Received: 12 September 2016 | Accepted: 26 April 2017

DOI: 10.1002/mrd.22824

RESEARCH ARTICLE

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

Keisuke Niikura1 | M. Shahanoor Alam1 | Masahiro Naruse1 |

Mitsuru Jimbo2 | Hideaki Moriyama3 | Adrian Reich4 | Gary M. Wessel4 |

Midori Matsumoto1

1Department of Biological Sciences and

Informatics, Keio University, Yokohama, Japan

2 School of Marine Biosciences, Kitasato

University, Sagamihara, Kanagawa, Japan

3 School of Biological Sciences, University of

Nebraska-Lincoln, Lincoln, Nebraska

4Department of Molecular Biology, Cellular

Biology and Biochemistry, Brown University,

Providence, Rhode Island

Correspondence

Midori Matsumoto, Department of Biological

Sciences and Informatics, Keio University, 3-14-1

Hiyoshi, Kouhoku-ku, Yokohama 223-8522,

Japan.

Email: mmatsumo@bio.keio.ac.jp

Funding information

Brown University, Grant number: Japanese

Students-Brown support; Grant-in-Aid for

Scientific Research on Innovative Areas from

the Ministry of Education, Culture, Sports,

Science and Technology of Japan (MEXT),

Grant number: 22112520; National Institutes

of Health, Grant number: HD028152;

National Science Foundation, Grant number:

IOS-1120972

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction,

which is immediately followed by the actin polymerization-dependent extension of an

acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large

proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal

sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome

reaction. Asterosap induces a transient elevation of intracellular cGMPandCa2+ levels,

and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+.

Yet, the contribution of signalingmolecules downstream of cAMP andCa2+ in inducing

AVexocytosis andAPextension remain unknown. Amodified acrosome reaction assay

was used here to differentiate between AV exocytosis and AP extension in starfish

sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP

extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of

target proteins occurs, and these substrates localize at the base of the AP,

demonstrating that PKA activation regulates an AP extension step during the

acrosome reaction. The major PKA substrate was further identified, from A. amurensis

and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation

motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin

polymerization during the acrosome reaction.

K E YWORD S

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

1 | INTRODUCTION

The acrosome reaction is essential for sperm to fuse with an egg

in most animals. In echinoderm sperm, the acrosome reaction

involves exocytosis of acrosomal vesicle (AV) contents and

extension of the acrosomal process (AP) by actin polymerization

(Dan, 1954; Hoshi et al., 1994). An obvious AP can be observed in

starfish sperm; it is approximately 25 μm long, penetrates the egg

jelly, and binds to the vitelline layer (Tilney, Kiehart, Sardet, &

Tilney, 1978; Vacquier, Swanson, & Hellberg, 1995). Although

exocytosis and actin polymerization are known to occur

sequentially in the AR, mechanistic relationships between these

processes remain unclear (Levine & Walsh, 1979; Ikadai & Hoshi,

1981).

Abbreviations: AP, acrosomal process; AV, acrosomal vesicle; PKA, Protein kinase A; PKC,

Protein kinase C.

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

614 | © 2017 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/mrd Mol Reprod Dev. 2017;84:614–625.

The acrosome reaction in Asterias amurensis is induced by

cooperative action of three components within the egg jelly: a

sulfated proteoglycan-like molecule designated as acrosome

reaction-inducing substance (ARIS); a group of sulfated steroidal

saponins, named cofactor for ARIS (Co-ARIS); and the sperm-

activating peptide, asterosap (Koyota, Wimalasiri, & Hoshi, 1997;

Matsui, Nishiyama, Hino, & Hoshi, 1986; Nishigaki, Chiba, Miki, &

Hoshi, 1996; Nishiyama, Matsui, Fujimoto, Ikekawa, & Hoshi,

1987). ARIS is the main factor that induces the acrosome reaction,

and its activity depends on sugar chains (Koyota et al., 1997;

Matsui et al., 1986). Co-ARIS alters the microdomain of the sperm

membrane, facilitating the binding between ARIS and the ARIS

receptor that surrounds the AV (Naruse et al., 2010). Asterosap

binds to its receptor, a guanylate cyclase present on the flagella

(Matsumoto et al., 2003; Nishigaki, Chiba, & Hoshi, 2000), that

transiently raises the concentrations of intracellular cGMP

([cGMPi]), intracellular pH ([pHi]), and intracellular Ca2+ ([Ca2þi ]),

together inducing sperm chemotaxis (Kawase, Minakata, Hoshi, &

Matsumoto, 2005; Nishigaki et al., 2000; Shiba et al., 2006).

