Membrane rafts regulate sperm acrosome reaction via cAMP-dependent pathway in chickens (Gallus gallus domesticus)†

Abstract

Both transcriptionally and translationally inactive sperm need preassembled pathways into specific cellular compartments to function. Although initiation of the acrosome reaction (AR) involves several signaling pathways including protein kinase A (PKA) activation, how these are regulated remains poorly understood in avian sperm. Membrane rafts are specific membrane regions enriched in sterols and functional proteins and play important roles in diverse cellular processes, including signal transduction. Our recent studies on chicken sperm demonstrated that membrane rafts exist and play a role in multistage fertilization. These, combined with the functional importance of membrane rafts in mammalian sperm AR, prompted us to investigate the roles of membrane rafts in signaling pathways leading to AR in chicken sperm. Using 2-hydroxypropyl-β-cyclodextrin (2-OHCD), we found that the disruption of membrane rafts inhibits PKA activity and AR without affecting protein tyrosine phosphorylation; however, these inhibitions were abolished in the presence of a cyclic 3,5-adenosine monophosphate (cAMP) analog. In addition, biochemical experiments showed a decrease in cAMP content in 2-OHCD-treated sperm, suggesting the involvement of soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclase (tmAC). Pharmacological experiments, combined with transcriptome analysis, showed that sAC and tmAC are present and involved in AR induction in chicken sperm. Furthermore, stimulation of both isoforms reversed the inhibition of PKA activity and AR in 2-OHCD-treated sperm. In conclusion, our results demonstrated that membrane rafts play an important role in AR induction by regulating the cAMP-dependent pathway and that they provide a mechanistic insight into membrane regulation of AR and sperm function in birds.

Introduction

After leaving the testis, the membranes of sperm undergo various changes to enable them to fertilize an egg. One of the most important steps during the process is the acrosome reaction (AR) in which the plasma membranes fuses with the outer acrosome membrane, enabling sperm passage through the zona pellucida (ZP) and subsequent fusion with the oocyte plasma membrane. Capacitation is a prerequisite for mammalian sperm to undergo AR [1]. Several studies for in vitro characterization of this process have shown that the removal of sterols from the plasma membrane triggers the activation of the signaling cascades inherent to capacitation and potentiates responsiveness to initiate AR [2]; however, unlike in mammalian sperm, capacitation is not necessary for avian sperm to undergo AR [3]. Avian sperm undergo AR immediately after exposure to the inner perivitelline layer (IPVL), which is analogous to the mammalian ZP [4]. These functional differences suggest the distinction in the cellular cascades inherent in AR induction between mammalian and avian sperm.

Membranes are important for cells to excite various functions as needed. Membrane rafts are specific membrane domains enriched with proteins and lipids, such as sterols and ganglioside G_M1. They play important roles in the regulation of cellular processes in diverse cell types [5]. Studies by our group have demonstrated that membrane rafts in mammalian sperm are present in the plasma membrane overlying the acrosome (APM), where membrane fusion occurs to precede AR [6–8]. Several attempts have been made to characterize the function of membrane rafts in mammalian sperm and showed its involvement in capacitation [9], binding to ZP [10] and AR [11]. Considering the previous findings that these domains play multiple roles in the sperm of marine species during fertilization [12–14], the functional importance of membrane rafts might be conserved within the animal kingdom. This was corroborated by our recent study that demonstrated that chicken sperm contain membrane rafts in the plasma membrane overlying the sperm head region [15]. More recently, we performed comprehensive characterization of chicken sperm rafts using biochemical and proteomic approaches, which demonstrated that these membrane domains might play functional roles in multistage of fertilization, including binding to IPVL [16]. These, together with the localization of these domains into the membrane regions including APM of chicken sperm, raise a question of the role of membrane rafts in avian sperm AR.

Of the cellular functions regulated by membrane rafts, signal transduction events that traverse the plasma membranes have been intensively investigated in other cells [5]. Pharmacological studies on chicken sperm have demonstrated the involvement of several signaling molecules, such as protein kinase A (PKA), protein kinase C, phosphoinositide 3-kinase, mitogen-activated protein kinase 3/1, and several protein phosphatases in AR [17–19]; however, it is not known how signaling pathways that include these molecules are regulated.

