Connexin 43 and plakophilin-2 as a protein complex that regulates blood testis barrier dynamics

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1 onnexin 43 nd plkophilin-2 s protein complex tht regultes lood testis rrier dynmics Michelle W. M. Li, Dolores D. Mruk, Will M. Lee, nd. Yn heng, enter for iomedicl Reserch, Popultion ouncil, New York, NY 65; nd School of iologicl Sciences, The University of Hong Kong, Hong Kong, hin Edited y Ryuzo Yngimchi, University of Hwii, Honolulu, HI, nd pproved pril 24, 29 (received for review Ferury 3, 29) The lood testis rrier (T) formed y djcent Sertoli cells is composed of coexisting tight junction (TJ), sl ectoplsmic speciliztion (ES), nd desmosome-like junction. Desmosome-like junctions disply structurl fetures of desmosome nd gp junctions, ut its function t the T remins unknown. Herein, we demonstrte tht connexin 43 (x43), gp junction integrl memrne protein, structurlly intercts with desmosoml protein plkophilin-2 (), sl ES proteins N-cdherin nd -ctenin, nd signling molecule c-src, ut not with the TJ proteins occludin nd ZO- in the seminiferous epithelium of dult rts. The locliztion of x43 in the seminiferous epithelium during (i) the norml epithelil cycle of spermtogenesis nd (ii) nchoring junction restructuring t the Sertoli spermtid interfce induced y djudin which mimics junction restructuring events during spermtogenesis hve suggested tht x43 is involved in cell dhesion. The knockdown of x43 y RNi technique using specific sirn duplexes ws performed in primry Sertoli cell cultures with n estlished TJ permeility rrier tht mimicked the T in vivo. This knockdown of x43 ffected neither the TJ rrier function nor the stedy-stte levels of junction proteins of TJ, sl ES, nd desmosome-like junction. However, fter the knockdown of oth x43 nd, the Sertoli cell TJ rrier function ws pertured trnsiently. This perturtion ws concomitnt with mislocliztion of occludin nd ZO- from the cell cell interfce. In summry, x43 nd form protein complex within the desmosome-like junction to regulte cell dhesion t the T, prtly through its effects on the occludin/zo- complex, so s to fcilitte the trnsit of primry preleptotene spermtocytes. desmosome-like junction seminiferous epithelil cycle Sertoli cell spermtogenesis In dult mmmls, including rodents nd humns, the lood testis rrier (T) is unique nd importnt ultrstructure in the seminiferous epithelium for mintining spermtogenesis. Unlike other lood tissue rriers, such s the lood rin rrier, the T is constituted y djcent Sertoli cells ner the sement memrne in the seminiferous epithelium, insted of t the endothelil tight junction (TJ) rrier of microvessels in the interstitium (, 2). In the rt testis, the T confers the fence function tht monitors the prcellulr influx of wter, electrolytes, hormones, nd iomolecules etween djcent Sertoli cells nd mintins cell polrity (3). Furthermore, the T cretes specilized microenvironment in the picl comprtment of the seminiferous epithelium nd segregtes the entire event of postmeiotic germ cell development from the systemic circultion (3). lthough the T is considered to e one of the tightest lood tissue rriers in mmmls (4), it must undergo extensive restructuring t stges VIII nd IX of the seminiferous epithelil cycle in dult rt testes. s such, junction restructuring fcilittes the trnsit of primry preleptotene spermtocytes through the T while they differentite into leptotene nd zygotene spermtocytes. However, the immunologicl rrier must e mintined nd remin intct (5). The mechnisms y which this timely T restructuring event is regulted hve remined oscure for lmost hlf century (3, 6). In other epitheli, the TJ rrier tht confers the rrier function is supported structurlly y discrete dherens junction (J) plques locted ehind the TJ firils, followed y the desmosome, forming the junction complexes (7). Interestingly, the T is constituted y coexisting TJ, sl ectoplsmic speciliztion (sl ES, n