Differential expression of cell-cell junction proteins in the testis, epididymis, and ductus deferens of domestic turkeys (Meleagris gallopavo) with white 
and yellow semen 
L.Pardyak,∗ <BR>A. Kaminska,∗ <BR>M. Brzoskwinia,∗ <BR>A. Hejmej,∗ <BR>M.Kotula-Balak,† <BR>J. Jankowski,‡ <BR>

∗,1 

A. Ciereszko,§ and B. Bilinska 
∗Department of Endocrinology, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, 30-387 Krakow, Poland; †University Centre of Veterinary Medicine, University of Agriculture in Krakow, 30-059 Krakow, Poland ; ‡Department of Poultry Science, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, 10-957 Olsztyn, Poland; and §Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-243 Olsztyn, Poland 
ABSTRACT Tight, adherens, and gap junctions are occludin and β-catenin mRNA(P < 0.05) and protein involved in the regulation of reproductive tissue func-(P < 0.05 and P < 0.01, respectively) and upregulation in male mammals. In birds, including domestiction of N-and E-cadherin mRNA(P < 0.001, P < turkeys, intercellular interactionsperformedby junc-0.05, P < 0.01, respectively) and protein(P < 0.01, tionalnetworkshavenotyetbeen studied.Furthermore,P < 0.05, and P < 0.05, respectively). In contrast, ZO-1the cellularand molecular basisofyellow semen syn-andCx43mRNAand proteinwere upregulatedinYSS drome(YSS)in the turkeypopulation remainspoorly testis(P < 0.05 and P < 0.001, respectively) but not understood. Thus, the aim of the present study wasin epididymis and ductus deferens;both mRNAs and2-fold: first,toprovide new information onthelocal-proteinsweredownregulated(P < 0.05) compared to ization and expression of cell-cell junction proteins in the respective WNS epididymis and ductus deferens. the testis, epididymis, and ductus deferens of domesticAltered staining intensity of immunoreactive proteinsturkeys and second, to compare expression of junctional in YSS vs. WNS reproductive tissue sections confirmed protein genes between 2 turkey population, one that the gene expression results. The present study is theproduces white normal semen(WNS)and the other first to demonstrate altered levels of junctional pro-thatproducesyellowabnormal semen. Expressionof oc-tein gene expression in reproductivetissues of male YSS cludin, zonula occludens-1 (ZO-1), connexin 43 (Cx43), turkeys. These findings maysuggest that subtlechangesN-and E-cadherin,and β-catenin genes were investi-in junctional protein expression affect the microenvigated using3 complementary techniques: quantitative ronmentinwhichspermatozoadevelopand matureandreal-time PCR, western blot, and immunohistochem-thus mayhave an impact on the appearance ofyellow istry. Compared to WNS testis, epididymis, and duc-semen in domestic turkeys. tus deferens, YSS tissues exhibited downregulation of 
Key words: cell-cell junctional proteins, testis-epididymis-ductus deferens, domestic turkey,yellow semen syndrome 
2020PoultryScience 99:555–566http://dx.doi.org/10.3382/ps/pez494 

INTRODUCTION ityand when used for insemination leads to decreased fertility and hatchability compared to the white nor-Yellow semen syndrome(YSS), endemic within do-mal semen (WNS)that is produced by most turkeys mesticturkeypopulations,was detectedasearlyasthe (HessandThurston, 1984). Althoughthe totalnum<BR>1980sby identificationof seminal plasma withayel-berofspermandpercentageof motilespermatozoado 
low colorandahigh protein concentration(Thurstonnot differbetweenwhiteandyellow semen(Hessetal., et al., 1982);the cellular and molecular basis of this syn-1976;Slowinska et al.,2011), there are apparentabnor<BR>dromehasnotbeenintensively studiedand, therefore,malspermatidsandspermwithlower motilityandhyremainspoorly understood.Yellowsemenislower qual-pertrophied epithelial cellsin ductuli deferentesof YSS males (Hess et al., 1982; Thurston and Korn, 1997).Other differences relate to proteomic profiling of white ReceivedSeptember5,2018.andyellow seminalplasma(Slowinskaetal., 2015). AcceptedAugust 10, 2019.We recently reported markedly increased expres
1Corresponding author: barbara.bilinska@uj.edu.pl 

