Expression of multiple connexins in the rat epididymis indicates a complex regulation of gap junctional communication

Julie Dufresne, Kenneth W. Finnson, Mary Gregory, and Daniel G. Cyr

Institut National de la Recherche Scientifique-Institut Armand Frappier, Université du Québec, Pointe-Claire, Québec, Canada H9R 1G6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Primers used in RT-PCR for the identification of rat epididymal Cxs

Total RNA was isolated from adult rat epididymides by using the guanidinium isothiocyanate method (4). The isolated RNA was treated with DNAse to remove any possible contamination by genomic DNA (1 U/µg of RNA; deoxyribonuclease I, amplification grade; Canadian Life Technologies, Burlington, ON, Canada). Resulting RNA was reverse transcribed by using oligo d(T)16-18 primers (Amersham Pharmacia Biotech, Baie D'Urfe, QC, Canada) and M-MLV reverse transcriptase (Canadian Life Technologies) according to the supplier's instructions. The cDNA templates (250 ng) were amplified by using 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 60 s, and elongating at 72°C for 90 s. A final extension at 72°C for 15 min was done to create 3'A-overhangs. The F1R1 and F2R2 RT-PCR products were separated on either a 1.5 or 2% agarose gel and visualized by ethidium bromide staining. The F1R1 RT-PCR product was extracted from the gel (QIAEX II Gel Extraction Kit; Qiagen, Mississauga, ON, Canada), cloned, and sequenced by using an automated sequencer (Sheldon Biotechnology Center, Montreal, QC, Canada).

F2R2 PCR products were further characterized by restriction enzyme digest by using 10 µl of PCR product. The restriction analysis was based on the digestion of PCR products with Cx-specific restriction enzymes. Published cDNA sequences of the different rat Cxs were obtained from Genbank. The predicted F2R2 amplicon size of each Cx was determined by using the Oligo software (version 5.0; Molecular Biology Insights, Cascade, CO). Restriction sites were identified for each Cx whose amplicon size corresponded to the molecular weight of the F2R2 PCR products. The Cx amplicon, restriction enzyme, and the predicted digestion fragment sizes are shown in Table 2. RT-PCR products were treated with specific restriction enzymes (Amersham Pharmacia Biotech) for 90 min according to the manufacturer's instructions. RT-PCR products incubated with buffer alone were used as negative controls. Digestion products were resolved on a 2% agarose gel and visualized by ethidium bromide staining.

                              
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Table 2.   Cx transcripts amplified by the F2R2 primer set

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.

Total cellular RNA from the proximal and distal regions of epididymides from 7- to 91-day-old rats was extracted by using the guanidinium isothiocyanate method (4). Total cellular RNA (10 µg/lane) was separated by electrophoresis on 1.2% agarose-formaldehyde gel and transferred onto a charged nylon membrane (Genescreen Plus; NEN Life Science Products, Boston, MA). The membranes were then hybridized with specific Cx cDNA probes according to the methods outlined by Viger and Robaire (41). For each blot (n = 3), RNA from paired epididymides was pooled from 3 individual rats of 7, 14, or 21 days of age, whereas RNA pooled from two rats was used for day 28. RNA from paired epididymides from individual rats was used for rats of older ages. Each blot contained RNA from distinct pools or individual rats.

Cx-specific cDNA probes were obtained as generous gifts [Cx26 from B. Nicholson (SUNY) (42); Cx31.1 from K. Willecke (Univ. of Bonn); Cx32 from D. Paul (Harvard Univ.) (27); and Cx43 from E. Beyer (Univ. Chicago) (1)] and used to hybridize the blots of epididymal RNA from rats of different postnatal ages. Fragments were excised from plasmids with the appropriate restriction enzymes, purified on agarose gel using a purification kit (QIAEX II Gel Extraction Kit; Qiagen), and labeled by random priming with [32P]-dCTP (Oligo Labeling Kit; Amersham Pharmacia Biotech). Northern blots were normalized for the amount of RNA loaded in each lane by stripping the membranes and reprobing with an end-labeled oligonucleotide probe recognizing the 18S ribosomal RNA sequence (5). Membranes were exposed to an X-ray film, and the resulting unsaturated autoradiograms were scanned by using a Fluor-S MultiImager (Bio-Rad Laboratories, Mississauga, ON, Canada). Each Cx signal was standardized against that of the 18S rRNA to determine relative levels of mRNAs. Three different blots composed of RNA from different rats were used to determine the relative Cx mRNA levels in each region of the epididymis.

