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Am J Physiol Cell Physiol 290: C733-C740, 2006. First published October 19, 2005; doi:10.1152/ajpcell.00106.2005
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EXTRACELLULAR MATRIX, CELL INTERACTIONS

Opposite regulation of connexin33 and connexin43 by LPS and IL-1{alpha} in spermatogenesis

Celine Fiorini,1 Xavier Decrouy,2 Norah Defamie,1 Dominique Segretain,2 and Georges Pointis1

Institut National de la Santé et de la Recherche Médicale (INSERM) U 670, 1Faculté de Médecine, Nice cedex; and 2Université de Paris V René Descartes, INSERM U 670, Paris, France

Submitted 9 March 2005 ; accepted in final form 11 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The gap junction proteins, connexins (Cxs), are present in the testis, and among them, Cx43 play an essential role in spermatogenesis. In the present study, we investigated the testicular expression and regulation of another Cx, Cx33, previously described as a negative regulator of gap junction communication. Cx33 mRNA was present in testis and undetectable in heart, liver, ovary, and uterus. In the mature testis, Cx33 was specifically immunolocalized in the basal compartment of the seminiferous tubules, whereas Cx43 was present in both seminiferous tubule and interstitial compartments. During stages IX and X of spermatogenesis, characterized by Sertoli cell phagocytosis of residual bodies, Cx43 was poorly expressed within seminiferous tubules, while Cx33 signal was strong. To evaluate the role of phagocytosis in the control of Cx33 and Cx43 expression, the effect of LPS was analyzed in the Sertoli cell line 42GPA9. We show herein that phagocytosis activation by LPS concomitantly stimulated Cx33 and inhibited Cx43 mRNA levels. These effects appear to have been mediated through IL-1{alpha}, because the exposure of Sertoli cells to the IL-1 receptor antagonist partly reversed these effects. IL-1{alpha} enhanced and reduced, respectively, the levels of Cx33 and Cx43 mRNA in a time- and dose-dependent manner. These data reveal that Cx33 and Cx43 genes are controlled differently within the testis and suggest that these two Cxs may exert opposite and complementary effects on spermatogenesis.

Sertoli cell; germ cell proliferation

INTERCELLULAR COMMUNICATION through gap junctions is an important mechanism for the coordination of cell metabolism and function. They are implied in the growth and differentiation of organs and tissues (for review, see Ref. 25). Gap junction channels allow direct exchange of signaling molecules at <1 kDa between adjacent cells. They are composed of proteins called connexins (Cxs). To date, at least 25 Cxs have been identified, and most organs express several Cxs (47). The combination of different Cxs allows the modulation of gap junction permeability and selectivity (17, 25).

In the testis, within the seminiferous tubules, the Sertoli cell is the only cell type in contact with germ cells. Germ cell proliferation and differentiation is controlled locally by Sertoli cells through paracrine factors and direct intercellular junctions. Homologous gap junctions are found between two adjacent Sertoli cells, whereas heterologous gap junctions are present between Sertoli cells and germ cells (3, 11, 34). Within seminiferous tubules, at least 11 different connexins (Cx26, Cx31, Cx31.1, Cx32, Cx33, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50) are presumably translated (33). However, their functional implications in the control of the spermatogenic process are still unclear (for review, see Ref. 32).

The role of Cxs in spermatogenesis is established by the study of the fertility of mice invalidated for Cx genes. Although Cx37-deficient mice demonstrate female infertility (8, 37), targeted disruption of different Cx genes (Cx31, Cx32, Cx37, Cx40, Cx46, and Cx50) does not affect male fertility (46, 48). Conversely, the null mutant of the Cx43 gene, the predominant Cx in the testis, results in a deficiency of primordial germ cells in the fetal gonads of both sexes (24, 36). The essential role of Cx43 in the control of spermatogenesis was definitively proved by the demonstration that transgenic knock-in mice in which the coding region of the Cx43 gene was replaced by the coding region of Cx32 or of Cx40 exhibited impaired spermatogenesis that resulted in Sertoli cell-only syndrome (31).

In contrast to Cx43, which is ubiquitous, Cx33 is found specifically in the testis (19, 43). In addition, Cx33 has been reported to exert a connexin-specific inhibitory effect on gap junction intercellular communication in the paired Xenopus oocyte system (9). In Sertoli cells, we recently demonstrated that the inhibitory effect of Cx33 could be mediated through sequestration of Cx43 within the cytoplasmic compartment (15). However, the physiological functions of Cx33 in the spermatogenic process has not been clearly established.

In the present study, we have demonstrated that Cx33 mRNA was expressed during testicular development. In addition, high levels of Cx33 were detected in seminiferous tubules at stages IX and X of spermatogenesis, stages at which Sertoli cells initiate phagocytosis of residual bodies. We further have shown that Sertoli cell Cx33 transcription was stimulated in vitro by phagocytosis of LPS and IL-1{alpha}. These data emphasize a physiological role of Cx33 in the control of germ cell proliferation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue and cell preparation. Rat fetal testes (18.5 days fetal age) were a gift from R. Habert (INSERM U 566, Fontenay aux Roses, France). Wistar rats were killed 14, 22, and 90 days postpartum by administering CO2 asphyxiation, and the testes were immediately collected. Testes and other tissues were frozen at –80°C and then processed for RNA extraction. Experiments on animals were conducted according to the standard ethical guidelines approved by the Institut National de le Santé et de la Recherche Médicale (INSERM) animal care committee.

