Protein Kinase a Activity Leads to the Extension of the Acrosomal Process in Starfish Sperm

Received: 12 September 2016 | Accepted: 26 April 2017 DOI: 10.1002/mrd.22824

RESEARCH ARTICLE

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm

1 Department of Biological Sciences and Informatics, Keio University, Yokohama, Japan Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, 2 School of Marine Biosciences, Kitasato which is immediately followed by the actin polymerization-dependent extension of an University, Sagamihara, Kanagawa, Japan acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large 3 School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal 4 Department of Molecular Biology, Cellular sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome Biology and Biochemistry, Brown University, reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, Providence, Rhode Island and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+. Correspondence Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing Midori Matsumoto, Department of Biological Sciences and Informatics, Keio University, 3-14-1 AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay Hiyoshi, Kouhoku-ku, Yokohama 223-8522, was used here to differentiate between AV exocytosis and AP extension in starfish Japan. Email: [email protected] sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of Funding information Brown University, Grant number: Japanese target proteins occurs, and these substrates localize at the base of the AP, Students-Brown support; Grant-in-Aid for demonstrating that PKA activation regulates an AP extension step during the Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, acrosome reaction. The major PKA substrate was further identified, from A. amurensis Science and Technology of Japan (MEXT), and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation Grant number: 22112520; National Institutes of Health, Grant number: HD028152; motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin National Science Foundation, Grant number: polymerization during the acrosome reaction. IOS-1120972

KEYWORDS acrosome reaction, actin polymerization, Asterias amurensis, protein kinase A

1 | INTRODUCTION extension of the acrosomal process (AP) by actin polymerization (Dan, 1954; Hoshi et al., 1994). An obvious AP can be observed in Theacrosomereactionisessentialforspermtofusewithanegg starfish sperm; it is approximately 25 μm long, penetrates the egg in most animals. In echinoderm sperm, the acrosome reaction jelly, and binds to the vitelline layer (Tilney, Kiehart, Sardet, & involves exocytosis of acrosomal vesicle (AV) contents and Tilney, 1978; Vacquier, Swanson, & Hellberg, 1995). Although exocytosis and actin polymerization are known to occur Abbreviations: AP, acrosomal process; AV, acrosomal vesicle; PKA, Protein kinase A; PKC, sequentially in the AR, mechanistic relationships between these Protein kinase C. processes remain unclear (Levine & Walsh, 1979; Ikadai & Hoshi,

Keisuke Niikura and Shahanoor M. Alam contributed equally to this work. 1981).

The acrosome reaction in Asterias amurensis is induced by 2 | RESULTS cooperative action of three components within the egg jelly: a sulfated proteoglycan-like molecule designated as acrosome 2.1 | Phalloidin-modified acrosome reaction assay reaction-inducing substance (ARIS); a group of sulfated steroidal Erythrosine staining was originally used to quantify starfish sperm AP saponins, named cofactor for ARIS (Co-ARIS); and the sperm- extension in an acrosome reaction assay (Kawase et al., 2005; Matsui activating peptide, asterosap (Koyota, Wimalasiri, & Hoshi, 1997; et al., 1986; Naruse et al., 2010). This novel phalloidin-modified assay, Matsui,Nishiyama,Hino,&Hoshi,1986;Nishigaki,Chiba,Miki,& modified from a method developed for visualizing the acrosome Hoshi, 1996; Nishiyama, Matsui, Fujimoto, Ikekawa, & Hoshi, reaction of sea urchin sperm (Vacquier & Hirohashi, 2004), was 1987). ARIS is the main factor that induces the acrosome reaction, developed to distinguish between AV exocytosis and AP extension and its activity depends on sugar chains (Koyota et al., 1997; steps. Hoechst 33342 staining revealed the presence or absence of Matsui et al., 1986). Co-ARIS alters the microdomain of the sperm the intact AV, which protrudes into the nucleus prior to the acrosome membrane, facilitating the binding between ARIS and the ARIS reaction (as seen by a Hoechst-null area) but is lost following the receptor that surrounds the AV (Naruse et al., 2010). Asterosap release of the AV (Figure 1a–c). Similar to the assay developed for sea binds to its receptor, a guanylate cyclase present on the flagella urchin sperm, staining with fluorescently labeled phalloidin allowed (Matsumoto et al., 2003; Nishigaki, Chiba, & Hoshi, 2000), that for visualization of the actin filaments in the AP, which is facilitated by transiently raises the concentrations of intracellular cGMP the additional length of the AP in starfish sperm. 2+ 2þ ([cGMPi]), intracellular pH ([pHi]), and intracellular Ca ([Cai ]), together inducing sperm chemotaxis (Kawase, Minakata, Hoshi, & Matsumoto, 2005; Nishigaki et al., 2000; Shiba et al., 2006). 2.2 | PKA inhibitors block AP extension but not AV Continual accumulation of intracellular cyclic AMP ([cAMPi]) and exocytosis 2þ [Cai ] requires both ARIS and asterosap (Islam, Kawase, Hase, Hoshi, & Matsumoto, 2006; Kawase et al., 2005). cAMP functions Pretreatment of sperm with a PKA-inhibitor prevents the as a second messenger, via Protein kinase A (PKA) activation. acrosome reaction in a concentration-dependent manner (Islam Studies conducted using starfish sperm showed that et al., 2006), but whether this PKA signaling causes AP extension or AV exocytosis is unknown. We therefore used the starfish [cAMPi] increases only in the presence of both ARIS and asterosap, and that the inhibition of cAMP-dependent protein kinase activity acrosome reaction assay to investigate the effects of PKA (e.g., PKA) suppresses the acrosome reaction (Islam et al., 2006). inhibition on each event. An intact AV and the actomere, the Therefore, PKA activation is important for the ARIS- and site of actin filament nucleation, were observed in an acrosome- – asterosap-induced acrosome reaction. intact sperm head (Figure 2a d). In contrast, control egg jelly- PKA plays a prominent role during the acrosome reaction in the induced acrosome-reacted sperm underwent AV exocytosis, and sperm of many organisms—from mammals to echinoderms. In sea the AP was easily observed via actin polymerization. We further urchins, fucose sulfate polymer, an acrosome reaction-inducing noted a coordinated loss of the unstained AV region within polysaccharide, activates adenylate cyclase, leading to an increase in the nucleus and the appearance of a stained, extended AP (Figure 2e–h). Starfish sperm pretreated with a PKA inhibitor, [cAMPi] levels (Garbers & Kopf, 1980; Garbers, Tubb, & Kopf, 1980; Su, Chen, Zhou, & Vacquier, 2005; Watkins, Kopf, & Garbers, 1978). KT5720, followed by egg jelly treatment resulted in loss of the How PKA initiates the acrosome reaction is still unknown. The AP of unstained region (AV), similar to that of control acrosome-reacted sea urchin sperm is very short, around 1 μm, so definitively observing sperm, whereas actin filaments remained aggregated within a – the acrosome reaction in these sperm is a challenge when using a spotatthetopofthespermhead(Figure2il). Quantitative light microscope for longitudinal studies (Dan, Ohori, & Kushida, analysis of these results revealed no significant differences 1964). Polymerization of actin filaments can be observed by between the sperm treated with or without KT5720 for AV fluorescence staining, which is the basis of an acrosome reaction exocytosis rate (Figure 2m), versus significant reduction in AP assay for sea urchin sperm; observation of AV exocytosis, however, extension rate (p < 0.001) (Figure 2n). requires antibodies and additional immunolabeling approaches (Vacquier & Hirohashi, 2004). In contrast, the acrosome reaction 2.3 | Identification of the PKA target substrate in starfish sperm can be investigated simply using erythrosine staining to visualize the 25-μm-long AP (Kawase et al., 2005; Matsui We next sought to identify the proteins phosphorylated by PKA et al., 1986; Naruse et al., 2010). during the acrosome reaction, using an anti-phospho-PKA sub- In this study, we modified the erythrosine-staining assay used strate monoclonal antibody that recognizes RRXT/RRXS sequences for measuring starfish sperm so that AV exocytosis and AP containing phosphorylated Thr/Ser residues that comprise a part of extension can be evaluated separately. This allowed us to the motifs phosphorylated by PKA. We detected a ∼60-kDa band investigate the role of PKA signaling during the acrosome reaction, that increased in intensity in lysate from acrosome-reacted sperm as well as to identify PKAS1, a novel PKA substrate in starfish compared to acrosome-intact sperm (Figure 3a). Pretreating sperm. starfish sperm with PKA inhibitors, followed by induction of the 616 | NIIKURA ET AL.

