The transgenic C1 sAC null mouse line, in which exons 2–4 are deleted, was generated at Lexicon Genetics (The Woodlands, TX). The transgenic mouse line whose sperm express green fluorescent protein (GFP) in their acrosome and red fluorescent protein (RFP) in their mitochondria (B6D2F1-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb) (Hasuwa et al., 2010) is from M. Okabe (Osaka U, Japan) via the RIKEN Institute. Animal experiments were approved by Weill Cornell Medicine’s Institutional Animal Care and Use Committee (IACUC).

3-Isobutyl-1-methylxanthine (IBMX), bovine serum albumin (BSA), NaHCO₃ and Progesterone were purchased from Sigma-Aldrich (St. Louis, MO, United States). 8-Bromo-cAMP (sodium salt) and cAMP Elisa kit from Cayman Chemical (Ann Arbor, MI, United States). β-mercaptoethanol from Thermo Fisher Scientific (Waltham, MA, United States). PBS buffer was purchased from Corning (Radnor, PA, United States). Rabbit monoclonal anti-phospho-PKA substrates (anti-pPKA substrates) (clone 100G7E) and Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG, were purchased from Cell Signaling Technology (Danvers, MA). Mouse anti-phospho Tyrosine (anti-pTyr) (clone 4G10) was from EMD Millipore.

Crossing sAC KO females with Acr-EGFP/Su9-DsRed2 males generated Acr-EGFP/Su9-DsRed2/sAC^−/− mice. The presence of the transgenes expressing fluorescent tags as well as disruption of sAC gene in the germline were confirmed by PCR analysis.

Mouse sperm were isolated by incision of the cauda epididymis of male mice or from the female genital tract after copulation, followed by a swim-out in 500 μL Toyoda Yokoyama Hoshi (TYH) medium (in mM: 119.3 NaCl, 4.7 KCl, 1.7 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 5.6 glucose, 0.5 pyruvate, 20 HEPES, pH 7.4 adjusted with NaOH), prewarmed at 37°C and counted using a hematocytometer. For capacitation, sperm were incubated in TYH containing 5 mg/mL BSA and 20 mM NaHCO₃ or 0.5 mM 8Br-cAMP and 0.5 μM IBMX at 37°C.

Aliquots of 3−4 x 10⁶ WT or sAC^−/− double transgenic sperm were incubated for 15 min in the presence of 0.5 μM IBMX in non-capacitating (TYH-) or capacitating (TYH + HCO₃/BSA) buffer. Sperm were sedimented by centrifugation at 2000 x g for 3 min and lysed in 50–100 μL of 0.1 M HCl for 10 min. Sperm lysates were centrifuged at 2000 x g for 3 min and the cAMP in the supernatant was acetylated and quantified using the Direct cAMP ELISA Kit (Cayman) according to the manufacturer’s instructions.

Aliquots of 1−2 x 10⁶ WT or sAC^−/− double transgenic sperm were incubated for 60 min in TYH buffer in the absence or presence of 5 mg/mL BSA and 20 mM NaHCO₃ or 0.5 mM 8Br-cAMP and 0.5 μM IBMX. Sperm were collected by centrifugation for 3 min at 2000 x g, washed twice with 1 mL TBS, and resuspended with Laemmli sample buffer without β-mercaptoethanol, vortexed for 10 s and boiled for 3 min. After centrifugation, 5% β-mercaptoethanol was added to the supernatants and boiled again for 5 min. Protein extracts equivalent to 0.5−1 x 10⁶ sperm/lane were subjected to SDS-PAGE and electrotransferred to PVDF membranes at 250 mA for 90 min. Membranes were blocked with 3% BSA in TBS containing 0.1% Tween-20 (T-TBS). Antibodies were diluted in T-TBS as follows: 1/5,000 for anti-pTyr and 1/3,000 for anti-pPKA substrates. Secondary antibodies were diluted 1/10,000 in T-TBS and the membranes analyzed using an enhanced chemiluminescence detection kit (SuperSignal West Femto Maximun Sensitivity Substrate, Thermo Scientific) according to manufacturer’s instructions.

Sperm suspensions were loaded on a 100-μm deep Leja slides and placed on a microscope stage at 37°C. Sperm movements were examined using an IVOSII Hamilton Thorne. Parameters used were as follows: 45 frames acquired, frame rate of 60 Hz, and cell size of 25–200 μm². At least 5 microscopy fields and a minimum of 500 sperm were analyzed in each experiment. The following parameters were measured: mean path velocity (VAP, μm/sec), curvilinear velocity (VCL, μm/sec), straight-line velocity (VSL, μm/sec), linearity (LIN, %), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz) and straightness (STR, %). Sperm were considered hyperactivated when presenting VCL ≥271 μm/s, LIN <50%, and ALH ≥3.5 μm and progressive when presenting VAP ≥50 μm/s and STR >80%.

After incubating WT and sAC^−/− sperm under non-capacitating or capacitating (+HCO₃ ⁻/BSA or +8Br-cAMP/IBMX) conditions for 60 min, sperm were incubated for an additional 30 min in the absence or presence of 25 μM progesterone (Pg). Sperm were sedimented by centrifugation at 1,000 x g for 5 min, washed twice with PBS, and seeded on 8-well glass slides. After adhesion, sperm were incubated with PBS containing DAPI for 10 min. Before mounting, samples were washed with PBS (four times for 5 min each time). Epifluorescence microscopy was used to assess acrosomal status, distinguished by the presence or absence of green fluorescence of intact acrosomes. At least 300 sperm were analyzed in each condition.

