3ba282b8750923c3dffc83a0a3eba4bd pmc_11395232.pdf 5d80fffc5d6d3d882ed5f948717fc762ecc5c4bd pmc_11395232.pdf 0e2c49357e695896e8eba8386c606278f3f16ab38f6871eb044b4acc3b78408e pmc_11395232.pdf Title: Aquaporin-3a Dysfunction Impairs Osmoadaptationin Post-Activated Marine Fish Spermatozoa Subject: 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 (-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 -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. Keywords: fish; sperm motility; volume regulation; DFP00173 Author: François Chauvigné, Júlia Castro-Arnau, Noelia López-Fortún, Alejandro Sánchez-Chardi, Michael Rützler, Giuseppe Calamita, Roderick Nigel Finn and Joan Cerdà Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Sep 4 16:29:20 2024 CEST ModDate: Wed Sep 4 16:41:28 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2476634 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IUYWSY+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 16 0 WCSEWR+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 NWQUPR+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 DCYZWS+URWPalladioL-Ital Type 1 Custom yes yes yes 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of Seabream Aqp3a Channel Function Destination: subsection.2.2 Item: Title: Aqp3a Inhibition Impairs Seabream Sperm Motility Destination: subsection.2.3 Item: Title: Aqp3a Dysfunction Induces Tail Deformations in Post-Activated Spermatozoa Destination: subsection.2.4 Item: Title: A Role of Aqp3a during Seabream Sperm Osmoadaptation Destination: subsection.2.5 Item: Title: Materials and Methods Destination: section.3 Children: Item: Title: Animals and Sample Collection Destination: subsection.3.1 Item: Title: Chemicals and Antibodies Destination: subsection.3.2 Item: Title: Oocyte Swelling Assays Destination: subsection.3.3 Item: Title: Sperm Motility Measurements Destination: subsection.3.4 Item: Title: Protein Extraction and Immunoblotting Destination: subsection.3.5 Item: Title: Immunofluorescence Microscopy Destination: subsection.3.6 Item: Title: Electron Microscopy Destination: subsection.3.7 Item: Title: Statistical Analysis Destination: subsection.3.8 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: appendix.A. Info: Title: Aquaporin-3a Dysfunction Impairs Osmoadaptationin Post-Activated Marine Fish Spermatozoa Author: François Chauvigné, Júlia Castro-Arnau, Noelia López-Fortún, Alejandro Sánchez-Chardi, Michael Rützler, Giuseppe Calamita, Roderick Nigel Finn and Joan Cerdà Subject: 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 (-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 -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. 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