FIGURE 1 Evaluation of the exocytosis of the AV and AP extension through nucleus and actin filament staining. (a) Illustration of acrosome-intact sperm with an AV (a′) and an acrosome-reacted sperm undergoing AV exocytosis (b′) and AP extension (c′). (b and c) Acrosome-intact (b) and acrosome-reacted sperm (c) stained with Hoechst 33342 and Alexa Fluor 488 phalloidin. Scale bars, 1 μm. Ac, actomere, AP, acrosomal process; AV, acrosome vesicle; F, flagella; M, mitochondria; N, nucleus acrosome reaction with egg jelly, resulted in a significant decreased 2.5 | Identification of the PKA substrates in signal of the 60-kDa phosphorylated band; this phenotype was Gel electrophoresis and purification of the 60-kDa protein was performed to observed following pretreatment with two different PKA inhib- determine the sequence of this polypeptide using hydroxylamine cleavage itors, KT5720 (Figure 3a) and Rp-8-Br-cAMPS (Figure 3b). Two- methodology. The N-terminal sequence of a fragment derived from this dimensional gel electrophoresis and Western blot analyses in excised, 60-kDa A. amurensis protein was determined to be NH2-(N) acrosome-intact sperm showed a faint spot at 60 kDa and at an GKHXEMAQQS-CO2H; this sequence was not similar to any known protein. isoelectric point (pI) 5.61 that is distinct from the spot in acrosome- A similar 60-kDa band was detected in the sperm lysate of Asterias reacted sperm, which appeared as a band at 60 kDa that stretched forbesi (Figure 5a), a species whose ovary transcriptome database has from a pI of 3.71–5.12 (Figure 3c). This change in pattern indicates been assembled (Reich, Dunn, Akasaka, & Wessel, 2015). This protein that the immunodetected protein is phosphorylated during the was then purified from A. forbesi acrosome-reacted sperm lysate by acrosome reaction. immunoprecipitation with the anti-phospho-PKA substrate monoclonal antibody, and analyzed by tandem mass spectrometry using the A. forbesi ovary transcriptome database as a reference (Table 1). The 2.4 | Localization of the PKA substrate in starfish most suitable candidate that matched the PKA substrate fingerprint was acrosome-reacted sperm Af005000T1, which contains six motifs recognized by the anti- Immunocytochemistry using egg jelly-treated sperm revealed that the phospho-PKA substrate antibody (one RRXT and five RRXS sites) putative PKA target substrate resides at the base of the AP, in the same (Figure 5b). Af005000T1 and the A. amurensis homolog were designated region as the actin filaments (Figure 4a–f). as AfPKAS1 and AaPKAS1, respectively, for PKA substrate 1. NIIKURA ET AL. | 617

FIGURE 2 Inhibition of PKA blocks AP extension but not AV exocytosis. (a–d) Acrosome-intact sperm. (e–h) Acrosome-reacted sperm. (i–l) Sperm pre-incubated with 10 μM KT5720 before the egg jelly (EJ)-induced acrosome reaction. All sperm were stained with Hoechst 33342 (a, e, i) and Alexa Fluor 488 phalloidin (b, f, j). Fluorescent images were merged (c, g, k), and overlaid onto the phase-contrast images (d, h, l). Scale bars, 1 μm. (m and n) Quantitative determinations of AV exocytosis (m) and AP extension rate (n) in starfish sperm. Results are expressed as mean ± standard deviation. *p < 0.001 from three replicates; each experiment was calculated using 100 sperms in all samples

