Chemicals were obtained from the following sources: Bovine Serum Albumin (BSA) fatty acid-free (#A7906), adenosine 3-phosphate (ATP, #A7699), Cyclic AMP (cAMP, #A9501), from Sigma-Aldrich. Myristoylated PKI 14–22 amide (sPKI, #2546) from Tocris. PhosStop protease inhibitor (#4906837001) and cOmplete EDTA-free protease inhibitor cocktail (#4693132001) from Roche. Rabbit monoclonal anti-phospho-PKA substrates (⍺-pPKAs, clone 100G7E, #9624S) from Cell Signalling Technology (Danvers, MA). Horseradish peroxidase-conjugated -mouse and -rabbit IgG, and anti-mouse (#211-032-171) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-tubulin beta antibody was purchased from DSHB (clone E7, AB_2315513). The traditional non-fluorescent Kemptide (sequence LRRASLG) was purchased from AnaSpec (#22594) while the fluorescent version of the Kemptide (termed “Kemptide-FITC”) was custom-synthesized by Biomatik with the sequence LRRASLGK-FITC (FITC fluorophore is conjugated to the C-terminal of the peptide sequence). All other chemicals were purchased from Cayman Chemicals (Ann Arbor, MI).

cAMP, DTT, ATP, Kemptide, Kemptide-FITC and inhibitor dilutions were prepared fresh the day of the experiment. sPKI was dissolved in dimethylsulfoxide (DMSO) which was also included as a vehicle control when needed. Kemptide-FITC should always be kept in dark to prevent quenching of the fluorophore.

A starter Luria Bertari (LB) broth culture supplemented with 100 μg/mL ampicillin was inoculated with Escherichia coli BL21 previously transformed with the pET15b_Prkaca vector. pET15b PKA Cat was a gift from Susan Taylor (Addgene plasmid # 14921; http://n2t.net/addgene:14921; RRID: Addgene_14921). This plasmid codifies for the PKA catalytic subunit alpha of Mus musculus fused to 6xHis tag at the N-terminal. Culture was grown overnight at 37°C and then transferred to 1,000 mL of LB medium supplemented with 100 μg/mL ampicillin. The culture was allowed to grow until an optical density of 0.4 and 0.5 mM IPTG was added to induce protein expression. Overexpression was done overnight at 18°C. Cells were harvested by centrifugation at 5,000 g, for 30 min at 4°C and the pellet resuspended in 20 mL of cold Bacterial Lysis Buffer (20 mM Tris-HCl pH = 8.0, 50 mM KH₂PO₄, 100 mM NaCl, 5 mM β-mercapthoethanol, 1 mM PMSF). Cells were sonicated on ice 10 times for 7 s at 30.5% sonicator power (Branson, U.S.A.). Cell debris was removed by centrifugation at 9,500 g for 30 min at 4°C. For every 2 mL of the supernatant, 250 μL of Dynabeads™ His-Tag Isolation and Pulldown (Invitrogen™, Carlsbad, CA, USA) were added, and the mixture was agitated at 4°C for 30 min. The flow-through was removed and the Dynabeads™ were washed with 20 mL of Bacterial Lysis Buffer. The attached PKAc-His protein was sequentially eluted with 2 mL of Bacterial Lysis Buffer supplemented with either 25, 50, 250 and 1,000 mM imidazole. All eluted fractions were saved and analyzed by SDS-PAGE to trace the PKAc-His rich fractions. The purified enzyme was concentrated, and the buffer was changed to 20 mM KH₂PO₄, 100 mM NaCl, 10 mM MgCl₂, pH = 7.0, using a 10 K MWCO filter Pierce™ Protein Concentrators (Fisher Scientific).

Quantification was performed by absorbance at 280 nm, considering an extinction molar coefficient (ε) of 53860 as estimated by the “ProtParam” tool of the free-access software “Expasy” (www.expasy.org) for the specific recombinant protein sequence.

C57BL/6 male mature (10–15 weeks old) male mice were used. In all cases, mice housing and all experimental procedures were conducted in accordance with Animal Care and Use Committee of the Facultad de Ciencias Bioquímicas y Farmacéuticas de Rosario (UNR), Argentina (protocol approved #434/2023). The Guide for Care and Use of Laboratory Animals approved by the National Institutes of Health (NIH) was strictly met.

