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research-article |
INRS-Institut Armand Frappier,3 Université du Québec, Pointe Claire, Quebec, Canada H9R 1G6 Department of Biological Sciences,4 University of Idaho, Moscow, Idaho 83844-3051 ISMER,5 Université du Québec à Rimouski, Rimouski, Quebec, Canada G5L 3A1
ABSTRACT
Certain fish, such as rainbow trout (Oncorhynchus mykiss), are seasonal breeders. Spermatogenesis in rainbow trout is synchronous; therefore, at any time point during this process, germ cells are predominantly at the same stage of development. As such, rainbow trout represent an excellent model in which to study spermatogenesis. Gap junctions are composed of connexons, which are themselves formed by six transmembrane proteins termed connexins (Cxs). The objectives of this study were to assess which Cxs are expressed in the rainbow trout testis, and if their expression was stage specific during gonadal maturation. Rainbow trout were killed at various stages of maturation, and total cellular RNA was isolated from the testes. RT-PCR using degenerate primers recognizing all vertebrate Cxs indicates that there are several different Cxs in trout testes. Amplicons were cloned and sequenced. Homology comparisons indicate that these were cx43, cx43.4, cx31, and cx30. Immunolocalization of these Cxs indicate that Cx43 was localized primarily to Sertoli cells, while Cx43.4 was localized along the lateral plasma membranes between adjacent spermatocytes. Cx30 was localized to the interstitial Leydig cells, and Cx31 was localized primarily to the endothelium of interstitial blood vessels. The expression of each Cx varied as a function of the stage of spermatogenesis, suggesting that the expression of these proteins is highly regulated. Together, these results indicate that intercellular communication in the testis is complex, involves several different Cxs, and is a highly regulated process.
cellular communication, connexin, Leydig cells, rainbow trout, Sertoli cells, spermatogenesis, testis
Spermatogenesis requires highly coordinated cellular interactions and communication between differentiating germ cells and Sertoli cells. Gap junctions are responsible for mediating direct intercellular communication between neighboring cells [1]. These junctions are comprised of integral transmembrane proteins, termed connexins (Cxs), which form a connexon. Connexons from adjacent cells align with each other to form intercellular pores that will permit the exchange of small molecules, including secondary messengers, between cells [1–3]. Gap junctions were first described in the mammalian testis by Dym and Fawcett [4], who showed by electron microscopy that gap junctions were present between adjacent Sertoli cells. Risley et al. [5] first reported the presence of Cx43 (also known as Gja1) in gap junctions between Sertoli cells of the seminiferous tubules, as well as between the Leydig cells of the interstitium. It has been reported that there are at least 12 different Cxs expressed in the mammalian testis, suggesting the presence of selective communication between the different cell types of the testis [6]. Gap junctional communication is essential for spermatogenesis, as testes from Cx43 knockout mice display arrested spermatogenesis [7].
The expression and cellular localization of Cxs have been shown to be regulated by several hormones, including testosterone [8], thyroid hormones [9], and retinoids [10]. These hormones have also been shown to influence testicular functions and maturation in mammals [11–13]. While we are only now beginning to understand the role and regulation of Cxs in the mammalian testis, there is little information on the comparative aspect of gap junctional communication in other vertebrate groups, and whether or not these may represent useful animal models to assess the role of gap junctions in male fertility.
In salmonid fish, spermatogenesis occurs in testicular cysts referred to as spermatocysts. Spermatocysts contain both a Sertoli cell and spermatogonia, which are themselves derived by mitotic division of a single spermatogonium [14]. Salmonids are seasonal breeders, and gonadal maturation involves differentiation and maturation of a pool of germ cells every year [15]. Spermatogenesis occurs in a synchronous pattern, and, thus, the germ cells of the testis are all at the same stage of development. As such, this represents an ideal model in which to study the regulation of stage-dependent processes, such as intercellular communication, which occur during spermatogenesis.