Single-caged double transgenic DsRed2/Acr3-EGFP WT or sAC^−/− males were combined with WT females in estrous and vaginal plug formation was checked every 30 min. Once plug formation was confirmed, the male was separated from the female. The uteri and oviducts were excised various times after coitus (1.5, 4, and 8 h), transferred to slides, and examined for the presence of fluorescent sperm expressing mitochondrial DsRed2 and/or acrosomal EGFP markers in the female reproductive tract (IX73 Olympus). The experiments were repeated at least 3 times with 3 different males.

To be able to study sAC null sperm in vivo, we crossed transgenes which label the acrosome and mitochondria (located in the midpiece) of spermatozoa with green and red fluorescent proteins, respectively (DsRed2/Acr3-EGFP) (Hasuwa et al., 2010), into sAC^−/− (KO) mice (Esposito et al., 2004; Hess et al., 2005) as well as into our isogenic C57Bl/6 strain. The resultant double transgenic sAC KO mouse line exhibited no overt developmental abnormalities. As previously reported, sAC KO males were infertile despite producing epididymal spermatozoa with the same count and morphology as wild type (WT) males (Supplementary Figures S1A, B) and showing normal mating behavior with successful ejaculation and vaginal plug formation (Supplementary Figure S1C).

Also similar to spermatozoa from sAC KO mice, epididymal spermatozoa from DsRed2/Acr3-EGFP/sAC KO males did not display molecular hallmarks normally associated with capacitation when incubated in the presence of HCO₃ ⁻: they did not increase cAMP levels and they did not produce the prototypical pattern of phosphorylation of PKA substrates and tyrosines. As expected, addition of membrane-permeable cAMP, the product of sAC, rescued the phosphorylation of PKA substrates and tyrosines (Figure 1), confirming that any defects observed in sperm from double transgenic sAC KO mice come from the absence of sAC. These results validate that the double transgenic, fluorescently tagged sAC KO line phenotypically reflect previously characterized sAC KO mice (Esposito et al., 2004; Hess et al., 2005; Xie et al., 2006).

Sperm surgically extracted from the epididymis are isolated prior to ejaculation so they have never been exposed to the HCO₃ ⁻ in seminal plasma. Like other WT epididymal sperm, epididymal sperm from double transgenic WT males are motile, and the fraction of sperm exhibiting hyperactivated motility increases in the presence of HCO₃ ⁻ (Figure 2; Supplementary Movies S1, S2). To compare epididymal sperm with ejaculated mouse sperm, we mated double transgenic males with WT females and recovered sperm from the uterus post-copulation. As expected, ejaculated WT sperm were motile, but remarkably they did not display the bicarbonate-induced increases in total and progressive motility seen in epididymal sperm. In addition, they showed only a slight increase in hyperactivation (Figure 2; Supplementary Movies S1, S2). Presumably ejaculated sperm are less sensitive to bicarbonate because they have already been exposed to bicarbonate-dependent stimulation upon mixing with seminal plasma and fluids in the female tract.

We used the same methods to isolate and characterize ejaculated sAC null sperm. When we mated sAC^−/− double transgenic males with WT females, we observed bona fide plugs and plumped uteri after copulation (Supplementary Figure S2), and we successfully obtained sAC null sperm from uteri of receptive females. We recovered the same amount of ejaculated sperm from the female tract after mating with WT and sAC KO males (Supplementary Figure S2), confirming that sAC ablation does not affect mating or ejaculatory behaviors. In agreement with previous reports for epididymal sAC KO sperm (Esposito et al., 2004; Hess et al., 2005; Marquez and Suarez, 2008), both epididymal and ejaculated double transgenic sAC^−/− sperm were immotile, showing only vibratory movements (Figure 2; Supplementary Movies S1, S2). Motility was restored by addition of a cell-permeable cAMP analog, proving the motility defect is due to the absence of sAC. Thus, double-transgenic sAC KO sperm are immotile even after ejaculation.

Another hallmark of capacitation is the ability to undergo a physiologically induced acrosome reaction (AR). WT epididymal sperm exhibited progesterone (Pg) induced AR following incubation with bicarbonate or after addition of exogenous membrane permeable cAMP (Figure 3A). In contrast, with epididymal sAC KO sperm, we only observed the Pg-induced AR in the presence of exogenous cAMP. These data demonstrate that there is a cAMP-dependent component to enable acrosomal responsiveness, which is mediated via sAC following bicarbonate stimulation. In ejaculated WT sperm, which were already exposed to bicarbonate in seminal plasma and fluids in the female reproductive tract, acrosomal responsiveness to Pg was observed in non-capacitating media (Figure 3B). Addition of bicarbonate enhanced the Pg-induced increase in AR (Supplementary Figure S3). Surprisingly, ejaculated sAC null sperm displayed a degree of Pg responsiveness in both non-capacitating and capacitating media (Figure 3). Thus, there also appears to be a sAC-independent mechanism regulating AR associated with ejaculation or residence in the female reproductive tract. As expected, epididymal and ejaculated sAC KO sperm lost any bicarbonate-induced increase in Pg-stimulated AR (Supplementary Figure S3), but in both cases, Pg responsiveness was increased by cell-permeable cAMP, corroborating the idea that there are both cAMP-dependent and cAMP-independent mechanisms for increasing the response to Pg.

Finally, to explore the in vivo roll of sAC, we studied sperm migration through the female reproductive tract following matings between WT or sAC KO double transgenic males with receptive WT females. Sperm ejaculated from WT mice migrated from the uterus through the uterotubular junction (UTJ) into the oviduct within an hour post-copulation, and by 4 h sperm were observed all the way from the oviduct to the ampulla. In contrast, while sperm ejaculated from sAC^−/− double transgenic mice were present inside the uterus, few were infrequently observed in the UTJ, and none successfully progressed into the oviduct even 8 h after copulation (Figure 4; Supplementary Figures S4, S5).