Institut National de la Recherche Scientifique-Institut Armand Frappier, Université du Québec, Pointe-Claire, Québec, Canada H9R 1G6
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ABSTRACT |
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In the epididymis, Cx43 forms gap junctions between principal and basal cells but not between adjacent principal cells. Cx30.3, 31.1, and 32 were identified in adult rat epididymis by RT-PCR, whereas Cx26 was present in young rats. Postnatal development studies indicate that Cx26 mRNA was detectable only in the caput-corpus region of the epididymis and that levels increased by fivefold during the first 4 wk postnatally, when epithelial cells differentiate, and decrease to nondetectable levels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout the epididymis in young rats and began to increase in the second and third weeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32 were localized to the lateral plasma membranes between adjacent epithelial cells of the epididymis. Colocalization studies indicate that Cx26 and Cx32 exist either independently of one another or can colocalize along the lateral plasma membrane of epithelial cells in young rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.
gap junctions; cell differentiation; development
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INTRODUCTION |
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THE FORMATION OF MATURE SPERMATOZOA involves not only their differentiation during spermatogenesis in seminiferous tubule of the testis but also their maturation during epididymal transit where they acquire both fertilizing ability and progressive motility (18, 26, 33). The epididymis is generally subdivided into four distinct regions based on both morphological appearance of the tubule and differences in physiological function. These regions include the initial segment, the caput, the corpus, and the cauda epididymidis (6). Segment-specific differences in epididymal physiology are suggestive of a complex coordination of function by the epididymal epithelium. Thus cellular communication appears to be crucial to epididymal function (7). In the epididymis, as in other epithelia, cellular communication is regulated by gap junctions.
Gap junctions were identified by freeze fracture electron microscopy between adjacent epididymal principal cells at their apical and lateral margins (11). Gap junctions form transmembrane channels between adjacent cells that permit bidirectional communication between cells by selectively allowing the passage of small molecules (<1 kDa), including secondary messengers (38). Gap junctions are formed by a family of integral proteins termed connexins (Cxs). Fifteen different Cxs have been identified and are named according to their molecular mass (13, 37). Cx subunits oligomerize in the trans-Golgi network to form hemichannels or connexons, which consist of six Cxs arranged radially around a central pore. Gap junctional intercellular communication has been implicated in diverse physiological processes including cellular proliferation and differentiation, growth control (tumor suppression), and development. In the epididymis, gap junctions containing Cx43 were localized between principal and basal cells (8). This observation suggests that other Cxs are also present in epididymal principal cells and mediate cellular communication between adjacent principal cells. The epithelium of the epididymis undergoes substantial postnatal differentiation. During the first 2 wk postnatally, epididymal epithelial cells are undifferentiated. These cells then begin to differentiate between 16 and 44 days postnatally to form several different cell types including principal, narrow, clear, and basal (34). Changes in gap junctional communication between differentiating epididymal epithelial cells may represent an essential component of cellular differentiation within the epididymis.
Several studies have indicated the presence of multiple Cxs in a given tissue. Davies et al. (10) have identified six different Cx mRNAs in the mouse blastocyst that contribute to gap junctions in preimplantation and development. In the testis, Risley (30) has reported the presence of 11 different Cx transcripts in seminiferous tubules, 10 of which were present on polysomes and presumably translated. Multiple Cxs have also been identified in kidney (16, 40) and skin (3). Clearly, the expression of individual Cxs in different cell types and at different times during development suggests an essential role for coordinating cellular growth and differentiation.
The objectives of this study were to determine 1) which Cxs are present in the rat epididymis by using a combination of RT-PCR and restriction enzyme mapping to identify each Cx, 2) whether or not the mRNA levels for these Cxs are modulated during postnatal development, and 3) the cellular localization of two specific Cxs, Cx26 and Cx32, in both prepubertal and adult rat epididymis.
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MATERIAL AND METHODS |
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Animals. Male and timed-gestation pregnant female Sprague-Dawley rats were purchased from Charles River Laboratories, (St-Constant, Canada). Rats were maintained under a constant photoperiod of a 12:12-h light-dark cycle. Rats received food and water ad libitum. All animal protocols used in this study were approved by the University Animal Care Committee.
Identification of epididymal Cxs.
To identify epididymal Cxs, a RT-PCR strategy was employed by using two
pairs of degenerate oligonucleotide primers (F1R1 and F2R2; Table
1). The primers were designed
according to highly conserved regions of the amino-terminal cytoplasmic
domain and the two extracellular loops of the Cx multigene family
(19).
