Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca 2+ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon.

Nanoplastics pollution is a rising environmental concern whose impacts on biodiversity and human health are far from being understood. This is particularly salient in aquatic ecosystems, where the majority of species depend on external fertilization for reproduction. Here we evaluated the effects of a short-term exposure to engineered polystyrene nanoplastics (NPs) in the zebrafish germline to further explore their impact on reproduction. To this end, zebrafish (Danio rerio) were exposed to 5 mg/L of 45 nm polystyrene (PS)-NPs via water for 96 h. We show that, in males, nanoplastics induced testicular histological alterations with abnormal sperm clustering and chromatin compaction, resulting in viable spermatozoa but with reduced motility. Moreover, in females we observed an alteration in oocyte stages frequencies during oogenesis, possibly reflecting alterations in oocyte growth. RNA-sequencing analysis in male testis links nanoplastic induced alterations in the expression of genes involved in chromatin structure, meiosis and DNA double-strand break formation and repair progression, and gametes recognition. Flow cytometry analysis revealed that the observed effects in males were directly due to nanoplastics penetrating the testicular barrier and being internalized within germline cells. Overall, our results demonstrate that acute exposure to NPs can compromise reproductive fitness, underscoring the environmental and health impacts of NPs pollution.

Translational silence of spermatozoa has long been considered the norm in animals. However, studies in mammals have shown that the mitochondrial ribosomal machinery is selectively activated during capacitation in the female reproductive tract, while cytosolic ribosomes remain inactive. Here, using quantitative proteomics in a piscine model species, we show that proteins involved in mRNA processing and cytoplasmic translation are predominantly accumulated in immature spermatozoa within the extratesticular excurrent ducts, while those related to flagellar motility are enriched in ejaculated (mature) sperm. Based upon in vitro incubation of isolated spermatozoa, motility assays and polysome profiling, we further show that 80S cytoplasmic and 55S mitochondrial ribosomes are actively involved in the translation of motility- and osmoadaptation-related proteins. These findings thus reveal that post-testicular piscine spermatozoa can maintain de novo protein synthesis through both mitochondrial and cytoplasmic ribosomal activity, which is necessary for the acquisition of full sperm function.

Special Issue New Insights into Aquaporins: 2nd Edition.-- 18 pages, 7 figures, supplementary information https://www.mdpi.com/article/10.3390/ijms27093937/s1.-- Data Availability Statement: All relevant data can be found within the article and its supplementary information; DIGITAL.CSIC https://doi.org/10.20350/digitalCSIC/18331, AQP8-type water channels are expressed superficially in the plasma membrane or intracellularly in the inner mitochondrial membrane, where they respectively function in osmohomeostasis or as peroxiporins to alleviate oxidative stress. To date only single-copy AQP8 or AQP16 genes are known in tetrapods and two binary gene clusters composed of aqp8aa-aqp8ab and aqp8ba-aqp8bb in teleost fishes. Here, using phylogenomic and synteny analyses, we revise this view and show that bony fish aqp8aa, -ab, -ba and -bb genes are non-canonical co-orthologs that independently arose at chromosomal breakpoints. Conversely, canonical orthologs of tetrapod AQP8 are now detected in all vertebrate classes except hagfishes. In cartilaginous fishes, intact aqp8 orthologs and linked pseudogenes exist in squalomorph sharks and only fractionated aqp8-like pseudogenes in galeomorph sharks. Some isolated aqp8-like exons are detected in batoid ray genomes, while no aqp8-type coding sequences are currently found in holocephalan genomes. In the actinopterygian (ray-finned fish) lineage, the canonical ortholog of tetrapod AQP8 is estimated to have undergone gene translocation in their common ancestor ~400 million years ago but was subsequently inactivated or lost in many descendant lineages. In close temporal proximity to this gene translocation event, the actinopterygian aqp8aa-aqp8ab binary gene cluster was generated in the original syntenic locus, potentially as a result of meiotic recombination. Our data support a model of total chromosomal replication for the generation of AQP16 genes and the teleost aqp8ba-aqp8bb gene cluster. We further uncover additional duplicates in Strepsirrhini primates that provide an eminent example of the stochastic nature of neofunctionalization. The present data thus suggest that that common ancestral genome duplications combined with lineage-level chromosomal translocation, recombination and replication events contributed to the diversification of vertebrate AQP8-type genes, R.N.F. was supported by the University of Bergen (Norway). This work was also sup-ported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033), Grant no. PID2022-138066OB-I00 (to J.C.), and the Agency for Management of University and Research Grants (Government of Catalonia), Grant no. 2021 SGR 00068 (to J.C.). The authors also acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI) 10.13039/501100011033, Peer reviewed