PKA activation is mediated by cyclic adenosine 3΄,5΄-monophosphate (cAMP), which is synthesized by the two isoforms, adenylyl cyclase (AC)—soluble (sAC) and transmembrane (tmAC). They are known to differ in terms of tissue distribution, subcellular localization, and regulatory responsiveness, which suggests a distinction in functional roles. Previous studies using mutant mice devoid of sAC demonstrated that this isoform plays an important role in flagellar motility, but is not involved in AR induction [20, 21]. On the other hand, a recent biochemical study using the same type of mutant mice showed that active tmAC is present in murine sperm and regulates AR induction [22]. Despite the functional difference between these isoforms, sAC and tmAC are tethered to plasma membranes by unknown interactions and transmembrane linkage, respectively. In fact, it is known that the regulatory module of cAMP signaling in other cells is spatially restricted to discrete subcellular locations [23, 24]. Several studies on the mechanisms by which cAMP production is regulated in other cells demonstrated that tmAC is spatially and functionally regulated by membrane rafts [25, 26]. In addition, sperm sAC in marine species was also found to be associated with membrane rafts [27]. These reports suggest the functional involvement of membrane rafts in the cAMP-dependent pathway. Given these findings, we chose to investigate the roles of membrane rafts in AR induction in chicken sperm, focusing on cAMP-dependent pathways involving PKA. Our findings demonstrated for the first time that membrane rafts play an important role in AR induction by regulating cAMP production and provide insight into the mechanisms by which membranes regulate avian sperm functions.

Materials and methods

Reagents

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. Fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA) was obtained from J-Oil Mills, Inc. (Tokyo, Japan). Monoclonal antibody to phospho-PKA substrate protein was purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibodies to phospho-tyrosine and α-tubulin were obtained from EMD Millipore Corporation (Temecula, CA, USA). The Amplex Red Cholesterol Assay Kit and cholera toxin subunit B (CTB) conjugated with HRP were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Cyclic AMP Select EIA Kit and KH7 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Fluo 3-AM was obtained from Dojindo Laboratories (Kumamoto, Japan), and forskolin was purchased from Focus Biomolecules (Plymouth Meeting, PA, USA). MDL-12,330A HCl (MDL) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antiserum against ZPC protein was generously gifted from Dr Tomohiro Sasanami, Shizuoka University, Japan [28].

Semen collection

Semen were collected from sexually mature fertile male Rhode Island Red (RIR) roosters that were raised at the Agricultural and Forestry Research Center, University of Tsukuba, Japan, using the dorsal–abdominal massage method previously described [29]. Semen from at least four birds were pooled to use for each experiment. All animal work was performed with approval from the Institutional Animal Care and Use Committee of the University of Tsukuba (approval no. 15–018).

Perivitelline layer separation

IPVL was obtained from freshly laid, nonfertilized eggs of RIR hens using the method previously described [30]. Briefly, the yolks were separated from the egg white and incubated in 100 mL 0.01 N HCl for 1 h at 37°C. The yolks were then collected from the medium and punctured to drain the inner material and collect the membranes. The membranes were carefully spread out in 1% NaCl in a petri dish at 4°C. The two membrane layers were then easily separated using tweezers, and the IPVL was rinsed with distilled water and homogenized in TES-NaCl buffer (150 mmol NaCl and 20 mmol TES N-Tris[hydroxymethyl]methyl-2-amino-ethanesulfonic acid, pH7.4) to be used to stimulate AR in chicken sperm.

Sperm incubation

The collected semen sample was washed in TES-NaCl buffer by centrifugation at 1000 × g for 5 min to remove seminal plasma. Sperm (1.0 × 10⁷ cells) were incubated for 1 h at 39°C in 300 μL NaCl-TES buffer supplemented with 2 mM Ca²⁺ and other chemicals as required for each experiment. In experiments wherein the nature of the membrane rafts was altered, 2-hydroxypropyl-β-cyclodextrin (2-OHCD), a sterol acceptor, concentrations were supplemented [31] and later was fixed at 1 mM.

Sterol quantification

Sterol content in sperm membranes was quantified as described previously [32]. Briefly, sperm (1.0 × 10⁷ cells) were incubated with 0 or 1 mM 2-OHCD for 1 h, centrifuged at 10,000 × g for 10 min and resuspended in a reaction buffer (0.1 M potassium phosphate, 0.05 M NaCl, 5 mM cholic acid, 0.1% Triton X-100). Samples were homogenized with a Dounce tissue grinder, sonicated, and centrifuged at 10,000 × g for 10 min. The supernatants containing membranes were subjected to sterol assay using Amplex Red Cholesterol Assay Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

Evaluation of acrosome status

After incubation, the samples were centrifuged, resuspended in 200 μL homogenized IPVL lysate, and incubated in the presence of 2 mM Ca²⁺ for 30 min at 39°C. Samples were centrifuged at 1000 × g for 1 min and treated with 100 μg/mL PNA-FITC in TES-NaCl buffer in dark for 10 min, as previously described [33]. Samples were washed again with TES-NaCl buffer and observed under a fluorescence microscope equipped with an AF6000 imaging system (Leica Microsystems, Wetzlar, Germany). At least 200 sperm from each sample were assessed for acrosome status.