Continual accumulation of intracellular cyclic AMP ([cAMPi]) and

[Ca2þi ] requires both ARIS and asterosap (Islam, Kawase, Hase,

Hoshi, & Matsumoto, 2006; Kawase et al., 2005). cAMP functions

as a second messenger, via Protein kinase A (PKA) activation.

Studies conducted using starfish sperm showed that

[cAMPi] increases only in the presence of both ARIS and asterosap,

and that the inhibition of cAMP-dependent protein kinase activity

(e.g., PKA) suppresses the acrosome reaction (Islam et al., 2006).

Therefore, PKA activation is important for the ARIS- and

asterosap-induced acrosome reaction.

PKA plays a prominent role during the acrosome reaction in the

sperm of many organisms—from mammals to echinoderms. In sea

urchins, fucose sulfate polymer, an acrosome reaction-inducing

polysaccharide, activates adenylate cyclase, leading to an increase in

[cAMPi] levels (Garbers & Kopf, 1980; Garbers, Tubb, & Kopf, 1980;

Su, Chen, Zhou, & Vacquier, 2005; Watkins, Kopf, & Garbers, 1978).

How PKA initiates the acrosome reaction is still unknown. The AP of

sea urchin sperm is very short, around 1 μm, so definitively observing

the acrosome reaction in these sperm is a challenge when using a

light microscope for longitudinal studies (Dan, Ohori, & Kushida,

1964). Polymerization of actin filaments can be observed by

fluorescence staining, which is the basis of an acrosome reaction

assay for sea urchin sperm; observation of AV exocytosis, however,

requires antibodies and additional immunolabeling approaches

(Vacquier & Hirohashi, 2004). In contrast, the acrosome reaction

in starfish sperm can be investigated simply using erythrosine

staining to visualize the 25-μm-long AP (Kawase et al., 2005; Matsui

et al., 1986; Naruse et al., 2010).

In this study, we modified the erythrosine-staining assay used

for measuring starfish sperm so that AV exocytosis and AP

extension can be evaluated separately. This allowed us to

investigate the role of PKA signaling during the acrosome reaction,

as well as to identify PKAS1, a novel PKA substrate in starfish

sperm.

2 | RESULTS

2.1 | Phalloidin-modified acrosome reaction assay

Erythrosine staining was originally used to quantify starfish sperm AP

extension in an acrosome reaction assay (Kawase et al., 2005; Matsui

et al., 1986; Naruse et al., 2010). This novel phalloidin-modified assay,

modified from a method developed for visualizing the acrosome

reaction of sea urchin sperm (Vacquier & Hirohashi, 2004), was

developed to distinguish between AV exocytosis and AP extension

steps. Hoechst 33342 staining revealed the presence or absence of

the intact AV, which protrudes into the nucleus prior to the acrosome

reaction (as seen by a Hoechst-null area) but is lost following the

release of the AV (Figure 1a–c). Similar to the assay developed for sea

urchin sperm, staining with fluorescently labeled phalloidin allowed

for visualization of the actin filaments in the AP, which is facilitated by

the additional length of the AP in starfish sperm.

2.2 | PKA inhibitors block AP extension but not AVexocytosis

Pretreatment of sperm with a PKA-inhibitor prevents the

acrosome reaction in a concentration-dependent manner (Islam

et al., 2006), but whether this PKA signaling causes AP extension

or AV exocytosis is unknown. We therefore used the starfish

acrosome reaction assay to investigate the effects of PKA

inhibition on each event. An intact AV and the actomere, the

site of actin filament nucleation, were observed in an acrosome-

intact sperm head (Figure 2a–d). In contrast, control egg jelly-

induced acrosome-reacted sperm underwent AV exocytosis, and

the AP was easily observed via actin polymerization. We further

noted a coordinated loss of the unstained AV region within

the nucleus and the appearance of a stained, extended AP

(Figure 2e–h). Starfish sperm pretreated with a PKA inhibitor,

KT5720, followed by egg jelly treatment resulted in loss of the

unstained region (AV), similar to that of control acrosome-reacted

sperm, whereas actin filaments remained aggregated within a

spot at the top of the sperm head (Figure 2i–l). Quantitative

analysis of these results revealed no significant differences

between the sperm treated with or without KT5720 for AV

exocytosis rate (Figure 2m), versus significant reduction in AP

extension rate (p < 0.001) (Figure 2n).