sion of aromatase and altered androgen and estrogen © 2019 The Authors. Published by Elsevier on behalf of Poultry Science Association Inc.  This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 
555 
concentrations in the testis, epididymis, and ductus deferens of YYS compared to WNS males (Pardyak et al., 2018). In many mammalian species, the bal-ance between testosterone and estradiol is crucial for spermatogenesis, normal sperm maturation within the epididymis, and proper functioning of the ductus deferens (Sharpe, 1998; Hess et al., 2001; O’Donnell et al., 2001;Carreau and Hess,2010).Besides endocrine regulation, local intercellular interactions, performed by complex junctional networks, are also involved in male mammalian reproductive tissue function (Mrukand Cheng, 2015;Hejmej and Bilinska,2018). Cell-cell junctions, i.e., tight, adherens, and gap junctions, regulate paracellular transport, cell adhesion and maintenance of tissue integrity, and metabolic coupling be-tween neighboring cells (Goossens and van Roy, 2005; MrukandCheng, 2010;Pointisetal.,2010).Analysesof junctional protein gene expression in turkey testis, epididymis, and ductus deferens may contribute new data that helps elucidate cellular and molecular differences between males with white andyellow semen.
In the turkey testis (Deviche et al., 2011), similar to that of otherpoultry species, rodents, and manyothermammals, developing germ cells remain in close contact with Sertoli cells using dynamic junctional complexes that form the blood-testis barrier(BTB), composed ofcoexisting tight, adherens, and gap junctions(Pelletier, 1990;MrukandCheng, 2004;Koperaet al.,2010).As in mammals, the avian BTB divides the seminiferous epitheliuminto2compartments (adluminal and basal), separates advanced germ cells within the testis from theinfluence of the immune system, and allows Sertoli cells toprovideaspecific microenvironmentinthe adluminal compartmentof the epithelium (Cheng and Mruk, 2002; Mruk and Cheng, 2015). 
The turkey epididymis and ductus deferens arebothlined with a pseudostratified columnar epithelium and are therefore morphologically similar (Hess et al., 1976). It is likely that as in the mammalian excurrent duct system, the turkey epididymal and ductal epithelial cells communicate with one anotherbyspecialized in-tercellular junctions that form the blood-epididymis barrier(BEB)(Cyr et al.,1995). The BEB regulates the microenvironment within the duct to allow for sperm maturation and sequesters autoantigenic spermatozoa from the immune system, while a blood-vasdeferens barrier secludesspermatozoa during their transit to the exterior (Robaire and Viger, 1995;Cyr, 2011).Both the BTB and BEB createaunique anatom-ical, physiological, and immunological microenvironment for proper germ cell development into fully functional sperm (Mital et al., 2011). 
Several rodent models have recentlybeen proposed to study the role for tight junction proteins (e.g.,claudinsandoccludin)forspermatogenesis completion and homeostasis maintenance within rat excurrentduct epithelia (Levy and Robaire, 1999;Wong and Cheng, 2005; Cyr et al., 2007). Studies on mice that lacked BTB proteins demonstrated that compromised BTBfunction resulted in reduced fertility(Cheng and Mruk, 2002;MrukandCheng,2004,2015;Koperaetal.,2010). The cadherin/catenin complexwas described as impor-tant to support cell adhesion and integrity in the tissue epithelia (Rowlands et al., 2000), while gap junc<BR>tions, formedbyconnexins (e.g., Cx43),were reportedas involved in tissue homeostasis maintenance through direct intercellular communication (Saez et al., 2003). Compared with extensive studies on mammals, little workwasperformed on the cell-cell junctionsin non-mammalian vertebrates (Bergmann et al., 1984; Izzo et al., 2006)and scarce information is available related to the presence and expression of intercellular junctionproteins in the testis of domestic birds (Osman et al., 1980;Pelletier,1990;Banerjee and Chaturvedi,2018). 
Since the network of cell-cell junctions providedbytight, adherens, and gap junctions coordinates cellu-lar functions within reproductive tissues and whole or-gans in mammals, we selected the representative protein or the protein complex for each junctional type and aimed to evaluate the expression of occludin/ zonula occludens-1 (ZO-1)-based tight junctions, cadherin/catenin-based adherens junctions, and con-nexin 43 (Cx43)-based gap junctions in the testis, epididymis, and ductus deferens of WNS and YSS turkeys. 
As mentioned above, we previously showed that anaugmented aromatization process and high estrogen levels are characteristic in the testis, epididymis, andductus deferens of YSS turkeys (Pardyak et al., 2018). Wehypothesized that these factors may act on Ser-toli, germ, and secretory epithelial cells of the excurrentduct and induce changes in occludin, ZO-1, Cx43, N-and E-cadherin,and β-catenin gene expression. Thesechanges, in turn, may alter the luminal microenvironment, crucial for the functional maturation and pro-tection of spermatozoa. If so, the YSS may rely, atleast partly, on discrete alterations of intercellular junctions presentbetweenvarious cellularplayersinvolvedin spermatogenesis and sperm maturation. 
MATERIAL AND METHODS 

Animals and Tissue Preparation 
In all 38-wk-old domestic turkeys (Meleagris gal-lopavo) with WNS (n = 6) and with YSS (n = 6) wereobtained from the Turkey’s Testing Farm of the De-partmentofPoultry Science(UniversityofWarmiaand Mazury in Olsztyn). Semen of male turkeys producingwhiteandyellow semenwas monitoredto37wkofage (Slowinskaetal., 2015)and criteriaforwhiteandyellowsemen classificationwereusedas describedbyThurston et al.(1982). Semen with a white color and a low seminal plasma protein concentration(≤20 mg/mL) was classified as normal white whereasyellow-colored semen with a high seminal plasma protein concentra-tion(>20 mg/mL)was classified as abnormalyellow.
Fragments (0.5cm× 0.5 cm) of testes, epididymides, and ductuli deferentes of both populations of males were obtained immediately post mortem. The tissue samples were fixed in Bouin’s fixative (saturated picric 
Table 1. Sequences of forward and reverse primers. 
Product size Annealing temp.Genes Primers (5&#2;–3&#2;) (bp) (°C) 
occludin 5&#2;-GAGCCCAGACTACCAAAGCAA-3&#2; 68 55 5&#2;-GCTTGATGTGGAAGAGCTTGTTG-3&#2; 
ZO-1 5&#2;-CCGCAGTCGTTCACGATCT-3&#2; 63 54 5&#2;-GGAGAATGTCTGGAATGGTCTGA-3&#2; 
Cx43 5&#2;-GTCTTCATGCTGGTAGTGTCTTTGGTGT-3&#2; 1558 61 5&#2;-CTGTGGGAGTAGGGGTCGGTTTTTC-3&#2; 
N-cad 5&#2;-GAATGGGACAGTTCCTGAAGGAT -3&#2; 75 56 5&#2;-GCATCGATGGCAGTAACAGTCATTA-3&#2; 
E-cad 5&#2;-GACAGGGACATGAGGCAGAA -3&#2; 120 54 5&#2;-GTACAACAACTGCACAAATAG -3&#2; 
Β-cat 5&#2;-ATGGCAATCAAGAAAGTAAGC-3&#2; 61 52 5&#2;-AGCCATCCCTCCTTCGCACA-3&#2; 
β-actin 5&#2;-AAGTACCCCATTGAACACGG -3&#2; 257 54 5&#2;-ATCACAATGCCAGTGGTACG-3&#2; 
acid, formaldehyde, glacial acetic acid at 15:5:1 proportion) for 24 h, dehydrated in an increasing gradient ofethanol, and embedded in paraffin. After mounting on slides, all sections (5 μm) were cleared in xylene, rehydratedinaseriesof ethanol grade,andwashedinwater(Slowinska et al., 2014;Bilinska et al.,2018). Other tis<BR>sue fragments were frozen in liquid nitrogen and stored at −80°C for RNA isolation and protein extraction. 

