help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2002-0139
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by DeBellefeuille, S.
Right arrow Articles by Cyr, D. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by DeBellefeuille, S.
Right arrow Articles by Cyr, D. G.
Endocrinology Vol. 144, No. 11 5040-5049
Copyright © 2003 by The Endocrine Society

Catenins in the Rat Epididymis: Their Expression and Regulation in Adulthood and during Postnatal Development

Sophie DeBellefeuille, Louis Hermo, Mary Gregory, Julie Dufresne and Daniel G. Cyr

Institut National de la Recherche Scientifique-Institut Armand Frappier (S.D., M.G., J.D., D.G.C.), Université du Québec, Pointe Claire, Québec, Canada H9R 1G6; and Department of Anatomy and Cell Biology, McGill University (L.H., D.G.C.), Montréal, Québec, Canada H3A 2B2

Address all correspondence and requests for reprints to: Dr. Daniel G. Cyr, Institut National de la Recherche Scientifique-Institut Armand Frappier, Université du Québec, 245 Hymus boulevard, Pointe Claire, Québec, Canada H9R 1G6. E-mail: daniel.cyr{at}inrs-iaf.uquebec.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tight and adhering junctions are important in maintaining the integrity of the epididymal epithelium and formation of the blood epididymal barrier, which are crucial for sperm maturation and storage. The composition of the catenin-adhering junctional family of proteins and their relationship with tight junctions remain to be established in the epididymis. In the normal adult rat epididymis, immunostaining for three anticatenin antibodies ({alpha}, ß-, and p120ctn) was noted along the lateral plasma membranes (LPM) between adjacent epithelial cells. Although {alpha}-catenin and ß-catenin were maximally expressed in the corpus and cauda epididymis, p120 expression was intense and similar in all epididymal regions. Bilateral orchidectomy of adult rats indicated that the expression of p120 at the LPM was not altered compared with that in control animals. On the other hand, staining at the LPM for {alpha}- and ß-catenin was markedly reduced, concomitant with an increased cytoplasmic reaction in each epididymal region. As the staining pattern for {alpha}- and ß-catenin returned to that seen in control animals after testosterone supplementation, it is suggested that their localization and targeting to the LPM are regulated by androgens. This is confirmed by postnatal studies in which maximal expression at the LPM for each catenin occurs by d 49, when androgen levels are adult-like. Immunolocalization of zona occludens-1 along with immunoprecipitation of epididymal homogenates of the initial segment/caput region of the epididymis revealed that zona occludens-1 is an integral part of the adhering junctional complex in young rats and coprecipitates with ß-catenin at the level of the apical tight junctions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FORMATION OF mature spermatozoa involves not only their development in the testis, but also their maturation in the epididymis, where they acquire progressive motility and fertilizing ability (1, 2). The maturation of spermatozoa in the epididymis is dependent on a number of factors, including the interaction of spermatozoa with proteins and lipids secreted in a merocrine or apocrine manner, the glycosylation of proteins by Golgi saccules present in cytoplasmic droplets of sperm, as well as establishing a specific microenvironment of the lumen of the epididymis (3, 4, 5, 6, 7). The development and maintenance of a luminal microenvironment necessary for sperm maturation are created by the blood-epididymal barrier, which is comprised of tight junctions between adjacent principal cells that border the lumen of the epididymal duct (8, 9).

The formation of tight junctions between adjacent epithelial cells requires that cells first adhere to one another and form adhering junctions (9, 10, 11, 12). Tight junctions are composed of occludin, claudins, and a number of cytoplasmic plaque proteins, including proteins that contain PDZ domains (e.g. zonula occludens, membrane-associated guanylate kinase inverted protein, and ALL-1 gene fusion partner), non-PDZ proteins (e.g. cingulin, symplekin, ZO-1-associated nucleic acid binding protein, and achaetic-scute homolog-1), cytoskeletal proteins, and GTP-binding proteins (13). Adhering junctions are composed of cadherins and cytoplasmic proteins known as catenins, which link cadherins to the cytoskeleton (14). Adhering junctions not only enable the cells to stick to one another, but also play a role in intracellular signaling, and they appear to be involved in the positioning and assembly of tight junctions (15, 16).

Cadherins are calcium-dependent, single-pass, transmembrane proteins involved in cell adhesion. At present, there are over 50 members of the cadherin family derived from a multigene family (17). In the adult rat epididymis, Cyr and collaborators (18, 19) first reported the presence of both E- and P-cadherin in the epididymis. E-cadherin was localized to the lateral plasma membranes (LPM) of adjacent epithelial principal cells (20). Postnatal developmental studies demonstrated that mRNA levels for E-cadherin correlated well with the formation of the blood-epididymal barrier. Furthermore, it was shown that E-cadherin mRNA levels were androgen dependant in all segments of the epididymis (19).

On the other hand, catenins are a small multigene family of cytoplasmic proteins that act as linker proteins for cadherins in the adhering junctions (14, 21, 22, 23, 24). To date, several members of the catenin family of proteins have been described. However, although studies of the expression and regulation of cadherins in the epididymis (19, 20) have been documented, at present little information exists regarding the expression and regulation of catenins in the epididymis.

From a functional point of view, it has been proposed that the formation of tight junctions first involves the formation of cadherin-based cell adhesion (10, 14). This is followed by the recruitment of the tight junctional cytoplasmic protein zona occludens-1 (ZO-1) to the LPM of the cells via direct interaction with the catenins ({alpha} and ß) of the adhering junction.

It was therefore of interest to examine the expression of catenins in the adult epididymis, follow their expression during postnatal development, determine whether they are regulated by androgens, and assess whether catenins associate with ZO-1 during the formation of the blood-epididymal barrier during postnatal development and remain as such in adult rats. This study was accomplished employing light microscope immunocytochemistry, immunoprecipitation, and Western blot analysis of epididymal tissues of adult and postnatal rats of different ages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague Dawley rats were purchased from Charles River Canada (St. Constant, Canada). Rats were maintained under a photoperiod of 12 h of light, 12 h of darkness and received food and water ad libitum. All protocols used in this study were approved by the university animal care committee.

