BIOLOGY OF REPRODUCTION 57, 367-376 (1997)
Differential Distribution of the Tight-Junction-Associated Protein ZO-1 Isoforms a+ and a- in Guinea Pig Sertoli Cells: A Possible Association with F-Actin and G-Actin' R.-Marc Pelletier,2,3 Yuji Okawara, 3 Maria Leiza Vitale, 3 and James M. Anderson4 Department of Anatomy,3 Faculty of Medicine, Universit6 de Montreal, Montreal, Quebec, Canada H3T 1J4 Department of Internal Medicine and Cell Biology,4 Division of Digestive Diseases, Yale School of Medicine, New Haven, Connecticut 06520-8019 ABSTRACT +
To elucidate the significance of a- and a isoforms of the tight-junction-associated protein ZO-1 in Sertoli cell tight junction regulation, taking into consideration that different isoforms are expressed in cells with different junctional morphologies, we investigated whether ae- and ea+ are differentially associated with junctions forming the continuous occluding zonules responsible for the blood-testis barrier, and/or with junctions forming the focal discontinuous occluding zonules. In addition, since Sertoli cells contact Sertoli cells and germ cells, we investigated whether each isoform is differentially associated with distinct classes of germ cells. Our immunoblot analyses of isolated seminiferous tubules, using affinity-purified polyclonal antibodies recognizing rat and human a- and ea+, showed that guinea pig testis contained the two ZO-1 isoforms initially described in rat and human kidneys, and that a + and a- were predominantly expressed during puberty and adulthood, respectively, indicating that a + was predominant during periods of increased junction assembly/disassembly. We used the same antibodies and immunoperoxidase labeling on fetal, neonatal, pubertal, and adult guinea pig testes sections. Both isoforms were expressed at the site of Sertoli cell-Sertoli cell and Sertoli cellgerm cell junctions in the seminiferous epithelium, before and after birth, and both were localized in continuous and in discontinuous tight junctions. However, the distribution of at and a + was not the same in different locations of the tight junctions. Only a- was incorporated into junctions joining the Sertoli cells to all classes of germ cells. The ea+ involved junctions joining Sertoli cells to particular classes of germ cells, suggesting that Sertoli cell expression of ZO-1 isoforms could be regulated by unique germ cell-Sertoli cell contacts. Conversely, we found a correspondence between the distribution of F-actin and ZO-l a+, indicating that the spatial organization of the subsurface actin accompanying cell junctions may affect ea+/a -plasma membrane association. INTRODUCTION The Sertoli cell tight junction unit is made up of three constitutional elements: 1) fusion sites joining adjacent plasma membranes, 2) a subsurface or cortical actin filament network, and 3) accompanying cisternae of endoplasmic reticulum. In the testis, focal tight junctions develop between Sertoli cells during fetal life [ 1, 2]. Around puberty Accepted April 4, 1997. Received February 6, 1997. 'Supported by grants CAFIR and MT-11160 from Medical Research Council of Canada to R.M.P. and by grant MT-12879 from Medical Research Council of Canada to M.L.V. M.L.V. is also funded by a scholarship from Fonds de la Recherche en sant6 du Qu6bec. 2Correspondence: R.-Marc Pelletier, R-808, Department of Anatomy, Faculty de Medecine, Pavilion Principal, Universit6 de Montreal, 2900 Edouard Montpetit blvd., Montr6al, P. Quebec, Canada H3T 1J4. FAX: (514) 343-2459; e-mail:
[email protected]
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the Sertoli cell-Sertoli cell focal junctions organize themselves into an impermeable occluding zonule around each supporting cell and thus create a selective barrier in the seminiferous tubules [3-5]. The barrier is not an "impervious seal," but it helps to build a gradient from underlying interstitial fluids to the lumenal fluids [6]. In the adult, this blood-testis barrier divides the seminiferous epithelium into basal and lumenal cellular compartments. Meiosis and completion of spermatogenesis occur in the lumenal compartment. During their development, germ cells in the basal compartment must translocate beyond the junctional barrier into the lumenal compartment without significant leakage of blood-borne substances. Additional focal Sertoli cellgerm cell junctions are assembled and disassembled in harmony with the germ cell migration toward the apex of the epithelium [7]. The junction modulation occurs in a basoapical direction and follows germ cell migration [4, 8, 9]. The factors ensuring that Sertoli cell junction assembly occurs in harmony with the passage of germ cells from one cellular compartment of the epithelium to the other are still the subject of intense research. Identification of protein components of the tight junctions, such as the membrane-associated guanylate kinase homolog (MAGUK) family of proteins, contributed significantly to the knowledge of the intracellular mechanisms involved in the regulation of tight junction assembly/disassembly. The tight-junction-associated protein zonula occludens 1 (ZO-1) is a 220-kDa protein component of epithelial and endothelial cell tight junctions [10, 11]. Both ZO-1 and a second tight-junction-associated protein of 160 kDa, zonula occludens 2 (ZO-2), which coimmunoprecipitates with ZO-1 [12], are members of the MAGUK family of proteins, which are implicated in signal transduction at the sites of cell-to-cell contact and in the regulation of interaction between the plasma membrane and the cortical cytoskeleton [13, 14]. ZO-1 has been reported to colocalize with cadherins [15, 16] whereas ZO-2 appeared to be restricted to tight junctions [17]. Immunolocalization of ZO-1 has been achieved in 5-day-old neonatal and adult mouse testis along the Sertoli cell-Sertoli cell and Sertoli cell-elongated spermatid cellular junctions [18]. Immunogold labeling of ZO-1 has allowed specific localization of the protein to the cytoplasmic surface of tight junctions in sheets of rat Sertoli cell plasma membranes [18]. Recently, two distinct isoforms of ZO-1, oa and a+, have been described and shown to result from an alternative splicing of an mRNA encoded by a single gene in the human and the rat [14, 19]. The primary structure of human and rat ZO-lt+ cDNA initially described [14, 19] contains a 240-base sequence encoding 80 amino acids called motif a, which is not present in ZO-la-. Different isoforms of ZO-1 have been associated with different morphologies.
