Journal of Reproduction and Development, Vol. 56, No. 2, 2010, 09-180N —Original Article— Evidence of the Existence of Adenylyl 10 (ADCY10) Ortholog Proteins in the Heads and Connecting Pieces of Boar Spermatozoa Shunsuke TATE1)*, Kazumi NAKAMURA2)*, Chihiro SUZUKI3)*, Taichi NODA2), Jibak LEE1)# and Hiroshi HARAYAMA1–3) 1)Graduate School of Science and Technology, 2)Graduate School of Agricultural Science and 3)Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan #Present: Dynamics Laboratory, RIKEN Advanced Science Institute, Saitama 351-0198, Japan Abstract. The aim of this study is to provide evidence of the existence of the 10 (ADCY10) ortholog proteins in boar spermatozoa. Experiments with RT-PCR techniques, nucleotide sequence analyses and Northern blot analyses revealed that boar testes exclusively express approximately 5.1-kbp RNA, the nucleotide sequence of which is highly similar to that of human ADCY10. Database analyses with CDART suggested that pig ADCY10 ortholog proteins conserve two catalytic domains of adenylyl cyclase. Western blot techniques and indirect immunofluorescence with a specific antiserum to pig recombinant ADCY10 ortholog proteins showed that 48-kDa and 70-kDa truncated forms of pig ADCY10 ortholog proteins are localized in the equatorial segments and connecting pieces of boar ejaculated spermatozoa. Finally, cell imaging techniques with fluo-3/AM indicated that incubation with sodium bicarbonate (an ADCY10 activator) can initiate the calcium influx in the boar sperm heads that is controlled via the cyclic AMP signaling cascades. These results are consistent with the suggestion that functional ADCY10 ortholog proteins exist in the heads of boar spermatozoa. This is the first direct evidence of the existence of ADCY10 proteins in the heads of mammalian spermatozoa. Key words: Bicarbonate, Calcium, Cyclic AMP, Pig, Sperm (J. Reprod. Dev. 56: 271–278, 2010)

hile mammalian spermatozoa are transported through the spermatozoa is an adenylyl cyclase 10 (ADCY10, formerly called epididymis, they gradually acquire potentials to move pro- soluble adenylyl cyclase) that is distinguished from the other iso- gressively and fertilize oocytes. This process is termed sperm forms by -independent activation and lack of a maturation [1]. In the terminal region of the epididymis (caudal membrane-binding domain. Binding of bicarbonate to ADCY10 epididymides), stored spermatozoa are temporally quieted by the obviously stimulates catalysis of adenosine 5’-triphosphates (ATP) influence of the acid-base status of the luminal fluid [2, 3] and to cyclic adenosine 3’,5’-monophosphate (cAMP) [22, 23], which interaction with membrane stabilizing factors [4, 5]. Immediately is an important second messenger that can activate the protein after ejaculation, spermatozoa initiate flagellar beating in response kinase A (PKA)-mediated signaling cascades [11, 24, 25] and to bicarbonate, which originates from the male accessory genital exchange protein directly activated by cAMP (Epac)-mediated sig- glands [6, 7]. In the female reproductive tract, a relatively higher naling cascades, leading to the expression of sperm fertilizing concentration of bicarbonate in the luminal fluid also promotes a ability [26–29]. Moreover, the PKA-mediated signaling cascades series of sperm changes that are required for the expression of fer- are also modulated by the protein phosphatase 1 [30, 31]. tilizing ability, including phospholipid changes in the plasma The ADCY10 of rodent spermatozoa is being investigated in membrane [8, 9], changes in surface glycoconjugates [10], several laboratories and is well characterized [22, 32–36]. For increases of phosphorylated proteins [11, 12], increases of intracel- instance, two controversial hypotheses have been suggested con- lular calcium [13, 14], activation of phospholipase A2 [15] and cerning the mechanisms for generation of the truncated form of polymerization and subsequent depolymerization of F-actin [16]. ADCY10 so far. In the primary report, it is considered that this These molecular changes enable the spermatozoa to undergo the cyclase is originally synthesized mainly in the testis as a 189-kDa acrosome reaction in the heads and hyperactivation in the flagella. precursor (full-length form) with two catalytic domains in the N- Extracellular bicarbonate enters spermatozoa through the terminal region and that it then changes to the 48-kDa truncated plasma membrane by the actions of carbonic anhydrase [3, 17, 18], form during the sperm maturational process. However, it has lately sodium-bicarbonate cotransporter [19] and bicarbonate/chloride been reported that both the full-length and truncated forms of exchanger [20, 21]. The intracellular acceptor for bicarbonate in ADCY10 are generated from the same Adcy10 in the testis by alternate splicing. In any case, the resultant truncated form consists Received: October 7, 2009 almost exclusively of two conserved catalytic domains. The spe- Accepted: December 20, 2009 Published online in J-STAGE: January 27, 2010 cific cyclase activity of the truncated form is approximately 20-fold ©2010 by the Society for Reproduction and Development higher than that of the full-length form. This cyclase is also stimu- Correspondence: H Harayama (e-mail: [email protected]) lated by direct binding with not only bicarbonate but also calcium. *S Tate, K Nakamura and C Suzuki contributed equally to this work. In mature spermatozoa, the ADCY10 is localized in the middle 272 TATE et al.

