REPRODUCTIONRESEARCH

Expression of Atp8b3 in murine testis and its characterization as a testis specific P-type ATPase

Eun-Yeung Gong, Eunsook Park, Hyun Joo Lee and Keesook Lee School of Biological Sciences and Technology, Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea Correspondence should be addressed to K Lee; Email: [email protected]

Abstract

Spermatogenesis is a complex process that produces haploid motile sperms from diploid spermatogonia through dramatic morphological and biochemical changes. P-type ATPases, which support a variety of cellular processes, have been shown to play a role in the functioning of sperm. In this study, we isolated one putative androgen-regulated gene, which is the previously reported sperm-specific aminophospholipid transporter (Atp8b3, previously known as Saplt), and explored its expression pattern in murine testis and its biochemical characteristics as a P-type ATPase. Atp8b3 is exclusively expressed in the testis and its expression is developmentally regulated during testicular development. Immunohistochemistry of the testis reveals that Atp8b3 is expressed only in germ cells, especially haploid spermatids, and the protein is localized in developing acrosomes. As expected, from its primary amino acid sequence, ATP8B3 has an ATPase activity and is phosphorylated by an ATP-producing acylphosphate intermediate, which is a signature property of the P-Type ATPases. Together, ATP8B3 may play a role in acrosome development and/or in sperm function during fertilization. Reproduction (2009) 137 345–351

Introduction the removal of testosterone by hypophysectomy or ethane dimethanesulfonate (EDS) treatment in rats leads to the Spermatogenesis is one of most complex processes of cell failure of spermatogenesis (Russell & Clermont 1977, differentiation in animals, in which diploid spermatogo- Bartlett et al. 1986). Within the seminiferous tubules, only nia undergo dramatic morphological and biochemical Sertoli cells and somatic cells that support the develop- changes to produce haploid motile spermatozoa. The ment of germ cells, seem to possess the androgen process of spermatogenesis consists of three phases. In receptor (AR) and, thus, are targets of the testosterone the first, the mitotic phase, spermatogonia are replen- signal that regulates spermatogenesis (Sharpe et al. 2003). ished through mitotic divisions of the stem cells. In the Recent studies with a Sertoli cell-specific knockout of AR second, the meiotic phase, the segregation of genetic in the testis showed that the testosterone signal is material results in the formation of haploid spermatids. In important for the development and survival of spermato- the third, the transformation phase, also referred to as cytes and spermatids (Chang et al. 2004, De Gendt et al. spermiogenesis, the morphogenetic events that are 2004, Tsai et al. 2006, Wang et al. 2006). needed to produce flagellated spermatozoa take place. P-type ATPases, as integral membrane proteins, The germ cells at different developmental stages are mediate the ATPase-dependent transport of substrates systematically arranged in the seminiferous tubules of the such as ions across biological membranes (Axelsen & testis, and move towards the lumen as their development Palmgren 1998, 2001). The P-type ATPase superfamily is proceeds. It has been shown that a large number of genes a large group of proteins the members of which are widely are involved in these processes, and their expressions are expressed from bacteria to humans (Catty et al. 1997, developmentally regulated in a stage-specific manner. Palmgren & Axelsen 1998). This superfamily is divided The process of spermatogenesis is regulated by a into five subfamilies based on phylogenetic analysis complex interplay of hormonal signals. One of the major according to substrate specificity and structural features. control hormones is testosterone that is produced in The subfamilies are designated Type 1 (heavy metal C C C C C testicular Leydig cells by the stimulation of the LH that is pumps), Type II (Na /K -, H /K -, and Ca2 -pumps), C C secreted from the pituitary. Testosterone is essential for Type III (H - and Mg2 -pumps), Type IV (phospholipid the initiation of spermatogenesis at puberty and for its pumps), and Type V (pumps with no assigned substrate; maintenance during adulthood (McPhaul et al. 1993, Peedersen & Carafoli 1987, Ardetzky 1996, Møller et al. Quigley et al. 1995, Walker & Cheng 2005). For example, 1996). The P-type ATPases show a common topological