RNA Isolation and Reverse Transcription 
Total RNA was extracted from testes, epididymides, and ductuli deferens (pooled after collection from theindividual males) using TRIzol reagent (Life Tech-nologies, Gaithersburg, MD, USA) as described by Pardyak et al.(2018).To remove contaminating DNA and the DNase from RNA preparations, the RNA samples were incubated with reagents from TURBODNase free Kit (Ambion,Austin, TX, USA). The yield and quality of the RNA were assessed by measuringthe A260:A280 ratio in a NanoDrop ND2000 Spec-trophotometer (Thermo Scientific, Wilmington, DE, USA) andby electrophoresis. The purified total RNAwas used to generate total cDNA. A volume equivalent to 1 μg of total RNA was reverse transcribed using high-capacity cDNA Reverse Transcription Kit(Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s protocol.Total cDNAwas prepared ina20 μLvolume using the random primers, dNTPmix,RNAse inhibitor, and reverse transcriptase(RT).For a negative control, the same reactions without adding ofRTwereperformed simultaneously(1μLof RNase-free waterwas addedin placeofRT). TheRT+ andRT− samples thenwere subjected to PCR amplificationperformedinaVeriti Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA). 

Real-Time Quantitative RT-PCR 
To determine alterations in occludin, ZO-1, Cx43, N-and E-cadherin,and β-catenin mRNA expression, quantitative real-time polymerase chain reaction (qPCR)analyseswereperformed. 
Real-timeRT-PCRwasperformed using the StepOne Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) and optimized standard conditions as described in detail (Gorowska et al., 2014). Primersets (Institute of Biochemistry and Biophysics,Polish Academy of Sciences, Warsaw, Poland) are listed in Table 1. Detection of amplification products for indi<BR>vidual geneswasperformed with10ng cDNA, 0.5mM primers, and SYBRGreen master mix (Applied Biosystems, Carlsbad, CA, USA) in a final volume of 20 mL. Amplifications were performed as follows: 55°C for 2 min, 94°C for 10 min, followed by annealing tem-peraturefor30s(Table1)and45s 72°Cto determine the cycle threshold for quantitative measurement. Tomonitor DNA contamination, control reactions without theRNA templatewereperformedin triplicateand one reaction without the RT enzyme was carried out pertissue sample. 
Relative quantification (RQ) was obtained using the 
2−ΔΔCt 
method, adjusting the occludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin mRNAs expression to the expression of β-actin mRNA. Relative levels of the transcripts in YSS testicular, epididymal, and ductalsamples were compared with WNS values, which were arbitrarily set as1(RQ = 1) (Livak and Schmittgen, 2001).
Three independentexperimentswereperformed, each in triplicate. All PCR products were analyzed by gel electrophoresis on 1.5 to 2.5% agarose gels with ethidium bromide together withaready-to-load 100-bp DNA ladder marker (Promega, Southampton, UK) and fol-lowedbyfluorescence digitization usinga Bio-RadGel-Doc XR system (Bio-Rad Labs., Hercules, CA, USA). 


Western Blot Analysis 
Western blot analyses were performed to assess changes in the levels of occludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin in YSS and WNS testes, epididymides, and ductuli deferentes. 