Immunoblotting

After incubation, the samples were centrifuged 10,000 × g for 5 min and processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis as previously described [34]. Transferring and blocking were largely performed as previously described [32]. Dilutions were 1:10,000 for anti-phospho-PKA substrate protein, anti-phosphotyrosine, and anti-α-tubulin antibodies and the seconday antibodies. Immunoreactivity was visualized by chemiluminescence and imaged by ChemiDoc XRS + imaging system (Bio-Rad, Hercules, CA, USA). The membranes used to detect phospho-PKA substrate protein were reused to detect the presence of phosphotyrosine residue or α-tubulin after applying Western blotting stripping buffer (TaKaRa Bio Inc., Kusatsu, Japan). The density of protein band was quantified using Image J 1.51 software downloaded from the NIH website (http://imagej.nih.gov/ij/). α-Tubulin was used as loading control.

For quality control of IPVL, protein composition and presence of ZPC protein were examined. Briefly, IPVL, outer perivitelline layer, and membrane consisting of these two layers (1 cm × 1 cm) were processed for SDS-PAGE. Proteins were separated and visualized with SYPRO Ruby (Thermo Fisher Scientific) [35]. Immunoblotting with ZPC antibody (1:10,000 dilution) was performed as described above. These experiments confirmed our ability to isolate intact IPVL freed from outer perivitelline layer (Supplementary Figure S1).

Motility assay

Sperm (1.0 × 10⁷ cells) were treated with 0, 1, 5, and 10 mM 2-OHCD in TES-NaCl buffer containing 2 mM Ca²⁺ for 1 h at 39°C and utilized for motility assay as described previously [37]. Briefly, sperm were observed under a microscope equipped with a warm plate. Sperm motility was recorded by digital videomicroscopy, and then categorized into either motile or immotile. At least 200 sperm for each sample were utilized for the evaluations.

Measurement of intracellular calcium ([Ca²⁺]i) content

Sperm (1.0 × 10⁷cells) were treated with 5 μM fluorescent Ca²⁺ indicator dye Fluo 3-AM in TES-NaCl containing 2 mM Ca²⁺ for 30 min at 39°C and centrifuged at 1,000 × g for 3 min to remove any extracellular dye. The sperm were resuspended in the medium and incubated with 0, 0.1, and 1 mM 2-OHCD for 30 min. Positive control was prepared by adding 1 μM calcium ionophore A23187 (A23187) to sperm suspensions.The fluorescence intensity in the sperm was measured using the DTX 800 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) with a 485 nm excitation filter and a 535 nm emission filter. No difference was observed in terms of fluorescence intensity among the media supplemented with different concentrations of only 2-OHCD.

Preparation of detergent-resistant membrane and G_M1 quantification

Sperm (1.0 × 10⁸ cells) were incubated with 0 or 1 mM 2OHCD in TES-NaCl for 1 h and then subjected to isolation of detergent-resistant membrane (DRM) which is biochemically correlated with membrane rafts [36]. Briefly, sperm membranes were isolated by Dounce homogenization and sonication in phosphate-buffered saline (PBS) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and centrifuged 10,000 × g for 10 min. Supernatant was further centrifuged at 20,000 × g for 2 h, separating membranes and cytosol. Resultant membrane pellet was treated with 0.5% Triton X-100 in PBS for 15 min at 4 ˚C and then centrifuged at 20,000 × g for 2 h to fractionate membranes based on insolubility. Soluble and DRM fractions were subjected to slot blotting for G_M1 quantification as described previously [32]. Briefly, samples were blotted using a Slot Blot Manifold (Hoefer, San Francisco, CA, USA) onto a PVDF membrane. The membrane was blocked and then incubated with 0.5 μg/mL CTB-HRP for 1 h at room temperature. The G_M1 expression was detected by chemiluminescence using ChemiDoc XRS + (Bio-Rad), and the resulting bands were subjected to densitometry as described above.

Cytosolic cAMP quantification

Sperm (1.0 × 10⁷ cells) were treated with 0, 0.1, and 1 mM 2-OHCD in TES-NaCl buffer containing 2 mM Ca²⁺ for 1 h at 39°C. Cytosolic cAMP was quantified using the Cyclic AMP Select EIA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. Briefly, after incubation, sperm from the same treatment were pooled (1.0 × 10⁸ cells), centrifuged at 10,000 × g for 5 min, and incubated in 0.1 M HCl for 20 min at room temperature. After additional centrifugation, the supernatant was subjected to the assay. The enzymatic reactions following the acetylcholinesterase competitive enzyme-linked immunosorbent assay were measured using a spectrophotometer at an excitation wavelength of 405 nm, and cAMP concentration in each sample was determined using the equation obtained from the standard curve plot.