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.

For Cx32 immunocytochemistry, rats were anesthetized and the epididymides were fixed by retrograde perfusion through the dorsal aorta with saline, followed by 100% Saint-Marie's fixative (95% ethanol/glacial acetic acid, 99:1). Epididymides were dissected out, dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin. Immunolocalization of Cx32 was done on 5-µm sections mounted on glass slides coated with 5% gelatin, baked at 60°C, and stored at room temperature. Immunocytochemical localization of Cx32 was done by using a rabbit polyclonal Cx32 antiserum (10 µg/ml; Zymed Laboratories) and the DAKO Catalyzed Signal Amplification System (DAKO, Carpenteria, CA). Antibody binding to Cx32 was detected with a horseradish peroxidase-conjugated secondary antiserum as outlined by the manufacturer (DAKO). Slides incubated with normal rabbit antiserum were used as a negative control.

For Cx26 and Cx32 colocalization studies, frozen sections were prepared as previously described and incubated with the Cx26 primary antibody and a Texas red-conjugated anti-rabbit secondary antibody (1:250 dilution; Jackson Immunoresearch), as described above, and washed for 5 min with PBS. Sections were then incubated with Cx32 primary antiserum (10 µg/ml; Chemicon International) for 1 h at room temperature and subsequently incubated with FITC-conjugated anti-mouse secondary antiserum (1:250; Jackson Immunoresearch). Sections were then washed three times in PBS for 5 min at room temperature, mounted with Vectastain mounting medium (Vectastain Laboratories) containing 4',6'-diamidino-2-phenylindole (DAPI) to visualize the nuclei, and stored at 4°C. Sections were viewed with a Leica fluorescent microscope, and the images were digitalized, merged, and analyzed by using the ImagePro Plus software (version 4.0; Media Cybernetics, Silver Spring, MD).

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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   RT-PCR amplification of connexin (Cx) mRNAs from the adult rat epididymis. A single 639-base pair (bp) product was amplified with the F1R1 primers identified as Cx43 by cloning and sequencing. F2R2 primers generated products between 358 and 379 bp that were then analyzed with Cx-specific restriction enzymes. WC, water control.

RT-PCR of epididymal RNA with the F2R2 primers generated PCR products of 375 bp and a second product(s) of ~360 bp (Fig. 1). Our initial attempts to clone the 360-bp product indicated that several transcripts of similar molecular weights had been amplified. Due to the close molecular weights of these amplicons, it was not possible to isolate them from the gel for cloning and sequencing. Therefore, the identification of these Cxs was determined by using a restriction enzyme digest approach (Table 2, Fig. 2). Based on the digestion pattern of the F2R2 amplification products, we identified the presence of transcripts for Cx30.3, Cx31.1, and Cx32 in adult epididymis. The identities of these transcripts were confirmed either by cloning and sequencing or by Northern blot analysis as described in MATERIALS AND METHODS. To determine whether there were other Cxs expressed in young rats, RT-PCR was also done with the F2R2 primers by using RNA from 21-day-old rats. The amplification and restriction enzyme digestion patterns were identical to adults, with the exception that a faint DNA smear with the PCR product observed with Pvu II, RcaI, or DraI digestion was suggestive of the presence of Cx26 and Cx30 (data not shown). The presence of a faint smear with DraI may be suggestive of another Cx present in the epididymis. Northern blot analysis for both Cx30 and Cx26 using epididymal RNA from 21-day-old rats indicated the presence of a transcript for Cx26 in the caput-corpus epididymis of young rats, although Cx30 was undetectable (not shown).


