0013-7227/92/1301-0353-03.00/o Endocrinology Copyright 0 1992 hy The

Distribution Messenger Localization Epididymis”

Vol.

Endocrine Society

Printed

130, No. 1

in U.S.A.

and Regulation of Epithelial Cadherin Ribonucleic Acid and Immunocytochemical of Epithelial Cadherin in the Rat

DANIEL G. CYRP, LOUIS BERNARD ROBAIRE

HERMO,

OREST

W. BLASCHUK,

AND

Departments of Pharmacology and Therapeutics (D.G.C., B.R.) and Anatomy (L.H., 0. W.B.) and the Centre for the Study of Reproduction (D.G.C., L.H., B.R., 0. W.B.), McGill University, Montreal, Quebec H3G 1 Y6; and the Department of Urology, Royal Victoriu Hospital (0. W.B.), Montreal, Quebec H3A lA1, Canada

entire epididymis. The relative intensities of the immunoreactivity suggested that the E-Cad protein concentration was highest in the corpus, followed by the caput, cauda, and initial segments of the epididymis. There was no reaction over the epithelial basal and clear cells or intraepithelial halo cells. Three days after bilateral orchidectomy, E-cad mRNA was decreased by 75% in the caput epididymidis. A dose-dependent maintenance of mRNA concentration for E-Cad was observed throughout the epididymis of orchidectomized rats after replacement with testosterone. Fourteen days after unilateral orchidectomy, no differences were observed in the concentrations of epididymal E-Cad mRNA between control and unilaterally orchidectomized rats. Together, these data demonstrate that mRNA for E-Cad is present and translated in the rat epididymis, is differentially distributed along this tissue, and can be regulated by circulating androgens. (Emfocrim~b~gy 130: 353-363,1992)

ABSTRACT. The epithelium of the epididymis possesses an elaborate network of tight junctions between principal cells which is altered as a function of postnatal age. Cadherins are implicated in the formation of tight junctions. The objective of the present study was to determine whether RNA transcripts for cadherins were present in the epididymis, and if so, how they were hormonally regulated. Using specific cDNA probes for epithelial cadherin (E-Cad) and neural cadherin (N-Cad), Northern blot analysis was used to study steady state levels of cadherin mRNAs. A major E-Cad mRNA species of 4.7 kilobases and a weaker 4.3-kilobase species were observed in the epididymis. No signal for N-Cad was detected. Steady state mRNA levels for E-Cad were highest in the caput and corpus epididymidis and were almost 4 times higher than those in the initial segments and cauda epididymidis; no signal was detected in the vas deferens. Light microscopic immunocytochemical localization of E-Cad revealed a reaction over the principal cells of the

T

bilateral orchidectomy of 20-day-old mice results in a diminution of the tight junction network in the epididymis within 2-4 days. This mesh network can be spontaneously reinitiated in the days following gonadectomy. Cadherins (CADS) have been implicated in the formation of tight junctions between epithelial cells of the liver, kidney, and intestine (7-12). These cell surface glycoproteins are mediators of calcium-dependent homophilic cell adhesion (7-10, 12, 13). This family is composed of several different members, which are usually named after the tissue from which they were originally isolated, e.g. epithelial cadherin (E-Cad) and neural cadherin (N-Cad) (9, 14-16). While a considerable amount of information currently exists demonstrating a spatiotemporal expression of cadherins during embryonic development, little information exists regarding the distribution, function, and regulation of cadherins in adult animals. The objectives of this study were to determine whether

HE EPITHELIUM of the epididymis is composed of an elaborate network of tight junctions (l-5). These junctions begin to form at the time of differentiation of the Wolffian duct (3). While the function of these junctions in the epididymis is not known, it is assumed that they are responsible for the formation of the bloodepididymal barrier. The maintenance of this barrier is necessary for creating the intraluminal environment believed to be important for sperm maturation (4-6). The formation and maintenance of tight junctions in the caput epididymidis appear to be regulated by gonadal hormones. Suzuki and Nagano (2) have reported that Received July 26, 1991. Address all correspondence and requests for reprints to: Dr. Bernard Robaire. Denartment of Pharmacoloav and Theraneutics, McGill University, ‘365’5 Drummond Street W&, Montreac Quebec, H3G lY6 Canada. *This work was supported by grants from the Medical Research Council of Canada (to B.R., L.H., and O.W.B.). t Medical Research Council-Ciba Geigy Postdoctoral Fellow. 353

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354

CADHERIN

mRNA

IN THE

RAT EPIDIDYMIS

Endo. 1992 Vol 130. No 1

there are mRNA transcripts for cadherins in the epididymis and, if present, how these transcripts are regulated. We have selected to study the steady state mRNA concentrations of E-Cad and N-Cad. Our rationale for selecting these two cadherins is based on previous observations on the localization of these proteins. The epidi-

six groups. There was one control and one unilaterally orchidectomizedgroup for eachof three postsurgerytimes: 3, 7, and 14 days. Surgery was performed under ether anesthesia;right testes were removed via an incision through the peritoneal cavity. Controls were shamoperated.