Table 1. Primer sets designed to amplify pig adenylyl cyclase 10 (ADCY10) ortholog

Primer Forward primers Reverse primers Expected Set # Sequential Sequential molecular size Nucleotide sequences No. Nucleotide sequences No. of PCR product 1 CTGACATGGCACTTCTGCTG 3– 22 CTCCTGGGTCTCAAACAATCC 587– 567 585 2 TGCCTTGAACATGAACCCTC 176– 195 GTAGGGGGCGGTTTTAAGA 853– 835 678 3 TCTTAAAACCGCCCCCTAC 835– 853 CTCATTAAGGCCCAAACACTG 1586– 1566 752 4 GTCAACATAGCTGCCAGGA 1416– 1434 GAACAGGTTATTCCAGGTCAC 2486– 2466 1071 5 TCTGCCTAGACCTCAGTGTCA 2305– 2325 GGTGCATGGCTTTCTTCTGG 3030– 3011 726 6 GCTGGAAAGCCAGGTGATTG 2924– 2943 ACCATCTGGCCCATGTTG 3622– 3605 699 7 CCTCTGGCTCACCACTTTCTG 3387– 3407 TTTGAACCACACGCCCTG 4004– 3987 618 8 TCCAGATCGTTAAGGCCTACC 3931– 3951 CCAGGAGCATTCCACTTTGGAA 4446– 4425 516 9 GAGCACATCTTCAGCAAGGC 4293– 4312 CCATGATGGGAGATTCAGGA 4928– 4909 636 10 GCGTCGACaCCCTCGAAGAGAAGAATCACAG 191– 212 CGCAAGCTTbAAATTTTAAGATGTCTCCTCC 473– 453 300 11 GGAATTCcACCTCCTGAGGCTGGCTTGC 925– 944 GGCAAGCTTbGGCATCCTGAATGGCTGAACC 1136– 1116 228 a The underlined sequence indicates the restriction site for SalI. b The underlined sequence indicates the restriction site for HindIII. c The underlined sequence indicates the restriction site for EcoRI.