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ZO-la + has been reported in tight junctions of the epithelium of the rat kidney tubule; however, only ZO-la- was reported in the endothelium and slit diaphragms of glomerular epithelial cells [20, 21]. Furthermore, modifications of the ZO-1 protein relate to different periods in the life of the junction. ZO-1 was not heavily phosphorylated in normal adult rat kidney but underwent tyrosine phosphorylation during tight junction formation and remodeling [22]. The use of affinity-purified polyclonal antibodies against ot and et+ should provide a more precise identification of the protein components of Sertoli cell tight junctions than that documented to date, thus offering a better chance to understand the factors regulating their dynamics in the blood-testis barrier. Therefore, we investigated whether ZO-1 isoforms a and o+ were differentially associated with the tight junctions forming continuous occluding zonules near the base of the Sertoli cells and with the focal tight junctions forming discontinuous occluding zonules joining Sertoli cells to germ cells in the middle and near the apex of the seminiferous epithelium. In addition, because the actin filaments formed hexagonally packed bundles of filaments parallel to each other and to the cell surface next to the tight junctions in the base and in the apex but not in the middle of the epithelium, we examined whether a- and ot+ were differentially associated with a distinct degree of organization of the subsurface actin. We used affinity-purified polyclonal antibodies against at and ca+ and immunoperoxidase labeling on fetal, neonatal, pubertal, and adult guinea pig testis sections and immunoblot analyses of isolated seminiferous tubules. In addition, we recorded the localization of the filamentous (F-) and of the nonfilamentous (G-) actin in adult testes. Our data showed that ot+ localized into junctions joining the Sertoli cells to particular classes of germ cells but that the distribution of a- and ct+ was not the same in different locations of the tight junctions. However, we found a positive correlation between the distribution of F-actin and ZO-lot+ and between G-actin and ZO-l-. MATERIALS AND METHODS Animals We used the testes from fetal, 1- and 7-day-old neonatal; 11-, 14-, 21-, 28-, 35-, and 42- to 45-day-old pubertal; and 800-g adult guinea pigs. All animals were from the Hartley strain. The fetuses were obtained from gestating females (Charles River, Quebec, PQ, Canada) aborted surgically and under anesthesia at 55-62 days postcoitum. The neonates and pubertal animals were delivered naturally in our animal facilities. These precautions were taken to ensure that the recordings of the distribution and expression of a given isoform of ZO-1 were made with reference to a precise time period during testicular development. In the adult, recordings were made with reference to the twelve stages of the cycle of the seminiferous epithelium, using the identification method of Clermont [23]. Fetuses and neonates were anesthetized with a halothane oxygen mixture. Pubertal animals and adults were anesthetized by intraperitoneal injection of 0.9 ml/kg BW of sodium phenobarbital (Somnotol; MTC Pharmaceutical, Mississauga, ON, Canada) and 0.15 ml/kg BW of a solution of 0.3 g/ml chloral hydrate in sterile saline. Five animals were used per age group. Antibodies against ZO-1 We used three distinct antibodies. 1) The affinity-purified polyclonal antibody #585, which recognizes only the
ZO-la isoform, was raised in a guinea pig against a human peptide antigen corresponding to ten residues at the splice junction and has already been characterized [24]. 2) The affinity-purified rabbit polyclonal antibody #8040, which recognizes exclusively the ZO-lot+ isoform, was produced against the at domain of a recombinant human fusion protein as previously described [19]. 3) The affinity-purified polyclonal antibody #7446 was raised in rabbit against human ZO-1 and recognizes both isoforms of ZO-1, - and c . It has been previously characterized [19]. Electrophoresis and Immunoblotting The presence of ZO-1 isoforms in adult guinea pig kidney and interstitial cells and in seminiferous tubules of 14-day-old and adult guinea pigs was evaluated by electrophoresis followed by immunoblotting. Testes obtained from decapitated guinea pigs were decapsulated in cold germcell Minimum Essential Medium (GC-MEM; Gibco BRL, Oakville, ON, Canada). Each decapsulated testis was transferred to a small beaker containing 50 ml of GC-MEM, 25 mg of collagenase Type I (Worthington Biochemical Corporation, Freehold, NJ) [25, 26]. The incubation was carried out at 37C for 20 min with agitation at 100 cycles/ min [26]. The seminiferous tubules were allowed to settle by gravity while the interstitial cells remained in the supernatant. The tubules were washed first in GC-MEM, then twice in PBS. The kidneys were decapsulated. Tissues were homogenized in PBS containing 1 M phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). Samples (100-Rg of