Aquaporin-Mediated Water and Hydrogen Peroxide Transport Is Involved in Normal Human Spermatozoa Functioning

Abstract

Different aquaporins (AQPs) are expressed in human sperm cells and with a different localization. Their function has been related to cell volume control in response to the osmotic changes encountered passing from the epididymal fluid to the cervical mucus or involved in the end stage of cytoplasm removal during sperm maturation. Recently, AQPs have also shown hydrogen peroxide (H₂O₂) permeability properties. Here, we investigate the expression, localization and functioning of AQPs in human sperm cells with particular attention to their role as peroxiporins in reactive oxygen species (ROS) scavenging in both normospermic and sub-fertile human subjects. Western blotting and immunocytochemistry were used to confirm and clarify the AQPs expression and localization. Water and H₂O₂ permeability was tested by stopped flow light scattering method and by the CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate, acetyl ester) H₂O₂ fluorescence probe, respectively. AQP3, -7, -8, and -11 proteins were found in human sperm cells and localized in the head (AQP7), in the middle piece (AQP8) and in the tail (AQP3 and -11) in both the plasma membrane and in intracellular structures. Sperm cells showed water and H₂O₂ permeability which was reversibly inhibited by H₂O₂, heat stress and the AQP inhibitor HgCl₂. Reduced functionality was observed in patients with compromised basal semen parameters. Present findings suggest that AQPs are involved in both volume regulation and ROS elimination. The relationship between sperm number and motility and AQP functioning was also demonstrated.

Keywords: aquaporins-7; aquaporins-8; oxidative stress; sperm motility; sterility; water channel.

Figure 1

Aquaporin-3 (AQP3), -7 (AQP7), -8 (AQP8) and -11 (AQP11) protein expression in human ejaculated semen from normospermic subjects. Blots representative of three were shown. Lanes were loaded with 40 μg of proteins, probed with anti-AQP3, -7, -8 and -11 rabbit polyclonal antibodies and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-beta-2-microglobulin (B2M) polyclonal antibody, as housekeeping. Major bands of about 31 kDa (monomer) and 62 kDa (dimer) were observed.

Figure 2

Immunocytochemical localization of the AQP3, -7, -8 and -11 proteins in human ejaculated semen from normospermic subjects: (A) AQP3 immunoreactivity was observed in the principal piece of the sperm tail membrane and in 3% of sperms in granules present in the head and in the midpiece (arrowhead); (B) intense AQP7 staining was observed in the plasma membrane region of the sperm head; (D) AQP8 labeled the midpiece of the spermatozoa, apparently in the mitochondria; and (E) AQP11 protein was localized into granules and vesicles of soma (arrowheads) and in the tail. Controls in which the primary antibody was substituted with non-immune serum show an absence of labeling (Neg.; (C)). Scale bar, 10 μm.

Figure 3

Representative traces of stopped-flow osmotic water permeability measurements obtained from human ejaculated semen of normospermic (A,C) and sub-fertile (B,D) subjects. Sperm cells were exposed to a 150 mOsm osmotic gradient in two different conditions: untreated cells (Control; (A,B)) and cells treated for 45 min with 50 μM H₂O₂ to induce an oxidative stress condition (H₂O₂-treated; (C,D)). k relative values of single curves are also shown.

Figure 4

Effect of oxidative stress on the water permeability of human ejaculated semen. (A) Effect of hydrogen peroxide (H₂O₂). Sperm cells were exposed to a 150 mOsm osmotic gradient in two different conditions: untreated cells (Control) and cells treated for 45 min with 50 μM H₂O₂ to induce an oxidative stress condition. The H₂O₂-sensitive water permeability was obtained by subtracting H₂O₂ insensitive water permeability from total water permeability. a, p < 0.05 vs. normospermic (Student’s t test); b, p < 0.05 vs. Control and c, p < 0.05 vs. H₂O₂-sensitive (ANOVA, followed by Newman–Keuls’s Q test); (B) Temperature dependence: The osmotic water permeability of human ejaculated semen from normospermic subjects was measured after cells exposure for three hours to different temperature (from 21 to 42 °C). a, p < 0.05 vs. 21 °C (ANOVA, followed by Newman–Keuls’s Q test); (C,D) Reversible effect of hydrogen peroxide (H₂O₂) and mercury chloride (HgCl₂). (C) Sperm cells were exposed to a 150 mOsm osmotic gradient in three different conditions: untreated cells (Control), cells treated for 45 min with 50 μM H₂O₂, cells treated with H₂O₂ followed by 15 min treatment with 5 mM dithiothreitol (DTT); (D) Sperm cells were exposed to a 150 mOsm osmotic gradient in three different conditions: normal untreated cells (Control), cells treated for 15 min with 100 μM HgCl₂ (HgCl₂), cells treated with HgCl₂ followed by 15 min treatment with 5 mM DTT. a, p < 0.05 vs. Control and DTT (ANOVA, followed by Newman–Keuls’s Q test). Bars represent the osmotic water permeability of sperm cells expressed as k relative (A) or percent of k relative (B–D). Values are means ± SEM of 4–15 single shots for each of 8–9 different experiments.