pieces and is involved in the systems of ATP synthesis. Moreover, ome.org/cgi-bin/web-primer). In addition, we designed the PCR it also plays a critical role in the regulation of cAMP-dependent primers with flanking restriction sites, and these were indicated as protein tyrosine phosphorylation and motility activation. Thus, set #10 (SalI and HindIII for the forward and reverse primers, male mice lacking ADCY10 are infertile, as their spermatozoa are respectively) and set #11 (EcoRI and HindIII for the forward and immotile. To our knowledge, however, only limited data are avail- reverse primers, respectively) in Table 1. The PCR products that able regarding ADCY10 ortholog proteins in spermatozoa from were amplified with the primer sets #10 and #11 were expected to other species of mammals, although existence of this cyclase is be partial cDNA fragments for the truncated form of pig ADCY10 postulated in the models of cAMP-signaling cascades leading to the ortholog. expression of sperm fertilizing ability [e.g., 21]. The aim of this Total RNAs were extracted from the testes, livers and kidneys of study was to provide evidence of the existence of functional three mature Meishan boars (1–3 years old) using ISOGEN RNA ADCY10 ortholog proteins in boar spermatozoa by reverse tran- extraction reagent (Nippon Gene, Tokyo, Japan), and then they scription (RT)-polymerase chain reaction (PCR), Northern were reverse-transcribed using a SuperScript First Strand Synthesis blotting, immunodetection and bioassay for the biological function System for RT-PCR (Invitrogen, Carlsbad, CA, USA). The of the ADCY10 ortholog proteins. obtained cDNAs were used as templates for PCR to amplify cDNA fragments of pig ADCY10 ortholog with PCR Master Mix Materials and Methods (Promega Corporation, Madison, WI, USA) and the above-men- tioned primer sets (Table 1). The PCR products were subjected to Animal use ethics statement electrophoresis in 1% agarose gel containing 100 ng/ml ethidium The Committee of Laboratory Animal Experiments of Kobe bromide (Wako Pure Chemical Industries, Osaka, Japan). University investigated our research plan and the feeding condi- We commissioned Hokkaido System Science (Sapporo, Japan) tions of our animals. We undertook the following experiments to do the cDNA sequence analyses of PCR products that were with the approval of this committee (document Nos.16-04-08 and amplified with primer sets #1–9 (http://www.hssnet.co.jp/). 19-5-11). Preparation of cRNA probes and Northern blot analysis Design of PCR primers, RT-PCR and sequence analysis The PCR products that were amplified with primer set #10 were To obtain a cDNA sequence of pig ortholog of ADCY10, the subcloned into the SalI/HindIII site of pSPT19 plasmids (Roche HTGS and EST of Sus scrofa were searched with the cross species Diagnostics, Manheim, FRG). Using a DIG RNA Labeling Kit megaBLASTN 2.2.18 of the BLAST Pig Sequences (http:// (Roche), digoxigenin (DIG)-labeled antisense and sense cRNA www.ncbi.nlm.nih.gov/projects/genome/seq/BlastGen/Blast- probes were synthesized from the plasmids. Northern blot analyses Gen.cgi?pid=10718) [37] using human ADCY10 (NM_018417.2) were performed as described previously [28]. In brief, 2.6 μg of as a query. Sequences showing significant homology were found total RNAs from the testes, livers and kidneys were loaded in each in the Sus scrofa chromosome 4 clone CH242-165E10 lane, electrophoresed in formaldehyde agarose gel and transferred (CU582795), Sus scrofa chromosome 4 clone CH242-281L8 to a nylon membrane (Hybond N+, GE Healthcare UK, Bucking- (CU462923), PDUts1003A12 Porcine testis cDNA library I Sus hamshire, UK) in 10 × saline sodium citrate (SSC) overnight. The scrofa cDNA clone (CV864231), 108744 MARC 1 PIG Sus scrofa membrane was prehybridized in a hybridization buffer (DIG Easy cDNA 5-, mRNA sequence (AW656468) and PDUts2051H11 Por- Hyb, Roche) at 50 C for 60 min. Subsequently, the membrane was cine testis cDNA library II Sus scrofa cDNA clone (CX060972). hybridized with either an antisense or sense DIG-labeled RNA The obtained sequences were combined and then used to design probe (600 ng) in the hybridization buffer at 50 C overnight. After PCR primer sets (Table 1) using Web Primer (http://seq.yeastgen- sequential washing, the membrane was blocked with 1x blocking BOAR SPERM ADCY10 ORTHOLOG 273 buffer (Roche) for 60 min and then treated with alkaline phos- serum to pig recombinant ADCY10 ortholog proteins (1:30). After phatase-conjugated anti-DIG Fab-fragments (1:10,000, Roche) for washing twice with PBS and blocking with PBS-BSA, they were 40 min. After washing, the alkaline phosphatase activity was visu- treated with fluorescein isothiocyanate (FITC)-conjugated rabbit alized with CDP-Star (Roche) and Hyperfilm-ECL (GE anti-mouse immunoglobulins (Dako, 1:30) for 120 min and then Healthcare). washed three times with PBS. The immunostained spermatozoa were put onto glass slides with VECTASHIELD Mounting Preparation of antiserum Medium (Vector Laboratories, Burlingame, CA, USA) and then Production of an antiserum was performed according to methods covered with coverslips. The preparations were observed under a described previously [38]. Briefly, the above-mentioned PCR differential interference microscope equipped with epifluorescence products that were amplified with primer set #11 were digested (U-MNIBA2 mirror unit composed of BP470–490 excitation filter, with EcoRI and HindIII and then subcloned into the E. coli expres- DM505 dichroic mirror and BA510–550 emission filter, Olympus, sion vector pGEX-KG. Mouse polyclonal antiserum was raised Tokyo, Japan). against pig ADCY10 ortholog proteins by injection of a mixture of the recombinant protein fragments (first injection, 0.3 mg protein; Detection of free calcium in spermatozoa second injection, 0.1 mg protein) twice with Titer Max Gold (Titer The procedures for detection of free calcium in spermatozoa Max, Norcross, GA, USA) with an interval of 37 days and was were described previously [39, 40]. In brief, the incubation recovered 17 days after the second injection. medium was a modified Krebs-Ringer Hepes medium (mKRH:

94.60 mM NaCl, 4.78 mM KCl, 1.19 mM MgSO4, 1.19 mM Sodium dodecyl sulfate (SDS)-polyacrylamide gel KH2PO4, 1.71 mM CaCl2, 25.07 mM Hepes, 5.56 mM glucose, electrophoresis (PAGE) and Western blot analysis 0.50 mM sodium pyruvate, 21.58 mM sodium lactate, 50 μg/ml Sperm-rich fractions from ejaculates were collected from two streptomycin sulfate, 100 IU/ml potassium penicillin G and 2 μg/ml mature Meishan boars (older than one year) by a manual method phenol red, pH 7.4) containing 0.1% PVA (mKRH-PVA). The and used for the following experiments within 60 min after ejacula- washed spermatozoa were loaded at 38.5 C in the dark for 30 min- tion. The spermatozoa were washed once in a two-step gradient of utes with a cell-permeable calcium indicator, fluo-3/AM (Dojindo 2 ml of 90% and 5 ml of 45% isotonic Percoll (GE Healthcare) that Laboratories, Kumamoto, Japan; final concentration: 5 μM), in was prepared with phosphate-buffered saline (PBS: 136.9 mM mKRH-PVA containing 0.02% Pluronic F127 (Sigma-Aldrich).

NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4) and Subsequently, the loaded spermatozoa were washed twice in PBS- then twice in PBS containing 0.1% polyvinyl alcohol (PVA, PVA and incubated in mKRH-PVA supplemented with either Sigma-Aldrich, St. Louis, MO, USA; PBS-PVA) by centrifugation, sodium bicarbonate (5–20 mM) or a cell-permeable cAMP analog as described previously [39]. The sperm suspension was diluted [Sp-5,6-cBiMPS (cBiMPS), 0.1 mM, Biomol Research Laborato- with an equal volume of double-strength Laemmli buffer and then ries, Plymouth Meeting, PA, USA] at a sperm concentration of 1.0 boiled for 5 min. After centrifugation to remove sperm debris, the × 108 cells/ml at 38.5 C for 60 min in the dark. Immediately before sperm extracts were subjected to SDS-PAGE using 10% polyacry- and after the 60-min incubation, aliquots of each sperm sample lamide gels. After electrotransfer onto a polyvinylidene fluoride were recovered, placed onto prewarmed glass slides and examined (PVDF) membrane (Immobilon P, Millipore, Bedford, MA, USA), under a differential interference microscope equipped with epifluo- the separated proteins were probed with the mouse antiserum to the rescence (U-MNIBA2 mirror unit). In the experiments to examine pig recombinant ADCY10 ortholog proteins (1:1,000) diluted with side effects of sodium bicarbonate (e.g., increases in pH and osmo- 5% (v/v) fetal calf serum (FCS, Dainippon Sumitomo Pharma, larity of the media), mKRH-PVA was supplemented with 1 N Osaka, Japan) in PBS containing 0.1% (v/v) Tween 20 (Wako, NaOH (to adjust the pH to 7.6–8.0) or NaCl (to increase the NaCl PBS-Tween) for 180 min. This was followed by treatment with concentration by 5–15 mM). In the experiments to examine roles horseradish peroxidase (HRP)-conjugated goat anti-mouse immu- of extracellular calcium, mKRH-PVA lacking CaCl2 and supple- noglobulins (1:2,500, DakoCytomation Denmark A/S, Glostrup, mented with ethylenediamine-N,N,N’,N’-tetraacetic acid trisodium Denmark) for 60 min. After washing, the peroxidase activity was salt trihydrate (EDTA, Dojindo, 1 mM) was used. In the experi- visualized using Western Blotting Luminol Reagent (SuperSignal ments to examine the relationship between bicarbonate and cAMP West Femto Maximum Sensitivity, Thermo Fisher Scientific, signaling, mKRH-PVA was supplemented with sodium bicarbon- Rockford, IL, USA) and Hyperfilm-ECL (GE Healthcare). ate (10 mM) and Rp-cAMPS (a cell-permeable cAMP antagonist, Sigma-Aldrich, 1 mM). Indirect immunofluorescence The washed spermatozoa were separated from PBS by centrifu- Results gation and then fixed with 3% paraformaldehyde (Wako) in PBS for 30 min. After washing twice in PBS, the spermatozoa were Detection of ADCY10 ortholog RNA in boar testes treated with 1% (v/v) Triton X-100 (Nacalai Tesque, Kyoto, Japan) Fig. 1 panel A shows results of RT-PCR using total RNAs from in PBS for 5 min and then washed three times in PBS. The fixed boar testes, livers and kidneys and with primer sets #1–11, which and membrane-permeated spermatozoa were blocked with 5% were designed to amplify almost all parts of pig ADCY10 ortholog bovine serum albumin (BSA, Wako) in PBS (PBS-BSA) for 120 cDNA (Table 1). In all experiments with primer sets #1–11, RT- min and subsequently treated overnight at 4 C with the mouse anti- PCR products were successfully amplified with the RNAs from the 274 TATE et al.