2.3 | Identification of the PKA target substrate

We next sought to identify the proteins phosphorylated by PKA

during the acrosome reaction, using an anti-phospho-PKA sub-

strate monoclonal antibody that recognizes RRXT/RRXS sequences

containing phosphorylated Thr/Ser residues that comprise a part of

the motifs phosphorylated by PKA. We detected a ∼60-kDa band

that increased in intensity in lysate from acrosome-reacted sperm

compared to acrosome-intact sperm (Figure 3a). Pretreating

starfish sperm with PKA inhibitors, followed by induction of the

NIIKURA ET AL. | 615

acrosome reaction with egg jelly, resulted in a significant decreased

in signal of the 60-kDa phosphorylated band; this phenotype was

observed following pretreatment with two different PKA inhib-

itors, KT5720 (Figure 3a) and Rp-8-Br-cAMPS (Figure 3b). Two-

dimensional gel electrophoresis and Western blot analyses in

acrosome-intact sperm showed a faint spot at 60 kDa and at an

isoelectric point (pI) 5.61 that is distinct from the spot in acrosome-

reacted sperm, which appeared as a band at 60 kDa that stretched

from a pI of 3.71–5.12 (Figure 3c). This change in pattern indicates

that the immunodetected protein is phosphorylated during the

acrosome reaction.

2.4 | Localization of the PKA substrate in starfishacrosome-reacted sperm

Immunocytochemistry using egg jelly-treated sperm revealed that the

putative PKA target substrate resides at the base of the AP, in the same

region as the actin filaments (Figure 4a–f).

2.5 | Identification of the PKA substrates

Gelelectrophoresis andpurificationof the60-kDaproteinwasperformed to

determine the sequence of this polypeptide using hydroxylamine cleavage

methodology. The N-terminal sequence of a fragment derived from this

excised, 60-kDa A. amurensis protein was determined to be NH2-(N)

GKHXEMAQQS-CO2H;thissequencewasnotsimilartoanyknownprotein.

A similar 60-kDa band was detected in the sperm lysate of Asterias

forbesi (Figure 5a), a species whose ovary transcriptome database has

been assembled (Reich, Dunn, Akasaka, & Wessel, 2015). This protein

was then purified from A. forbesi acrosome-reacted sperm lysate by

immunoprecipitationwith the anti-phospho-PKA substratemonoclonal

antibody, and analyzed by tandem mass spectrometry using the

A. forbesi ovary transcriptome database as a reference (Table 1). The

most suitable candidate thatmatched thePKA substrate fingerprintwas

Af005000T1, which contains six motifs recognized by the anti-

phospho-PKA substrate antibody (one RRXT and five RRXS sites)

(Figure 5b). Af005000T1and theA. amurensishomologweredesignated

as AfPKAS1 and AaPKAS1, respectively, for PKA substrate 1.

FIGURE 1 Evaluation of the exocytosis of the AV and AP extension through nucleus and actin filament staining. (a) Illustration ofacrosome-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

616 | NIIKURA ET AL.

2.6 | Identification of AfPKAS1 and AaPKAS1transcripts from starfish testes

The complete cDNAsequences coding forAaPKAS1 andAfPKAS1were

obtained from A. amurensis and A. forbesi testes, respectively, using

reverse-transcription PCRand5′-rapid amplificationof cDNAends. The

full-length cDNA (1,645 bp) encodes a protein containing 457 amino

acid residues (calculated molecular weight of 54.3 kDa) with no

previously reported domains recognized by BlastP or Interproscan.