Table 2. Primary antibodies used in this study. 
Antibody Host species Vendor Catalog no Application(s)/dilution(s) 
occludin Rabbit Invitrogen 71-1500 IHC (1:50)WB (1:500) ZO-1 Rabbit Invitrogen 61-7300 IHC(1:50)WB (1:500) Cx43 Rabbit Sigma–Aldrich C6219 IHC(1:400)WB (1:8000) N-cad Mouse Thermo Fisher 33-3900 IHC (1:50)WB (1:500) E-cad Rabbit Invitrogen PA5-19479 IHC(1:100)WB (1:1000) β-cat Rabbit Invitrogen 71-2700 IHC(1:100)WB (1:1000) β-actin Mouse Sigma–Aldrich A2228 WB (1:3000) 
First, the tissues collected from the individual males were pooled and samples of testes, epididymides and ductuli deferens were homogenized on ice with a cold Radio-Immuno-precipitation Assay Buffer (RIPA, pH 8.0; Thermo Scientific; Inc., Rocheford, Il, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Second, the tissue sampleswere sonicated and centrifuged at 10,000gfor 20 min at 4°C as describedpreviously (Hejmej et al., 2012). Aliquots were assayed for protein by the Lowry dye-binding with bovine serum albumin as a standard (Bio-Rad Labs, GmbH,Mchen, Germany). Next, 50 μgofproteinwassolubilized in a sample buffer (Bio-Rad Labs) and heated at 99.9°C for5min. After denaturation, proteinswereseparatedbySDS-PAGEin6to10%(vol/vol) resolving gels under reducing conditions. Separated proteinswere transferred on topolyvinylidene difluoride membranes(Merck Millipore, Darmstadt, Germany) using a wet blotterinthe GenieTransfer Buffer(pH8.4)for90min at a constant current of 250 mA. Nonspecific binding sites were blocked with a solution of non-fat dry milk (5%, wt/v) containing 0.1% Tween 20 (vol/vol), andthe membrane was incubated with respective primary antibody against occludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin (Table 2)at4°Covernight.All primary antibodies were commercially available and recommended for chicken. Thereafter, the membranes were washed with TBS buffer with Tween 20 (0.05M Tris-HCl, 0.15-M NaCl, pH 7.6 containing 0.1% Tween 20) and incubated in a goat anti-rabbit or horseanti-mouse IgG conjugated to horseradish peroxidase (1:3,000; Vector Labs, Burlingame, CA, USA) for 1 hr at room temperature. Immunoreactive proteins were detected using chemiluminescence with western blotting luminol reagent (Santa Cruz Biotechnology),and imageswere captured witha ChemiDocTM XRS+ System (Bio-Rad Laboratories). All immunoblotswere stripped with stripping buffer containing 62.5 mM Tris-HCL, 100 mM 2-mercaptoethanol and 2% SDS (wt/vol) (pH 6.7) at 50°C for 30 min, and incubated in an antibody against β-actin (dilution, 1: 3000; Sigma-Aldrich), which served as the loading control. The bands were densitometrically scanned using Image LabTM 2.0 (Bio-Rad Labs) and the values obtained for occludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin were normalized against the corresponding β-actin. The molecular weights of target proteins were estimated by reference to standard proteins (Color-Burst Electrophoresis Marker, Sigma-Aldrich). Protein levels from WNS tissues were arbitrarily set as 1, against whichstatistical significance was analyzed. 

Immunohistochemistry 
To achieve antigen retrieval, slides were immersed in 10 mM citrate buffer (pH 6.0) and/or in Tris-EDTA buffer(10mMTris containing1mMEDTAand 0.05%Tween20;pH 9.0),and heatedfor3to5mininamicrowaveoven (750W)as described previously(Pardyaket al.(2018).In brief, sectionswere incubated with the same antibodies as for western blotting (Table 2), at 4°C overnight. Subsequently, sections were incubated with biotinylated secondary antibody goat anti-rabbit or horse anti-mouse IgG (1: 500; Vector Labs). Aftereachstep of these procedures sections were rinsed with TBS. The staining was developed using avidin biotinylated horseradish peroxidase complex (ABC/HRP; 1:100; Vectastain Elite ABC Reagent, Vector Lab.) for 30 min. Bound antibody was visualized with TBScontaining 0.05% 3,3 -diaminobenzidine tetrachloride (DAB; Sigma-Aldrich) and 0.07% imidazole for 4 to 5 min. Thereafter, sections were washed and counterstained with Mayer’s hematoxylin, dehydrated andmounted using DPX mounting media (Sigma-Aldrich). Tovalidate specificity of primary antibodies used forimmunohistochemistrywestern blottingwasperformed (Bordeaux et al. 2010). Control sections included omission of the primary antibody and substitutionbyan irrelevant IgG. The sections were examined with a Leica DMR microscope (Leica, Microsystems GmBHWetzlar,Wetzlar, Germany). 

Qualitative and Quantitative Evaluation of the Immunohistochemical Reactions 
Immunohistochemical staining for all antigens, occludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin was evaluated qualitatively in at least 20 serial sectionsfrom each tissue. The slides were processed immunohistochemically at the same time with the same treatmentso thatthe stainingintensities couldbe compared (Kotula-Balak et al., 2008). The tissue cells were con-sidered immunopositive if brown reaction product waspresentat the testicular, epididymal, and ductal epithelia; the cells without anyspecific immunostaining wereconsidered immunonegative. All immunohistochemical stainingswererepeatedat least3times. 
For quantitative analysis of the staining intensity,digital color images of testis, epididymis, and ductus deferens sections were obtained using a CCD VideoCamera (KYF55, JVC) mounted on an optical micro-scope (Microphot, Nikon, Japan) and connected to a video capture card (PV BT878P1, Prolink, Nei-Hu,Taipei,Taiwan) installedinapersonal computer. Image processing and analyseswereperformed using Im-ageJ software (National Institutes of Health, Bethesda, MD, USA). The intensity of immunostainings was ex-pressed as relative optical densityof diaminobenzidinebrown reaction products and calculated using the formula describedbySmolen(1990).Atotalnumberof60 tissue sections (n = 10per tissue;a random selectionof YSS and WNS tissue sections)were subjected to imageanalysis and results of 10 separate measurements were expressed as mean ± SD. 

Statistical Analysis 
Each variable was tested using the Shapiro–Wilk W-test for normality. The homogeneityofvariancewas as-sessed with Levene’s test. All statistical analyses wereperformed using non-parametric Mann–Whitney U-test to determine which YSS values differed significantly from WNS values that served as controls. The analysiswas made using Statistica 10 (Statsoft,Poland). Data were presented as mean ± SD. Data were considered statistically significant at P < 0.05. 
RESULTS 