Ultrastructural studies have demonstrated the presence of gap junctions between adjacent Sertoli cells and between germ cells and Sertoli cells of teleost fish [16–18]. Batlouni et al. [16] first reported the presence of Cx32 in the catfish (Pseudoplatystoma fasciatum) testis. Using immunogold labeling, they were able to demonstrate that Cx32 was present in both germ cells and Sertoli cells, suggesting a role for this Cx in testicular gap junctions. Given the complexity of intercellular communication in the mammalian testis, and the large number of Cxs that are implicated in modulating this communication, it appears likely that there are several other Cxs expressed in the fish testis that may modulate various testicular functions.
The objectives of this study were to assess, by RT-PCR and Western blots, which Cxs are expressed in rainbow trout (Oncorhynchus mykiss) testis, to determine, using immunocytochemistry, whether the expression of these Cxs varies as a function of spermatogenesis, and to identify which cell types of the testis expressed which Cx.
Male rainbow trout (O. mykiss, House Creek strain) were sampled from a population maintained at the University of Idaho's Aquaculture Experiment Station in Hagerman, Idaho. The fish were held in an outdoor raceway under ambient conditions of light and temperature. Ten males at each stage of spermatogenesis (total of 60 fish) were anesthetized (Finquel; Argent, Redmond, WA) and humanely killed, according to a protocol approved by the University of Idaho Animal Care and Use Committee, on five separate occasions between August 1999 and May 2000. The testes were removed from these fish and samples either frozen in liquid and stored at –80°C, or immersion-fixed in Histochoice (Amresco, Solon, OH). These samples were used to provide the six different stages of spermatogenesis found in the rainbow trout [19]. Stages of maturation were defined as follows: Stage 1, testes contained numerous spermatogonia; Stage 2, the testicular cysts are increased in size—spermatogonia and spermatocytes are present; Stage 3, the testis contains spermatogonia, spermatocytes and spermatids; Stage 4, presence of spermatozoa; Stage 5, start of spermiation—tubules are filled with spermatozoa; Stage 6, the testis is in regression, the structure of testicular cysts is disorganized, and vacuoles are present.
Identification of Testicular Cxs
To identify testicular Cxs, an RT-PCR strategy was employed 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 mammalian Cx multigene family [20]. We have previously used this approach to identify epididymal Cxs [21].
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Testes were staged histologically and total RNA was isolated from rainbow trout testes at six different stages of spermatogenesis using Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA). The isolated RNA was treated with DNase (1 U/µg of RNA; deoxyribonuclease I, amplification grade; Canadian Life Technologies, Burlington, ON, Canada) to remove any contaminating genomic DNA. The resulting RNA was reverse transcribed using oligo d(T) 16–18 primers (Amersham Pharmacia Biotech, Baie D'Urfe, PQ, Canada) and M-MLV reverse transcriptase (Canadian Life Technologies), according to the suppliers' instructions. The resulting cDNA templates (250 ng) were amplified using two different primer combinations: F1R1 and F2R2 (Table 1). Amplification by PCR was done using 30 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 60 sec, and elongation at 72°C for 90 sec. 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 a 1.5% agarose gel and visualized by ethidium bromide staining. The RT-PCR products were extracted from the gel (QIAEX II Gel Extraction Kit; Qiagen, Mississauga, ON, Canada), cloned, and sequenced using an automated sequencer (Sheldon Biotechnology Center, Montreal, PQ, Canada). The sequences of the resulting RT-PCR products were compared to other known Cxs using BLAST homology comparisons (GenBank, National Center for Biotechnology Information, Bethesda, MD). Experiments were repeated once on rainbow trout as well as in brook trout (Salvelinus fontinalis) testes (data not shown).