proteins, measured by the method of Bradford [27]), from each homogenate were subjected to electrophoresis on 10% acrylamide gels. The gels were run for 20 h at 60 V. Proteins were electrotransferred onto nitrocellulose membranes [28]. Membranes were blocked with 5% nonfat dry milk in PBS for 60 min and then incubated for 60 min at 37°C either with a 1:125 dilution of anti-ZO-l1 (Ab# 7446) or with specific ZO-1 isoform antibodies: 1:40 dilution of antiZO-1a- (Ab #585) or 1:80 dilution of anti-ZO-la + (Ab #8040). Membranes were thoroughly rinsed and incubated with an alkaline phosphatase-conjugated secondary antibody-an anti-rabbit IgG in the case of Ab#7446 and Ab #8040, and an anti-guinea pig IgG in the case of Ab #585. Color was developed by treatment with a mixture of pnitroblueterazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt. Immunolocalization of ZO- 1 Isoforms Testes were perfused-fixed through the testicular artery [29], first with PBS and then with Bouin's fixative, and were immersed for 36 h in the same fixative at room temperature (RT). Tissues were dehydrated in a graded series of ethanols (70%, 95%, and 100%) and in xylenes, and were paraffinized. Sections (5 tIm thick) were mounted on glass slides coated with 3-aminopropyltriethoxysilane (Sigma). Sections were deparaffinized and rehydrated in xylene and ethanols. Tissue sections were exposed to 0.6% hydrogen peroxide (H20 2 ) in 70% ethanol for 5 min to inhibit potential endogenous peroxidase activity. Sections were immersed for 5 min in TBSTw (Tris-buffered saline [TBS: 140 mM NaCl, 50 mM Tris HCI, pH 7.4] containing 0.1% Tween-20). Immunolabeling. Residual picric acid was inactivated with 1% lithium carbonate in 70% ethanol, and free aldehydes were blocked by immersing tissue sections in a 300 mM glycine aqueous solution (pH 7.4) for 5 min after re-
THE DISTRIBUTION OF ZO-1 ISOFORMS a + AND a- AND SERTOLI CELL ACTIN hydration [30]. The unspecific binding was blocked by incubating the sections for 30 min at 37°C with 1-2% nonfat dry milk or 6% BSA + 6% normal guinea pig serum in TBSTw. After blocking, sections were incubated with the primary antibody for 90 min at 37°C or overnight at RT. Then sections were incubated for 40 min at RT, first with (1:1000) biotinylated anti-rabbit (Amersham Bio/Can Scientific, Mississauga, ON, Canada ) or anti-guinea pig (Chemicon, Burlington, ON, Canada) IgG, and next with (1:200) horseradish-peroxidase (HRP)-conjugated streptavidin (Amersham). Sections were washed in TBSTw after each incubation and, finally, were exposed to TBS (pH 7.7) containing 0.01% H2 0 2 , 0.05% diaminobenzidene tetrachloride, and 10 mM imidazole [31] for 10 min at RT. The primary and the secondary antibody and the HRP-conjugated streptavidin were diluted in 1-2% nonfat dry milk or 2% BSA + 2% normal guinea pig serum in TBSTw. The sections were washed in water and counterstained in a 0.05% aqueous methylene blue dye, rinsed in water, dehydrated, and mounted in Permount (Fisher Scientific, Pittsburgh, PA). Controls. Specificity of ZO-1 immunolocalization was tested in fetus and adult guinea pig kidneys as a positive control. Additional controls included use of the preimmune serum or the primary or second antibody alone. The best results were obtained with the following dilutions for each antibody tested: 1:80 for Ab #585 and 1:140 for Ab #8040. Localization of F-Actin Cryostat sections (5 $xm thick) of unfixed adult guinea pig testes were air-dried, immersed in PBS, fixed for 10 min in a 4% formaldehyde solution, and rinsed twice in PBS. Localization of F-actin was carried out as before except that biotin-XX-phalloidin (Molecular Probes, Eugene, OR) replaced the primary antibody. The sections were incubated for 45 min with biotin-XX-phalloidin (1:9) in PBS containing 0.1% Tween (PBSTw) at RT. Washing of sections between incubations was done with PBSTw. Localization of G-Actin Cryostat sections of unfixed adult guinea pig testes were labeled with Gc-globulin according to the method of Cao et al. [32] with the following modifications. Sections were fixed with formaldehyde and treated as described for F-actin localization. Subsequently, they were incubated at RT, first in 10 ,ug/ml of human Gc-globulin (Calbiochem, La Jolla, CA) for 60 min, second in 1:500 rabbit anti-human Gc-globulin (Daco Corp., Carpenteria, CA) for 40 min, third in 1:1000 biotinylated goat anti-rabbit IgG for 40 min, fourth in 1:2000 HRP-conjugated streptavidin (Molecular Probes) for 40 min, and fifth in diaminobenzidine tetrachloride and H2 02 as described above for visualization of the reaction product. Controls included use of the second or third antibody alone. A total of 100-120 light micrographs were taken for each experimental group under study and for each labeling at a final magnification of x780. RESULTS Immunoblot Analysis Immunoblots performed with antibody #7446, which recognizes rat and human ZO-la+ and ZO-la-, revealed the presence of only two ZO-1 isoforms in guinea pig kidney, seminiferous tubules, and interstitial cells (Fig. la).