Testicular RNA analysis

Testicular tissues were collected from matured RIR males and stored in liquid nitrogen. Total RNA was isolated using TRI reagent. For RT-PCR, 0.5 μg testis RNA was used for cDNA synthesis using SuperScript III (Invitrogen). Complementary DNA (100 ng) was used with Go Taq Green Master Mix (Promega). PCR products were separated in 1% agarose gel containing GelRed and visualized by ChemiDoc XRS + (Bio-Rad). Following primers were used for determination of the presence of AC isoforms. ADCY 1: forward 5΄-GGAGGATTGCC TGGGAAAGT-3΄, and reverse 5΄-CAGAGGCTGTCCTTAACGCT-3΄. ADCY 2: forward 5΄-CCTCCGGACTGGAAATGGAA-3΄, and reverse 5΄- AGATGCAGAGGTCAAGACGC-3΄. ADCY 3: forward 5΄-CTGCGCATAGGCATGAACAAG-3΄, and reverse 5΄-TGGTTTCAGATCCAGACCTCAG-3΄. ADCY 5: forward 5΄-GGCCATCTCATTAGTCCGGG-3΄, and reverse 5΄-CTGCCGGTTCATCTTGGCTA-3΄. ADCY 7: forward 5΄-CAGGCCTACCTGTGTCCTTG-3΄, and reverse 5΄-TGCACTGTTTGGACCTTGGA-3΄. ADCY 8: forward 5΄-CACTCTGGCTGCCCTAACAA-3΄, and reverse 5΄-GACCATTGGTATCACCCTGGA-3΄. ADCY 9: forward 5΄-GCCCTTCATGTAGTACCACCC-3΄, and reverse 5΄-CCTTCATGAGGCTGTCCTCT-3΄. ADCY 10: forward 5΄-TTGAAAGAGATTGCGCTGGC-3΄, and reverse 5΄-GCACAAAGGTGTGTGAGCAG-3΄.

Data analyses

STAT Plus (AnalystSoft Inc., Walnut, CA, USA) was used to analyze the data collected from at least four replicates of each experiment. Multiple comparisons were conducted using Tukey's honest significant difference test when normality assumptions and the equal variance were confirmed.

Results

Sterol removal inhibits phosphorylation of protein kinase A substrate protein and acrosome reaction

When chicken sperm were incubated with 0, 1, 5, 10, and 15 mM 2-OHCD, phosphorylation of PKA substrate proteins was inhibited in a dose-dependent manner with sharp difference in protein bands around 10, 50, and 100 KDa (Figure 1A). In contrast, there was no difference in the profile of tyrosine-phosphorylated proteins when the concentration of 2-OHCD was varied (Figure 1B). In sperm not incubated with 2-OHCD, IPVL stimulation significantly increased AR (32.2% ± 1.5%) compared with sperm without stimulation (7.2% ± 1.2%); however, when sperm were incubated with 0.1, 1, and 5 mM 2-OHCD, AR in response to IPVL stimulation was significantly decreased (21.0% ± 1.8%, 15.6% ± 0.2%, and 18.2% ± 1.2%, respectively) compared with that in the sperm incubated with no 2-OHCD. These was no difference in percentage of motile sperm among 2-OHCD concentrations (Supplementary Figure S2). 2-OHCD treatment alone increased spontaneous AR, which was induced without IPVL stimulation, in a dose-dependent manner (0.1 mM: 13.6% ± 0.6%, 1 mM: 16.2% ± 2.3%, 5 mM: 19.0% ± 2.9%), which resulted in no difference in the percentage of AR between those treated with and without IPVL stimulation (Figure 1C). A previous study of the signaling pathways associated with capacitation in hamster sperm demonstrated that [Ca²⁺]i plays a key role in spontaneous AR induction [38]; therefore, we measured [Ca²⁺]i in sperm incubated with 2-OHCD using a fluorescent calcium indicator dye and found that 2-OHCD treatment increased [Ca²⁺]i in a dose-dependent manner although no difference was observed among 2-OHCD concentrations when treated with A23187 (Figure 1D). Taken together, these results demonstrated that 2-OHCD–induced sterol removal suppressed AR in response to IPVL stimulation as well as stimulated spontaneous AR, most likely through Ca²⁺-dependent mechanisms, and suggested the role of membrane rafts in AR induction. In support with this view, our sterol and G_M1 quantification with sperm membranes demonstrated that 1 mM 2-OHCD treatment significantly reduced membrane sterol and G_M1 content of DRM (Figure 1E and F). These and the complete inhibition of IPVL-induced AR in sperm treated with ≥ 1 mM 2-OHCD allowed us to use it at 1 mM to induce sterol removal in subsequent experiments.