q 2009 Society for Reproduction and Fertility DOI: 10.1530/REP-08-0048 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/25/2021 10:49:31AM via free access 346 E-Y Gong and others feature. They have 8 or 10 transmembrane domains with that were not treated with EDS and in the EDS-treated a large intracellular loop containing P-type ATPase- testis respectively (Jeong et al. 2004). One ATP8B3 of specific sequences and an ATP-binding site. One of the putative androgen regulated genes was moderately P-type ATPase consensus motifs within the loop consists induced in the EDS-treated testis compared with the of seven amino acids, DKTGT (L,I,V,M) (T,I,S). During the control testis or EDS-treated testis with 4 day testosterone catalytic cycle of the protein, the aspartic acid residue supplement (Fig. 1), which suggests that the gene is (‘D’) in the motif is phosphorylated by ATP, and is negatively regulated by androgen in the testis. Sequence subsequently dephosphorylated following substrate analysis of the cDNA revealed it as the ‘Atp8b3’ gene binding and transport (Tang et al. 1996, Cronin et al. (GenBank accession number: AY364445), which was 2002, Schultheis et al. 2004). recently reported to be required for normal fertilization P-type ATPases support a variety of cellular processes by sperm (Wang et al. 2004). by generating transmembrane electrochemical ion gradients. Recent studies showed that they also play diverse roles in successful male reproduction. For Expression of Atp8b3 is developmentally regulated in C example, the P-type Ca2 -ATPase isoform 4 of the murine testis plasma membrane (Atp2b4), which is localized to Wang et al.(2004)previously showed that Atp8b3 is the principal piece of the sperm tail, is responsible for exclusively expressed in the testis by RNA dot-blot the hyperactivated motility of sperms, causing male analysis. To examine further, the expression pattern of infertility in ATP2B4-null mice (Mansharamani et al. C C Atp8b3, northern blot analysis was performed with various 2001). The a-4 isoform of the Na /K -ATPase, which is mouse tissues. The Atp8b3 transcript was exclusively localized in the mid-piece of the tail, is also suggested to detected in the testis (Fig. 2A), which indicates that Atp8b3 be responsible for sperm motility (Wilson et al. 2001). In is expressed in a testis-specific manner. Investigation of addition, an ATP-dependent aminophospholipid trans- Atp8b3 expression during testis development showed porter that is in another subfamily of P-type ATPase and is that Atp8b3 messages were first detected at around localized in the acrosomal region of the sperm is 17 days after birth. Atp8b3 expression in the testis was probably critical for phospholipid distribution in the later upregulated up to adulthood and maintained bilayer, for sperm binding and penetration of the zona thereafter (Fig. 2B). ATP8B3 protein expression was pellucida of the egg (Mansharamani & Chilton 2001). also investigated by western blot analyses using an In this study, we isolated a testis-specific P-type ATPase affinity-purified anti-ATP8B3 antibody raised against that was previously cloned and designated sperm- specific aminophospholipid transporter (Atp8b3, pre- viously known as Saplt; Wang et al. 2004), as a candidate of androgen-regulated genes in murine testis. Atp8b3 is exclusively expressed in germ cells, especially haploid spermatids, and the protein is localized in developing acrosomes. ATP8B3 has ATPase activity, and reveals the characteristics of the P-Type ATPase. Taken together, ATP8B3 may play a role in acrosome development and/or in sperm function during fertilization.

Results Isolation of Atp8b3 as a putative androgen-regulated gene We have independently isolated the ATP8B3 (Wang et al. 2004) from a previous attempt to clone androgen- regulated genes in the testis; this process was conducted by PCR-select cDNA subtractions with mRNA samples Figure 1 Isolation of Atp8b3 as a putative androgen-regulated gene. prepared from the EDS-treated and androgen-supplied Northern blot analyses were performed with testis total RNAs isolated rat testis against those from EDS only-treated testis (Jeong from mice injected either with vehicle only (Control), with EDS (EDS) or et al. 2004). EDS is a cytotoxic compound that destroys with EDS, and supplemented with testosterone for 4 days (EDSCT differentiated Leydig cells in adult rats, causing the 4 days). Hsd3b was used as a Leydig cell-specific marker gene. Ngfr depletion of androgen (Kerr et al. 1985, Sharpe et al. that is expressed in Sertoli cells and negatively regulated by androgen was used as an indicator of androgen depletion. Atp8b3 that was 1992). Hsd3b, a Leydig cell-specific marker gene, and obtained by PCR-select cDNA subtraction screening was induced by Ngfr, a gene that is negatively regulated by androgen in testosterone depletion. Expression of Gapdh was used as an internal Sertoli cells, were expressed only in the control testes control.