Therefore,weusedScansite to find short sequencemotives inAaPKAS1

and AfPKAS1, and identified several consensus PKA, Protein kinase C

(PKC), Casein kinase, andAKTkinase phosphorylation sites, aswell as an

ERK D-domain and PIP3-binding PH sequences. Six of the identified

sites (Thr109, Ser125, Ser201, Ser314, Ser362, and Ser451) are

potentially phosphorylated by PKA (Figure 5b).

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

are expressed in both of these tissues. The transcripts were detected in

both testes and in ovaries, although AaPKAS1 was more highly

expressed in the testis than in the ovary of A. amurensis (Figure 5c).

2.7 | AaPKAS1 represents a PKA substrate

Finally, to determine if AaPKAS1 and AfPKAS1 are the same molecules

identified as 60-kDa bands in Western blot analyses, we generated a

polyclonal antibody in the serum of guinea pigs, with a fusion protein

composedofglutathione-S-transferase fused toapartialAfPKAS1protein

fragment (Figure S1) to determine ifAaPKAS1 andAfPKAS1 are the same

molecules identified as 60-kDa bands in immunoblot analyses. This serum

antibody recognized a 60-kDa band, similar to the one recognized by the

anti-phospho-PKA substrate monoclonal antibody (Figure 6).

The signal of the anti-phospho-PKA substratemonoclonal antibody

and the anti-AfPKAS1 serum co-localized in both acrosome-intact and

acrosome-reacted sperm of A. amurensis (Figure 7). In acrosome-intact

sperm, these signals were detected in the outer membrane of the

acrosome on the nucleus side, while in the acrosome-reacted sperm

cells, these signals were detected at the base of the AP, with some

staining also detected in the flagella. We attempted to detect AaPKAS1

in the immatureormatureeggsofA.amurensisusinganti-AfKAS1serum,

but we did not observe any distinct signals (data not shown).

2.8 | AaPKAS1 phosphorylated sites during AR

The amino acid sequence of AaPKAS1 contained six potentially

phosphorylated sites by PKA (Thr109, Ser125, Ser201, Ser314,

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

phosphorylation sites, such as Thr109 and Thr117 inAaPKAS1 (Table 2;

Figure S2). The consensus of these results suggests that Thr109may be

the important phosphorylation site ofAaPKAS1 that is targeted byPKA.

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 Hoechst33342 (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 areexpressed as mean ± standard deviation. *p < 0.001 from three replicates; each experiment was calculated using 100 sperms in all samples

NIIKURA ET AL. | 617

3 | DISCUSSION

PKA signaling regulates a wide variety of biological events, including

diverse metabolic processes, vasodilation, and calcium sequestration.

Differences in the functions of PKA within cells derive from how cells

are stimulated to accumulate cAMP, which eventually activates PKA,

resulting in substrate phosphorylation and active signaling. For

example, PKA phosphorylation of LIMK1 in cultured cells results in

the aggregation of F-actin (Nadella et al., 2009).

Sperm behavior critically depends on PKA in all species studied to

date. In mammalian sperm, activated PKA participates in capacitation

events that precede the acrosome reaction, and may induce actin

polymerization through a tyrosine kinase intermediate (Breitbart &

Etkovitz, 2011; Visconti et al., 2005). cAMP/PKA-dependent phos-

phorylation of LIMK1 and cofilin is also essential for mammalian sperm

acrosome exocytosis (Romarowski et al., 2015). A brief elevation of

intracellularCa2+ is sufficient toactivate the cAMP/PKApathwayduring

capacitation and the acrosome reaction (Tatenoa et al., 2013). The exact

FIGURE 3 Western blot of sperm lysates using anti-phospho-PKA substrate antibody. (a–b) Western blot detection of a ∼60-kDa band inacrosome-intact sperm that was intensified in acrosome-reacted sperm lysate induced by egg jelly (EJ). This effect was abolished in spermpretreated 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) to3.71–5.12 (white arrows) after the acrosome reaction

618 | NIIKURA ET AL.

stimulus that triggers the PKA-dependent acrosome reaction in

mammalian sperm remains controversial, and likely includes more

factors than proposed in the classically defined ZP3 model (Inoue,

Satouh, Ikawa, Okabe, & Yanagimachi, 2011; Jin et al., 2011;

Yanagimachi, 2011).