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Postnatal development.
Pups obtained from pregnant females were sexed on the day of birth and
random litters of 10 male pups were placed with each lactating female.
Rats were weaned at 24 days of age. For Northern blot analyses, rats
were killed by CO2 asphyxiation at postnatal ages 7, 14, 21, 28, 35, 42, 49, 56, 63, 77, and 91 days. Epididymides were divided
into a caput-corpus (initial segment, caput, and corpus epididymidis)
and a cauda (cauda epididymidis) region. Tissues were frozen in liquid
nitrogen and stored at 86°C.
Amplification of Cx26 and Cx30.3. RT-PCR using specific primers (Table 1) was performed to detect mRNAs encoding Cx26 and Cx30.3 in the epididymis of both 21-day-old and adult rats. Primers were designed with the Oligo software (Molecular Biology Insights). Conditions for RT-PCR were the same as those described for the F1R1 and F2R2 primer sets, with the exception that 30 amplification cycles were used with annealing temperatures of 56°C (Cx26) and 59°C (Cx30.3). RT-PCR products were analyzed on a 1.2% agarose gel, cloned, and sequenced to confirm their identity.
Cx26 immunoblots.
Total proteins were extracted from proximal and distal regions of
epididymides removed from 13 21-day-old and 2 adult rats. Pooled
tissues were ground in liquid nitrogen and homogenized in four volumes
of buffer (10 mM Tris · HCl, pH 7.5, 0.25 M sucrose, 2 µg/ml
leupeptin, 2 µg/ml aprotinin, 100 µg/ml PMSF, 1 µg/ml pepstastin,
and 2 µg/ml antipain) using a motor driven pestle (10 strokes).
Samples were centrifuged at 10 000 g for 10 min at 4°C and
the supernatant was collected. Protein concentrations were determined
using the Bio-Rad protein assay (Bio-Rad Laboratories) and stored at
20°C. Samples were resolved in Laemmli buffer on a 12% SDS
polyacrylamide gel and transferred onto a nitrocellulose membrane
(Bio-Rad Laboratories) at 100 V for 45 min at 4°C in the presence of
2 mM of CaCl2. Blots were stained with Ponceau red S to
evaluate transfer efficiency, rinsed, and blocked overnight at 4°C in
×1 TBST buffer (20 mM Tris · HCl, 500 mM NaCl, and 0.05% Tween 20, pH 7.5) containing 5% milk. Membranes were incubated with an
anti-Cx26 antibody (1.5 µg/ml; Chemicon International, Temecula, CA)
diluted in the same buffer for 90 min at room temperature. The blots
were washed three times for 5 min in ×1 TBST at room temperature and
subsequently incubated with an alkaline phosphatase-conjugated secondary antibody (0.4 µg/ml; Santa Cruz Biotechnology, Santa Cruz,
CA) in ×1 TBST containing 5% milk and then washed as described. Complexed anti-Cx26 was revealed by using the Bio-Rad blotting detection kit (Bio-Rad Laboratories).
Immunolocalization of Cx26 and Cx32.
Cx26 immunocytochemistry was done by using frozen sections.
Epididymides were carefully dissected out and placed in OCT-cryomatrix (Fisher Scientific, Ottawa, ON, Canada) and frozen on dry ice. Solidified blocks of tissues were then stored at 86°C until cut. Sections (10-µm) were cut and mounted onto glass slides and stored at
20°C until immunocytochemistry. Slides were fixed in methanol for
20 min at
20°C, allowed to air dry, and rehydrated with
phosphate-buffered saline (PBS) for 30 min at room temperature and then
blocked in buffer (PBS, 3% bovine serum albumin, and 5% goat serum)
for 20 min at room temperature. This was followed by three 5-min washes in PBS. Immunocytochemical localization of Cx26 was done by using rabbit polyclonal Cx26 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 FITC-conjugated
anti-rabbit secondary antibody (1:250; Jackson Immunoresearch, West
Grove, PA). The sections were subsequently washed three times in PBS
and mounted with Vectashield containing propidium iodide (Vectastain
Laboratories, Burlington, ON, Canada). Slides incubated with normal
rabbit antiserum were used as a negative control because
immunoabsorption was not possible due to the lack of Cx antigen.
