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Research ArticleSLC9A3 Affects Vas Deferens Development and Associates withTaiwanese Congenital Bilateral Absence of the Vas Deferens

Yi-NoWu1 Kuo-Chiang Chen12 Chien-ChihWu34

Ying-Hung Lin 5 and Han-Sun Chiang 567

1 School of Medicine Fu Jen Catholic University New Taipei City Taiwan2Department of Urology Cathay General Hospital Taipei Taiwan3School of Medicine Taipei Medical University Taipei Taiwan4Department of Urology Taipei Medical University Hospital Taipei Taiwan5Graduate Institute of Biomedical and Pharmaceutical Science Fu Jen Catholic University New Taipei City Taiwan6Division of Urology Department of Surgery Cardinal Tien Hospital New Taipei City Taiwan7Department of Urology Fu Jen Catholic University Hospital New Taipei City Taiwan

Correspondence should be addressed to Ying-Hung Lin 084952mailfjuedutw and Han-Sun Chiang yino58hotmailcom

Received 29 October 2018 Revised 6 January 2019 Accepted 3 February 2019 Published 10 March 2019

Academic Editor George Seki

Copyright copy 2019 Yi-No Wu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background The pathophysiology of Taiwanese congenital bilateral absence of the vas deferens (CBAVD) is different from thatin Caucasians In particular major cystic fibrosis transmembrane conductance regulator (CFTR) mutations and cystic fibrosis areabsent in the former Instead deficiency in solute carrier family 9 sodiumhydrogen exchanger isoform 3 (SLC9A3) may play arole by generating obstructive azoospermia and degraded epithelial structure in the reproductive tract Objectives The objectiveof the study was to test whether SLC9A3 variants cause Taiwanese CBAVDMaterials and Methods Six-month-old Slc9a3minusminusmalemice were used to evaluate the effect of long-term SLC9A3 loss on the reproductive system A case-control cohort of 29 men withCBAVD and 32 fertile men were genotyped for SLC9A3 variants Results SLC9A3 was expressed and localized in the apical borderof the epithelium of human vas deferens and glandular epithelium of the seminal vesicle SLC9A3 deficiency specifically inducesatrophy of vas deferens and unfolding of seminal vesicle mucosa in mice Loss of SLC9A3 increased the incidence of CBAVD inhumans from 31 to 379 (p lt 0001) Up to 759 of CBAVD patients carry at least one variant in either SLC9A3 or CFTRDiscussion Our findings build upon previous data associated with CBAVD pathogenesis Here we now report for the first time anassociation between CBAVD and loss of SLC9A3 and propose that specific defects in the reproductive duct due to SLC9A3 variantsdrive CBAVD development ConclusionThe data implicate loss of SLC9A3 as a basis of Taiwanese CBAVD and highlight SLC9A3function in reproduction

1 Introduction

Congenital bilateral absence of the vas deferens (CBAVD)accounts for 1 to 2 of cases of male infertility [1] andis the most frequent cause of obstructive azoospermia [2]Strikingly CBAVD is also associated with mild cystic fibrosis[1 3] a progressive lung disease due to mutations in cysticfibrosis transmembrane conductance regulator (CFTR) [4]and is the most common autosomal recessive disorder inCaucasians Indeed CFTR variants have been identified in50 to 74 of alleles in CBAVD patients [5ndash7]

Remarkably CFTR lesions are not commonly observed ininfertile Asians [8] Cystic fibrosis is also generally very rarein Asians overall and has not been observed in Asians withCBAVD Accordingly the CFTR-mutation spectrum among63 infertile Taiwanese men with CBAVD is narrow with apolythymidine (Tn) variant in intron 8 (IVS8-5T) accountingfor 913 (4246) of the variants observed although mostof the identified variants (2763 429) were indeterminate[9] Nevertheless a large percentage of these patients (2663413) carried one defective CFTR allele whereas Caucasianmen with one variant CFTR copy do not develop CBAVD

HindawiBioMed Research InternationalVolume 2019 Article ID 3562719 10 pageshttpsdoiorg10115520193562719

2 BioMed Research International

Table 1 Reproductive-organ weight in 6-month-old wild-type and Slc9a3minusminus male mice

Group Organ weight (mg)Vas deferens Testis Epididymis Seminal vesicle

Wild Type 265plusmn23 878plusmn13 54plusmn12 162plusmn25Slc9a3minusminus 89plusmn21lowast 508plusmn28lowast 296plusmn11lowast 912plusmn43lowastValues represent the mean plusmn standard error of the mean (n = 3 per genotype)lowastp lt 001 vs wild type according to Studentrsquos t test

[9 10] Therefore other as-yet-unknown genetic or environ-mental factors might cause CBAVD in Taiwan

Mutations in the solute carrier family 9 sodiumhydro-gen-exchanger isoform 3 gene (SLC9A3NHE3) have beenidentified as a possible basis of Taiwanese CBAVD [11]SLC9A3 encodes an ion channel regulated by CFTR andaccumulates at the apicalmembrane of renal proximal tubulesand intestinal epithelial cells to mediate NaCl and HCO3

minus

absorption [12ndash15] Homozygous Slc9a3ndashndash mice are viablebut develop persistent slight diarrhea and mild acidosis andHCO3

minus and fluid absorption are sharply reduced in thekidney as are blood pressure and absorption in the intestine[16] Additionally female Slc9a3ndashndash mice are fertile whereasmales are infertile [17] Notably loss of estrogen receptor 120572increases the luminal pH in themouse epididymis and causessperm defects by suppressing SLC9A3 carbonic anhydraseXIV and SLC4 proteins [17] The C-terminal PDZ motif inCFTR interacts with and regulates the activity of SLC9A3 asdemonstrated inCr (ΔF508)mutantmice [18]Our previousresearch showed that SLC9A3 deficiency causes obstructiveazoospermia degrades the epithelium of the reproductivetract and drastically suppresses CFTR expression [19]

Despite this evidence the role of SLC9A3 in humanreproduction remains somewhat elusive One possibility isthat SLC9A3 is crucial for male reproductive function Inthis study we first evaluated the effect of long-term SLC9A3deficiency in mice on the structure and function of thereproductive tract to determine its possible role in CBVADBecause SLC9A3 is abundantly expressed in mouse repro-ductive organs [19] we first focused on whether long-termloss of SLC9A3 impairs development of the vas deferensSLC9A3 expression and distribution were also comparedin normal human and rodent reproductive tissues and wefurther assessed the potential relationship between SLC9A3copy number variations (CNVs) and CBAVD in a case-control study through genetic analysis of a cohort of menin Taiwan Collectively these data can help to reinforce andcontextualize clinical observations and illuminate the basisof Taiwanese CBAVD as well as the possible general role ofSLC9A3 in human reproduction

2 Materials and Methods

21 Animals FVB129(Cg)-Slc9a3tm1GesJ mice were pur-chased from Jackson Laboratory All animal studies wereapproved by the Fu Jen Laboratory Animal Care and UseCommittee (IACUC number A10580) The genotype of eachmale mouse was assayed by extracting genomic DNA from

the tail and by performing polymerase chain reaction (PCR)using the following primers