Androgen regulation
To assess whether epididymal catenins were regulated by testicular factors or androgens, adult rats (Sprague Dawley, 350–400 g), obtained from Charles River Canada were anesthetized with an ip injection of ketamine/xylazine (50/10 mg/kg). Four sham-operated rats were used as controls. Four rats were bilaterally orchidectomized via an abdominal incision and killed 14 d later. Eight other orchidectomized rats were implanted with either an empty 2.5-cm capsule (four rats) or 18.6-cm capsules (three, measuring 6.2 cm each; four rats) containing testosterone. Testosterone-filled polydimethylsiloxane capsules were prepared according to the method outlined by Stratton et al. (25) and have well characterized steroid release rates (26). The latter mimic epididymal (18.6 cm) testosterone levels, which are 10 times greater than blood levels. To ensure that the newly made capsules would have a constant testosterone release rate and that the initial surge of testosterone release would be complete at the time of implantation in orchidectomized rats, additional carrier rats were implanted with the testosterone implants before the start of the experiment. These implants were removed from the carrier rats 3 d later, cleaned, and inserted sc on the backs of experimental animals at the time of orchidectomy. All of these rats were killed 14 d after surgery. At the completion of each experimental design, the epididymides were fixed by perfusion with Bouin’s fixative, removed from the rats, and subsequently processed as described below for immunocytochemical analyses.

Postnatal development
To determine the postnatal developmental expression of {alpha}-catenin, ß-catenin, and p120ctn in the epididymis, timed gestation female rats were purchased from Charles River Canada, Inc. At the time of birth, the sex of each pup was determined, and random litters of 10 male pups were placed with each lactating mother. Rats were weaned at 24 d of age. Rats were anesthetized with somnitol, and the epididymides of 7-, 21-, 29-, 49-, 56-, and 91-d-old rats were then fixed by retrograde perfusion through the abdominal aorta with Bouin’s fixative as previously described (19). Fixed epididymides were dehydrated in graded ethanol, cleared in xylene, and mounted in paraffin for light microscope immunocytochemical analysis.

Immunocytochemical analysis
Immunoperoxidase staining of epididymal sections was performed according to previously published protocols (27). Immunolocalization of catenins was performed using goat polyclonal antisera raised against {alpha}-catenin, ß-catenin, or a rabbit polyclonal anti-p120ctn (2.7, 2.7, and 2.0 µg/ml, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibody binding to each catenin was detected using either an antigoat or an antirabbit horseradish peroxidase-conjugated secondary antibody (1:250; Sigma-Aldrich Corp., Mississauga, Canada). To demonstrate the specificity of the catenin antisera, some slides were incubated with both the catenin primary antibody and the corresponding blocking peptide (Santa Cruz Biotechnology, Inc.). For these analyses, the blocking peptide was diluted with buffer such that the final concentration of blocking peptide was 2 mg/ml. The mixture of diluted blocking peptide and catenin antisera was preincubated at 37 C for l h before incubation with the tissue, according to standard protocol. Negative control slides, in which there was no primary antibody, were also performed concurrently.

ZO-1 immunocytochemical localization in the adult rat epididymis was performed using frozen sections. Epididymides were carefully dissected out, placed in OCT-Cryomatrix (Fisher Scientific, Ottawa, Canada), and frozen on dry ice. Solidified blocks of tissue were then stored at -86 C until needed. Ten-micron-thick sections were cut, mounted onto glass slides, and stored at -20 C until needed for immunocytochemistry. Slides were fixed in methanol for 20 min at -20 C, allowed to air-dry, rehydrated with PBS for 30 min at room temperature, and then blocked in buffer (PBS, 3% BSA, and 5% goat serum) for 20 min at room temperature. This was followed by three 5-min washes in PBS. Immunocytochemical localization of ZO-1 was performed using rabbit polyclonal ZO-1 antisera (5 µg/ml; Zymed Laboratories, South San Francisco, CA). Sections were incubated for 90 min in a hydrated chamber with the primary antibody at room temperature. The sections were then washed in PBS and incubated for 45 min with a fluorescein isothiocyanate-conjugated antirabbit secondary antibody (1:250; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The sections were subsequently washed three times in PBS and mounted with Vectashield (Vectastain Laboratories, Inc., Burlington, Canada). Slides incubated with normal rabbit antiserum were used as a negative control, because immunoabsorption was not possible due to the lack of antigen.

For ß-catenin and ZO-1 colocalization studies, frozen sections were prepared as described above, incubated with the ß-catenin primary antibody (1:200; Chemicon International, Inc., Temecula, CA) and a Texas Red-conjugated antirabbit secondary antibody (1:250 dilution; Jackson ImmunoResearch Laboratories, Inc.) as described above for ZO-1, and washed for 5 min with PBS. Sections were then incubated with ZO-1 mouse monoclonal primary antiserum (1:200; Chemicon International, Inc.) for 1 h at room temperature and subsequently incubated with fluorescein isothiocyanate-conjugated antimouse secondary antiserum (1:250; Jackson ImmunoResearch Laboratories, Inc.). Sections were then washed three times in PBS for 5 min each time at room temperature, mounted with Vectastain Mounting Medium (Vectastain Laboratories, Inc) containing 4',6-diamido-2-phenylindole hydrochloride to visualize the nuclei, and stored at 4 C. Sections were viewed with a Leica fluorescent microscope (Deerfield, IL). The images were digitalized, merged, and analyzed using ImagePro Plus software (version 4.0, Media Cybernetics, Silver Spring, MD).