Statistical analysis. To determine whether differences in Cx mRNA levels during development were significant, normality of the data was assessed by using the Kolmogorov-Smirov tests, whereas the Levine median test was done for equal variance. Statistical differences between groups were determined by ANOVA followed a posteriori by a Student-Newman-Keuls test for multiple comparisons between experimental groups. Significance was established at P < 0.05. All analyses were done by using SigmaStat computer software (Jandel Scientific Software, San Rafael, CA).
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RESULTS |
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Identification of epididymal Cxs.
RT-PCR amplification with F1R1 primers generated a 639-base pair (bp)
product from adult rat epididymal mRNA (Fig.
1). Sequencing of this cDNA followed by
Blast search in Genbank confirmed that the amplified product was Cx43.
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Developmental patterns of epididymal Cxs. To assess whether or not epididymal Cx mRNA levels were altered as a function of development, the patterns of Cx mRNA were compared as a function of postnatal development in the proximal (initial segment, caput, and corpus) and distal (cauda) regions of the epididymis.
Northern blot analysis of Cx26 mRNA levels in the proximal epididymis indicate that Cx26 mRNA transcripts are detectable in rat proximal epididymis as early as day 7 (Fig. 3A). Cx26 mRNA levels subsequently increased to peak at day 28 and decreased to below detection by day 35. Cx26 mRNA levels remained low thereafter. In the distal region of the epididymis, Cx26 mRNA was not detectable by Northern blot analysis at any of the ages sampled.
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Amplification of Cx26 and Cx30.3.
To determine the presence of transcripts for Cx26 and Cx30.3 in
the epididymis, we designed specific primers for each cDNA and
amplified these with epididymal RNA isolated from either 21-day-old or
adult rats (Fig. 5). Using this approach,
we amplified a Cx26 cDNA transcript in both the caput-corpus and cauda
epididymidis of 21-day-old rats. In the adult, an amplification product
was observed in the caput-corpus epididymidis, and a barely detectable band was also present in the distal epididymis. Subdividing the adult
epididymis into four separate segments, initial segment, caput, corpus,
and cauda epididymidis, resulted in mRNA levels for Cx26 that were low
throughout the adult epididymis but that were somewhat more prominent
in the initial segment (Fig. 5A). Cx30.3 transcripts were
detectable by RT-PCR in both the caput-corpus and cauda epididymidis of
both 21- and 91-day-old rats. Furthermore, a Cx30.3 mRNA transcript was
amplified in all four regions of the adult epididymis (Fig.
5B). The identity of both the Cx26 and Cx30.3 transcripts
was confirmed by sequencing and Blast search in Genbank.
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Immunoblot of Cx26.
To determine whether or not Cx26 was expressed at the protein level in
the epididymis, and in particular in the adult epididymis when Cx26
mRNA levels are low, an immunoblot was done using total proteins
isolated from the caput-corpus or cauda epididymidis of either 21- or
91-day-old rats (Fig. 6). In adult rat
liver, which was used as a positive control, a single protein band of ~26 kDa was identified. In the epididymis of 21-day-old rats, Cx26
was present in both the caput-corpus and cauda epididymidis, despite
the fact that Cx26 mRNA levels were detectable only by RT-PCR in the
cauda epididymidis. In the epididymis of adult rat, Cx26 was also
present in both the caput-corpus and cauda epididymidis. Whereas the
protein loading appeared to be similar according to the Ponceau red S
staining of the nitrocellulose membrane (data not shown), Cx26 protein
levels appear to be lower in the epididymis of adult rats compared with
levels in the 21-day-old rat epididymis.
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Immunolocalization of Cx26 and Cx32.
Cx26 in 21-day-old rats was localized to epithelial cells that line the
lumen of the epididymis. The immunostaining was punctate and appeared
to be more intense in the basal region of the epithelial cells (Fig.
7A). The intensity of the Cx26
immunostaining appeared to be equivalent throughout the
epididymis, including the cauda epididymidis. In adult rats, Cx26
immunostaining appeared to be less intense and was localized to the
epithelial principal cells (Fig. 7, B-D).
The Cx26 immunostaining was similar in the different regions of the
epididymis.