Based on the nucleotide sequences of trout testicular Cxs, we assessed the similarity of these sequences with other vertebrates using JellyFish software (LabVelocity Inc., Los Angeles, CA; Table 2). The nucleotide sequences of the Cxs were converted to their amino acid sequences. Based on these sequences, we compared peptide sequences used by commercial companies to generate antisera against mammalian Cxs, and identified those antisera developed against peptides whose sequence was also found in the trout Cx. Using this approach, two antisera were identified as being likely to recognize trout Cxs: Cx43 and Cx43.4. For trout Cx43, an anti-Cx43 (Cx43 H-150; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used. Trout Cx43.4 has a high degree of homology with zebra fish Cx43.4. It had been reported that anti-human CX45 antisera (also known as GJA7) generated against a conserved region of the N-terminal of the protein could recognize zebra fish Cx43.4 (Cx45 N-19; Santa Cruz Biotechnology). Because there were no existing antisera against peptides or proteins whose sequences shared homology with trout Cx30 and Cx31, we generated custom-made peptides and antisera against these Cxs.
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Synthesis of Cx30 and Cx31 Antisera
For both Cx30 and Cx31, the hydropathicity of the predicted amino acid sequences was assessed using the method of Kyte and Doolittle [22] to identify extracellular and intracellular regions of each Cx. Two peptides were synthesized corresponding to different extracellular regions of Cx30 (amino acids from 62 to 76) and Cx31 (amino acids from 62 to 85) (Table 3). Peptides Cx30 (62–76) and Cx31 (62–85) were synthesized using a solid-phase procedure based on fluorenylmethyloxycarbonyl (Fmoc) chemistry, with a Rink-AM-amide resin (Chem-Impex International, Wood Dale, IL) as the solid support. N-
-Fmoc-protected amino acids (Matrix Innovation Inc., PQ, Canada) were introduced into the peptide chain following a benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate coupling strategy, and each coupling reaction was monitored through a ninhydrin test. Most coupling reactions were completed within 1 hr, but, when required, the coupling step was repeated. Cleavage from the resin to obtain crude peptides was achieved with a mixture of trifluoroacetic acid (TFA)/ethanedithiol/triisopropylsilane/water (92.5/2.5/2.5/2.5; 20 ml/g). After TFA evaporation, peptides were precipitated using diethylether.
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Disulfide bond formation for the Cx peptides was performed with air oxidation by leaving the crude peptide solutions at 4°C overnight. Cyclic crude peptides were then purified by RP-HPLC following two different methods, since Cx30 (62–76) and Cx31 (62–85) had distinct solubility properties. Cx30 (62–76) was dissolved in 15% acetonitrile (ACN)/H2O and 0.06% TFA before being injected onto a Flanged MODCOL column (25 x 3.5 cm) packed with Jupiter C18 resin (15 µm, 300 Å; Phenomenex, Torrance, CA). The purification step was carried out using a Waters Prep 590 pump system connected to a Waters model 441 absorbance detector. The flow rate was maintained at 20 ml/min and the detection level at 229 nm. The peptide was eluted from the column with a solvent gradient from 15% to 55% ACN/H2O and 0.06%TFA in 2 hr.
Cx31 (62–85) was dissolved in 20% isopropanol/H2O containing 1 M urea before being injected onto the same column and system used for Cx30 (62–76). The flow rate was maintained at 8 ml/min for this purification. A gradient from 20% to 80% isopropanol/H2O over 2 hr was used to obtain a purified Cx31 (62–85) peptide. The purity of the collected fractions was evaluated using analytical RP-HPLC, and the product was characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Voyager DE; Applied Biosystems, Foster City, CA).
Homogeneous fractions corresponding to the desired peptides were pooled and lyophilized. The purified peptides were linked to the keyhole limpet hemocyanin protein and used to generate rabbit polyclonal antisera (PolyQuik method; Zymed Laboratories, San Francisco, CA). Two antisera were produced against each Cx-specific peptide.