2.6 | Identification of AfPKAS1 and AaPKAS1 polyclonal antibody in the serum of guinea pigs, with a fusion protein transcripts from starfish testes composed of glutathione-S-transferase fused to a partial AfPKAS1 protein fragment (Figure S1) to determine if AaPKAS1 and AfPKAS1 are the same The complete cDNA sequences coding for AaPKAS1 and AfPKAS1 were molecules identified as 60-kDa bands in immunoblot analyses. This serum obtained from A. amurensis and A. forbesi testes, respectively, using antibody recognized a 60-kDa band, similar to the one recognized by the reverse-transcription PCR and 5′-rapid amplification of cDNA ends. The anti-phospho-PKA substrate monoclonal antibody (Figure 6). full-length cDNA (1,645 bp) encodes a protein containing 457 amino The signal of the anti-phospho-PKA substrate monoclonal antibody acid residues (calculated molecular weight of 54.3 kDa) with no and the anti-AfPKAS1 serum co-localized in both acrosome-intact and previously reported domains recognized by BlastP or Interproscan. acrosome-reacted sperm of A. amurensis (Figure 7). In acrosome-intact Therefore, we used Scansite to find short sequence motives in AaPKAS1 sperm, these signals were detected in the outer membrane of the and AfPKAS1, and identified several consensus PKA, Protein kinase C acrosome on the nucleus side, while in the acrosome-reacted sperm (PKC), Casein kinase, and AKT kinase phosphorylation sites, as well as an cells, these signals were detected at the base of the AP, with some ERK D-domain and PIP3-binding PH sequences. Six of the identified staining also detected in the flagella. We attempted to detect AaPKAS1 sites (Thr109, Ser125, Ser201, Ser314, Ser362, and Ser451) are in theimmature or mature eggs of A. amurensis usinganti-AfKAS1 serum, potentially phosphorylated by PKA (Figure 5b). but we did not observe any distinct signals (data not shown). AaPKAS1 and AfPKAS1 were amplified in the samples obtained both from the ovaries and from the testes of A. amurensis and A. forbesi, respectively, by reverse-transcription PCR, in order to determine if they 2.8 | AaPKAS1 phosphorylated sites during AR are expressed in both of these tissues. The transcripts were detected in The amino acid sequence of AaPKAS1 contained six potentially both testes and in ovaries, although AaPKAS1 was more highly phosphorylated sites by PKA (Thr109, Ser125, Ser201, Ser314, expressed in the testis than in the ovary of A. amurensis (Figure 5c). Ser362, and Ser451), so we attempted to identify PKA-mediated phosphorylation sites in AaPKAS1 during the acrosome reaction (Figure 5b). Tandem mass spectrometry analysis identified potential 2.7 | AaPKAS1 represents a PKA substrate phosphorylation sites, such as Thr109 and Thr117 in AaPKAS1 (Table 2; Finally, to determine if AaPKAS1 and AfPKAS1arethesamemolecules Figure S2). The consensus of these results suggests that Thr109 may be identified as 60-kDa bands in Western blot analyses, we generated a the important phosphorylation site of AaPKAS1 that is targeted by PKA. 618 | NIIKURA ET AL.

FIGURE 3 Western blot of sperm lysates using anti-phospho-PKA substrate antibody. (a–b) Western blot detection of a ∼60-kDa band in acrosome-intact sperm that was intensified in acrosome-reacted sperm lysate induced by egg jelly (EJ). This effect was abolished in sperm pretreated with 10 μM KT5720 (a) and 100 μM Rp-8-Br-cAMPS (b), which are both PKA inhibitors. Left, SDS–PAGE; right, Western blot. (c) Two-dimensional Western blot revealing the change in isoelectric point of the ∼60-kDa band from approximately 5.61 (black arrows) to 3.71–5.12 (white arrows) after the acrosome reaction

3 | DISCUSSION Sperm behavior critically depends on PKA in all species studied to date. In mammalian sperm, activated PKA participates in capacitation PKA signaling regulates a wide variety of biological events, including events that precede the acrosome reaction, and may induce actin diverse metabolic processes, vasodilation, and calcium sequestration. polymerization through a tyrosine kinase intermediate (Breitbart & Differences in the functions of PKA within cells derive from how cells Etkovitz, 2011; Visconti et al., 2005). cAMP/PKA-dependent phos- are stimulated to accumulate cAMP, which eventually activates PKA, phorylation of LIMK1 and cofilin is also essential for mammalian sperm resulting in substrate phosphorylation and active signaling. For acrosome exocytosis (Romarowski et al., 2015). A brief elevation of example, PKA phosphorylation of LIMK1 in cultured cells results in intracellular Ca2+ is sufficient to activate the cAMP/PKA pathway during the aggregation of F-actin (Nadella et al., 2009). capacitation and the acrosome reaction (Tatenoa et al., 2013). The exact NIIKURA ET AL. | 619