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DISCUSSION |
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The presence and physiological roles of gap junctions in the epididymis and in sperm maturation are poorly understood. We have previously demonstrated that gap junctions containing Cx43 are present between basal and principal cells (8). Morphological studies have shown that gap junctions are also present along the lateral margins between adjacent principal cells (29). In the present study, we demonstrate that there are at least five different Cxs present in the epididymis. Although we do not know whether or not these are all expressed in principal cells or whether they are expressed in other specific cell types (e.g.. basal, narrow, apical, etc.), the large number of Cxs present in the epididymis is surprising and suggestive of a highly organized and complex intercellular communication within the epididymis. The number of Cxs in the epididymis is lower than in the testis where 11 Cxs have been identified; however, the cellular makeup of the epididymis is much less heterogeneous than in the testis, where germ cells of different stages must interact, or communicate, with other germ cells or with Sertoli cells. This is in addition to cells that surround the seminiferous tubule or that are present in the interstitium (30, 40). It has been reported that other tissues express variable numbers of Cxs; for example, mouse keratinocytes express four different Cxs, whereas adult rodent hepatocytes express only two Cxs (21, 28).
Different Cx-containing gap junctions allow molecules of different size or charge to pass between neighboring cells (24, 25, 29). As such, it is feasible that changes in intercellular communication during developmental processes can be regulated by gap junctions if they selectively allow passage of regulatory molecules between cells in any given epithelium.
In the epididymis, there are several well-defined events that occur during postnatal development. In addition to the formation of the blood-epididymal barrier, epididymal principal cells become fully differentiated between days 16 and 44 (17, 39), spermatozoa enter the epididymis on day 45 and are present throughout the epididymis by day 56, and serum androgen levels begin to increase by day 28 and peak by 42 days of age (32, 33). The developmental patterns of epididymal Cx mRNA levels in the caput-corpus and cauda epididymidis varied among the different Cxs, suggesting that these may have different roles in the development and function of the epididymis.
Developmental changes in Cx26 mRNA levels in the caput-corpus
epididymidis indicate that levels for this Cx were particularly elevated in young animals until day 35, when levels decrease
dramatically and remain low thereafter. The decrease in Cx26 is well
correlated with the completion of epididymal principal cell
differentiation (30, 31). Cx26 expression has been
reported to be elevated in differentiating keratinocytes
(2). When these cells differentiate, they switch from
producing Cx26 to either Cx31 or Cx31.1. A similar type of regulation
may also be occurring in the caput-corpus epididymidis, because Cx31.1
mRNA levels increase as Cx26 levels are decreasing (Fig.
3A). The decrease in Cx26 in the caput-corpus epididymidis is well correlated with increasing circulating androgen levels, as well
as with increasing epididymal 4-ene steroid-5--reductase levels
(2). Whether or not Cx26 is androgen repressed remains to
be established. Other studies, however, have reported that sex steroid
hormones, such as combinations of progesterone and estradiol, can
decrease the expression of Cx26 in the uterus (15).
In the cauda epididymidis, Cx26 and Cx43 mRNA levels appear to be regulated quite differently from the caput-corpus. In this segment of the epididymis, a Cx26 transcript was not detectable by Northern blot, although it could be amplified by RT-PCR using Cx26 specific primers. Furthermore, Cx26 protein is expressed in the cauda epididymidis of both 21- and 91-day-old rats, as shown by immunoblotting (Fig. 6). Clearly, however, Cx26 protein levels were much less abundant in the adult epididymis, particularly in the cauda region. Segment-specific expression and regulation of epididymal genes have been reported for several genes (9, 12, 14, 20). The immunolocalization of Cx26 in epididymides of 21-day-old rats suggests that Cx26 is present between adjacent epithelial cells that line the lumen of the epididymis. In the adult, however, Cx26 immunostaining was less intense. The fact that Cx26 protein levels in 21-day-old rats did not appear to be different between epididymal regions either by Western blot or immunocytochemistry, whereas mRNA levels were much lower in the cauda epididymidis, suggests that Cx26 may be regulated at the posttranscriptional level. Studies by Krem et al. (22) indicate that in hepatocytes of regenerating liver, Cx32 and Cx26 are both regulated at the posttranscriptional level. Further studies will be needed to elucidate the exact mechanism and regulation of Cx26 in the epididymis.
Interestingly, Cx43 mRNA levels appear to follow a similar expression pattern as Cx26, but these levels do not decrease as dramatically as those of Cx26 after postnatal day 28. These data indicate that cellular communication in the epididymis varies as a function of development and that there appears to be a switch in Cx expression in the caput-corpus epididymidis.