Figure 4

Effect of oxidative stress on the water permeability of human ejaculated semen. (A) Effect of hydrogen peroxide (H₂O₂). Sperm cells were exposed to a 150 mOsm osmotic gradient in two different conditions: untreated cells (Control) and cells treated for 45 min with 50 μM H₂O₂ to induce an oxidative stress condition. The H₂O₂-sensitive water permeability was obtained by subtracting H₂O₂ insensitive water permeability from total water permeability. a, p < 0.05 vs. normospermic (Student’s t test); b, p < 0.05 vs. Control and c, p < 0.05 vs. H₂O₂-sensitive (ANOVA, followed by Newman–Keuls’s Q test); (B) Temperature dependence: The osmotic water permeability of human ejaculated semen from normospermic subjects was measured after cells exposure for three hours to different temperature (from 21 to 42 °C). a, p < 0.05 vs. 21 °C (ANOVA, followed by Newman–Keuls’s Q test); (C,D) Reversible effect of hydrogen peroxide (H₂O₂) and mercury chloride (HgCl₂). (C) Sperm cells were exposed to a 150 mOsm osmotic gradient in three different conditions: untreated cells (Control), cells treated for 45 min with 50 μM H₂O₂, cells treated with H₂O₂ followed by 15 min treatment with 5 mM dithiothreitol (DTT); (D) Sperm cells were exposed to a 150 mOsm osmotic gradient in three different conditions: normal untreated cells (Control), cells treated for 15 min with 100 μM HgCl₂ (HgCl₂), cells treated with HgCl₂ followed by 15 min treatment with 5 mM DTT. a, p < 0.05 vs. Control and DTT (ANOVA, followed by Newman–Keuls’s Q test). Bars represent the osmotic water permeability of sperm cells expressed as k relative (A) or percent of k relative (B–D). Values are means ± SEM of 4–15 single shots for each of 8–9 different experiments.

Figure 5

Effect of mercury chloride (HgCl₂) treatment on the hydrogen peroxide permeability of human ejaculated semen from normospermic and sub-fertile subjects. Hydrogen peroxide permeability was measured by loading the human ejaculated semen with CM-H2DCFDA reagent and incubating with 50 μM H₂O₂ as described in Materials and methods. Bars represent the H₂O₂ permeability of sperm cells expressed as k relative. Values are mean ± SEM of two time courses for each of eight different experiments. a, p < 0.05 vs. Ctr sub-fertile, HgCl₂ normospermic, HgCl₂ sub-fertile (ANOVA, followed by Dunnett t test test). Ctr, controls.

Figure 6

Relationship between osmotic water permeability and sperm number (A) or progressive motility (B) of human ejaculated semen from all normospermic and sub-fertile subjects. Water permeability of human ejaculated semen was measured by exposure to a 150 mOsm osmotic gradient. Values, expressed as k relative, are means of at least 15 single shots. Overall linear regression (black line) is presented. p and r² values are shown.

Figure 7

Schematic model of aquaporins (AQP) functioning under normal and oxidative stress conditions: (A) under normal condition, water enters through AQP7 and exits through AQP3. H₂O₂ produced in low amount exits from mitochondria through AQP8; and (B) under oxidative stress condition, H₂O₂ reduces water inflow and outflow through AQP7 and AQP3, thus affecting sperm motility. H₂O₂ reduces also AQP8 permeability that hampers H₂O₂ wasting; this leads to H₂O₂ accumulation into mitochondria, reduced AQP production and thus to reduced sperm motility. Intracellular AQP11 could be involved in the end stage of cytoplasm removing.