The specificity of both the commercial antisera as well as the trout-specific antisera was assessed by Western blot analyses. A membrane-enriched protein fraction was extracted from mature rainbow trout testis. Tissues were ground with a mortar and pestle in liquid nitrogen and homogenized in buffer (7 mM Tris-HCl, pH 6.8, 0,04 mM CaCl2, 2 µg/ml leupeptin, 2 µg/ml aproptonin, 100 µg/ml PMSF, 1 µg/ml pepstatin, 2 µg/ml antipain) using a motor-driven pestle. Samples were centrifuged at 2200 x g for 30 min at 4°C, and the supernatant was then removed and centrifuged at 30 000 x g for 30 min at 4°C. The resulting protein pellet was resuspended in homogenization buffer and the protein concentration determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Samples were stored at –86°C until electrophoresis. Samples were heated at 95°C for 2 min and resolved in Laemmli buffer on a 12.5% SDS polyacrylamide gel. Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories) at 100 V for 90 min at 4°C. The resulting blots containing the proteins were stained with Ponceau red S to evaluate the transfer efficiency. The blots were then rinsed and blocked overnight at 4°C in Tris-buffered saline-Tween (TBST) buffer (20 mM Tris-HCl, 500 mM NaCl, and 0.05% Tween 20, pH 7.5) containing 5% non-fat powdered milk. Membranes were incubated with the corresponding anti-Cx antisera (anti-Cx43 [800 ng/ml], anti-Cx43.4 [800 ng/ml], anti-Cx30 [1 µl/ml], and anti-Cx31 [1.33 µl/ml]) in TBST buffer for 75 min at room temperature. The blots were washed four times for 15 min in TBST at room temperature and subsequently incubated for 60 min with an appropriate alkaline phosphatase-conjugated anti-rabbit (Cx30, Cx31, Cx43) or anti-goat (Cx43.4) secondary antibody (400 ng/ml; Santa Cruz Biotechnology) in TBST containing 5% non-fat milk, and subsequently washed as described above. Complexed antibodies were revealed using the Bio-Rad blotting detection kit (Bio-Rad Laboratories). Western blots were repeated twice (n = 3) on separate individuals.
Immunolocalization of the Different Cxs
Small pieces (0.5 cm2) of the middle portion of one testis were fixed in Histochoice MB (Amresco, Solon, OH). After 2 weeks of fixation at 4°C, the tissues were stored in 70% ethanol at room temperature. Tissues were subsequently dehydrated with graded ethanols and embedded in paraffin. Tissues sections (4 µm) were mounted onto glass slides coated with poly-L-lysine for immunohistochemistry. Testes from four different fish (n = 4) at each stage of spermatogenesis were used for immunolocalization of each Cx. Tissue sections were deparaffinized in HistoClear (Fisher Scientific, Ottawa, ON, Canada) and rehydrated by immersion in a series of graded ethanols. Immunocytochemistry was done using the DAKO Catalyzed Signal Amplification System (DAKO, Carpenteria, CA) according to the manufacturer's instructions. Tissue sections were incubated at 37°C with each of the anti-Cx antisera: anti-Cx43 (800 ng/ml for 60 min), anti-Cx43.4 (800 ng/ml for 90 min), anti-Cx30 (1 µl/ml for 90 min), and anti-Cx31 (1.33 µl/ml for 60 min). Antibody binding to each Cx was detected using either anti-rabbit or anti-goat horse radish peroxidase-conjugated secondary antiserum according to the manufacturer's instructions. Sections were counter stained with methylene blue. Tissue sections incubated with normal rabbit serum were used as a negative control.
Identification of Testicular Cxs
Amplification of testicular mRNA by RT-PCR with the F1R1 primers yielded a single amplicon of 649 bp (Fig. 1a). Sequencing of this cDNA, followed by BLAST search in Genbank, confirmed that the amplified product was cx43. cx43 mRNA was expressed at all stages of spermatogenesis (Fig. 1).
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In contrast, amplification with the F2R2 primers resulted in the amplification of three different amplicons of 506, 398, and 357 bp (Fig. 1). Sequencing results indicated that these cDNAs were cx43.4, cx30, and cx31. Unlike cx43, not all of these amplified Cxs were expressed throughout spermatogenesis. cx43.4 was expressed only in stages 1 and 2 of spermatogenesis, while cx30 was present at all stages of spermatogenesis. Finally, cx31 was present in stages 1–5, and was undetectable at stage 6. Data were reproducible and similar to results observed in brook trout testis (data not shown), suggesting that these results may be consistent between salmonids.