testicular RNAs were used for sequential analyses. The obtained sequence data were combined, and the missing parts of the nucle- otide sequences were supplemented with sequential data from databases. The total sequence of cDNA (sequential Nos. 1-5021) was subjected to alignment analyses with human ADCY10 cDNA (NM_018417.2) using the ClustalW2 tool provided by EMBL-EBI (http://www.ebi.ac.uk/Tools/clustalw2/). The CDS of this sequence [from 186–188, ATG (start codon), to 5016–5018, TAA (stop codon)] was also translated into the amino acid sequence (Table 2) and then subjected to alignment analyses with human ADCY10 protein (NP_060887.2) using BLAST (blastx, http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi). The sequential homology rate (identity) of pig ADCY10 ortholog protein against human ADCY10 (NM_018417.2 and NP_060887.2) was 79% at the amino acid level (additionally, positives at the amino acid level= 1439/1612: 89%, data not shown). Furthermore, database analyses with the conserved domain architecture retrieval tool (CDART; http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi? cmd=rps) predicted that the pig ADCY10 ortholog protein con- tained two catalytic domains of adenylyl cyclase in the N-terminal region (Table 2). Additional analyses with BLASTN 2.2.18+ against the pig build 1 genome database [reference assembly only (gpipe/9823/ ref_conting)] revealed that most parts of the nucleotide sequences of pig ADCY10 ortholog (sequential Nos. 1–443, 445–676, 678– 3064 and 3066–4667) were included in the genome sequence of Sus scrofa chromosome 4 (NW_001886233.1), although three SNPs were observed out in sequential Nos. 444, 677 and 3065. This indicates that pig has an ortholog gene of ADCY10 in the genome at chromosome 4.

Immunodetection of ADCY10 ortholog proteins in boar Fig. 1. Detection of expression of ADCY10 ortholog RNA in boar testes. Panel A (RT-PCR, representative of three replicates): Total ejaculated spermatozoa RNAs from testes, livers and kidneys were reverse-transcribed As shown in Fig. 2 panel A, the mouse antiserum to pig recom- and amplified by PCR as described in the Materials and Methods binant ADCY10 ortholog proteins (a portion of truncated form) section. RT-PCR products were separated in 1% agarose gel recognized proteins with molecular masses of 48 kDa and 70 kDa containing ethidium bromide. G3PDH was used as the control sample. Panel B (Northern blot analyses, representative of three from boar ejaculated spermatozoa in the Western blots. The immu- replicates): Each lane of the gel was loaded with RNA from nodetection levels were almost equal in these proteins. When this testes, livers and kidneys, and the RNA was separated by antiserum was used for indirect immunofluorescence of boar ejacu- electrophoresis and blotted onto a membrane. The blotted membranes were hybridized to either an antisense or sense DIG- lated spermatozoa, a fluorescence signal of FITC was observed in labeled cRNA probe. Bands of ribosomal RNAs on the blotted the equatorial segment of the heads and the connecting pieces (Fig. membrane are shown below each lane. 2, panel B). In addition, the reactivity of the antiserum that was preabsorbed with antigenic recombinant proteins was reduced (Fig. 2, panels A and B). This proves the specificity of this antiserum to testes but not with those from the livers and kidneys. Moreover, the pig ADCY10 ortholog proteins. Northern blot analyses with the probes to the RT-PCR products amplified with primer set #10 detected an approximately 5.1-kbp Observation of the effects of bicarbonate on the increase of free transcript intensively in the testes but barely in the livers and kid- calcium in the sperm heads neys (Fig. 1 panel B). The molecular size of this testicular Our previous paper [40] showed that the cAMP signaling cas- transcript was almost equivalent to that of human ADCY10 (5061 cades are linked to the increases of intracellular calcium in the bps, NM_018417.2) and that of mouse Adcy10 (5209 bps, heads of boar spermatozoa. In this study, we observed the effects NM_173029.2). These results indicate that pig ADCY10 ortholog of bicarbonate (a physiological activator for ADCY10) on the RNA is exclusively expressed in the testes. Such an exclusive increase of intracellular free calcium concentration in order to expression of ADCY10 in the testes has been reported in mice and obtain physiological data supporting the existence of bicarbonate- rats [23, 41]. sensitive adenylyl (ADCY10 ortholog proteins) in the RT-PCR products that were amplified with primer sets #1–9 and boar sperm heads. Calcium imaging with fluo-3/AM (Fig. 3) BOAR SPERM ADCY10 ORTHOLOG 275