FIGURE 4 Immunocytochemical analyses of acrosome-reacted A. amurensis sperm using an anti-phospho-PKA substrate antibody. (a–c) Antibody localization of PKA substrate(s) around the base of the acrosomal process. (d–f) Control treated with IgG. Acrosome-reacted sperm was stained with Hoechst 33342 (a and d) and Alexa Fluor 488 goat anti-rabbit IgG (b and e). Fluorescence images were merged and overlaid onto the phase-contrast images (c and f). Scale bars, 1 μm stimulus that triggers the PKA-dependent acrosome reaction in reaction. This PKA substrate, identified as AaPKAS1, resides at mammalian sperm remains controversial, and likely includes more the base of the AP, suggesting that it contributes to AP extension by factors than proposed in the classically defined ZP3 model (Inoue, regulating actin polymerization. AaPKAS1 was found to contain six Satouh, Ikawa, Okabe, & Yanagimachi, 2011; Jin et al., 2011; PKA phosphorylation motifs (RRXT/RRXS), implying that AaPKAS1 Yanagimachi, 2011). and AfPKAS1 may be regulated by various kinases and could recruit The acrosome reaction has been studied extensively in both signaling factors during the acrosome reaction. For example, starfish and sea urchin sperm, in which PKA also plays an essential and AaPKAS1 is localized around the actomere, where it may contribute conserved role (Yanagimachi, 2011). We previously showed that PKA to AP extension by interaction with kinases such as PKA. The inhibitors suppressed the acrosome reaction of starfish sperm in a Scansite program also identified motifs in AaPKAS1 that may be dose-dependent manner (Islam et al., 2006)—although the exact role of phosphorylated by PKA, PKC, Casein kinase, and AKT kinase, as well PKA activation during the acrosome reaction remains unclear. We as ERK D-domain and PIP3-binding PH sequences. The ERK D- therefore developed a novel approach to dissect and quantitatively domain is involved in the interaction between ERK and its substrate evaluate the key steps that comprise the acrosome reaction in starfish (Fernandes & Allbritton, 2009) while PIP3-binding PH sequences sperm, that is, AV exocytosis and AP extension. We additionally interact with PIP2 or PIP3 within cell membranes (Harlan, Hajduk, analyzed the effects of PKA inhibitors on the various steps of the Yoon, & Fesik, 1994). starfish acrosome reaction using two different PKA inhibitors: AaPKAS1 differs from PKA substrates previously identified in sea KT5720, which interferes with ATP binding to the catalytic subunits urchin sperm—such as Adenylate kinase 1, Phosphodiesterase 5, and of PKA (Engh, Girod, Kinzel, Huber, & Bossemeyer, 1996; Kase et al., EPS8 (Su et al., 2005)—and the sea urchin genome does not encode an 1987), and the cAMP analog Rp-8-Br-cAMPS, a potent competitor of AaPKAS1 ortholog. The closest functional homology for PKAS1 is cAMP binding to PKA regulatory subunits (Gjertsen et al., 1995). These EPS8, which regulates actin dynamics through end capping (via its distinct PKA inhibitors elicited similar acrosome-reaction phenotypes C-terminal amphipathic helix H1) and bundling of actin filaments on starfish sperm, suggesting that PKA activation contributes (through its H2-H5 helices) (Hertzog et al., 2010). Might differences in selectively to AP extension by regulating actin polymerization. AP length between starfish and sea urchin help explain the A key to understanding the function of PKA in sperm regulation evolutionary selection for these distinct modes of PKA-dependent is the identification of PKA substrates. We discovered a 60-kDa actin polymerization in sperm of sea urchin (EPS8) versus starfish protein phosphorylated by PKA during the starfish acrosome (AaPKAS1)? Among Echinoderms, Crinoidea (crinoids) and sea urchins 620 | NIIKURA ET AL.

FIGURE 5 Identification of AfPKAS1 and AaPKAS1. (a) Western blot analyses of A. forbesi sperm lysate, using anti-phospho-PKA substrate antibody. (b) The amino acid sequence of A. amurensis and A. forbesi Af005000T1. Letters indicate motifs identified by Scansite using “High Stringency” conditions. The sequence determined by hydroxylamine cleavage method is underlined. A, phosphorylated by Casein kinase 2; B, Erk D-domain; C, D, E, H, I, J, phosphorylated by PKA; F, PIP3-binding PH; G, phosphorylated by PKC; E, phosphorylated by PKA, PKC, and Akt kinase. (c) AfPKAS1 gene expression analysis, using total RNA obtained from testis and egg

possess a short AP whereas starfish, Holothuroidea (sea cucumbers), from Woods Hole Marine Biological Laboratory, USA. Dry sperm from and Ophiuroidea possess long APs (Naruse et al., 2011). Furthermore, the adults were collected as previously described (Matsui et al., 1986). Hemichordata, Cephalochordata, and Ctenophora sperm also form Egg jelly and ARIS were prepared as previously described (Koyota et al., long APs (Colwin & Colwin, 1963; Morisawa, Mizuta, Kubokawa, 1997; Matsui et al., 1986). Kinase inhibitors KT5720 and Rp-8-Br- Tanaka, & Morisawa, 2004). Therefore, how actin polymerization of cAMPS were purchased from Sigma–Aldrich (St. Louis, MO) and PKA substrates occurs may be conserved in many species; we predict dissolved in dimethylsulfoxide (DMSO). that PKAS1 orthologs function in their sperm as well. AaPKAS1 is present in both testes and ovaries of starfish, and is 4.2 | Acrosome reaction assay and fluorescence likely used by embryos as well. Although regulation of PKAS1 in these imaging different cell types is likely through distinct mechanisms, we hypothesize that this protein is a more general regulator of diverse The acrosome reaction assay was performed as previously described actin dynamics employed in multiple starfish cell types. Our novel (Naruse et al., 2010) with some modifications. Briefly, 1 μl of dry sperm findings thus provide a foundation for further research exploring PKA was resuspended in 1 ml of ice-cold artificial seawater (ASW) (423 mM function in an array of cells types. NaCl, 9 mM KCl, 9 mM CaCl2, 25 mM MgSO4, 23 mM MgCl2,10mM EPPS, NaOH pH 8.2), and incubated on ice for 10 min with or without inhibitors. The sperm suspension (20 µl) was added to 80 μl of egg jelly 4 | MATERIALS AND METHODS (1 mg sugar/ml), incubated on ice for 5 min to induce the acrosome reaction, and then 20 μl of 5% (v/v) glutaraldehyde in ASW were added 4.1 | Materials to each sample to fix the sperm. Fixed sperm (100 μl) were placed onto A. amurensis samples were collected from the Otsuchi Bay and Tokyo glass slides and incubated for 30 min to bind to the glass. After washing Bay, Japan, and Sandy Bay, Australia. A. forbesi samples were collected with ASW, 100 μl of Staining Solution (0.05% [v/v] Triton X-100, 0.4 U NIIKURA ET AL. | 621