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Fig. 2.   Restriction enzyme analysis of F2R2 PCR product. Epididymal amplification product was digested with Cx-specific restriction enzymes as listed in Table 2. Fragment sizes that were generated allowed us to identify Cx30.3 (NruI), Cx31.1 (XhoI), and Cx32 (SmaI). Cx26 and Cx30 were not identified in the adult rat.

Based on these analyses, we conclude that in the epididymis there are mRNA transcripts for Cx43, Cx32, Cx31.1, and Cx30.3. Furthermore, although we could not confirm the presence of Cx26 in adult rat epididymis, Cx26 was shown to be present in the epididymis of young (21-day-old) rats.

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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Fig. 3.   Cx mRNA levels in the rat epididymis during postnatal development. Total cellular RNA was isolated from the proximal epididymis (PE; initial segment, caput, and corpus) of rats ranging from 7- to 91-days old. Transcripts for Cx26 (A), 31.1 (B), 32 (C), and 43 (D) were detected by Northern blot analysis. RNA loading was standardized by hybridizing the membranes with an oligonucleotide probe recognizing the 18S rRNA. Relative Cx mRNA levels were determined densitometrically by expressing the Cx mRNA levels relative to the 18S rRNA. Data were normalized with the ratio obtained for the 91-day-old adult rat to allow blot to blot comparisons and are expressed as means ± SE from 3 separate pools of tissue. * Significant difference between ages.

Unlike Cx26, Cx31.1 mRNA levels in the caput-corpus epididymidis were low on days 7, 14, and 21. Levels increased thereafter to peak at day 49 and subsequently decreased until day 91. In the caput-corpus epididymidis, Cx31.1 mRNA levels increased as a function of age from day 7 until day 28, and levels remained constant thereafter (Fig. 3B). Levels in the cauda epididymidis were low at day 7, increased to peak by day 28, and remained constant until the rats reached adulthood (Fig. 4A). Cx32 mRNA levels in the caput-corpus epididymidis were low on days 7 and 14 and increased ~3.5-fold on day 21; levels remained constant thereafter (Fig. 3C). In cauda epididymidis, Cx32 mRNA levels increased as a function of age and peaked between days 49 and 56, when sperm enter the epididymidis (Fig. 4B); Cx32 mRNA levels remained constant afterwards.


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Fig. 4.   Cx mRNA levels in the rat epididymis during postnatal development. Total cellular RNA was isolated from the cauda epididymidis of rats ranging from 7- to 91-days old. Transcripts for Cx31.1 (A), 32 (B), and 43 (C) were detected by Northern blot analysis. Cx26 mRNA was at undetectable levels. RNA loading was standardized by hybridizing the membranes with an oligonucleotide probe recognizing the 18S rRNA. Relative Cx mRNA levels were determined densitometrically by expressing the Cx mRNA levels relative to the 18S rRNA. Data were normalized with the ratio obtained for the 91-day-old adult rat to allow blot to blot comparisons and are expressed as means ± SE from 3 separate pools of tissue. * Significant difference between these ages and 7- to 14-day-old rats. CA, cauda.

Cx43 mRNA levels in the caput-corpus epididymidis increased in a linear fashion from days 7 to 28. Cx43 mRNA levels then decreased rapidly almost twofold and were fivefold lower by day 91 (Fig. 3D). Relative Cx43 mRNA levels in the cauda epididymis did not change significantly during postnatal development (Fig. 4C). Cx30.3 mRNA levels were below the level of detection by Northern blot analysis in both the caput-corpus and cauda epididymidis.

