Cyclic AMP is downstream of the effect of Cl^– on capacitation. Sperm were incubated in media without Cl^– in the absence or presence of IBMX (0.2 mm) and different cAMP analogs: dibutyryl cyclic AMP (db; 5 mm), sp-cAMPS (sp; 1 mm), or 8-bromo-cAMP (8Br; 0.5 mm). The increase in tyrosine phosphorylation (A) and the percentage of hyperactivation (B) were evaluated. In IVF assays (C), gamete co-incubation was conducted in drops with the same composition as the capacitation media. The percentage of fertilization in the control was 85 ± 6. Each experiment was repeated at least three times. For hyperactivation and IVF experiments, Tukey's multiple comparison tests were performed. The means of groups that have different letters differ significantly (p < 0.01) (B and C).

Effect of bumetanide on capacitation-associated events. A and B, bumetanide, an NKCC inhibitor, blocks the increase in tyrosine phosphorylation and hyperactivation with no effect on the percentage on sperm motility. Sperm were incubated under noncapacitating (NC) or capacitating (C) conditions in the presence of increasing bumetanide concentrations. Subsequently, Western blotting with anti-PY antibodies (A, upper panel) and CASA (B) were carried out. Membranes were stripped and reblotted with an anti-β-tubulin antibody for loading control (A, lower panel). C, bumetanide inhibits the ZP-induced acrosome reaction. Bumetanide was added to sperm during capacitation or at the same time as the ZP. Subsequently, ZP-induced acrosome reaction was evaluated. D, bumetanide inhibits IVF. Eggs were inseminated with sperm capacitated in the presence of different concentrations of bumetanide. The inhibitor was also added to each co-incubation drop at the same concentration as during capacitation. The percentage of fertilization in the control was 80 ± 9. Each experiment was repeated at least three times. For hyperactivation experiments (B), *, p < 0.01 versus control (0 mm bumetanide plus DMSO) using two-tailed Student's t test; for acrosome reaction (C), *, p < 0.05 comparing basal with ZP-induced acrosome reaction for each bumetanide concentration and using Bonferroni post tests; for IVF experiments, Tukey's multiple comparison test was performed. The means of groups that have different letters differ significantly (p < 0.05) (D).

Cyclic AMP agonists rescue the inhibition of capacitation by bumetanide. Capacitation was carried out in the presence or absence of bumetanide (1 mm), IBMX (0.2 mm), and dibutyryl cyclic AMP (db cAMP; 5mm). Then, protein tyrosine phosphorylation (A), sperm hyperactivation (B), and fertilization (C) were evaluated. The percentage of fertilization in the control was 78 ± 7. Each experiment was repeated at least three times. For hyperactivation and IVF experiments, Tukey's multiple comparison tests were performed. The means of groups that have different letters (a and b) differ significantly (p < 0.01) (B and C).

NKCC is present in mouse sperm. A, NKCC mRNA is present in spermatogenic cells. Reverse transcription-PCR of spermatogenic cells was carried out using oligonucleotides for NKCC1 and cDNA of pachytene, round cells, and elongated spermatids (+); negative controls (–) without reverse transcriptase are also shown for each PCR. B, NKCC1 is present in mature mouse sperm. Total kidney and sperm extracts were analyzed by Western blotting with a monoclonal anti-NKCC1 antibody (left). In addition, sperm membrane purifications were carried out as described under “Experimental Procedures” and SDS extracts from 100,000 × g pellet were analyzed using anti-NKCC antibodies (right). C, NKCC1 localizes to different compartments in sperm including anterior head, mid and principal piece. For immunolocalization assays, mouse sperm were air-dried, fixed, permeabilized, and probed with anti-NKCC1 antibodies. Peanut agglutinin (PNA) was used to follow the acrosomal status. An NKCC1-specific signal in the head is not observed when peanut agglutinin is negative (arrows). Each experiment was repeated at least three times.

Cl^– is necessary for capacitation-associated events and for fertilization. A, capacitation-associated tyrosine phosphorylation is inhibited in Cl^–-free medium. Mouse sperm were incubated for 60 min in media that do not support (–BSA, –)(NC) or support capacitation (+BSA, )(C) in the presence or absence of Cl^– (replaced by gluconate). Subsequently, aliquots from each condition were processed for Western blotting with anti-PY antibodies (upper panel). The increase in tyrosine phosphorylated proteins was observed only when sperm were capacitated in the presence of Cl^–. The stripped membranes were reblotted with an anti-β-tubulin antibody for loading control (lower panel). B, the increase in tyrosine phosphorylation is dependent on Cl^– concentration. Sperm were incubated in Whitten's media containing BSA, , and different Cl^– concentrations. Sperm proteins were analyzed by Western blots with anti-PY antibodies (upper panel). After being stripped, membranes were reblotted with an anti-β-tubulin antibody for loading control (lower panel). C, the percentage of hyperactivated sperm is reduced in the absence of Cl^–. After capacitation in the presence or the absence of Cl^–, motility parameters were analyzed by CASA. The percentage of motile cells (upper panel) and the percentage of hyperactivation (lower panel) were compared between treatments as explained under “Experimental Procedures.” D, Cl^– is needed for the induction of acrosome reaction. Sperm were incubated with different Cl^– concentrations in capacitation media, and then solubilized ZP was added and incubated for induction of acrosome reaction. After fixation and staining with Coomassie Blue, the status of the acrosome was assessed in at least 100 sperm/treatment. E, IVF is inhibited in the absence of Cl^–. Sperm incubated in capacitating media with different Cl^– concentrations were used in IVF assays. The insemination drops contained 10–20 eggs and had the same Cl^– concentration as the capacitation media. F, the absence of Cl^– during the co-incubation does not affect the interaction between gametes. Eggs were co-incubated in the absence of Cl^– with sperm capacitated in control media. The percentage of fertilization in the control was 75 ± 8. Each experiment was repeated at least three times. Hyperactivation experiments: *, p < 0.05 versus control (100 mm Cl^–) using two-tailed Student's t test. Acrosome reaction: *, p < 0.05 comparing basal with ZP-induced acrosome reaction for each Cl^– concentration using Bonferroni post tests. IVF experiments: Tukey's multiple comparison test was performed. The means of groups that have different letters differ significantly (p < 0.05).

