SEPT12–NDC1 Complexes Are Required for Mammalian Spermiogenesis

International Journal of Molecular Sciences

Article SEPT12–NDC1 Complexes Are Required for Mammalian Spermiogenesis

Tsung-Hsuan Lai 1,2,3, Ying-Yu Wu 4, Ya-Yun Wang 5, Mei-Feng Chen 6, Pei Wang 2, Tsung-Ming Chen 7, Yi-No Wu 4, Han-Sun Chiang 4, Pao-Lin Kuo 8 and Ying-Hung Lin 4,*

1 Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei 106, Taiwan; [email protected] 2 School of Medicine, Fu Jen Catholic University, New Taipei City 242, Taiwan; [email protected] 3 Institute of Systems Biology and Bioinformatics, National Central University, Jhongli City, Taoyuan Country 320, Taiwan 4 Graduate Institute of Biomedical and Pharmaceutical Science, Fu Jen Catholic University, New Taipei City 242, Taiwan; [email protected] (Y.-Y.W.); [email protected] (Y.-N.W.); [email protected] (H.-S.C.) 5 Department of Chemistry, Fu Jen Catholic University, New Taipei City 242, Taiwan; [email protected] 6 Bone and Joint Research Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; [email protected] 7 Department and Graduate Institute of Aquaculture, National Kaohsiung Marine University, Kaohsiung 811, Taiwan; [email protected] 8 Department of Obstetrics & Gynecology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-2-2905-3399; Fax: +886-2-2905-3415

Academic Editor: William Chi-shing Cho Received: 8 September 2016; Accepted: 7 November 2016; Published: 16 November 2016

Abstract: Male factor infertility accounts for approximately 50 percent of infertile couples. The male factor-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile sperm, immature sperm, abnormally structured sperm, and sperm with nuclear damage. Our knockout and knock-in mice models demonstrated that SEPTIN12 (SEPT12) is vital for the formation of sperm morphological characteristics during spermiogenesis. In the clinical aspect, mutated SEPT12 in men results in oligozoospermia or teratozoospermia or both. Sperm with mutated SEPT12 revealed abnormal head and tail structures, decreased chromosomal condensation, and nuclear damage. Furthermore, several nuclear or nuclear membrane-related proteins have been identiﬁed as SEPT12 interactors through the yeast 2-hybrid system, including NDC1 transmembrane nucleoporin (NDC1). NDC1 is a major nuclear pore protein, and is critical for nuclear pore complex assembly and nuclear morphology maintenance in mammalian cells. Mutated NDC1 cause gametogenesis defects and skeletal malformations in mice, which were detected spontaneously in the A/J strain. In this study, we characterized the functional effects of SEPT12–NDC1 complexes during mammalian spermiogenesis. In mature human spermatozoa, SEPT12 and NDC1 are majorly colocalized in the centrosome regions; however, NDC1 is only slightly co-expressed with SEPT12 at the annulus of the sperm tail. In addition, SEPT12 interacts with NDC1 in the male germ cell line through coimmunoprecipitation. During murine spermiogenesis, we observed that NDC1 was located at the nuclear membrane of spermatids and at the necks of mature spermatozoa. In male germ cell lines, NDC1 overexpression restricted the localization of SEPT12 to the nucleus and repressed the ﬁlament formation of SEPT12. In mice sperm with mutated SEPT12, NDC1 dispersed around the manchette region of the sperm head and annulus, compared with concentrating at the sperm neck of wild-type sperm. These results indicate that SEPT12–NDC1 complexes are involved in mammalian spermiogenesis.

Keywords: male infertility; SEPT12; NDC1

Int. J. Mol. Sci. 2016, 17, 1911; doi:10.3390/ijms17111911 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1911 2 of 10

1. Introduction

1.1. Male Infertility Between 2% and 12% of couples globally are affected by low fertility, and the cause in approximately half of these cases can be traced to the men [1]. Over the last 20 years, advanced intracytoplasmic sperm injection has been a technique used in the treatment of numerous subfertile men, who still produce small amounts of sperm that can be used for injection treatment [2]. Nevertheless, although intracytoplasmic sperm injection (ICSI) created a breakthrough in assisted reproduction, many infertile cases are still unable to achieve paternity, even when combined with testicular sperm extraction. The sperm-related causes of ICSI failure include immotile or immature sperm, sperm with structural defects, the absence of sperm, and sperm with premature chromosomal condensation or DNA damage [3].