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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Fig. 5.   Detection of Cx26 (A) and Cx30.3 (B) mRNAs by RT-PCR in different regions of the adult (91-day-old) and immature (21-day-old) rat epididymis. Epididymides were divided into caput-corpus (initial segment, caput, and corpus epididymidis) (P) and cauda epididymidis (D) from 21- (21D) and 91-day-old rats (91D). Adult rat epididymides were also divided into the initial segment (IS), caput (CT), corpus (CS), and CA segments. Liver (L) and kidney (K) were used as positive controls.

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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Fig. 6.   Western blot analysis of Cx26 expression in the P (IS, CT, and CS epididymidis) and D from 21D and 91D rats. Cx26 protein was detected in the adult (91D) and the immature (21D) rat epididymis. L was used as a positive control.

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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Fig. 7.   Immunolocalization of Cx26 in the epididymis of 21D and 91D rat. Cryosections (10 µm) were incubated with Cx26 antiserum and localized with a FITC-conjugated secondary antibody. Nuclei were stained with propidium iodide. In 21D rats (A), Cx26 (arrows) was localized to the epithelial cells (E) that line the lumen (Lu) of the caput epididymidis (magnification ×400). The immunolocalization of Cx26 was similar throughout the different regions of the epididymis (not shown). In the CT epididymidis of the adult rat, Cx26 was localized to epithelial principal cells (P) near the base of the epithelium between principal cells (arrows). Similar immunostaining was observed in the CS (B) and CA (D) epididymidis. Magnification ×650. IT, interstitial space; B, basal cells; n, nuclei.

Cx32 in 21-day-old rat epididymis was localized along the lateral plasma membrane of adjacent epithelial cells that line the epididymal lumen (Fig. 9B). In adult rats, Cx32 was detected in all regions of the adult rat epididymis (Fig. 8, A-D). Cx32 was localized along the lateral plasma membrane of adjacent principal cells and appeared as punctate staining along the entire length of the cells. Cx32 was also present between apical cells and principal cells, as well as between narrow cells and principal cells and between clear and principal cells (Fig. 8, A-D). The intensity of the Cx32 immunostaining appeared to be similar in the different regions of the epididymis, with the exception of the distal cauda epididymidis, in which Cx32 levels were low.


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Fig. 8.   Cx32 immunolocalization in the adult rat epididymis. Sections (5 µm) were incubated with Cx32 antiserum and localized with a horseradish peroxidase-conjugated secondary antibody. In the initial segment of the epididymis (A), Cx32 is present along the lateral plasma membrane of adjacent principal cells (black arrows) and between apical (A) and principal cells (white arrows), as well as between narrow cells (N) and principal cells. An immunoreactive product is also present at the base of the epithelium and appears to be localized between basal and principal cells (yellow arrows). In the CT (B), CS (C), and CA (D) epididymidis, Cx32 was localized along the lateral plasma membrane of adjacent P, and at the base of the epithelium between principal cells and basal cells. Immunostaining was also present between principal cells and clear cells (not shown). Magnification ×900. S, spermatozoa.

Colocalization studies in young (14-day-old) rats revealed that Cx26 and Cx32 are colocalized along the lateral plasma membrane (Fig. 9, A-C). In adult rats, however, Cx26 and Cx32 were either colocalized along the lateral plasma membrane between adjacent principal cells or were present independently of one another (Fig. 9, D-F).


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Fig. 9.   Immunolocalization of Cx26 and Cx32 in the epididymis of 14D and 91D rats. Cryosections (10 µm) were incubated with both Cx26 and Cx32 antiserum and localized with a Texas red or FITC-conjugated secondary antibody. In 21D rats (A), Cx26 and Cx32 were localized to the epithelial cells that line the lumen of the CT epididymis (arrows). In merged photomicrographs, both Cx26 and Cx32 were colocalized along the lateral plasma membrane of E. In adult rats, Cx26 and Cx32 were localized between adjacent principal cells. Colocalization studies indicate that both Cxs can colocalize (white arrow) or be present independently of each other (yellow arrow). Magnification ×650.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha -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.


    ACKNOWLEDGEMENTS

Dr. L. Hermo and S. DeBellefeuille are thanked for their assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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