F1 (51015840-CATACAACATAGGACTAGCC-31015840)R1 (51015840-CACTACTAGTCAGGCACTCT-31015840) andR2 (51015840-CACTACTAGTCAGGCACTCT-31015840) The primer

ratio of F1 R1 and R2 was 211 Mice of each genotype weresacrificed at 6 months of age by anesthesia with isofluraneand their organs including the testes epididymis and vasdeferens were collected

22 CBAVD Patients and Control Participants Subjects wererecruited among infertile men referred to us for diagnosis atTaipei Medical University Hospital (Taipei Taiwan) CBAVDwas diagnosed based on physical examination of scrotalcontents In particular men without palpable vas deferens onboth sides but with normal testes size (long axis gt 2 cm)were further screened as previously described [11] To confirmCBAVD semen samples were analyzed by standard tests aswell as for pH level and fructose content Some patients wereexamined by magnetic resonance imaging (MRI) to evaluatethe intra-abdominal segment of the vas deferens and sem-inal vesicle [20] Renal ultrasonography was used to assessboth kidneys whereas hormonal assays and chromosomalanalyses were used to rule out testicular azoospermia Bloodsamples were collected to obtain genomic DNA All studieswere approved by the TMU-Joint Institutional Review BoardTaipei Taiwan (trial registration number 201207028) andinformed consent was obtained from each participant

23 Array Based Comparative Genomic Hybridization DataA total of 66 CNVs with high aberrant scores (ltminus1 or gt1)were previously found by array based comparative genomichybridization [11] Of these seven were considered candidateCBAVD-related genes based on a 2-fold decrease in copynumber in at least two of seven patients (Table 1)

24 CFTR Genotyping DNA was extracted from peripheralleukocytes using the Puregene DNA purification kit (GentraSystems Minneapolis MN USA) Exon 9 in CFTR and the50-bp intron 8 upstreamwere sequenced in both directions todetermine the length of IVS8Tn andTGdinucleotide repeatsas previously described [9]

25 Quantitative Real-Time PCR The fold change in SLC9A3copy number between cases and controls was determinedby quantitative real-time PCR using the iQ SYBR Greensupermix kit and the CFX96 Touch real-time PCR detec-tion system (Bio-Rad Laboratories Hercules CA USA)

BioMed Research International 3

SV

EP

VD VD VDSV

SV

EP EP

T T T

Wild type

1 cm

Slc9a3+- Slc9a3--

Figure 1 Gross morphology of the reproductive organs of 6-month-old male wild-type mice and mice with heterozygous or homozygous ofSlc9a3 deficiency Intact reproductive organs including the seminal vesicle (SV) vas deferens (VD) T (testis) and epididymis (EP)

Targets were amplified by preincubation at 95∘C for 10min followed by over 40 cycles of 95∘C for 30 s 57∘Cfor 15 s and 72∘C for 10 s The mean threshold-cyclenumber for each gene in each sample was obtained fromtriplicate experiments Patients with consistently atypi-cal PCR results from two different primer sets weredeemed to harbor an SLC9A3 variant [11] The primersequences were the following SLC9A3 intron 451015840-AGC-CAGGTCTTCCTGAGACA-31015840 and 51015840-TGGATCCCTCAC-TCTCTTGG-31015840 SLC9A3 exon 13 51015840-ATCCCGCAGTAC-AAGCATCT-31015840 and 51015840-AGCTTGGTCGACTTGAAGGA-31015840 and ATP2B4 51015840-CCACGAACACCACTCCTG-31015840 and 51015840-ACCCTAGTCCCAAACTTAGAAGCC-31015840

26 RNA Extraction and Reverse-Transcription PCR TotalRNA from normal adult human and rat tissues were obtainedfrom Xing-Yi Biotechnologies Company (Taipei Taiwan)and 10 120583L of RNA was used to generate cDNA at 55∘C usingSuperscript III RT enzymes (Invitrogen Breda Netherlands)in a final volume of 20 120583l Targets were amplified over 30cycles of denaturation at 95∘C for 1 min annealing for 1 minand extension at 72∘C for 1 min followed by final extensionat 72∘C for 5 min PCR products were mixed with 10 EZvision (Amresco Inc Solon OH USA) and analyzed byelectrophoresis on a 2 agarose gel The four primers usedto amplify human SLC9A3 human GAPDH rat Slc9a3 andrat Gapdh were as follows

SLC9A3 5-GGAGTCCTTCAAGTCGACCA-31015840 and 51015840-AAGAAGGTGCCGGGAGAGTAG-31015840 Slc9a3 51015840- ACCC-CGCCCATCTACAGT -31015840 and 51015840- CACAGAAGCGGAG-GAATAGC -31015840 GAPDH 51015840-TGGCGTCTTCACCACCAT-31015840 and 51015840-CACCACCCTGTTGCTGTA-31015840 and Gapdh 51015840-TCAACGGGAAACCCATCA -31015840 and 51015840- TGATGGGTGT-GAACCACGAG -31015840

27 Immunofluorescence Thedeferent ducts epididymis andseminal vesicle were dissected from an adult rat Paraffin-embedded normal adult human reproductive tissues wereobtained from Xing-Yi Biotechnologies Company Sections

were treated with 01 Triton X-100 washed twice withphosphate-buffered saline (PBS) and probed for 60 minat room temperature with a 1100 dilution of a polyclonalantibody against SLC9A3 (Santa Cruz Biotechnology DallasTX USA) Subsequently sections were washed with PBSlabeled for 60 min at room temperature with goat anti-rabbitIgG conjugated to Alexa Fluor 488 and goat anti-mouse IgGconjugated to Alexa Fluor 594 (Molecular Probes CarlsbadCA USA) and washed again with PBS 410158406-Diamidino-2-phenylindole (DAPI) was used as counterstain to visualizenuclei

28 Statistical Analysis Statistical analyses were performedusing SPSS version 120 for Windows (IBM Corp ChicagoIL USA) The McNemar test was used to assess the relation-ship between SLC9A3 copy number variations and CBAVDwith p lt 005 considered significant

3 Results

31 Long-Term Slc9a3 Deficiency Leads to Gradual Atrophyof the Vas Deferens and Seminal Vesicle in Mice BecauseSLC9A3 is abundantly expressed in mouse reproductiveorgans [19] we tested whether long-term loss of SLC9A3impairs development of the vas deferens We found thatthe vas deferens was significantly shorter in 6-month-oldSlc9a3minusminus mice (21 cm) relative to that in Slc9a3 wild-typemice (35 cm n = 3 p lt 005) (Figure 1) The weight ofthe seminal vesicle was also significantly lower (Table 1)Interestingly these changes were absent in young Slc9a3minusminus

mice These data suggested that Slc9a3minusminus mice exhibitedfeatures similar to those in men with CBAVD as observedby MRI [20]