dymis is composed mainly of epithelial cells, which line the convoluted tubule (17). It has been observed that ECad is present in epithelial cells of different tissues, thereby suggesting that it may be present in the epididymis (7,8,12,17). In contrast, N-Cad has been localized

mainly to nervous and muscle tissue (7, 15, 16, 18-20). Since the epididymis is highly innervated and contains an outer smooth muscular layer, this suggested the pos-

At the end of each experiment, rats were killed by decapitation under ether anesthesia.Ventral prostate, seminalvesicles, and epididymides were dissectedfree of other tissues.Epididymides were subdivided into four different sections: initial segment (section l), caput (sections 2-3), corpus (section 4), and cauda (sections5-6), as previously described(23). Tissues were immediately frozen in liquid nitrogen and subsequently stored at -80 C.

sibility

RNA isolation

that N-Cad

Experiments

was also present

were also performed

in the epididymis.

to study both the lon-

gitudinal distribution and endocrine regulation of steady state mRNA concentrations for cadherins in the epididymis as well as the immunocytochemical localization of the protein for E-Cad along the epididymis. Materials

and Methods

Epididymal segmentsfrom three or four animals were used for each RNA isolation. Frozen tissueswere pulverized under liquid nitrogen using a mortar and pestle. Total cellular RNA was isolatedusing the guanidinium isothiocyanate method and centrifugation in cesiumchloride, as describedby Sambrooket al. (24). The concentration of RNA recoveredfrom thesetissues wasdetermined spectrophotometrically. Northern

AnimaLs

Male Sprague-Dawleyrats (300-325 g) were purchasedfrom Charles River Canada, Inc. (St. Constant, Quebec, Canada). Ratsweremaintained under a 14-h light, 10-h dark photoperiod and received food and water ad libitum. Experimental

procedures

distribution. The longitudinal distribution of cadherins in the epididymis was determined using tissuespooled from three or four animals.The experiment wasrepeatedthree times (n = 3).

Longitudinal

orchidectomy. For the first bilateral orchidectomy study, two groups of four animals were used (n = 2). One (control) was sham orchidectomized, while the other was orchidectomized,under ether anesthesia,via a peritoneal incision. For the secondbilateral orchidectomy study, eight animals were assignedto each of four groups: control, sham-operated (C), orchidectomized implanted with an empty 2.5-cm polydimethylsiloxane capsule(0), and orchidectomized implanted SC with testosterone-filled capsulesmeasuringeither 2.5 cm (2.5) or 18.6 cm (18.6). Surgery was performed under ether anesthesia; testeswere removedvia an incision through the peritoneal cavity. Testosterone-filled polydimethylsiloxane (Silastic) implants were preparedaccording to the method of Stratton et al. (21) and have well characterized steroid releaserates (22); for the 18.6~cmgroup, three implants of 6.2 cm were used. Carrier rats were implanted for 3 days before surgery so that the initial surgeproduced by the newly made implants would be complete,and implants would be releasingtestosteroneat a constant rate. The experiment was repeated three times (n = Bilateral

3). Unilateral

Tissue isolation

orchidectomy.

Forty-eight animalswere divided into

blot transfers

RNA samples(lo-15 rg) were fractionated by electrophoresison 1.2% agarose-formaldehydegelsat 30 V for 20 h in 5 x running buffer (80 mM boric acid, 16 mM sodiumborate, and 1 mM EDTA, pH 8.3). After electrophoresis,the gel was soaked in sterile water for 5 min and placed on an LKB vacuumtransfer apparatus (Montreal, Quebec,Canada). The gel was hydrolyzed in 50 mM NaOH and 10 mM NaCl for 30 min with a vacuum of 50 cm Hz0 and then neutralized in 0.1 M Tris-Cl (pH 7.4) for 30 min. The RNA wastransferred for 90 min in a 10 x sodiumcitrate buffer (3 M NaCl and 30 mM sodiumcitrate, pH 7.0; SSC) onto a nylon membrane(Genescreen-Plus,New England Nuclear, Missassauga,Ontario, Canada). After the RNA transfer, the gel was removed, and the membranewas washedfor 5 min in 5 X SSC. The membranewasthen baked at 80 C under vacuum for 2 h. cDNA probes