Cell culture. The 42GPA9 Sertoli cell line was maintained in DMEM (Life Technologies/GIBCO-BRL, Cergy Pontoise, France) containing 10% FCS at 32°C (6). Cells were washed twice with DMEM without serum and incubated for 12 h in serum-free defined medium (SFDM) composed of DMEM-Ham's F-12 mixture (vol/vol) supplemented with EGF (10 ng/ml), insulin (10 mg/ml), transferrin (10 mg/ml), and testosterone (0.1 mM). The medium was then replaced by fresh SFDM and cells were cultured, when indicated, in the presence or absence of 20 µg/ml LPS (Sigma, St. Louis, MO) (a dose known to affect Sertoli cell function) (42), increasing concentrations of IL-1{alpha} (1–1,000 pg/ml, Sigma), 1 ng/ml FSH (ovine FSH-20; National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD), or 0.5 mM 8-bromoadenosine 3',5'-cyclic monophosphate (Sigma) for an additional 0, 3, 6, or 18 h. To verify that the effect of LPS was mediated through IL-1{alpha}, Sertoli cells were treated for 6 h with LPS and IL-1 receptor antagonist (IL-1ra; R&D Systems, Minneapolis, MN) at a concentration (1 µg/ml) previously reported to prevent IL-1 action (29).

RT-PCR. Total RNA was extracted from tissue fragments or 42GPA9 Sertoli cells using RNeasy (Qiagen, Courtaboeuf, France). RNA was treated with RNase-free DNase I (Life Technologies/GIBCO-BRL) at 22°C for 5 min. Briefly, RNA was reverse transcribed into cDNA using oligo(dT)12–18 as primers (Roche Diagnostics, Meylan, France) and SuperScript II reverse transcriptase (Life Technologies/GIBCO-BRL). To confirm that there was no genomic DNA contamination within the total RNA, RT-PCR experiments were performed under identical conditions with the exception that SuperScript II was omitted in the cDNA synthesis step. From the RT-PCR product, 4 µl were used as a template for PCR with a PTC-100 DNA thermal cycler (MJ Research/Bio-Rad Laboratories, Waltham, MA) used according to the manufacturer's instructions. The nucleotide sequences of oligonucleotide primer for Cx33 (19), Cx43 (4), IL-1{alpha} (28), and beta-actin (44) are shown in Table 1. The cycling parameters for PCR were as follows: denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and elongation at 72°C for 1 min, for a total of 27 cycles. PCR products were run on 1.5% agarose gel (Eurobio, Les Ulis, France), stained with ethidium bromide, and photographed using a Polaroid camera. The sizes of the expected amplification products were 267 bp for Cx43, 380 bp for Cx33, 453 bp for IL-1{alpha}, and 649 bp for beta-actin. The identity of each product was determined by performing sequencing.


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  		Table 1. PCR primer sets used for analysis of mRNA for beta-actin, Cx43, Cx33, and IL-1{alpha}

 
Semiquantitative RT-PCR was used to examine relative changes in Cx33 and Cx43 mRNA during testicular development and after exposition of Sertoli cells to LPS, IL-1{alpha}, or IL-1ra. Changes in Cx43 and Cx33 PCR products were compared with beta-actin used as an internal standard. The amount of amplified products was quantified for each sample using the ImageMaster VDS and Image Master 1D software, version 3.00 (Amersham Pharmacia Biotech, Orsay, France). Each value was determined as the mean of three densitometric readings. The results are expressed as average ratios of the relative optical densities of Cx33 and Cx43 PCR products to that of beta-actin.

Western blot analysis. Sertoli cells were washed twice with DMEM without serum and incubated for 12 h in SFDM composed of DMEM supplemented with 0.1% BSA (no. A7030; Sigma). The medium was then replaced with fresh SFDM, and cells were cultured in the presence or absence of 20 µg/ml LPS or 100 pg/ml IL-1{alpha} for an additional 0, 5, 15, 30, 60, 180, or 360 min. Sertoli cells were then solubilized in Nonidet P (NP)-40/Brij buffer [50 mM Tris·HCl, pH 7.5, 1% NP-40, 1% Brij 96 (Fluka, St. Quentin Fallavier, France), 1 mM Na3VO4, 10 mM beta-glycerophosphate, 10 mM NaF, 2 mM EDTA, and protease inhibitors (Complete; Roche Diagnostics)]. Cell lysates were studied using Western blot analysis as previously described (12) with antibodies directed against the phosphorylated active forms of ERK (anti-phospho-Thr202/Tyr204 ERK, 1:2,000 dilution; New England BioLabs, Boston, MA). After the blots were stripped, equal loading of proteins was verified by reprobing the same blots with anti-ERK antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The presence of primary antibody was revealed using horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000 dilution; Dako, Trappes, France) and visualized using an ECL detection system (Amersham, Little Chalfont, UK).