32 SLC9A3 Depletion Induces Obstruction of the Vas Defer-ens and Unfolding of Seminal Vesicle Mucosa Macroscopicinspection of the vas deferens in 6-month-old Slc9a3minusminusmiceshowed the obstruction in the proximal lumen (Figures 2(a)and 2(b)) and the distal lumen (Figures 2(c) and 2(d))


Wild type

Proximal vas deferens

(a)

Slc9a3--6-month-old mice


100m

(b)

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20m

(c)


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20m

(d)

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Seminal vesicle

(e)


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20m

(f)

Figure 2 Hematoxylin-eosin staining of the proximal vas deferens of 6-month-old (a) wild-type and (b) Slc9a3minusminus mice distal vas deferensof 6-month-old (c) wild-type and (d) Slc9a3minusminusmice and seminal vesicle of (e) 6-month-old wild-type and (f) Slc9a3minusminusmice Scale bar100120583m and 20 120583m respectively

Additionally significant differences between Slc9a3-deficientandwild-typemicewere observed inmucosal-fold length andthe volume of seminal fluid (Figures 2(e) and 2(f)) Theseobservations suggested that Slc9a3 deficiency caused atrophyof the vas deferens and seminal vesicle via degeneration ofsecretory columnar epithelial cells

33 Expression of the SLC9A3 Gene in Human and Rat Repro-ductive Tissues Weused semi-quantitative reverse transcrip-tion PCR with glyceraldehyde 3-phosphate dehydrogenase(GAPDH) as an internal control to confirm the abundantexpression of SLC9A3mRNA in the vas deferens epididymisprostate and testis of human patients (Figure 3(a)) as wellas in the vas deferens epididymis and seminal vesicles ofrats (Figure 3(b)) highlighting the significance of SLC9A3 inreproductive tissues

34 SLC9A3 Localization and Function in the Vas Defer-ens Immunofluorescence assays indicated accumulation ofSLC9A3 at the apical and basal layers of the pseudostratifiedcolumnar epithelium of the rat vas deferens but not inthe associated smooth muscle Importantly costaining forSLC9A3 and pancytokeratin which are markers of epithelialcells indicated that SLC9A3 is specifically localized to thestereocilia cells of the mucosa external to the epitheliumSimilarly SLC9A3 was detected at the apical border of

the pseudostratified columnar epithelium in human vasdeferens specifically in stereocilia cells (Figure 3(c)) Thesedata implied that SLC9A3 mediates ion exchange in the vasdeferens

35 SLC9A3 Expression and Localization in the Human Epi-didymis and Seminal Vesicle In epididymal ducts in rat theapical borders of epithelial and ciliated cells but not smoothmuscle cells were strongly stained for SLC9A3 (Figure 4(a)panels (A)ndash(E)) as was the apical glandular epithelium ofthe seminal vesicle (Figure 4(a) panels (F)ndash(I)) SimilarlySLC9A3 was detected in the apical borders of epithelial andciliated cells in the epididymis of healthy volunteers but notin the associated smooth muscle (Figure 4(b) panels (K) (I)(N) and (P)) SLC9A3 also colocalized with pancytokeratinan epithelial marker (Figure 4(b) panels (M) (O) and (Q))Furthermore in seminal vesicles SLC9A3 was specificallyobserved in the glandular epithelium (Figure 4(b) panels (R)and (S))

36 SLC9A3 Variants Might Cause Taiwanese CBAVD Toevaluate the significance associated with loss of SLC9A3 copynumber we recruited 29 subjects with CBAVD (patientsP1ndashP29) Based onnormalized gene-dosage ratios fromquan-titative real-time PCR (Figures 5(a) and 5(b)) one SLC9A3copywas lost in patients P1 P11 P13 P15-16 P22-P26 andP28


SLC9A3

GAPDH

HumanTe

stis

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idym

is

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efer

ens

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Gapdh

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icle

(b)

SLC9A3Control IgG

DAPISMA

SLC9A3

DAPISMA

SLC9A3

DAPI

PancytokeratinSLC9A3

DAPIPancytokeratin

SLC9A3

DAPI

SLC9A3Control IgG

DAPI

SLC9A3SMA

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SLC9A3SMA

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Control IgG

DAPI

SLC9A3Pancytokeratin

DAPI

Pancytokeratin


DAPI

Rat

Human100m 100m

100m

100m

100m 100m

100m

50m

50m

50m

50m

(c)

Figure 3 Expression of SLC9A3mRNA in humans and rats (a) SLC9A3 is enriched in human male reproductive organs (b) In rats Slc9a3transcripts are expressed in the vas deferens and seminal vesicle GAPDH and Gapdhwere used as controls (c) In human vas deferens cross-sections upper panels show strong staining for SLC9A3 in the epithelium (red) The layers of circular and longitudinal muscle fibers wereidentified by immunoreactivity against 120572-smoothmuscle actin (120572-SMA) (green) whereas nuclei were stained with DAPI (blue) Lower panelsshow SLC9A3 immunostain (red) costaining with pancytokeratin (green) In rat vas deferens cross-sections SLC9A3 (green) was clearlyobserved at the apical plasma membrane of epithelial cells but not in smooth muscle cells (red) However the pseudostratified epitheliumat the apex was strongly costained with SLC9A3 and pancytokeratin (red) an epithelial marker SLC9A3 was found to accumulate in thestereocilia of the vas deferens No apical or basal reactivity was observed in control mouse or rabbit serum


Pancytokeratin SLC9A3 DAPI Merge Merge

Epididymis

Seminal vesicle

50 m

(A)

(F)

(B)

(G)

(C)

(H)

(D)

(I)

(E)

(J)

50 m 50 m

50 m 50 m

50 m 50 m

50 m 20 m

20 m

(a)

SMA SLC9A3


DAPI


DAPI

(K) (L)

SMASLC9A3

DAPI

(M)

SMASLC9A3

DAPI

(N)

Pancytokeratin

(O) (P)

(Q)

SMASLC9A3

DAPI

(R)

SLC9A3

(S)

100m 100m 100m

100m100m 50m

50m 50m500m

(b)

Figure 4 Immunofluorescence staining of rat epididymis and seminal vesicles (aAndashE) At upper panels the apical borders of the epitheliumin the epididymal duct were strongly immunoreactive for SLC9A3 (green) SLC9A3 was localized in ciliated cells (arrows) as showed in(a) (E) Pancytokeratin (red) was used as markers of epithelial cells respectively (FndashI) The glandular epithelium of the seminal vesicle wasalso uniformly stained for SLC9A3 (green) (J) SLC9A3 (green) was observed at the plasma membrane of glandular epithelial cells andcolocalized (arrows) with pancytokeratin (b) Immunofluorescence staining of human epididymis and seminal vesicles (KndashO) The apicalborder of epithelial cells in the epididymal duct was strongly immunoreactive for SLC9A3 (green) (PndashQ) Ciliated cells were clearly stainedfor SLC9A3 (400times)120572-Smoothmuscle actin and pancytokeratin (red) were used asmarkers of smoothmuscle and epithelial cells respectively(R) Low-magnification imaging of the seminal vesicle revealed that SLC9A3 (green) accumulated at glandular epithelial cells (S) A higher-magnification image showed SLC9A3 (green) at the plasma membrane of these cells 120572-Smooth muscle actin was used to mark smoothmuscle