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FIG. 1. Presence of ZO-1 isoforms a and a+ in kidney, seminiferous tubules, and interstitial cells as shown by immunoblot analysis. a) Homogenates from adult guinea pig kidney (k), seminiferous tubules (st) and interstitial cells (ic) were subjected to SDS-PAGE followed by immunoblotting using Ab #7446, which recognizes both ZO-1 isoforms. Two bands corresponding to a- and a ZO-1 isoforms were detected in the samples (arrows). b) Homogenates of seminiferous tubules from 14-dayold (y) and adult (a)guinea pigs testes were subjected to SDS-PAGE followed by immunoblot analysis with specific ZO-1 isoform antibodies: antibody #585, which recognizes ZO-lc-, and antibody #8040, which recognizes ZO-la +. The position of each ZO-1 isoform is indicated by an arrow.
Calculation of the molecular size of guinea pig ZO-1 isoforms indicated a mass of 225 + 3 kDa (n = 6) for the higher molecular-mass band and a mass of 216 + 3.5 kDa (n = 6) for the lower molecular-mass band. These results show that 1) the difference in molecular size between the two bands is close to the molecular mass of the a motif; and 2) the molecular sizes of guinea pig ZO-1 isoforms closely resemble the molecular masses of rat ZO-1 isoforms a+ and a- [24]. The experimental evidence thus suggests that guinea pig testis and kidney express two ZO- 1 isoforms and that these two isoforms are a+ and or-. Additional studies using antibodies specifically directed against each ZO-1 isoform indicated that a + and a- isoforms are differentially expressed during guinea pig testicular development. Antibody #585, directed against human ZO-la- isoform, recognized only one band in guinea pig seminiferous tubule homogenates (Fig. 1). Immunoblots performed with this antibody showed that ZO-la- was predominant in seminiferous tubules of the adult (Fig. lb, a +, lane a). Antibody #8040, raised against motif ac and therefore recognizing ZO-l +, showed the presence of only one band in guinea pig seminiferous tubule homogenates; the band had a molecular mass that corresponded to ZO-loa+. Immunoblots performed with antibody #8040 showed that a + isoform was predominant in seminiferous tubules from 14-day-old guinea pigs (Fig. lb, a +, lane y). Immunohistochemistry Controls using preimmune serum or the primary or secondary antibody alone on adult testicular tissue (Fig. 2a)
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FIG. 2. Controls. Negative controls in adult testis (a) and fetal (b) and adult kidney (c) showed no labeling when the second antibody was used alone. in both fetal and Positive controls for the specificity of the localization of ZO-1 - isoforms in fetal (d) and adult kidney (e) revealed the presence of adult renal glomeruli (arrows). f, g) Positive controls for the specificity of the localization of ZO-la' isoforms (arrows) in fetal and adult kidney. In the fetus, a+ was present in the glomerulus, whereas in the adult, the visceral and parietal layers of the Bowman's capsule were labeled. 780.