Effect of Cl^– channel inhibitors in capacitation. A and B, CFTR inhibitors do not inhibit capacitation-associated increase in tyrosine phosphorylation, motility, or hyperactivation. Sperm were incubated in complete capacitating media in the presence of two different CFTR inhibitors: inh-172 (250 μm) or DPC (1 mm), and the increase in tyrosine phosphorylation (A, upper panel) and percentage of motile sperm and hyperactivation (B) were analyzed. C, upper panel, Cl^– channel inhibitors with no effect on tyrosine phosphorylation. Sperm were incubated under noncapacitating (NC) or capacitating (C) conditions in the presence of different anionic channel blockers: picrotoxin (1 mm), bicuculline (1 mm), indanyloxyacetic acid 94 (IAA-94; 0.2 mm), propionic acid (1 mm), 9-(hydroxymethyl)anthracene (AC-9; 0.25 mm), chlorotoxin (0.01 mm), thiazide (1 mm), R(+)-butylindazone (DIO-A; 0.2 mm), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 0.2 mm); and niflumic acid (NFA; 0.5 mm). Then, Western blotting was conducted using anti-PY antibodies. D, upper panel, DIDS and SITS block capacitation-associated increase in tyrosine phosphorylation. Sperm were incubated under noncapacitating or capacitating conditions in the presence or absence of DIDS or SITS (disulfonic stilbene derivatives), two broad anionic channel blockers. Next, Western blotting was conducted using anti-PY antibodies. E and F, DIDS and SITS block hyperactivation and IVF. Sperm incubated in capacitating media in the presence or absence of DIDS or SITS (1 mm) were used to assay for hyperactivation (E) and IVF (F). The inhibitors were present also during the gametes co-incubation. The percentage of fertilization in the control was 78 ± 13. All membranes were stripped and reblotted with an anti-β-tubulin antibody for loading control (A, C, and D, lower panels). Each experiment was repeated at least three times. For motility, hyperactivation and IVF experiments, two-tailed Student's t test (B) or Tukey's multiple comparison test (E and F) were performed. The means of groups that have different letters differ significantly (p < 0.01) (E and F).

Analysis of sperm capacitation in the absence of K⁺. A and B, the lack of K⁺ during capacitation does not affect either the increase in tyrosine phosphorylation or sperm hyperactivation. Sperm were incubated under noncapacitating (NC) or capacitating (C) medium with or without K⁺, and the increase in tyrosine phosphorylation (A, upper panel), the percentage of motility (B, upper panel), and hyperactivation (B, lower panel) were evaluated. Membranes were stripped and reblotted with an anti-β-tubulin antibody for loading control (A, lower panel). C, K⁺ ions are necessary for the ZP-induced acrosome reaction to occur. Sperm were incubated in control or K⁺-free capacitating media, and ZP-induced acrosome reaction was assayed. Each experiment was repeated at least three times. For hyperactivation (B), *, p < 0.01 versus control, using two-tailed Student's t test. For acrosome reaction (C), *, p < 0.01 comparing basal with ZP-induced acrosome reaction, using Bonferroni post tests.

Capacitation-associated increase in cAMP levels are prevented in the absence of Cl^– or in the presence of bumetanide. Capacitation was carried out in the presence or absence of Cl^– or bumetanide (1 mm). Then, sperm were centrifuged and resuspended in HCl (0.1 mm) for 20 min. After that, sperm were centrifuged again and the supernatant was used to measure cAMP levels (A). A standard curve was run for each assay. A typical standard curve is depicted (B). Tukey's multiple comparison tests were performed. The means of groups that have different letters (a and b) differ significantly (p < 0.05) (A).

Model for the involvement of Cl^– during capacitation. A, initial events during capacitation. At first, an acute Cl^–-independent increase in cAMP levels involving sAC activation by and mediated through a cotransporter occurs allowing the consequent activation of PKA. Subsequently, PKA phosphorylation exerts a negative feedback on cAMP levels. B, later events during sperm capacitation. Because of the need of cAMP for later events, it is possible to hypothesize that the negative feedback mechanism on the cAMP levels exerted by PKA phosphorylation is released at some point during the capacitation process. Our working hypothesis is that inward translocation of Cl^– (through NKCC and other Cl^– transporters) is coupled to the activity of a antiporter. Inward fluxes of through this exchanger would stimulate sAC and raise the cAMP levels necessary for the posterior increase in tyrosine phosphorylation. Also, NKCC-dependent Cl^– currents would be necessary to prepare sperm for ZP-induced acrosome reaction. In addition, Cl^–-dependent unknown mechanisms occurring in sperm are essential for fertilization to occur.