ARTICLE |
Correspondence to:
Georges Pointis, INSERM CJF 95/04, IFR 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 2, France. E-mail:
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Summary |
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Connexin43 (Cx43) is one of the most predominant gap junction proteins found in the testis. We used in situ hybridization and indirect immunofluorescence to study the distribution of Cx43 mRNA and protein in the rodent seminiferous epithelium. During mouse testis maturation, Cx43 mRNA and its corresponding protein were first detected in the adluminal compartment of the growing seminiferous tubules (early postnatal age: Day 12) to become progressively located in the basal compartment at later ages (Days 16, 19, 27). In seminiferous tubules of sexually mature animals, the intensity of the hybridization signal was stage-dependent, with a maximum at Stage VII compared with Stages V and IX of the spermatogenic cycle (p<0.05). The highest expression of Cx43 mRNA was observed in the supporting Sertoli cells and, to a lesser extent, in the most basally located and less mature germ cells (spermatogonia and spermatocytes). Consistent with these observations, in situ dye coupling was observed between Sertoli cells and basal germ cells. In a mutant mouse deficient for the retinoid X receptor ß, which exhibited abnormal spermatogenesis due to altered Sertoli cell function, Cx43 transcripts were markedly decreased in the seminiferous epithelium (p<0.01). The immunoreactive signal for Cx43 was significantly reduced in seminiferous tubules of the 3-month-old mutant mice (p<0.05) and undetectable in older animals. These data provide new information about the precise localization of Cx43 mRNA and protein in seminiferous tubules of immature and mature rodent testes. Moreover, they suggest that retinoids, through the RXRß receptors, could be involved in the control of Cx43 gene expression in Sertoli cells. (J Histochem Cytochem 48:793805, 2000)
Key Words: connexin43, Sertoli cell, germ cell, gap junction, retinoids, spermatogenesis
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Introduction |
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Most cells in normal tissues communicate via gap junctions. Gap junctions are composed of transmembrane channels that directly link the cytoplasm of adjoining cells and are formed by a family of homologous transmembrane proteins termed connexins (for review see
It is now established that Cx can be modulated at the transcriptional and translational levels or by alterations in the gating channels (for review see
Immunoreactive Cx43 has been mainly detected in the basal compartment of the seminiferous tubules (
In this study, the cellular localization of Cx43 was determined in the developing and functional mature testis at the mRNA and protein level by in situ hybridization (ISH) and indirect immunofluorescence, respectively. In addition, the presence of Cx43 transcripts and Cx43 protein was examined during the normal spermatogenic cycle and in mutant mice deficient for the retinoid X receptor ß with abnormal spermatogenesis. These results point to the essential roles of gap junction protein in the maturation of fertile male germ cells.
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Materials and Methods |
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Animals
Male LongEvans rats and male Swiss mice were housed in a daynight-cycled room, with food and water provided ad libitum. Mice were sacrificed at 4, 8, 12, 16, 19, 27, or 60 days postpartum and rats at 3 months. RXRß-/- mice were obtained by homologous recombination (
Indirect Immunofluorescence
Testes were frozen in OCT embedding compound (Tissue Tek; Miles, Naperville, IL) and cryosectioned. Testicular sections (5 µm) were applied to 3-aminopropyltriethoxysilane-coated slides and treated as previously reported (
In Situ Hybridization
Testes were dissected out and immediately fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.4) for 4 hr before standard paraffin embedding. Tissue sections (5 µm thick) were collected on 3-aminopropyltriethoxysilane-coated slides and ISH was performed as previously described (
In Situ Dye Coupling Experiments
Cell-to-cell coupling in seminiferous tubules was measured by the cut end-loading method originally described by
Semiquantitative Evaluation
Hybridization intensity was quantified using a computer-assisted image analysis system Visilog 4. 15 (Noesis; Les Ulis, France), consisting of an IMC 500 camera and an IMC 500 digitizer. The intensity of hybridization was expressed as percentage of pixels within a marked area occupied by silver grains that was above a set gray threshold level. Measurements were taken from 10 seminiferous tubules from three different testis sections for each animal group. For each group, two serial sections were analyzed. One was probed with the antisense RNA probe and the remaining section was probed with the sense probe. Both serial sections were analyzed for quantitation. Semiquantitative evaluation of Cx43 immunostaining was performed by use of a computerized image analysis system as previously described (
Statistical Analysis
A one-way analysis of variance (ANOVA) followed by a StudentNewmanKeuls post hoc test was used to test for differences among groups. Values were considered statistically different when p<0.05.