Cauda epididymal mouse sperm were collected from adult male mice (10–13 weeks old). Each minced cauda epididymis was placed in 600 μL of HEPES-buffered TYH medium (H-TYH) containing 119.3 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5.6 mM glucose, 0.5 mM sodium pyruvate, 1.7 mM Ca²⁺, and 20 mM HEPES (pH 7.3), accounting for non-capacitating medium (“NC medium”). After 15 min of incubation at 37°C (“swim-out”), epididymides were removed and the suspension was adjusted with NC medium to a final concentration of 1–2 x10⁶ cells/mL. For capacitation, BSA and NaHCO₃ were added to final concentrations of 5 mg/mL and 20 mM respectively (“CAP medium”). In the experiments where endogenous PKA activity was assessed, sperm were incubated in either NC or CAP medium at 37°C for 15 min, sufficient to trigger PKA activation (Baro Graf et al., 2020; Balbach et al., 2021). Note that unless otherwise stated, 7.5 × 10⁶ sperm/mL (3 × 10⁶ sperm in 400 µL) were treated in each condition (so that every 10 µL of total sperm extract added to the PKA kinase reaction would represent an extract from 300,000 sperm cells, see “PKA kinase reaction”). In cases where PKA inhibitors (sPKI or H89) were used, sperm were pre-incubated with the respective reagents in NC medium for 10 min prior to the incubation in the CAP medium further supplemented with the inhibitors.

For preparation of total sperm extracts used to quantify endogenous PKA activity in cells incubated in either NC or CAP media, sperm were pelleted by centrifugation at 1,000 g for 3 min at RT, and a precise volume of the supernatant was discarded from each reaction tube, leaving 30 µL of supernatant above the pelleted cells. Cells were re-suspended in 70 µL of cold Triton Lysis Buffer (final concentrations 25 mM Tris-HCl, 150 mM NaCl, 1X EDTA-free protease inhibitor mixture, 1X PhosSTOP cocktail inhibitor, 1% Triton X-100, 10 mM DTT, pH 7.4) to achieve a final volume of 100 µL. Note that re-suspending cells to equal final volumes in all treatments is critical to get consistent results. If these steps are not done carefully, differences in PKA apparent activity may arise due to different dilution factors of the sperm extracts. To achieve cell lysis, sperm were incubated for 30 min on ice with frequent gentle pipetting. Samples were then either immediately used in PKA kinase reactions or nitrogen-frozen and stored at −80°C for future use in 50% Glycerol.

For the preparation of insoluble sperm extracts, used as source of PKA holoenzyme to measure cAMP concentrations, sperm were processed as detailed elsewhere (Wertheimer et al., 2013). In brief, sperm cells were directly diluted in NC medium after swim-out to achieve a final concentration of 7.5 × 10⁶ sperm cells/mL (i.e., 3 × 10⁶ sperm in 400 µL). Cells were pelleted by centrifugation at 10,000 g for 3 min at RT and 30 µL of supernatants were left before adding 70 µL of ice-cold Triton Lysis Buffer (final concentrations 25 mM Tris-HCl, 150 mM NaCl, 1X EDTA-free protease inhibitor mixture, 1X PhosSTOP cocktail inhibitor, 1% Triton X-100, 10 mM DTT, pH 7.4) to a final volume of 100 µL. This total extract was further centrifuged at 10,000 g for 10 min at 4°C, and the pelleted cells re-suspended in a final volume of 100 µL of ice-cold Triton Lysis Buffer to obtain the insoluble fraction that contains the PKA holoenzyme.

Each kinase reaction was made by mixing 5 µL of 5X Kinase Buffer (1 M Tris-HCl pH 7.4; 50 mM MgCl₂; 1 mM ATP, 50 mM DTT, 5 X cOmplete EDTA-free protease inhibitor cocktail and 5X PhosSTOP); 2.5 µL of 0.4 µg/µL Kemptide-FITC (equivalent to 30 µM); 2.5 µL of 0.24 μg/μL non-fluorescent Kemptide (equivalent to 30 µM) and a variable volume (1–10 µL) of either total sperm extract or insoluble fraction (depending on the experiment). MiliQ water was added to achieve a final volume of 25 µL. Detection of NC and CAP status of PKA requires 10 µL of sperm total fraction, equivalent to 300,000 sperm cells. For cAMP determination assays, 10 µL of sperm insoluble fraction equivalent to 300,000 cells were used in each reaction.