typicl testis-specific J), nd desmosome-like junction (). We envisioned tht these coexisting junctions must hve physiologicl significnce t the T other thn their structurl role in contriuting to the rrier integrity. The desmosome-like junction in the seminiferous epithelium ws reported to possess the ultrstructurl fetures of oth the desmosome nd gp junction (GJ) when exmined y electron microscopy (8). There is ccumulting evidence tht desmosome cn serve s pltform for signl trnsduction (9) nd tht connexin 43 (x43) cn medite cell cell communiction (). We thus sought to exmine the physiologicl significnce of the desmosome-like junction t the T. We report herein protein complex consisting of x43 nd plkophilin-2 () t the desmosome-like junction nd descrie how it regultes T dynmics through its effects on the TJ protein complex occludin/zo-. Results Stge-Specific Expression of x43 t the lood Testis rrier in Rt Testes. The specificity of the nti-x43 ntiody is shown in Fig.. single nd of 39 kd ws detected y immunolot nlysis using lystes of seminiferous tuules, nd n dditionl phosphorylted isoform of 4 kd ws detected using lystes of Sertoli cells (see lso supporting informtion (SI) Fig S). This ntiody ws lso used to loclize x43 in the seminiferous epithelium of dult rt testes (Figs. nd nd Tle S). The stge-specific expression of x43 is shown in Fig., compred with the control shown in Fig. D. In vrious stges of the seminiferous epithelil cycle, x43 ws detected ner the picl ES t the Sertoli cell elongting spermtid interfce nd predominntly t the T (Fig. ). Its expression t the picl ES nd the T ws highest from stge V to erly stge VIII, ut it diminished gretly t oth sites t lte stge VIII (Fig. ). The decline in x43 t lte stge VIII coincided with spermition, which involves the disssemly of the picl ES to relese the fully developed spermtids into the tuule lumen, nd with the restructuring t the T tht fcilittes the trnsit of preleptotene spermtocytes. These findings implicte the likely involvement of x43 in cell dhesion t the picl ES nd the T. The Decline in x43 During djudin-induced nchoring Junction Restructuring in the Seminiferous Epithelium. To study the restructuring of nchoring junctions in the seminiferous epithelium, n estlished in vivo model ws used. dult rts were fed single dose of djudin (5 mg/kg ody weight) to induce germ cell loss, most uthor contriutions:.y. designed reserch; M.W.M.L. nd.y.. performed reserch; D.D.M., W.M.L., nd.y.. contriuted new regents/nlytic tools; M.W.M.L., D.D.M., nd.y.. nlyzed dt; nd M.W.M.L. nd.y.. wrote the pper. The uthors declre no conflict of interest. This rticle is PNS Direct Sumission. To whom correspondence should e ddressed. E-mil: y-cheng@popcr.rockefeller.edu. This rticle contins supporting informtion online t 976/DSupplementl. DEVELOPMENTL IOLOGY cgi doi.73 pns.976 PNS June 23, 29 vol. 6 no

2 Time fter tretment with djudin (5 mg/kg.w.) onnexin 43, 39 kd ctin, 42 kd x43, 39-4 kd ctin, 42 kd II IV V VI Time fter tretment with djudin VII ltevii VIII lteviii : trl i : i c: ii e: iii f: H h: iv i: v k: 4 D l: Rit IgG ii IX X XI XII d: H iii XIII XIV D Rit IgG g: D v iv Fig.. Stge-dependent expression of x43 in the seminiferous epithelium of dult rt testes. () Immunolot nlysis illustrtes the specificity of the rit nti-x43 ntiody using lystes of seminiferous tuules (ST) nd Sertoli cells (S). () Immunohistochemicl locliztion of x43 in the seminiferous epithelium of dult rt testes. The expression of x43, which ppers s reddish-rown precipittes, in the epithelium is stge dependent. () The locliztion of x43 in the epithelium t different stges of the epithelil cycle. x43 is detected t the T nd the picl ES, ut its expression is more predominnt t the sl region. The expression of x43 t oth the T nd picl ES is highest from stge VI to erly stge VIII; it diminishes drsticlly t oth sites t lte stge VIII. (D) In the negtive control, rit IgG ws sustituted for the primry