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Centre de Recherche en Santé Humaine, INRS-Institut Armand Frappier (M.G., J.D., D.C.), Université du Québec, Pointe Claire, Québec, Canada H9R 1G6; and Department of Anatomy and Cell Biology, McGill University (L.H., D.C.), Montréal, Québec, Canada H3A 2B2
Address all correspondence and requests for reprints to: Dr. Daniel G. Cyr, INRS-Institut Armand Frappier, Université du Québec, 245 boulevard Hymus, Pointe Claire, Québec, Canada H9R 1G6. E-mail: daniel.cyr{at}INRS-sante.uquebec.ca
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Introduction |
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Little information exists on the proteins that comprise epididymal tight junctions. In some tissues, tight junctions are composed of a variety of peripheral membrane proteins, including zona occludens-1, -2, and -3 (ZO-1, -2, and -3), symplekin, cingulin, 7H6 antigen, cytoskeletal elements (fodrin and actin), as well as integral transmembrane proteins such as occludin (12, 13, 14, 15). Previous studies have reported that tight junctions between adjacent epithelial cells contain occludin (16, 17). In the mouse epididymis, occludin was immunolocalized between adjacent epithelial cells of the epididymis as early as embryonic day 13.5. In the adult, occludin was localized in the apical region among epithelial principal cells of the caput, corpus, and cauda epididymidis. In the initial segment, however, occludin immunostaining was associated only with narrow cells (16). As tight junctions in the initial segment are extensive, this suggested the presence of other tight junctional proteins in the epididymis.
Recently, a new family of transmembrane proteins named claudins has been identified (18). To date, there are 18 claudins, and their tissue distribution is variable. Studies have indicated that they are colocalized with occludin; however, it has also been reported that in the absence of occludin, claudins are still present and are recruited to form tight junctions (18, 19, 20). Transfection of MDCK cells with claudin complementary DNAs (cDNAs) indicate that these are transported to the tight junctions between adjacent cells, supporting the idea that these proteins are tight junctional proteins (18). There are no studies regarding the presence of claudins in the epididymis and their role.
The objectives of the present study were to determine whether claudin-1 (Cl-1) and claudin-2 (Cl-2) are expressed in the rat epididymis and to assess their localization using light and electron microscopy and Northern blot analysis. This study also determined the expression of claudin with respect to the different cell types of the adult epididymis and its various regions as well as its expression during postnatal development and its regulation by testicular factors.
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Materials and Methods |
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At the time of sampling, rats were killed with CO2. Epididymides were dissected, weighed, and subdivided into four separate regions (initial segment, caput, corpus, and cauda epididymidis). Tissues were frozen in liquid nitrogen and stored at -80 C.
Cl-1 and Cl-2 expression in the epididymis. The presence of Cl-1 and Cl-2 mRNA transcripts in the epididymis was determined by RT-PCR using RNA isolated from adult rat epididymal segments. Total RNA (500 ng) was reverse transcribed using an oligo(deoxythymidine)16 primer. Cl-1 was amplified using the specific forward primer 5'-ACGCAGGAGCCTCGCCCCGCAGCTGCA-3' and the reverse primer 5'-CAGCCAAGGCCTGCATAGCCATGG-3'; Cl-2 was amplified using the forward primer 5'-ACGACAAGCAAACAGGCTCCGAAG-3' and the reverse primer 5'-TATAGTCCCAGCCACTAC-3' (18). Testis was used as a positive control. The cDNA was amplified using 30 cycles of denaturation at 94 C, annealing at 60 C, and elongation at 72 C (90 sec) followed by 10 min at 72 C to add single deoxyadenosine (A) overhangs. The resulting PCR product was loaded onto a 1.25% agarose gel prepared in Tris-borate EDTA buffer and stained with ethidium bromide.