For negative PKA activity control, Lysis Buffer replaced either sperm extract or recombinant PKAc, according to the experiment. For measurement of maximum PKA activity (used as positive activity control), 10 µL of either the CAP or the NC total sperm extract and 1 µM exogenous cAMP were added to the kinase reaction.

All reaction components were combined and kept on ice until incubated for 25 min at 37°C in the dark. Reactions were stopped by heating at 95°C for 1 min, before mixing with loading buffer 10X (63%–70% Glycerol; 1% Bromophenol blue; 5% Tween-20, 5 mM DTT). Finally, samples were centrifuged at 10,000 g for 1 min at 4°C and supernatants were kept on ice or stored at −80°C until assayed by agarose electrophoresis.

Determination of cAMP concentration through PKA activity was performed by mixing the sperm insoluble fraction (as a source of the PKA holoenzyme) with both Kemptide and Kemptide-FITC, as described in the “PKA Kinase Reaction” section of Materials and Methods. Briefly, 10 µL of the insoluble sperm fraction equivalent to the protein content of 300 × 10³ cells were mixed with 5 µL of 5X Kinase Buffer (1 M Tris-HCl pH 7.4; 50 mM MgCl²; 300 µM ATP, 50 mM DTT, 5X cOmplete EDTA-free protease inhibitor cocktail and 5X PhosSTOP); 2.5 µL of 0.4 μg/μL Kemptide-FITC, 2.5 µL of 0.24 μg/μL Kemptide and 1–5 µL known amounts of cAMP for the standard curve (or unknow for cAMP determinations), and water as needed to complete a final volume of 25 µL. A standard curve using known concentrations of cAMP should be performed for each assay. Then, a plot correlating PKA activity with known cAMP concentrations on a logarithmic scale can be used to determine unknown cAMP concentrations by interpolation, as described previously (Wertheimer et al., 2013). Kinase reactions were started by incubating reaction mixtures for 25 min at 37°C. After the incubation period, reactions were stopped by 1 min at 100°C. Samples were centrifuged at 10,000 g for 1 min at 4°C and the supernatants were assayed by agarose electrophoresis.

Kinase reaction samples were loaded and subjected to electrophoresis (140 V for 40–60 min) on a horizontal 1.5% agarose gel in 50 mM Tris (pH 10.0). Note that the 50 mM Tris Buffer was used both for diluting the agarose and as the running electrophoresis buffer. When possible, an empty lane was left between samples to account for diffusion effects. Electrophoresis was stopped before the Bromophenol blue dye reached the bottom of the gel.

Non-phosphorylated and phosphorylated Kemptide-FITC signals were visualized by UV transillumination. Fluorescence was assessed with a Typhoon-FLA 700 spectrofluorometer under excitation/emission of 473/580 nm. Note that detection settings could be adapted to other image acquisition systems available.

Scanned images of gel electrophoresis were analysed with ImageJ (Schneider et al., 2012) to quantify band densitometries. Raw results were expressed in arbitrary fluorescence units (AFU) for non-phosphorylated (“Kemptide”) and for the phosphorylated Kemptide-FITC (“pKemptide”). For rapid assessment of PKA activity, the percentage of product generated (i.e., % phosphorylation) was directly calculated. For this, total fluorescence in a particular lane (i.e., Kemptide + pKemptide) was established as 100%. Then, the percentage of phosphorylated Kemptide in the gel could be calculated as: % pKemptide = pKemptide AFU /(pKemptide AFU+ non-pKemptide AFU)*100

For kinase activity determination, the following steps were taken:

1. Calculate loaded pmoles of Kemptide in the gel. For example, considering a 60 μM Kemptide present in the Kinase Reaction, a gel loaded volume of 4 μL represents 240 pmoles of total Kemptide.

Note 1

total pKemptide accounts for both non-fluorescent and fluorescent pKemptide in the Kinase Reaction tube.

Note 2

Since one phosphate is transferred from a molecule of ATP to a molecule of Kemptide, results are expressed as pmoles of ATP instead of pmoles of Kemptide so it can be directly compared with measures performed elsewhere.