ntiody. (Scle r, 5 m in nd D nd 3 m in.) notly elongting spermtids, from the epithelium eginning t dy posttretment (3). The T remined intct despite the disruption of dhesion etween Sertoli nd germ cells (3) when ssessed y n in vivo integrity ssy (). time-dependent decline in x43 in the testes ws detected following the disruption of nchoring junctions induced y djudin (Fig. 2 nd ). Results from representtive immunohistochemicl experiment re shown in Fig. 2. In norml stge VII tuule, the stining of x43 ws very strong ner elongting spermtids t the picl ES (Fig. 2). t the time of djudin-induced depletion of elongting spermtids, x43 stining ner these res diminished (compre Fig. 2 nd h). t the T, the intensity of x43 stining diminished in some dmged tuules y h nd dy posttretment (compre Fig. 2 d i nd c). x43 stining lso ws more diffuse nd ws less restricted to the T. When the drug ws metolized y 2 to 4 dys (2) nd more germ cells hd left the epithelium, x43 stining considerly wekened in the seminiferous epithelium (Fig. 2 j nd k). These findings further support the notion tht x43 is involved in the germ cell dhesion in the seminiferous epithelium. x43 s n Integrl omponent of the lood Testis rrier. Using dul-leled immunofluorescent nlysis, x43 ws shown to 24 兩 j: 2 D Fig. 2. The expression nd locliztion of x43 during djudin-induced restructuring of nchoring junctions in dult rt testes. dult rts were treted with djudin, nd testes were otined for immunolot nlysis (using lystes) (, ) nd immunohistochemistry (frozen sections) (). The stedy-stte protein level of x43 decresed fter djudin tretment (, Upper Lne), with ctin serving s the protein loding control (, Lower Lne). () The r grph shows the results of 3 experiments., P.5;, P.. () hnges in the locliztion of x43 in the seminiferous epithelium were studied in testes collected h (H) nd, 2, nd 4 dys (D) fter djudin tretment. The oxed res in, d, nd g re mgnified nd shown in, c, e, h, nd i. t h nd dy fter djudin tretment, immunorective x43 t the sl region remined t the T ut diminished grdully, nd its expression t the picl ES lso ws considerly reduced compred with controls ( d f, nd g i versus c). In the negtive control (l), norml rit IgG replced the primry ntiody. (Scle r, 2 m in, d, g, j, k, nd l, m in f; 5 m in, c, e, h, nd i.) co-loclize with oth ZO-, TJ dptor (Fig. 3 d), nd N-cdherin, sl ES integrl memrne protein (Fig. 3 e h), t the T of the seminiferous epithelium. The structurl interction of x43 with some selected junction proteins ws detected y co-immunoprecipittion. These proteins include N-cdherin nd -ctenin from the sl ES, the GJ protein connexin 26 (x26), desmosoml protein, nd c-src, non-receptor protein kinse tht ws shown to e component of the T (Fig. 3) (3). Even though x43 co-loclized with ZO- nd N-cdherin t the T (Fig. 3), x43 ws shown to interct structurlly with the N-cdherin/ -ctenin protein complex of the sl ES insted of with the TJ-sed occludin/ ZO- protein complex (Fig. 3). Li et l.

3 onnexin 43 Merge DPI c d I: connexin 43 ZO- IP ntiody N-cdherin Fig. 3. The co-locliztion nd interction of x43 with other known constituent proteins t the T. () Prtil co-locliztion of x43 (FIT) with either ZO- (Y3) ( d) or N-cdherin (Y3) (e h) t the T ws detected, showing tht x43 is component of the T. Nuclei were stined with DPI. (Scle r, 5 m.) () Interctions of x43 with other junction proteins, including -ctenin, c-src, ZO-, occludin, nd N-cdherin, which were reported to e present t the T, were exmined y co-immunoprecipittion using lystes of seminiferous tuules from dult rts. -tenin, c-src, N- cdherin, connexin 26, nd, ut not ZO-, occludin, nd lminin 3, interct with x43. Rit IgG nd got IgG served s negtive controls. These findings re representtive dt from 3 independent experiments. Knockdown of x43 y RNi Did Not ffect Sertoli ell dhesion t the lood Testis rrier. The significnce of x43 in Sertoli cell dhesion t the T ws ssessed y RNi in primry