Expression of Occludin, ZO-1, N-and E-cadherin, β-catenin, and Cx43 mRNA in Turkey Reproductive Tissues 
Electrophoresis revealed PCR-amplified products ofthe following predicted size: 68 base pairs(bp)for occludin, 63 bp for ZO-1, 1,558 bp for Cx43,75bpfor N-cadherin, 120 bp for E-cadherin,61bpfor β-catenin, and 257 bp for β-actin in YSS and WNS testes, epididymides, and ductuli deferentes (Figure 1A–E; see respective bands). 
Quantitative real-time PCR analysis revealed down-regulation of occludin and β-catenin mRNA (Figure 1A andE)in all YSS tissues(P < 0.05), upregulation of N-and E-cadherin in YSS testis, epididymis, and ductus deferens(P < 0.001, P < 0.05, and P < 0.01, respectively; Figure 1D), while ZO-1 and Cx43 mRNA were upregulated(P < 0.05 and P < 0.001, respectively) in YSStestis(Figure1BandC)butnotin epididymisandductus deferens,in whichboth mRNAsweredownregulated(P < 0.05; Figure 1B andC) compared to therespective WNS tissues (Figure 1A–E) 

Expression of Occludin, ZO-1, N-and E-cadherin, β-catenin, and Cx43 Protein in Turkey Reproductive Tissues 
Immunodetectable proteins were observed as singlebands near 65 kDa (occludin), 210 kDa (ZO-1), 43 kDa (Cx43), 135 kDa (N-and E-cadherin), 92 kDa (β-catenin), and 42 kDa(β-actin) (Figure 1A’–E’; see re<BR>spective bands). Protein band intensities were either markedly reduced or increased in YSS vs. WNS tissuesamples as measured quantitatively. 
Quantitative analysisof3separate experiments indicated thatoccludin and β-catenin expression decreased (P < 0.05; P < 0.01) and N-and E-cadherin increased (P < 0.01; P < 0.05)inallYSS tissues (Figure 1A’,D’, and E’) compared to the respective WNS tissues. ZO-1 and Cx43 protein levels,however,were elevated(P < 
0.05 and P < 0.001, respectively) in YSS testis but notin the epididymis and ductus deferens (Figure 1B’ and C’). In the latter tissues, ZO-1(P < 0.05) and Cx43 (P < 0.01) were significantly reduced compared to therespective WNS tissues (Figure 1B’ and C’). 


Immunohistochemical Detection of Occludin, ZO-1, Cx43, N-and E Cadherin, and β-Catenin in Turkey Reproductive Tissues 
Alterationsin thelocalization and immunoexpressionof junctional proteins were characterized using qualitative and quantitative analyses. Immunohistochemistry revealed positive staining for all examined junctionalproteins in YSS and WNS testes, epididymides, and ductuli deferentes (Figures 2A–C and 3A–B). Detaileddescriptions for each junctional protein are presented below. 
Occludin and ZO-1 In YSS testis,occludinwas de-localizedbetween Sertoli and germ cells; the signal ran perpendiculartothe basementmembrane, exhibited“aspoke-like” pattern, and failed to localize to the BTB. Comparatively, in WNS testis, occludin signal ran par-allel to the basement membrane, consistentwith its localizationattheBTB (Figure 2A).InYSS epididymis, occludin appeared as discontinuous lines at the BEB region.More precisely, this staining patternwas prominent at the apical-lateral border of the principal epithelial cells vs. continuous lines observed close to the luminalborder of the WNS epididymal epithelium. Of note, this altered signal pattern was observed over theentire YSS epididymis length (Figure 2A; compare in-serts at higher magnification). In YSS ductus deferens, 


Figure 1. Occludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin mRNA (A to E) and protein (A’ to E’) expression in yellow semen syndrome (YSS) and white normal semen (WNS) testes, epididymides, and ductuli deferentes. Representativequantitativereal-time PCR analysis of occludin (A), ZO-1 (B), Cx43 (C), N-and E-cadherin (D), and β-catenin (E) mRNA expression in YSS and WNS testes, epididymides, and ductuli deferentes. As aninternal control, β-actin mRNA level was measured. Relative quantification (RQ) is expressed as mean ± standard deviation (SD). Datawere obtained from3separate experiments. Asterisks indicate statistically significant differences(*P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U-test). YSS turkeys (n = 6) and WNS turkeys (n = 6). Representative western blots and relative expression of occludin (A’), ZO-1 (B’), Cx43 (C’), N-and E-cadherin (D’), and β-catenin (E’) proteins in YSS and WNS testes, epididymides, and ductuli deferentes. Densitometric analysis of protein content was normalized against the corresponding β-actin. Protein levels within the WNS tissues were arbitrarysetas1. Data obtained from3separate analyses are expressedas mean ± SD. Asterisks indicate statistically significantdifferences (*P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U-test).N = 5eachfor YSS and WNS tissues. 