4. Finally, the amount of ATP per unit time was calculated considering 25 min Kinase Reaction periods and normalized to the amount of either sperm extract or purified protein.

After incubation under the experimental treatments, sperm were collected by centrifugation at 800 g for 4 min at RT and washed twice in 700 μL of TBS. The cell pellet was re-suspended in Laemmli sample buffer (Laemmli, 1970) without β-mercaptoethanol, vortexed for 15 s and boiled for 5 min. After centrifugation at 13,400 g for 3 min, 5% β-mercaptoethanol was added to the supernatants and boiled for 5 min. Protein extracts equivalent to 1 × 10⁶ sperm/lane were subjected to SDS-PAGE and transferred to PVDF membranes (Bio-Rad) at 250 mA for 90 min on ice. Membranes were blocked with 3% BSA in TBS containing 0.1% Tween-20 (T-TBS).

Membranes were first developed using a 1/3,000 dilution of anti-pPKA substrates antibody in T-TBS containing 1% BSA. Secondary HRP-conjugated anti-IgG rabbit and anti-IgG mouse antibodies were diluted 1/10,000 and 1/20,000 respectively in T-TBS containing 1% fat-free milk and developed using an enhanced chemiluminescence detection kit (Biolumina, Kalium Tech, Argentina) according to manufacturer’s directions. PVDF membranes were stripped at 60°C for 15 min in 2% SDS, 0.74% β-mercaptoethanol, and 62.5 mM Tris (pH 6.5) and washed twice for 10 min each time in T-TBS. Then, membranes were developed using anti-β-Tubulin antibody diluted in 1% BSA in T-TBS in a 1/10,000 dilution.

Statistical analyses were performed using Prism 9.5.0 (GraphPad, Boston, MA, USA). All data are shown as mean ± SEM. Statistical significance between two groups was determined using two-tailed, paired t-tests, and statistical significance between multiple groups using matched one-way ANOVA with Tukey post-comparison tests. Significant differences are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

To address the linearity of increasing fluorescence signals of FITC conjugated Kemptide, different amounts of the tagged peptide were loaded on a 1.5% agarose gel at pH 10. The non-phosphorylated Kemptide-FITC has net negative charge at pH 10, evidenced by migration towards the anode. The fluorescence signal was quantified by optical density and exhibited direct proportionality to the quantity of labelled peptide loaded. The linear response spanned from 50 to 300 pmoles, with a linear regression coefficient of R ² = 0.9286, saturation at 600 pmoles and experimental detection limit of 50 pmoles (Figure 1).

This analysis allowed us to set 300 pmoles as an appropriate amount of Kemptide-FITC to be loaded on the agarose gels after the kinase reaction for our particular detection settings. Note that linearity values might change depending on the imaging settings and should be determined for each specific equipment.

Different concentrations of recombinant catalytic PKA subunit (recPKAc) were used along with 60 µM of total Kemptide in kinase reactions. Enzymatic reactions were carried out at 37°C for 25 min and then resolved by agarose gels electrophoresis. As anticipated, two distinct signals were clearly observed migrating towards the anode. A low-mobility band corresponding to the non-phosphorylated Kemptide-FITC and a high-mobility band as the result of Kemptide phosphorylation (Figure 2). A linear response between 53 ng/mL and 533 ng/mL of recPKAc was observed at 37°C for a reaction time of 25 min (R ² = 0.9974).