Sertoli cell cultures hving n estlished TJ permeility rrier tht mimics the T in vivo. knockdown of x43 y 5% in Sertoli cells using specific sirn duplexes versus non-trgeting control sirn duplexes showed tht reduction of x43 did not ffect the stedy-stte levels of vrious integrl memrne or regultory proteins t the T (Fig. 4 nd ). Dul-leled immunofluorescence nlysis reveled no oservle chnges in the distriution of TJ proteins occludin nd ZO- t the Sertoli Sertoli cell interfce (see lter sections). Using iotinyltion technique to ssess chnges in proteins t the cell surfce following RNi of x43 versus controls, we found tht knockdown of x43 did not ffect the levels of 4 integrl memrne proteins t the cell surfce (Fig. 4). These proteins were the TJ proteins occludin nd JM-, the sl ES-protein N-cdherin, nd the hemidesmosome protein -integrin. Furthermore, knockdown of x43 filed to pertur the Sertoli cell TJ permeility rrier (see lter discussion). ollectively, knockdown of x43 lone, nd thus the suppression of connexons comprising x43, hd n insignificnt effect on the T integrity. In short, x43 lone my not e required for the mintennce of the T etween Sertoli cells. Knockdown of x43 nd Perturs TJ Permeility rrier Function nd ell dhesion in Sertoli ells. esides existing y itself in the testis, the GJ is lso known integrl component of the desmosome-like junction, which hs the ultrstructurl fetures of oth the desmosome nd GJ (4). ecuse x43 intercted with the desmosoml protein (Fig. 3), we sought to exmine if concurrent knockdown of x43 nd would ffect the T function. knockdown of x43 nd, y 4% t the protein level nd y 6% t the RN level, e f g h RNi trl x43 onnexin 43, GJ 39-4 kd - onnexin 26, 26 kd - Occludin, 65 kd TJ - JM-, 36 kd - ZO-, 2 kd - N-cdherin, 3 kd sl ES - α-ctenin, 2 kd - β-ctenin, 92 kd DJ HD Protein kinse ytoskeleton iotinylted cell surfce protein - γ-ctenin, 82 kd - β-integrin, 4 kd - c-src p6, 6 kd - ctin, 42 kd RNi -ve trl x43 - Occludin, 65 kd - JM-, 36 kd - N-cdherin, 3 kd - β-integrin, 4 kd Reltive protein level (ritrry unit) Reltive protein level (ritrry unit) Occludin JM- N-cdherin β-integrin respectively, ws detected in primry Sertoli cell culture (Fig. 5 nd nd Fig. 5nd ). This knockdown of x43 nd lso cused decline in the stedy-stte level of ZO- (Fig. 5 nd ) nd mild ut significnt decline in the levels of N-cdherin nd oxsckie virus nd denovirus receptor (R) t the Sertoli cell surfce using the iotinyltion technique (Fig. 5 nd ), suggesting possile increse in endocytosis of N-cdherin nd R. surge in the protein level of the TJ protein cludin- ws oserved, ut its level t the cell surfce remined unchnged (Fig. 5 nd nd Fig. 5 nd ). The findings shown in Fig. 6 re consistent with results of Fig. 6, wherein the Sertoli cell TJ permeility rrier ws disrupted with simultneous knockdown of oth x43 nd ut not when the expression of either x43 or lone ws suppressed (Fig. 6). redistriution of ZO- nd occludin t the cell cell interfce ws lso oserved following knockdown of oth x43 nd ut not with the knockdown of x43 lone (Fig. 6 nd ). However, knockdown of lone ws sufficient to pertur the distriution of ZO-, ut not occludin, t the Sertoli Sertoli cell interfce. These findings show tht the desmosome-like junction is functionlly constituted y the GJ protein x43 nd desmosoml protein. Furthermore, this x43/ complex is linked functionlly to the occludin/zo- complex. disruption of the x43/ cn ffect the integrity of the TJ permeility rrier vi mislocliztion of occludin nd ZO-, perhps medited y enhnced endocytosis of occludin t the T. Discussion x43 lone Does Not onfer ell dhesion in the Seminiferous Epithelium. x43, GJ protein, ws shown to express stge specificity in the seminiferous epithelium of rt testes. sed on its onnexin 43 trl sirn x 43 sirn ell surfce protein level trl sirn x43 sirn Fig. 4. functionl study to ssess the role of x43 in T function y RNi in primry Sertoli cell cultures. Sertoli cells