Immunoblotting
Proteins were isolated from frozen segments (initial segment, caput, corpus, and cauda) of three pools of four adult epididymis and crushed under liquid nitrogen using a mortar and pestle. To determine epididymal ß-catenin levels at different developmental ages, epididymides of 7 (n = 13), 21 (n = 6), 42 (n = 3), or 91 (n = 3)-d-old rats were pooled. The tissue was then homogenized with a Polytron (Brinkmann Instruments, Inc., West Orange, NJ) at 4 C in lysis buffer [1:3, wt/vol; 60 mM Tris-Cl (pH 6.8), 2 mM CaCl2, 40 mM ß-octylglucopyranoside, and 20 µg/ml pepstatin A; Sigma-Aldrich Corp.] supplemented with a protease inhibitor cocktail (Sigma-Aldrich Corp.). Samples were transferred to microcentrifuge tubes and centrifuged at 2,200 x g for 30 min at 4 C. The supernatant, containing the plasma membrane fraction, was then centrifuged at 30,000 x g for 30 min at 4 C, and the resulting pellet was resuspended in the same buffer. Protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Mississauga, Canada).

Membrane protein samples (50 µg) were diluted in loading buffer (Laemmli buffer), boiled for 5 min, and cooled on ice. The samples were loaded onto either a 6.5% (for ZO-1 immunoblotting) or a 7.5% polyacrylamide gel (28). Electrophoresis was performed at 80 V for 1.5–2 h until the dye front reached the end of the gel. Proteins were subsequently transferred onto a nitrocellulose membrane using a Bio-Rad Transblot apparatus at 100 V for 1 h. Colored molecular weight markers were used to assess the efficiency of the transfer. The blots were blocked overnight in TBST [20 mM Tris-HCl, 500 mM NaCl (pH 7.5), and 0.1% Tween 20] containing 5% powdered milk and then incubated for 60 min at room temperature with an antiactin primary antibody in the same buffer (2.0 µg/ml; Santa Cruz Biotechnology, Inc.). The blots were rinsed briefly in TBST and incubated for 60 min at room temperature with the primary antibodies against {alpha}-catenin, ß-catenin, or p120ctn (1.0 µg/ml, respectively; Santa Cruz Biotechnology, Inc.) or ZO-1 (1.75 µg/ml; Zymed Laboratories, South San Francisco, CA) in blocking buffer. After the incubation, the membranes were washed three times for 10 min each time with TBST and then incubated for 60 min at room temperature with an antigoat or an antirabbit alkaline phosphatase-conjugated secondary antibody (0.4 µg/ml; Santa Cruz Biotechnology, Inc.). The blots were washed three times for 10 min each time with TBST, and the presence of each protein was revealed using the AP Conjugate Substrate Kit (BioRad Laboratories, Inc.). Each catenin level was quantified with a Fluor Image analyzer (Bio-Rad Laboratories, Inc.) and standardized against the signal for the actin protein.

Immunoprecipitation
Epididymal proteins were isolated from 7-, 18-, and 91-d-old rats (four per age group) as described above and used to immunoprecipitate ß-catenin and ZO-1. For the ZO-1 immunoprecipitation, a rat monoclonal antibody to ZO-1 (American Tissue Type Culture, Washington, D.C.) was used. Epididymal proteins (800 µg) were immunoprecipitated with 5 or 15 µl undiluted primary antiserum (ß-catenin and ZO-1, respectively) in 1 ml PBS. The proteins and antisera were incubated for 1 h at 4 C in a rotating tube. At the end of the incubation, 20 µl protein G plus agarose conjugate (Santa Cruz Biotechnology, Inc.) were added to the proteins and incubated overnight at 4 C in a rotating tube. Samples were then centrifuged at 1000 x g for 5 min at 4 C to recover the agarose beads. The supernatant was discarded, and the pellet of agarose beads was washed once in PBS, three times in wash buffer I [500 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.05% Nonidet P-40, and 0.2% BSA], and twice in wash buffer II [50 mM Tris-HCl (pH 7.5)]. Agarose beads were recovered after each wash by centrifugation at 1000 x g for 5 min at 4 C. The resulting pellets were then boiled in sample buffer for 5 min and subjected to electrophoresis and immunoblotting as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of catenins in the adult rat epididymis
Although the three isoforms of catenin were expressed in the adult rat epididymis, a variable degree of immunostaining was observed for each depending on the region of the epididymis in which they were expressed. With all three antibodies, a reaction was noted throughout the epididymis along the LPM of adjacent epithelial principal cells (Fig. 1Go, A–C), suggesting their role as part of the adhering junctions. Furthermore, in the initial segment of the epididymis, these three proteins were also present at the lateral interface between adjacent principal, narrow, and apical cells, whereas in the caput, corpus, and cauda epididymis, staining was observed between adjacent principal and clear cells (not shown). Although evenly distributed along the LPM in the caput, corpus, and cauda epididymis, ß-catenin immunoreaction appeared to be most intense in the upper or apical part of the lateral plasma membrane in the initial segment of the epididymis (Fig. 1BGo). In the case of p120ctn, an immunoreaction was observed in all epididymal regions (Fig. 1CGo), whereas the {alpha}-catenin immunoreaction appeared to be distributed evenly along the lateral plasma membrane of epithelial cells lining the epididymal lumen (Fig. 1AGo). Nevertheless, the fact that all three junctional proteins colocalized together in varying degrees suggests that they form complex interconnecting components of the adhering junctions between adjacent epithelial cells.



View larger version (122K):
[in this window]
[in a new window]
  		FIG. 1. A–D, Immunolocalization of {alpha}-catenin (A), ß-catenin (B), p120ctn (C), and ZO-1 (D) in the initial segment of the adult rat epididymis. Of the three catenin antibodies, p120ctn (C) shows the most intense reaction in the initial segment, where it is localized along the LPM (arrows) of adjacent principal cells (P), with a moderate immunoperoxidase reaction being noted for {alpha}-catenin (A). Immunostaining for ß-catenin (B) is also observed along the LPM of adjacent principal cells; however, the reaction is seen to be more intense apically (arrows). ZO-1 (D) is localized to the apical region (arrows) of principal cells (P) and not along the LPM. Epididymal sections were blocked with peptides for each catenin to establish the specificity of the immunoreaction. A ß-catenin blocking peptide was used to block the anti-ß-catenin antisera (E). Similar results were obtained for each of the other antisera. IT, Intertubular space; L, lumen; n, nuclei; S, spermatozoa. Magnification, x640.