R.N.F. was supported by the University of Bergen (Norway). This work was also sup-ported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033), Grant no. PID2022-138066OB-I00 (to J.C.), and the Agency for Management of University and Research Grants (Government of Catalonia), Grant no. 2021 SGR 00068 (to J.C.). The authors also acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI) 10.13039/501100011033, Peer reviewed

10 pages, 4 figures, supporting information https://doi.org/10.1073/pnas.2537571123.-- Data, Materials, and Software Availability: RNA sequencing, whole genome sequence, and assembly data have been deposited in National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (GSE313210) (50) and BioProject (PRJNA1393770) (57). All other data are included in the manuscript and/or supporting information, Neural crest-derived cells offer valuable opportunities to dissect mechanisms of cell fate specification and differentiation and the underpinnings of cell type diversification over evolutionary time. Particularly useful for such analyses are pigment cells of ectothermic vertebrates that arise from neural crest cells or via latent neural crest-derived stem cells. Among these are white cells, leucophores, present in a variety of species that contribute to patterns on the body or ornamentation on the fins. To better understand developmental and evolutionary origins of these cells, we examined leucophores harboring deposits of yellow/orange carotenoids-xantholeucophores-of zebrafish and leucophores of white cloud minnow. We show that white phenotypes of both cell types require sepiapterin reductase and an accumulation of pale and colorless pteridines. We further demonstrate that xantholeucophores of zebrafish develop from yellow, sepiapterin-rich xanthophore-like cells and that this transition requires both gap junctional communication and the aquaglyceroporin/peroxiporin channel Aquaporin 3, revealing similarities and differences in differentiation and patterning compared to pigment cells on the body. These findings identify xantholeucophores of zebrafish and leucophores of white cloud minnow as distinct developmentally, genetically, and biochemically from other white cells of zebrafish-melanoleucophores-that develop directly from melanophores and depend on guanine crystals, as well as white cells of medaka fish and anemonefish. Our results highlight remarkable convergences and parallelisms in the acquisition of white cell phenotypes within and between phylogenetic lineages and identify this as a rich system for enquiries into the evolutionary individuation of novel cell types, This work was supported by NIH R35 GM122471 (to D.M.P.), with support for Aqp3 channel analyses from Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) and the European Regional Development Fund (European Union) Grant No. PID2022-138066OB-I00 to J.C, This work contributes to the Institut de Ciències del Mar "Severo Ochoa Centre of Excellence" accreditation CEX2024-001494-S funded by AEI 10.13039/501100011033 of the Spanish Ministry of Science and Innovation, Peer reviewed