Figure 1.

Protein phosphorylation and AR in chicken sperm after sterol removal. Sperm were treated with different concentrations of 2-OHCD in TES-NaCl containing 2 mM Ca²⁺ medium for 1 h at 39°C and subjected to immunoblotting to detect phosphorylation of PKA sustrate and protein tyrosine residue or AR assay. (A) Phosphorylation of PKA substrate protein was inhibited by 2-OHCD treatment in a dose-dependent manner. (B) Protein tyrosine phosphorylation did not differ, even after sterol removal. (C) Sterol removal inhibited AR in response to IPVL stimulation (black bar) and stimulated AR that spontaneously occurred without IPVL stimulation. (D) [Ca²⁺]i increased after sterol removal. (E) Membrane sterol content in sperm decreased in response to 1 mM 2-OHCD treatment. (F) G_M1 content in sperm DRM decreased in response to 1 mM 2-OHCD treatment. Data are presented as mean ± SEM (n = 5). ^a-cP < 0.05.

Membrane rafts regulate acrosome reaction through the protein kinase A pathway

To identify an upstream mechanism involved in membrane raft regulation of PKA substrate protein phosphorylation, we incubated sperm with 1 mM 2-OHCD in the presence of 0, 0.5, 1, 5, and 10 mM dibutyryladenosine 3΄,5΄-cyclic monophosphate (dbcAMP). To compare degree of protein phosphorylation, we performed densitometry for relative abundance of 100 KDa phosphorylated proteins to α-tubulin. This is because proteins around 100 KDa are prominently phosphorylated in response to PKA activation in murine sperm [39] and, in contrast to this, phosphorylation profile of PKA substrate proteins has not been characterized in avian sperm. Treatment with 1 mM 2-OHCD alone diminished phosphorylation of PKA substrate proteins (Figure 2A and B) and AR in response to IPVL stimulation (13.0% ± 2.3%, Figure 2C) compared to that in the control (33.0% ± 2.0%); however, when the culture media was supplemented with ≥ 0.5 mM dbcAMP, the inhibition of both phosphorylation of PKA substrate proteins and AR was abolished (23.8% ± 2.3%, 23.4% ± 3.5%, 26.4% ± 3.3%, and 30%.4 ± 3.7%, respectively) in a dose-dependent manner. However, when sperm were treated with dbcAMP alone, there was no difference in phosphorylation of PKA substrate proteins and AR among concentrations (Figure 2D–F).These results demonstrated that membrane rafts are involved in AR induction by regulating cAMP-dependent pathways, including PKA.

Figure 2.

PKA substrate protein phosphorylation and AR in sperm in the presence of dbcAMP. Sperm were treated with or without 1 mM 2-OHCD and different concentrations of dbcAMP in TES-NaCl containing 2 mM Ca²⁺ medium for 1 h at 39°C and subjected to immunoblotting to detect the phosphorylation of PKA sustrate proteins and AR assay. (A) dbcAMP supplementation restored the inhibition of PKA substrate protein phosphorylation due to sterol efflux in a dose-dependent manner. (B) This was confirmed by densitometry for relative expression of 100 KDa phosphorylated proteins to α-tubulin. (C) Comcomitantly, the pecentage of sperm acrosomes that reacted was restored. (D and E) dbcAMP treatment alone did not change protein phosphorylation of PKA substrate. (F) AR percentage also did not change in response to dbcAMP treatment alone. Data are presented as mean ± SEM (n = 5). ^{a, b}P < 0.05.

Sterol removal inhibits cytosolic cAMP production

To determine the role of membrane rafts in the regulation of cAMP concentration in sperm, cytosolic cAMP was quantified in sperm incubated with 0, 0.1, and 1 mM 2-OHCD. 2-OHCD treatment decreased cAMP concentration (0.1 mM: 18.2 ± 2.8 pmol/mL and 1 mM: 14.4 ± 2.4 pmol/mL) compared with that in the control (26.3 ± 2.7 pmol/mL), and a significant difference was detected between the treatment with 1 mM 2-OHCD and the control (Figure 3). These results demonstrated that membrane rafts regulate cAMP concentration in sperm.

Figure 3.