Figure 2. Qualitative (A to C) and quantitative (D to F) analysis of immunohistochemical staining for occludin (A), ZO-1 (B), and Cx43 (C). Representative micrographs fromyellowsemen syndrome (YSS) and white normal semen (WNS) testes, epididymides, and ductuli deferentes. Counterstainingwasperformed withMayer’s haematoxylin. Scale bars are10 μm.Frames indicate thelocationof the higher magnification view.
(A)Positive staining foroccludinwas visiblein YSS and WNS testes, epididymides, and ductuli deferentes.Aspoke-like patternbetween Sertoli andgermcells(arrows),ofdiscontinuouslinesattheblood-epididymisbarrier(BEB)(arrows),andattheapical epithelium(arrows)ofYSStestis epididymis, and ductus deferens, instead of the parallel linebetween Sertoli and germ cells (arrows) and of the continuous lines at the luminar epithelium(arrows)oftherespectiveWNS tissuesectionswere observed.Notethe alteredoccludinstaining patternathigher magnificationviews (inserts in A) and reduced staining intensityin all YSS tissue sections. (B) The ZO-1 signal was either dispersed at the base of the seminiferous epithelium(open arrows)or visibleinthe lateral contactsbetween Sertoli cells(arrows)and closetothe luminalborderof epididymaland ductal epithelium(arrows) ofYSS and WNS tissues.Note increased ZO-1 staining intensityinthe YSStestis and reduced staining in YSS epididymis and ductus deferens. Note also sloughed germ cells admixed with maturespermatozoainYSS epididymal lumen (asterisksinAandB).(C) Punctate pattern of Cx43 was visible in the basal and adluminal compartment of the testis (arrows and open arrows, respectively), while a strong, linear patternwas presentbetween neighboring Leydig cells in the interstitial tissue (arrowheads). In the epididymal epithelium, thesignalwas observedbetween principal cells(arrows)and principaland basal cells(open arrows).Notea mixedCx43 patternoffocior linesat the higher magnification views. In the ductus deferens, weak Cx43 signal was seen in the YSS apical epithelium (arrows) and strong staining was observed in the WNS stroma layer (cross). Note increased Cx43 staining intensityin the YSS testis and reduced intensityin YSS epididymis and ductus deferens. (D to F) Histograms of occludin (D), ZO-1 (E), and Cx43 (F) staining intensity expressed as relative optical density (ROD) of diaminobenzidine brown reaction product. Data are expressed as mean ± SD of3 independent experiments (each in triplicate). Significant differences from WNS values are denoted as *P < 0.05, **P < 0.01. (D’-F’)Immunoblots thatshowthespecificityofthe respectiveantibodies againstoccludin(D’),ZO-1(E’),andCx43(F’)in tissue lysates(50µg protein).The relativepositionofthe proteinband correspondingtothe ColorBurst Electrophoresis Marker(Sigma-Aldrich)is notedtothe left.Abbreviations:T – testis,E – epididymis,D – ductus deferens,SE – seminiferous epithelium,IT –interstitial tissue,EE – epididymal epithelium,DE – ductal epithelium,SL –stromalayer,MW – molecularweight. 
occludin immunoreactivityappeared as a discreet, dis-continuous band at the apical epithelium (Figure 2A). In all YSS reproductive tissues, changes in the staining patternand orientationofoccludinwere accompanied by a reduction in the immunostaining intensity as confirmedby optical density quantitative measurement(Figure 2D). The signalwas significantly reduced (P < 0.05) in YSS vs. WNS tissues. In the epididy-mal epithelium, the differencebetween YSS and WNSsampleswas even more pronounced(P < 0.01). 
ZO-1 staining was frequently dispersed throughoutthe Sertoli cell cytoplasm at the base of the epithelium inboth WNS and YSS testes. However, in the latter, a diffuse staining pattern colocalized with ZO-1 in lat-eral contactsbetween adjacent Sertoli cellsperpendicular to the basal lamina (Figure 2B). Such a stainingpatternwas also apparentbetween adjacent epithelial cells, closetothe luminalborderofYSS epididymisand ductus deferens, while in WNS samples, ZO-1 stainingwas diffuse and/or localized to the plasma membranes of adjacent epithelial cells (Figure 2B). Of note, stain<BR>ing intensity increased in YSS testes(P < 0.05) anddecreasedintheYSS excurrentductsystem(P < 0.05) compared to the respective WNS tissues (Figure 2E). 
Cx43 Inboth YSS and WNS testes, Cx43 signal ap-peared as distinct foci or lines localized in the basal compartment close to the basal lamina and in the apical compartment close to the lumen (Figure 2C). Closer examination revealed that stainingoccurredbetween adjacentSertoli cells orbetween Sertoli and germcells, as shown at higher magnification (Figure 2C). In both compartments, signal intensity was strongerin YSS compared to WNS testis. In the interstitium, Cx43 immunoreactivity was observed between neighboring Leydig cells. Further, in YSS testis, there wasa very strong and linear Cx43 signal at the plasma membrane, whereas in WNS testis it was clearly re-duced (Figure 2C).In YSS epididymis, Cx43was ap-parent as distinct foci and/or discreet lines localized along the lateral plasma membranes of adjacentepithelial principal cells andbetween principal and basal cells (Figure2C), whereasa similar staining patternof ap-parently stronger intensity was observed in the respective WNS epididymis. Inboth YSS and WNS ductus deferens, Cx43 staining was localized to the apical cy-toplasm of the columnar epithelium and the stroma layer. However, there was a significant difference in stainingintensityinthe stroma.Cx43signalwasapparently reduced in the YSS compared to the WNS stroma (Figure2C). The results of quantitative evaluation ofthe staining intensityconfirmed the immunohistochemical observations. Therewere significantdifferencesbetween the YSS and WNS testicular interstitium, epididymal epithelium, and stromal layer, (P < 0.01, P < 0.01, and P < 0.05 respectively; Figure 2F). 
N-and E-cadherin and β-catenin In YSS and WNS testis, N-cadherin and β-catenin signals were observed in the basal and adluminal compartment of the seminiferous epithelium (Figure 3Aand B). Thor-ough analysis revealed increased N-cadherin and de-creased β-catenin intensity dispersed in the cell cytoplasmand/orintheformof discontinuouslinesbetweenSertoli and germ cells at the BTB of YSS vs. WNS testis (Figure3AandB; see higher magnification views).In YSS epididymis, E-cadherinwaslocalizedalongthelateral plasma membranes of adjacent epithelial principal cells andbetween principal and basal cells (Figure 3A). β-cateninlocalizationwas similarto thatof E-cadherin (Figure3B);however, E-cadherin stainingintensitywas increased, while that for β-catenin was reduced in YSS vs. WNS epididymis (Figure 3Aand B). Along the epi<BR>didymis, staining intensity was consistently strong, as shown in inserts at higher magnification (Figure 3A andB), while stromal cellswereweakly stainedinboth YSSandWNS epididymides (Figure 3AandB).Inthepseudostratified columnar epithelium of the YSS ductus deferens, E-cadherin and β-catenin signals were mainly associated with the cell surface and localized along thelateral plasma membranes of adjacent columnar cells andbetween columnar and basal cells (Figure 3Aand B). Of note, sloughed germ cells admixed with mature spermatozoa were observed in the lumen of YSS epididymis (Figures 2AandBand3AandB, asterisks).
Quantitative evaluation of N-and E-cadherin and β-catenin staining reflected the qualitative results (Figure3Cand D). Therewasa statistically significantincrease in cadherin signal in YSS testis, epididymis, and ductus deferens vs. WNS tissues(P < 0.05, P < 0.01, and P < 0.05, respectively). In contrast, β-catenin signals were significantly reduced in YSS compared to WNS reproductive tissues(P < 0.001, P < 0.01, and P < 0.05, respectively). Interestingly, compared to WNS, in YSS ductus deferens, the E-cadherin signal was significantly higher in the stromal layer(P < 0.05), while β-catenin was significantly reduced in the ductal epithelium(P < 0.05). 
Negative control YSS and WNS tissue sections showed no immunopositive staining foroccludin, ZO1, Cx43, N-and E-cadherin, and β-catenin when incubationwasperformed without the respective primaryantibodies (upper inserts of Figure 2A-C). 
In summary, quantitative measurement junctionalprotein immunohistochemical staining confirmed qPCR (Figure1A–E) andwestern blot (Figure 1A’–E’) analy<BR>ses and indicated pronounced expressionchangesinoccludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin in YSS vs. WNS testis, epididymis, and ductus deferens (for details, see Figures 2D–F and 3C and D).Specificityofallantibodies usedforimmunohistochemistry were validated by western blot, as shown in Figures2D’–F’ and 3C’ and D’. 