Dataset S1. Differential expression analysis from bulk RNA-Seq of XL vs ~X (Fig. 3A).
Dataset S2. Enrichment for signaling and pigment synthesis pathways from bulk RNA-Seq of XL vs. ~X (Fig. 3A).
Dataset S3. Gene enrichment analyses (GO terms, KEGG pathways; Fig. 3A).
Dataset S4. Pteridine pathway summary; pteridine synonyms and properties (Fig. 4A) (refs. 6, 13, 22, 41-57).
Dataset S5. LC-MS compound discovery (rgmb, rgmb/+; ltk-/- zebrafish; Fig. 4B, SI Appendix, Fig. S8C).
Dataset S6. Multivariate analysis of LC-MS compound discovery data, one-tailed tests (rgmb, rgmb/+; ltk-/- zebrafish; Fig. 4B).
Dataset S7. Multivariate analysis of LC-MS compound discovery data, two-tailed tests (rgmb, rgmb/+; ltk-/- zebrafish; SI Appendix, Fig. S8C).
Dataset S8. LC-MS standards-verified pteridines (rgmb, rgmb/+; ltk-/- zebrafish; Fig. 4C).
Dataset S9. LC-MS compound discovery for wild-type zebrafish juvenile (14 SSL; SI Appendix, Fig. S8D).
Dataset S10. Cell-type contrasts for LC-MS compound discovery data from wild-type zebrafish juvenile (14 SSL; SI Appendix, Fig. S8D).
Dataset S11. LC-MS compound discovery for spra-/- mosaic vs. wild-type zebrafish (Fig. 4D, SI Appendix, Fig. 8F).
Dataset S12. Analysis of LC-MS compound discovery data for spra-/- mosaic zebrafish vs. wild type (Fig. 4D, SI Appendix, Fig. 8F).
Dataset S13. LC-MS compound discovery for spra-/- mosaic Tanichthys vs. wild-type (Fig. 4D, SI Appendix, Fig. 8F).
Dataset S14. Analysis of LC-MS compound discovery data for spra-/- mosaic zebrafish vs. wild type (Fig. 4D, SI Appendix, Fig. 8F).
Dataset S15. LC-MS guanine and urate in Tanichthys fin samples (SI Appendix, Fig. 8E), This work was supported by NIH R35 GM122471 (to D.M.P.), with support for Aqp3 channel analyses from Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) and the European Regional Development Fund (European Union) Grant No. PID2022-138066OB-I00 to J.C, This work contributes to the Institut de Ciències del Mar "Severo Ochoa Centre of Excellence" accreditation CEX2024-001494-S funded by AEI 10.13039/501100011033 of the Spanish Ministry of Science and Innovation, Peer reviewed

16 pages, 5 figures, supplementary materials https://www.mdpi.com/article/10.3390/ijms25179604/s1.-- Data Availability Statement: All related data are included in either the manuscript or Supplementary Information. Other data are available from the corresponding author upon request, Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca2+ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon, This work was supported by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033) and FEDER “A way of making Europe”, European Union, Grants no. AGL2016-76802-R and PID2022-138066OB-I00 (to J.C.). J.C.-A. was supported by a predoctoral contract from Spanish MCIN (BES-2017-080778). R.N.F was supported by the University of Bergen. This work also acknowledges the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S) funded by AEI 10.13039/501100011033, Peer reviewed

12 pages, 6 figures, supplementary material https://doi.org/10.1016/j.jhazmat.2024.136529.-- Data availability: RNA-sequencing data generated during this study can be found at NCBI Gene Expression Omnibus (GEO) under accession number GSE279916, Nanoplastics pollution is a rising environmental concern whose impacts on biodiversity and human health are far from being understood. This is particularly salient in aquatic ecosystems, where the majority of species depend on external fertilization for reproduction. Here we evaluated the effects of a short-term exposure to engineered polystyrene nanoplastics (NPs) in the zebrafish germline to further explore their impact on reproduction. To this end, zebrafish (Danio rerio) were exposed to 5 mg/L of 45 nm polystyrene (PS)-NPs via water for 96 h. We show that, in males, nanoplastics induced testicular histological alterations with abnormal sperm clustering and chromatin compaction, resulting in viable spermatozoa but with reduced motility. Moreover, in females we observed an alteration in oocyte stages frequencies during oogenesis, possibly reflecting alterations in oocyte growth. RNA-sequencing analysis in male testis links nanoplastic induced alterations in the expression of genes involved in chromatin structure, meiosis and DNA double-strand break formation and repair progression, and gametes recognition. Flow cytometry analysis revealed that the observed effects in males were directly due to nanoplastics penetrating the testicular barrier and being internalized within germline cells. Overall, our results demonstrate that acute exposure to NPs can compromise reproductive fitness, underscoring the environmental and health impacts of NPs pollution, This work was supported by the Spanish Ministry of Science and Innovation (PID2020-112557GB-I00 founded by AEI/10.13039/501100011033 to A.R.-H.; PID2021-126710OB-C21 to N.R.A; and PID2022-138066OB-I00 to J.C. and PID2020-113221RB-I00 to M.T), the Agència de Gestió d′Ajuts Universitaris i de Recerca, AGAUR (2021SGR00122 to A.R.-H. and 2021SGR00068 to J.C) and the Catalan Institution for Research and Advanced Studies (ICREA) to A.R.-H. G.P. and L.A.-G. were supported by FPI predoctoral fellowships from the Ministry of Economy and Competitiveness (PRE-C-2021–0083 and PRE-2018–083257, respectively). L.M.-G. was supported by an FPU predoctoral fellowship from the Ministry of Science, Innovation and University (FPU18/03867). MT was supported by a Ramon y Cajal contract (ref. RYC2019–026841-I), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Peer reviewed