Immunocytochemical procedures. Fresh testes were embedded in optimum cutting temperature compound (Tissue-Tek; Miles, Naperville, Illinois), frozen at –20°C, and then stored at –80°C. Frozen sections (7 µm) were cut on a cryostat, applied to 3-aminopropyltriethoxysilane-coated slides, and then stored at –20°C. Frozen sections were fixed for 10 min with methyl alcohol at –20°C, washed with PBS, and then incubated 5 min in 0.1% Tween 20-PBS. Sections were incubated for 2 h with a cocktail containing the anti-Cx43 antibody (1:100 dilution; Transduction Laboratories, Lexington, KY) and the anti-Cx33 antibody (1:100 dilution) in PBS containing 3% BSA as previously reported (15). Subsequently, slides were incubated in a secondary antibody mixture containing rhodamine tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG (1:200 dilution) and FITC-conjugated anti-rat IgG (1:200 dilution in PBS containing 3% BSA; Jackson ImmunoResearch Laboratories, Baltimore, UK). Immunoreactive signals for Cx43 and Cx33 were examined with a confocal laser-scanning microscope (model LSM 510; Zeiss, Oberkochen, Germany) fitted with a 488-nm or 543-nm krypton-argon laser, which permitted simultaneous analysis of fluorescein and rhodamine chromophores. One representative section was chosen in the total series of 20 optical sections. In controls, primary antibody was omitted. Stages of the seminiferous epithelium were identified as previously described (27).

Data analysis. Data are expressed as means ± SE. Statistical analysis was performed using Student's t-test or one-way ANOVA and Duncan's new multiple-range test. Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Tissue specificity of Cx33 gene expression. The transcription of the Cx33 gene was evaluated in different tissues of mature rats (testis, heart, liver, ovary, and uterus) and compared with that of Cx43 (Fig. 1A). A band at the expected size of 380 bp for Cx33 was detected in testis only while no signal was present in other tissues studied. In contrast, a band at 267 bp corresponding to Cx43 was detected in all tissues analyzed (Fig. 1A). Sequencing confirmed that the amplified sequences were identical to the published sequences (data not shown).


Figure 1
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  		Fig. 1. Analysis of connexin (Cx)33 tissue specificity and gene expression during testis development. A: total RNA was prepared from testis, heart, liver, ovary, and uterus tissue samples from mature rats. Samples were subjected to RT-PCR as described in MATERIALS AND METHODS. The reaction products were analyzed by performing SDS-PAGE on 1.5% agarose gels and staining with ethidium bromide. The expected sizes of the PCR products were 380 bp for Cx33, 267 bp for Cx43, and 649 bp for beta-actin. No amplified DNA fragments were identified in the absence of an RNA sample or in RNA samples incubated without RT (data not shown). B: semiquantitative analysis of Cx33 and Cx43 mRNA levels during testis maturation from 18.5 days postcoitus (pc) to 90 days postpartum (pp). Amounts of amplified products were quantified for each sample using densitometric analysis. Results are expressed as average ratios of relative optical densities (ROD) of Cx33 or Cx43 PCR products to those of beta-actin and are means ± SE of 3 densitometric readings in 3 separate experiments. *P < 0.05 and **P < 0.01 vs. 18.5-day-old fetal testis.

 
Analysis of Cx33 gene expression during testicular development. The transcription of the Cx33 gene was evaluated in the testis at different stages of development (18.5 days postcoitus and 14, 22, and 90 days postpartum) and compared with that of Cx43 (Fig. 1B). Semiquantitative RT-PCR using beta-actin as an endogenous standard indicated that the levels of Cx33 mRNA increased during testicular development from fetal to adult stages. The relative Cx33 mRNA expression was significantly higher in the testis of mature (90 days old; P < 0.01) or immature rats (22 days old; P < 0.05) compared with fetal rats. The Cx43/beta-actin ratio was not significantly modified during the same developmental period (Fig. 1B), although a redistribution of Cx43 signal within growing seminiferous tubules was previously described (3, 7).

Inverted stage-dependent expression of Cx33 and Cx43. Figure 2 shows that the intensity of the Cx33 and Cx43 immunoreactive signals was dependent on the stage of spermatogenesis identified by 4',6'-diamidino-2-phenylindole staining (Fig. 2, A and B). Dual immunofluorescence analysis with the two antibodies revealed that the higher staining levels for Cx33 and Cx43 were not observed at the same stage of spermatogenesis (Fig. 2, C and D). High magnification showed that the anti-Cx33 antibody detected a marked Cx33 signal within seminiferous tubules at stages IX and X of spermatogenesis (Fig. 2E), whereas the Cx43 immunosignal was undetectable in the same seminiferous tubule section (Fig. 2F). As previously reported, a signal for Cx43 was present in the adjacent interstitial compartment (Fig. 2F). In contrast, no Cx33 immunoreactivity was detected in this compartment (Fig. 2E).