 
In the adult rat epididymis, ZO-1 was localized exclusively to the apical region of the epithelium in the area of the epididymal tight junctions and not along the lateral plasma membrane (Fig. 1DGo). Blocking peptides for each of the three catenins were used as negative controls. For each antiserum, the immunostaining was abolished with the peptides, as shown for ß-catenin (Fig. 1EGo).

Composition of epididymal adhering junctions
To corroborate the finding of each catenin in the different epididymal regions, Western blot analysis was performed on homogenized epididymides of adult rats. Western blot analysis of ZO-1 was also performed, as this protein has been shown to be present in the adult epididymis and to associate in vitro with adhering junctions during the assembly of tight junctions. A single protein band of 102 kDa was obtained for {alpha}-catenin in each epididymal segment (Fig. 2AGo). {alpha}-Catenin levels were similar in the different epididymal regions (Fig. 2AGo). In comparison, there was a single band of 92 kDa for ß-catenin, with the most intense band being present in the corpus and cauda region (Fig. 2BGo). In the case of p120ctn (Fig. 2CGo), several protein bands, ranging from 115–125 kDa, which represent the multiple phosphorylated forms of p120ctn, were obtained, with the most intense bands being present in the corpus epididymis. A single 225-kDa band was obtained for ZO-1, and levels appeared to be most abundant in the corpus epididymis, whereas levels were similar in the initial segment, caput, and corpus epididymis (Fig. 2DGo).



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 2. Western blot analysis of {alpha}-catenin (A), ß-catenin (B), p120ctn (C), and ZO-1 (D) in the initial segment (IS), caput (CT), corpus (CS), and cauda (CA) epididymis of adult rats. A 50-µg aliquot of membrane-enriched epididymal proteins was isolated by centrifugation and separated by PAGE. Proteins were then transferred onto a nitrocellulose membrane and visualized using primary antisera as well as actin. Data are expressed as a percentage of the IS ± SEM from three blots of protein pooled from epididymal tissue of four rats.

 
Immunoprecipitation of ß-catenin was performed to demonstrate whether all three catenins were associated with one another in the epididymis (Fig. 3Go). Using the initial segment/caput region of the epididymis of adult rats, membrane-enriched protein fractions were isolated and immunoprecipitated with the ß-catenin antiserum. Western blots were subsequently performed on electrophoretically separated immunoprecipitated proteins with {alpha}-catenin and p120ctn. To confirm that these proteins were part of the adhering junctions, E-cadherin was also measured in the immunoprecipitated proteins. Results from these experiments revealed that {alpha}-catenin and p120ctn as well as E-cadherin, all at their appropriate molecular weights, are associated with ß-catenin in the adult epididymis.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3. Immunoprecipitation of ß-catenin in the initial segment/caput epididymis of adult rats. ß-Catenin was immunoprecipitated as described in Materials and Methods, and immunoprecipitated proteins were separated by PAGE. Proteins were then transferred onto a nitrocellulose membrane. Blots were screened for E-cadherin (E-Cad), {alpha}-catenin, ß-catenin, and p120ctn using specific antisera for each of these proteins. The results demonstrated that each of these proteins, which comprise the adhering junctions, coprecipitated with ß-catenin.

 
Regulation of catenins by androgens
Bilateral orchidectomy of adult rats sampled 14 d later revealed that the immunostaining pattern for {alpha}- and ß-catenin was dramatically altered in the absence of testicular factors (Fig. 4Go, A and B). In the case of {alpha}- and ß-catenin, the reaction along the LPM of adjacent epithelial cells was markedly reduced compared with that in control animals (Fig. 4Go, A and B). In addition, an intense cytoplasmic reaction was observed in each epididymal region. Interestingly, the staining for p120ctn did not appear to be altered along the LPM, and no conspicuous cytoplasmic reaction was noted in any epididymal region (Fig. 4CGo). The immunostaining pattern for {alpha}- and ß-catenin was comparable to that noted for control rats in those animals that received immediate testosterone replacement at the time of surgery (Fig. 4DGo). Blocking peptides for each of the three catenins were used as negative controls. For each antiserum the immunostaining in sections from orchidectomized rats was abolished with the peptides, as shown for ß-catenin (Fig. 4EGo).



View larger version (132K):
[in this window]
[in a new window]
  		FIG. 4. Regulation of catenins in adult rat epididymis; only the caput epididymis is illustrated. Rats were orchidectomized and sampled 14 d after surgery. Immunostaining for {alpha}-catenin (A) and ß-catenin (B) was altered in orchidectomized rats. In these animals, {alpha}-catenin and ß-catenin immunostaining was less prominent along the LPM (arrows) of adjacent principal cells (P). However, an intense cytoplasmic reaction was observed in each of the four epididymal segments in orchidectomized animals. Unlike {alpha}-catenin and ß-catenin immunostaining, p120ctn (C) appeared to be unaltered by orchidectomy in any of the four epididymal segments. Orchidectomized rats that received immediate testosterone implants revealed staining along the LPM for {alpha}-catenin (D) comparable to that observed in intact control rats. A ß-catenin-blocking peptide was used to block the anti-ß-catenin antiserum in orchidectomized rats (E). Similar results were obtained for {alpha}-catenin and P120ctn (not shown). IT, Intertubular space; L, lumen; n, nucleus; S, spermatozoa. Magnification: A–D, x640; E, x1024.

 
Modulation of catenin expression during postnatal development
Postnatal immunocytochemical studies revealed that all three catenins were already expressed by postnatal d 7 (Fig. 5Go, A–C). At this age, localization was present along the LPM between the adjacent undifferentiated epithelial cells. Differences in immunostaining between each catenin were not apparent, nor were differences among the various epididymal regions for a given catenin. However, the intensity of the immunoperoxidase reaction product appeared to increase as a function of age and was similar to that in adults by postnatal d 49 (Fig. 5DGo), when circulating androgen levels become markedly elevated.