Cytosolic cAMP quantification in sperm after sterol removal. Cyclic AMP content was quantified in sperm treated with 0, 0.1, and 1 mM 2-OHCD in TES-NaCl containing 2 mM Ca²⁺ medium for 1 h at 39°C. Cyclic AMP was decreased in response to sterol removal. Data are given as the mean ± SEM (n = 6). ^{a, b}P < 0.05.

Two adenylyl cyclase subtypes regulate protein kinase A substrate phosphorylation and the acrosome reaction

In sperm, cAMP synthesis is mainly mediated by the AC subtypes sAC and tmAC. Multiple lines of evidence from kinetic studies on cAMP synthesis showed specific stimulation of sAC and tmAC by NaHCO₃ and forskolin and inhibition by KH7 and MDL in mammalian sperm [40]. To identify the regulation mechanism and functional roles of cAMP, sperm were incubated with 2-OHCD, NaHCO₃, forskolin, KH7, and MDL. Supplementation with 10 and 25 mM NaHCO₃, and 10 and 50 μM forskolin showed tendency of the increase in phosphorylation of PKA substrate proteins (Figure 4A and B). However, neither stimulator treatment increased AR (ranging from 29.0% ± 0.5% to 33.8% ± 0.6%) over that in the control (31.6% ± 0.8%). In contrast, incubation with 25 μM KH7 and 50 μM MDL dramatically decreased protein phosphorylation, whereas no differences were observed from that in the control at 10 μM. Similarly, KH7 (10 μM: 18.8% ± 0.6%, 25 μM: 13.4% ± 0.4%) and MDL (10 μM: 17.8% ± 0.6%, 50 μM: 10.6% ± 0.5%) treatments significantly decreased AR in a dose-dependent manner (Figure 4C). Based on these findings, we performed transcriptome analysis of AC isoforms in chicken testis, showing prominent expression of ADCY 1, 3, 5, 7, 8, 9 of tmAC and 10 known as sAC (Figure 4D). These results demonstrated that the two AC subtypes are present in chicken sperm and play important roles in AR induction though the PKA pathway.

Figure 4.

PKA substrate protein phosphorylation and AR in sperm treated with specific stimulators and inhibitors of sAC and tmAC. Sperm were treated with 1 mM 2-OHCD and different concentrations of NaHCO₃, forskolin, KH7, and MDL in TES-NaCl containing 2 mM Ca²⁺ medium for 1 h at 39°C and subjected to immunoblotting for detection of phosphorylation of PKA sustrate proteins and AR assay. (A and B) PKA substrate protein phosphorylation was slightly stimulated, but not significant, by NaHCO₃ and forskolin treatments, although greatly inhibited by KH7 and MDL treatments. (C) AR was not stimulated by either NaHCO₃ or forskolin, but was inhibited by KH7 and MDL treatments. (D) Transcriptome analysis of AC isoforms in chicken testis showed the expression of ADCY 1, 3, 5, 7, 8, 9 of tmAC and ADCY 10 sAC. Data are presented as mean ± SEM (n = 5). ^{a, b}P < 0.05.

Sterol removal inhibits adenylyl cyclase activity

Our findings of the involvement of membrane rafts in cAMP regulation and the roles of sAC and tmAC in chicken sperm suggested that the inhibition of PKA substrate protein phosphorylation and AR in response to sterol removal might be a result of diminishment of AC activity. To test this, sperm were incubated with 2-OHCD in the presence of either 10 or 25 mM NaHCO₃ or 10 or 50 μM forskolin. Incubation with 2-OHCD alone decreased protein phosphorylation and AR (13.6 ± 0.6%) compared with that in the control (31.2% ± 1.3%), which is the same as the above results (Figure 5A–C); however, the presence of NaHCO₃ (10 mM: 24.2% ± 1.2%, 25 mM: 24.4% ± 1.8%) or forskolin (10 μM: 27.4% ± 1.4%, 50 μM: 36.8% ± 1.8%) restored both of them, and there was no difference in the percentage of AR between the results of the forskolin treatments and the control. These results demonstrated that membrane rafts are involved in cAMP synthesis through functional regulation of AC activities.

Figure 5.

PKA substrate protein phosphorylation and AR in sperm after sterol removal in the presence of specific sAC and tmAC stimulators. Sperm were treated with 1 mM 2-OHCD at different concentrations of NaHCO₃ and forskolin in TES-NaCl containing 2mM Ca²⁺ medium for 1 h at 39°C and subjected to immunoblotting to detect the phosphorylation of PKA sustrate proteins and AR assay. (A and B) Inhibition of PKA substrate protein phosphorylation was abolished in the presence of NaHCO₃ and forskolin. (C) The percentage of sperm acrosomes that reacted was restored after treatment with the stimulators. Data are presented as mean ± SEM (n = 5). ^{a, b}P < 0.05.