The acrosome reaction has been studied extensively in both

starfish and sea urchin sperm, in which PKA also plays an essential and

conserved role (Yanagimachi, 2011). We previously showed that PKA

inhibitors suppressed the acrosome reaction of starfish sperm in a

dose-dependentmanner (Islam et al., 2006)—although the exact role of

PKA activation during the acrosome reaction remains unclear. We

therefore developed a novel approach to dissect and quantitatively

evaluate the key steps that comprise the acrosome reaction in starfish

sperm, that is, AV exocytosis and AP extension. We additionally

analyzed the effects of PKA inhibitors on the various steps of the

starfish acrosome reaction using two different PKA inhibitors:

KT5720, which interferes with ATP binding to the catalytic subunits

of PKA (Engh, Girod, Kinzel, Huber, & Bossemeyer, 1996; Kase et al.,

1987), and the cAMP analog Rp-8-Br-cAMPS, a potent competitor of

cAMP binding to PKA regulatory subunits (Gjertsen et al., 1995). These

distinct PKA inhibitors elicited similar acrosome-reaction phenotypes

on starfish sperm, suggesting that PKA activation contributes

selectively to AP extension by regulating actin polymerization.

A key to understanding the function of PKA in sperm regulation

is the identification of PKA substrates. We discovered a 60-kDa

protein phosphorylated by PKA during the starfish acrosome

reaction. This PKA substrate, identified as AaPKAS1, resides at

the base of the AP, suggesting that it contributes to AP extension by

regulating actin polymerization. AaPKAS1 was found to contain six

PKA phosphorylation motifs (RRXT/RRXS), implying that AaPKAS1

and AfPKAS1 may be regulated by various kinases and could recruit

signaling factors during the acrosome reaction. For example,

AaPKAS1 is localized around the actomere, where it may contribute

to AP extension by interaction with kinases such as PKA. The

Scansite program also identified motifs in AaPKAS1 that may be

phosphorylated by PKA, PKC, Casein kinase, and AKT kinase, as well

as ERK D-domain and PIP3-binding PH sequences. The ERK D-

domain is involved in the interaction between ERK and its substrate

(Fernandes & Allbritton, 2009) while PIP3-binding PH sequences

interact with PIP2 or PIP3 within cell membranes (Harlan, Hajduk,

Yoon, & Fesik, 1994).

AaPKAS1 differs from PKA substrates previously identified in sea

urchin sperm—such as Adenylate kinase 1, Phosphodiesterase 5, and

EPS8 (Su et al., 2005)—and the sea urchin genome does not encode an

AaPKAS1 ortholog. The closest functional homology for PKAS1 is

EPS8, which regulates actin dynamics through end capping (via its

C-terminal amphipathic helix H1) and bundling of actin filaments

(through its H2-H5 helices) (Hertzog et al., 2010). Might differences in

AP length between starfish and sea urchin help explain the

evolutionary selection for these distinct modes of PKA-dependent

actin polymerization in sperm of sea urchin (EPS8) versus starfish

(AaPKAS1)? Among Echinoderms, Crinoidea (crinoids) and sea urchins

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 spermwas stained with Hoechst 33342 (a and d) and Alexa Fluor 488 goat anti-rabbit IgG (b and e). Fluorescence images were merged and overlaidonto the phase-contrast images (c and f). Scale bars, 1 μm

NIIKURA ET AL. | 619

possess a short AP whereas starfish, Holothuroidea (sea cucumbers),

and Ophiuroidea possess long APs (Naruse et al., 2011). Furthermore,

Hemichordata, Cephalochordata, and Ctenophora sperm also form

long APs (Colwin & Colwin, 1963; Morisawa, Mizuta, Kubokawa,

Tanaka, & Morisawa, 2004). Therefore, how actin polymerization of

PKA substrates occurs may be conserved in many species; we predict

that PKAS1 orthologs function in their sperm as well.

AaPKAS1 is present in both testes and ovaries of starfish, and is

likely used by embryos as well. Although regulation of PKAS1 in these

different cell types is likely through distinct mechanisms, we

hypothesize that this protein is a more general regulator of diverse

actin dynamics employed in multiple starfish cell types. Our novel

findings thus provide a foundation for further research exploring PKA

function in an array of cells types.