cultured t 6 cells/cm 2 on Mtrigel-coted dishes were used on dy 4 when the functionl TJ permeility rrier ws formed. ells were trnsfected with 5 nm of either non-trgeting control duplexes or specific x43 sirn duplexes, using Rio- Juice trnsfection regent. ells were wshed 24 h lter, nd cultures were terminted fter 48 h to otin cell lystes. The stedy-stte protein levels of selected mrkers were investigted (). esides knockdown of x43 y 5% (), none of the proteins exmined showed significnt chnges in their stedy-stte levels. The levels of selected integrl memrne proteins t the cell surfce were then exmined to see if the knockdown of x43 would ffect their levels (). Immunolots shown in () nd () re representtive results of 3 independent culture experiments (ech in triplicte), which re summrized in () nd ()., P. versus control sirn. DEVELOPMENTL IOLOGY Li et l. PNS June 23, 29 vol. 6 no

4 GJ TJ sl ES ytoskeleton HD Signling kinses 653 p 57 p 453 p 394 p 298 p Reltive RN level (ritrry unit) trl trl RNi 2 D 3 D x43 + Plkophilin-2 trl sirn trl RNi - 2D x43 + trl x43 + x43 + (x43 + ) sirn onnexin 43, 39-4 kd - onnexin 26, 26 kd - Occludin, 65 kd - ludin-, 22 kd - R, 46 kd - ZO-, 2 kd - N-cdherin, 3 kd - α-ctenin, 2 kd - β-ctenin, 92 kd - β-integrin, 4 kd - ERK, 44 kd - ERK2, 42 kd - perk, 44 kd - perk2, 42 kd - Vimentin, 55 kd - ctin, 42 kd - Plkophilin-2, 56 p - S6, 385 p Reltive protein level (ritrry unit) iotinylted cell surfce protein Reltive level of cell surfce protein (ritrry unit) onnexin 43 ludin- ZO- trl 2D - trl sirn 2D - (x43 + ) sirn 3D - trl sirn 3D - x43 + sirn RNi - 2D x43 + trl ludin- x43 + -ve R - ludin-, 22 kd - R, 46 kd - N-cdherin, 3 kd N-cdherin trl sirn (x43 + ) sirn Fig. 5. functionl study to ssess the role of the x43/ complex in the T y RNi in primry Sertoli cell cultures. On dy 4, when the Sertoli cell TJ rrier ws estlished, cells were trnsfected with sirn duplexes for 24 h, either non-trgeting control or specific x43 nd sirn pool, t finl concentrtion of nm using RioJuice sirn trnsfection regent. ultures were terminted 48 or 72 h therefter. Lystes were otined for immunolotting, nd RN ws extrcted for RT-PR. () knockdown of x43 y 4% on dy 2 (2 D) nd y 3% on dy 3 (3 D) fter trnsfection ws detected. n increse in TJ integrl memrne protein cludin- of % nd decline in the TJ dptor protein ZO- of % were detected 3 dys (3 D) fter trnsfection. Other selected trget proteins t the T remined unchnged fter RNi of x43 nd. ()Ondy2(2 D) fter RNi of x43 nd, there ws decline in t the RN level of 6% when ssessed y RT-PR. () ell surfce proteins were iotinylted on dy 2 (2 D) fter trnsfection with the corresponding sirn duplexes to ssess the effect of RNi of x43 nd on the stedy-stte level of integrl memrne proteins versus control sirn duplexes. Results shown here re representtive results of 3 experiments., P.5;, P.. locliztion in norml nd djudin-treted testes, it seems to e ssocited with the restructuring of junctions. t stge VIII of the epithelil cycle, fully developed spermtids leve the epithelium t spermition. The T undergoes restructuring to fcilitte the trnsit of preleptotene spermtocytes, nd considerle loss of x43 t the picl ES nd the T ws detected immunohistochemiclly. Similrly, when elongting spermtids re induced to deprt from the epithelium premturely y djudin in tuules other thn those t stge VIII, the stining of x43 is reduced drsticlly t the picl ES. ollectively, these findings suggest tht x43 is involved in conferring cell dhesion in the seminiferous epithelium. We further investigted the role of x43 in cell cell dhesion using loss-of-function pproch y RNi using primry Sertoli cells cultured in vitro with n estlished TJ permeility rrier tht mimicked the T in vivo. The knockdown of only x43 in Sertoli cells did not cuse ny chnges in the integrity of the TJ rrier or in the distriution nd levels of junction proteins, such s occludin nd ZO-, t or ner the cell surfce. These results