Western blot analyses were done to assess the specificity of the antisera for rainbow trout Cxs. Anti-Cx43 antisera recognized a single protein band of 43 kDa from the rainbow trout testis (Fig. 2). Rat epididymis was used as a positive control for Cx43, and negative controls, in which there was no primary antiserum, did not show any specific protein bands of this molecular weight. The Cx43.4 antisera recognized a single 44 kDa protein (Fig. 2). Rat brain was used as positive control for Cx43.4. Antisera generated from synthesized peptides for Cx30 and Cx31 also recognized a single protein of the appropriate molecular weight (30 and 31 kDa, respectively; Fig. 2). Incubation of protein blots with preimmune serum did not reveal any bands corresponding to these molecular weights.
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Immunolocalization of Testicular Cxs
We observed a unique immunostaining pattern for each Cx through immunolocalization of rainbow trout Cxs, suggesting that each Cx is involved in cell-specific intercellular communication. Cx43 was localized in linear arrays at the periphery of the Sertoli cell plasma membranes (Fig. 3A) in rainbow trout testis. No immunostaining was observed in either Leydig cells or other cells of the interstitium (Fig. 3, A–C). A weak immunoreaction was also observed between developing spermatocytes at stages 1 and 2 of spermatogenesis (Fig. 3B). The immunolocalization of Cx43 was not altered as a function of the stage of spermatogenesis between stages 1 and 5, although the immunoreaction appeared to be less intense in testes of stage 1 (stages 1 and 3 are shown in Fig. 3, B and C). The localization of Cx43 appeared to change somewhat at stage 6 of spermatogenesis, where a more cytoplasmic reaction was observed (Fig. 3D). This suggests that there may be a loss of Cx43 gap junctional coupling at this stage of spermatogenesis.
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Cx43.4 was localized between spermatogonia, type I spermatocytes, and type II spermatocytes at stages 1 and 2 of spermatogenesis (Fig. 3, E–G). The immunoreaction was particularly intense along the plasma membrane of type II spermatocytes in stage 2 testes. Cx43.4 was not detectable by immunocytochemistry at any other stage of spermatogenesis (Fig. 3H).
Cx30 was localized between interstitial Leydig cells of the testis. Cx30 immunoreaction appeared as an intense reaction between Leydig cells (Fig. 4A, 1000x). There was no immunostaining within the spermatocysts. While Cx30 immunostaining did not vary dramatically between stages 1 and 5, the immunoreaction appeared to be more intense at stages 1 and 3 of spermatogenesis (Fig. 4, B and C). In stage 6 testes, the localization of Cx30 varied, and appeared be localized along the base of the spermatocysts (Fig. 4D, 400x).
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Cx31 was localized to the spermatocytes, as well as to the endothelium of interstitial blood vessels (Fig. 4E). Cx31 immunoreaction appeared more intense at stage 1 as compared with stages 2–5 of spermatogenesis (Fig. 4, F and G). At stage 6 of spermatogenesis, Cx31 was localized to the base of the spermatocysts (Fig. 4H), as well as between spermatogonia and spermatocytes.
Gap junctional intercellular communication in the testis has been shown to be essential for testicular development and spermatogenesis in mammals [7, 23–25]. In mammals, there are at least 12 different Cxs that are expressed in the testis and that appear to mediate complex intercellular communications implicated in testicular functions [26]. There is little information on the presence of Cxs in the testis of other vertebrates. In the present study, we have shown that there are at least four different Cxs that are expressed in the salmonid testis: cx43, cx43.4, cx30, and cx31. Other studies have reported that cx48.5 transcripts are expressed in the testis of adult zebra fish [27]. Thus, these results indicate that, as in mammals, adult fish testis express a variety of Cxs. This suggests that there may be different signals that are exchanged between different cell types of the salmonid testis.
Our observations indicate that Cx43 is expressed throughout spermatogenesis and is localized in Sertoli cells. Marina et al. [18] reported that the Sertoli cells of the spotted ray are coupled by gap junctions. Our results suggest that these gap junctions are likely composed of Cx43. Previously, cx43 has been identified in rodent testis, and its absence results in arrested spermatogenesis in the mouse; it has also been linked to infertility in humans [10, 28, 29]. Decrouy et al. [30], using in situ hybridization, reported that, in the rat, cx43 participates in the coupling of neighboring Sertoli cells. Plum et al. [31] have demonstrated that substitution of cx43 by cx32 (also known as Gjb1) or cx40 (also known as Gja5) induces altered spermatogenesis, suggesting not only that cx43 is essential for the control of spermatogenesis, but that there are gap junctions composed of different Cxs that allow different messages to be exchanged between cells. It has been suggested that homologous communication between adjacent Sertoli cells provides a mechanism whereby these cells could synchronize gonadal maturation [30, 32]. Based on the cellular distribution of Cx43 within the testis, it would appear that the role of cx43 in the testis is highly conserved in evolution, and likely plays a similar role in fish as it does in mammals.
Recent studies have indicated that the expression of Cxs varies with different stages of spermatogenesis in the mouse [33]. Using cDNA arrays, it was observed that mRNA levels for cx26 (also known as Gjb2) and cx40 increase as spermatogonia pass from type A to type B, and that cx26 mRNA decreases when type B spermatogonia develop into preleptotene spermatocytes. We have also observed that the expression of Cx transcripts varies as a function of spermatogenesis. In this case, cx43.4 was expressed only in stages 1 and 2 of spermatogenesis. Immunolocalization of Cx43.4 indicates the presence of a weak immunoreaction in spermatogonia and a more pronounced immunostaining reaction in spermatocytes. Based on the histology of the cells, we could identify two main populations of spermatocytes in stage 1 and 2 testes of the trout: preleptotene (type I) and pachytene (type II) spermatocytes. Our results indicate that Cx43.4 is expressed exclusively in type II spermatocytes. Cx43.4 was not observed at other stages of spermatogenesis. This is consistent with RT-PCR data that indicate that cx43.4 is expressed only in stages 1 and 2 of spermatogenesis. cx43.4 Is an ortholog of the human and avian Cx45, and has also been identified in the zebra fish [34]. In zebra fish, cx43.4 is expressed during gastrulation with the first definitive assignments of axial cell fate, and during tail formation cx43.4 was not expressed in the most posterior region of the embryonic shield or in the overlying primary ectoderm during the late stages of gastrulation [34]. In the mouse, Cx45 has also been identified in the testis [23, 35]. Mok et al. [36] have also reported that Cx31 (also known as Gjb3) is expressed in spermatogonia and primary spermatocytes of rat testes, as determined by both in situ hybridization and immunohistochemistry. These results suggest that, in rainbow trout, Cx43.4 may play a similar role to that of Cx31 in the mammalian testis. The specific spatial and temporal expression of Cx43.4 in germ cells suggest that Cx43.4 function is tightly regulated and required during a restricted window of cellular differentiation at early stages of spermatogenesis.
In our experiments, we observed that Cx30 was localized between Leydig cells of the testis. Cx30 expression appeared to increase between stages 1 and 2 of spermatogenesis, which is consistent with the notion that gap junctional communication is involved in the control of androgen secretion [18, 37, 38]. This observation is consistent with RT-PCR data, which suggest an increase in cx30 mRNA levels between stages 1 and 2. Whether or not cx30 is androgen dependant, or if it is regulated by hypophyseal factors implicated in steroidogenesis, remains to be determined. In rodents, it has been shown that Cx43 is present between Leydig cells of the testis [5, 8, 39]. Whether or not cx30 in rainbow trout has a homologous function also remains to be established. Mammalian studies have shown that Cx30 (also known as Gjb6) is distributed in different tissues, such as the brain, uterus, testis, and kidney [40, 41]. While Dahl et al. [40] have detected Cx30 in the mouse testis by Northern blot analyses, they did not localize it to a particular cell type. It is, therefore, not possible at the moment to establish whether or not cx30 has a cellular distribution in mammalian testis similar to that in the trout.