4 | MATERIALS AND METHODS

4.1 | Materials

A. amurensis samples were collected from the Otsuchi Bay and Tokyo

Bay, Japan, and Sandy Bay, Australia. A. forbesi samples were collected

fromWoods Hole Marine Biological Laboratory, USA. Dry sperm from

the adults were collected as previously described (Matsui et al., 1986).

Egg jelly andARISwere prepared as previously described (Koyota et al.,

1997; Matsui et al., 1986). Kinase inhibitors KT5720 and Rp-8-Br-

cAMPS were purchased from Sigma–Aldrich (St. Louis, MO) and

dissolved in dimethylsulfoxide (DMSO).

4.2 | Acrosome reaction assay and fluorescenceimaging

The acrosome reaction assay was performed as previously described

(Naruse et al., 2010) with somemodifications. Briefly, 1 μl of dry sperm

was resuspended in 1ml of ice-cold artificial seawater (ASW) (423mM

NaCl, 9 mM KCl, 9 mM CaCl2, 25mM MgSO4, 23mM MgCl2, 10mM

EPPS, NaOH pH 8.2), and incubated on ice for 10min with or without

inhibitors. The sperm suspension (20 µl) was added to 80 μl of egg jelly

(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 ASWwere added

to each sample to fix the sperm. Fixed sperm (100 μl) were placed onto

glass slides and incubated for 30min to bind to the glass. After washing

with ASW, 100 μl of Staining Solution (0.05% [v/v] Triton X-100, 0.4 U

FIGURE 5 Identification of AfPKAS1 and AaPKAS1. (a) Western blot analyses of A. forbesi sperm lysate, using anti-phospho-PKA substrateantibody. (b) The amino acid sequence of A. amurensis and A. forbesi Af005000T1. Letters indicate motifs identified by Scansite using “HighStringency” 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, andAkt kinase. (c) AfPKAS1 gene expression analysis, using total RNA obtained from testis and egg

620 | NIIKURA ET AL.

Alexa Fluor 488 phalloidin [Life Technologies, Bethesda, MD],

10 μg/ml Hoechst 33342 [Sigma–Aldrich] in ASW) were added and

incubated on ice for 1–2 hr in the dark. After further washing, sperm

samples were observed and scored using an IX70 and DP72

fluorescence microscope (Olympus, Tokyo, Japan) at 1,000× magnifi-

cation. One hundred sperm cells were scored per sample.

4.3 | Protein extraction from starfish sperm

Dry sperm (20 μl) were resuspended in 1ml of ice-cold ASW, and

incubated on ice for 10min with or without inhibitors. Sperm

suspension (200 μl) were added to 800 μl of egg jelly (1 mg

sugar/ml), and incubated on ice for 5 min to induce the acrosome

reaction. Each sperm sample was centrifuged (1,000g, 5 min) and

washed with ASW. Following the second wash, the supernatants were

removed and replaced with 1ml of RIPA buffer (1% [v/v] NP-40, 1%

[w/v] deoxycholic acid, 150mM NaCl, 50mM Tris–HCl pH 7.4, 2mM

EDTA, 50mM NaF, 0.2mM Na3VO4, 100 nM okadaic acid [Wako,

Osaka, Japan], and 1:100 dilution of protease inhibitor cocktail [Nacalai

Tesque, Kyoto, Japan]). Sperm pellets were homogenized using

Microtube Pestles (Scientific Specialties, Hanover, MD). After

centrifugation (12,000g, 15 min), the supernatant containing the

sperm lysate was collected from each sample. The concentration of

each sperm lysate was measured using a BCA Protein Assay Kit

(Thermo Scientific, Waltham, MA) and stored at −30°C.

4.4 | Preparation of serum containing polyclonalantibody against AfPKAS1

Polyclonal antibodies against AfPKAS1 (amino acid residues 92- 458)

were generated in guinea pigs. Guinea pig serum containing anti-

AfPKAS1 antibodies was then used for Western blot and immunocy-

tochemistry. Peroxidase-AffiniPure goat anti-guinea pig IgG (H + L)

(Jackson ImmunoResearch Laboratories, West Grove, PA) and a highly

cross-adsorbed Alexa Fluor 568 goat anti-guinea pig IgG (H + L) (Life

Technologies) were used as secondary antibodies for the detection of

anti-AfPKAS1 antibodies in the respective assays.