N1

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Gain Gain 219

Normal Loss

0

20

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f ind

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(c)

Figure 5 Quantitative real-time PCR targeting SLC9A3 Fold change in SLC9A3 copy number relative to the endogenous reference geneATP2B4 was compared for (a) fertile controls (N1ndashN32 set 1) and (b) CBAVD patients (P1ndashP29) Fold change was interpreted as normal(05ndash15 grey bar) loss (lt05 black bar) or gain (gt15 white bar) (c) Distribution of copy number variants in CBAVD patients and controlsWhite grey and black bars represent gain no change and loss of copy number respectively

(1129 379) but in only one of 32 unaffected volunteers (N231) Additionally Slc9a3 loss incidence was significantlyhigher in CBAVD patients than in control individuals (p lt0001 Figure 5(c))

37 SLC9A3 and CFTR Variants Co-Contribute to TaiwaneseCBAVD Strikingly we found that in almost all CFTR IVS8-5T patients the mutant allele was associated with either TG12or TG13 repeats with only one 5T allele linked to TG11 Bycontrast 18 14 and 1 7T alleles were associated with TG11TG12 and TG13 respectively (Table 2) Additionally six of 29CBAVD patients (207) were homozygous for IVS8-5T andnine of 29 patients (31) were either heterozygous for IVS8-5T or harbored invariant CFTR whereas at least one variantin either SLC9A3 or CFTR was detected in 22 of 29 patients(759 Figure 6)

4 Discussion

The interaction between SLC9A3 and CFTR modifies theseverity of cystic fibrosis [21] In this study we showed thatlong-term SLC9A3 deficiency induced obstruction in the vasdeferens and abnormal secretion in the seminal vesicle aswell as structural defects in the epithelia of these tissuesWe also found that SLC9A3 mRNA was abundantly andspecifically expressed in the epithelial apex of the vas defer-ens Importantly we found that loss of SLC9A3 significantlyincreased CBAVD risk and that Taiwanese CBAVD is likelydue to the cumulative effects of CFTR and SLC9A3 variantsCollectively these data provided direct evidence that SLC9A3is a novel causative gene of Taiwanese CBAVD

41 e Male Reproductive Tract in Slc9a3minusminus Mice andCBAVD Patients In classic CBAVD absence or hypoplasia


Table 2 CFTR genotypes IVS8 and SLC9A3 copy number in Taiwanese CBAVD patients

PID CFTR Genotype IVS8 Tn (TG)m SLC9A3 copy number17 IVS8-5Tminus (TG)125T(TG)127T 169 IVS8-5Tminus (TG)115T(TG)117T 370 minusminus (TG)117T(TG)127T 371 IVS8-5Tminus (TG)135T(TG)117T 372 IVS8-5Tminus (TG)125T(TG)127T 373 IVS8-5T IVS8-5T (TG)135T(TG)135T 374 minusminus (TG)117T(TG)117T 275 IVS8-5Tminus (TG)135T(TG)127T 376 IVS8-5Tminus (TG)135T(TG)137T 278 minusminus (TG)117T(TG)127T 380 minusminus (TG)117T(TG)117T 181 minusminus (TG)117T(TG)117T 282 IVS8-5T IVS8-5T (TG)125T(TG)125T 184 IVS8-5Tminus (TG)125T(TG)117T 287 minusminus (TG)117T(TG)127T 289 IVS8-5Tminus (TG)135T(TG)127T 190 minusminus (TG)117T(TG)127T 195 IVS8-5T IVS8-5T (TG)125T(TG)135T 296 IVS8-5T IVS8-5T (TG)125T(TG)135T 297 minusminus (TG)117T(TG)127T 298 minusminus (TG)117T(TG)117T 399 IVS8-5T IVS8-5T (TG)135T(TG)135T 2100 IVS8-5Tminus (TG)125T(TG)117T 1101 minusminus (TG)117T(TG)127T 1102 IVS8-5Tminus (TG)125T(TG)127T 1105 IVS8-5T IVS8-5T (TG)135T(TG)135T 1107 IVS8-5Tminus (TG)135T(TG)127T 1110 IVS8-5Tminus (TG)125T(TG)127T 2111 IVS8-5Tminus (TG)125T(TG)127T 1

of seminal vesicles might be observed likely because seminalvesicles form as a diverticulum from the ampulla of the vasdeferens in the embryo In a previous MRI survey of 12 menwith CBAVD four were found to have bilateral seminal-vesicle agenesis whereas unilateral hypoplasia and seminal-vesicle remnants were observed in the remaining eightThese results strongly associated seminal-vesicle atrophywithCBAVD [20] although CBAVD pathogenesis has remaineda puzzle The basis of atrophic or absent vas deferens andseminal vesicles in CBAVD likely differs from that of thedysregulated respiratory and pancreatic tissues in cysticfibrosis which excessively secrete fluid and thereby causesecondary bacterial infection

SLC9A3 is a critical cell-membrane protein that facilitatesH+ secretion and Na+ absorption and regulates intracellularpH in intestinal epithelial tissue [16] Previously we foundthat the vas deferens in 2-month-old Slc9a3minusminus mice produceelevated and aberrant secretions although the weight of theorgan is only slightly reduced [19] In the present study weobserved that 6-month-old Slc9a3minusminusmice present atrophicvas deferens and seminal vesicles similar to clinical casesand perhaps as a result of imbalances in intracellular pH

Based on the observed pathology at 2 and 6 months inmice it is possible that adult patients with CBAVD mightharbor intact vas deferens as newborns or children as wepreviously hypothesized [20] Collectively these findingsstrongly indicated a direct association between SLC9A3 andTaiwanese CBAVD

42 Functional Roles of SLC9A3 in Fluid Reabsorption andSecretion in the Vas Deferens Previous reports demonstratedthat in rats SLC9A3mRNA is found in the kidney stomachintestine [22] epididymal duct [23] and efferent duct [24]We now report that SLC9A3 mRNA is also expressed in thehuman vas deferens and seminal vesicles Additionally weobserved SLC9A3 accumulation in stereocilia cells externalto the epithelium in rats and at the apical border of thepseudostratified columnar epithelium of the vas deferensin humans These results implied that SLC9A3-mediatedfluid and electrolyte reabsorption and secretion in the vasdeferens might shape the glandular epithelium of seminalvesicles and that its loss causes atrophy of the vas deferensand agenesis of seminal vesicles In addition we found theexpression of the SLC9A3 in testis is different between the


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1 SLC9A3+- and CFTR5T5T2 SLC9A3+- and CFTR5T7T3 SLC9A3+- and CFTR7T7T4 SLC9A3++ and CFTR5T5T5 SLC9A3++ and CFTR5T7T6 SLC9A3++ and CFTR7T7T