and on fetal (Fig. 2b) and adult (Fig. 2c) kidney tissue sections were negative. Positive controls included immunolocalization of the ZO-lat isoform in fetal (Fig. 2d) and adult (Fig. 2e) renal glomeruli. Additional controls included detection of the ZO-la+ isoform over the capillaries of the glomerulus in fetal kidney (Fig. 2f) and along the visceral and parietal layers of the Bowman's capsule in the adult (Fig. 2g). ZO- la- Ab# 585 The ZO-lat isoform was detected along the lining of the blood vessels of the interstitial space and within the tubules of the testis of all the age groups studied (Fig. 3). In the fetus, a was localized along the Sertoli cell plasma membrane (Fig. 3a). By Day 7 after birth, Sertoli cell nuclei had migrated from the center of the tubule to the limiting membrane (Fig. 3b). By Days 11 and 14, labeling occurred all along the Sertoli cell plasma membrane, including membrane segments that faced adjacent Sertoli cells and those that faced spermatogonia and leptotene spermatocytes (Fig. 3, c and d). A lumen had begun to develop in 21-day-old guinea pigs. Labeling occurred in the Sertoli cell plasma membrane segments facing adjoining Sertoli cells and in those surrounding all germ cells, including pachytene spermatocytes in the 28-day-old animals (Fig. 3e) and round spermatids in the 35-day-old animals (Fig. 3f). In the 42-day-old guinea pigs, step 15 spermatids were the most advanced germ cells found. In the adult, the apical and lateral Sertoli cell plasma membranes contacting adjoining Sertoli cells and germ cells of all classes were labeled; however, labeling did not seem to be stage-related, as it appeared to be similar from one tubule to the next (Fig. 3, g and h).
ZO-la + Ab# 8040 The ZO-la + isoform was present in fetal, pubertal, and adult testes. It was absent from Leydig cells but appeared in both the blood vessels of the interstitial space and the seminiferous tubules (Fig. 4, a-k). Within the seminiferous epithelium of the fetus and 1- to 3-day-old guinea pigs, labeling appeared as a series of small dots aligned along adjoining lateral Sertoli cell plasma membranes; minute Sertoli cell plasma membrane segments facing gonocytes were also sporadically labeled (Fig. 4, a and b). This distribution of the isoform coincided with that of the focal minute tight junctions we reported to be developing on the Sertoli cell plasma membranes in this species during the same period [3]. By Day 14 after birth, the entire lateral Sertoli cell plasma membrane from the base of the epithelium to the center of the tubule was labeled (Fig. 4c). This distribution of at+ persisted after the establishment of a lumen by 21 days after birth (Fig. 4d). In the 28- and the 35-day old guinea pig tubules, labeling occurred along the Sertoli cell plasma membrane segments facing spermato-
FIG. 3. Immunolocalization of ZO-lo . The (x (arrows) was localized lining the blood vessel (bv; see panel g) of the interstitial space and along the Sertoli cell (S) plasma membrane regardless of whether the membrane segment faced gonocytes (G) in fetal testis (a), or spermatogonia, leptotene (L) or pachytene spermatocytes (P), or even round spermatids in pubertal testes (b, 7-day-old; c, 11-day-old; d, 14-day-old; e, 28-day-old; and f, 35-day-old). In adult testis (g, h), labeling is identified by arrows at the site of the cellular junctions in the blood-testis barrier situated immediately above the spermatogonia (g) (see lowest arrow in h) and also along Sertoli cell plasma membrane segments facing other Sertoli cell segments or pachytene spermatocytes (P) and round and elongated spermatids (s). Leydig cells (I) were not labeled (g). The Roman numerals at the top of g and h indicate the stage of the cycle. x780.
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a+ was localized in minute dots along the lateral Sertoli cell (S)membrane segments whether these faced FIG. 4. Immunolocalization of ZO-1 a +. The o another Sertoli cell (arrows) or a gonocyte (G; arrowheads) in fetal (a)and neonatal (b; 1-day-old) testes. By 14 days after birth (c), (I (arrows in c-k) was observed along the lateral Sertoli cell plasma membrane from the base of the epithelium to the center of the tubule. A lumen was present in the tubule by 21 days after birth (d). In the 28- (e)and 35-day-old testes (f), the isoform was present along Sertoli cell plasma membrane segments facing leptotene spermatocytes, but not along segments facing pachytene spermatocytes (P)and round spermatids (s). In the adult (g-k), Roman numerals indicate the stage of the cycle of the seminiferous epithelium. During stages I-VIII (g,h), a was localized at the site of the tight junctions that constitute the blood-testis barrier+ immediately above spermatogonia (g)and preleptotene (pl) and leptotene (L)spermatocytes in the basal third of the epithelium. During stage IX (i), a was present along Sertoli cell plasma membrane segments facing leptotene spermatocytes (L) that were migrating toward the lumenal compartment. Upon completion of the translocation of early zygotene spermatocytes (Z)from the basal to the lumenal cellular compartment of the epithelium during stage X (j), a+ was localized at the site +of the tight junctions that had re-formed basally to the zygotene spermatocytes (Z), next to the limiting membrane of the tubule. This distribution of a in the tight junctions next to the tubular limiting membrane and immediately above the spermatogonia was maintained during the remaining stages of the cycle of the epithelium (i.e., stage XI-VI Ig]). Asterisks indicate the presence of reaction product in the blood vessels but not in the Leydig cells (I; e and f). Additionally, a was localized along the plasma membrane of Sertoli cell apical cytoplasmic processes surrounding step 8-15 spermatids (s)(h-k). The reaction product was evident along the Sertoli cell plasma membrane segment that faced the spermatid acrosome (s)(h, i) particularly the tip (k). Furthermore, in k, the reaction product (arrows) was labeled along a Sertoli cell plasma membrane segment facing type A and type B spermatogonia (see A and B in f). x780.
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FIG. 5. Localization of F-actin (a)and G-actin (b) in 5-1.m-thick frozen sections. a) In this cross section of a stage VII seminiferous tubule, F-actin was localized (open arrow) within the Sertoli cell cytoplasmic processes accompanying the tight junctions that form the blood-testis barrier above the spermatogonia (G)and preleptotene spermatocytes (pl), and in the thin processes facing the elongated mature spermatids (closed arrow). No reaction product was found in the Sertoli cell processes surrounding the pachytene spermatocytes (P)and round spermatids (s). This micrograph was taken with a Nomarsky microscope. b) Cross section of a stage IV seminiferous tubule. G-actin was localized (arrowheads) in the Sertoli cell cytoplasmic processes that surround each class of germ cells including spermatogonia, pachytene spermatocytes (P), and round (s) and elongated (S) spermatids. The limiting membrane was also positive x680.
gonia, preleptotene, and leptotene spermatocytes (Fig. 4,e and f). However, labeling occurred neither in Sertoli cell membrane segments that faced zygotene, pachytene (Fig. 4, e and f), diplotene, or secondary spermatocytes, or round spermatids, nor in the adjoining Sertoli cell plasma membrane segments between these germ cells (Fig. 4, e and f). In the adult, o + was present during the twelve stages of spermatogenesis, and changes in the distribution of this isoform coincided with the modulation of tight junctions in the Sertoli cell junctional blood barrier during the stage of the cycle of the seminiferous epithelium (Fig. 4, g-k). In the basal third of the epithelium, during stages I-VIII, labeling was present on the Sertoli cell plasma membrane segments surrounding spermatogonia and preleptotene and leptotene spermatocytes, and in short Sertoli cell membrane segments involved in the continuous occluding zonules immediately above these classes of germ cells (Fig. 4, g and h). No labeling was present in the Sertoli cell plasma membrane segments lumenal to the aforementioned classes of germ cells or surrounding older classes of spermatocytes and round spermatids (Fig. 4, g and h). During stage IX, ot+ was weakly labeled on the Sertoli cell plasma membrane segments surrounding the leptotene spermatocytes that had migrated at some distance from the tubular limiting mem-
brane and were in transit from the basal to the lumenal cellular compartment (Fig. 4i). During stage X, labeling was present along Sertoli cell tight junctions that had reformed between adjacent Sertoli cell plasma membranes, basal to the zygotene spermatocytes, and next to the limiting membrane after their translocation into the lumenal compartment (Fig. 4j). In the apical third of the epithelium, the Sertoli cell cytoplasmic processes surrounding the acrosome of the elongating step 8-15 spermatids were positive (Fig. 4, h-k). The reaction product was particularly evident on Sertoli cell plasma membrane segments that faced the tip of the spermatids' acrosome (Fig. 4k). Labeling was also apparent in residual bodies (not shown). F-Actin In the adult, F-actin was present during the twelve stages of spermatogenesis. Its distribution was stage-related similar to the distribution described for o +. That is, during stages I-VIII, F-actin was localized in the basal third of the epithelium-within the Sertoli cell cytoplasmic processes accompanying the tight junctions situated above the classes of younger germ cells, and near the apex-in the thin processes facing the elongating step 8-15 spermatids (Fig. 5a).
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No reaction product was found in the Sertoli cell processes surrounding pachytene spermatocytes and round spermatids that occupied the middle of the epithelium (Fig. 5a). F-actin was localized in the Sertoli cell plasma membrane segments facing leptotene spermatocytes during stage IX, and basal to the zygotene spermatocytes and next to the limiting membrane during stage X (not shown). G-Actin In the adult, the distribution of G-actin (Fig. 5b) was similar to the distribution we reported for o-. G-actin was localized in the same sites as F-actin, but in addition, the monomeric actin was found in the Sertoli cell cytoplasmic processes that surrounded each class of germ cell, including spermatogonia, pachytene spermatocytes, and round and elongated spermatids (Fig. 5b). DISCUSSION Our immunoblot analyses indicate that guinea pig kidney and testis express two isoforms of the tight-junction-associated protein ZO-1. Guinea pig ZO-1 isoforms are recognized by antibodies directed against rat and human ZO-lxt+ and ZO-la- [24]. The molecular masses of guinea pig ZO-1 isoforms are similar to those of rat and human ZO-la + and ZO-lox . Furthermore, the difference in molecular mass between the two guinea pig ZO-1 isoforms closely corresponds to the molecular weight of the 80 amino acids of the motif a+. All this suggests that ZO-1 isoforms observed in guinea pig kidney and testis are indeed the ot+ and oa isoforms reported earlier [24]. To elucidate the significance of the two ZO-1 isoforms in Sertoli cell tight junction regulation, taking into consideration that different ZO-1 isoforms are expressed in cells with different junctional morphologies [20], we investigated whether ZO-1 isoforms o- and xt+ were differentially associated with the tight junctions forming the continuous occluding zonules responsible for the blood-testis barrier, and with the focal tight junctions forming the discontinuous occluding zonules. In addition, since any given Sertoli cell is in contact with adjacent Sertoli cells and germ cells, and since focal tight junctions have been reported to sometimes join adjacent Sertoli cells and at other times join Sertoli cells to germ cells [8, 9, 18], we also investigated whether each isoform was differentially localized in focal tight junctions involving distinct classes of germ cells. The difference in structural plasticity reported in adult rat kidney and testis between ot+- and a--junctions-i.e., ZO-la- expressed in structurally dynamic junctions and ZO-lot expressed in less dynamic junctions-has been said to suggest a differential linkage to the cell cytoskeleton [24]. Therefore, we examined whether cx and a + were differentially associated with a distinct degree of organization of the subsurface actin, i.e., filamentous vs. nonfilamentous. Only one other published report had documented the distribution of ZO-1 in the testis [33]. This work used rat antiZO-1 hybridoma supernatant (R26: 3Ig), which did not allow distinct labeling of each ZO-1 isoform. The report concludes that ZO-1 is present in testes from 5-day-old mice in the absence of recognizable tight junctions [33]. This argument is inconsistent with previous observations that tight junction formation has been reported between Sertoli cells in the mouse embryo [1], and in fetal rats [2] and guinea pigs [4]. The formation of focal tight junctions between Sertoli cells and their development into a competent blood-testis barrier are not synchronous events. Develop-
ment of focal tight junctions occurs during fetal life, but it is around puberty that the junctions organize themselves around the base of each Sertoli cell into a continuous belt, called a continuous occluding zonule, which creates a selective barrier in the seminiferous tubule [3, 4, 5, 9]. The present study is the first to localize the two distinct ZO-1 isoforms ox and ot+ morphologically and biochemically in the fetal, pubertal, and adult guinea pig testis. It is also the first to document the presence of the two isoforms in fetal kidney. We found that the distribution of ZO-lot1 changed during the development and maturation of the kidney: during fetal life, ot+ was present in the glomerulus; during adulthood, it was localized over the Bowman's capsule and tubular epithelium. This change in the distribution of ot+ may reflect the appearance, between the foot processes of glomerular cells, of slit diaphragms [20, 21, 34] that are responsible for a change in the functional state of the kidney and for establishment of glomerular filtration shortly after birth. Our finding that a' and isoforms were present in fetal testis agrees with the report that both isoforms are expressed in mouse embryos [35], although they are expressed in a temporal sequence during cleavage: a- mRNA is transcribed around the 4-cell stage, while xt mRNA is transcribed later, at the 16-cell stage [35]. We found that ZO-1 isoforms a- and ot+ were both expressed in the testis during late embryonic life. There could nevertheless be a temporal sequence in the appearance of each isoform, either during sexual differentiation, resulting in testicular cord formation, or during adulthood, when one isoform may be required for adequate expression of the other. Our immunoblot analyses showed that a + was predominantly expressed during puberty, a period when the initiation of spermatogenesis results in the production of many young germ cells, and when the development of a lumen in the seminiferous tubule is accompanied by an increase in Sertoli cell tight junction modulation, i.e., assembly/disassembly [4, 9, 18]. We found that ot was predominant in seminiferous tubules of normal adult guinea pigs, and this is in agreement with the observation that ZO-l predominates in the adult rat testis [24]. Interestingly, in the rat kidney, while both isoforms are expressed in typical epithelial tight junctions, only ot is found in slit diaphragms, in which the intercellular spaces are normally open, and in endothelial junctions, which are readily opened by physiologic signals, suggesting that ot is typical of labile junctions [20]. Our finding that ot was predominant in normal adult guinea pig tubules and t+ was predominant in pubertal tubules may indicate, under normal conditions and in the adult, two conclusions. First, the Sertoli cell tight junctions may be sealed to permeability tracers [3-6] and still exhibit significant amounts of flux through a size- and ion-selective pericellular pathway, thus normally being partially open. The difference in concentrations of a variety of ions and other small molecules between the tubular lumen and the systemic circulation reveals that the seminiferous epithelium has selective transport and permeability properties [36, 37]. Second, the rearrangement or assembly of tight junctions after migration and translocation of germ cells into the lumenal compartment occupies only a short period of the complete spermatogenic cycle, i.e., two (IX and X) out of the twelve stages [3, 4, 9], and consequently, significant expression of ct might be required for only brief durations during the cycle. In addition, that ot+ is associated with Sertoli cell tight junction modulation was suggested by the finding that this isoform was
THE DISTRIBUTION OF ZO-1 ISOFORMS a + AND a- AND SERTOLI CELL ACTIN selectively increased after experimentally induced assembly/disassembly of tight junctions (unpublished results). Our data showed that a- and a+ were incorporated into both discontinuous and continuous junctions, suggesting that the type of tight junction has little impact on the a+/ c--plasma membrane association. However, only a- was incorporated into junctions joining the Sertoli cells to all classes of germ cells. The a'+ involved junctions joining Sertoli cells to particular classes of germ cells, suggesting that Sertoli cell expression and localization of ZO-1 isoforms could be regulated by unique germ cell-Sertoli cell contacts. Interestingly, in the Madin-Darby Canine Kidneystrain 2-cell line, the endogenous occludin, a transmembrane component of tight junctions, forms a continuous junctional ring [38], which we believe could be related to ZO-lo-. Nevertheless, the localization of a+' in some tight junctional membrane segments and not in others, and even in junctional membrane segments other than tight, strongly suggests that something else in the Sertoli cell tight junction unit besides the fusion site itself affects the ea+-plasma membrane association, since the distribution of neither of the two ZO-1 isoforms was perfectly concurrent with that of the tight junctions. We propose that the spatial organization or the state (filamentous vs. nonfilamentous) of the subsurface actin ac-companying Sertoli cell junctions could regulate the +/ca plasma membrane association. Actin filaments detected by monoclonal antibodies along most of the Sertoli cell subsurface [39-41] were documented to display a characteristic organization similar to that found in the microvilli of the intestine in only two regions of the supporting cell. They form hexagonally packed bundles of filaments parallel to each other and to the cell surface next to the basal Sertoli cell-Sertoli cell tight junctions and next to the apical Sertoli cell-spermatid junctions [3, 4, 8, 40, 41]. The present study substantiates these findings and, in addition, reports that F-actin and G-actin distributions were concurrent with those of ZO-1 isoforms a'+ and a-, respectively. We observed the existence of two actin pools along the Sertoli cell plasma membrane: a pool of F-actin concentrated next to the continuous and the discontinuous tight junctions in, respectively, the base and apex of the cell, and a pool of G-actin distributed in the base, the apex, and the middle third of the cell. ZO-1 is believed to regulate interaction between the plasma membrane and the cortical cytoskeleton at the site of tight junctions. The use of cytochalasin D [42] and/or of ZO-toxin of cholera, two actin depolymerizing agents, induces a disruption of perijunctional actin filaments followed by a breakage of the pericellular barrier, suggesting a functional link between actin organization and the tight junction [43]. ZO-1 tyrosine phosphorylation has been reported to occur at the same time as accumulation of ZO-1 in the perijunctional area in A431 cells [44]. The pretreatment with cytochalasin D blocks both the epidermal growth factor-induced ZO-1 rearrangement and tyrosine phosphorylation [44]. Interestingly, the only discontinuous junctions of the seminiferous epithelium that were reported intact after cytochalasin D treatment were situated in the middle of the seminiferous epithelium [45, 46] and correlate with regions of the Sertoli cell in which we localized nonfilamentous actin. Additionally, Sertoli cells are accompanied by subsurface actin and intermediate filaments on both sides of the cellular contact, whereas in all Sertoli cellgerm cell contacts, except Sertoli cell-elongated spermatid contacts, intermediate filaments are only in the cytoplasm of the Sertoli cell [18]. The presence of intermediate fila-
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ments in preferential cortical regions of the Sertoli cell may also influence the actin organization along the cell subsurface. In conclusion, our data showed that guinea pig testis contained ZO-1 isoforms a'+ and a- initially described in rat and human kidney, and these were expressed before and after birth at the site of Sertoli cell-Sertoli cell and Sertoli cell-germ cell junctions. ZO-lu+ and ZO-lo- were predominantly expressed during puberty and adulthood, respectively, indicating that a+ was predominant during periods of increased junction assembly/disassembly. Both isoforms were localized in continuous and in discontinuous tight junctions. However, the distribution of a- and a' was not the same in different locations of the tight junctions in the seminiferous epithelium. The findings indicate that the type of tight junction bears little relation to the a'+/a--plasma membrane association. However, a'+ was localized to junctions that join the Sertoli cells to particular classes of germ cells, suggesting that Sertoli cell expression and localization of ZO-1 isoforms could be regulated by unique germ cell-Sertoli cell contacts. Conversely, we found a correspondence between the distribution of F-actin and ZO-loa+ and between the distribution of G-actin and ZO-1a-, indicating that the spatial organization of the subsurface actin accompanying cell junctions may affect a+/ a--plasma membrane association. ACKNOWLEDGMENTS The authors are thankful to Drs. R. Day and M. Marcinkiewics for the use of microscopes, to Dr. M. Marcinkiewics for useful advice during the course of this study, to Dr. L. Dong for technical assistance, and to J. Lveilld for photographic assistance.
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