suggest tht the loss of x43 function t the GJ nd the desmosome-like junction does not ffect the T integrity nd show tht x43 lone is not necessry for the mintennce of the T integrity. It is likely tht other connexins (5) cn supersede the trnsient loss of x43 t the T conferred y Sertoli cells. Regultion of the ell dhesion y the x43/ Protein omplex t the lood Testis rrier. ecuse x43 ws shown to interct structurlly with the desmosoml protein, s demonstrted y co-immunoprecipittion, the functionl role of this x43/ complex in the desmosome-like junction ws exmined. The simultneous knockdown of oth x43 nd y RNi, ut not the single knockdown of either x43 or, ws shown to disrupt the Sertoli cell TJ permeility function. The knockdown of oth x43 nd lso ws shown to induce redistriution of the TJ proteins occludin nd ZO- from TJ firils t the Sertoli cell surfce. s compred with controls, significnt decline in the level of iotinylted cell surfce proteins, including N-cdherin nd R, ws detected t the T following the knockdown of x43 nd. These findings show tht the x43/ protein complex is puttive functionl GJ/desmosome protein complex tht regultes Sertoli cell TJ rrier function through its effects on the occludin/zo- protein complex t the T. This oservtion lso is in greement with n emerging concept tht the desmosome cn serve s the pltform for signl trnsduction (9), so its disruption t the desmosome-like junction cn pertur the TJ rrier function t the T. Possile Mechnism y Which the x43/ omplex Induces lood Testis rrier Restructuring. The desmosome is n intermedite filment-sed nchoring junction, wheres the GJ is communiction junction (3, 7). Thus, the desmosome-like junction is expected to confer cell dhesion (3). In this study, trnsient loss of the functionl desmosome-like junction vi knockdown of x43 nd ws shown to cuse disruption of the Sertoli cell TJ rrier prtly vi redistriution of occludin nd ZO- t the cell cell interfce. The ERK MPK pthwy, which ecomes ctivted during vrious junction restructuring events (6), is not involved in this event. The non-receptor protein tyrosine kinse c-src is likely to e involved. c-src intercts with x43, nd it hs een reported to e component of the T in the rt testis (3). Studies from other 26 cgi doi.73 pns.976 Li et l.

5 c e g ZO- d f h ZO-/ sirn/dpi Trnsfection Time in culture (Dy) Occludin Occludin/ sirn/dpi Fig. 6. functionl study to ssess the role of x43 nd in T function y RNi in primry Sertoli cell cultures. () The TJ rrier function ws monitored y quntifying TER cross the cell epithelium. Sertoli cells were trnsfected for 24 h with the corresponding sirn duplexes t finl concentrtion of 8 nm., P.5;, P.. (, ) Fluorescence stining of ZO- (Y3) nd occludin (Y3) ws performed 48 h fter trnsfection to investigte the distriution of the trget proteins fter the knockdown of x43 nd/or. In the trnsfection step of the experiment, 52.5 nm of control (trl), x43, nd/or sirn ws co-trnsfected with 7.5 nm of siglo Green (FIT), cting s trnsfection indictor, t finl concentrtion of 7 nm. The green stining surrounding nuclei indictes positive trnsfection. fter knockdown of oth x43 nd, there ws less ZO- nd occludin t the Sertoli Sertoli cell interfce thn in controls ( nd nd nd vs. gnd h nd gnd h, respectively). decline in ZO- t the cell cell interfce ws oserved fter the knockdown of only, ut not of x43 (e nd f nd c nd d vs.nd ). There ws no chnge in the distriution of occludin in the single knockdown of x43 or (c fvs. nd ). Results shown re the representtive dt of 3 experiments. (Scle rs, 4 m.) epitheli hve shown tht occludins loclized t TJ firils (7) re phosphorylted t oth Ser/Thr nd Tyr residues (8, 9). minor pool of less phosphorylted occludins is not ssemled into TJ firils nd is restricted to the solterl region (8, 2). The modultion of the phosphoryltion sttus of occludins thus provides unique mechnism to ssemle nd disssemle TJ firils in epitheli rpidly in response to chnges in