TABLE 1 List of significant PKA target proteins, obtained by MS/MS Protein IDs

Accession Protein Unique #of #of Grp number Protein name score PSMa RRXT RRXS 1 Af005000T1 None 1,747.73 37 1 5 2 Af000909T4 Glutamate_dehydrogenase 1,129.63 23 0 0 3 Af002783T6 T-complex protein 1 subunit theta 436.7 9 0 0 4 Af001362T12 T-complex protein 1 subunit alpha 383.32 10 0 0 5 Af001704T6 Actin 241.44 7 0 0 6 Af001904T14 T-complex protein 1 subunit beta 236.2 7 0 0 7 Af016711T1 Spermatogenesis associated 18 202.27 5 1 0 8 Af014085T6 none (chromosome segregation protein: SMC domain) 198.15 4 0 0 9 Af007517T1 Delta-1-pyrroline-5-carboxylate_dehydrogenase 192.69 4 0 0 10 Af000863T22 T-complex protein 1 subunit eta 162.28 4 0 0 11 Af000196T10 ATP_synthase alpha_subunit 158.68 3 0 0 12 Af032762T1 Adenylate_kinase 128.17 3 0 0 13 Af000828T7 T-complex protein 1 subunit zeta 115.21 2 0 0 14 Af000524T1 Atlastin-1 95.83 2 0 0 15 Af028663T5 FAS-associated factor 2 93.12 2 0 0 16 TBA_XENLA Tubulin alpha chain (OS = Xenopus laevis GN = tuba PE = 2 SV = 2) 85.25 2 0 0 17 Af001656T4 Intraflagellar transport protein 46 78.15 2 1 0 18 Af001844T5 Acyl-dehydrogenase 70.55 2 0 0 19 Af001272T5 Methylmalonate-semialdehyde_dehydrogenase 61.33 2 0 0 20 Af008818T1 Fumarate_class_I 60.89 2 0 0 21 SRA3_CAEEL Serpentine receptor class alpha-3 (OS = Caenorhabditis elegans 59.51 2 0 0 GN = sra-3)

PSM, peptide spectrum match; RRXS and RRXT, PKA phosphorylation site sequences.

Alexa Fluor 488 phalloidin [Life Technologies, Bethesda, MD], centrifugation (12,000g, 15 min), the supernatant containing the 10 μg/ml Hoechst 33342 [Sigma–Aldrich] in ASW) were added and sperm lysate was collected from each sample. The concentration of incubated on ice for 1–2 hr in the dark. After further washing, sperm each sperm lysate was measured using a BCA Protein Assay Kit samples were observed and scored using an IX70 and DP72 (Thermo Scientific, Waltham, MA) and stored at −30°C. fluorescence microscope (Olympus, Tokyo, Japan) at 1,000× magnification. One hundred sperm cells were scored per sample. 4.4 | Preparation of serum containing polyclonal antibody against AfPKAS1 4.3 | Protein extraction from starfish sperm Polyclonal antibodies against AfPKAS1 (amino acid residues 92- 458) Dry sperm (20 μl) were resuspended in 1 ml of ice-cold ASW, and were generated in guinea pigs. Guinea pig serum containing anti- incubated on ice for 10 min with or without inhibitors. Sperm AfPKAS1 antibodies was then used for Western blot and immunocy- suspension (200 μl) were added to 800 μl of egg jelly (1 mg tochemistry. Peroxidase-AffiniPure goat anti-guinea pig IgG (H + L) sugar/ml), and incubated on ice for 5 min to induce the acrosome (Jackson ImmunoResearch Laboratories, West Grove, PA) and a highly reaction. Each sperm sample was centrifuged (1,000g, 5 min) and cross-adsorbed Alexa Fluor 568 goat anti-guinea pig IgG (H + L) (Life washed with ASW. Following the second wash, the supernatants were Technologies) were used as secondary antibodies for the detection of removed and replaced with 1 ml of RIPA buffer (1% [v/v] NP-40, 1% anti-AfPKAS1 antibodies in the respective assays. [w/v] deoxycholic acid, 150 mM NaCl, 50 mM Tris–HCl pH 7.4, 2 mM EDTA, 50 mM NaF, 0.2 mM Na VO , 100 nM okadaic acid [Wako, 3 4 4.5 | Electrophoresis Osaka, Japan], and 1:100 dilution of protease inhibitor cocktail [Nacalai Tesque, Kyoto, Japan]). Sperm pellets were homogenized using One-dimensional SDS–PAGE was performed on a 10% acrylamide Microtube Pestles (Scientific Specialties, Hanover, MD). After gel using the Laemmli method (Laemmli, 1970), and then stained 622 | NIIKURA ET AL.

incubated for 30 min at room temperature with Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H + L) or Peroxidase-AffiniPure goat anti-guinea pig IgG (H + L) (Jackson ImmunoResearch Laboratories), and then washed again. The bound antibody was detected using ECL Plus (GE Healthcare) on a Molecular Imager FX (Bio-Rad Laboratories).