Postnatal development. To determine the postnatal developmental expression of Cl-1 in the epididymis, timed gestation female rats were purchased from Charles River Laboratories, Inc. At the time of birth, the sexes of the pups were determined and random litters of 10 male pups were placed with each lactating mother. Rats were weaned at 24 days of age. For Northern blot analyses, epididymides were sectioned into caput-corpus and cauda epididymidis as previously described (21, 22). For Northern blot analyses, rats were anesthetized with CO2 and killed at 7, 14, 21, 29, 35, 42, 49, 56, 63, 77, and 91 days of age. Tissues were frozen in liquid nitrogen and subsequently stored at -80 C. For immunocytochemistry, rats were killed on days 7, 21, 29, 39, 56, and 91.
Androgen regulation. To assess whether epididymal claudins were regulated by testicular factors or androgens, adult rats were anesthetized with an ip injection of 50 mg/kg ketamine/10 mg/kg xylazine and orchidectomized via a scrotal incision. Testosterone-filled polydimethylsiloxane capsules were prepared according to the method outlined by Stratton et al. (23) and have well characterized steroid release rates (24). Orchidectomized rats were implanted with an empty 2.5-cm capsule or with 18.6 cm (three capsules of 6.2 cm each) testosterone. The latter mimic epididymal (18.6 cm) testosterone levels, which are 10 times greater than blood levels. Carrier rats were implanted with testosterone pellets for 3 days before the start of the experiment, so that the initial surge of testosterone release would be complete, ensuring that the newly made capsules had a constant testosterone release rate. The implants were removed from the carrier, cleaned, and inserted sc on the backs of experimental animals at the time of orchidectomy. Rats were killed 14 and 21 days after surgery, and the epididymides were fixed for immunocytochemistry according to the procedures described below.
Cloning of the rat Cl-1
Rat Cl-1 was amplified by RT-PCR as described above, and the
product was separated on an agarose gel. The 893-bp PCR product was
isolated from the gel using the Qiaex II extraction kit
(QIAGEN, Valencia, CA) and cloned into the T/A cloning
site of the pCR 2.1 plasmid (Invitrogen, Palo Alto, CA).
The plasmid was then used to transform competent bacteria, and positive
clones were color selected. The insert was isolated by restriction
enzyme digest and agarose gel electrophoresis and was purified using
the QIAex II extraction kit (QIAGEN, Valencia, CA). Rat
Cl-1 was sequenced using an automated DNA sequencer (PE Biosystems,
Palo Alto, CA) and was identified by sequence homology with other
claudins (GenBank, NCBI, Washington DC).
Immunocytochemistry
Immunocytochemical localization of Cl-1 was performed using a
rabbit polyclonal Cl-1 antiserum (10 µg/ml; Zymed Laboratories, Inc., Seattle, WA). Antibody binding to Cl-1 was detected with
the horseradish peroxidase method as outlined by Cyr et al.
(12), using a horseradish peroxidase-conjugated antirabbit
secondary antibody (1:250; Sigma, St. Louis, MO). Slides
incubated with normal rabbit antiserum were used as a negative control,
because immunoabsorption was not possible due to the lack of Cl-1
antigen.
For immunocytochemistry at the electron microscope level, small pieces (1 mm) of epididymal tissue of each region were fixed with 4% paraformaldehyde plus 0.1% glutaraldehyde and then processed according to the methods outlined by Cyr et al. (21). Tissues were immersed in fixative for 2–4 h and subsequently washed twice in 0.15 M PBS containing 4% sucrose and 50 mM NH4Cl for 1 h at 4 C. Tissue blocks were dehydrated in graded methanol and embedded in Lowicryl K4M (21). Immunogold labeling was performed on ultrathin sections using Cl-1 antisera (1:10 dilution), according to the methods outlined by Hermo et al. (13).