Figure 6 The distribution of the CFTR IVS8-polyT genotype andSLC9A3 copy number variants in 29 CBAVD patients

human and rat In fact the expression of SLC9A3 at the apicalmembrane of epithelial cells is modulated by several mech-anisms The function and role of SLC9A3 in testis are stillunclear

43 SLC9A3 Variants Link CBAVD to Specific Defects inthe Reproductive Duct The 5T variant accounts for themajority of CFTR alleles in Taiwanese CBAVD patients [2526] Indeed 413 of Taiwanese CBAVD patients carry onevariant CFTR allele although intriguingly Caucasian menwith one CFTR variant do not develop CBAVD [9] The roleof SLC9A3 in reproduction and fertility was discovered inSlc9a3minusminus mice which are infertile due to defective watertransport during sperm maturation in the epididymis [17]Loss of an SLC9A3 allele was also recently reported tosuppress the hyperproliferation of goblet cells in the intestineof mice with cystic fibrosis [27] We now report for the firsttime an association between CBAVD and loss of SLC9A3 andpropose that specific defects in the reproductive duct due toSLC9A3 variants drive CBAVD development

44 Taiwanese CBAVD Is due to Cumulative Effects ofCFTR and SLC9A3 Variants SLC9A3 is coexpressed withand regulated by CFTR in intestinal and lung epithelialcells [18 28] Indeed SLC9A3 expression is diminishedby sim53 in the pancreatic duct of homozygous CFTR(ΔF508) mice [18] In the present study we demonstratedthat CFTR expression is also suppressed in the reproduc-tive ducts of Slc9a3minusminus mice Additionally Slc9a3minusminus micepresent obstructed azoospermia-like phenotypes possiblyattributable to lower CFTR expression as observed in Cr-knockoutmice [19] Remarkably 759 of Taiwanese CBAVD

patients tested carry at least one variant in SLC9A3 or CFTRsuggesting that CBAVD likely arises from independent orcumulative effects of CFTR and SLC9A3 deficiency

45 SLC9A3 Could Be a Novel erapeutic Target in CysticFibrosis Interestingly loss of one or both copies of Slc9a3 inCr-null mice promotes intestinal-fluid secretion preventsobstruction formation rescues gastrointestinal phenotypesand enhances survival [27] One possible explanation is thatthe interaction between SLC9A3 and CFTR alters the regu-lation of intestinal fluids To our knowledge this representsthe first report of the physiological significance of the inter-action between SLC9A3 and CFTR in CrSlc9a3 double-homozygous mice A potential issue concerns whetherCrSlc9a3 double-heterozygous mice exhibit the phenotypeand pathology of Cr-knockout or slc9a3-knockout miceOur results suggested SLC9A3 as a novel causative geneof Taiwanese CBAVD and highlighted SLC9A3 function inreproduction We anticipate that this study will stimulatefurther investigation of the basis of Taiwanese CBAVD andultimately identify a novel therapeutic strategy and target forcystic fibrosis

Data Availability

The original data used to support the findings of this studyare included within the article

Disclosure

The funders had no role in study design data collectionand analysis decision to publish or preparation of themanuscript An earlier version of this study was presented asan abstract in EAU18 -33rd Annual EAU Congress 2018

Conflicts of Interest

The authors report no potential conflicts of interest

Acknowledgments

This work was supported by the Chi Mei Hospital and FuJen Catholic University Education and Development Coop-eration Project (104-CM-FJU-01) the Cardinal Tien Hospitaland Fu Jen Catholic University Education and DevelopmentCooperation Project (106-CTH-FJU-06) and the CathayGeneral Hospital and Fu Jen Catholic University Educationand Development Cooperation Project (106-CGH-FJU-03)Yi-No Wu and Han-Sun Chiang received research fundingfrom both Fu Jen Catholic University School ofMedicine andCathay General Hospital

References

[1] D S Holsclaw A D PerlmutterH Jockin and H ShwachmanldquoGenital abnormalities in male patients with cystic fibrosisrdquoe Journal of Urology vol 106 no 4 pp 568ndash574 1971


[2] J P Jarow M A Espeland and L I Lipshultz ldquoEvaluation ofthe azoospermic patientrdquoe Journal of Urology vol 142 no 1pp 62ndash65 1989

[3] A DeMeeus C Guittard M Desgeorges S Carles J DemailleandM Claustres ldquoLinkage disequilibrium between theM470Vvariant and the IVS8 polyT alleles of theCFTRgene inCBAVDrdquoJournal of Medical Genetics vol 35 no 7 pp 594ndash596 1998

[4] J R Riordan J M Rommens B-S Kerem et al ldquoIdentificationof the cystic fibrosis gene cloning and characterization ofcomplementary DNArdquo Science vol 245 no 4922 pp 1066ndash1073 1989

[5] D Dayangac H Erdem E Yilmaz et al ldquoMutations of theCFTR gene in Turkish patientswith congenital bilateral absenceof the vas deferensrdquoHumanReproduction vol 19 pp 1094ndash11002004

[6] R N Josserand F Bey-Omar J Rollet et al ldquoCystic fibrosisphenotype evaluation and paternity outcome in 50 males withcongenital bilateral absence of vas deferensrdquoHuman Reproduc-tion vol 16 no 10 pp 2093ndash2097 2001

[7] Z Wang J Milunsky M Yamin T Maher R Oates and AMilunsky ldquoAnalysis by mass spectrometry of 100 cystic fibrosisgene mutations in 92 patients with congenital bilateral absenceof the vas deferensrdquo Human Reproduction vol 17 no 8 pp2066ndash2072 2002

[8] L J Wong O M Alper B T Wang M H Lee and S Y LoldquoTwo novel null mutations in a Taiwanese cystic fibrosis patientand a survey of East Asian CFTRmutationsrdquo American Journalof Medical Genetics Part A vol 120A pp 296ndash298 2003

[9] HS Chiang Lu JF CH Liu Wu YN and CC Wu ldquoCFTR(TG)m(T)n polymorphism in patients with CBAVD in a pop-ulation expressing low incidence of cystic fibrosisrdquo ClinicalGenetics vol 76 pp 282ndash286 2009

[10] L B Baskin F H Wians Jr and F Elder ldquoPreconceptionand prenatal screening for cystic fibrosisrdquo Medical LaboratoryObserver vol 234 pp 8ndash12 2002

[11] C H Lee C C Wu Y N Wu and H S Chiang ldquoGene copynumber variations in Asian patients with congenital bilateralabsence of the vas deferensrdquo Human Reproduction vol 24 pp748ndash755 2009

[12] C Bookstein AM DePaoli Y Xie et al ldquoNa+H+ exchangersNHE-1 and NHE-3 of rat intestine Expression and localiza-tionrdquo Journal of Clinical Investigation vol 93 pp 106ndash113 1994