Figure 2
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  		Fig. 2. Stage-dependent expression of Cx33 and Cx43 within seminiferous tubules. A and B: 4',6'-diamidino-2-phenylindole-stained nuclei of testicular tissue sections. Immunolocalization of Cx33 (C and E) and Cx43 (D and F) in sections of mature rat testis. C: note that the Cx33 signal was present only within the seminiferous tubules, whereas Cx43 labeling was detected in both interstitial and tubular compartments (D) and that the two signals appeared to be stage dependent. Bars in A–D, 80 µm. E and F: high magnification revealing strong Cx33 signal detected in seminiferous tubule at stages IX and X of spermatogenesis (E), whereas a faint signal for Cx43 was located in the same tubular region (F). In contrast, Cx43 staining was present mainly in the adjacent interstitial compartment. Bars in E and F, 25 µm.

 
Effects of LPS on IL-1{alpha} mRNA and on ERK signaling pathway in Sertoli cells. Similarly to native Sertoli cells, the 42GPA9 Sertoli cell line exhibited strong phagocytic activity (5). The results presented in Fig. 3A show that 42GPA9 Sertoli cells responded to LPS (used to activate phagocytosis) by increasing IL-1{alpha} gene expression. A band corresponding to IL-1{alpha} mRNA was present 3 and 6 h after cell exposure to 20 µg/ml LPS, whereas IL-1{alpha} transcripts were not detected in the absence of LPS. As shown in Fig. 3B, exposure of 42GPA9 Sertoli cells to IL-1{alpha} (100 pg/ml) or LPS (20 µg/ml) induced ERK activation. Stimulation of ERK phosphorylation by IL-1{alpha} was evident by 15 min, with a maximal response observed at 30-min exposure. As expected, the effect of LPS on ERK phosphorylation was observed later. Slight signals for both p44 ERK and p42 ERK were detected by 30 min, with a peak response observed at 1 h. High levels of ERK activation were maintained for both effectors by 6 h. In untreated cells, the presence of ERK phosphorylation was undetectable, whereas the total amount of ERK was stably expressed in all experimental conditions studied (Fig. 3B, bottom).


Figure 3
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  		Fig. 3. Time-dependent effects of LPS on IL-1{alpha} mRNA (A) and of LPS and IL-1{alpha} on ERK signaling pathway (B) in Sertoli cells. A: cells were cultured for increasing times (0, 3, 6, and 18 h) in the presence or absence of 20 µg/ml LPS as described in MATERIALS AND METHODS. IL-1{alpha} mRNA were analyzed by performing RT-PCR. B: Sertoli cells were cultured in the presence of 100 pg/ml IL-1{alpha} or 20 µg/ml LPS for increasing times (0, 5, 15, 30, 60, 180, and 320 min). Phospho-p42/44 ERK (top) and p42/44 ERK (bottom) were identified using Western blot analysis. Arrowheads indicate 44- and 42-kDa isoforms. Representative blots of 3 different experiments are shown.

 
Inverted control of Cx33 and Cx43 gene expression by LPS and IL-1{alpha}. Stimulation of phagocytosis by LPS exposure resulted in concomitant time-dependent inhibition of Cx43 and stimulation of Cx33 gene transcription. The intensity of the band corresponding to Cx43 mRNA was strongly reduced as a function of time exposure to 20 µg/ml LPS (Fig. 4). In contrast, Cx33 mRNA was first detected 6 h after LPS exposure, was highly expressed 18 h afterward, and was undetectable in control cells and in cells treated with LPS for 3 h.


Figure 4
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  		Fig. 4. Time-dependent effect of Sertoli cell phagocytosis on Cx33 and Cx43 mRNA. Sertoli cells were cultured for increasing times (0, 3, 6, and 18 h) in the presence or absence of 20 µg/ml LPS. Cx33, Cx43, and beta-actin mRNA were analyzed using RT-PCR. Representative gels from 3 different experiments are shown.

 
As indicated in Fig. 5, IL-1{alpha} stimulated Cx33 mRNA but decreased Cx43 mRNA in a time- and dose-dependent manner (Fig. 5, left and right columns, respectively). Semiquantitative analysis of Cx33 levels indicated high, significant increases in Cx33 transcription levels after exposure to IL-1{alpha} for 6 h (P < 0.05) and 18 h (P < 0.01) compared with control cells incubated in the absence of IL-1{alpha} (Fig. 5, left column). Inversely, cytokine reduced Cx43 mRNA levels in a time-dependent manner with a maximum effect after 18-h exposure (P < 0.05). As shown in Fig. 5 (right column), the stimulatory effect of IL-1{alpha} on Cx33 gene expression was dose dependent, with a maximal response (P < 0.01) for doses up to 100 pg/ml IL-1{alpha}. In contrast, IL-1{alpha} was able to inhibit Cx43 gene expression in a dose-dependent manner. The higher effect was observed for the concentrations of 100 and 1,000 pg/ml IL-1{alpha} (P < 0.01). It is likely that the effect of LPS on Cx33 and Cx43 mRNA could be mediated mainly through IL-1{alpha}, because Sertoli cell treatment with IL-1ra (1 µg/ml) significantly suppressed these effects (Fig. 6). Indeed, the presence of IL-1ra significantly prevented the stimulatory effect of LPS on Cx33 mRNA (P < 0.01) and partially restored Cx43 mRNA levels (P < 0.05). IL-1ra alone had no effect on Cx33 and Cx43 mRNA levels (data not shown).