4.7 | Peptide sequencing

Gels used for protein sequencing were transferred onto Sequi-Blot PVDF Membrane after SDS–PAGE. The blotted protein was stained with Coomassie Brilliant Blue and cut from the membrane. After washing and destaining, the blotted protein was sequenced using automated Edman degradation with a Procise 492 (PerkinElmer, Waltham, MA) protein sequencing system at the Instrumental Analysis Division at the Creative Research Institution of Hokkaido University, Japan. The N-terminal sequence the target protein could not be FIGURE 6 Anti-AfPKAS1 polyclonal antibody recognizes the analyzed by peptide sequencing, so the purified protein was chemically same protein as anti-phospho-PKA substrate monoclonal antibody. Immune-precipitation of sperm extracts using anti-phospho-PKA digested by the hydroxylamine cleavage method, which cleaves substrate antibody and anti-AfPKAS1 antibody, before and after peptides at Asn-Gly sites for peptide sequencing (Breitbart & Etkovitz, induction of the acrosome reaction. Western blot detection used an 2011), as previously reported (Crimmins, Mische, & Denslow, 2005). anti-phospho-PKA substrate antibody. A distinct band of ∼60 kDa was detected in acrosome-reacted sperm lysate induced by egg jelly (EJ). This 60-kDa band indicates the phosphorylation of 4.8 | Immunocytochemistry AaPKAS1 protein. Both antibodies recognize the same protein. Left, SDS–PAGE results; right, Western blot analysis Sperm were fixed with 4% paraformaldehyde in ASW for 15 min. Fixed sperm (100 μl) were placed onto a glass slide and incubated for 30 min to bind to the glass. After blocking with 3% BSA in ASW for 30 min, the with Coomassie Brilliant Blue (Rapid Stain CBB kit; Nacalai samples were incubated overnight at 4°C with 3% BSA containing Tesque) or processed for Western blot (see section 4.6). phospho-PKA substrate (RRXS/T) (100G7E) rabbit monoclonal anti- Two-dimensional electrophoresis was performed as follows: body or guinea pig anti-AfPKAS1 in ASW. After several washes with protein samples (100 μg) were used to rehydrate 11-cm Immobiline ASW, the samples were incubated for 3 h at 4°C with 3% BSA DryStrip (pH range, 3–10) (GE Healthcare, Waukesha, WI) overnight in containing Alexa Fluor 488 goat anti-rabbit IgG (H + L) or Alexa Fluor rehydration buffer (8 M urea, 2 M thiourea, 2% [v/v] CHAPS, 1.5 M Tris– 568 goat anti-guinea pig IgG (H + L) (Life Technologies) and 10 μg/ml HCl [pH 8.8], bromophenol blue, 0.35% dithiothreitol, 0.5% IPG buffer Hoechst 33342 in ASW. After an additional wash, the samples were [pH 3–10]) (GE Healthcare). Rehydrated strips were then subjected to observed under an IX70 and DP72 fluorescence microscope at 1,000× isoelectric focusing using a Multiphor Electrophoresis Unit Immobiline magnification. DryStrip Kit (GE Healthcare) at a constant temperature of 20°C; the gel was run at 200 V for 1 min, then at a linear gradient up to 3,500 V for 1 h 4.9 | Immunoprecipitation 30 min, and finally at 3,500 V for 1 h. Strips were then equilibrated for 30 min in equilibration buffer (1.5 M Tris–HCl [pH 8.8], 6 M urea, 1% Phospho-PKA substrate (RRXS/T) (100G7E) rabbit monoclonal anti- [w/v] DTT, 30% [v/v] glycerol, 2% [w/v] SDS, with bromophenol blue) body was added to 200 μl of each sperm lysate, and each sample was for electrophoretic separation by SDS–PAGE. incubated overnight at 4°C under rotation. Activated 50% COSMOGEL Ig-Accept Protein A (20 μl; Nacalai Tesque) was added to each sample, and incubated for 1 hr at 4°C under rotation. The supernatant was then 4.6 | Western blot removed following centrifugation (1,500g, 5 min), and the COSMOGEL Gels were transferred onto polyvinylidene difluoride (PVDF) mem- polymer was washed five times with 200 μl of RIPA buffer. Proteins branes (Hybond-P, GE Healthcare) using a Trans-Blot SD Semi-Dry were eluted from the beads using 30 μl of 2 × SDS sample buffer, boiled Electrophoretic TransferCell (Bio-Rad, Hercules, CA), and run at 20 V for for 5 min, and then separated by gel electrophoresis. 35 min. Membranes were blocked for 1 h with TBS-T (Tris-buffered saline plus 0.1% Tween-20) containing 5% skim milk, and then incubated 4.10 | Identification of PKA target protein by mass overnight at 4°C with TBS-T containing 5% bovine serum albumin (BSA) spectrometry and phospho-PKA substrate (RRXS/T) (100G7E) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA) or guinea pig anti- Immunoprecipitated samples were separated by SDS–PAGE, and AfPKAS1 (see section 4.4). Membranes were washed with TBS-T, protein bands were digested with the In-Gel Tryptic Digestion Kit NIIKURA ET AL. | 623

FIGURE 7 Immunocytochemical analyses of acrosome-intact or acrosome-reacted sperm samples. (a–f) Acrosome-intact sperm. (g–l) Acrosome-reacted sperm. Green, Alexa Fluor 488 anti-rabbit IgG (anti-phosphorylated PKA substrate monoclonal antibody [a, g] or normal rabbit IgG [d, j]); red, Alexa Fluor 568 anti-guinea pig IgG (anti-AfPKAS1 serum [c, h] or normal guinea pig IgG [e, k]). Merged images are presented in c, f, i, and l. Scale bars, 1 μm

(Thermo Scientific). Digested peptides were separated and identified Science, v1.9.0) with an A. forbesi ovary transcriptome database by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at maintained by Brown University (http://www.echinobase.org/ the Proteomic Facility of Brown University, USA. Database searches of Echinobase/OvaryAbout), which combines entries from TrEMBL/ the acquired MS/MS spectra were performed using Mascot (Matrix SWISSPROT (Boeckmann et al., 2003; Wu & Han, 2006) and 624 | NIIKURA ET AL.