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Results |
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Localization of Cx43 mRNA and Protein in Mature Testis
In the mature testis, darkfield images reveal a high accumulation of silver grains at the base of seminiferous tubules and in the interstitial compartment of both rat (Fig 1A) and mouse (Fig 2A). Brightfield examinations indicate that the intensity of the ISH signal was changed throughout the spermatogenic cycle of rat (Fig 1D, Fig 1F, and Fig 1H) and mouse (Fig 2D, Fig 2F, and Fig 2H) testes, suggesting that the accumulation of Cx43 transcripts might be dependent on the stage of the seminiferous epithelium cycle. The seminiferous tubules corresponding to Stage VII appeared the most strongly labeled (Fig 1F and Fig 2F). Semiquantitative analysis indicated that the intensity of the signal, measured with the antisense probe, was significantly greater (p<0.05) at Stage VII compared to Stages V and IX in the rat seminiferous epithelium (Fig 3). Control sections incubated with the sense probe did not reveal any specific hybridization. Similar results were obtained in the mouse (not shown).
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Immunofluorescence studies showed that Cx43 signal was detected in the basal compartment of seminiferous tubules, and in interstitial cells of both species (Fig 1C and Fig 2C). As observed for Cx43 mRNA, the pattern of Cx43 protein distribution appeared stage-dependent. From Stage I to Stage V of spermatogenesis, faint immunostaining for Cx43 was detected in the basal compartment of rat (Fig 1E) and mouse (Fig 2E) seminiferous tubules. At Stage VII of spermatogenesis, the intensity of the immunostaining, localized in the basal compartment of seminiferous tubules was increased (Fig 1G and Fig 2G). Finally, at Stage IX, the basal immunoreactivity was decreased in both species (Fig 1I and Fig 2I). In controls, no immunoreactivity could be detected (not shown).
The cellular distribution of Cx43 mRNA was examined at the same stages. Transcripts for Cx43 were mainly expressed in the cytoplasm of Sertoli cells (Fig 1D, Fig 1F, Fig 1H, Fig 2D, Fig 2F, and Fig 2H). This localization was clearly evidenced by the columnar distribution of silver grains, a pattern typical for Sertoli cells (Fig 1F and Fig 2F). In addition to Sertoli cells, ISH signals were also present in the basally located germ cells of rat and mouse at all stages considered (Fig 1D, Fig 1F, Fig 1H, Fig 2D, Fig 2F, and Fig 2H). Higher magnification of the basally located ISH signal in both species indicates that silver grains were detected in spermatogonia (Fig 4A), preleptotene spermatocytes (Fig 4B and Fig 4C), and in leptotene and zygotene spermatocytes (not shown). In pachytene spermatocytes, accumulation of Cx43 transcripts appeared to be correlated with their position in the seminiferous epithelium (Fig 4A and Fig 4D). No distinct signal was observed in round and elongated spermatids at any stage of the mouse and rat seminiferous epithelium cycle (Fig 1D, Fig 1F, Fig 2D, and Fig 2F).
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In Situ Dye Coupling in Mature Rat Seminiferous Tubule
Dye coupling experiments were developed to determine if functional coupling correlates with the distribution of Cx43 mRNA and of the protein within the seminiferous epithelium. As shown in Fig 5, a selective intercellular transfer of Lucifer yellow was observed between cells basally located in the seminiferous tubules and identified by DAPI staining (Fig 5A). In the seminiferous tubule section selected, representative of about 20% of sections examined, dextranrhodamine fluorescence was detected in Sertoli cells, identified by the specific columnar distribution of the fluorescence (Fig 5B). No transfer of this dye molecule through germ cells could be observed. In contrast, diffusion of the Lucifer yellow occurred from Sertoli cells to spermatogonia and early spermatocytes (Fig 5C). In the other sections analyzed, dye coupling from germ cell to Sertoli cell was never observed. Preincubation of testes in the presence of 3 mM heptanol reduced dye coupling between Sertoli and basal germ cells by 90%, indicating that diffusion of the Lucifer yellow is gap junction-mediated (not shown).