Figure 5
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  		Fig. 5. Analysis of Cx33 and Cx43 gene expressions in Sertoli cells exposed to IL-1{alpha}. Left: time-dependent effect of IL-1{alpha} on Cx33 and Cx43 mRNA levels. Sertoli cells were cultured in the presence (solid bars) or absence (open bars) of 100 pg/ml IL-1{alpha}, and Cx33 and Cx43 mRNA were analyzed using a semiquantitative RT-PCR procedure. Representative gels are shown at top. Right: dose-dependent effect of IL-1{alpha} on Cx33 and Cx43 mRNA levels. Sertoli cells were cultured for 18 h in the presence or absence of increasing concentrations of IL-1{alpha} (0, 1, 10, 100, and 1,000 pg/ml) and Cx33 (open bars) and Cx43 (filled bars) mRNA were analyzed using a semiquantitative RT-PCR procedure. Representative gels are shown at top. Amounts of amplified products were quantified for each sample using densitometric analysis. Results are expressed as the average ratios of the ROD of Cx33 or Cx43 PCR product to that of beta-actin and are means ± SE of 3 densitometric readings from 3 separate experiments. *P < 0.05 and **P < 0.01 vs. cells cultured in presence of IL-1{alpha} for 3 h (left) or in absence of IL-1{alpha} (right).

 

Figure 6
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  		Fig. 6. Effect of IL-1 receptor antagonist (IL-1ra) treatment on Cx33 and Cx43 gene expression in Sertoli cells exposed to LPS. Sertoli cells were cultured for 6 h in the presence or absence of 20 µg/ml LPS plus 1 µg/ml IL-1ra. Cx33 and Cx43 mRNA were analyzed using a semiquantitative RT-PCR procedure with beta-actin as an internal standard. Results represent means ± SE from 3 different experiments and are expressed as %Cx mRNA levels.*P < 0.05 and **P < 0.01 vs. cells exposed to LPS.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although it has been reported that at least 11 Cxs are presumably translated in the testis, their respective physiological roles in spermatogenesis are still undetermined (33). In the present study, we addressed two Cxs: Cx43, the predominant Cx in the testis, and Cx33, a specific testicular Cx previously described to exert a specific inhibitory effect on Cx43-based gap junctions (9, 15). Our data indicate that Cx33 and Cx43 were differentially expressed and regulated in the testis. This finding is supported by three major observations. First, Cx33 was present only within the seminiferous tubules, whereas Cx43 was localized in both tubular and interstitial compartments. Second, Cx33 and Cx43 were differentially expressed in seminiferous tubules according to specific stages of spermatogenesis. Third, the two Cxs were inversely regulated in response to known effectors of Sertoli cells: LPS, used to activate the phagocytic activity, and IL-1{alpha} (for review, see Ref. 20).

Conflicting results have been reported regarding the tissue specificity of Cx33 gene expression. Using Northern blot analysis, other researchers have reported that Cx33 mRNA was found specifically in the testis and undetected in other tissues analyzed (9, 19). In contrast, using RT-PCR, which has a higher sensitivity than Northern blot analysis, other authors reported the presence of Cx33 mRNA not only in the testis but also in the brain, ovary, lung, uterus, and thyroid (10, 33). The results of the present study have demonstrated that Cx33 mRNA was present mainly in testis and undetectable in other tissues analyzed. The testis specificity of Cx33 gene expression is strongly supported by recent findings demonstrating that Cx33 protein was localized specifically within seminiferous tubules, mainly in Sertoli cells (Refs. 15 and 16, in addition to the present study).

There is evidence that FSH and its second messenger cAMP control gap junction coupling (18, 30) and Cx43 expression in Sertoli cells (26). It also has been reported previously that FSH can induce Cx43 in granulosa cells, which are homologous to testicular Sertoli cells in females (39, 49). Together, these observations led to the hypothesis that Cx43 could be a FSH-responsive gene. In contrast, the present study does not provide evidence that Cx33 expression is controlled by FSH and cAMP (data not shown). Thus other mechanisms likely could be responsible for the regulation of Cx33 gene expression. One obvious possibility is that Cx33 mRNA levels are dependent on local controls in Sertoli cells. This hypothesis is in agreement with the present observations that Cx33 and Cx43 genes were differentially regulated during specific stages of spermatogenesis by phagocytosis and locally produced factors, such as IL-1{alpha}. It is also consistent with our findings in the present study that the increased testicular Cx33 during pubertal development occurs concomitantly with elevated intratesticular IL-1 levels described previously (41).