TABLE 2 Phosphorylation status of specific sites in AaPKAS1 Breitbart, H., & Etkovitz, N. (2011). Role and regulation of EGFR in actin remodeling in sperm capacitation and the acrosome reaction. Asian Number of sites phosphorylated Journal of Andrology, 13, 106–110. Phosphorylated site of Acrosome- Acrosome-intact Colwin, A. L., & Colwin, L. H. (1963). Role of the gamate membranes AaPKAS1 reacted sperm sperm in fertilization in Saccoglossus kowalevskii (Enteropneusta). I. The Thr109 and/or Thr117 2 Not Phosphorylated acrosomal region and its changes in early stages of fertilization. Journal of Cell Biology, 19, 477–500. Ser125 Not detected 1 Crimmins, D. L., Mische, S. M., & Denslow, N. D. (2005). Chemical cleavage Ser201 and/or Ser204 11 of proteins in solution. Current Protocols in Protein Science, Chapter 11: and/or Thr207 Unit 11.4. https://doi.org/10.1002/0471140864.ps1104s40 Dan, J. C. (1954). Studies on the acrosome. II. Acrosome reaction in starfish Thr310 and/or Ser313 1, 2, and 3 1 and 3 spermatozoa. Biological Bulletin, 107, 203–218. and/or Ser314 Dan, J. C., Ohori, Y., & Kushida, H. (1964). Studies on the acrosome. VII. Thr353 and/or Ser362 2 2 Formation of the acrosomal process in sea urchin spermatozoa. Journal – Ser442 and/or Ser447 11 of Ultrastructure Research, 11, 508 524. and/or Ser451 Engh, R. A., Girod, A., Kinzel, V., Huber, R., & Bossemeyer, D. (1996). Crystal structures of catalytic subunit of cAMP-dependent protein kinase in complex with isoquinolinesulfonyl protein kinase inhibitors H7, H8, and H89. Structural implications for selectivity. Journal of Biology and GENBANK (Benson, Karsch-Mizrachi, Lipman, Ostell, & Wheeler, Chemistry, 271, 26157–26164. Fernandes, N., & Allbritton, N. L. (2009). Effect of the DEF motif on 2007). phosphorylation of peptide substrates by ERK. Biochemical and Biophysical Research Communications, 387, 414–418. Garbers, D. L., & Kopf, G. S. (1980). The regulation of spermatozoa by 4.11 | Phosphorylation site identification calcium cyclic nucleotides. Advances in Cyclic Nucleotide Research, 13, 251–306. The digested peptides were bound to a ZipTip C18 pipette tip (Merck- Garbers, D. L., Tubb, D. J., & Kopf, G. S. (1980). Regulation of sea urchin Millipore, Billerica, MA), and washed with 5% MeCN (acetonitrile) sperm cyclic AMP-dependent protein kinases by an egg associated – and 0.1% TFA (trifluoroacetic acid). After elution with 1 μl of 80% factor. Biology of Reproduction, 22, 526 532. Gjertsen, B. T., Mellgren, G., Otten, A., Maronde, E., Genieser, H. G., Jastorff, MeCN and 0.1% TFA, the eluate was mixed with 5 mg/ml of 4- B., ...Døskeland, S. O. (1995). Novel (Rp)-cAMPS analogs as tools for chloro-a-cyanocinnamic acid in 90% MeCN and 0.1% TFA, and spotted inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity and dried on an MTP 384 target plate (ground steel T F, Bruker modulates interleukin-1 beta action. Journal of Biology and Chemistry, – Daltonics, Bremen, Germany) (Leszyk, 2010). The mass was measured 270, 20599 20607. Harlan, J. E., Hajduk, P. J., Yoon, H. S., & Fesik, S. W. (1994). Pleckstrin using a MALDI-TOF MS AutoFlex III (Bruker Daltonics) in reflectron homology domains bind to phosphatidylinositol-4,5-bisphosphate. mode. Angiotensin II (Sigma–Aldrich) and Insulin B chain (Sigma– Nature, 371, 168–170. Aldrich) were used as standard peptides. Hertzog, M., Milanesi, F., Hazelwood, L., Disanza, A., Liu, H., Perlade, E., ... Scita, G. (2010). Molecular basis for the dual function of Eps8 on actin dynamics: Bundling and capping. PLoS Biology, 8, e1000387. ACKNOWLEDGMENTS Hoshi, M., Nishigaki, T., Ushiyama, A., Okinaga, T., Chiba, K., & Matsumoto, M. (1994). Egg-jelly signal molecules for triggering the acrosome This work is supported by Grant-in-Aid for Scientific Research on reaction in starfish spermatozoa. International Journal of Developmental – Innovative Areas (22112520 to M.M.) from the Ministry of Education, Biology, 28, 167 174. Ikadai, H., & Hoshi, M. (1981). Biochemical studies on the AR of the starfish, Culture, Sports, Science and Technology of Japan (MEXT). Research in Asterias amurensis, I. Factors participating in the AR. Development the Wessel lab was supported by the National Institutes of Health Growth & Differentiation, 23,73–80. (HD028152) and the National Science Foundation (IOS-1120972). We Inoue, N., Satouh, Y., Ikawa, M., Okabe, M., & Yanagimachi, R. (2011). gratefully acknowledge the help and support of Dr. Mamiko Yajima Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs. Proceedings of the National Academy of during the course of this project. Her humanitarian efforts following Sciences of the United States of America, 108, 20008–20011. the devastating Fukushima Tsunami of 2011 helped many, including Islam, M. S., Kawase, O., Hase, S., Hoshi, M., & Matsumoto, M. (2006). PKA us, and one result was the initiation of this collaborative work. We are activation in concert with ARIS and asterosap induces the acrosome grateful to her for this. reaction in starfish. Zygote, 14, 329–340. Jin, M., Fujiwara, E., Kakiuchi, Y., Okabe, M., Satouh, Y., Baba, S. A., ... Hirohashi, N. (2011). Most fertilizing mouse spermatozoa begin their REFERENCES acrosome reaction before contact with the zonapellucida during in vitro fertilization. Proceedings of the National Academy of Sciences of the Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., & Wheeler, United States of America, 108, 4892–4896. D. L. (2007). GenBank. Nucleic Acids Research, 35(Database issue), Kase, H., Iwahashi, K., Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., D21–D25. ...Kaneko, M. (1987). K-252 compounds, novel and potent inhibitors of Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., protein kinase C and cyclic nucleotide-dependent protein kinases. Gasteiger, E., ... Schneider, M. (2003). The SWISS-PROT protein Biochemical and Biophysical Research Communications, 142, 436–440. knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Kawase, O., Minakata, H., Hoshi, M., & Matsumoto, M. (2005). Asterosap- Research, 31, 365–370. induced elevation in intracellular pH is indispensable for ARIS-induced NIIKURA ET AL. | 625