[13] S Hihnala M Kujala J Toppari J Kere C Holmberg andP Hoglund ldquoExpression of SLC26A3 CFTR and NHE3 inthe human male reproductive tract Role in male subfertilitycaused by congenital chloride diarrhoeardquo Molecular HumanReproduction vol 12 no 2 pp 107ndash111 2006

[14] W A Hoogerwerf S C Tsao O Devuyst et al ldquoNHE2 andNHE3 are human and rabbit intestinal brush-border proteinsrdquoAmerican Journal of Physiology-Gastrointestinal and Liver Phys-iology vol 270 no 1 pp G29ndashG41 1996

[15] M Kujala S Hihnala J Tienari et al ldquoExpression of iontransport-associatedproteins in human efferent and epididymalductsrdquo Reproduction vol 133 no 4 pp 775ndash784 2007

[16] P J Schultheis L L Clarke P Meneton et al ldquoRenal andintestinal absorptive defects in mice lacking the NHE3Na+H+exchangerrdquo Nature Genetics vol 19 no 3 pp 282ndash285 1998

[17] Q Zhou L Clarke R Nie et al ldquoEstrogen action and malefertility Roles of the sodiumhydrogen exchanger-3 and fluidreabsorption in reproductive tract functionrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 98 no 24 pp 14132ndash14137 2001

[18] W Ahn K H Kim J A Lee et al ldquoRegulatory interactionbetween the cystic fibrosis transmembrane conductance regu-lator andHCO3- salvage mechanisms inmodel systems and themouse pancreatic ductrdquoe Journal of Biological Chemistry vol276 no 20 pp 17236ndash17243 2001

[19] Y Y Wang Y H Lin Y NWu et al ldquoLoss of SLC9A3 decreasesCFTR protein and causes obstructed azoospermia in micerdquoPLoS Genetics vol 13 Article ID e1006715 2017

[20] H-S Chiang Y-H Lin Y-N Wu C-C Wu M-C Liu andC-M Lin ldquoAdvantages of magnetic resonance imaging (MRI)of the seminal vesicles and intra-abdominal vas deferens inpatients with congenital absence of the vas deferensrdquo Urologyvol 82 no 2 pp 345ndash351 2013

[21] S V-N Pereira J D Ribeiro C S Bertuzzo and F A LMarson ldquoAssociation of clinical severity of cystic fibrosis withvariants in the SLC gene family (SLC6A14 SLC26A9 SLC11A1and SLC9A3)rdquo Gene vol 629 pp 117ndash126 2017

[22] J Orlowski R A Kandasamy and G E Shull ldquoMolecularcloning of putative members of the NaH exchanger genefamily cDNA cloning deduced amino acid sequence andmRNA tissue expression of the rat NaH exchanger NHE-1and two structurally related proteinsrdquo e Journal of BiologicalChemistry vol 267 no 13 pp 9331ndash9339 1992

[23] C Bagnis M Marsolais D Biemesderfer R Laprade and SBreton ldquoNa+H+-exchange activity and immunolocalization ofNHE3 in rat epididymisrdquo American Journal of Physiology-RenalPhysiology vol 280 no 3 pp F426ndashF436 2001

[24] K M Kaunisto and H J Rajaniemi ldquoExpression and localiza-tion of the Na+H+ exchanger isoform NHE3 in the rat efferentductsrdquo Journal of Andrology vol 23 no 2 pp 237ndash241 2002

[25] C C Wu O M Alper J F Lu et al ldquoMutation spectrum ofthe CFTR gene in Taiwanese patients with congenital bilateralabsence of the vas deferensrdquo Human Reproduction vol 20 pp2470ndash2475 2005

[26] C C Wu H M Hsieh-Li Y M Lin and H S Chiang ldquoCysticfibrosis transmembrane conductance regulator gene screeningand clinical correlation in Taiwanese males with congenitalbilateral absence of the vas deferensrdquoHuman Reproduction vol19 no 2 pp 250ndash253 2004

[27] E M Bradford M A Sartor L R Gawenis L L Clarke andGE Shull ldquoReduced NHE3-mediated Na+ absorption increasessurvival and decreases the incidence of intestinal obstruc-tions in cystic fibrosis micerdquo American Journal of Physiology-Gastrointestinal and Liver Physiology vol 296 no 4 pp G886ndashG898 2009

[28] M Park S B H Ko J Y Choi et al ldquoThe cystic fibrosistransmembrane conductance regulator interacts with and reg-ulates the activity of the HCO3- salvage transporter humanNa+-HCO3- cotransport isoform 3rdquo e Journal of BiologicalChemistry vol 277 no 52 pp 50503ndash50509 2002

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


Table 1 Reproductive-organ weight in 6-month-old wild-type and Slc9a3minusminus male mice

Group Organ weight (mg)Vas deferens Testis Epididymis Seminal vesicle

Wild Type 265plusmn23 878plusmn13 54plusmn12 162plusmn25Slc9a3minusminus 89plusmn21lowast 508plusmn28lowast 296plusmn11lowast 912plusmn43lowastValues represent the mean plusmn standard error of the mean (n = 3 per genotype)lowastp lt 001 vs wild type according to Studentrsquos t test

[9 10] Therefore other as-yet-unknown genetic or environ-mental factors might cause CBAVD in Taiwan

Mutations in the solute carrier family 9 sodiumhydro-gen-exchanger isoform 3 gene (SLC9A3NHE3) have beenidentified as a possible basis of Taiwanese CBAVD [11]SLC9A3 encodes an ion channel regulated by CFTR andaccumulates at the apicalmembrane of renal proximal tubulesand intestinal epithelial cells to mediate NaCl and HCO3

minus

absorption [12ndash15] Homozygous Slc9a3ndashndash mice are viablebut develop persistent slight diarrhea and mild acidosis andHCO3

minus and fluid absorption are sharply reduced in thekidney as are blood pressure and absorption in the intestine[16] Additionally female Slc9a3ndashndash mice are fertile whereasmales are infertile [17] Notably loss of estrogen receptor 120572increases the luminal pH in themouse epididymis and causessperm defects by suppressing SLC9A3 carbonic anhydraseXIV and SLC4 proteins [17] The C-terminal PDZ motif inCFTR interacts with and regulates the activity of SLC9A3 asdemonstrated inCr (ΔF508)mutantmice [18]Our previousresearch showed that SLC9A3 deficiency causes obstructiveazoospermia degrades the epithelium of the reproductivetract and drastically suppresses CFTR expression [19]

Despite this evidence the role of SLC9A3 in humanreproduction remains somewhat elusive One possibility isthat SLC9A3 is crucial for male reproductive function Inthis study we first evaluated the effect of long-term SLC9A3deficiency in mice on the structure and function of thereproductive tract to determine its possible role in CBVADBecause SLC9A3 is abundantly expressed in mouse repro-ductive organs [19] we first focused on whether long-termloss of SLC9A3 impairs development of the vas deferensSLC9A3 expression and distribution were also comparedin normal human and rodent reproductive tissues and wefurther assessed the potential relationship between SLC9A3copy number variations (CNVs) and CBAVD in a case-control study through genetic analysis of a cohort of menin Taiwan Collectively these data can help to reinforce andcontextualize clinical observations and illuminate the basisof Taiwanese CBAVD as well as the possible general role ofSLC9A3 in human reproduction