1.2. SEPTINs (SEPTs) and Spermatogenesis SEPTINs (SEPTs) are highly preserved polymerizing GTP-binding proteins that belong to the fourth component of the cytoskeleton [4]. SEPTs participate in membrane compartmentalization, cytoskeletal remodeling, cell polarity, and spermatogenesis through interaction with cytoskeletal proteins [5]. SEPTs are involved in the pathogenesis of various conditions, including Alzheimer disease, hereditary neuralgic amyotrophy, leukemia, ovarian tumors, breast cancer, and male infertility [5,6]. During mammalian spermiogenesis, SEPT4 is located at the annulus, which is a ring-like structure connecting the midpiece and principal piece of the flagellum, and is required to maintain annulus integration [7,8]. Clinically, most spermatozoa from asthenozoospermia patients lost the SEPT4 signal [7,9,10]. In our groups, SEPT12 was identified as a potential sterile gene by employing a cDNA microarray analysis of the testicular tissue, which determined that SEPT12 was expressed in postmeiotic male germ cells during mammalian spermiogenesis [11,12]. Furthermore, sperm from SEPT12-mutated mice exhibited unique morphological defects (e.g., immature sperm head, bent tail, premature chromosomal condensation, and nuclear damage) [13]. In humans, SEPT12 mutations in infertile men result in teratozoospermia and oligozoospermia [14–16]. In addition, several nuclear or nuclear membrane-related proteins were identified as SEPT12 interactors through the yeast 2-hybrid system; one of these interactors is NDC1 [17].

1.3. Nuclear Pore Complexes and NDC1 In eukaryotic cells, the nuclear envelope (NE) defines a boundary between the nucleus and cytoplasm and is composed of an outer and an inner nuclear membrane [18]. Outer and inner nuclear membranes are fused to form nuclear pore complexes (NPCs), which mediate the nucleocytoplasmic transport. The NPC comprises approximately 30 nucleoproteins (Nups), which can be subdivided into four classes: (1) transmembrane ring Nups; (2) core scaffold (inner and outer ring Nups); (3) linker Nups; and (4) nuclear Phe-Gly (FG) Nups [19,20]. NPCs are anchored to the NE through transmembrane ring Nups, which are composed of three proteins: nucleoporin 210 (NUP210), NDC1, and POM121 transmembrane nucleoporin (POM121) in vertebrate cells [20]. NDC1 and POM121 are critical for nuclear assembly, whereas NUP210 is a major molecule for efficient nuclear pore complex disassembly and NE breakdown [21–23]. Akiyama et al. indicated that mutated NDC1 (also called Transmembrane Protein 48; TMEM48) causes gametogenesis defects and skeletal malformations in mice, and this strain was spontaneously detected at The Jackson Laboratory in skeletal fusions with sterility (sks) in mice [24]. To better understand the molecular mechanism explaining the association of SEPT12 mutation with teratozoospermia, a yeast 2-hybrid approach was conducted, allowing the identification of NDC1 as a potential SEPT12 interactor. In this study, we observed that NDC1 was expressed around the NE in round spermatids and at the neck of mature spermatozoa. Ectopic overexpressed NDC1 restricted SEPT12 localization at the nucleus and repressed SEPT12 filament formation. Int. J. Mol. Sci. 2016, 17, 1911 3 of 9 around the NE in round spermatids and at the neck of mature spermatozoa. Ectopic overexpressed NDC1Int. J. Mol. restricted Sci. 2016, 17SEPT12, 1911 localization at the nucleus and repressed SEPT12 filament formation.3 of In 10 SEPT12-mutated spermatozoa, the location of NDC1 was disturbed. According to these results, we suggestIn SEPT12-mutated that SEPT12–NDC1 spermatozoa, complexes the location are involved of NDC1 in was sperm disturbed. head and According tail formation to these during results, mammalianwe suggest thatspermiogenesis. SEPT12–NDC1 complexes are involved in sperm head and tail formation during mammalian spermiogenesis. 2. Results 2. Results 2.1. NDC1 as a SEPT12 Interactor 2.1. NDC1 as a SEPT12 Interactor In our previous study, SEPT12 mutations in human and mice spermatozoa were demonstrated to causeIn our teratozoospermia previous study, SEPT12 (e.g., nuclear mutations damage, in human premature and mice spermatozoachromosomal were condensation, demonstrated and to abnormalcause teratozoospermia morphologies (e.g.,of sperm nuclear heads damage, and prematuretails) [13–15,25]. chromosomal NDC1 was condensation, identified andas a abnormal SEPT12 interactormorphologies through of sperm the headsyeast and2-hybrid tails) [system13–15,25 by]. NDC1using wasa human identified testicular as a SEPT12 cDNA interactor library through[17]. In micethe yeast models, 2-hybrid mutated system NDC1 by using also aaffected human testicularmammalian cDNA spermatoge library [17nesis]. In [25]. mice We models, suggest mutated that mutatedNDC1 also SEPT12 affected disturbed mammalian the spermatogenesisspermiogenesis th [25rough]. We suggestNDC1. thatFirst, mutated to test SEPT12whether disturbed SEPT12 interactsthe spermiogenesis with NDC1 throughin human NDC1. male germ First, cells, to test we whether conducted SEPT12 an immunofluorescence interacts with NDC1 assay in human (IFA) andmale coimmunoprecipitation germ cells, we conducted assay an immunofluorescence (Co-IP). In mature human assay (IFA) spermatozoa, and coimmunoprecipitation SEPT12 and NDC1 assay are majorly(Co-IP). Incolocalized mature human in the spermatozoa,centrosome regions; SEPT12 andhowever, NDC1 NDC1 are majorly is only colocalized slightly co-expressed in the centrosome with SEPT12regions; at however, the annulus NDC1 of isthe only sperm slightly tail (Figure co-expressed 1A). In withmouse SEPT12 spermatozoa, at the annulus colocalization of the sperm patterns tail of(Figure SEPT121A). and In mouse NDC1 spermatozoa, are similar to colocalization those of human patterns spermatozoa of SEPT12 and[26]. NDC1 Second, are NTERA-2 similar to thosecl.D1 (NT2D1)of human cells, spermatozoa a pluripotent [26]. Second,human NTERA-2testicular embryonal cl.D1 (NT2D1) carcinoma cells, a pluripotentcell line, were human cotransfected testicular withembryonal pFLAG-NDC1 carcinoma and cell pGFP-SEPT12 line, were cotransfected vectors and with then pFLAG-NDC1 subjected to Co-IP and pGFP-SEPT12 with anti-GFP vectors antibody. and Thethen Co-IP subjected assays to performed Co-IP with using anti-GFP the antibody.anti-FLAG The antibody Co-IP assaysrevealed performed that FLAG-NDC1 using the was anti-FLAG pulled downantibody with revealed SEPT12-GFP that FLAG-NDC1 (Figure 1B, was Lane pulled 3: downAnti-GFP with SEPT12-GFPantibody). These (Figure results1B, Lane indicated 3: Anti-GFP that SEPT12antibody). interacts These with results NDC1 indicated in human that SEPT12male germ interacts cells. with NDC1 in human male germ cells.

FigureFigure 1. ColocalizationColocalization and and interaction interaction between SEPT12 and NDC1.NDC1. ( (AA)) Immunofluorescence Immunoﬂuorescence detectiondetection in aa maturemature human human spermatozoon spermatozoon of (ofa) ( SEPT12;a) SEPT12; (b) NDC1;(b) NDC1; and (andc) merged (c) merged SEPT12 SEPT12 and NDC1. and NDC1.White arrowsWhite indicatearrows indicate the centrosome the centrosome regions ofregi theons sperm. of the Insetssperm. reveal Insets control reveal IgGcontrol staining. IgG staining.Scale bar Scale = 5 µ mbar;(B =) coimmunoprecipitation5 μm; (B) coimmunoprecipitation assay of FLAG-NDC1 assay of FLAG-NDC1 and SEPT12-GFP. and SEPT12-GFP. Lysates from Lysatestransfected from cells transfected were immunoprecipitated cells were immunoprecipit with an anti-GFPated with antibody an anti-GFP (Lane 3) antibody or a nonspeciﬁc (Lane 3) control or a nonspecificIgG (Lane 2),control followed IgG by(Lane IB with2), followed an anti-GFP by IB (indicated with an anti-GFP by black arrow)(indicat ored anti-FLAGby black arrow) antibody or anti-FLAG(indicated byantibody black arrow). (indicated An inputby black protein arrow). (5%) An was input used protein as the (5%) control was in used IB of as the the transfected control in cell IB oflysates the transfected (Lane 1). cell lysates (Lane 1). Int. J. Mol. Sci. 2016, 17, 1911 4 of 10 Int. J. Mol. Sci. 2016, 17, 1911 4 of 9

2.2. Dynamic Expression of NDC1 during Murine Spermiogenesis To definedefine the dynamic expression patterns of NDC1NDC1 during mouse spermiogenesis, the murine testicular germgerm cellcell populations populations were were separated, separated, and and IFAs IFAs were were performed. performed. We determined We determined that NDC1 that wasNDC1 located was aroundlocated thearound nuclear the membrane nuclear membrane in the round in spermatidthe roundstage spermatid (Figure stage2A). Furthermore,(Figure 2A). Furthermore,in the elongating in the spermatid elongating stage, spermatid NDC1 was stage, expressed NDC1 was in the expressed manchette in regionsthe manchette of the developing regions of thesperm developing head (Figure sperm2B,C), head and (Figure was 2B,C), finally and localized was finally in the localized neck region in the of neck mature region spermatozoa of mature (Figurespermatozoa2D,E). (Figure These findings2D,E). These suggested findings that suggest NDC1ed is that involved NDC1 inis sperminvolved head in sperm and tail head formation and tail formationduring murine during spermiogenesis. murine spermiogenesis.

Figure 2. Localization of N NDC1DC1 during murine spermiogenesis.spermiogenesis. Immunofluorescence Immunoﬂuorescence assay assay (IFA) (IFA) results revealed multiple localizations of NDC1NDC1 signals:signals: ( A) round spermatids; ( B,C) elongating spermatids; ( D)) elongated spermatids; and and ( E)) mature spermatozoa. Anti-NDC1 Anti-NDC1 antibody antibody (“NDCI” (“NDCI” panels), DAPI antibody (“DAPI” panels), and a combination of anti-NDC1 and DAPI antibodies (“Merge” panels) were used as stains.stains. The arrows indicate NDC1 signals. NDC1: NDC1: red; red; DAPI: DAPI: blue. blue. MagnificationMagniﬁcation = 1000×. 1000× .

2.3. Effect of NDC1 on SEPT12 Localizatio Localizationn and Filamental Structure in a Cell Model Figure1 1 illustrates illustrates the the interaction interaction of of NDC1 NDC1 with with SEPT12 SEPT12 and and Figure Figure2 illustrates 2 illustrates the the expression expression of ofNDC1 NDC1 during during sperm sperm head head and and tail tail formation; formation; specifically, specifically, this this NDC1 NDC1 expression expression was was similar similar to the to theSEPT12 SEPT12 patterns patterns found found previously previously [25]. Thus, [25]. we Thus, suggest we thatsuggest NDC1 that regulates NDC1 SEPT12 regulates localization SEPT12 localizationand polymerization and polymerization structure. First, structure. pFLAG-NDC1 First, pFLAG-NDC1 was transfected was transfected into NT2D1 into cells NT2D1 to determine cells to whetherdetermine NDC1 whether localizes NDC1 at thelocalizes nuclear at membranethe nuclear in membrane a male germ in cella male line. germ NDC1 cell was line. located NDC1 around was locatedthe NE (Figurearound3 ).the The NE cells (Figure transfected 3). The with cells SEPT12 transfected alone showedwith SEPT12 three typicalalone patterns:showed three (1) localized typical patterns:at cytoplasm (1) (62.41%);localized (2) at filament cytoplasm structure (62.41%); (25.73%); (2) andfilament (3) dot-like structure structure (25.73%); (11.86%) and (Figure (3) dot-like4A,B). structureThe expressional (11.86%) types (Figure of SEPT12 4A,B). inThe the expressional cell line are similartypes of to SEPT12 the physiological in the cell patternsline are similar at the various to the physiologicalstages of postmeiotic patterns male at the germ various cells stages (e.g., filament of postmeiotic structure male around germ the cells manchette (e.g., filament structure structure of the aroundelongating the spermatidmanchette andstructure dot-like of structurethe elongating located spermatid at the necks and ofdot-like mature structure sperm) [11located]. However, at the necksafter cotransfection of mature sperm) with NDC1[11]. However, and SEPT12, after the co NDC1transfection restricted with the NDC1 SEPT12 and to theSEPT12, nucleus the (69.12%) NDC1 restrictedand repressed the SEPT12 the formation to the nucl of theeus filament (69.12%) structure and repressed (0%) (Figure the formation4C,D). This of the result filament indicated structure that (0%)NDC1 (Figure modulates 4C,D). the This SEPT12 result localization indicated that in the NDC1 male modulates germ cell line. the SEPT12 localization in the male germ cell line. Int. J. Mol. Sci. 2016, 17, 1911 5 of 10 Int. J. Mol. Sci. 2016, 17, 1911 5 of 9 Int. J. Mol. Sci. 2016, 17, 1911 5 of 9

Figure 3. NDC1 location near the nuclear envelope in the male germ cell line. IFA results depicting Figure 3. NDC1 location near the nuclear envelope in the male germ cell line. IFA results depicting Figurethe signals 3. NDC1 of ( locationA) (a) FLAG-NDC1 near the nuclear using envelope NDC1 in the(red), male LAMN germB1 cell (green), line. IFA DAPI results (blue), depicting and the a the signals of (A) (a) FLAG-NDC1 using NDC1 (red), LAMNB1 (green), DAPI (blue), and a signalscombination of (A)( ofa )NDC1, FLAG-NDC1 LAMN, using and DAPI NDC1 (“Merge” (red), LAMNB1 panel) stains; (green), and DAPI (b) control (blue), andIgG. aMagnification combination combination of NDC1, LAMN, and DAPI (“Merge” panel) stains; and (b) control IgG. Magnification of= 400×. NDC1, IFA LAMN, results anddepicting DAPI the (“Merge” signals panel)of (B) FLAG-NDC1 stains; and (b using) control NDC1 IgG. (red Magniﬁcation), Phalloidin =(green), 400×. = 400×. IFA results depicting the signals of (B) FLAG-NDC1 using NDC1 (red), Phalloidin (green), IFADAPI results (blue), depicting and thea combination signals of (B ) FLAG-NDC1of NDC1, Phal usingloidin, NDC1 and (red), DAPI Phalloidin (“Merge” (green), panel) DAPI stains. (blue), DAPI (blue), and a combination of NDC1, Phalloidin, and DAPI (“Merge” panel) stains. andMagnification a combination = 400×. of NDC1, Phalloidin, and DAPI (“Merge” panel) stains. Magniﬁcation = 400×. Magnification = 400×.

Figure 4.4.Effects Effects of of NDC1 NDC1 overexpression overexpression on SEPT12 on SEPT12 localization. localization. (A) Western (A) blottingWestern of blotting cells transfected of cells Figure 4. Effects of NDC1 overexpression on SEPT12 localization. (A) Western blotting of cells withtransfected SEPT12-EGFP; with SEPT12-EGFP; (B) IFA results showing(B) IFA SEPT12-EGFPresults showing patterns: SEPT12-EGFP (a) localized patterns: at cytoplasm, (a) localized (b) fibrous, at transfected with SEPT12-EGFP; (B) IFA results showing SEPT12-EGFP patterns: (a) localized at orcytoplasm, (c) dot-like; (b) ( Cfibrous,) Western or blotting(c) dot-like; of cells (C) with Western coexpressed blotting SEPT12-EGFP of cells with andcoexpressed pFLAG-NDC1; SEPT12-EGFP (D) IFA cytoplasm, (b) fibrous, or (c) dot-like; (C) Western blotting of cells with coexpressed SEPT12-EGFP resultsand pFLAG-NDC1; showing SEPT12-EGFP (D) IFA results patterns: showing (a) localized SEPT12-EGFP at cytoplasm, patterns: (b) dot-like, (a) localized or (c) localized at cytoplasm, at nuclear (b) and pFLAG-NDC1; (D) IFA results showing SEPT12-EGFP patterns: (a) localized at cytoplasm, (b) rim;dot-like, (B,D )or signals (c) localized from EGFP at proteinnuclear (green),rim; (B anti-FLAG,D) signals antibody from EGFP (red), prot DAPIein (blue), (green), and anti-FLAG combined dot-like, or (c) localized at nuclear rim; (B,D) signals from EGFP protein (green), anti-FLAG EGFP,antibody anti-FLAG, (red), DAPI and DAPI (blue), (“Merge” and combined panels). Magnification EGFP, anti-FLAG, = 1000× .and DAPI (“Merge” panels). antibody (red), DAPI (blue), and combined EGFP, anti-FLAG, and DAPI (“Merge” panels). Magnification = 1000×. Magnification = 1000×. 2.4. Effect of Mutated SEPT12 on NDC1 Localization In Vivo 2.4. Effect of Mutated SEPT12 on NDC1 Localization In Vivo 2.4. EffectThe SEPT12D197Nof Mutated SEPT12 (Asp197Asn; on NDC1 Localization D197N) mutation In Vivo has been isolated in infertile men with The SEPT12D197N (Asp197Asn; D197N) mutation has been isolated in infertile men with teratozoospermiaThe SEPT12D197N and oligozoospermia (Asp197Asn; D197N) [14,17]. mutation In addition, has knock-in been isolated mice with in infertile the SEPT12D197N men with teratozoospermia and oligozoospermia [14,17]. In addition, knock-in mice with the SEPT12D197N mutation,teratozoospermia which were and usedoligozoosperm to mimic infertileia [14,17]. men In with addition, SEPT12D197N, knock-in exhibitedmice with a the high SEPT12D197N percentage of mutation, which were used to mimic infertile men with SEPT12D197N, exhibited a high percentage disorganizedmutation, which sperm were annuli used andto mimic necks infertile [27]. To me determinen with SEPT12D197N, whether SEPT12 exhibited also affects a high NDC1 percentagein vivo, of disorganized sperm annuli and necks [27]. To determine whether SEPT12 also affects NDC1 in IFAof disorganized was performed sperm in the annuli sperm. and In thenecks bent-tail [27]. To sperm determine isolated whether from the SEPT12 SEPT12D197N also affects knock-in NDC1 mice, in vivo, IFA was performed in the sperm. In the bent-tail sperm isolated from the SEPT12D197N NDC1vivo, IFA was was dispersed performed around in thethe manchettesperm. In regionthe bent-tail of the spermsperm headisolated and from annulus, the whereasSEPT12D197N in the knock-in mice, NDC1 was dispersed around the manchette region of the sperm head and annulus, wild-typeknock-in mice, mice, NDC1 NDC1 was was dispersed concentrated around at the the sperm manchette neck and region slightly of the at thesperm annulus head (Figureand annulus,5A,B). whereas in the wild-type mice, NDC1 was concentrated at the sperm neck and slightly at the Furthermore,whereas in the some wild-type of the sperm mice, isolated NDC1 from was SEPT12D197N concentrated miceat the with sperm a disabled neck neckand wereslightly separated at the annulus (Figure 5A,B). Furthermore, some of the sperm isolated from SEPT12D197N mice with a betweenannulus (Figure the sperm 5A,B). head Fu andrthermore, tail, and some also of revealed the sperm substantial isolated nonlocalized from SEPT12D197N NDC1 signals mice with at the a disabled neck were separated between the sperm head and tail, and also revealed substantial spermdisabled annulus neck (Figurewere separated5C). These between results indicated the sperm that head loss ofand function tail, and because also ofrevealed SEPT12 substantial mutations nonlocalized NDC1 signals at the sperm annulus (Figure 5C). These results indicated that loss of innonlocalized vivo also affected NDC1 dynamicsignals at localization the sperm ofannulus NDC1, (F andigure may 5C). affect These precise results sperm indicated formation. that loss of function because of SEPT12 mutations in vivo also affected dynamic localization of NDC1, and may function because of SEPT12 mutations in vivo also affected dynamic localization of NDC1, and may affect precise sperm formation. affect precise sperm formation. Int. J. Mol. Sci. 2016, 17, 1911 6 of 10 Int. J. Mol. Sci. 2016, 17, 1911 6 of 9

Figure 5.5. Localization of NDC1 in sperm fromfrom SEPT12D197NSEPT12D197N mice.mice. IFA results showing multiplemultiple localizations of NDC1NDC1 signalssignals inin ((AA)) wild-typewild-type spermsperm andand ((BB,,CC)) SEPT12D197NSEPT12D197N sperm.sperm. Anti-NDC1Anti-NDC1 antibody (“NDC1” panels), MitoTracker (“Mito” panels),panels), DAPI (“DAPI” panel), and a combinationcombination of the anti-NDC1 antibody and MitoTracker (“Merge”(“Merge” panels)panels) werewere usedused asas stains.stains. ( A–C)) NDC1: red; Mito: green; green; DAPI: DAPI: blue. Arrows Arrows = = sperm sperm neck; neck; Arrow Arrow head head = = annulus; annulus; * * = = sperm sperm head. MagniﬁcationMagnification == 1000 1000×;×; ScaleScale bar bar = 5=µ 5m; μ (m;D) ( aD cartoon) a cartoon model model of SEPT12D197N of SEPT12D197N affecting affecting NDC1 location. NDC1 Bluelocation. area: Blue Sperm-head; area: Sperm-head; green-line: mito-tracker; green-line: blackmito-tracker; line: principal black piece; line: arrow:principal sperm-neck; piece; arrow: arrow head:sperm-neck; annulus. arrow head: annulus.

3. Discussion Discussion

3.1. SEPT12 Interaction with Nuclear and Nuclear-Related Proteins We previouslypreviously identiﬁedidentified SEPT12SEPT12 interactors,interactors, one of whichwhich waswas nuclearnuclear oror nuclearnuclear membranemembrane proteins. Notably, Notably, this this protein protein group group contains contains (1) (1) NDC1 NDC1 tran transmembranesmembrane nucleoproteins nucleoproteins (NDC1); (NDC1); (2) (2)sperm-associated sperm-associated antigen antigen 4 proteins 4 proteins (SPAG4); (SPAG4); and and (3) (3) protamine-2 protamine-2 proteins proteins (PRM2) (PRM2) [17]. [17]. PRM2 PRM2 and PRM1 areare majormajor spermsperm proteinsproteins involved involved in in nuclear nuclear packing packing [28 [28],], whereas whereas SPAG4 SPAG4 (SUN4) (SUN4) belongs belongs to theto the SUN SUN family. family. Additionally, Additionally, SUN1 SUN1 is one is one of the of well-knownthe well-known nuclear nuclear membrane membrane proteins, proteins, which which links withlinks LAMINwith LAMIN and the and cytoplasmic the cytoplasmic cytoskeleton cytoskeleton [29]. In [29]. our previousIn our previous study, westudy, found we that found SEPT12 that interactsSEPT12 interacts with SPAG4, with butSPAG4, that thebut mutated that the SEPT12mutated disrupts SEPT12 this disrupts interaction this interaction [17]. NDC1 [17]. belongs NDC1 to thebelongs transmembrane to the transmembrane ring Nups, whichring Nups, are major which proteins are major in NPCs proteins anchored in NPCs to the anchored NE, and to is vitalthe NE, for nuclearand is vital membrane for nuclear assembly membrane [21–23]. assembly Moreover, [21 the–23]. SUN Moreover, protein, Msp3,the SUN in yeast protein, cells Msp3, controls in NDC1 yeast distributioncells controls and NDC1 function distribution in the nuclear and function membrane in the [30 nuclear]. The presentmembrane study [30]. is theThe ﬁrst present to reveal study the is associationthe first to reveal between theSEPTs association and the be nucleoproteintween SEPTs and NDC1. the nucleoprotein NDC1.

3.2. Male Reproductive Roles of SEPT12 and NDC1 The sks in micemice werewere spontaneouslyspontaneously detected at The Jackson Laboratory [[24].24]. Akiyama Akiyama et et al. al. indicated that NDC1 mutation induces abnormal sp splicinglicing and exon 6 skipping in sks micemice throughthrough linkage mappingmapping and and splicing splicing assay assay [26]. [26]. Furthermore, Furthermore, they demonstrated they demonstrated that NDC1 that were NDC1 expressed were inexpressed pachytene in spermatocytespachytene spermatocytes and following and the following sperm head the in sperm round head and elongatingin round spermatidsand elongating [26]. Inspermatids addition, [26]. mutated In addition, NDC1 inducedmutated meiosisNDC1 indu arrestced inmeiosis pachytene arrest spermatocytes in pachytene andspermatocytes increased DNAand increased double-strand DNA breaks.double-strand In a previous breaks. In study, a pr weevious demonstrated study, we thatdemonstrated SEPT12 proteins that SEPT12 were expressedproteins were at the expressed sperm head at the during sperm spermatid head duri elongation,ng spermatid and atelongation, the neck and and annulus at the ofneck mature and annulus of mature sperm [25]. Furthermore, sperm containing a mutated SEPT12 allele exhibited an immature sperm head, bent tail, premature chromosomal condensation, and nuclear damage [13]. Int. J. Mol. Sci. 2016, 17, 1911 7 of 10 sperm [25]. Furthermore, sperm containing a mutated SEPT12 allele exhibited an immature sperm head, bent tail, premature chromosomal condensation, and nuclear damage [13]. During murine spermatogenesis, NDC1 was majorly expressed at the spermatocyte and the subsequent spermatid stages, whereas SEPT12 was specifically expressed at the postmeiotic male germ cell stages. Moreover, NDC1 and SEPT12 were co-expressed at the spermatid stage. In the present study, we observed dynamic expressions of NDC1 around the NE in round spermatids, and at the manchette region in elongating spermatids (Figure2). During spermiogenesis, numerous proteins are involved in sperm morphogenesis [31,32]. After the final step, some proteins migrate to the sperm neck and sperm tail for other functions (e.g., SPAG4); others move into the residual body and are thrown out along with the excess cytoplasm. The results of NDC1 expression are similar to those of SEPT12 expression in our previous study, specifically in the manchette region of the sperm head and tail formation [25]. Thus, we suggest that NDC1 is a regulator of SEPT12 function during sperm head and tail formation.

3.3. Effect of Mutated SEPT12 on Cellular Localization of NDC1 During mammalian spermiogenesis, the precise regulation of the manchette dynamic is critical for sperm head formation [31,32]. As depicted in Figures1 and2, the SEPT12 and NDC1 proteins in this study formed complexes, and expressed similar patterns at the manchette and neck region during terminal differentiation of male germ cells. In humans, the mutated SEPT12D197N (Asp197Asn), SEPT12T89M (Thr89Met), and SEPT12Del (c.474G/A induced truncated form) caused teratozoospermia and oligozoospermia [14–16]. To mimic the effect of SEPT12D197N in vivo, we generated knock-in mice [27], for which, as Figure5 illustrates, the NDC1 patterns were irregular and dispersed around the manchette region of the sperm head and tail. This phenomenon may be attributed to the following: (1) the disrupted SEPT12 affects NDC1 localization or (2) the degradation of mutated SEPT12 affects the sperm formation resulting in the mislocalization of NDC1. In this study, we could not disregard the possibility of (2). We hypothesized that SEPT12–NDC1 dynamic localization and functionalism facilitate precise sperm head and tail formation and prevent abnormally shaped spermatozoa.

4. Materials and Methods

4.1. Immunoﬂuorescence Assay Human semen collection from fertile men (n = 3) for health evaluation was approved by the Institutional Review Board of Cathay General Hospital. More than 100 sperm were stained per man. The animal studies were approved by the Institutional Animal Care and Use Committee of Fu Jen Catholic University (A10153; 22 November 2012). Three knock-in mice (more than 50 sperm per mice) were evaluated. For an immunoﬂuorescence assay (IFA), sperm and cells transfected with vectors were treated with 0.1% Triton X-100, washed twice with Tris-buffered saline (TBS), and subsequently incubated with a primary antibody (NDC1: sc-161929, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; SEPT12:H00124404-B01P, Abnova, Taipei, Taiwan; LAMINB1: ab16048, Abcam, Cambridge, MA, USA) and secondary antibody (Alexa Fluor-568 donkey anti-goat IgG antibody: cat no. A-11057, Invitrogen, Carlsbad, CA, USA; Alexa Fluor-488 donkey anti-rabbit IgG antibody: cat no. A-21206, Invitrogen; Alexa Fluor-488 donkey anti-mouse IgG antibody; cat no. A-21202, Invitrogen). The procedure for IFA analysis was described in our previous study [33].

4.2. Cloning, Transfection, and Coimmunoprecipitation Assay Human SEPT12 and NDC1 were ampliﬁed from a human RNA panel (Clontech, Mountain View, CA, USA) and cloned into pEGFP-N1 and pFLAG-CMV2 vectors, as described previously [33]. NT2D1 cells were transfected with the vectors through lipofection. Overexpression of NDC1 or co-transfection with SEPT12 was replicated three times and more than 100 cells were counted per assay. The cells lysates and the antibodies (Anti-GFP antibody: sc-9996, Santa Cruz Biotechnology Inc.; anti-FLAG Int. J. Mol. Sci. 2016, 17, 1911 8 of 10 antibody: F1804, Sigma-Aldrich, St. Louis, MO, USA; Anti-NDC1 antibody: sc-161929, Santa Cruz Biotechnology Inc.) were used in the Co-IP and IB assays, by employing the procedure described in our previous study [33].

4.3. Separation of the Murine Testicular Germ Cell Populations and Sperm Preparation The spermatogenic cells isolated from mice (n = 3) were separated on the basis of the density of various germ cell types by using a centrifugal system, as described previously [33].

5. Conclusions In this study, we demonstrated that SEPT12 binds with NDC1 and forms a complex during sperm head and tail formation. Overexpression of NDC1 affected the localization of SEPT12 in the cell. Moreover, mutated SEPT12 disturbed the localization of NDC1. Thus, we conclude that SEPT12–NDC1 function is involved in the morphogenesis of male germ cells.

Acknowledgments: This study was supported by grants from the Ministry of Science and Technology in the Republic of China (MOST 104-2314-B-030-001; MOST 104-2314-B-006-001; MOST 105-2314-B-006-001; and MOST 105-2314-B-030-001) and Cathay General Hospital (102-CGH-FJU-03). Author Contributions: Conceived and designed the experiments: Ying-Yu Wu, Tsung-Hsuan Lai, Pei Wang, Yi-No Wu, Han-Sun Chiang, and Pao-Lin Kuo. Performed the experiments: Ying-Yu Wu, Ya-Yun Wang, Mei-Feng Chen. Analyzed the data: Ying-Yu Wu, Ying-Hung Lin. Contributed reagents/materials/analysis tools: Tsung-Hsuan Lai, Yi-No Wu, Han-Sun Chiang, Pao-Lin Kuo. Wrote the paper: Ying-Hung Lin, Tsung-Hsuan Lai, Mei-Feng Chen, and Tsung-Ming Chen. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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