sustained increase in intracellular Ca2+ and following acrosome Shiba, K., Tagata, T., Ohmuro, J., Mogami, Y., Matsumoto, M., Hoshi, M., & reaction in starfish spermatozoa. Zygote, 13,63–71. Baba, S. A. (2006). Peptide-induced hyperactivation-like vigorous Koyota, S., Wimalasiri, K. M., & Hoshi, M. (1997). Structure of the main flagellar movement in starfish sperm. Zygote, 14,23–32. saccharide chain in the acrosome reaction-inducing substance of the starfish, Su, Y. H., Chen, S. H., Zhou, H., & Vacquier, V. D. (2005). Tandem mass Asterias amurensis. Journal of Biology and Chemistry, 272, 10372–10376. spectrometry identifies proteins phosphorylated by cyclic AMP- Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly dependent protein kinase when sea urchin sperm undergo the of the head of bacteriophage T4. Nature, 227, 680–685. acrosome reaction. Developmental Biology, 285, 116–125. Leszyk, J. D. (2010). Evaluation of the new MALDI matrix 4-chloro-alpha- Tatenoa, H., Krapf, D., Hino, T., Sanchez-Cardenas, C., Darszon, A., cyanocinnamic acid. Journal of Biomolecular Techniques, 21,81–91. Yanagimachi, R., & Viscontie, P. E. (2013). Ca2+ ionophore A23187 can Levine, A. E., & Walsh, K. A. (1979). Involvement of an acrosin-like enzyme make mouse spermatozoa capable of fertilizing in vitro without in the acrosome reaction of sea urchin sperm. Developmental Biology, activation of cAMP-dependent phosphorylation pathways. Proceedings 72, 126–137. of the National Academy of Sciences of the United States of America, 46, Matsui, T., Nishiyama, I., Hino, A., & Hoshi, M. (1986). Induction of the 18543–18548. acrosome reaction in starfish. Development Growth & Differentiation, 28, Tilney, L. G., Kiehart, D. P., Sardet, C., & Tilney, M. (1978). Polymerization of 339–348. actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane Matsumoto, M., Solzin, J., Helbig, A., Hagen, V., Ueno, S., Kawase, O., ... fusion in the acrosomal reaction of echinoderm sperm. Journal of Cell Weyand, I. (2003). A sperm-activating peptide controls a cGMP-signaling Biology, 77, 536–550. pathway in starfish sperm. Developmental Biology, 260, 314–324. Vacquier, V. D., & Hirohashi, N. (2004). Sea urchin spermatozoa. Methods in Morisawa, S., Mizuta, T., Kubokawa, K., Tanaka, H., & Morisawa, M. (2004). Cell Biology, 74, 523–544. Acrosome reaction in spermatozoa form the amphioxus Branchiostoma Vacquier, V. D., Swanson, W. J., & Hellberg, M. E. (1995). What have we belcheri (Cephalochordata, Chordata). Zoological Science, 21, learned about sea urchin sperm bindin? Development Growth & 1079–1084. Differentiation, 37, 110. Nadella, K. S., Saji, M., Jacob, N. K., Pavel, E., Ringel, M. D., & Kirschner, L. S. Visconti, P. E., Moore, G. D., Bailey, J. L., Leclerc, P., Connors, S. A., Pan, (2009). Regulation of actin function by protein kinase A-mediated D., ...Kopf, G. S. (2005). Capacitation of mouse spermatozoa. II. Protein phosphorylation of Limk1. EMBO Reports, 10, 599–605. tyrosine phosphorylation and capacitation are regulated by a cAMP- Naruse, M., Suetomo, H., Matsubara, T., Sato, T., Yanagawa, H., Hoshi, M., & dependent pathway. Development, 121, 1139–1150. Matsumoto, M. (2010). Acrosome reaction-related steroidal saponin, Watkins, H. D., Kopf, G. S., & Garbers, D. L. (1978). Activation of sperm Co-ARIS, from the starfish induces structural changes in microdomains. adenylate cyclase by factors associated with eggs. Biology of Reproduc- Developmental Biology, 347, 147–153. tion, 19, 890–894. Naruse, M., Ishikawa, R., Sakaya, H., Moriyama, H., Hoshi, M., & Matsumoto, Wu, L., & Han, D. K. (2006). Overcoming the dynamic range problem in mass M. (2011). Novel conserved structural domains of acrosome reaction- spectrometry-based shotgun proteomics. Expert Review of Proteomics, inducing substance are widespread in invertebrates. Molecular Repro- 3, 611–619. duction and Development, 78,57–66. Yanagimachi, R. (2011). Mammalian sperm acrosome reaction: Where does Nishigaki, T., Chiba, K., & Hoshi, M. (2000). A 130-kDa membrane protein of it begin before fertilization? Biology of Reproduction, 85,4–5. sperm flagella is the receptor for asterosaps, sperm-activating peptides of starfish Asterias amurensis. Developmental Biology, 219, 154–162. Nishigaki, T., Chiba, K., Miki, W., & Hoshi, M. (1996). Structure and function SUPPORTING INFORMATION of asterosaps, sperm-activating peptides from the jelly coat of starfish eggs. Zygote, 4, 237–245. Additional Supporting Information may be found online in the Nishiyama, I., Matsui, T., Fujimoto, Y., Ikekawa, I., & Hoshi, M. (1987). supporting information tab for this article. Correlation between the molecular structure and biological activity of Co-ARIS, a cofactor for acrosome reaction-inducing substance. Development Growth & Differentiation, 29, 171–176. Reich, A., Dunn, C., Akasaka, K., & Wessel, G. M. (2015). Phylogenetic analyses of echinodermata support the sister groups of Asterozoa and How to cite this article: Niikura K, Alam MS, Naruse M, Echinozoa. PLoS ONE, 10, e0119627. et al. Protein kinase A activity leads to the extension of the Romarowski, A., Battistone, M. A., La Spina, F. A., Molina, L. D. C. P., Luque, acrosomal process in starfish sperm. Mol Reprod Dev. G. M., Vitale, A. M., ... Buffone, M. G. (2015). PKA-dependent 2017;84:614–625. https://doi.org/10.1002/mrd.22824 phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis. Developmental Biology, 405, 237–249.