 10 pg/ml, while its crossreaction with Oxt was follicles were fixed for immunofluorescence microscopy, frozen in calculated to be 8.43 ± 1.72%. liquid nitrogen and stored at -80°C for further protein extraction, or processed for water uptake assays using radiolabeled water as previously described (12). 4.11 Immunofluorescence microscopy Isolated ovarian follicles were processed for immunofluorescence 4.10 ELISA determination of Avp in treated microscopy as previously described (65, 66). Control sections were ovarian explants in vitro incubated with antibodies (1:400 dilution) preadsorbed with the corresponding peptides used for immunization (for aquaporin To determine the Avp levels in ovarian explants incubated in antibodies), or with the Avp or Oxt synthetic peptides. Secondary vitro, a competitive ELISA was developed using the specific anti- antibodies were sheep anti-rabbit IgG Cy3 (Merck #C2306) or goat Avp antiserum and the synthetic Avp to create the standard curve anti-chicken IgY Alexa Fluor 488 (Invitrogen #A11039) at a 1:1,000 (Supplementary Figure S3). For this, polystyrene ELISA microtiter dilution. Sections were counterstained with DAPI (1:10,000), and plates (F96 Maxisorp Nunc Immuno Plates; Nunc) were coated mounted with Fluoromount™ Aqueous Mounting Medium (Merck with 100 µl/well of 50 ng Avp/ml carbonate buffer (50 mM sodium #F4680). Immunofluorescence was observed and documented with a carbonate, pH 9.6) and incubated overnight at 4°C. For the blank Zeiss Axio Imager Z1/Apotome fluorescent microscope (Carl Zeiss and the non-specific binding, two wells were either not coated or Corp.). Changes in Aqp1ab1 and Aqp1ab2 in the oocyte plasma coated with 100 µl/well of 50 ng BSA/ml carbonate buffer, membrane after Avp treatment were evaluated using the ImageJ respectively. Ovarian explants were homogenized on ice with a software v.1.52o (https://imagej.nih.gov/ij/) as previously glass dounce tissue homogenizer in water containing 0.25% glacial described (12). acetic acid in a proportion of 500 µl/100 mg tissue. After incubation of the homogenate at 100°C for 5 min, the samples were centrifuged a 10,000 × g for 5 min at 4°C, and the 4.12 Protein extraction and supernatant recovered in a new tube. The standards (0.01-500 immunoblotting ng Avp/ml) and experimental samples were diluted in PBST (PBS plus 0.05% Tween 20) with 0.5% normal goat serum (NGS), The total and plasma membrane fractions of X. laevis oocytes containing the Avp antibody diluted at 1:15,000, and were pre- (n = 10), and total membrane fractions of seabream ovarian follicles incubated overnight at 4°C. Displacement curves for ovarian (n = 50), were isolated as described previously (12) and resuspended samples were obtained by serial dilutions from 1:2 to 1:16 to in Laemmli sample buffer. For immunoblotting, Laemmli-mixed confirm the parallelism with the standard curves. For the protein samples were denatured at 95°C for 10 min and subjected to measurement of the experimental samples a 1:4 dilution was 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis applied, and to evaluate the cross-reactivity of the assay with (SDS-PAGE) and blotted onto Immun-Blot nitrocellulose 0.2 mm Oxt, the same standard curve as for Avp was created (0.01-500 ng/ membranes (Bio-Rad Laboratories, Inc.), as described previously ml). All standards and samples were processed in duplicate. After (67). Membranes were blocked with 5% nonfat dry milk in TBST coating, the plates were washed 3 times with 200 µl/well of PBST, (20 mM Tris, 140 mM NaCl, 0.1% Tween, pH 8) for 1 h at room and blocked with 200 µl/well of PBST buffer containing 2% NGS temperature, and subsequently incubated overnight at 4°C with the for 1 h at room temperature. Then, 100 µl of samples and seabream-specific Aqp1aa, Aqp1ab1 or Aqp1ab2 antisera, or HA standards preincubated with the antibody were added to each antibodies, diluted (1:1,000) in TBST with 5% milk. Bound well, and plates were incubated at 4°C for another 24 h. The plates antibodies were detected with horseradish peroxidase-coupled Frontiers in Endocrinology 16 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 anti-rabbit (Bio-Rad Laboratories, Inc., #172-1019) or anti-chicken Union Council Guidelines (86/609/EU). The procedure for surgical (Thermo Fisher Scientific, #A16054) secondary antibodies diluted laparotomy of female frogs was approved by the Ethics Committee 1:5000 as above, and reactive protein bands were revealed using for Animal and Human Experimentation from UAB and the ImmobilonTM Western chemiluminescent HRP substrate Catalan Government (Direcció General de Polı́tiques Ambientals (Millipore, #WBKLS). i Medi Natural; Project no. 10985). 4.13 Co-immunoprecipitation Author contributions X. laevis oocytes and seabream follicles (n = 10) were homogenized Conceptualization: JC. Investigation: AF, FC and MG. in the lysis buffer from the Pierce™ Crosslink Magnetic IP/Co-IP Kit Formal analysis: AF, FC, MG, EK, RF and JC. Supervision: JC (Pierce Thermo Fisher Scientific, #88805) with a bouncer on ice, and and EK. Funding acquisition: JC and RF. Visualization: JC. centrifuged at 14,000 × g for 1 min. An aliquot (10%) of the protein Writing-original draft preparation: AF. Writing- review & extract was collected as “input” and mixed with 2 × Laemmli sample editing: JC, RF and EK. All authors approved the submitted buffer, whereas the remaining extract was incubated overnight at 4°C version of the manuscript. under constant agitation with magnetic beads previously coated with anti-Aqp1ab1 or anti-Aqp1ab2 antibodies. For rabbit primary antibodies, the immunoprecipitation was carried out using the kit Funding indicated above, and final eluted proteins in 50 µl of elution buffer were mixed with 4 × Laemmli sample buffer supplemented with protease This work was supported by the Spanish Ministry of Science and phosphatase inhibitors. For chicken antibodies, the and Innovation (MCIN/AEI/10.13039/501100011033), the immunoprecipitation was performed using Dynabeads™ M-280 European Regional Development Fund (ERDF) “A way of making Tosylactivated (Invitrogen Thermo Fisher Scientific, #14203) Europe” (European Union), Grant no. AGL2016-76802-R (to JC), following the manufacturer’s instructions. The input and and the Norwegian Research Council (RCN) Grant no. 294768/E40 immunoprecipitated samples were immunoblotted as indicated above. (to RF). AF was recipient of a predoctoral contract from Spanish MCIN (BES-2014-068745). RF was also supported by the University of Bergen (Norway). 4.14 Statistical analyses Comparisons between two independent groups were made by the Conflict of interest two-tailed unpaired Student’s t-test. The statistical significance among multiple groups was analyzed by one-way ANOVA, followed by the The authors declare that the research was conducted in the Tukey’s multiple comparison test, or by the non-parametric Kruskal- absence of any commercial or financial relationships that could be Wallis test and further Dunn’s test for nonparametric post hoc construed as a potential conflict of interest. comparisons. Percentages were square root transformed previous analyses. Statistical analyses were carried out using the GraphPad Prism v9.1.2 (226) (GraphPad Software). Publisher’s note All claims expressed in this article are solely those of the authors Data availability statement and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the The original contributions presented in the study are included reviewers. Any product that may be evaluated in this article, or in the article/Supplementary Material. Further inquiries can be claim that may be made by its manufacturer, is not guaranteed or directed to the corresponding author. endorsed by the publisher. Ethics statement Supplementary material The animal study was reviewed and approved by Procedures The Supplementary Material for this article can be found online at: relating to the care and use of fish and sample collection were https://www.frontiersin.org/articles/10.3389/fendo.2023.1222724/ approved by the Ethics Committee of IRTA, following the European full#supplementary-material Frontiers in Endocrinology 17 frontiersin.org Ferré et al. 10.3389/fendo.2023.1222724 References 1. Huang HF, He RH, Sun CC, Zhang Y, Meng QX, Ma YY. Function of aquaporins 25. Mayasich SA, Clarke BL. The emergence of the vasopressin and oxytocin in female and male reproductive systems. Hum Reprod Update (2006) 12(6):785–95. hormone receptor gene family lineage - Clues from the characterization of vasotocin doi: 10.1093/humupd/dml035 receptors in the sea lamprey (Petromyzon marinus). Gen Comp Endocrinol (2016) 2. Cerdà J, Chauvigné F, Finn RN. 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