4.5 | Electrophoresis

One-dimensional SDS–PAGE was performed on a 10% acrylamide

gel using the Laemmli method (Laemmli, 1970), and then stained

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

Protein IDs

GrpAccessionnumber Protein name

Proteinscore

UniquePSMa

# ofRRXT

# ofRRXS

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

GN = sra-3)

59.51 2 0 0

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

NIIKURA ET AL. | 621

with Coomassie Brilliant Blue (Rapid Stain CBB kit; Nacalai

Tesque) or processed for Western blot (see section 4.6).

Two-dimensional electrophoresis was performed as follows:

protein samples (100 μg) were used to rehydrate 11-cm Immobiline

DryStrip (pH range, 3–10) (GE Healthcare, Waukesha, WI) overnight in

rehydrationbuffer (8Murea, 2Mthiourea, 2% [v/v]CHAPS, 1.5MTris–

HCl [pH 8.8], bromophenol blue, 0.35% dithiothreitol, 0.5% IPG buffer

[pH 3–10]) (GE Healthcare). Rehydrated strips were then subjected to

isoelectric focusing using a Multiphor Electrophoresis Unit Immobiline

DryStrip Kit (GE Healthcare) at a constant temperature of 20°C; the gel

was run at 200V for 1min, then at a linear gradient up to 3,500V for 1 h

30min, and finally at 3,500V for 1 h. Strips were then equilibrated for

30min in equilibration buffer (1.5M Tris–HCl [pH 8.8], 6M urea, 1%

[w/v] DTT, 30% [v/v] glycerol, 2% [w/v] SDS, with bromophenol blue)

for electrophoretic separation by SDS–PAGE.

4.6 | Western blot

Gels were transferred onto polyvinylidene difluoride (PVDF) mem-

branes (Hybond-P, GE Healthcare) using a Trans-Blot SD Semi-Dry

ElectrophoreticTransferCell (Bio-Rad,Hercules,CA), and runat20 V for

35min. Membranes were blocked for 1 h with TBS-T (Tris-buffered

salineplus0.1%Tween-20) containing5%skimmilk, and then incubated

overnight at 4°Cwith TBS-T containing 5%bovine serum albumin (BSA)

and phospho-PKA substrate (RRXS/T) (100G7E) rabbit monoclonal

antibody (Cell Signaling Technology, Danvers, MA) or guinea pig anti-

AfPKAS1 (see section 4.4). Membranes were washed with TBS-T,

incubated for 30min 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

analyzed by peptide sequencing, so the purified proteinwas chemically

digested by the hydroxylamine cleavage method, which cleaves

peptides at Asn-Gly sites for peptide sequencing (Breitbart & Etkovitz,

2011), as previously reported (Crimmins, Mische, & Denslow, 2005).

4.8 | Immunocytochemistry

Spermwere fixed with 4% paraformaldehyde in ASW for 15min. Fixed

sperm (100 μl) were placed onto a glass slide and incubated for 30min

to bind to the glass. After blocking with 3%BSA in ASW for 30min, the

samples were incubated overnight at 4°C with 3% BSA containing

phospho-PKA substrate (RRXS/T) (100G7E) rabbit monoclonal anti-

body or guinea pig anti-AfPKAS1 in ASW. After several washes with

ASW, the samples were incubated for 3 h at 4°C with 3% BSA

containing Alexa Fluor 488 goat anti-rabbit IgG (H + L) or Alexa Fluor

568 goat anti-guinea pig IgG (H + L) (Life Technologies) and 10 μg/ml

Hoechst 33342 in ASW. After an additional wash, the samples were

observed under an IX70 and DP72 fluorescence microscope at 1,000×

magnification.