DISCUSSION 

In the present study, we sought to assess the possible changes in mRNA and protein levels of the se-lected junctional proteins that are present at the testicular, epididymal, and ductal epithelia of YSS vs. 

Figure 3. Qualitative(AandB)and quantitative(CandD) analysisofimmunohistochemicalstainingforN-and E-cadherin(A)and β-catenin (B). Representative micrographsofyellowsemen syndrome(YSS)and white normal semen(WNS) testes, epididymides,and ductuli deferentes. Counterstainingwasperformed withMayer’s haematoxylin. Scale bars are10 μm.Frames indicate thelocationof the higher magnification view. (A and B) Similar staining pattern for N-and E-cadherin (A) and β-catenin (B) were observed in the basal compartment at the blood-testis barrier(BTB)(arrows)andinthe adluminal compartmentofseminiferous epithelium(open arrows)aswellasinthe lateralplasmamembranesof adjacentepididymalprincipalcells(arrows)andbetweenprincipalandbasalcells(openarrows),andinthelateralplasmamembranesofadjacent ductalcolumnarcells(arrows)andbetweencolumnarandbasalcells(openarrows)ofYSSandWNS epididymidesandductulideferentes.Note increased N-and E-cadherin and reduced β-catenin stainingintensity attheBTBofYSS testis(see higher magnification viewsinAandB) and of YSS epididymis and ductus deferens sections (see higher magnification views in B). Note weaker staining for β-cateninbetween epithelial principal cells (arrows) and principal and basal cells (open arrows) along the entire YSS epididymis length (see higher magnification inserts in B).Notealsosloughedgermcells admixedwith maturespermatozoainYSS epididymallumen (asterisksinAandB).(CandD) HistogramsofN-and E-cadherin (C) and β-catenin(D)stainingintensityexpressedasRODof diaminobenzidinebrown reactionproduct. Data are expressed as mean ± SDof3 independent experiments (eachin triplicate). Significant differences from WNSvalues are denoted as*P < 0.05, **P < 0.01, ***P < 0.001. (C’-D’) Immunoblots thatshow thespecificityof the respective antibodies againstN-and E-cadherin (C’) and β-catenin (D’)in tissue lysates(50µg protein).The relativepositionofthe proteinband correspondingtothe ColorBurst ElectrophoresisMarker (SigmaAldrich)is notedtothe left.Abbreviations:T – testis,E – epididymis,D – ductus deferens,SE – seminiferous epithelium,IT –interstitialtissue, EE – epididymal epithelium, DE – ductal epithelium, SL – stroma layer, MW – molecular weight. 
WNS turkeys.It shouldbe added that the molecu-coincidewellwith alteredoccludinimmunostainingpat-lar composition of intercellular junctions in the repro-terns found at the BTB of YSS testis and the BEB re-ductive tissuesof domestic turkey has notyetbeengionofYSS epididymis.It shouldbe stressed thatindemonstrated. YSS epididymis suchan altered expression pattern was 
We observed downregulation of occludin mRNA and observed along the entire tissue length (compare higher protein in all YSS reproductive tissues. These results magnification micrographsin Figure 2A). Both findings clearly indicate impaired functionality of reproductiveepithelia in YSS turkeys. 
It is tempting to speculate that changes in ZO-1and Cx43 expression in YSS reproductive tissues may also contribute to disturbed functionality. This notion is supported by the following observations: increasedZO-1 and Cx43 mRNA and protein expression in YSS testis and reduced expression of these proteins in YYSepididymis and ductus deferens as compared to the respective WNS tissue.Moreover, similar expression pat-ternsofboth junctional proteins may indicate apossible relationship between ZO-1 and Cx43 expression in turkey reproductive tissues. Similar physical associationofCx43withZO-1aswellasZO-1-regulated gap junction organization was reported in cardiac my-ocytes (Toyofuku et al., 1998), osteoblastic cells (Laing et al., 2001), and Sertoli cells (Segretain et al., 2004). Itis relevantto note that altered ZO-1localizationwas also observed in rat testes exposed to various toxicants (Wong et al., 2004; Sobarzo et al., 2009)thatled to the loss of functional BTB integrity as a consequence of ZO-1 protein mislocalization. 
In turkey testes, Cx43 signal was observed at the BTB in the basal epithelium andbetween Sertoli and germ cells in the adluminal compartment, more precisely at the Sertoli cell-elongated spermatid interface.Interestingly, the staining intensity at the BTB was stronger in YSS compared to WNS tissue. Increased Cx43immunoexpressionattheBTBofYSS testismayresult, at least partly, from increased ZO-1 expression in this region. A similar relationship between im-paired Cx43 and ZO-1 protein distribution at the BTB was demonstrated in adult rat testis exposed to flutamide(Chojnackaetal., 2016).It shouldbenotedthatelevated Cx43 mRNA and protein in YSS testis are presumably causedbyincreased Cx43 expressionintheinterstitial space,asshownbyquantitativeanalysisofimmunohistochemical staining. This pattern may reflect disturbed functionalityof YSS Leydig cells, as reportedinour previousstudy(Pardyaketal., 2018)thatshowed increased testosterone and estradiol concentrations in YSS vs. WNS testes. It is interesting to note here that the absence or apparent decrease of Cx43 expression within seminiferous tubules, and the loss of Cx43 from Sertoli cellsinrodentsandhumanswith impaired testis functionality,resultsin Leydig cellhyperplasia(Kotula-Balaketal., 2007;for review see Kidder and Cyr,2016). 