Table 2. Amino acid sequence of pig ADCY10 ortholog protein

The amino acid sequence of pig ADCY10 ortholog protein was translated from cDNA sequence data. Amino acids of human ADCY10 (NP_060887.2) that are the same as those of pig ADCY10 ortholog protein are indicated with asterisks. The underlined sequences indicate the domains of adenylyl cyclases suggested by the database analyses with CDART. 276 TATE et al.

Fig. 2. Immunodetection of ADCY10 ortholog proteins in boar ejaculated spermatozoa. Panel A (Western blot analyses, representative of three replicates): An aliquot of each sperm suspension (2.5 × 105 cells/lane) was used for SDS-PAGE and transblotting to a membrane, treated with an antiserum to pig recombinant ADCY10 ortholog proteins (1:1,000) and subsequently treated with horseradish peroxidase-conjugated anti-mouse immunoglobulins (1:2,500). Panel B (indirect immunofluorescence, representative of three replicates): The balance of each sperm suspension was fixed with 3% paraformaldehyde, treated with the anti-ADCY10 antiserum (1:30) and subsequently treated with fluorescein isothiocyanate- conjugated anti-mouse immunoglobulins (1:30). In the photographs, the equatorial segments and connecting pieces are indicated by arrowheads and arrows, respectively. For control experiments, either the antiserum preabsorbed with the antigens or normal mouse serum (NMS) was used instead of the antiserum.

revealed that addition of sodium bicarbonate (final concentrations: present study, the antiserum to pig recombinant ADCY10 ortholog 5–20 mM) increased free calcium in the sperm heads after a 60-min proteins equally recognized 48-kDa and 70-kDa boar sperm pro- incubation (Fig. 3, panel A). Additionally, in order to examine teins, as shown by the Western blotting techniques (Fig. 2, panel whether this increase of free calcium is due to side effects of the A). The molecular mass of the former corresponds to that of the addition of sodium bicarbonate, such as increases of pH and osmo- truncated form of rodent ADCY10 with catalytic domains in the N- larity, we used media with the pH ranging from 7.4 to 8.0 or media terminal region [22, 23], whereas the latter may be another trun- with higher concentrations of NaCl in the absence of sodium bicar- cated form that is limited to boar spermatozoa. Moreover, this bonate. In these experiments, the increase of intracellular free antiserum strongly reacted to the antigens of the equatorial seg- calcium in the sperm heads was barely detected (Fig. 3, panels B ments and connecting pieces of boar ejaculated spermatozoa (Fig. and C). Furthermore, when either calcium-deficient medium or 2, panel B). These results indicate that the pig ADCY10 ortholog cAMP antagonist [Rp-cyclic adenosine 3’,5’-monophosphorothio- proteins are potentially related to the functions of both the heads ate triethylammonium salt (Rp-cAMPS)]-containing medium was and flagella. Thus, there are likely differences in the regulatory used, the increase of intracellular free calcium in the sperm heads system for cAMP synthesis in the sperm heads between mice and was reduced (Fig. 3, panels D and E). These results were inter- boars. preted as showing that bicarbonate induced the cAMP signaling- dependent calcium influx. Roles of ADCY10 ortholog proteins in sperm function Previous articles [43, 44] have already shown that bicarbonate Discussion can induce an increase of intracellular cAMP in boar spermatozoa. Our previous papers [39, 40] have also reported that the boar sperm Localization of ADCY10 ortholog proteins heads have the cAMP-dependent signaling cascades leading to ele- In mouse spermatozoa [34, 36], ADCY10 is localized in the vation of intracellular calcium. In the present study, when boar middle pieces and is involved in the generation of ATP and the spermatozoa were incubated in the presence of bicarbonate, the flagellar beating but not in the functions of the heads, such as level of intracellular free calcium clearly increased in the heads. acrosomal exocytosis and fusion with the oocyte plasma mem- This increase was not due to side effects of the addition of bicar- brane. In sea urchin spermatozoa [42], however, the ADCY10 bonate, such as increases in the pH and osmolarity of the incubation ortholog proteins are present in the heads and are involved in the medium (Fig. 3). Thus, bicarbonate may enter the sperm heads by acrosomal exocytosis that is stimulated by bicarbonate. In the the action of bicarbonate transporter [19, 20] and then stimulate BOAR SPERM ADCY10 ORTHOLOG 277

study, however, ADCY10 ortholog proteins were not detected in this segment of the boar spermatozoa (Fig. 2). Thus, how do boar sperm principal pieces acquire a sufficient amount of the cAMP synthesized by bicarbonate-activated ADCY10 ortholog proteins? As a possible answer to this question, we have speculated that bicarbonate activates ADCY10 ortholog proteins of the connecting pieces and synthesized cAMP molecules are then rapidly dispersed to the principal pieces through the middle pieces. Otherwise, the principal pieces might include other forms of ADCY10 ortholog proteins that are not recognized by our antibody. The connecting piece in boar spermatozoa is an important seg- ment where cAMP-dependent signaling cascades modulate hyperactivation through activation of spleen tyrosine (Y) kinase (SYK), phospholipase Cγ and protein kinase C [25, 45, 46]. Thus, it is reasonable to suggest that ADCY10 ortholog proteins exist in this segment of boar ejaculated spermatozoa (Fig. 2) and may regu- late the above-mentioned cAMP signaling cascades leading to hyperactivation. This suggestion is supported by reports in other species. Specifically, bicarbonate stimulates motility and hyperac- tivation through the activation of ADCY10 and tyrosine phosphorylation of AKAP3 in human spermatozoa [47]. More- over, bicarbonate/ADCY10-induced protein phosphorylation is likely important in the cAMP signaling cascades that lead to hyper- activation in mouse [48] and bull spermatozoa [49]. Based on the data obtained in the present study, we would like to emphasize that functional ADCY10 ortholog proteins exist in the heads of boar ejaculated spermatozoa. This fact enables us to account for the effects of bicarbonate on the fertilization-related events in the boar sperm heads. Moreover, it is possible that proper modulation of sperm head ADCY10 ortholog proteins with bicar- bonate contributes to the suppression of polyspermy in in vitro fertilization and precocious acrosome reaction during the storage in vitro. On the other hand, it is very interesting that there are differ- Fig. 3. Detection of free calcium in boar ejaculated spermatozoa treated ences in the localizations of ADCY10 between boar (Fig. 2) and with NaHCO3. Washed boar spermatozoa were loaded with a cell-permeable calcium indicator fluo-3/AM (5 μM) and mouse spermatozoa [34]. This study has provided the first direct subsequently incubated with an ADCY10 activator NaHCO3 (5– evidence of the existence of ADCY10 proteins in the heads of 20 mM, panel A) for 60 min. For control experiments to mammalian spermatozoa. examine the side effects of the addition of NaHCO3, the incubation media were supplemented with NaCl (to increase the osmolarity, panel B) or with 1 N NaOH (to adjust the pH in four Acknowledgments steps between 7.4 and 8.0, panel C). The medium lacking CaCl2 and supplemented with EDTA (1 mM) was used in the This work was supported in part by a Grant-in-Aid (20580310) experiments to examine the roles of extracellular calcium (panel D). The medium was supplemented with NaHCO3 (10 mM) and from the Japan Society for the Promotion of Science to HH. We Rp-cAMPS (1 mM) in the experiments to examine the thank Ms Y Hirashima for her technical support in production of relationship between bicarbonate and cAMP signaling (panel E). the mouse antiserum.

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