24 pages, 10 figures, 1 table, supplemental information https://doi.org/10.1016/j.isci.2024.111537, Translational silence of spermatozoa has long been considered the norm in animals. However, studies in mammals have shown that the mitochondrial ribosomal machinery is selectively activated during capacitation in the female reproductive tract, while cytosolic ribosomes remain inactive. Here, using quantitative proteomics in a piscine model species, we show that proteins involved in mRNA processing and cytoplasmic translation are predominantly accumulated in immature spermatozoa within the extratesticular excurrent ducts, while those related to flagellar motility are enriched in ejaculated (mature) sperm. Based upon in vitro incubation of isolated spermatozoa, motility assays and polysome profiling, we further show that 80S cytoplasmic and 55S mitochondrial ribosomes are actively involved in the translation of motility- and osmoadaptation-related proteins. These findings thus reveal that post-testicular piscine spermatozoa can maintain de novo protein synthesis through both mitochondrial and cytoplasmic ribosomal activity, which is necessary for the acquisition of full sperm function, This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) and the European Regional Development Fund (ERDF) (European Union), Grants no. AGL2016-76802-R and PID2022-138066OB-I00 (to J.C.), and by the Agency for Management of University and Research Grants (Government of Catalonia) Grant 2021 SGR 00068 (to J.C.). J.C.-A. was recipient of a predoctoral contract from Spanish MICIU (BES-2017-080778), and RNF. was supported by the University of Bergen (Norway). The authors also acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI) 10.13039/501100011033, Peer reviewed

9th International Workshop on the Biology of Fish Gametes, 15-18 July 2024, León, Spain, Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3 and -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses. In contrast to mammals, the ejaculated spermatozoa of marine teleosts experience a high hypertonic shock in seawater, resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. However, the potential involvement of other aquaporins in cell volume regulatory mechanisms in post-activated marine spermatozoa are unknown. Using the gilthead seabream (Sparus aurata) as a model of marine teleosts and a paralog-specific antibody, we show that the AQP3 ortholog Aqp3a is highly accumulated in the mid posterior region of the spermatozoon flagella. Aqp3a-mediated water conductance is specifically inhibited by a recently developed AQP3 antagonist (DFP00173) as well as by the Aqp3aspecific seabream antibody (¿-Aqp3a) when expressed in Xenopus laevis oocytes. Both DFP00173 and ¿-Aqp3a impair post-activated sperm motility kinetics (% motility, % progressivity, and curvilinear velocity) with respect to the controls at 30 s post activation in a dose-dependent manner. Interestingly, in a close resemblance to the phenotypes of AQP3-deficient murine sperm, field emission scanning and transmission electron microscopy revealed that both Aqp3a inhibitors induce an abnormal sperm tail morphology, including local swelling and tail angulation, with complete coiling of the flagella in some cases. These data suggest that Aqp3a may function as an osmosensor to regulate cell volume under a high hypertonic stress, or that the channel is required for the efflux of specific solutes which can inhibit sperm motility, Supported by the Spanish Ministry of Science, Innovation and Universities (PID2022-138066OB-I00 to J.C., BES-2017-080778 to J.C-A., and CEX2019-000928-S to ICM), Peer reviewed