As reported earlier (Fiorini et al., 2004;Brehm et al., 2007), Cx43 represents a functional marker for Sertolicells and contributes to coexisting tight and adherens junctions to organize the BTB.To date, however, thephysiological role of Cx43-mediated gap junctions in processesthatinvolvethefinal maturationofspermatozoa during their transit along the epididymal duct is notfully elucidated, although gap junctionsbetween adjacentprincipal cellsof rat epididymiswere identifiedbyfreeze-fracture electron microscopyin 1972 (Friend and Gilula, 1972). It ispossible that reduced Cx43 expres-sion in the BEB andbetween the principal and basal cells might lead to altered epithelial integrityand dis-turbed cellular homeostasis in YSS epididymis (Cyr, 2011;Kidder and Cyr,2016). More than tenyears ago,the Cyr group demonstrated that the BEB is critical for the maintenance and composition of the luminalenvironment in the epididymal duct (Cyr et al., 2007). Interestingly, analysis of male reproductive function in mice withmutationsin Cx43 revealed thatspermatozoaqualityand motilityin mutant mice were significantly decreased while epididymal morphologywas not altered(Gregory et al., 2011). 
Further, strong Cx43 staining in the WNS ductus deferens stromal cells may indicate a putative role ofconnective tissue cells in the cell-to-cell communication that mediates signals to the excurrentduct epithelium.If this conjecture is true, a distinct reduction of the Cx43 signal in the YSS stroma maysuggest an adverse impact on direct intercellular communication and ex-change of small growth-regulatory molecules that are indispensable for the luminal microenvironment and, consequently, proper sperm maturation. This supposition agrees with the widely accepted idea that expres-sion and localization of connexins are altered duringcellular distress, related to disease or chemical insult, and lead to abnormal intercellular communication (for further details seeMesnil et al., 2005).
Finally, our results suggest that adherens junctionconstituent protein levels might influence the ductusepididymis lumen microenvironment in YSS turkeys. We detected upregulation of N-and E-cadherin and downregulation of β-catenin genes in YSS testis and ex-current duct system compared to the respective WNS tissues. N-cadherin is expressed in the seminiferous epithelium, whereas E-cadherin is present in excurrent duct epithelia (Cyr et al., 1993;Andersson et al.,1994). Here, we found that N-cadherin and β-catenin dis-tribution in YSS testis was altered predominantly at the BTB region (compare higher magnification micro-graphsinFigure 3AandB),andthis findingmaysug-gest dysregulated trafficking or enhanced endocytosisof the adherens junction proteins (Fiorini et al., 2008; Lietal., 2009).Asignificantincreasein E-cadherinand decrease in the β-catenin staining in the YSS excurrentduct, as detected by immunohistochemistry, supports thishypothesis. The above results are in line with the idea that affected fertility in domestic poultry mightbe associated with epididymal abnormalities (Deviche et al., 2011)and our observation that sloughed germcells (admixed with mature spermatozoa) and dense epididymal fluid were present in the lumen of the epididymis of YSS turkey.
Taken together, the most prominent alterations in cell-cell junction protein gene expression, their distribution,andthe stainingintensitywere observedinYSSepididymis and ductus deferens compared to WNS tissues. The gene and protein expression data from tissue homogenateswere confirmedbysubcellular distribution of occludin, ZO-1, Cx43, N-and E-cadherin, and β-catenin in YSS reproductive tissue samples, as visualized with lightmicroscopy. In our previous study,we reported a positive correlation between quantitatively measured immunolocalization and expression ofjunction proteins in the rat (Zarzycka et al., 2015). The present results are the first that demonstrate altered levels of junctional protein mRNA and protein expres-sion couldbe relatedtotheappearanceofyellow semen in domestic turkeys. 

ACKNOWLEDGMENTS 
Supported by a grant PRELUDIUM13, 

2017/25/N/NZ9/00585 from the National Science 
Centre. 

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