Two cDNA probes, a mouseE-Cad probe and a mouseNCad probe, were obtained from M. Takeichi, Kyoto University. The E-Cad cDNA clone is a full-length cDNA, asdescribedby Nagafuchi et al. (25). The N-Cad cDNA clone was identical to the mn-2 clone describedby Hatta and Takeichi (15). Each of the denatured probes (50 rig/reaction) was labeledby random priming (OligolabelingKit, Pharmacia, Montreal, Quebec,Canada) using 32P-labeleddeoxycytosine triphosphate ( [32P]dCTP; SA, 3000 Ci/mmol; Amersham Canada, Inc., Mississauga,Ontario, Canada). The labeledprobe was separatedfrom nonincorporated free phosphorususing a Sephadexcolumn (Sephadex G-50, Pharmacia) equilibrated in a Tris-EDTA buffer (24). l&5’ rRNA probe

Northern blots were standardized for the amount of RNA loaded in each lane using a synthetic oligonucleotideprobe

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CADHERIN

mRNA

IN THE RAT EPIDIDYMIS

recognizing the 18s rRNA sequence (a kind gift from M. Szyf, McGill University). The probe was prepared according to the sequence outlined by Szyf et al. (26). The probe was labeled with y-ATP (SA, 3000 Ci/mmol; Amersham Canada, Inc.), using the end-labeling method described by Sambrook et al. (24. Hybridizations Hybridizations on nylon membranes with the cadherin cDNA probes and the 18s rRNA oligonucleotide probe were performed using the procedures outlined by Viger and Robaire (27). RNA quantification Radioautograms were scanned using an LKB laser densitometer, and the integrated area under the curve for each signal was standardized against the signal for the 18s rRNA synthetic oligonucleotide probe. The linearity of the densitometric scan was determined for the apparatus, and all readings presented in these experiments are within the linear range for absorbance of the signal. Data are expressed as the relative concentration of steady state mRNA, defined as the mRNA concentration in the tissue at the time the tissue was removed. Immunocytochemistry Four adult rats (350-450 g) were anesthesized with sodium pentobarbitol, and their reproductive tracts were fixed by perfusion for 10 min with Bouin’s fixative. Two types of perfusions were performed in order to ensure proper fixation of the different segments of the epididymis. The first was a retrograde perfusion through the abdominal aorta (28) to perfuse the initial segment and caput, and the second was a prograde perfusion to perfuse the corpus and cauda epididymidis. Once the tissues were fixed, epididymides were cut along their longitudinal plane so that each section contained one or more segments of the epididymis. The tissues were immersed in Bouin’s solution for 24 h and were subsequently dehydrated and embedded in paraffin. Epididymal sections of 5 pm were cut and mounted on glass slides. Sections were hydrated, and residual picric acid was inactivated by a 5-min immersion in 70% ethanol containing 1% lithium carbonate; endogenous peroxidase activity was inactivated with a 5-min immersion in 1% (vol/vol) hydrogen peroxide. The sections were then incubated for 5 min in 300 mM glycine to block free aldehydes and washed in 20 mM TrisCl saline containing 1% BSA (TBS) at pH 7.4. The antibody to E-Cad (uvomorulin), kindly provided by R. Kemler, has been described previously (10, 11). Before E-Cad antibody incubation, the tissue sections were blocked for 15 min with 10% goat serum (Sigma Chemical Co., St. Louis, MO) in TBS. Sections were then incubated for 1.5 h at 35 C with the anti-E-Cad antiserum at dilutions ranging from 1:120 to 1:30 in TBS. After the incubation, the tissue sections were washed four times (2 min each) in TBS containing 0.1% Tween20 (TWBS; Sigma), blocked for 10 min with 10% goat antiserum, and incubated for 30 min with a goat antirabbit immunoglobulin G conjugated to horseradish peroxidase (Sigma) at a concentration of 1:500 (vol/vol) in TBS.

355

After thorough washings in TWBS, tissue sections were incubated with 0.03% hydrogen peroxide and 0.05% diaminobenzidine tetrachloride (Sigma) in TBS containing 0.1 M imidazole at pH 7.6 (29). Epididymal sections were then washed with H20, counterstained for 5 min in 0.1% methylene blue, dehydrated in solution containing graded concentrations of ethanol, immersed in xylene, and mounted in Permount. Normal rabbit serum was used as a negative control.