Stage-dependent expression of Cx43 and of Cx43 gap junction communication was reported previously by others (35) and by our group (2, 3, 11). The present immunofluorescence results show that the intensity of the immunoreactive signal for Cx33 was sustained in seminiferous tubules at stages IX and X, whereas it was undetectable for Cx43. The reasons for such a discrepancy are presently unknown. An interesting feature is that around these stages of spermatogenesis, residual bodies (i.e., the excess of germ cell cytoplasm) are phagocytosed by Sertoli cells (38), and it has been suggested that this process may constitute a signal for the initiation of a new wave of spermatogenesis (for review, see Ref. 22). There is also clear evidence that the effect of residual body phagocytosis could be mediated through IL-1{alpha} secretion, which reached maximum levels at stages IX and X (40, 41). This cytokine is known to exert pleiotropic effects and that among them are mitogenic effects on a wide variety of cells (38), including spermatogonia (20, 29). However, the fine mechanisms by which IL-1{alpha} triggers this process have not been identified. The present study clearly demonstrates that LPS regulated Cx33 positively and Cx43 negatively via IL-1{alpha} in Sertoli cells, suggesting that Cx genes may be potential target genes in the response of Sertoli cells to residual body phagocytosis. Such an effect of IL-1 on gap junctions and Cx expression has been reported in other cell types or tissues, such as endothelial cells (21), kidney and lung (14), articular chondrocytes (45), and fetal astrocytes (13, 23).

There is now strong evidence that gap junctions and Cx43 actively participate in the developing process of germ cells and that alteration of such control leads to a decreased number of germ cells in males and females (1, 24, 31, 37). Within the seminiferous epithelium, the Sertoli cell is the only cell type able to communicate with germ cells through gap junctions (34). A recent study (11) demonstrated that Cx43 was able to form functional channels between Sertoli and basally located germ cells, such as spermatogonia. In Sertoli cells, Cx33 has been demonstrated to inhibit gap junction communication through sequestration of Cx43 into the intracellular compartment (15). However, the precise role of Cx33 in the spermatogenic process remains unclear. On the basis of the present study's results, it is tempting to speculate that Sertoli cell phagocytosis of residual bodies could control germ cell proliferation by at least stimulating IL-1{alpha}. The induction of Cx33 by IL-1{alpha} could overcome the control exerted by Sertoli cells through Cx43-based gap junctions on germ cell proliferation. This control, which is stage dependent, occurred punctually and was finely regulated in the seminiferous epithelium.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), a grant from Pfizer (to C. Fiorini), and a grant from Sanofi-Aventis (to X. Decrouy).


   ACKNOWLEDGMENTS
 
We thank C. Avondet for technical assistance and the Service d'Imagerie de l'Institut Curie (Orsay, France).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Pointis, Faculté de Médecine, INSERM U 670, 28 Ave. de Valombrose, 06107 Nice cedex 2, France (e-mail: pointis{at}unice.fr)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Ackert CL, Gittens JEI, O'Brien MJ, Eppig JJ, and Kidder GM. Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev Biol 233: 258–270, 2001.[CrossRef][ISI][Medline]

2. Batias C, Defamie N, Lablack A, Thepot D, Fénichel P, Segretain D, and Pointis G. Modified expression of testicular gap-junction connexin 43 during normal spermatogenic cycle and in altered spermatogenesis. Cell Tissue Res 298: 113–121, 1999.[CrossRef][ISI][Medline]

3. Batias C, Siffroi JP, Fénichel P, Pointis G, and Segretain D. Connexin43 gene expression and regulation in the rodent seminiferous epithelium. J Histochem Cytochem 48: 793–805, 2000.[Abstract/Free Full Text]

4. Beyer EC, Paul DL, and Goodenough DA. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105: 2621–2629, 1987.[Abstract/Free Full Text]

5. Bourdon V, Defamie N, Fénichel P, and Pointis G. Regulation of tissue-type plasminogen activator and its inhibitor (PAI-1) by lipopolysaccharide-induced phagocytosis in a Sertoli cell line. Exp Cell Res 247: 367–372, 1999.[CrossRef][ISI][Medline]

6. Bourdon V, Lablack A, Abbe P, Segretain D, and Pointis G. Characterization of a clonal Sertoli cell line using adult PyLT transgenic mice. Biol Reprod 58: 591–599, 1998.[Abstract/Free Full Text]

7. Bravo-Moreno JF, Díaz-Sánchez V, Montoya-Flores JG, Lamoyi E, Saéz JC, and Pérez-Armendariz EM. Expression of connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development. Anat Rec 264: 13–24, 2001.[CrossRef][Medline]

8. Carabatsos MJ, Sellitto C, Goodenough DA, and Albertini DF. Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence. Dev Biol 226: 167–179, 2000.[CrossRef][ISI][Medline]

9. Chang M, Werner R, and Dahl G. A role for an inhibitory connexin in testis? Dev Biol 175: 50–56, 1996.[CrossRef][ISI][Medline]