4.9 | Immunoprecipitation

Phospho-PKA substrate (RRXS/T) (100G7E) rabbit monoclonal anti-

body was added to 200 μl of each sperm lysate, and each sample was

incubated overnight at 4°C under rotation. Activated 50%COSMOGEL

Ig-Accept Protein A (20μl; Nacalai Tesque) was added to each sample,

and incubated for 1 hr at 4°C under rotation. The supernatant was then

removed following centrifugation (1,500g, 5 min), and the COSMOGEL

polymer was washed five times with 200 μl of RIPA buffer. Proteins

were eluted from the beads using 30 μl of 2 × SDS sample buffer, boiled

for 5min, and then separated by gel electrophoresis.

4.10 | Identification of PKA target protein by massspectrometry

Immunoprecipitated samples were separated by SDS–PAGE, and

protein bands were digested with the In-Gel Tryptic Digestion Kit

FIGURE 6 Anti-AfPKAS1 polyclonal antibody recognizes thesame protein as anti-phospho-PKA substrate monoclonal antibody.Immune-precipitation of sperm extracts using anti-phospho-PKAsubstrate antibody and anti-AfPKAS1 antibody, before and afterinduction of the acrosome reaction. Western blot detection used ananti-phospho-PKA substrate antibody. A distinct band of ∼60 kDawas detected in acrosome-reacted sperm lysate induced by eggjelly (EJ). This 60-kDa band indicates the phosphorylation ofAaPKAS1 protein. Both antibodies recognize the same protein. Left,SDS–PAGE results; right, Western blot analysis

622 | NIIKURA ET AL.

(Thermo Scientific). Digested peptides were separated and identified

by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at

the Proteomic Facility of BrownUniversity, USA. Database searches of

the acquired MS/MS spectra were performed using Mascot (Matrix

Science, v1.9.0) with an A. forbesi ovary transcriptome database

maintained by Brown University (http://www.echinobase.org/

Echinobase/OvaryAbout), which combines entries from TrEMBL/

SWISSPROT (Boeckmann et al., 2003; Wu & Han, 2006) and

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] ornormal 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 imagesare presented in c, f, i, and l. Scale bars, 1 μm

NIIKURA ET AL. | 623

GENBANK (Benson, Karsch-Mizrachi, Lipman, Ostell, & Wheeler,

2007).

4.11 | Phosphorylation site identification

The digested peptides were bound to a ZipTip C18 pipette tip (Merck-

Millipore, Billerica, MA), and washed with 5% MeCN (acetonitrile)

and 0.1% TFA (trifluoroacetic acid). After elution with 1 μl of 80%

MeCN and 0.1% TFA, the eluate was mixed with 5mg/ml of 4-

chloro-a-cyanocinnamic acid in 90%MeCNand 0.1%TFA, and spotted

and dried on an MTP 384 target plate (ground steel T F, Bruker

Daltonics, Bremen, Germany) (Leszyk, 2010). The mass was measured

using a MALDI-TOF MS AutoFlex III (Bruker Daltonics) in reflectron

mode. Angiotensin II (Sigma–Aldrich) and Insulin B chain (Sigma–

Aldrich) were used as standard peptides.

ACKNOWLEDGMENTS

This work is supported by Grant-in-Aid for Scientific Research on

Innovative Areas (22112520 to M.M.) from the Ministry of Education,

Culture, Sports, Science and Technology of Japan (MEXT). Research in

the Wessel lab was supported by the National Institutes of Health

(HD028152) and theNational Science Foundation (IOS-1120972).We

gratefully acknowledge the help and support of Dr. Mamiko Yajima

during the course of this project. Her humanitarian efforts following

the devastating Fukushima Tsunami of 2011 helped many, including

us, and one result was the initiation of this collaborative work. We are

grateful to her for this.

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TABLE 2 Phosphorylation status of specific sites in AaPKAS1

Number of sites phosphorylated

Phosphorylated site ofAaPKAS1

Acrosome-reacted sperm

Acrosome-intactsperm

Thr109 and/or Thr117 2 Not Phosphorylated

Ser125 Not detected 1

Ser201 and/or Ser204and/or Thr207

1 1

Thr310 and/or Ser313and/or Ser314

1, 2, and 3 1 and 3

Thr353 and/or Ser362 2 2

Ser442 and/or Ser447and/or Ser451

1 1

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How to cite this article: Niikura K, Alam MS, Naruse M,

et al. Protein kinase A activity leads to the extension of the

acrosomal process in starfish sperm. Mol Reprod Dev.

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