Immunoblots
Frozen caput (including initial segment), corpus, and cauda
epididymidis from adult rats were homogenized in buffer (60
mM Tris-Cl, 2 mM CaCl2, 1
mM phenylmethylsulfonylfluoride, 40 mM
ß-octylglucopyranoside, 20 µg/ml aprotinin, 20 µg/ml trypsin
inhibitor, 20 µg/ml pepstatin A, 40 µg/ml antipain, and 20 µg/ml
leupeptin, pH 6.8) and centrifuged at 2,500 x g for 30
min at 4 C to remove the nuclear fraction. The postnuclear fraction was
obtained by centrifuging the supernatant at 33,000 x g
for 30 min at 4 C. The resulting pellet was resuspended in buffer, and
its protein content was determined using a protein assay (Bio-Rad Laboratories, Inc., Missassauga, Canada). Membrane protein
samples (50 µg) were diluted in loading buffer (Laemmli buffer),
boiled for 5 min, and cooled at room temperature. The sample was loaded
onto a 15% polyacrylamide gel with a 5% stacking gel
(14). Electrophoresis was done at 75 V for 1.5–2 h until
the dye front reached the end of the gel.
The gel was removed from the glass plates, and the proteins were transferred onto a nitrocellulose membrane using a Bio-Rad Laboratories, Inc., Transblot apparatus at 30 V for 16 h and 70 V for 3 h at 4 C. At the end of the transfer, the membrane was removed. The transfer of colored mol wt markers was used to assess the efficiency of the transfer. The presence of Cl-1 was determined using the Cl-1 antiserum and the Bio-Rad Laboratories, Inc., Blotting Detection Kit, which uses a streptavidin-alkaline phosphatase-conjugated secondary antibody.
Northern blot analysis
Northern blot analysis was performed on total cellular RNA
isolated from the caput-corpus and cauda region of the epididymis. RNA
was isolated using the guanidinium isothiocyanate method
(15), and a 10-µg aliquot of total RNA was separated by
electrophoresis in a 1.2% agarose-formaldehyde gel. The RNA was then
transferred to a charged nylon membrane (GeneScreen Plus, DuPont
Chemicals, Missassauga, Canada) as previously described
(25).
The rat Cl-1 cDNA was labeled by random priming with [32P]deoxy (d)-CTP (Oligonucleotide Labeling Kit, Amersham Pharmacia Biotech, Baie D’Urfe, Canada) (13). Each Northern blot was standardized for RNA loading by hybridizing the membranes with an end-labeled oligonucleotide probe recognizing the 18S ribosomal RNA (rRNA) (13). Hybridizations of the Cl-1 cDNA and 18S rRNA probes were performed as previously described (25). The resulting unsaturated radioautograms were scanned using a Bio-Rad Laboratories, Inc. Fluor Image analyzer, and the integrated area under the curve for each signal was standardized against the signal for the 18S rRNA to determine the relative levels of Cl-1 mRNA.
Statistical analyses
To determine whether differences in Cl-1 mRNA levels during
development were significantly different, normality of the data was
determined using the Kolmogorov-Smirov test, whereas the Levine median
test was performed to determine equal variance. Statistical differences
between groups were determined by ANOVA followed a
posteriori by Student-Newman-Keuls test for multiple comparisons
between experimental groups. Significance was established at
P < 0.05. All analyses were performed using SigmaStat
computer software (Jandel Scientific Software, San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Primers for Cl-2
amplified a predicted 750-bp product in testis, but did not amplify any
product in epididymis (data not shown). The sequencing of the rat Cl-1
indicated a full-length cDNA encoding the 748-bp coding sequence that
was 93% homologous to mouse Cl-1 and 91% homologous to human
Cl-1/SEMP-1 (GenBank access no. AF195500).
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). Along the entire length of the
epididymis, Cl-1 immunostained the lateral and basal margins of
principal cells (Fig. 2, B–E). At high magnification staining was
observed between the lateral plasma membranes of adjacent principal
cells and apical regions containing tight junctions of the
blood-epididymal barrier (Fig. 2C). In addition, Cl-1 was noted at the
interface between principal and basal cells (Fig. 2, B–E). Staining
was also present along the basal plasma membrane of the epithelium and
the area of the basement membrane. No staining was observed in the
interstitial space or on maturing spermatozoa. Sections incubated with
normal rabbit serum (negative control) did not show any immunoreactive
product (data not shown).
