Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Int J Mol Sci
2024 Sep 04;2517:. doi: 10.3390/ijms25179604.
Show Gene links
Show Anatomy links
Aquaporin-3a Dysfunction Impairs Osmoadaptation in Post-Activated Marine Fish Spermatozoa.
Chauvigné F
,
Castro-Arnau J
,
López-Fortún N
,
Sánchez-Chardi A
,
Rützler M
,
Calamita G
,
Finn RN
,
Cerdà J
.
???displayArticle.abstract??? 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 spermtail 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.
AGL2016-76802-R and PID2022-138066OB-I00 Ministerio de Ciencia, Innovación y Universidades, BES-2017-080778 Ministerio de Ciencia, Innovación y Universidades
Figure 1. Aqp3a protein expression is upregulated during seabream sperm maturation. (A–D) Bright field (BF, left) and epifluorescence images (right) of Aqp3a localization in seabream intratesticular sperm (A), sperm from the efferent duct (SPZED), ejaculated sperm (SPZEJ) diluted in non-activating medium (NAM; immotile) and activated sperm in seawater (SW; motile), using a seabream-specific Aqp3a chicken antibody (α-SaAqp3a). In (A), sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and fluorophore-coupled lectin wheat germ agglutinin (WGA; red), whereas in (B–D) the sections were stained with DAPI alone. The right panels in (A–D) show control sections incubated with the primary antibody preadsorbed by the antigenic peptide to test for specificity. In (A), the arrows indicate the spermatozoa, while in (B–D) the arrows indicate the spermatozoon flagella. Scales bars: 10 µm (A), 5 µm (B–D). (E) Immunoblot of protein extracts from immotile and activated sperm probed with α-SaAqp3a. The right panel shows a duplicate blot that was run in parallel and incubated with the preabsorbed antisera. The asterisks indicate potential post-translational modifications of the Aqp3a channel. (F) Immunoblot of Aqp3a in SPZED and SPZEJ from three different male fish. Prohibitin (Phb) was used as loading control. In (E,F), molecular mass markers (kDa) are on the left. (G) Densitometric analysis of Aqp3a accumulation in spermatozoa normalized to Phb. Data (mean ± SEM; n = 3 males) were statistically analyzed by the unpaired Student’s t-test (*, p < 0.05). Uncropped blots are shown in Supplementary Figure S1.
Figure 2. The DFP00173 specifically inhibits seabream Aqp3a (SaAqp3a) water conductance in X. laevis oocytes. (A) Inhibition of Pf of X. laevis oocytes noninjected (Uninj, light blue) or expressing SaAqp3a (blue) and exposed to 0.5% DMSO alone or containing increasing concentrations of DFP00173. Oocytes expressing human (HsAQP3) were used as positive controls (red). (B) Effect of DFP00173 (10 µM) or vehicle (0.5% DMSO) on the Pf of noninjected oocytes or expressing different seabream aquaporins that are expressed in the seabream spermatozoa. In (B,C) data are the mean ± SEM (n indicated above each bar) and were statistically analyzed by one-way ANOVA or an unpaired Student’s t-test, respectively (**, p < 0.01; ***, p < 0.001; with respect to oocytes not exposed to the inhibitor, or as indicated in brackets). ns, statistically not significant. (C) Immunolocalization of SaAqp3a in noninjected oocytes and oocytes expressing SaAqp3a and treated or not with DFP00173 (10 µM). The plasma membrane, indicated by an arrow, was stained with wheat germ agglutinin (WGA; green). Scale bars, 10 µm.
Figure 3. Immunological inhibition of seabream Aqp3a in X. laevis oocytes. (A) Water permeabilities of uninjected oocytes or oocytes expressing SaAqp3a in the presence of 0.5% DMSO alone (light blue) or with increasing amounts of the seabream Aqp3a-specific antibody (α-SaAqp3a, blue). Immunoglobulin Y (IgY) was used as the negative control (red). The data are presented as the mean ± SEM (n indicated above each bar) and were statistically analyzed by one-way ANOVA (***, p < 0.001, with respect to non-treated Aqp3a-expressing oocytes). (B) Immunolocalization of SaAqp3a in noninjected oocytes or expressing SaAqp3a and treated or not with 200 nM of α-SaAqp3a. Sections from Uninj oocytes and oocytes expressing SaAqp3a and not treated with the α-SaAqp3a, were probed with the primary (α-SaAqp3a) and secondary (α-IgY) antibodies, whereas oocytes treated with the α-SaAqp3a antibody in vitro were probed only with the secondary α-IgY. The plasma membrane (arrows) was stained with wheat germ agglutinin (WGA; green). The arrowheads indicate partial cytoplasmic retention of Aqp3a in the presence of the antibody. Scale bars, 10 µm.
Figure 4. Inhibition of Aqp3a impairs seabream sperm motility. (A–F) Dose-response inhibition of the percentage of motility and progressivity (% MOT and % PROG, respectively) and curvilinear velocity (VCL) at 5, 30 and 60 s post activation induced by DFP00173 (A–C) and the seabream Aqp3a antibody (α-SaAqp3a) (D–F). Control spermatozoa were treated with 0.5% DMSO (vehicle) or 200 nM IgY. In all panels, the data (n = white dots above each bar, corresponding to one ejaculated per male) are presented as the mean ± SEM. Statistical differences within each time point were measured by one-way ANOVA, or Kruskal-Wallis test, followed by Dunn’s multiple comparisons test (*, p < 0.05; **, p < 0.01; ***, p < 0.001, with respect to DMSO-treated sperm). (G) Sperm treated with IgY or α-SaAqp3a, further activated for ~30 s, and subsequently stained only with anti-chicken secondary antibodies, confirmed the specific binding of α-SaAqp3a to its target protein (green) in the spermatozoon flagellum (arrows). The nuclei of the spermatozoa were counterstained with DAPI (blue). Scale bars, 5 µm.
Figure 5. Chemical and immunological inhibition of Aqp3a induces abnormal spermtail morphology. (A–B) Representative immunostaining of α-tubulin in seawater-activated spermatozoa treated with vehicle (0.5% DMSO), 20 µM DFP00173, or 200 nM IgY or α-SaAqp3a at ~30 s post activation time, showing that both Aqp3a inhibitors generate spermtail bending and local swelling in the spermatozoon. Scale bars, 5 µm. (C) Percentage of spermatozoa showing spermtail structural defects. Data (n = white dots above each bar, corresponding to one ejaculated per male) are presented as the mean ± SEM, and were statistically analyzed by an unpaired Student t-test (***, p < 0.001, with respect to vehicle- or IgY-treated sperm). (D,E) Field emission scanning electron microscope (FESEM; (D1–17)) and transmission electronic microscopy (TEM, (E1–7)) revealed that activated sperm treated with α-SaAqp3a show expanded intracellular space in the flagella compared with the IgY-exposed sperm, as well as a progressive volume expansion and tail bending. In some cases, the spermtail also becomes strongly coiled. Scale bars are indicated in each panel.