the cellulr environment, such s during the trnsit of preleptotene spermtocytes t the T. The c-src in the x43//c-src complex is in proximity to the TJ-sed occludin nd ZO-, s demonstrted y their co-locliztion with x43. Following trnsient knockdown of c e g d f h x43 nd, c-src my fil to mintin the phosphoryltion sttus of occludin t the TJ firils t the T, leding to decline in occludin t the Sertoli cell interfce. This chnge in distriution of occludin, such s moving from the Sertoli Sertoli cell interfce to cell cytosol, thus perturs the Sertoli cell TJ rrier. Is the Desmosome-Like Junction Involved in the Preleptotene Spermtocyte Trnsit t the lood Testis rrier? It ws postulted tht junction proteins, such s R, residing on primry spermtocytes my interct with those on Sertoli cells to fcilitte cell movement t the T while mintining the immunologicl rrier during spermtogenesis (2). There is unidirectionl movement of primry spermtocytes from the sl to the picl comprtment of the epithelium while differentiting into lte-stge spermtocytes (3). Desmosome-like junctions, s well s those formed etween Sertoli cells, lso re formed etween Sertoli nd spermtocytes/ round spermtids ut not elongting nd elongted spermtids (8). We propose herein tht primry preleptotene spermtocytes in trnsit t the T proly use the desmosome-like junction to fcilitte cell movement while mintining the immunologicl rrier. t stges VIII nd IX of the epithelil cycle, the interction of desmosome-like junction protein on the Sertoli cell with one on primry spermtocyte in trnsit, insted of with one on nother Sertoli cell, my cuse loclized reduction in the numer of functionl desmosome-like junctions etween Sertoli cells. It hs een reported tht GJ communiction from germ cells to Sertoli cells differs from tht etween Sertoli cells (5, 22). chnge of x43 x43 ssocition from 2 Sertoli cells to Sertoli cell spermtocyte my destilize the occludin/zo--sed TJ firils t the T, possily t the picl region of the spermtocyte, fcilitting cell migrtion t the T. Other studies hve shown tht the formtion of n N-cdherin-sed J is prerequisite for the ssemly of GJ (23, 24) nd desmosome (25). ecuse the knockdown of x43 nd led to loss of the sl ES protein N-cdherin t the cell surfce (Fig. 7), the disruption of sl ES might prohiit the ssemly of desmosome-like junction t the picl region of spermtocyte in trnsit. There proly is feedck loop tht promotes the ssemly of TJ, sl ES, nd/or desmosome-like junction in the sl region of spermtocyte in trnsit to ssemle new TJ firils to mintin the immunologicl rrier. This feed-ck loop might e medited y prcrine fctors produced loclly t the T, which re known to regulte T dynmics (3,, 26). Mterils nd Methods nimls. Sprgue-Dwley rts were otined from hrles River Lortory. The use of nimls ws pproved y the niml Use nd re ommittee of the Rockefeller University (protocol numer 68). Primry Sertoli ell ulture. Sertoli cells were isolted from testes of 2-dy-old Sprgue-Dwley rts nd were cultured in F2/DMEM supplemented with growth fctors nd citrcin (27). The functionl TJ permeility rrier, which mimicked the T in vivo morphologiclly nd functionlly, ws estlished on dy 3 or 4 when ssessed y trnsepithelil electricl resistnce (TER) cross the cell epithelium (27) nd y electron microscopy (28). Ultrstructures of TJ, ES, nd desmosome-like junction were oserved. In this in vitro system, virtully no picl ES ws present, ecuse the cultured Sertoli cells were contminted with negligile germ cells, including elongting nd elongted spermtids. Trnsient Trnsfection of sirn Duplexes nd TJ Permeility rrier ssessment. Sertoli cells were cultured on Mtrigel (D iosciences)-coted culture pltes t 6 cells/cm 2, Millicell icmerl units t. 6 cells/cm 2,or coverglsses t.8. 6 cells/cm 2. ells were trnsfected with sirn duplexes (Dhrmcon, Thermo Fisher Scientific), nmely the ON-TRGET plus non-trgeting sirn control pool (D-8 ), the sirn pool specificlly trgeting x43 (J-64 9, -, -) or (J , -, -2), nd siglo green trnsfection indictor (D-63 ), t finl concentrtion of 7 nm using RioJuice sirn trnsfection regent (Novgen, EMD io- DEVELOPMENTL IOLOGY Li et l. PNS June 23, 29 vol. 6 no