Most PKA activity assays are designed to detect the activity of either extract purified enzyme or recombinant PKA (Araujo et al., 2016; Stival et al., 2018; 2020). However, they do not reflect the state of PKA within the cells which depends on intracellular levels of cAMP. Four cAMP molecules bind to a pair of regulatory PKA subunits to release active PKAc (Taylor et al., 2013; Baro Graf et al., 2020). When analysing cellular PKA activity, it is then intended to maintain as much as possible the actual physiological state of the enzyme. To develop an assay that would reflect the activity of endogenous PKA under different treatments, the first step was to obtain cell lysates to be used for kinase reactions, as described in the previous section. As recently shown, maximal PKA activity during capacitation is achieved at 15 min of incubation in capacitating conditions (Baro Graf et al., 2020; Balbach et al., 2021). To assess this physiological activation of PKA by KiMSA, mouse sperm cells were incubated in either non-capacitating or capacitating media for 15 min. Extracts from 50, 100, 200 or 300 × 10³ sperm cells in final kinase reaction volumes of 25 µL were used to optimize the amount of starting cells. All tested cell extracts showed significant differences in PKA activity between sperm incubated in non-capacitating versus capacitating media, as the amount of phosphorylated Kemptide (lower bands) increased upon capacitation. As expected, the higher the number of cells used in the kinase reaction, the more phosphorylated Kemptide-FITC in both types of media. From all tested quantities, 300 × 10³ sperm cells was selected as starting material to be used in the kinase reaction protocol for quantitation of native sperm PKA activity, based on improved signal differences (Figures 3A, B, see also Figure 4). However, our results show that this assay can quantitatively and reproducibly address both basal and stimulated PKA activity in different sperm cells concentrations.

To discard the effect of kinases other than PKA acting on the Kemptide-FITC, cells were incubated with either 15 µM sPKI or 30 μM H89, two different PKA inhibitors with different modes of action. Inhibitors were present during incubation in either non-capacitating or capacitating media. Then, cells were centrifuged, and inhibitors washed away as standard protocol was followed, as explained above, to obtain total cell extracts. Kinase reactions were performed without further addition of inhibitors, showing impaired in vitro PKA activity (Figures 4A, B). Thus, KiMSA reflects the effect of in vivo incubation rather than the effect of inhibitors added to the reaction mixture. Note that differences that raised from capacitation of sperm cells in terms of PKA activity (NC and CAP lanes, Figures 4A, B), as well as when extracts deriving from NC cells were challenged with 1 µM cAMP (Figures 4A, B, lane NC cAMP). In parallel to KiMSA, equally treated cell samples were analysed for PKA activity through traditional Western blot detection of phosphorylated PKA substrates (pPKAs, Figures 4C, D), as an indirect marker of PKA activity in sperm (Krapf et al., 2010). The results showed that the Kemptide-FITC substrate is not phosphorylated in the presence of PKA inhibitors. sPKI is considered as highly specific and allows discarding promiscuous phosphorylation effects by other kinases (Liu et al., 2020). Furthermore, KiMSA was able to reflect the in vivo status of PKA, since inhibitors were added only to the incubation media and removed before lysis.

Traditional activation of PKA relies on cAMP binding to the regulatory subunits. These regulatory subunits are tethered to Triton X-100 insoluble structures both in somatic (Bramson et al., 1982; Mucignat-Caretta and Caretta, 2001) and sperm cells (Vijayaraghavan et al., 1997; Visconti et al., 1997; Baker et al., 2007). Binding of cAMP results in a conformational change that releases the active catalytic subunits of PKA, which can then be collected in the soluble fraction. Therefore, inactive PKA holoenzyme can be recovered from the insoluble sperm fraction and remains sensitive to cAMP levels. This allowed us to adapt the KiMSA assay to indirectly measure the amounts of cAMP by assessing the extent of PKA activation recovered from the insoluble fraction of sperm cells when challenged with unknown concentrations of cAMP. When assessing cAMP levels in cellular extracts, holoenzyme is prepared fresh the day of the experiment, and a concentration curve using known amounts of cAMP needs to be constructed for every assay. The resulting plot is used to determine intracellular cAMP levels in sperm cells by interpolation (Wertheimer et al., 2013). To this end, sperm cells were immediately lysed in Triton X-100 buffer after swim-out, and the insoluble fraction was recovered to use in KiMSA experiments (see Materials for further details). The kinase reaction was performed over different known concentrations of cAMP in a final volume of 25 µL and the samples analysed in an agarose gel (Figure 5A). The PKA activity values obtained were used to generate a standard curve on a logarithmic scale (Figure 5B).

This method was able to reproducibly detect cAMP concentrations ranging from 3 nM to 100 nM, equivalent to 0.12–4 pmoles (in 25 μL). This detection scale is optimized for detection of cAMP in mouse sperm samples (and other mammalian species), compared to the reported values of intracellular cAMP in mouse sperm cells ranging from 0.5-1 pmoles in 1 × 10⁶ sperm cells incubated in non-capacitating medium to 1–3 pmoles upon capacitating (Balbach et al., 2021).