10. Chung SS, Lee WM, and Cheng CY. Study on the formation of specialized inter-Sertoli cell junctions in vitro. J Cell Physiol 181: 258–272, 1999.[CrossRef][ISI][Medline]

11. Decrouy X, Gasc JM, Pointis G, and Segretain D. Functional characterization of Cx43 based gap junctions during spermatogenesis. J Cell Physiol 200: 146–154, 2004.[CrossRef][ISI][Medline]

12. Defamie N, Mograbi B, Roger C, Cronier L, Malassine A, Brucker-Davis F, Fénichel P, Segretain D, and Pointis G. Disruption of gap junctional intercellular communication by lindane is associated with aberrant localization of connexin43 and zonula occludens-1 in 42GPA9 Sertoli cells. Carcinogenesis 22: 1537–1542, 2001.[Abstract/Free Full Text]

13. Duffy HS, John GR, Lee SC, Brosnan CF, and Spray DC. Reciprocal regulation of the junctional proteins claudin-1 and connexin43 by interleukin-1beta in primary human fetal astrocytes. J Neurosci 20: RC114.1–RC114.6, 2000.

14. Fernandez-Cobo M, Gingalewski C, and De Maio A. Expression of the connexin 43 gene is increased in the kidneys and the lungs of rats injected with bacterial lipopolysaccharide. Shock 10: 97–102, 1998.[ISI][Medline]

15. Fiorini C, Mograbi B, Cronier L, Bourget I, Decrouy X, Nebout M, Ferrua B, Malassine A, Samson M, Fénichel P, Segretain D, and Pointis G. Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration. J Cell Sci 117: 4665–4672, 2004.[Abstract/Free Full Text]

16. Fischer P, Brehm R, Konrad L, Hartmann S, Kliesch S, Bohle RM, and Bergmann M. Connexin 33: a rodent-specific member of the gap junction protein family? J Androl 26: 75–84, 2005.[Abstract/Free Full Text]

17. Goldberg GS, Lampe PD, and Nicholson BJ. Selective transfer of endogenous metabolites through gap junctions composed of different connexins. Nat Cell Biol 1: 457–459, 1999.[CrossRef][ISI][Medline]

18. Grassi F, Monaco L, Fratamico G, Dolci S, Iannini E, Conti M, Eusebi F, and Stefanini M. Putative second messengers affect cell coupling in the seminiferous tubules. Cell Biol Int Rep 10: 631–639, 1986.[CrossRef][ISI][Medline]

19. Haefliger JA, Bruzzone R, Jenkins NA, Gilbert DJ, Copeland NG, and Paul DL. Four novel members of the connexin family of gap junction proteins: molecular cloning, expression, and chromosome mapping. J Biol Chem 267: 2057–2064, 1992.[Abstract/Free Full Text]

20. Hedger MP and Meinhardt A. Cytokines and the immune-testicular axis. J Reprod Immunol 58: 1–26, 2003.[CrossRef][ISI][Medline]

21. Hu VW and Xie HQ. Interleukin-1{alpha} suppresses gap junction-mediated intercellular communication in human endothelial cells. Exp Cell Res 213: 218–223, 1994.[CrossRef][ISI][Medline]

22. Jégou B, Stéphan JP, Cudicini C, Gomez E, Bauché F, Piquet-Pellorce C, and Touzalin AM. The Sertoli cell-germ cell interactions and the seminiferous tubule interleukin-1 and interleukin-6 system. Results Probl Cell Differ 28: 53–68, 2000.[Medline]

23. John GR, Scemes E, Suadicani SO, Liu JSH, Charles PC, Lee SC, Spray DC, and Brosnan CF. IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels. Proc Natl Acad Sci USA 96: 11613–11618, 1999.[Abstract/Free Full Text]

24. Juneja SC, Barr KJ, Enders GC, and Kidder GM. Defects in the germ line and gonads of mice lacking connexin43. Biol Reprod 60: 1263–1270, 1999.[Abstract/Free Full Text]

25. Kumar NM and Gilula NB. The gap junction communication channel. Cell 84: 381–388, 1996.[CrossRef][ISI][Medline]

26. Lablack A, Bourdon V, Defamie N, Batias C, Mesnil M, Fénichel P, Pointis G, and Segretain D. Ultrastructural and biochemical evidence for gap junction and connexin 43 expression in a clonal Sertoli cell line: a potential model in the study of junctional complex formation. Cell Tissue Res 294: 279–287, 1998.[CrossRef][ISI][Medline]

27. Leblond CP and Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci 55: 548–573, 1952.[ISI][Medline]

28. Nishida T, Nishino N, Takano M, Sekiguchi Y, Kawai K, Mizuno K, Nakai S, Masui Y, and Hirai Y. Molecular cloning and expression of rat interleukin-1{alpha} cDNA. J Biochem (Tokyo) 105: 351–357, 1989.[Abstract/Free Full Text]

29. Petersen C, Boitani C, Fröysa B, and Söder O. Interleukin-1 is a potent growth factor for immature rat Sertoli cells. Mol Cell Endocrinol 186: 37–47, 2002.[CrossRef][ISI][Medline]

30. Pluciennik F, Joffre M, and Délèze J. Follicle-stimulating hormone increases gap junction communication in Sertoli cells from immature rat testis in primary culture. J Membr Biol 139: 81–96, 1994.[ISI][Medline]

31. Plum A, Hallas G, Magin T, Dombrowski F, Hagendorff A, Schumacher B, Wolpert C, Kim JS, Lamers WH, Evert M, Meda P, Traub O, and Willecke K. Unique and shared functions of different connexins in mice. Curr Biol 10: 1083–1091, 2000.[CrossRef][ISI][Medline]

32. Pointis G and Segretain D. Role of connexin-based gap junction channels in testis. Trends Endocrinol Metab 16: 300–306, 2005.[CrossRef][ISI][Medline]

33. Risley MS. Connexin gene expression in seminiferous tubules of the Sprague-Dawley rat. Biol Reprod 62: 748–754, 2000.[Abstract/Free Full Text]

34. Risley MS, Tan IP, and Farrell J. Gap junctions with varied permeability properties establish cell-type specific communication pathways in the rat seminiferous epithelium. Biol Reprod 67: 945–952, 2002.[Abstract/Free Full Text]

35. Risley MS, Tan IP, Roy C, and Saéz JC. Cell-, age- and stage-dependent distribution of connexin43 gap junctions in testes. J Cell Sci 103: 81–96, 1992.[Abstract/Free Full Text]

36. Roscoe WA, Barr KJ, Mhawi AA, Pomerantz DK, and Kidder GM. Failure of spermatogenesis in mice lacking connexin43. Biol Reprod 65: 829–838, 2001.[Abstract/Free Full Text]

37. Simon AM, Goodenough DA, Li E, and Paul DL. Female infertility in mice lacking connexin 37. Nature 385: 525–529, 1997.[CrossRef][Medline]

38. Söder O, Syed V, Callard GV, Toppari J, Pöllänen P, Parvinen M, Fröysa B, and Ritzén EM. Production and secretion of an interleukin-1-like factor is stage-dependent and correlates with spermatogonial DNA synthesis in the rat seminiferous epithelium. Int J Androl 14: 223–231, 1991.[ISI][Medline]

39. Sommersberg B, Bulling A, Salzer U, Fröhlich U, Garfield RE, Amsterdam A, and Mayerhofer A. Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone. Biol Reprod 63: 1661–1668, 2000.[Abstract/Free Full Text]

40. Stéphan JP, Syed V, and Jégou B. Regulation of Sertoli cell IL-1 and IL-6 production in vitro. Mol Cell Endocrinol 134: 109–118, 1997.[CrossRef][ISI][Medline]

41. Svechnikov K, Petersen C, Sultana T, Wahlgren A, Zetterström C, Colón E, Bornestaf C, and Söder O. The paracrine role played by interleukin-1{alpha} in the testis. Curr Drug Targets Immune Endocr Metabol Disord 4: 67–74, 2004.[CrossRef][Medline]

42. Syed V, Stéphan JP, Gérard N, Legrand A, Parvinen M, Bardin CW, and Jégou B. Residual bodies activate Sertoli cell interleukin-1{alpha} (IL-1{alpha}) release, which triggers IL-6 production by an autocrine mechanism, through the lipoxygenase pathway. Endocrinology 136: 3070–3078, 1995.[Abstract]

43. Tan IP, Roy C, Saéz JC, Saéz CG, Paul DL, and Risley MS. Regulated assembly of connexin33 and connexin43 into rat Sertoli cell gap junctions. Biol Reprod 54: 1300–1310, 1996.[Abstract]

44. Tokunaga K, Taniguchi H, Yoda K, Shimizu M, and Sakiyama S. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal beta-actin mRNA. Nucleic Acids Res 14: 2829, 1986.[Free Full Text]

45. Tonon R and D'Andrea P. Interleukin-1beta increases the functional expression of connexin 43 in articular chondrocytes: evidence for a Ca2+-dependent mechanism. J Bone Miner Res 15: 1669–1677, 2000.[CrossRef][ISI][Medline]

46. White TW and Paul DL. Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol 61: 283–310, 1999.[CrossRef][ISI][Medline]

47. Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Güldenagel M, Deutsch U, and Söhl G. Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem 383: 725–737, 2002.[CrossRef][ISI][Medline]

48. Willecke K, Kirchhoff S, Plum A, Temme A, Thönnissen E, and Ott T. Biological functions of connexin genes revealed by human genetic defects, dominant negative approaches and targeted deletions in the mouse. Novartis Found Symp 219: 76–96, 1999.[ISI][Medline]

49. Yogo K, Ogawa T, Akiyama M, Ishida N, and Takeya T. Identification and functional analysis of novel phosphorylation sites in Cx43 in rat primary granulosa cells. FEBS Lett 531: 132–136, 2002.[CrossRef][ISI][Medline]





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