6 Fig. 7. schemtic drwing illustrting the regultion of T dynmics y desmosome-like junction. () simplified overview of different junctions t the Sertoli Sertoli interfce t the T in norml dult rt testes. () Following the knockdown of x43 lone, there is decline in the numer of connexons constituted y x43, possily in oth the GJ nd desmosome-like junction, ut the cell dhesion conferred y TJ nd J is unffected, perhps ecuse connexons constituted y other GJ proteins mintin the GJ communiction nd the T integrity. s such, the T integrity still remins intct. () The knockdown of oth x43 nd leds to decline in the levels of TJ proteins occludin, R, nd ZO- nd sl ES protein N-cdherin through yet-to-e defined signling pthwy(s). Nonetheless, this leds to T disruption. This mechnism, s depicted here, is likely utilized y the testis t stge VIII of the seminiferous epithelil cycle of spermtogenesis to induce T restructuring to fcilitte the trnsit of primry preleptotene spermtocytes t the T. J refers to the sl ES t the T. sciences) on dy 3 or 4 fter n intct cell epithelium ws estlished. In the group trnsfected with oth x43 nd sirn, equl mounts of ech sirn duplex pool were used so tht the finl concentrtion ws equivlent to the nontrgeting control sirn duplex. For tretment groups trnsfected with either x43 or sirn lone, n equl mount of trl sirn ws used together with x43 or sirn to otin the sme finl sirn concentrtion. ells then were incuted in the trnsfection mixture for 24 h nd replenished with fresh DMEM/ F2 therefter. In selected experiments where Sertoli cells were plted on icmerl units, the TJ rrier cross the cell epithelium ws monitored y TER s descried (27). Estimtion of the ell Surfce Protein Levels Following RNi y the iotinyltion Technique. For Sertoli cell cultures trnsfected with non-trgeting control sirn, specific sirn duplexes trgeting x43,, or x43 nd were used to ssess whether there were ny chnges in the level of junction proteins t the cell surfce following RNi. In short, proteins on the Sertoli cell surfce following trnsfection were iotinylted using sulfo-nhs-ss-iotin (Pierce/Thermo-Fisher Scientific) t 4 for 3 min, nd non-ound iotins were quenched with 5 mm mmonium chloride solution (26, 29). t this temperture, endocytosis nd endosome-medited protein degrdtion were inhiited. Thus, the level of cell surfce proteins following RNi in tretment versus control groups could e ssessed quntittively. ell lystes were prepred in Nonidet P-4 lysis uffer (26). iotinylted cell surfce proteins were isolted from cell lystes using Ultr- Link Immoilized Neutrvidin Protein Plus (Pierce/Thermo-Fisher Scientific) nd were resolved in SDS/PGE for immunolot nlysis using the corresponding specific ntiodies. Sttisticl nlyses. G-STT sttisticl nlysis softwre pckge (Version 7., Dynmic Microsystems) ws used to perform Student s t test nd -wy NOV, followed y the Tukey/Krmer procedure. Generl Methods. Informtion on the lyste preprtion, RT-PR, immunolot nlysis, immunohistochemistry, nd dul-leled immunofluorescence nlysis cn e found in the SI Text. KNOWLEDGMENTS. This study ws supported in prt y grnts from the Ntionl Institutes of Helth (NIHD, R HD5634, R3 HD552, nd U54 HD2999 Project 5) to.y.. nd Hong Kong Reserch Grnts ouncil (HKU 7693/7M) to W.M.L.. Wong H, heng Y (25) The lood testis rrier: Its iology, regultion, nd physiologicl role in spermtogenesis. urrent Topics in Developmentl iology 7: Setchell P (28) lood testis rrier, junctionl nd trnsport proteins nd spermtogenesis in Moleculr Mechnisms in Spermtogenesis, ed heng Y (Springer/Lndes ioscience, ustin, Texs), pp Mruk DD, runo S, heng Y (28) nchoring junctions s drug trgets: Role in contrceptive development. 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