View larger version (120K):
[in this window]
[in a new window]
  		FIG. 5. Immunocytochemical localization of catenins in the epididymis of rats on postnatal d 7 (caput epididymis is illustrated). Immunolocalization of {alpha}-catenin (A), ß-catenin (B), and p120ctn (C) reveals that all three catenins are already present along the LPM of adjacent undifferentiated principal cells (P) at this early age, as indicated by arrows; the intensity of the reaction is similar throughout the entire epididymis for each catenin. In addition, although the intensity of the reaction increases with age in each segment, the distribution of the reaction product does not change with age. By d 49, immunostaining in all segments and for each catenin is comparable to that in 90-d-old adult animals. As an example, the caput epididymis of a 49-d-old rat is shown in D. IT, Intertubular space; L, lumen. Magnification, x640.

 
To determine whether ß-catenin levels changed as a function of age in the entire epididymis, Western blot analyses were performed on pools of membrane-enriched proteins from epididymides of 7-, 21-, 42-, and 91-d-old rats (Fig. 6Go). The results indicated that ß-catenin levels were low on d 7 and increased approximately one third by d 21; levels remained constant thereafter.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 6. Western blot analysis of ß-catenin in epididymides of 7-, 21-, 42-, and 91-d-old rats. A 50-µg aliquot of membrane-enriched epididymal proteins was isolated by centrifugation and separated by SDS-PAGE. Proteins were then transferred onto a nitrocellulose membrane and visualized using primary antisera for ß-catenin as well as actin. The results revealed that ß-catenin levels in the epididymis increase as a function of age, particularly in 42- and 91-d-old rats. Data are expressed as the ratio of ß-catenin to actin for a pool of epididymides from 7 (n = 13), 21 (n = 6), 42 (n = 3), and 91 (n = 3)-d-old rats.

 
Association of ß-catenin and ZO-1 during formation of the blood-epididymal barrier
To determine whether ZO-1 and ß-catenin associate during the formation of the blood-epididymal barrier, immunoprecipitation of epididymal extracted proteins was performed with ZO-1 and ß-catenin (Fig. 7Go). The results demonstrated that ZO-1 was associated with ß-catenin in the epididymis. This association appeared to be more extensive in young rats (7 and 18 d old) than in adult rats, i.e. at a time during formation of the blood-epididymal barrier (Fig. 7Go, I-A and II-A). The presence of ZO-1 and ß-catenin in their respective immunoprecipitates indicates that these were present at each age and that the levels did not decrease as a function of age (Fig. 7Go, I-B and II-B). Therefore, these data suggest that ZO-1 and components of the adhering junctions associate with each other during epididymal tight junction assembly.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 7. Immunoprecipitation of ZO-1 (I) and ß-catenin (II) from protein samples isolated from enriched membrane fractions from the initial segment/caput region of postnatal 7-, 18-, and 91-d-old rats. ZO-1 immunoprecipitates were screened for ß-catenin (IA) and ZO-1(IB), whereas ß-catenin immunoprecipitates were screened for ZO-1 (IIA) and ß-catenin (IIB). The results demonstrate that ZO-1 and ß-catenin closely associate with each other during formation of the blood-epididymal barrier, as demonstrated by the intense immunoblot reactions for both ZO-1 and ß-catenin in the ß-catenin and ZO-1 respective immunoprecipitates. The association between ß-catenin and ZO-1 appears to decrease in older animals, as indicated by the fainter reactive bands for each protein in 91-d-old rats.

 
Immunofluorescent localization of ß-catenin (Fig. 8Go, A and D) and ZO-1 (Fig. 8Go, B and E) and their colocalization (Fig. 8Go, C and F) on postnatal d 21 (Fig. 8Go, A–C) and in 90-d-old adult rats (Fig. 8Go, D–F) revealed the following. On d 21, ß-catenin (Fig. 8AGo) and ZO-1 (Fig. 8BGo) were localized apically and laterally between adjacent epithelial cells, and merged photomicrographs of the two revealed that they colocalized together at these sites (Fig. 8CGo), supporting the observation that ß-catenin and ZO-1 colocalize in young rats. In adult rats, ß-catenin was localized apically and laterally between adjacent epithelial cells (Fig. 8DGo), whereas ZO-1 was noted apically (Fig. 8EGo). Colocalization of the two revealed their shared localization apically in the area of the tight junction, whereas only a pronounced ß-catenin immunostaining was noted below the tight junctional complex (Fig. 8FGo).



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 8. Immunofluorescent localization of ß-catenin (A and D), ZO-1 (B and E), and their colocalization (C and F) in the caput epididymis of postnatal d 21 (A–C) and 90-d-old adult (D–F) rats. Cryosections (10 µm) were incubated with both ß-catenin and ZO-1 antisera and localized with a Texas Red (red stain) or FITC-conjugated (green stain) secondary antibody, respectively. In 21-d-old rats, ß-catenin (A) and ZO-1 (B) outline the apical and lateral borders of the epithelial cells (E) lining the epididymal duct (arrows). In merged photomicrographs (C), both ß-catenin and ZO-1 colocalize with each other along the apical and LPM of the epithelial cells (yellow stain). In adult rats, ß-catenin (D) is localized apically and laterally (arrows), whereas ZO-1 (E) is seen apically (arrows) between adjacent principal cells (P). Merged photomicrographs (F) demonstrate that ß-catenin and ZO-1 colocalize apically (yellow stain, white arrows) in the area of the tight junction. However, pronounced ß-catenin immunostaining (red stain, yellow arrows) is also present along the LPM between adjacent epithelial cells below the tight junctional complex, independent of ZO-1 localization. n, Nuclei; Lu, lumen. Magnification, x650.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Formation of the blood-epididymal barrier creates a microenvironment essential for sperm maturation as well as for protecting sperm from the immune system, as they are antigenic. The formation of the blood-epididymal barrier is likely to involve both adhering junctions, which are necessary for cell adhesion and intercellular signaling, as well as tight junctions, which form the seal between adjacent epithelial cells. Therefore, an understanding of the composition and regulation of the various proteins involved in formation of the blood-epididymal barrier is a major concern of male reproductive biology.

