THE ANATOMICAL RECORD 230:199-208 (1991)
Effect of Tunicamycin, an Inhibitor of Protein Glycosylation, on Testicular Cord Organization in Fetal Mouse Gonadal Explants In Vitro YOSHIAKIRA KANAI, YOSHIHIRO HAYASHI, HAYATO KAWAKAMI, KUNIAKI TAKATA, MASAMICHI KUROHMARU, HIROSHI HIRANO, AND TAKA0 NISHIDA Department of Veterinary Anatomy, Faculty of Agriculture, University of Tokyo, Bunkyo-ku (Y.K., Y . H . ,M.K., T.N.) and Department of Anatomy, Kyorin University School of Medicine, Mitaka (H.K.,K.T., H.H.), Tokyo, Japan.
ABSTRACT The effect of tunicamycin (TM) on testicular cord organization in the fetal mouse was examined in vitro at light and electron microscopic levels, with special reference to the glycoprotein functions during Sertoli cell differentiation. In testicular explants treated with TM, testicular cord organization was inhibited. TM treatment affected basal lamina formation by Sertoli cells, resulting in a discontinuous basal lamina or none at all in certain areas. The disorganized Sertoli cells were amorphous in shape, exhibited poor epithelial polarity, and were irregularly arranged in the testicular parenchyma. Extracellular matrix and collagen fibers were often observed in the intercellular spaces between the disorganized Sertoli cells. Lectin histochemical observation revealed that the number of wheat germ agglutinin binding sites on the plasma membrane and basal lamina of disorganized Sertoli cells was significantly decreased by TM treatment. However, junctions were normally observed in the plasma membrane between disorganized Sertoli cells. Leydig cells showed a normal differentiation in the testicular parenchyma in the presence of TM. These observations suggest that basal lamina formation of Sertoli cells and/or the expression of their cell surface glycoconjugates may be crucial for the establishment of Sertoli cell polarity and/or the Sertoli-Sertoli cell interactions required for proper testicular cord formation. Sertoli cell organization into testicular cords and Leydig cell differentiation may be controlled by different regulatory mechanisms. INTRODUCTION
Felici, 1984; Faze1 et al., 1987; Kanai et al., 1989) and In mammalian gonads, sexual differentiation, in monoclonal antibodies (Stinnakre et al., 1981; Fox et general, occurs prenatally. The first morphological fea- al., 1981; Hahnel and Eddy, 1986). In our previous ture in the male is organization of testicular cords, fol- study (Kanai et al., 1990), we found that certain carlowed by the Leydig cell differentiation in the extra- bohydrate chains of glycoconjugates changed dramaticordal region. Since testicular cords are normally cally on the cell surface of Sertoli cells during the fororganized without germ cells (Merchant, 1975), Sertoli mation of testicular cords. Tunicamycin (TM), discovered by Takatsuki et al. cells most likely play an important role in testicular cord organization (Wachtel et al., 1975; Ohno et al., (1971) and Takatsuki and Tamura (1971), is a potent 1979; Burgoyne et al., 1988). Morphological character- inhibitor of the formation of dolicho1-diphospho-Nistics of this organizational process are the appearance acetylglucosamine, and thereby blocks the glycosylaof epithelial polarity and the formation of basal lamina tion of asparagine residues in glycoproteins (Kuo and in the differentiating Sertoli cells (Frojdman et al., Lampen, 1974; Tkacz and Lampen, 1975; Heifetz et al., 1979). Subsequently, many reports appeared on the ef1989). Recently, it has been shown that glycoconjugates on fect of TM on the morphogenesis and cell differentiathe cell surface change profoundly during cell differen- tion of various tissues (Webb and Duksin, 1981; Sarras tiation (Kawai et al., 1979; Takata and Hirano, 1983a; et al., 1981; Takata and Hirano, 1983b). In the present study, the effect of TM on testicular Noguchi et al., 1982; Fenderson et al., 1984) and have an important effect on the cell-to-cell interactions (Huang, 1978; Blackburn and Schnaar, 1983; Rauvala, 1983; Thorpe et al., 1988; Tulsiani et al., 1989; WasReceived June 22, 1990; accepted October 2, 1990. sarman, 1990). In fetal testes, carbohydrate chains of Address reprint requests to Yoshiakira Kanai, Department of Vetglycoconjugates that appear during fetal gonadal dif- erinary Anatomy, Faculty of Agriculture, University of Tokyo, Bunferentiation have been studied with lectin probes (De kyo-ku, Tokyo 113, Japan. 0
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Fig. 1. a-c: Light micrographs of semithin sections showing undifferentiated gonads taken on day 12 p.c. (a)and cultured for 3 days (b) or for 5 days ( c ) in control medium. Upper portion: coelomic side; TC, testicular cord; EC, extracordal region; M, mesenchyme x 250. Testicular cord organization is evident by the 3rd day of culture. Testicular explants on the 3rd day of culture (b)and those on the 5th day ( c ) morphologically corresponded to the in situ fetal testes on day 14 p.c. and 16 P.c., respectively.
Fig. 2. a,b Electron micrographs showing control testicular explants on the 3rd day of culture. G, germ cell; S, Sertoli cell; EC, extracordal region. a: x 4,600, b: x 18,000. The basal lamina (bl) is clearly present between Sertoli cells and extracordal region (a, b). The cuboidal Sertoli cells had epithelial polarity as evidenced by the hemidesmosome-like structure (arrowheads) and a n accumulation of intermediate filaments in their basal cytoplasm. Adjoining Sertoli cells were tightly connected with each other by their membrane infoldings and zonula adherens-like junctions (arrow).
cord organization was examined in vitro at light and electron microscopic levels, with special reference to glycoprotein functions on cell interactions and the development of epithelial polarity during Sertoli cell dif-
ferentiation. The effect of TM on the distribution of carbohydrate chains was quantitatively examined a t the electron microscopic level by the colloidal gold labeling method.
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E F F E C T OF TM ON FETAL TESTICULAR ORGANIZATION
TABLE 1. Effect of TM on testicular cord oreanization in vitro
Culture condition TM 0 pg/ml (control) TM 0.1 pgiml TM 0.25 pg/ml TM 0.5 pg/ml TM 1.0 pgiml TM 0.25 pgiml + 4 d culture without TM
Number of experiments 22 4 7
4 4
Number of exDeriments in which Inhibition of testicular organization (cord-like Inhibition of testicular explant Testicular arrangement partially cords developed observed) was noted growth was found 22 0 0 0 4 (4) 0 0 7 (3) 0 0 0 4
3
MATERIALS AND METHODS Preparation of Pregnant Animals
Female C57BL/6 strain mice were caged with male ICR strain mice overnight. The next day, on which the vaginal plug was found, was regarded as day 1 post coitum (P.c.). F1 fetuses were obtained from the mother’s bodies on day 12 P.c., half a day before sexual differentiation occurs morphologically. The undifferentiated gonadal primordia was used for organ culture a s described below. Culture Procedure for Undifferentiated Gonads
The culturing of fetal gonads was performed according to the procedure reported by Byskov (1978a,b) and Taketo and Koide (1981). In short, the undifferentiated gonads, with mesonephric region, were taken up on day 12 p.c. under a dissecting microscope and were supported on a Nucleopore filter (Nucleopore Corporation, Pleasanton, CA) with a pore diameter of 1.0 pm, and then were floated on RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd., Toyko) supplemented with 10% horse serum (GIBCO Laboratories, Grand Island, NY). One of each pair of fetal gonadal primordia was cultured for 3 days with TM (0.1-1.0 pg/ml, a gift from Dr. Gakuzo Tamura, University of Tokyo), while the other was left untreated as the control and for judgment of the sex. In order to examine the reversibility of the TM effect, we used control medium to wash some explants that had been cultured with 0.25 pg/ml TM for 3 days, and thereafter cultured them without TM for 4 days. These explants were used for morphological and histochemical observations. Morphological Observation by Light and Electron Microscopy
After culture, the explants were fixed in 2.5% glutaraldehyde-0.1 M phosphate buffer, pH 7.4, at 4°C for 1 h, and then washed with PBS for 2 h. They were postfixed in 1% O,O, in the same buffer, dehydrated through a series of graded ethanol, and embedded in Araldite 6005. Some explants, after fixation in the same solution, were dehydrated in dimethylformamide, and embedded in Lowicryl K4M (Altman et al., 1984) that was polymerized by UV irradiation at -20°C for 1 h. Semithin and ultrathin sections were cut from Araldite- and/or Lowicryl K4M-embedded specimens, and examined under light and electron microscopes.