Connexons consist of six Cxs arranged radially around a central pore and contain either a single type of Cx (homomeric) or different types of Cxs (heteromeric). Furthermore, adjacent homologous or heterologous cell types can contribute specific types of connexons to form either homotypic, heterotypic, or heteromeric intercellular channels (13). Homomeric and heteromeric connexons have distinct molecular permeabilities that contribute to the regulation of intercellular communication. In mouse mammary gland, Cx26 mRNA and protein levels increased from early pregnancy onwards, whereas Cx32 was detectable only during lactation (23). During this time, Cx26 and Cx32 colocalized to the same junctional plaques and were organized as either homomeric or heteromeric connexons. Thus the structural diversity in assembly of gap junctional hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that then influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells (35). In the present study, Cx32 and Cx26 along the lateral margins of principal cells are either colocalized or are localized independently of one another. This suggests that there are different types of connexons between principal cells and that these may regulate specific interactions between these cells. Interestingly, in younger rats, Cx26 and Cx32 are also colocalized, although at this age it does not appear that these Cxs form homotypic gap junctions. Further studies will be needed to determine whether the formation of homotypic connexons in the adult rat epididymis is developmentally regulated.
Results from the present experiments indicate that after 4 wk of postnatal development, relative mRNA levels of Cx26 and Cx43 decrease, whereas those of Cx31.1 and Cx32 increase in the caput-corpus epididymis, suggesting that Cxs may play a role in the differentiation of epithelial cells into narrow, principal, basal, and clear cells (13, 17, 34). In rat hepatic cell lines, characteristics of different stages of hepatic differentiation have revealed a correlation between the differentiated state of the cells and the Cxs they express (35). As hepatocytes differentiate, the proportions of Cx26 and Cx43 mRNA decrease, whereas Cx32 increases. It is therefore possible that alterations in Cxs may modulate intracellular messages associated with cellular differentiation in the epididymal epithelium.
In the cauda epididymidis, the developmental expression pattern of Cx31.1 is low at day 7 and increases rapidly to peak at approximately day 28. Cx32 mRNA levels, on the other hand, are not detectable at day 7 and peak by day 63. In contrast to the developmental patterns of Cx31.1 and Cx32 mRNA levels, Cx43 mRNA levels in the cauda epididymidis remain unchanged during development. These data indicate that the regulation of Cx43 appears to be region specific. Previous studies have reported that in the cauda epididymidis, Cx43 is present not only between principal and basal cells, as is the case in other epididymal segments, but in this region it is also present between myoid cells and may be important in expelling spermatozoa from the cauda epididymidis at the time of ejaculation. Differences in cell-specific expression of Cx43 may account, at least in part, for differences in the developmental expression pattern of Cx43 along the epididymis.
Cx30.3 could not be detected by Northern blot analyses but was amplified by RT-PCR. Previous studies in the kidney have also reported that the cellular levels of Cx30.3 were too low to be detected by Northern blot analysis (40). In both 21-day-old and adult rat epididymis, Cx30.3 appears to be present throughout the epididymis. Whereas in the adult the mRNA levels for Cx30.3 appear to be greater in the proximal regions (initial segment and caput) of the epididymis, further studies will be needed to clearly understand the distribution and regulation of this Cx in the epididymis.
Immunolocalization of Cx32 indicate that in the adult rat epididymis, it is expressed along the lateral margins of adjacent principal cells. This suggests that Cx32-mediated gap junctions are involved in direct communication between adjacent principal cells. The localization of Cx32 along the lateral plasma membrane supports previous observations of the presence of gap junctions between these cells, as determined by freeze fracture electron microscopy (11).
Data from these experiments indicate that the cellular communication in the epididymis is complex and involves a large number of Cxs. Whether or not these Cxs form homologous or heterologous gap junctions is presently unknown; however, the developmental patterns of Cx mRNA levels in the epididymis suggest that some Cxs, such as Cx26 and Cx43, may play a role in the differentiation of the epididymal epithelium and that there is a switch from Cx26 to Cx31.1 during the development of the epididymis.
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ACKNOWLEDGEMENTS |
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Dr. L. Hermo and S. DeBellefeuille are thanked for their assistance.
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FOOTNOTES |
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K. W. Finnson was the recipient of an Institut National de la Recherche Scientifique postdoctoral fellowship. This study was supported by National Sciences and Engineering Research Council in the form of a grant to D. G. Cyr.
Address for reprint requests and other correspondence: D. G. Cyr, INRS-Institut Armand Frappier, Univ. de Québec, 245 Hymus Blvd., Pointe-Claire, Québec, Canada H9R 1G6 (E-mail: daniel.cyr{at}INRS-IAF.uquebec.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 4, 2002;10.1152/ajpcell.00111.2002
Received 12 March 2002; accepted in final form 26 August 2002.
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