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Localization of Cx43 mRNA and Protein in Developing Mouse Testis
During the first wave of mouse spermatogenesis, Cx43 mRNAs were detected at all postnatal ages studied (Days 4, 8, 12, 16, 19, and 27). Because the localization of Cx43 mRNA was similar from Day 4 to Day 12, only the more representative data are presented here. At Day 12, Cx43 mRNA and Cx43 immunostaining were localized in the adluminal region of all seminiferous tubules (Fig 6A and Fig 6E). At Day 16, Cx43 transcripts became progressively present in the basal region of the tubules (Fig 6B). At this stage, Cx43 immunoreactivity was located in the basal third of the seminiferous tubules (not shown). At Days 19 and 27, Cx43 mRNA (Fig 6C and Fig 6D) and Cx43 immunoreactivity (Fig 6F) were located in the basal compartment of the seminiferous tubules. As in the mature seminiferous epithelium, silver grains were detected in cytoplasm of Sertoli cells and in basally located germ cells (spermatogonia and spermatocytes). For Sertoli cells, the ISH signal was detected at any postnatal age (Fig 6A6D), whereas for germ cells few silver grains were observed at Day 19 (Fig 6C) and Day 27 (Fig 6D). Cx43 mRNA and Cx43 protein were also present in interstitial and peritubular cells at all stages studied (not shown).
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Localization of Cx43 mRNA and Protein in the RXRß2/- Mutant Mouse Testis
In RXRß2/- mutant mice, darkfield illumination revealed faint ISH signal for Cx43 mRNA in the seminiferous tubules and in the interstitial compartment of 3- (Fig 7A) and 11-month-old animals (Fig 7C). The accumulation of silver grains at the periphery of the seminiferous tubules was markedly reduced in the 11-month-old mutant (Fig 7C) compared to the mutant at 3 months of age (Fig 7A) and to wild-type mice (Fig 7A). Brightfield illumination at 3 months showed, various germ cell types in the seminiferous tubules (Fig 7B), whereas at 11 months, different degrees of germ cell loss, specifically the elongated spermatids, could be observed (Fig 7D). The intensity of the hybridization signal was significantly reduced in seminiferous tubules of the 3-month-old (p<0.01) and 11-month-old (p<0.01) mutants without significant modification of the background levels revealed with the sense probe (Fig 8). A large decrease in the ISH signal was observed in Sertoli cells of the mutant at both ages studied (Fig 7E and Fig 7G, insets). In 3-month-old mutant mice, the immunoreactive Cx43 signal localized in the basal compartment of seminiferous tubules was reduced (compare Fig 7F and Fig 2G). Cx43 expression decreased by 47 ± 9% compared to wild-type mice (p<0.05). In the 11-month-old mutant, only a few silver grains were detected, but no distinct Cx43 immunostaining was observed (Fig 7H).
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Discussion |
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Previous studies by Northern blotting analysis have reported the presence in rat testes of mRNA for Cx43, the predominant gap junction protein in this organ (
The presence of Cx43 transcripts in Sertoli cells is supported by the columnar distribution of the ISH signal detected at the base of the seminiferous tubules which is typical of Sertoli cell localization. An identical distribution has been already reported for mRNA transcripts of Sertoli cell-specific genes (
Our present observations also reveal that Cx43 mRNA and protein expression is dependent on the stage of the spermatogenic cycle in the mature testis. In accordance with the results obtained in the protein analysis, which showed a strong immunoreactive signal at Stage VII (
In the developing mouse testis, Cx43 mRNAs were detected in seminiferous tubules at all postnatal ages, from Day 4 to Day 27. The distribution of Cx43 transcripts, first in the adluminal region of the seminiferous tubules (Days 412) and then in the basal region (later phases) probably result from Sertoli cell cytoplasm organization during development. At the earlier phase, the cytoplasm of palisaded Sertoli cells filled most of the lumen of the growing seminiferous tubules, whereas at the later phase it occupied a larger portion in the basal region of the tubules. This specific distribution of Cx43 mRNA during development coincided with the localization of immunodetectable Cx43 protein as previously observed (
Another interesting observation is that the expression of Cx may vary in response to various natural factors, such as toxic agents and cell proliferation and transformation (
In conclusion, the present data provide evidence that Cx43 mRNA and Cx43 protein, the predominant gap junction protein in the testis, are present in the supporting Sertoli cells and in specific basally located germ cells, spermatogonia and spermatocytes. This cell type-specific expression of Cx43 mRNA and the stage-specific expression of Cx43 protein and its messengers at restricted periods of spermatogenesis suggest that this gap junction protein is involved in a specific function during male germ cell differentiation. Intercellular communication via gap junctions is believed to be involved in the control of cell growth (
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Acknowledgments |
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Supported by INSERM, Ministère de la Recherche et de l'Enseignement Supérieur, and by grants from ARC (no. 98-80). CB was supported by grants from Hoechst Marion Roussel.
We thank Dr J-M Gasc and M-T Morin for their help in in situ hybridization experiments, N. Parseghian, F. Carpentier, and B. Decrossas for technical assistance, J.M. Lepecq for photographic illustrations, and Dr F. BruckerDavis for revising the English.
Received for publication September 14, 1999; accepted February 9, 2000.
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