2 Materials and Methods

21 Animals FVB129(Cg)-Slc9a3tm1GesJ mice were pur-chased from Jackson Laboratory All animal studies wereapproved by the Fu Jen Laboratory Animal Care and UseCommittee (IACUC number A10580) The genotype of eachmale mouse was assayed by extracting genomic DNA from

the tail and by performing polymerase chain reaction (PCR)using the following primers

F1 (51015840-CATACAACATAGGACTAGCC-31015840)R1 (51015840-CACTACTAGTCAGGCACTCT-31015840) andR2 (51015840-CACTACTAGTCAGGCACTCT-31015840) The primer

ratio of F1 R1 and R2 was 211 Mice of each genotype weresacrificed at 6 months of age by anesthesia with isofluraneand their organs including the testes epididymis and vasdeferens were collected

22 CBAVD Patients and Control Participants Subjects wererecruited among infertile men referred to us for diagnosis atTaipei Medical University Hospital (Taipei Taiwan) CBAVDwas diagnosed based on physical examination of scrotalcontents In particular men without palpable vas deferens onboth sides but with normal testes size (long axis gt 2 cm)were further screened as previously described [11] To confirmCBAVD semen samples were analyzed by standard tests aswell as for pH level and fructose content Some patients wereexamined by magnetic resonance imaging (MRI) to evaluatethe intra-abdominal segment of the vas deferens and sem-inal vesicle [20] Renal ultrasonography was used to assessboth kidneys whereas hormonal assays and chromosomalanalyses were used to rule out testicular azoospermia Bloodsamples were collected to obtain genomic DNA All studieswere approved by the TMU-Joint Institutional Review BoardTaipei Taiwan (trial registration number 201207028) andinformed consent was obtained from each participant

23 Array Based Comparative Genomic Hybridization DataA total of 66 CNVs with high aberrant scores (ltminus1 or gt1)were previously found by array based comparative genomichybridization [11] Of these seven were considered candidateCBAVD-related genes based on a 2-fold decrease in copynumber in at least two of seven patients (Table 1)

24 CFTR Genotyping DNA was extracted from peripheralleukocytes using the Puregene DNA purification kit (GentraSystems Minneapolis MN USA) Exon 9 in CFTR and the50-bp intron 8 upstreamwere sequenced in both directions todetermine the length of IVS8Tn andTGdinucleotide repeatsas previously described [9]

25 Quantitative Real-Time PCR The fold change in SLC9A3copy number between cases and controls was determinedby quantitative real-time PCR using the iQ SYBR Greensupermix kit and the CFX96 Touch real-time PCR detec-tion system (Bio-Rad Laboratories Hercules CA USA)


SV

EP

VD VD VDSV

SV

EP EP

T T T

Wild type

1 cm

Slc9a3+- Slc9a3--








3 Results





Wild type


(a)



100m

(b)

Wild type

Distal vas deferens

20m

(c)


Distal vas deferens

20m

(d)

Wild type

Seminal vesicle

(e)


Seminal vesicle

20m

(f)









SLC9A3

GAPDH

HumanTe

stis

Epid

idym

is

Vas d

efer

ens

Pros

tate

Ute

rus

Plac

enta

(a)

Slc9a3

Gapdh

Rat

Testi

s

Vas d

efer

ens

Epid

idym

is

Pros

tate

Sem

inal

ves

icle

(b)

SLC9A3Control IgG

DAPISMA

SLC9A3

DAPISMA

SLC9A3

DAPI


DAPIPancytokeratin

SLC9A3

DAPI

SLC9A3Control IgG

DAPI

SLC9A3SMA

DAPI

SLC9A3SMA

DAPI

Control IgG

DAPI


DAPI

Pancytokeratin


DAPI

Rat

Human100m 100m

100m

100m

100m 100m

100m

50m

50m

50m

50m

(c)




Epididymis

Seminal vesicle

50 m

(A)

(F)

(B)

(G)

(C)

(H)

(D)

(I)

(E)

(J)

50 m 50 m

50 m 50 m

50 m 50 m

50 m 20 m

20 m

(a)

SMA SLC9A3


DAPI


DAPI

(K) (L)

SMASLC9A3

DAPI

(M)

SMASLC9A3

DAPI

(N)

Pancytokeratin

(O) (P)

(Q)

SMASLC9A3

DAPI

(R)

SLC9A3

(S)

100m 100m 100m

100m100m 50m

50m 50m500m

(b)



N1

N2

N3

N4

N5

N6

N7

N8

N9

N10

N11

N12

N13

N14

N15

N16

N17

N18

N19

N20

N21

N22

N23

N24

N25

N26

N27

N28

N29

N30

N31

N32

0

1

2

3

4

5

6Fo

ld C

hang

e

(a)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

P26

P27

P28

P29

0

1

2

3

4

5

6

Fold

Cha

nge

(b)

Control CBAVD

Gain Gain 219

Normal Loss

0

20

40

60

80

100

Perc

enta

ge o

f ind

ivid

uals

(c)




4 Discussion











1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















SV

EP

VD VD VDSV

SV

EP EP

T T T

Wild type

1 cm

Slc9a3+- Slc9a3--








3 Results





Wild type


(a)



100m

(b)

Wild type

Distal vas deferens

20m

(c)


Distal vas deferens

20m

(d)

Wild type

Seminal vesicle

(e)


Seminal vesicle

20m

(f)









SLC9A3

GAPDH

HumanTe

stis

Epid

idym

is

Vas d

efer

ens

Pros

tate

Ute

rus

Plac

enta

(a)

Slc9a3

Gapdh

Rat

Testi

s

Vas d

efer

ens

Epid

idym

is

Pros

tate

Sem

inal

ves

icle

(b)

SLC9A3Control IgG

DAPISMA

SLC9A3

DAPISMA

SLC9A3

DAPI


DAPIPancytokeratin

SLC9A3

DAPI

SLC9A3Control IgG

DAPI

SLC9A3SMA

DAPI

SLC9A3SMA

DAPI

Control IgG

DAPI


DAPI

Pancytokeratin


DAPI

Rat

Human100m 100m

100m

100m

100m 100m

100m

50m

50m

50m

50m

(c)




Epididymis

Seminal vesicle

50 m

(A)

(F)

(B)

(G)

(C)

(H)

(D)

(I)

(E)

(J)

50 m 50 m

50 m 50 m

50 m 50 m

50 m 20 m

20 m

(a)

SMA SLC9A3


DAPI


DAPI

(K) (L)

SMASLC9A3

DAPI

(M)

SMASLC9A3

DAPI

(N)

Pancytokeratin

(O) (P)

(Q)

SMASLC9A3

DAPI

(R)

SLC9A3

(S)

100m 100m 100m

100m100m 50m

50m 50m500m

(b)



N1

N2

N3

N4

N5

N6

N7

N8

N9

N10

N11

N12

N13

N14

N15

N16

N17

N18

N19

N20

N21

N22

N23

N24

N25

N26

N27

N28

N29

N30

N31

N32

0

1

2

3

4

5

6Fo

ld C

hang

e

(a)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

P26

P27

P28

P29

0

1

2

3

4

5

6

Fold

Cha

nge

(b)

Control CBAVD

Gain Gain 219

Normal Loss

0

20

40

60

80

100

Perc

enta

ge o

f ind

ivid

uals

(c)




4 Discussion











1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Wild type


(a)



100m

(b)

Wild type

Distal vas deferens

20m

(c)


Distal vas deferens

20m

(d)

Wild type

Seminal vesicle

(e)


Seminal vesicle

20m

(f)









SLC9A3

GAPDH

HumanTe

stis

Epid

idym

is

Vas d

efer

ens

Pros

tate

Ute

rus

Plac

enta

(a)

Slc9a3

Gapdh

Rat

Testi

s

Vas d

efer

ens

Epid

idym

is

Pros

tate

Sem

inal

ves

icle

(b)

SLC9A3Control IgG

DAPISMA

SLC9A3

DAPISMA

SLC9A3

DAPI


DAPIPancytokeratin

SLC9A3

DAPI

SLC9A3Control IgG

DAPI

SLC9A3SMA

DAPI

SLC9A3SMA

DAPI

Control IgG

DAPI


DAPI

Pancytokeratin


DAPI

Rat

Human100m 100m

100m

100m

100m 100m

100m

50m

50m

50m

50m

(c)




Epididymis

Seminal vesicle

50 m

(A)

(F)

(B)

(G)

(C)

(H)

(D)

(I)

(E)

(J)

50 m 50 m

50 m 50 m

50 m 50 m

50 m 20 m

20 m

(a)

SMA SLC9A3


DAPI


DAPI

(K) (L)

SMASLC9A3

DAPI

(M)

SMASLC9A3

DAPI

(N)

Pancytokeratin

(O) (P)

(Q)

SMASLC9A3

DAPI

(R)

SLC9A3

(S)

100m 100m 100m

100m100m 50m

50m 50m500m

(b)



N1

N2

N3

N4

N5

N6

N7

N8

N9

N10

N11

N12

N13

N14

N15

N16

N17

N18

N19

N20

N21

N22

N23

N24

N25

N26

N27

N28

N29

N30

N31

N32

0

1

2

3

4

5

6Fo

ld C

hang

e

(a)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

P26

P27

P28

P29

0

1

2

3

4

5

6

Fold

Cha

nge

(b)

Control CBAVD

Gain Gain 219

Normal Loss

0

20

40

60

80

100

Perc

enta

ge o

f ind

ivid

uals

(c)




4 Discussion











1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















SLC9A3

GAPDH

HumanTe

stis

Epid

idym

is

Vas d

efer

ens

Pros

tate

Ute

rus

Plac

enta

(a)

Slc9a3

Gapdh

Rat

Testi

s

Vas d

efer

ens

Epid

idym

is

Pros

tate

Sem

inal

ves

icle

(b)

SLC9A3Control IgG

DAPISMA

SLC9A3

DAPISMA

SLC9A3

DAPI


DAPIPancytokeratin

SLC9A3

DAPI

SLC9A3Control IgG

DAPI

SLC9A3SMA

DAPI

SLC9A3SMA

DAPI

Control IgG

DAPI


DAPI

Pancytokeratin


DAPI

Rat

Human100m 100m

100m

100m

100m 100m

100m

50m

50m

50m

50m

(c)




Epididymis

Seminal vesicle

50 m

(A)

(F)

(B)

(G)

(C)

(H)

(D)

(I)

(E)

(J)

50 m 50 m

50 m 50 m

50 m 50 m

50 m 20 m

20 m

(a)

SMA SLC9A3


DAPI


DAPI

(K) (L)

SMASLC9A3

DAPI

(M)

SMASLC9A3

DAPI

(N)

Pancytokeratin

(O) (P)

(Q)

SMASLC9A3

DAPI

(R)

SLC9A3

(S)

100m 100m 100m

100m100m 50m

50m 50m500m

(b)



N1

N2

N3

N4

N5

N6

N7

N8

N9

N10

N11

N12

N13

N14

N15

N16

N17

N18

N19

N20

N21

N22

N23

N24

N25

N26

N27

N28

N29

N30

N31

N32

0

1

2

3

4

5

6Fo

ld C

hang

e

(a)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

P26

P27

P28

P29

0

1

2

3

4

5

6

Fold

Cha

nge

(b)

Control CBAVD

Gain Gain 219

Normal Loss

0

20

40

60

80

100

Perc

enta

ge o

f ind

ivid

uals

(c)




4 Discussion











1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Epididymis

Seminal vesicle

50 m

(A)

(F)

(B)

(G)

(C)

(H)

(D)

(I)

(E)

(J)

50 m 50 m

50 m 50 m

50 m 50 m

50 m 20 m

20 m

(a)

SMA SLC9A3


DAPI


DAPI

(K) (L)

SMASLC9A3

DAPI

(M)

SMASLC9A3

DAPI

(N)

Pancytokeratin

(O) (P)

(Q)

SMASLC9A3

DAPI

(R)

SLC9A3

(S)

100m 100m 100m

100m100m 50m

50m 50m500m

(b)



N1

N2

N3

N4

N5

N6

N7

N8

N9

N10

N11

N12

N13

N14

N15

N16

N17

N18

N19

N20

N21

N22

N23

N24

N25

N26

N27

N28

N29

N30

N31

N32

0

1

2

3

4

5

6Fo

ld C

hang

e

(a)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

P26

P27

P28

P29

0

1

2

3

4

5

6

Fold

Cha

nge

(b)

Control CBAVD

Gain Gain 219

Normal Loss

0

20

40

60

80

100

Perc

enta

ge o

f ind

ivid

uals

(c)




4 Discussion











1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















N1

N2

N3

N4

N5

N6

N7

N8

N9

N10

N11

N12

N13

N14

N15

N16

N17

N18

N19

N20

N21

N22

N23

N24

N25

N26

N27

N28

N29

N30

N31

N32

0

1

2

3

4

5

6Fo

ld C

hang

e

(a)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

P26

P27

P28

P29

0

1

2

3

4

5

6

Fold

Cha

nge

(b)

Control CBAVD

Gain Gain 219

Normal Loss

0

20

40

60

80

100

Perc

enta

ge o

f ind

ivid

uals

(c)




4 Discussion











1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















1 2 3 4 5 60

2

4

6

8

Num

ber o

f ind

ivid

uals








Data Availability


Disclosure




Acknowledgments


References


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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SLC9A3 Affects Vas Deferens Development and Associates ...downloads.hindawi.com/journals/bmri/2019/3562719.pdf · ResearchArticle SLC9A3 Affects Vas Deferens Development and Associates