Results Longitudinal

distribution

of E-Cad mRNA

Northern blot analysis of mRNA obtained from adult rat epididymides revealed a major E-Cad mRNA species of 4.7 kilobases (kb) and a weaker 4.3-kb mRNA transcript (Fig. 1A). Only the 4.7-kb transcript was used to

calculate steady state E-Cad mRNA concentrations. To determine the longitudinal distribution of E-Cad mRNA, epididymides were subdivided into four separate segments corresponding to the initial segment, the caput, the corpus, and the cauda. Standardizing against the amount of 18s rRNA (Fig. lB), a quantitative evaluation of the mRNA concentrations for E-Cad along the epididymis was performed (Fig. 1C). The concentrations were highest and of similar magnitude in the caput and corpus epididymidis; they were approximately one quarter of this concentration in the initial segment and the cauda epididymidis (Fig. 1C). No signal was detected in the vas deferens (data not shown). No signal for N-Cad was detected in the epididymis; only newborn brain was positive (Fig. 2). Immunocytochemistry

In the light microscope there was a complete absence of a reaction product over the epithelial cells of the entire epididymis in control treated tissues treated with normal rabbit serum (Fig. 3). Likewise, there was no reaction in the cells of the intertubular space or over spermatozoa in the lumen (Fig. 3). In tissues treated with the anti-E-Cad antibody, a distinct immunoperoxidase reaction was observed throughout the epididymis, but it varied in intensity. At low magnification, the overall immunoperoxidase-staining pattern in each epididymal segment was apparent (Fig. 4, a-d); it was present over the epithelium and increased progressively from the initial segment to caput (Fig. 4b) and peaked in the corpus (Fig. 4c) epididymidis.

There was an overall weaker reaction in the cauda epididymidis (Fig. 4d). At high magnification, it was apparent that the reaction was over the epithelial principal cells (Fig. 5, a-d). Neither the epithelial basal nor the intraepithelial halo cells showed any reaction. The reaction was over the entire cytoplasm of all principal cells (Fig. 5, a-d) and

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CADHERIN

356

IS CT CS

A

mRNA IN THE RAT EPIDIDYMIS

Endo. Vol130.

IS

CA

1992 No 1

CT CS CA B

A 4.3 +

B

:dj: 18s

,,

+

C

B 60

-

60

-

40

-

20

-

18S-0

C

is 500 5 450

e

400

w”

350

IS

UG; z-

300

8% z-48

250 200

2

150

3

100 50

FIG. 2. Northern blot analysis of the regional distribution for N-Cad steady state mRNA in adult rat epididymis. Total RNA (10 pg) isolated from newborn rat brain was used as a positive control. A, 32P-Labeled N-Cad probe was hybridized with the membrane containing RNA from the initial segment (IS), caput (CT), corpus (CS), and cauda (CA) epididymidis as well as newborn brain (B). B, After hybridization with the N-Cad probe, the membrane was stripped and hybridized to a synthetic oligonucleotide probe recognizing the 18s RNA. C, The radioautogram was scanned by laser densitometry, and the area under the curve was standardized for the amount of RNA loaded in each lane.

--

o-

A

1

0 1

1 IS

CS

CA

1. Northern blot analysis of the regional distribution of E-Cad steady state mRNA in adult rat epididymis. A, ‘*P-Labeled E-Cad probe was hybridized with the membrane containing RNA from the initial segment (IS), caput (CT), corpus (CS), and cauda (CA) epididymidis. B, After hybridization with the E-Cad probe, the membrane was stripped and hybridized to a synthetic oligonucleotide probe recognizing the 18s RNA. C, The radioautograms were scanned by laser densitometry, and the area under the curve for each signal was standardized for the amount of RNA loaded in the lane (n = 3). FIG.

CS

CA

B

LJ Jl

CT

CT

was most intense over the corpus epididymidis (Fig. 5~). In the cauda epididymidis, principal cells were reactive, but while the reaction was strong over the apical and supranuclear regions, it was weak over the basal region (Fig. 5d). In this respect, they differed from the principal cells of the other epididymal segments. The epithelial clear cells, identified by their large size and the absence of a brush border, were not immunoreactive along the

entire epididymis (Fig. 5, c and d). Likewise, no staining over the cells of the intertubular spermatozoa in the lumen (Fig. 5, a-d).

there was space or

Effects of bilateral orchidectomy Three days after rats had been bilaterally orchidectomized, the steady state E-Cad mRNA concentration was decreased by 75% in the caput epididymidis (Fig. 6). Waiting an additional 4 days after bilateral orchidectomy did not further decrease the mRNA for E-Cad in the caput epididymidis (Fig. 8C). Since bilateral orchidectomy lowers blood testosterone concentrations to undetectable levels, these results suggest that testosterone may play a role in the regulation of epididymal E-Cad mRNA.

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CADHERIN

mRNA IN THE RAT EPIDIDYMIS

357

tosterone were sufficient to maintain E-Cad mRNA concentrations. Rats were unilaterally orchidectomized at the start of the experiment, and tissues were excised 7 and 14 days postsurgery. Steady state mRNA concentrations from the initial segment, caput, corpus, and cauda epididymidis indicate that this surgical procedure did not alter the E-Cad mRNA concentrations at any time point, suggesting that circulating levels of androgens were sufficient to maintain normal concentrations of E-Cad mRNA. Data from all segments of the epididymis 7 days postsurgery are shown (Fig. 9); results obtained 3 and 14 days after unilateral orchidectomy could not be distinguished from those obtained at 7 days. FIG. 3. High power light micrograph of the caput epididymidis immunostained with normal rabbit serum. Epithelial principal cells (P), spermatozoa (S), and intertubular connective tissue space (IT) are shown. Magnification, ~512.