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). Immunogold particles were also
present in more distal sites between the interdigitating lateral plasma
membranes of adjacent principal cells (Fig. 3A) as well as between the
plasma membranes of principal and basal cells (Fig. 3B). Interestingly,
numerous gold particles were also present along the basal plasma
membrane in areas adjacent to the basement membrane of the epididymal
epithelium (Fig. 3, A and B). Sections incubated with normal rabbit
antiserum showed few gold particles consistent with background levels
(not shown).
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). A higher mol wt band (35 kDa)
in the corpus and cauda was also observed. The identification of this
band is unknown; however, given the increasing number of new claudins
that have been identified recently, it is possible that this band
corresponds to a different claudin. Further investigation is needed to
identify this protein; nonetheless, its presence does not detract from
the clear differences in localization observed in the initial segment
among control, orchidectomized, and orchidectomized plus
testosterone-treated rats.
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), but significantly
increased from days 7–21. Levels were significantly higher than those
in 7-day-old rats on both days 28 and 35, but not at any other age
(Fig. 5A). In the cauda epididymidis, the mRNA levels of the 4.0-kb
transcript were lowest on day 7, increased almost 2-fold by day 14, and
remained at this level until day 91 (Fig. 5B). The mRNA levels of the
1.5-kb transcript in the caput-corpus epididymidis increased
significantly from days 7–14 and then decreased to day 7 levels by day
35. Levels remained constant thereafter (Fig. 5A). The mRNA levels of
the 1.5-kb transcript in the cauda were lowest on day 7, increased by
more than 3-fold on day 14, and then decreased from day 14 to day 42,
when levels returned to those observed in 7-day-old rats (Fig. 5B), but
thereafter gradually increased until day 91 (Fig. 5B).
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). In
the corpus and cauda epididymidis, Cl-1 immunostaining was also
localized between the lateral plasma membranes of adjacent
undifferentiated principal cells, but was more pronounced at sites
distal to the areas containing tight junctions (Fig. 6, B and C). In
21-day-old rats, however, Cl-1 immunoreactive product was present along
the entire length of the lateral plasma membranes between adjacent
principal cells as well as between principal and basal cells, a
staining pattern identical to that observed in adult rats (Fig. 6D).
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). However, Cl-1 immunostaining at the
interface between basal and principal cells and along the basal plasma
membrane and basement membrane of the epithelium was not altered by
orchidectomy (Fig. 7B). In orchidectomized rats given testosterone
replacement, Cl-1 expression was maintained in the apical region of the
lateral plasma membranes between adjacent principal cells associated
with tight junctions, although there was still no staining in more
distal areas of the lateral plasma membranes (Fig. 7C). As with
orchidectomized rats, Cl-1 immunostaining between principal and basal
cells and along the basal plasma membrane of the epithelium was not
altered by testosterone replacement. In all other regions of the
epididymis, neither orchidectomy nor orchidectomy with testosterone
replacement altered Cl-1 immunostaining, as illustrated in the corpus
epididymidis of a 14-day orchidectomized rat (Fig. 7D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). At both the light and electron
microscope levels, the immunocytochemical localization of Cl-1 at the
apical region of the epididymal epithelium between adjacent principal
cells is consistent with areas where tight junctions have been
identified morphologically (27). Thus, Cl-1 appears to
play a role in epididymal tight junctions. In transfected fibroblasts,
Cl-1 and Cl-2 induce the formation of different types of tight
junctions (19). Cl-1-induced strands were largely
associated with the protoplasmic (P) face and were mostly continuous,
whereas Cl-2-induced strands were discontinuous at the P face with
complementary grooves at the extracellular (E) face (19).
In the epididymis the tight junctions are mostly continuous, supporting
the idea that these are mediated at least in part by Cl-1.