Cx31 was expressed at low levels throughout spermatogenesis at the mRNA and protein levels where it was localized to the endothelial cells of the capillary in the interstitium of the testis, as well as between spermatogonia and spermatocytes. While Cx31 has never been identified in teleosts, its localization in the testis appears to resemble that of Cx37 (also known as GJA4), which is localized primarily to endothelial cells [36, 42]. It has been reported in the literature that Cx31 is one of the earliest Cxs to be expressed in murine development, and is present as early as the morula stage and throughout the blastocyst [40]. The presence of Cx31 mRNA transcripts in the testis were first reported by Hennemann et al. [35]. As stated above, Mok et al. [36] identified Cx31 in the spermatogonia and early spermatocytes. Clearly, however, rainbow trout Cx31 has a different cellular distribution pattern in the testis than mammalian Cx31, indicating different functions for Cx31 gap junctions in rainbow trout.
The expression and localization of Cxs at stage 6 of spermatogenesis is complex. At the end of the reproductive cycle, it has been demonstrated that testis that have just finished spermatogenesis show the presence of certain cells that have already begun a new cycle of spermatogenesis [15]. At this stage, the lobules shrink and the Sertoli cells retract their processes, resulting in a more pronounced lobular membrane. Dziewulska and Domagala [15] have also observed the presence of large vacuoles in different species of salmonids at this stage. The vacuoles can remain in the Sertoli cells for a long time after the end of the reproduction cycle. The appearance of these vacuoles and Sertoli cells' retraction may explain the diffuse localization of Cx43 at stage 6. Certainly, it would appear that these cells are not forming specific gap junctions between Sertoli cells at this stage, since the Cx43 localization is cytoplasmic, as opposed to being localized on the plasma membranes where gap junctions are normally observed. Similarly, Leydig cells are also altered at the end of spermatogenesis, as determined by both the cytoplasmic and mitochondrial structures of the cells [43]. These changes are associated with a decrease in circulating sex steroids [44]. Furthermore, as steroid levels decrease at the end of the cycle, Leydig cells degenerate [43]. This phenomenon could explain the altered Cx30 localization in stage 6, where we observed the presence of Cx43 around Sertoli cells, spermatogonia, and some spermatocytes.
Together, the data from this study indicate that intercellular communication in the rainbow trout testis is complex and involves gap junctions comprised of different Cxs. Interestingly, each Cx is localized to different cell types of the testis, suggesting that different types of messages are likely exchanged between different cell types of the testis, and that these change as a function of the stage of spermatogenesis. While we have identified four testicular Cxs, it is likely that there are other Cxs in rainbow trout testis that may be implicated in communication between more mature spermatogenic cells. Nevertheless, this study shows that the localization of testicular Cxs in rainbow trout share many similarities with those of mammalian testis, thereby confirming the importance of this intercellular communication in the progression of spermatogenesis. Further studies on the endocrine regulation of these Cxs will allow a better understanding of the physiological role of these proteins in spermatogenesis and testicular development in fish.
ACKNOWLEDGMENTS
The authors thank J. Barthelemy for her assistance and M. Gregory for her helpful suggestions.
FOOTNOTES
1Supported by the AQUANET Network of Excellence, the SORDAC, and the VRQ-Network for Quebec Aquaculture. B.d.M. was the recipient of a studentship from the Armand-Frappier Foundation. 
Correspondence: 2 Daniel Cyr, INRS-Institut Armand Frappier, Université du Québec, 245 Hymus boul., Pointe Claire, PQ, Canada H9R 1G6. FAX: 514 630 8850; e-mail: daniel.cyr{at}iaf.inrs.ca
Received: 30 May 2006.
First decision: 26 June 2006.
Accepted: 12 September 2006.
REFERENCES
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