Building on these data, we propose a model to assess how membrane rafts support induction of AR in chicken sperm (Figure 6). The plasma membranes of sperm retain multiple membrane rafts that act as a scaffold/assembly platform for the functional regulation of sAC and tmAC. This interaction supports cAMP production, which enhances the phosphorylation of PKA substrate proteins, leading to AR induction in response to IPVL stimulation.

Figure 6.

Schematic model of membrane raft regulation of AR in chicken sperm. The plasma membranes of sperm consist of multiple membrane rafts that act as scaffold/assembly platforms for the functional regulation of sAC and tmAC. This interaction supports cAMP production, thereby enhancing PKA substrate protein phosphorylation, leading to AR induction in response to IPVL stimulation.

Discussion

Our previous findings on the presence of membrane rafts in the plasma membrane overlying the sperm head region and the functional importance of these domains in mammalian sperm motivated us to investigate the membrane raft's role in regulating the signaling pathways leading to AR in response to physiological stimulation. Our results showed that membrane rafts play an important role in AR induction through the regulation of the cAMP-dependent pathways, including PKA. These findings add to our knowledge of the mechanisms by which AR is induced in avian sperm, highlighting the similarities and differences between mammalian and avian sperm in terms of the behavior of the signal transduction events that precede AR.

In previous studies, sterol removal by sterol acceptors resulted in disorganization of membrane rafts [41, 42]. In this study, we found that phosphorylation of PKA substrate proteins and IPVL-induced AR were inhibited in response to sterol removal in chicken sperm. Previous pharmacological experiments on chicken sperm demonstrated that inhibition of PKA activity resulted in diminished AR without affecting the population of motile sperm [19]. In concert with these results, we confirmed no change in percentage of motile sperm after sterol removal. Together with the present finding that dbcAMP supplementation abolished the inhibition caused by sterol removal, our results suggest that the disorganization of the membrane rafts inhibits PKA activity, thereby reducing the AR ability in chicken sperm. 2-OHCD treatment is known to result in the elevation of acrosomal responsiveness to physiological stimulation by lowering sterol content in human [43, 44] and murine sperm [45]. This is contrary to the present finding that the ability to undergo AR was diminished by sterol removal. This discrepancy might be the result of a distinction between signal transduction pathways associated with AR induction in mammals and birds. In mammalian sperm, sterol removal stimulates the phosphorylation of protein tyrosine followed by PKA activation, leading to an increase in acrosomal responsiveness [45, 46]. In contrast, sterol removal in chicken sperm did not affect the phosphorylation of protein tyrosine, but inhibited both PKA activity and AR, suggesting that the activation of tyrosine kinase(s) is not involved in the cAMP-dependent pathway in avian sperm. This is supported by evidence that stimulators for the cAMP-dependent pathway did not increase protein tyrosine phosphorylation in quail sperm [47]. In mammalian sperm, protein tyrosine phosphorylation is a major event that signals capacitation and is largely regulated by sAC activity. Our results will provide a foundation for investigating why capacitation is not necessary for avian sperm to undergo AR.

Despite the distinction between mammalian and avian sperm in terms of the signaling pathways that precede AR, it is intriguing that, similar to that in mammalian sperm, sterol removal stimulated spontaneous AR in chicken sperm [11, 43]. A previous study on murine sperm showed that extracellular Ca²⁺ potentiates the initiation of spontaneous AR [48]. In addition, our results showed that [Ca²⁺]i significantly increases in response to sterol removal in chicken sperm. These results suggest that 2-OHCD–induced spontaneous AR results from an increase in [Ca²⁺]i.

A previous study performed on murine sperm demonstrated that PKA activity is suppressed by Ser/Thr phosphatases during the noncapacitated state [39]. Multiple Ser/Thr phosphatases were found in chicken sperm and appeared to be involved in AR [17, 49], which brought up the possibility of the involvement of Ser/Thr phosphatases in the inhibition of PKA substrate protein phosphorylation by sterol removal; therefore, changes in cytosolic cAMP content were examined in chicken sperm after sterol removal. The results showed that PKA inhibition was a result of a decrease in cAMP content. Cytosolic cAMP levels depend on the concerted action of both synthesis and degradation. In other cells, several isoforms of ACs were found to be tethered to membrane rafts and this is of importance for compartmentalized localization of regulatory modules for cAMP-dependent signaling pathways [50, 51]. A study on ciona sperm demonstrated that a disruption of the membrane rafts by sterol removal inhibited cytosolic cAMP synthesis by suppressing AC activity [14]. These reports and our results suggest that a regulatory module for the cAMP-dependent pathway might target the membrane rafts in chicken sperm.