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The cellular localization of Cl-1 in the adult rat epididymis is different from another tight junction protein, occludin (16), which is present only apically and between adjacent principal cells in the epididymis of most regions, in contrast to the extensive distribution of Cl-1 observed in the present study. In addition, occludin was associated exclusively with narrow cells and not between adjacent principal cells in the initial segment or around basal cells, indicating a complex pattern of expression of the different types of junctional proteins. Furthermore, although occludin was expressed apically between nonciliated cells of the efferent ducts, no reaction was noted for Cl-1 in this tissue.
The immunocytochemical localization of Cl-1 between principal and basal cells is interesting. Although there are no reports of tight junctions, Cyr et al. (29) reported the presence of the gap junction protein, connexin 43 (Cx43), between these cell types. However, unlike the punctate staining reaction observed for Cx43, the distribution of Cl-1 was uniform along the entire interface of these two cell types. These data suggest that Cl-1 may serve to adhere principal cells to basal cells.
One of the more unexpected observations was the Cl-1 immunoreaction observed along the basal plasma membrane of the epithelium in the area of the basement membrane. This suggests that Cl-1, unlike other junctional proteins such as occludin, cell adhesion molecules (cadherins), or integrins, may be involved not only in cell-cell interactions but also in the interactions between cells and molecules comprising the basement membrane.
The formation of tight junctions in the epididymis occurs during embryonic development and has been identified as early as embryonic day 12 in mice by freeze-fracture electron microscopy (30). Although at this age the tight junctions surround the entire circumference of the epithelial epididymal cells, at the juxtaluminal position a functional cellular barrier (blood-epididymal barrier) still does not exist (7, 10). This barrier is formed during postnatal development as the number of apically localized tight junctional strands increases up to 21 days of age, when the barrier becomes fully functional, as determined by lanthanum exclusion (10). In the present study Cl-1 mRNA levels in either the caput-corpus or cauda epididymidis appeared by day 7 and peaked between 14 and 21 days of age, at the time of blood-epididymal barrier formation. Light microscope immunocytochemistry revealed that in the initial segment and caput epididymidis, Cl-1 was already present along the entire lateral plasma membranes of adjacent epithelial principal cells by postnatal day 7. However, in the corpus and cauda epididymidis at this age, Cl-1 was present only at more distal sites of the lateral plasma membrane between adjacent principal cells by day 7 and not in the apical areas where tight junctions are formed. These data suggest that at this age the targeting of Cl-1 to the lateral plasma membranes of these regions is under different regulating factors. By day 21 the staining pattern of Cl-1 expression was identical to that of adult animals. As this is an age when androgens are not at peak levels, the data suggest that they are not a major factor regulating Cl-1 expression in the epididymis. Other studies have demonstrated that the formation of the blood-epididymal barrier also commences in the initial segment and caput epididymis and gradually progresses along the epididymis between postnatal days 18 and 21 (10). Thus, our observations on the coincidence of Cl-1 expression in areas of tight junctions during early development up until day 21 suggest a role for Cl-1 with respect to the blood-epididymal barrier; however, the nature of this relationship has yet to be determined. Moreover, Cl-1 expression in areas of opposing plasma membranes of epithelial cells other than where tight junctions are present implies other roles for Cl-1 in the epididymis. In the testis, the Sertoli cells of claudin-11 null mice lack intercellular tight junctions, providing direct evidence of the importance of this family of proteins in the formation of testicular tight junctions (20). However, the presence and importance of Cl-1 and other members of the claudin family have not as yet been demonstrated in the epididymis.
There is little information on the regulation of tight junctions of the
blood-epididymal barrier. Preliminary data by Suzuki and Nagano
(31) suggest that there is a loss of tight junctional
strands associated with orchidectomy in the epididymis. In the present
study, orchidectomy resulted in changes to the staining pattern of Cl-1
in the initial segment only. There was an absence of reaction along the
entire length of the lateral plasma membranes between adjacent
principal cells, but not at the interface between principal and basal
cells and along the basal plasma membrane of the epithelium (Fig. 8
).