Effects of testosterone replacement on steady state E-Cad mRNA

Rats were bilaterally orchidectomized at the start of the experiment and were simultaneously implanted SC with either empty capsules (2.5 cm) or testosterone-filled capsules (0,2.5, and 18.6 cm). Weights of ventral prostate and paired seminal vesicles (Fig. 7) were decreased by castration and increased in a dose-dependent manner, indicating that these tissues were exposed to increasing concentrations of biologically active circulating testosterone (Fig. 7). The changes in epididymal weight reflect the fact that this tissue is androgen dependent but does not respond by hypertrophy to high testosterone doses (Fig. 7). E-Cad mRNA concentrations in the caput, corpus, and cauda epididymidis increased in a dose-dependent manner with increasing testosterone concentrations. For bilaterally orchidectomized animals, mRNA concentrations for E-Cad in the caput epididymidis were less than 40% of those in the intact controls. In this segment, low dose testosterone replacement maintained the message at almost 75% of the control level, while high dose testosterone maintained the concentrations at intact control levels (Fig. 8). A similar pattern was observed for the cauda epididymidis. In the corpus epididymidis, mRNA concentrations for E-Cad were decreased by 90% after orchidectomy, and low dose testosterone could maintain E-Cad mRNA at only 40% of control levels; high dose testosterone administration increased mRNA concentrations for E-Cad to 120% of control levels (Fig. 8). Effects of unilateral orchidectomy

Experiments were performed with unilaterally orchidectomized rats to determine if circulating levels of tes-

Discussion For several tissues, the formation of epithelial tight junctions has been correlated with the presence of cadherins (7-12). Results from the present study indicate that the epididymis has E-Cad, but not N-Cad mRNA. These observations suggest the possibility that E-Cad may play a role in the formation and maintenance of tight junctions in the epididymis. Such a correlation is supported by the cellular localization of E-Cad in the epididymis, which is found exclusively in the principal cells. The tight junctions between these epithelial principal cells are responsible for the formation of the bloodepididymal barrier (l-3). Gumbiner and Simons (30) reported that an E-Cadlike protein is associated with the formation of an epithelial-occluding barrier in Mardin Darby canine kidney cell monolayers cultured on nitrocellulose filters. In their experiments they measured the electrical resistance across these epithelial cells as an indication of junctional integrity. They reported that the formation of junctions could be inhibited up to 54% by adding monoclonal antibodies to mouse E-Cad (Uvomorulin) in the medium. Since both kidney and epididymis are embryologically derived from the mesonephric duct, we may speculate that E-Cad serves a similar function in the epididymis as it does in these renal cells. Hybridization of total cellular RNA from the epididymis suggests that there may be more than one species of mRNA encoding E-Cad in the rat. This phenomenon has also been noted for the rat during fetal development (31). Previous studies in the mouse have indicated the presence of a single 4.7-kb E-Cad mRNA transcript (25, 32). While this is the first report identifying the presence of E-Cad mRNA in the epididymis, it also suggests that the rat epididymis may contain a RNA transcript slightly smaller than that of murine E-Cad. This smaller mRNA species may encode either a closely related isoform of ECad or a related cadherin. It is possible that this mRNA species arises from the intracellular processing of the

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CADHERIN

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Endo Voll30

l l

1992 No 1

FIG. 4. Low power light micrographs of initial segment (a), caput (b), corpus (c), and cauda (d) epididymidis. Epithelial principal cells (P), clear cells (C), intertubular connective tissue space (IT), lumen of the ducts (Lu), and spermatozoa (S) are shown. Magnification, ~128.

4.7-kb E-Cad transcript. Based on current observations, this 4.3-kb transcript appears to be regulated in the same fashion as the 4.7-kb transcript. The immunoperoxidase reaction observed along the epididymis establishes the presence of immunoreactive E-Cad in the principal cells. These data indicate that the epididymis not only contains E-Cad mRNA transcripts, but that the protein itself is made in this tissue. The localization of E-Cad in the principal cells suggests that these junctional proteins may play a role in maintaining the integrity of the blood-epididymal barrier. While the

histology of the blood-epididymal barrier is well characterized (4), this is the first report identifying a junctional protein in the rat epididymis. Light microscope immunocytochemistry revealed a regional difference in E-Cad intensity along the epididymis. The reaction was most intense in the principal cells of the corpus epididymidis and progressively weaker in the caput, cauda, and initial segment of the epididymis. These results correlate well with the results obtained for E-Cad mRNA levels in the different segments of the epididymis. In all regions of the epididymis, except the

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CADHERIN

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FIG. 5. High power light micrographs of initial segment (a), caput (b), corpus (c), and cauda (d) epididymidis. Epithelial principal cells (P), nuclei of principal cells (n), clear cells (C), intertubular connective tissue space (IT), lumen of the ducts (Lu), and spermatozoa (S) are shown. In a and b the brush borders of the principal cells are designated by asterisks, in c by arrows and in d by arrowheads. The basal regions of principal cells in d are designated by arrows. Magnification, X512.

cauda, immunolocalization was present throughout the cytoplasm. The weak reaction in the basal region of the cauda principal cells suggests that these cells may not be as actively involved in the synthesis of the E-Cad protein as in the other regions of the epididymis. Most surprising is the absence of staining in basal and clear cells of the epididymis. This suggests that these cells either do not synthesize E-Cad at all or make very little of it. This raises the question of the type of junctions and cell adhesion molecules existing either between these cells

and the principal cells or between adjacent clear cells. Not surprising is the absence of a reaction in halo cells, since these cells are migrating intraepithelial monocytes (17). The whole body tissue distribution of cadherins in the adult rat has not yet been reported. In other animals, ECad has been found in epithelial cells of mouse skin and kidney (10, 33) as well as in chicken and mouse liver cells (9, 31, 34). Since the epididymis is primarily composed of epithelial cells, it is not surprising to find E-

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CADHERIN

360

mRNA

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Endo.

Voll30. VENTRAL

700

A

1992 No 1

PROSTATE

1

600 500 400 300

4.71)

200 100 0 SEMINAL

700

VESICLES

600 500 400 300 200

B

100 0

18S-0

1000

,

EPIDIDYMIDES

600

100

-

80 TESTOSTERONE

(cm)

FIG. 7. Effects of testosterone replacement on the weights of paired epididymides, paired seminal vesicles, and ventral prostate of bilaterally orchidectomized rat. Bilaterally orchidectomized rats were given testoterone implants of 0,2.5, and 18.5 cm for 7 days. Control (C) rats were sham operated. Results are expressed as the mean f SEM (n = 8).

60

40

20

0

IMPLANT

-

Co Or FIG. 6. Steady state E-Cad mRNA in the caput epididymidis 3 days after bilateral orchidectomy. A, ‘*P-Labeled E-Cad probe was hybridized with RNA from sham-operated control (Co) and bilaterally orchi-

Cad mRNA present in this tissue. While the differential expression of E-Cad during embryonic development has been noted in the mouse (7,8), the factors regulating its expression are not known. Bilateral orchidectomy and testosterone replacement indicate that steady state mRNA concentrations for ECad in the adult are dependent on testosterone. Previous studies have demonstrated that a 2.5cm testosterone dectomized rats (Or). B, Blots were stripped and rehybridized with the 18s synthetic oligonucleotide probe. C, The radioautograms were scanned by laser densitometry (n = 2).

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CADHERIN

mRNA

IN THE

INITIAL SEGMENT m’c! 00crie

A

FIG. 8. Effects of bilateral orchidectomy and testosterone replacement on steady state E-Cad mRNA in all segments of the epididymis. Orchidectomized adult rats received testosterone implants of 0, 2.5, and 18.6 cm for 7 days. A, ‘*P-Labeled E-Cad probe was hybridized with the membrane containing RNA from intact controls (C) or those implanted with 0-, 2.5-, and 18.6~cm testosterone capsules. Blots were exposed for 24 h (caput and corpus epididymidis) or 4 days (initial segment and cauda epididymidis). B, Blots were stripped and rehybridized with the 18s synthetic oligonucleotide probe. C, The radioautograms were scanned by laser densitometry. Data are expressed as a percentage of the control value (mean + SEM; n = 3).

4.7+

RAT

EPIDIDYMIS

CAPUT lo’4

OOcu’$

361

CORPUS CD 00CUFse6

OOOi$

C 025

C 0 2.5 16.6

CAUDA m’4

:

B 18S-D

C

0 2.5

16.6

C

025

186

TESTOSTERONE

A

F1c.9. Effects of unilateral orchidectomy on steady state E-Cad mRNA in all segments of the epididymis. A, 32PLabeled E-Cad probe was hybridized with RNA from the left (L) and right (R) epididymal segments of control rats and left (L; contralateral side) and right (R; ipsilateral side) segments of unilaterally orchidectomized rat. B, Blots were stripped and rehybridized with the 18s synthetic oligonucleotide probe. C, The radioautograms were scanned by laser densitometry.

INITIAL SEGMENT Co Uni LR LR

CAPUT Co Uni LRLR

LR

LR

IMPLANT

166

km)

CORPUS Co Uni LRLR

CAUDA Co Uni LRLR ‘.‘ ” ;I ‘La, ., ia, .)

lSS+

Ii :

z E

CO

LR hi

implant can maintain circulating concentrations of testosterone similar to those normally found in blood (35). Our results would suggest that this concentration of testosterone is sufficient to maintain mRNA concentrations for E-Cad in all regions of the epididymis with the exception of the corpus segment. The reason why the

CO

LR Uni

LR CO

LR Uni

L

R

CO

L

R

Uni

corpus epididymidis should require a higher dose of testosterone to maintain its concentration of message for E-Cad is not clear, particularly in light of the unilateral orchidectomy study. It is well established that serum testosterone returns to control values within 1 day of unilateral orchidectomy

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(36-39). The results obtained after unilateral orchidectomy suggest that normal circulating concentrations of testosterone are sufficient to maintain control concentrations of mRNA for E-Cad. Similar results have been observed for the androgendependent epididymal protein B/C and proenkephalin. Garret et al. (40) have reported that the steady state mRNA concentrations for both proenkephalin and protein B/C were also decreased 7 days after castration and that testosterone replacement (2.5 cm) as well as unilateral orchidectomy could maintain mRNA concentrations for these two mRNA species. This is the first report indicating that cadherin mRNA concentrations (in this case E-Cad) can be regulated by testosterone. Previous studies have reported that an ECad-like protein in rat granulosa cells is stimulated in vitro by estradiol-induced folliculogenesis (41). Similar results have been reported in Xenopus, where progesterone-induced oocyte maturation can increase concentrations of cadherin-like protein, a protein thought to be the Xenopus analog of placental-cadherin (P-Cad) (42). Results from these studies clearly indicate that in the epididymis, the message for E-Cad is present, expressed, and regulated by testosterone. These observations are consistent with the hypothesis that the alterations in epididymal tight junctions that have been previously observed during orchidectomy (2) may be the result of alterations in the expression of E-Cad. Further studies will be needed to assess the role and regulation of E-Cad in maintaining the integrity of epididymal tight junctions. Acknowledgments We should like to thank Drs. M. Takeichi and R. Kemler for their generous gifts of cDNA probes and antiserum, respectively. R. Viger, F. Moussa, and J. Wright are thanked for their assistance.

References 1. Suzuki F, Nagano T 1978 Regional differentiation of cell junctions in the excurrent duct epithelium of the rat testis as revealed by freeze-fracture. Anat Ret 191:503-520 2. Suzuki F, Nagano T 1976 Changes in occluding junctions of epididymal epithelium in the develonina -- and -gonadectomized mammais. J Cell Biol 70:lOla 3. Suzuki F, Nagano T 1978 Development of tight junctions in the caput epididymal epithelium of the mouse. Dev Biol63:321-334 4. Agarwal A, Hoffer AP 1989 Ultrastructural studies on the development of the blood-epididymis barrier in immature rats. J Androl l&425-431 5. Hinton BT 1985 The blood-epididymis barrier. In: Lob1 TJ, Hafez ESE (edsl Male Fertilitv and Its Rezulation. MTP Press. Boston. pp 371-382 6. Hoffer AB, Hinton BT 1984 Morphological evidence for a blood epididymis barrier and the effects of gossypol on its integrity. Biol Reprod 30:991-1004 7. Takeichi M 1990 Cadherins: a molecular family important in selective cell-cell adhesion. Annu Rev Biochem 59:237-252

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8. Takeichi M 1987 Cadherins: a molecular family essential for selective cell-cell adhesion and animal morphogenesis. Trends in Genetics 3:213-217 9. Ogou SI, Yoshida-Noro C, Takeichi M 1983 Calcium-dependent cell-cell adhesion molecules common to hepatocytes and teratocarcinema stem cells. J Cell Biol97:944-948 10. Vestweber D, Kemler R, Ekblom P 1985 Cell-adhesion molecule uvomorulin during kidney development. Dev Biol112:213-221 11. Bollier K, Vestweber D, Kemler R 1985 Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells. J Cell Biol 100~327-332 12. Takeichi M 1988 The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development 102:639-655 13. Ringwald M, Shuh R, Vestweber D, Eistetter H, Lottspeich F, Engel J, Dolz R, Jahnig F, Epplen J, Mayer S, Muller C, Kemler R 1987 The structure of cell adhesion molecule uvomorulin. Insights into the mechanism of Ca-dependent cell adhesion. EMBO J 63647-3653 14. Hatta K, Nose A, Nagafuchi A, Takeichi M 1988 Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family. J Cell Biol 106873881 15. Hatta K, Takeichi M 1986 Expression of N-cadherin adhesion molecules associated with early morphogenic events in chick development. Nature 320:447-449 16. Miyatani S, Shimamura K, Hatta M, Nagafuchi A, Nose A, Matsunaga M, Hatta K, Takeichi M 1989 Neural cadherin: role in selective cell-cell adhesion. Science 245~631-635 17. Robaire B, Hermo L 1988 Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. In: Knobil E, Neil1 J (eds) The Physiology of Reproduction. Raven Press, New York, pp 999-1079 18. Rutishauser U 1989 N-Cadherin: a cell adhesion molecule in neural development. Trends Neurosci 12:275-276 19. Matsunaga M, Hatta K, Nagafuchi A, Takeichi M 1988 Guidance of optic nerve by N-cadherin adhesion molecules. Nature 334:6264 20. Dalsag AM, Andersson AM, Bock H 1990 Characterization of Ncadherin mRNA in chicken brain and heart by means of oligonucleotide probes. FEBS Letts 262234-236 21. Stratton IG, Ewing LL, Desjardins C 1973 Efficacy of testosteronefilled polydimethylsiloxane implants in maintaining plasma testosterone in rabbits. J Reprod Fertil35:235-244 22. Brawer JR, Schipper H, Robaire B 1983 Effects of long term androgen and estradiol exposure on the hypothalamus. Endocrinology 112:194-199 23. Hales BF, Hachey C, Robaire B 1980 The presence and longitudinal distribution of the glutathione S-transferases in rat epididymis and vas deferens. Biochem J 189135-142 24. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning-A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor 25. Nagafuchi A, Shirayoshi Y, Okazaki K, Yasuda K, Takeichi M 1987 Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature 329:341-343 26. Szyf M, Milstone DS, Schimmer BP, Parker KL, Seidman JG 1990 Cis modification of the steroid 21-hydroxylase gene prevents its expression in the mouse adrenocortical tumor cell line. Mol Endocrinol4:1144-1152 27. Viger RS, Robaire B 1991 Differential regulation of steady-state 4ene steroid 5cy-reductase messenger ribonucleic acid levels along the rat epididymis. Endocrinology 1282407-2414 28. Hermo L, Wright J, Oko R, Morales CR 1991 Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin). Biol Reprod 44:1113-1131 29. Straus W 1982 Imidazole increases the sensitivity of cytochemical reaction for peroxidase with diaminobenzidine at neutral pH. J Histochem Cytochem 30:491-493 __ --. I .. _^. 30. tiumbmer B, Yimons K 1986 A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide. J Cell Biol 102:457-468

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CADHERIN mRNA IN THE RAT EPIDIDYMIS 31. Chen B, Blaschuk OW, Hales BF 1991 Cadherin mRNAs during rat embryo development in viva and in vitro. Teratology 44:581590 32. Gallin WJ, Sorkin BC, Edelman GM, Cunnigham BA 1987 Sequence analysis of a cDNA clone encoding the liver cell adhesion molecule, L-CAM. Proc Nat1 Acad Sci USA 84~28082812 33. Hirai Y, Nose A, Koboyashi S, Takeichi M 1989 Expression role of E- and P-cadherin in embryonic hi&genesis. II. Skin morphogenesis. Development 105271-277 34. Sorkin BC, Hemperly JJ, Edelman GM, Cunningham BA 1988 Structure of the gene for the liver cell adhesion molecule, L-CAM. Proc Nat1 Acad Sci USA 85~7617-7621 35. Robaire B, Ewing LL, Irby DC, Desjardins C 1979 Interaction of testosterone and estradiol-17fi on the reproductive tract of the male rat. Biol Reprod 21:455-463 36. Lindgren S, Damber JE, Carsten H 1976 Compensatory testosterone secretion in unilaterally orchidectomized rats. Life Sci l&1203-

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1206 37. Mock EJ, Frankel AI 1982 Response of testosterone to hemicastration in the testicular vein of the mature rat. J Endocrinol92:231236 38. Moger WH, Anakwe 00 1986 Propanolol inhibits the compensatory increase in androgen secretion after unilateral orchidectomy in rats. J Reprod Fertil76:251-256 39. Robaire B 1979 Effect of unilateral orchidectomy on rat epididymal A’-5cY-reductase and 3cY-hydroxysteroid dehydrogenase. Can J Physiol Pharmacol57:999-1003 40. Garrett JE, Garrett SH, Douglass J 1990 A spermatozoa-associated factor regulates proenkephalin gene expression in the rat epididymis. Mol Endocrinol4:108-118 41. Blaschuk OW. Farookhi R 1989 E&radio1 stimulates cadherin expression in rat granulosa cells. Dev Biol 136564-567 42. Choi Y, Sehegal R, McCrea P, Gumbiner B 1990 A cadherin-like protein in eggs and cleaving embryos of Xenopus locvis is expressed in oocytes in response to progesterone. J Cell Biol 110:1575-1582

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