In the present study all three catenins ({alpha}-catenin, ß-catenin, and p120ctn) were present in the epididymis and were localized along the LPM of adjacent epithelial cells. However, region-specific differences were observed with LPM immunocytochemistry and with Western blot analysis. {alpha}-Catenin and ß-catenin were most prominent in the corpus region, whereas p120ctn appeared to be prominent in all epididymal regions, suggesting segment-specific requirements for catenins along the epididymis. The precise role of catenins in the different regions is unclear; however, electron microscopic analysis revealed that the tight junctions of the corpus and cauda were not as extensive as those seen in the initial segment/caput regions, suggesting a need for more adhesion in these regions (8). These data are further supported by the finding of significantly greater E-cadherin mRNA in the caput and corpus regions (19).

The immunoprecipitation data of the present study suggest that all three catenins interact with E-cadherin and form part of the adhering junctions in the epididymis. Previous studies have reported that in epithelial cells, catenins may play different roles, such as linking cadherins to the cytoskeleton or in intracellular signaling pathways. The interaction of the cadherin-catenin complex with the actin-based cytoskeleton through {alpha}-catenin is necessary for cadherin-based cell adhesion activity (14). {alpha}-Catenin associates with the COOH-terminal end of the cadherin cytoplasmic tail via ß-catenin (14). Thus, the present findings suggest that similar mechanisms are at play in the epididymis. Imamura et al. (29) demonstrated that there are three functional domains of {alpha}-catenin required for strong adhesion. These are a vinculin/{alpha}-actinin-binding domain, an adhesion-modulation domain, and a ZO-1 binding site. The latter will be discussed below.

In the present study p120ctn was noted along the epididymis between epithelial cells. This protein contains an armadillo repeat domain protein that shares structural similarities to ß-catenin and plakoglobin. p120ctn has unique binding partners and plays a distinct role in the cadherin-catenin complex. However, the role of p120ctn in mediating juxtamembrane domain function is controversial. Although studies have suggested that p120ctn is necessary for strong intercellular adhesion (30, 31), others have suggested that p120ctn may inhibit intercellular adhesion (32, 33). In any case, it is clear that p120ctn is a regulatory catenin that can directly modulate cadherin-cadherin interaction. Furthermore, the large number of phosphorylated isoforms of p120ctn, noted in other cells as well as in the epididymis of the present study, suggests that this protein may be highly regulated by various factors (34).

ß-Catenin has also been shown to have dual functions. It is necessary for cadherin-cadherin interaction as part of the adhering junctions, but ß-catenin is also an important member of the Wnt signaling pathway, which is involved in the cell cycle. Activation of the Wnt pathway results in the translocation of ß-catenin into the nucleus (35). In the present study we did not observe any immunolocalization of ß-catenin in epididymal nuclei. Although this suggests that the Wnt pathway may not be very active during postnatal development, we cannot rule out the possibility that this pathway may be important during embryonic development during epididymal differentiation.

Previous studies have shown that E-cadherin mRNA levels are androgen dependent in the epididymis (19). In the present study it was noted that orchidectomy resulted in a decreased staining of the LPM, but increased cytoplasmic staining for both {alpha}- and ß-catenin. The latter has also been reported in other cell types under different conditions (36). Results from the present study support the idea that androgens are necessary for maintaining the trafficking of catenins to the LPM, as both {alpha}-, and ß-catenin became localized to these sites in orchidectomized rats that received testosterone implants. Recent studies have shown that ß-catenin and E-cadherin associate shortly after the synthesis of E-cadherin in the cytoplasm, and that this complex is then transported to the basal-lateral membrane of cells (23, 37, 38). In fact, it has been proposed that ß-catenin acts as a chauffeur to facilitate the transport of E-cadherin (38). Therefore, a decrease in E-cadherin in the epididymis, as reported in orchidectomized animals (19), would result in a decrease in ß-catenin binding to E-cadherin and its targeting to the basal-lateral membrane, thus resulting in an increase in cytoplasmic localization of ß-catenin, as noted in the present study.

Interestingly, the intensity or localization of p120ctn was not altered by orchidectomy or by orchidectomy plus testosterone treatment. This suggests that p120ctn is regulated differently from {alpha}- and ß-catenin in the epididymis. Furthermore, it suggests that p120ctn may bind to other proteins that are not affected by orchidectomy. Papkoff (39), using transfected L cells, showed that there are different pools of catenins in the cell and that p120ctn is regulated differently from {alpha}- and ß-catenin. This difference appears to be in part because p120ctn does not bind as extensively to E-cadherin as do the other two catenins. Whether this is the case in epididymal cells is not known, but this may explain why p120ctn is regulated differently from {alpha}- and ß-catenin.

The development of epididymal tight junctions, which form the blood-epididymal barrier, is regulated in a segment-specific manner beginning on postnatal d 18 in the initial segment of the rat epididymis and is completely formed by 21 d of age in the cauda epididymis (40). Previous studies by Cyr et al. (19) reported that E-cadherin mRNA levels increase significantly during the formation of the blood-epididymal barrier. It has been suggested that ZO-1 first associates with the adhering junctions during the formation of tight junctions and that this protein can then dissociate from catenins and become localized to the area of the tight junctions, where it binds to the transmembrane tight junctional proteins, claudins and occludin (41).