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Lectin Histochemical Observation
In order to examine the effect of TM on the distribution and glycosylation of carbohydrate chains in glycoconjugates, lectin histochemistry at the electron microscopic level was carried out as follows: Lowicryl K4M-embedded ultrathin sections were mounted on nickel grids, incubated with 1% bovine serum albumin (BSA)-PBS for 10 min, and thereafter incubated with biotinylated concanavalin A (ConA) or wheat germ agglutinin (WGA) (12.5 pg/ml) in 0.1% BSA-PBS for 1 h. Both biotinylated ConA and WGA were obtained from Vector Laboratory (Burlingame, CA). After being washed with PBS, they were incubated with streptavidin-colloidal gold in 0.1% BSA-PBS, prepared as reported previously (Kanai et al., 1989). After a rinse in PBS and distilled water, they were stained with lead citrate and uranyl acetate, and observed with a JEM1200EX transmission electron microscope. The numbers of ConA- and WGA-binding sites were counted on the plasma membrane and the basal lamina of Sertoli cells and expressed as numbers of colloidal gold particles per pm. The data from TM-treated explants were compared with those of control explants. Statistical analysis was performed by Student’s t test. Control Experiments for Lectin Histochemistry
As control experiments for lectin binding specificity, the sections were pre-incubated with appropriate 0.2 M hapten sugars for each lectin (a-methyl-mannosidefor ConA, and N-acetyl glucosamine for WGA), and then incubated with biotinyl lectins in the presence of the hapten sugars. Non-specific staining was also checked by the incubation of sections with the streptavidin-colloidal gold conjugate only. RESULTS Light and Electron Microscopic Findings of the Control Explants
On day 12 P.c., testes could not be morphologically distinguished from ovaries. Testicular cords were not yet organized a t this stage (Fig. la). In the control culture system for undifferentiated gonads taken on day 12 P.c., the organization of testicular cords was recognized by the 3rd day of culture (Fig. lb). Testicular cords were tightly packed with both germ cells and Sertoli cells. The tunica albuginea was seen beneath the coelomic epithelium. The extracordal regions were formed in the outer area of the cords. On the 5th day of culture, the size of the testicular parenchyma increased
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Fig. 3. a , b Light micrographs of semithin sections showing explants cultured for 3 days with 0.1 pgiml (a) or 0.25 pgiml TM (b). Upper portion, coelomic side; M, mesenchyme. a , b x 230. In the explants treated with 0.1 pgiml TM and in some explants treated with
0.25 pgiml TM, testicular cord-like arrangements (TA) in contact with the rete testes (RT) were formed at the mesonephric sides of the testicular parenchyma (a).In other explants treated with 0.25 pgiml TM, testicular cord organization was blocked almost completely (b).
obviously. Testicular cords showed a uniform diameter at this time, and Sertoli cells were regularly located in basal regions of the cords (Fig. lc). From a morphological viewpoint, testicular explants on the 3rd day of culture and those on the 5th day of culture corresponded to in situ fetal testes on day 14 p.c. and those on day 16 P.c., respectively.
Under the electron microscope, control testicular explants on the 3rd day of culture had smooth outer walls of testicular cords and had established a basal lamina between Sertoli cells and extracordal regions (Fig. 2a,b). Differentiated cuboidal Sertoli cells exhibited epithelial polarity, for hemidesmosome-like structures and a n accumulation of intermediate filaments were
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Fig. 4. a,b: Electron micrographs showing testicular cord-like arrangements in explants cultured with 0.1 pg/ml TM. er, the dilated rough ER caused by TM. a: X 7,800, b: x 30,000. The basal lamina along testicular cord-like arrangements is partially discontinuous or completely absent in certain areas (a,b).Occasional Sertoli cell processes project into the extracordal region (open arrow). The basal surface of Sertoli cells in areas where the basal lamina is morphologically lacking exhibits an irregular outline. However, junctions (arrows) are normally observed between Sertoli cells.
Fig. 5. a,b Electron micrographs showing testicular explants treated with 0.25 pgiml TM. G, germ cell; er, the dilated rough ER caused by TM. a: x 3,600, b: x 12,000. The testicular cord organization was inhibited by TM. The disorganized Sertoli cells (S) are amorphous in shape, exhibit poor epithelial polarity, and are irregularly arranged in the testicular parenchyma (a,b). Extracellular matrix and collagen fibers were often observed in the intercellular spaces between the disorganized Sertoli cells (stars).On the other hand, junctions (arrows) are clearly seen between disorganized Sertoli cells (b).
observed in the basal cytoplasm of these Sertoli cells (Fig. 2b). Adjoining Sertoli cells were tightly connected with each other by their membrane infoldings, in which junctions had been formed. In the extracordal region, myoid cells were arranged around the testicular cords, and some Leydig cells were found to be differentiated.
treatment inhibited testicular cord organization. The effects of TM treatment on testicular cord organization are summarized in Table 1. In the case of explants treated with 0.1 pg/ml TM and in some explants with 0.25 pgiml TM, testicular cord-like arrangements were formed in contact with the rete testes at the mesonephric sides of the testicular parenchyma (Fig. 3a). In other explants exposed to TM at 0.25 pg/ml, testicular cord formation was almost completely blocked (Fig. 3b). Moreover, TM treatment partially inhibited the penetration of mesenchymal
Morphological Findings on TM-Treated Explants
In this study, the effect of TM on testicular cord organization was examined on the 3rd day of culture. TM
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rough ER (Figs. 4a,b, 5b, 8b,d). However, TM treatment did decrease the number of germ cells drastically. The testicular parenchyma of the TM-treated explants was found to contain pyknotic cells, some of which had been phagocytosed by Sertoli cells. We consider most of the pyknotic cells to be germ cells, since they were heavily labeled by monoclonal antibody against the Le" epitope (SSEA-11, a marker of primordial germ cells (data not shown). On the other hand, 0.5 pg/ml and 1.0 pg/ml TM inhibited the development and growth of testicular explants, which remained nearly equal in size to the gonadal primordia taken on day 12 p.c. The gonadal cells were oval in shape, and some of them, especially in the explants treated with 1.0 pg/ml TM, exhibited evidence of degeneration. When explants were treated with 0.25 kg/ml TM for 3 days followed by culture in the absence of TM for 4 days, the round-shaped and aggregated Sertoli cells and the Leydig cells remained randomly scattered throughout the testicular parenchyma, indicating no reversibility of the TM effect on testicular cord organization (Fig. 7a). However, the disorganized Sertoli cells partly exhibited normal differentiation characteristics. That is, they were surrounded by a basal lamina (Fig. 7b) and had endocytic-coated and uncoated vesicles on Fig. 6. Electron micrograph showing a Leydig cell in a n explant cultured with TM 0.25 pg/ml. x 9,300, inset: x 32,000. Leydig cells in their basal side. Also, the junctional complex occasionthe explants have differentiated normally. Morphological features in- ally was observed in the plasma membrane between dicative of steroid production, i.e., lipid droplets, well-developed Sertoli cells (Fig. 7c). Microtubules became clearly obsmooth ER, and mitochondria with tubular cristae, are evident. The served in the basal surface (Fig. 7d), compared with inset shows a higher magnification of mitochondria as indicated by those in the undifferentiated testes, and in the control the rectangle. and TM-treated explants on 3rd day of culture. The dilation of rough ER caused by TM disappeared in the Sertoli cells and the other gonadal cells, although most cells into the subepithelial region beneath the coelomic of the germ cells had degenerated. epithelium. The continuity between the coelomic epithelium and the gonadal cells remained. Lecfin Histochemical Findings on TM-Treated Explants Upon electron microscopic observation of the TMBoth in controls and in TM-treated explants, the distreated explants, the basal lamina along the testicular cord-like arrangements was discontinuous or com- tribution of carbohydrate chains of glycoconjugates in pletely lacking in certain areas (Fig. 4a,b). Occasional the Sertoli cells was examined at the electron microSertoli cell processes projected into extracordal regions, scopic level. and basal surfaces of Sertoli cells whose basal laminae In control explants, ConA binding was observed in were morphologically lacking showed a n irregular out- the nuclear membrane, rough ER, and plasma memline. In explants with testicular cord formation com- brane of the Sertoli cells, and also in the basal lamina pletely blocked, the disorganized Sertoli cells were along the testicular cords (Fig. 8a). In TM-treated examorphous in shape, exhibited poor epithelial polarity, plants, ConA binding also showed a distribution simiand were irregularly arranged in the testicular paren- lar to that in the control explants, except that the dichyma (Fig. 5a). Extracellular matrix and collagen fi- lated luminal spaces of the rough ER were strongly and bers were often observed in the intercellular spaces uniformly labeled with ConA (Fig. 8b). The number of between the disorganized Sertoli cells (Fig. 5a,b). Such ConA binding sites did not significantly change in the spaces may be equivalent to extracordal regions in con- plasma membrane and basal lamina of TM-treated Sertrol explants. On the other hand, junctions were clearly toli cells (Table 2). WGA binding was mainly localized in the plasma seen between adjacent disorganized Sertoli cells (Fig. membrane of Sertoli cells in the control explants (Fig. 5b). Leydig cells, which were found to differentiate in the 8c). TM treatment significantly ( P < .01) decreased the presence of TM, showed evidence of steroid production; number of WGA-binding sites in the plasma membrane i.e., they had morphological features such a s lipid drop- of Sertoli cells to a fifth of that found in controls (Fig. lets, well-developed smooth ER, and mitochondria with 8d; Table 2). WGA-binding sites were also decreased by TM treatment in the basal lamina along the Sertoli cell tubular cristae (Fig. 6 ) . The size of testicular explants treated with 0.1 kg/ml surface (P < .01). or 0.25 pg/ml TM was nearly equal to that of control The effects of TM on ConA and WGA binding to the explants, thus suggesting no toxic effects. Morpholog- other gonadal cells were similar to those seen in the ically, the Sertoli cells and other gonadal cells showed Sertoli cells. In fact, TM treatment decreased the numno cytotoxicity due to TM except for dilation of some ber of WGA-binding sites on the plasma membrane of
E F F E C T OF TM ON FETAL TESTICULAR ORGANIZATION
Fig. 7. a-d: Electron micrographs of explants cultured with 0.25 pgiml TM for 3 days followed by culture in the absence of TM for 4 days. Star, intercellular space. a: x 1,400, b: x 5,300, c: x 36,000, d: x 20,000. The effect of TM on testicular cord organization was irreversible, for the round-shaped and aggregated Sertoli cells ( S )and the Leydig cells (L) remained randomly scattered throughout the tes-
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ticular parenchyma (a).The disorganized Sertoli cells, however, are surrounded by a basal lamina (arrowheads) (b,d),and the junctional complex (arrow) is seen between Sertoli cells (c). Microtubles (arrows) are developed in the basal cytoplasm of some disorganized Sertoli cells
(d).
Sertoli cells. Sertoli cell organization into testicular cords is therefore independent of the functional role of junction formation between Sertoli cells. Also, Leydig cells showed a normal differentiation in the testicular parenchyma in the presence of TM. Interestingly, Taketo e t al. (1984) reported that cAMP analogues inhibited testicular organization in vitro without any influence on Leydig cell differentiation. Therefore, Sertoli cell organization into testicular cords and Leydig cell DISCUSSION differentiation might be controlled by different regulaThe present study shows that TM treatment pre- tory mechanisms. Electron microscopic observations showed that the vents testicular cord organization in vitro. In testicular explants treated with 0.25 pglml TM, cord formation basal lamina along the cord-like arrangement was parwas blocked, and the disorganized Sertoli cells were tially discontinuous or completely lacking in some aramorphous in shape and irregularly arranged in the eas in the TM-treated explants. Similar disintegration parenchyma. However, junctions were clearly seen in of the basal lamina along the testicular cords was rethe plasma membrane between adjacent disorganized ported when explants were cultured with cAMP ana-
germ cells to a tenth of that in controls (data not shown). Specific positive reactions for lectin staining as above were completely inhibited by the addition of appropriate hapten sugars. Furthermore, no specific positive reaction was detected in the case of the incubation with the streptavidin-colloidal gold conjugate alone.
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Fig. 8.a 4 Electron micrographs of Lowicryl KQM-embedded ultrathin sections showing Sertoli cells in control explants (a,c) and disorganized Sertoli cells in explants treated with 0.25 pgiml TM (b,d).N, nucleus of Sertoli cell; EC, extracordal region, er, the dilated rough ER caused by TM. a d x 25,000. a: ConA staining in a control explant. ConA binding is observed in the nuclear membrane, rough ER (arrows), and plasma membrane of the Sertoli cells, and also in the basal lamina along the testicular cords. b ConA staining in a TM-
treated explant. The binding shows a similar distribution as seen in the control (a), except that the dilated rough ER (arrows) is more stongly stained by ConA. c: WGA staining in a control explant. The plasma membrane of Sertoli cells is positive (arrows). d: WGA staining in a TM-treated explant. TM treatment significantly decreased the number of WGA-binding sites in the plasma membrane of Sertoli cells (arrows).
logues (Taketo et al., 1984). The basal lamina has been shown to be important for providing morphological stability to the epithelium during morphogenesis (Banerjee et al., 1977). In isolated Sertoli cells in vitro, the
basal lamina is needed for expression of the normal histotype, i.e., the polarized columnar shape (Tung and Fritz, 1984; Hadley et al., 1985). It is well known that the basal lamina is composed of glycoproteins, collagen
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not explain the reason for this differential effect at present, it may be partly due to the possible difference, under the influence of TM treatment, between the halflives of ConA-binding glycoproteins and those of WGAbinding glycoproteins. The effect of TM is reversible in a variety of cell types Number of CGIpm (Damsky et al., 1979; Ekblom et al., 1979; Pratt et al., PM (n = 10) BL (n = 5) Lectin Culture condition 1979). In the present experiment, however, the inhibConA Cont 0.98 t 0.01 1.82 ? 0.04 itory effect of TM on testicular cord organization was 2.05 ? 0.30 TM + 1.18 t 0.14 irreversible within the 4-day culture in the control me1.17 ? 0.13 WGA Cont 1.31 t 0.09 dium. However, each aggregate of several Sertoli cells TM + 0.24 ? 0.03* 0.39 2 0.08* was surrounded by a basal lamina and showed normal differentiation characteristics such as the formation of 'Each value is expressed as mean 2 SE. *P < .01,compared with the value of the corresponding control. specific junction-like structures, the active ingestion by endocytic vesicles on their basal side, and the development of microtubules in their basal cytoplasm. Thus, type IV, laminin, heparansulphate proteoglycan, and the effect of TM on Sertoli cell differentiation is partly fibronectin. Since TM treatment significantly (P < .01) reversible. The acquisition of such differentiation chardecreased the number of WGA-binding sites in the acteristics as above may not need the formation of the basal lamina, these molecules could be affected by TM, testicular cords, but it may require the enclosure by the which might result in the disintegration of the basal basal lamina. The irreversibility of the testicular cord lamina. Such changes in the basal lamina might affect organization may be due to the TM-induced irregular the establishment or maintenance of Sertoli cell polar- formation of the extracordal space or to the existence of a critical period for testicular cord organization. ity. Since Merchant (1975) showed that germ cells had no ACKNOWLEDGMENTS effect on testicular cord organization, Sertoli cells, in The authors wish to thank Dr. Teruko Taketo-Hosoparticular Sertoli-Sertoli cell interactions, may be important for this organization (Wachtel et al., 1975; tani (Department of Urology, McGill University) for Ohno et al., 1979). Our previous study also showed the her kind and helpful advice and discussion on the orappearance of WGA-binding glycoconjugates on the gan culture method. The authors also wish to thank Dr. plasma membrane between Sertoli cells a t the time of Yoshihiro Akimoto (Department of Anatomy, Kyorin the onset of testicular cord formation (Kanai et al., University School of Medicine) for his helpful advice 1990). This suggests that these particular cell surface during the course of this work, and Mr. Iwao Tsugglycoconjugates may be involved significantly in this iyama (Department of Veterinary Anatomy, Faculty of Agriculture, University of Tokyo) for his expert care in phase of gonadal development. It has been reported by many investigators that TM keeping the laboratory mice. This work was supported by Grant-in-Aid from the affects cell surface glycoconjugates (Duksin and Bornstein, 1977; Duksin et al., 1978; Sarras et al., 1981). Ministry of Education, Science and Culture of Japan. Bordy et al. (1979) reported that TM treatment reLITERATURE CITED sulted in the inhibition of FSH-induced aggregation of Altman, L.G., B.G. Schneider, and D.S. Papermaster 1984 Rapid emimmature rat Sertoli cells in primary culture. The bedding of tissues in Lowicryl K4M for immunoelectron microspresent study showed that TM treatment decreased the copy. J . Histochem. Cytochem., 32t1217-1223. number of WGA-binding sites on the cell surface of Banerjee, S.D., R.H. Cohn, and M.R. Bernfield 1977 Basal lamina of disorganized Sertoli cells. When testicular cord organiembryonic salivary epithelia: production by the epithelium and role in maintaining lobular morphology. J. Cell Biol., 73r445zation was completely blocked by TM, extracellular 463. matrix and collagen fibers were often observed in the Blackburn, C.C., and R.L. Schnaar (1983) Carbohydrate-specific cell intercellular spaces between the disorganized Sertoli adhesion is mediated by immobilized glycolipids. J. Biol. Chem., cells, suggesting the loss of the Sertoli-Sertoli cell in258r1180-1188. teractions. The inhibitory effect of TM on testicular Bordv. M.J.. S. Berger. C. Desiardins. and J.C. Davis 1979 Active cell aggregation biimmature rat Sertoli cells in primary culture: a cord organization may be partially due to the inhibirole for cell surface glycoproteins. J . Cell. Physiol., 99r175-182. tory effect of the drug on expression of certain cell sur- Burgoyne, P.S., M. Buehr, P. Koopman, J. Rossant, and A. McLaren face glycoproteins that are morphogenetically active 1988 Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX-XY chimaeric mouse testes. and lead to effective interactions between adjoining Development, 102t443-450. Sertoli cells. Byskov, A.G. 1978a Regulation on initiation of meiosis in fetal goThis study also showed that the dilated luminal renads. Int. J . Androl. [Suppl.],2:20-38. gion of rough ER caused by TM (Sarras et al., 1981; Byskov, A.G. 1978b The meiosis inducing interaction between germ cells and rete cells in the fetal mouse gonad. Ann. Biol. Anim. Takata and Hirano, 1983b) was heavily and uniformly Biochem. Biophys., 18~327-334. labeled with ConA. Thus, glycoconjugates containing Damsky, C.H., A. Levy-Benshimol, C.A. Buck, and L. Warren 1979 mannose and glucose residues, whose translocation Effect of tunicamycin on the synthesis, intracellular transport may be blocked by TM, accumulate in the rough ER; and shedding of membrane glycoproteins in BHK cells. Exp. Cell Res., 119tl-13. and consequently, the rough ER may dilate in the goM. 1984 Binding of fluorescent lectins to the surface of nadal cells. TM treatment did not significantly change De Felici, germ cells from fetal and early postnatal mouse gonads. Gamete the number of ConA binding sites in the plasma memRes., 10t423-432. brane and basal lamina of Sertoli cells, in contrast to Duksin, D., and P. Bornstein 1977 Changes in surface properties of normal and transformed cells caused by tunicamycin, a n inhibiits effect on the WGA-binding sites. Although we canTABLE 2. Number of colloidal gold particles representing ConA- and WGA-binding sites in the plasma membrane (PM) and basal lamina (BL) of Sertoli cells in testicular explants cultured with 0.25 udml TM (TM + ) or without TM (Cont)'
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