Testosterone replacement maintained expression apically between the
lateral plasma membranes of adjacent principal cells, but not in more
distal sites of these membranes (Fig. 7). No changes in Cl-1 expression
were noted in any other region of the epididymis (Fig. 7). Thus,
androgens appear to regulate Cl-1 expression not only in a
segment-specific manner, but also only in areas of the lateral plasma
membranes where tight junctions are present (9). The
absence of Cl-1 expression in more distal sites of the lateral plasma
membranes between adjacent principal cells in the initial segment, even
after testosterone replacement, suggests that testicular factors other
than androgens regulate or synergize with androgens to regulate Cl-1
expression in this region as well as at the interface between principal
and basal cells and at the basal plasma membrane of the epithelium.
Postnatal developmental studies also indicate that factors other than
androgens regulate Cl-1 expression in the initial segment and other
epididymal regions, as Cl-1 is present in the epithelium as early as
day 7, at a time when androgen levels are below detection
(32). Orchidectomy has also been shown to alter the
intracellular localization of the gap junctional protein Cx43 in the
initial segment only (29).
Studies have suggested that the carboxyl-terminal of Cl-1/SEMP-1 contains intracellular phosphorylation sites that may encompass a domain for initiating a signal transduction cascade (26). Furthermore, Itoh et al. (33) reported that the carboxyl-terminus of Cl-1 may bind to the MAGUK family of proteins, further supporting a role in signal transduction. In humans, SEMP-1/Cl-1 is thought to be a tumor suppressor gene in a variety of tissues, including breast, liver, kidney, and pancreas (26). Whether this is related to the role of tight junctions or cellular signaling remains to be established. However, the possibility that nonjunctional Cl-1 is involved in cell interactions in the epididymis cannot be discounted and requires further study to fully understand its role.
In the present study Cl-1 was also noted at the interface between principal and basal cells where tight junctions are not prominent as well as along the basal plasma membrane in relation to molecules of the basement membrane. Immunoblots of epididymal membrane preparations revealed that a single band corresponding to the predicted mol wt of Cl-1 protein is present in the epididymis, indicating the specificity of the antibody. It is thus suggested that Cl-1 may act as an adhesion molecule in the epididymis, serving to bind adjacent epithelial cells to each other as well as to the underlying basement membrane.
In conclusion, Cl-1 is present in the rat epididymis, where its localization was not restricted to areas of tight junctions. Cl-1 was located apically between the lateral plasma membranes of adjacent principal cells at sites occupied by tight junctions, at more distal sites of these membranes, as well as at the interface between principal and basal cells and at the basal plasma membrane of the epithelium. These data suggest that Cl-1 may act as an adhesion molecule, adhering epithelial cells to each other and to the basement membrane. Postnatal development studies using Northern blot analysis and immunocytochemistry indicate that Cl-1 is already present in the epididymis by 7 days of age and peaks by day 21, concomitant with the formation of the blood-epididymal barrier, suggesting a role for Cl-1 with regard to this barrier. These data also suggest that androgens, which only peak by day 39, are not a major factor regulating Cl-1 expression. These data are also consistent with orchidectomy studies in adult animals that revealed that androgens regulate Cl-1 expression only in a region-specific manner restricted to the initial segment and to specific domains of the lateral plasma membranes. Taken together, these data indicate complex interactions for Cl-1 in the epididymis and its regulation by androgens as well as other factors.
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Acknowledgments |
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Footnotes |
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Received June 22, 2000.
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References |
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-reductase messenger ribonucleic acid
levels along the rat epididymis. Endocrinology 128:2407–2414[Abstract]
4–5--reductase and 3ß-hydroxysteroid
dehydrogenase in the rat epididymis during development. Endocrinology 107:948–953[Abstract]
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N. St-Pierre, J. Dufresne, A. A. Rooney, and D. G. Cyr Neonatal Hypothyroidism Alters the Localization of Gap Junctional Protein Connexin 43 in the Testis and Messenger RNA Levels in the Epididymis of the Rat Biol Reprod, April 1, 2003; 68(4): 1232 - 1240. [Abstract] [Full Text] [PDF]
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