The present results indicate that ZO-1 and ß-catenin closely associate with each other during postnatal development and that this association decreases in adults after the barrier is complete. This suggests that ZO-1 associates with ß-catenin and adhering junctions during the formation of epididymal tight junctions. Previous studies by Rajaskaran et al. (42) reported that in MDCK cells, in which tight junctional assembly was initiated by a calcium switch, ZO-1 bound to catenins during the formation of tight junctions. Furthermore, several studies suggest that ZO-1 associates with catenins after the adherens junction is formed, thereby emphasizing the importance of adherens junctions in the assembly of tight junctions (43, 44, 45). In colocalization studies, ZO-1 and ß-catenin colocalized in the epididymis of young rats, supporting the immunoprecipitation data. In the epididymis of adult rats, however, ß-catenin was localized independently of ZO-1 along the LPM of neighboring principal cells. Surprisingly, ß-catenin colocalized with ZO-1 in the apical region of the epithelium. Whether this association occurs just below the tight junction or with the tight junction is not known. These data, however, support the fact that ß-catenin and ZO-1 coprecipitate in adult rats, although to a lesser extent than in young animals. The precise role and signaling pathways resulting from these associations with respect to the formation and maintenance of the epididymal tight junctions may provide new information about the factors and mechanisms responsible for the formation of epididymal tight junctions.

In summary, these studies demonstrate that catenins ({alpha}-catenin, ß-catenin, and p120ctn) are present along the LPM of adjacent epithelial cells throughout the epididymis, with each showing varying degrees of intensity dependent on the epididymal segment. In addition, in adult rats, {alpha}- and ß-catenin appear to be regulated by androgens in terms of their targeting to the LPM. The appearance of each catenin in the different epididymal segments by postnatal d 49, at a time when androgen levels are high, also indicates a role for androgens in their expression and subcellular localization. Finally, it was noted that ß-catenin and ZO-1 closely associate with each other during formation of the blood-epididymal barrier.


   Footnotes
 
This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada (to D.G.C.) and the Canadian Institutes of Health Research (to L.H.).

Abbreviations: LPM, Lateral plasma membranes; ZO-1, zona occludens-1.

Received December 12, 2002.