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Downloaded from Bioscientifica.com at 09/25/2021 10:49:31AM via free access Expression and characterization of ATP8B3 in murine testis 347

Figure 2 Developmentally regulated expression of Atp8b3 in murine testis. Northern blot analysis of total RNA (20 mg/lane) and western blot analysis of total protein (50 mg/lane) from mouse tissues. (A and B) Blots were hybridized with a 32P-labeled mouse Atp8b3 cDNA (nucleotides 3528–3777) probe as described in ‘Materials and Methods’. The expression of 28S ribosomal RNA (rRNA) was used to verify the integrity and quantity of RNA. (C and D) Blots were detected using affinity-purified anti-ATP8B3 antibody. The expression of b-actin was Figure 3 Localization of ATP8B3 protein in the developing acrosome of used to verify the integrity and quantity of protein. male germ cells. ATP8B3 protein was detected by immunohistochem- istry in mouse adult testis using the affinity-purified anti-ATP8B3 antibody. Dark brown colors (arrows) indicate positive signals from the amino acids 396–568 of the ATP8B3 protein. The acrosomes of spermatids within testicular tubes. (A) Seminiferous antibody detected a protein of approximate 160 kDa tubules detected with the affinity-purified anti-ATP8B3 antibody. that showed the same size with the in vitro-translated (B) Negative control detected with the preimmune sera. (C) ATP8B3 ATP8B3 from the full-length cDNA (data not shown). In expression in round spermatids. (D) ATP8B3 expression in elongated spermatids. Signals were detected using MetaFluor software for a light consistence with the data from northern blot analyses, microscope. ATP8B3 protein was detected only in mouse testis (Fig. 2C), and found from day 17 to adulthood during expressing green fluorescent protein (GFP)-fused testis development (Fig. 2D). ATP8B3 in Cos-7 cells that did not have an acrosome structure, but have cytoplasmic organells. GFP–ATP8B3 fusion protein was predominantly localized in the ATP8B3 protein is localized to the developing acrosome cytoplasmic compartment (Fig. 4A and B). Further of germ cells within seminiferous tubules analysis using ER-TrackerBlue-White distrene-plastici- zer-xylene (Blue; Fig. 4C) and WGA, Golgi-marker (Red; To examine the cell types that express ATP8B3 protein in Fig. 4D) suggested that the ATP8B3 proteins were the testis, we performed immunohistochemistry of adult associated with both the ER and Golgi apparatus mouse testis using affinity-purified anti-ATP8B3 (Fig. 4G and H). antibody. ATP8B3 protein was detected only in sperma- tids, both round and elongated, within seminiferous tubules at all stages. Interestingly, ATP8B3 protein was ATP8B3 is a P-type ATPase specifically localized to the developing acrosomes Analysis of the primary amino acid sequence suggests (Fig. 3). Some non-specific signals were weakly detected ATP8B3 as a Type IV P-type ATPase. To confirm the in the spermatogonia that did not appear during the ATPase activity of ATP8B3, we prepared a recombinant immunofluorescence detection method (data not protein of ATP8B3 fused to GFP, which was over- shown). Negative controls, preimmune serum (Fig. 3B), expressed in Cos-7 cells and purified by immunoprecipi- anti-His antibody, and secondary antibody gave no tation using anti-GFP antibody. In ATPase assays positive signals (data not shown). These results are conducted by the Malachite Green Method, GFP– consistent with the previously published data that ATP8B3 fusion protein exhibited high ATPase activity, ATP8B3 is localized in the acrosomes of mouse sperms while the control GFP protein itself did not (Fig. 5A). isolated from the epididymis (Wang et al. 2004). A signature property of the P-Type ATPases is that the Acrosomes are formed from the cytoplasmic orga- conserved aspartic acid residue in the DKTGT motif nelles such as endoplasmic reticulum (ER) and Golgi accepts the g-phosphate from ATP during the catalytic C apparatus (Griffiths et al. 1981, Tanii et al. 1992). To cycle in the presence of Mg2 , and forms a covalent further confirm ATP8B3 as an acrosome protein, we acylphosphate intermediate. To confirm the property of examined the subcellular localization of ATP8B3 by ATP8B3 as a P-type ATPase, we investigated the formation www.reproduction-online.org Reproduction (2009) 137 345–351

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Figure 5 The ATPase activity and the formation of the phosphoenzyme intermediate of ATP8B3. (A) The ATPase assay was performed as described in ‘Materials and Methods’ using the GFP–ATP8B3 fusion protein that was partially purified using an agarose A beads containing Figure 4 Localization of ATP8B3 protein in cytoplasmic organelles in anti-GFP antibody. Buffer, assay buffer only. GFP, immuno-purified GFP non-spermatid mammalian cells. (A and B) The cellular localization of protein. ATP8B3, immuno-purified GFP–ATP8B3 fusion protein. (B and m g 32 ATP8B3 was investigated with GFP–ATP8B3 fusion protein (Green) in C) The reaction was initiated by the addition of 50 M( - P)ATP at 8 Cos-7 cells. (C) Endoplasmic reticulum was stained with ER-Track- 4 C. Samples were analyzed on 8% polyacrylamide gels. erBlue-White distrene-plasticizer-xylene (Blue). (D) The Golgi appar- atus was stained with WGA (Red). (E and F) Nuclei were stained with Propidium iodide (Red) or DAPI (Blue). (G) Merged A, C and E. (H) However, Atp8b3 is mostly expressed in germ cells that Merged B, D and F. do not express AR (Chang et al. 2004, Holdcraft & Braun 2004). This suggests that Atp8b3 expression is likely of the phosphoenzyme using (g-32P) ATP. As shown in under the indirect control of androgen through Fig. 5B, immunoprecipitated ATP8B3 (GFP-ATP8B3) was interaction between germ cells and testicular somatic readily phosphorylated in the presence of ATP, and this cells such as Sertoli cells that express AR. phosphorylation was enhanced in the presence of Testosterone, a major physiological androgen in the phosphatase inhibitors, orthovanadate (Na3VO4), and NaF. The phosphoenzyme formed under these conditions testis, is a critical hormone for the spermatogenesis. has been shown to be slowly dephosphorylated following Some putative target genes have been reported (Meng the exhaustion of the low concentration of ATP, but not to et al. 2005, Denolet et al. 2006) in the testis, although be dephosphorylated in the presence of EDTA (Fan & only a few target genes have been confirmed as real Rosen 2002). As shown in Fig. 5C, the phosphorylated targets. One of the well-known androgen targets is the ATP8B3 accumulated and lasted longer in the presence of Rhox5 homeobox gene that is expressed in Sertoli cells. EDTA. All together, these results suggest that ATP8B3 is a However, mice with a targeted mutation in the Rhox5 functional P-type ATPase. gene shows normal reproduction with no detectable alteration in testicular development or function, which suggests that Rhox5 is dispensable for normal spemato- Discussion genesis (Pitman et al.1998). The pathway of the In the present study, Atp8b3 was isolated as one of androgen signal and roles of its target genes in germ putative androgen-regulated genes in the testis. cell development remain to be poorly understood.

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The testis specific P-type ATPase that we isolated from a residues 396–568) to the PvuII/EcoRI-digested pRESET A vector testis library turned out to be ATP8B3. ATP8B3 was found (Invitrogen). GFP–ATP8B3 was constructed by inserting the in the membrane fraction of testicular cells and the full-length ATP8B3 into the EcoRI site of the pEGFP-C1 vector acrosomal region of the sperm head. Disruption of the (Clontech). Atp8b3 gene in mice caused abnormal phospholipid distribution in the outer bilayer of sperm. Fewer Atp8b3K/K sperm bind tightly or penetrate the zona PCR-select cDNA subtraction cloning pellucida of the egg, and undergo acrosomal reactions, Adult male Sprague–Dawley rats (75–80 days old) were resulting in lower fertility than the wild-type (Wang et al. injected i.p. with EDS (75 mg/kg) in dimethylsulfoxide–water 2004). Besides ATP8B3, other P-type ATPases are shown to (1:3, vol/vol). At the same time, the animals also received an be involved in the functional ability of the sperm. The C injection of either 0.1 ml vehicle oil or 25 mg testosterone P-type Ca2 -ATPase isoform 4 of the plasma membrane C C esters (Sustanon; Organon Korea Ltd, Seoul, Korea). The latter (ATP2B4) and a-4 isoform of the Na /K -ATPase, both of treatment was repeated every 3 days. Animals were killed which are Type II P-type ATPases, are responsible for 4 days after the EDS injection. PCR-select cDNA subtraction sperm motility, localizing in the principal piece of the was performed using PCR-select cDNA subtraction kit sperm tail (Woo et al. 2000, Prasad et al. 2004). For (Clontech) according to the manufacturer’s instructions (Jeong fertilization, sperm must be capacitated and gain the et al. 2004). ability to move towards and penetrate the zona pellucida. Such processes may include changes in the concentrations of some ions, cytosolic pH, and membrane compositions, Screening of l-testis cDNA library which are easily accomplished through the functions of A l-ZAPa cDNA library from mouse testis was purchased from P-type ATPases. Thus, more P-type ATPase family Stratagene Inc. (La Jolla, CA, USA). A total of 2.5!105 clones members are expected to be involved in spermatogenesis. were screened according to the manual. Briefly, phage During the formation of acrosomes in mammalian particles on replica nylon membranes were denatured in spermatids, proacrosomal vesicles are formed at the 0.5 M NaOH, 1.5 M NaCl, and neutralized in 0.5 M Tris, pH concave of the Golgi apparatus, and several proacroso- 8.0, 1.5 M NaCl. After u.v. cross-linking, the filters were mal vesicles coalesce on the face of the nuclear envelope. prehybridized and hybridized at 42 8C in the presence of 50% In general, further proacrosomal vesicles are not formed formamide, 10% dextran sulfate, 5! standard saline citrate after the formation of a nucleus-adherent acrosomal (SSC), 1 mM EDTA, 10 mg/ml denatured salmon sperm DNA, vesicle. The acrosomal vesicle then spreads over the and a random-primed (32P) dCTP-labeled rat ATP8B3 cDNA anterior hemisphere of a nucleus in the form of a cap (Susi probe. Filters were washed and exposed on Kodak X-ray film at et al. 1971, Burgos & Gutierrez 1986). ATP8B3 protein is 70 8C. Secondary screening was repeated as described to localized in the acrosomes of spermatids, while it is isolate single, pure phage plaques. The cDNA inserts were associated with the cytoplasmic membrane organelles recovered from the phage particles as plasmids in the pBSaSK such as the Golgi apparatus and ER in non-spermatid vector by in vivo excision and sequenced (Jeong et al. 2004). mammalian cells. Thus, it is likely that ATP8B3 protein is first associated with the Golgi apparatus and/or proacro- somal vesicles, and is then concentrated in the acrosome Northern blot analysis during acrosome formation in spematids. However, it is Total RNA was extracted from dissected tissues using Tri- also possible that ATP8B3 is expressed and directly reagent solution (Molecular Research Center Inc., Cincinnati, associated with the acrosome during the CAP Phase. OH, USA). Twenty micrograms of total RNA were separated on In this study, we have detailed the expression and a 1.2% denaturing agarose gel, transferred onto a Zeta-probe localization of ATP8B3, a protein that is known to be nylon membrane in 10! SSC by capillary transfer, and then involved in the redistribution of acrosome phospholipids, immobilized under u.v. light. After prehybridization, the during germ cell development in the testis. We have also membrane was hybridized at 42 8C in a solution containing confirmed its characteristics as a P-type ATPase. Further 50% formamide, 10% dextran sulfate, 5! SSC, 1 mM EDTA, studies are needed to address the biological role of 10 mg/ml denatured salmon sperm DNA, and a random-primed phospholipid distribution of acrosomes by ATP8B3, and (32P)-labeled TSPA probe spanning nucleotides 3528–3777. to determine how ATP8B3 expression is regulated in germ After washing at 65 8C for 20 min in 0.2! SSC and 0.1% SDS cell-specific and developmental stage-specific manners. as a final stringency, the membrane was exposed on Kodak X-ray film at K70 8C(Chattopadhyay et al. 2006).

Materials and Methods Fluorescence Plasmids Cos-7 cells were plated onto gelatin-coated coverslips the day The pRESET-ATP8B3 (396–568) plasmid was constructed by before transfection. The cells were transiently transfected with conjugating the C-terminal portion of ATP8B3 (amino acid GFP–ATP8B3 or GFP-only using Effectene reagent (Qiagen). www.reproduction-online.org Reproduction (2009) 137 345–351

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After 48 h, the cells were washed with PBS and fixed with 2% without 200 mM ATP, incubated for 1 h in a water bath at 37 8C, formaldehyde for 5 min for fluorescent microscopy. Staining and centrifuged at 13 000 g for 1 min. Reactions were stopped was achieved by incubating cells in staining solution (for by the addition of 900 ml stop buffer (0.034 g malachite green, nucleus staining: 500 mg/ml propidium iodide (Sigma), for ER 1.1 g ammonium molybdate, 8.8 ml HCl, 40 ml tritonX-100 in staining: 1 mM/ml ER-Tracker Blue-White distrene-plasticizer- a final volume of 100 ml) into 100 ml collecting supernatant, xylene Molecular probe (Eugene, OR, USA). Cells were and were measured for absorbance at 650 nm after 10 min mounted onto microscope slides and were examined using of incubation at room temperature (Itaya & Ui 1966, Hartman the Olympus 1!70 fluorescent microscope (Tokyo, Japan). The et al. 1998). images were analyzed using MetaFluor software (Universal Imaging Corp., Downingtown, PA, USA). Detection of phosphoenzyme intermediate Agarose bead-purified ATP8B3 was incubated with 0.5 mM Production of ATP8B3 antibody and western blot assay (g-32P)ATP (106 cpm/pmol of ATP) at room temperature for the Anti-ATP8B3 antibody was produced using a ATP8B3 protein designated periods of time in a solution (25 mM Tris/MES, pH domain corresponding to amino acid residues 396–568 (a 7.5, 30 mM NaCl, 1 mM DTT, 10% glycerol, and 3 mM hydrophilic region of ATP8B3). His-tagged ATP8B3 was MgCl2) in the presence of 50 mM orthovanadate (Na3VO4) produced from pRESET-ATP8B3 (396–568) in BL21 bacterial and 50 mM NaF or 3 mM EDTA were indicated. The reaction cells and isolated with SDS-PAGE gel running. The ATP8B3 was stopped by the addition of SDS sample buffer, followed protein was vacuum-dried, powdered, and injected intramus- by SDS-PAGE and autoradiography (Ding et al. 2000, Fan & cularly into rabbits with complete Freund’s adjuvant one time Rosen 2002). for the first immunization and with incomplete Freund’s adjuvant five times for subsequent immunizations every 2 weeks. Serum was collected after the six immunizations. Declaration of interest Affinity purification of antibody was conducted using antigen The authors declare that there is no conflict of interest that immobilized on nitrocellulose filters. Western blot analysis was could be perceived as prejudicing the impartiality of the performed with the ATP8B3 antibody as reported previously research reported. (Chattopadhyay et al. 2006). Signals were then detected with an ECL kit (Amersham Pharmacia). Funding Immunohistochemistry This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2006-005- For immunostaining, paraffin-embedded sections of adult J03002 and KRF-2004-005-C00135). mouse testes were deparaffinized in Histoclear and rehydrated in an ethanol series. After blocking endogenous peroxidases with 10% hydrogen peroxide, the sections were processed for immunohistochemistry using the affinity-purified ATP8B3 References antibody and the Histostain-Plus kit (Zymed Laboratories Inc., Ardetzky OJ 1996 Simple allosteric model for membrane pumps. Nature South San Francisco, CA, USA) according to the protocol 211 969–970. suggested by the manufacturer. Slides were then mounted with Axelsen KB & Palmgren MG 1998 Evolution of substrate specificities in the GVA mounting solution (Zymed) and observed under a light P-type ATPase superfamily. Journal of Molecular Evolution 46 84–101. 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