Although multiple tmACs were shown to exist in mammalian sperm based on mRNA expression [22], their presence and roles remain controversial because there is a discrepancy in the effect of forskolin, an tmAC stimulator, on cAMP production in mammalian sperm [40]. On the other hand, sAC is insensitive to forskolin but is regulated by bicarbonate ion. A previous study performed using mutant mice devoid of sAC and a specific inhibitor demonstrated that sAC contributes to protein tyrosine phosphorylation and motility; however, we found no reports of studies on the presence of either tmAC or sAC in avian sperm. Previous studies reported in mammalian sperm that bicarbonate and forskolin elevated cytosolic cAMP [52, 53] as well as stimulated AR [54, 55]; however, we found that neither stimulators increased AR in response to physiological stimulation, suggesting that, unlike in mammalian sperm, an increase in cAMP is not necessary for chicken sperm to undergo AR. This suggestion is supported by a previous study that showed that stimulation of sAC by bicarbonate did not increase AR in chicken sperm [56]. This difference might be a result of a distinction in sperm functions. In mammalian sperm, cAMP regulates the initiation of capacitation, which then stimulates AR. Avian sperm does not undergo this process to prepare for AR induction; however, it is intriguing that inhibitors for both isoforms decreased AR, concomitant to the disappearance of the phosphorylation of PKA substrate proteins, in a dose-dependent manner. Considering that our transcriptome analysis of AC isoforms identified testicular expression of ADCY1, 2, 3, 5, 7, 8, 9, and 10, this suggests the involvement of both isoforms in chicken sperm AR. In mammalian sperm, several lines of evidence have implicated that AR does not appear to need sAC activity [21, 57], but does rely on tmAC. Recently, it was reported that the cAMP synthetic pathway is compartmentalized in sperm as follows: sAC in the sperm tail for motility and tmAC in the sperm head for AR [22]. Our results showed a distinction between mammalian and avian sperm functions and provide a foundation to promote the characterization of both AC isoforms in avian sperm.

In a previous study, sAC activity was found to be associated with the plasma membranes in mammalian sperm, but the mechanism of this activity remains unclear [58]. On the other hand, tmAC consists of nine members, five of which were found to be localized in membrane rafts through biochemical interactions in other cells [50], suggesting that membrane rafts might regulate tmAC function. These results, combined with our findings of the functional involvement of AC isoforms in chicken sperm, led to a hypothesis that inhibition of PKA substrate protein phosphorylation and AR in response to sterol removal might be restored by either bicarbonate or forskolin treatment. In agreement with this, we observed that both bicarbonate and forskolin abolished the inhibition of phosphorylation of PKA substrate proteins and AR in 2-OHCD-treated chicken sperm. Interestingly, we found that stimulation with forskolin restored AR more potently than bicarbonate. This is consistent with recent results that AR is predominantly regulated by tmAC and that sAC is not needed for AR in murine sperm [20, 22, 57]. In a previous study, the G protein subunit responsible for tmAC activation was localized in the plasma membrane overlying the acrosome [22]. In fact, we have shown, using live cell imaging, that this membrane region contains multiple membrane rafts [7, 8]. Similarly, membrane rafts were found in the plasma membrane overlaying the sperm head in chicken sperm [15]. These reports taken in conjunction with a strict linkage identified between sterol removal and phosphorylation of PKA substrate proteins in mammalian and bird sperm; our results suggest the need for further investigations to more fully characterize the functional and spatial distinctions between tmAC and sAC in avian sperm.

Supplementary data

Supplementary Figure S2. Protein composition analyses of IPVL. Egg membranes were separated into outer perivitelline layer (OPVL) and IPVL, and subjected to SDS-PAGE for immunoblotting for the presence of ZPC protein and dissection of whole protein composition. (A) Only total membranes and IPVL showed the expression of ZPC at predicted molecular weight. (B) IPVL were largely devoid of ovoalbumin (45 KDa) and ovotransferrin (76 KDa), major egg white proteins, while whole egg membranes and OPVL contained them.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Notes

Conference presentation: Presented at the Annual meeting of the Society for the Study of Reproduction 2017, 13–16 July 2017, Washington DC.

Edited by Dr. Monika A. Ward, PhD, University of Hawaii John A. Burns School of Medicine

Footnotes

†

Grant support: This work was supported by Japan Society of the Promotion of Science (JSPS) KAKENHI 15K07761 (to Atsushi Asano).