Accepted for publication August 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yeung CH, Cooper TG 2002 Acquisition and development of sperm motility upon maturation in the epididymis. In: Robaire B, Hinton B, eds. The epididymis: from molecules to clinical practice. New York: Plenum Press; 417–434
  2. Cornwall GA, Layere JJ, Matusik RJ, Hinton BT, Orgebin-Crist MC 2002 Gene expression and epididymal function. In: Robaire B, Hinton B, eds. The epididymis: from molecules to clinical practice. New York: Plenum Press; 169–200
  3. Oko R, Hermo L, Chan PT, Fazel A, Bergeron JJ 1993 The cytoplasmic droplet of rat epididymal spermatozoa contains saccular elements with Golgi characteristics. J Cell Biol 123:809–821[Abstract/Free Full Text]
  4. Hermo L, Robaire B 2002 Epididymal cell types and their functions. In: Robaire B, Hinton B, eds. The epididymis: from molecules to clinical practice. New York: Plenum Press; 81–102
  5. Dacheux JL, Dacheux F 2002 Protein secretion in the epididymis. In: Robaire B, Hinton B, eds. The epididymis: from molecules to clinical practice. New York: Plenum Press; 151–168
  6. Kirchoff C 2002 Specific gene expression in the human and non-human primate epididymis. In: Robaire B, Hinton B, eds. The epididymis: from molecules to clinical practice. New York: Plenum Press; 201–218
  7. Turner TT 2002 Necessity’s potion: inorganic ions and small organic molecules in the epididymal lumen. In: Robaire B, Hinton B, eds. The epididymis: from molecules to clinical practice. New York: Plenum Press; 131–150
  8. Cyr DG, Robaire B, Hermo L 1995 Structure and turnover of the junctional complexes between principal cells of the rat epididymis. Microsc Res Technol 30:54–66[CrossRef][Medline]
  9. Cyr DG, Finnson KW, Dufresne J, Gregory M 2002 Cellular interactions and the blood-epididymal barrier. In: Robaire B, Hinton B, eds. The epididymis: from molecules to clinical practice. New York: Plenum Press; 103–118
  10. Denker BM, Nigam SK 1998 Molecular structure and assembly of the tight junction. Am J Physiol 274:F1–F9
  11. West MR, Ferguson DJ, Hart VJ, Sanjar S, Man Y 2002 Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions. Cell Commun Adhes 9:29–44[CrossRef][Medline]
  12. Steinberg MS, McNutt PM 1999 Cadherins and their connections: adhesion junctions have broader functions. Curr Opin Cell Biol 11:554–560[CrossRef][Medline]
  13. Citi S 2001 The cytoplasmic plaque proteins of the tight junction. In: Cereijidio M, Anderson J, eds. Tight junctions. Boca Raton, FL: CRC Press; 231–284
  14. Gumbiner BM 2000 Regulation of cadherin adhesive activity. J Cell Biol 48:399–404
  15. Yap AS, Stevenson BR, Keast JR, Manley SW 1995 Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation. Endocrinology 136:4672–4680[Abstract]
  16. Stevenson BR, Keon B 1998 The tight junction: morphology to molecules. Annu Rev Cell Dev Biol 14:89–110[CrossRef][Medline]
  17. Berx G, Van Roy F 2001 The E-cadherin/catenin complex: an important gatekeeper in breast cancer tumorigenesis and malignant progression. Breast Cancer Res 3:289–293[CrossRef][Medline]
  18. Cyr DG, Robaire B 1991 Developmental regulation of epithelial and placental-cadherin mRNA in the rat epididymis. Ann NY Acad Sci 637:399–408[CrossRef][Medline]
  19. Cyr DG, Hermo L, Blaschuk OW, Robaire B 1992 Distribution and regulation of epithelial-cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin in the rat epididymis. Endocrinology 130:353–363[Abstract/Free Full Text]
  20. Cyr DG, Hermo L, Robaire B 1993 Developmental changes in epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin during postnatal epididymal development in the rat. Endocrinology 132:1115–1124[Abstract/Free Full Text]
  21. Hajra KM, Fearon ER 2002 Cadherin and catenin alterations in human cancer. Genes Chromosomes Cancer 34:255–268[CrossRef][Medline]
  22. Kintner C 1992 Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain. Cell 69:225–236[CrossRef][Medline]
  23. Ozawa M, Kemler R 1992 Molecular organization of the uvomorulin-catenin complex. J Cell Biol 116:989–996[Abstract/Free Full Text]
  24. Peifer M, Berg S, Reynolds AB 1994 A repeating amino acid motif shared by proteins with diverse cellular roles. Cell 76:789–791[CrossRef][Medline]
  25. Stratton IG, Ewing LL, Desjardins C 1973 Efficacy of testosterone-filled polydimethylsiloxane implants in maintaining plasma testosterone in rabbits. J Reprod Fertil 35:235–244[Abstract/Free Full Text]
  26. Brawer JR, Schipper H, Robaire B 1983 Effects of long term androgen and estradiol exposure on the hypothalamus. Endocrinology 112:194–199[Abstract/Free Full Text]
  27. Oko R, Clermont Y 1989 Light microscopic immunocytochemical study of fibrous sheath and outer dense fiber formation in the rat spermatid. Anat Rec 225:46–55[CrossRef][Medline]
  28. Cyr DG, Blaschuk OW, Robaire B 1992 Identification and developmental regulation of cadherin messenger ribonucleic acids in the rat testis. Endocrinology 131:139–145[Abstract/Free Full Text]
  29. Imamura Y, Itoh M, Maeno Y, Tsukita S, Nagafuchi A 1999 Functional domains of {alpha}-catenin required for the strong state of cadherin-based cell adhesion. J Cell Biol 144:1311–1322[Abstract/Free Full Text]
  30. Yap AS, Niessen CM, Gumbiner BM 1998 The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn. J Cell Biol 141:779–789[Abstract/Free Full Text]
  31. Thoreson MA, Anastasiadis PZ, Daniel JM, Ireton RC, Wheelock MJ, Johnson KR, Hummingbird DK, Reynolds AB 2000 Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion. J Cell Biol 148:189–202[Abstract/Free Full Text]
  32. Aono S, Nakagawa S, Reynolds AB, Takeichi M 1999 p120(ctn) Acts as an inhibitory regulator of cadherin function in colon carcinoma cells. J Cell Biol 145:551–562[Abstract/Free Full Text]
  33. Ohkubo T, Ozawa M 1999 p120(ctn) Binds to the membrane-proximal region of the E-cadherin cytoplasmic domain and is involved in modulation of adhesion activity. J Biol Chem 274:21409–21415[Abstract/Free Full Text]
  34. Oho S, Levansuo L, Montonen O, Kari C, Rodeck U, Uitto J 2002 Specific sequences in p120ctn determine subcellular distribution of its multiple isoforms involved in cellular adhesion of normal and malignant epithelial cells. J Cell Sci 115:1391–1402.[Abstract/Free Full Text]
  35. Akimoto S, Ochiai A, Inomata M, Hirohashi S 1998 Expression of cadherin-catenin cell adhesion molecules, phosphorylated tyrosine residues and growth factor receptor-tyrosine kinases in gastric cancers. Jpn J Cancer Res 89:829–836[CrossRef][Medline]
  36. Moon RT, Bowerman B, Boutros M, Perrimon N 2002 The promise and perils of Wnt signalling through ß-catenin. Science 296:1644–1646[Abstract/Free Full Text]
  37. Hinck L, Nathke IS, Papkoff J, Nelson WJ 1994 Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly. J Cell Biol 125:1327–1340[Abstract/Free Full Text]
  38. Chen YT, Stewart DB, Nelson WJ 1999 Coupling assembly of the E-cadherin/ß-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells. J Cell Biol 144:687–699[Abstract/Free Full Text]
  39. Papkoff J 1997 Regulation of complexed and free catenin pools by distinct mechanisms. Differential effects of Wnt-1 and v-Src. J Biol Chem 272:4536–4543[Abstract/Free Full Text]
  40. Agarwal A, Hoffer AP 1989 Ultrastructural studies on the development of the blood-epididymis barrier in immature rats. J Androl 10:425–431[Abstract/Free Full Text]
  41. Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S 1999 Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol 147:1351–1363[Abstract/Free Full Text]
  42. Rajasekaran AK, Hojo M, Huima T, Rodriguez-Boulan E 1996 Catenins and ZO-1 forms a complex during early stages in the assembly of tight junctions. J Cell Biol 132:451–463[Abstract/Free Full Text]
  43. Decaens C, Cassio D 2001 Spatiotemporal expression of catenins, ZO-1, and occludin during early polarization of hepatic WIF-B9 cells. Am J Physiol Cell Physiol 280:C527–C539
  44. Itoh M, Nagafuchi A, Moroi S, Tsukita S 1997 Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to {alpha} catenin and actin filaments. J Cell Biol 138:181–192[Abstract/Free Full Text]
  45. Itoh M, Morita K, Tsukita S 1999 Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occluding and {alpha} catenin. J Biol Chem 274:5981–5986[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
E. Dube, P. T.K. Chan, L. Hermo, and D. G. Cyr
Gene Expression Profiling and Its Relevance to the Blood-Epididymal Barrier in the Human Epididymis
Biol Reprod, June 1, 2007; 76(6): 1034 - 1044.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
V. Hyenne, J. C Harf, M. Latz, B. Maro, U. Wolfrum, and M.-C. Simmler
Vezatin, a ubiquitous protein of adherens cell-cell junctions, is exclusively expressed in germ cells in mouse testis
Reproduction, March 1, 2007; 133(3): 563 - 574.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. Dufresne, N. St-Pierre, R. S. Viger, L. Hermo, and D. G. Cyr
Characterization of a Novel Rat Epididymal Cell Line to Study Epididymal Function
Endocrinology, November 1, 2005; 146(11): 4710 - 4720.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by DeBellefeuille, S.
Right arrow Articles by Cyr, D. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by DeBellefeuille, S.
Right arrow Articles by Cyr, D. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals