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BOR - Papers in Press, published online ahead of print October 30, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.010504
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BIOLOGY OF REPRODUCTION 68, 1232–1240 (2003)
DOI: 10.1095/biolreprod.102.010504
© 2003 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Neonatal Hypothyroidism Alters the Localization of Gap Junctional Protein Connexin 43 in the Testis and Messenger RNA Levels in the Epididymis of the Rat1

Nancy St-Pierrea, Julie Dufresnea, Andrew A. Rooney3,a, and Daniel G. Cyr2,a

a INRS-Institut Armand-Frappier, Université du Québec, Montreal, Québec, Canada H9R 1G6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objectives of this study were to determine the effects of propylthiouracil (PTU)-induced neonatal hypothyroidism on the gap junctional protein Cx43 in rat testis and epididymis. PTU (0.02%) was administered via lactation from birth to Day 30, and the rats were sampled at 14, 18, 22, 26, 30, and 91 days of age. Testicular Cx43 was localized along the plasma membranes and cytoplasm of Sertoli cells until Day 22. At Day 30, the immunostaining was localized exclusively along the plasma membrane of Sertoli cells. In PTU-treated rats, Cx43 did not localize to the plasma membrane and was still cytoplasmic at 30 days of age. Occludin was present in tubules of treated rats, but was not localized to the blood-testis barrier in 30-day-old rats, as in controls. There were no differences in Cx43 immunostaining in the adult testis. In the proximal epididymis (initial segment, caput, corpus), Cx43 mRNA levels were lower in PTU-treated rats at 14, 18, and 22 days of age, but no differences were observed in the distal (cauda) epididymis at these ages. In 22- and 30-day-old rats, Cx43 was localized along the plasma membrane between principal and basal cells throughout the epididymis. In PTU-treated rats, Cx43 was not detectable in initial segment, caput, or corpus epididymidis. In the cauda epididymidis, however, Cx43 immunostaining in PTU-treated rats was similar to controls. These data suggest that thyroid hormones regulate Cx43-dependent gap junctional communication in the testis and epididymis.

developmental biology, epididymis, gene regulation, Sertoli cells, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intercellular communication in the testis and epididymis plays a crucial role in spermatogenesis and sperm maturation [1–3]. Gap junctions are responsible for direct communication between neighboring cells. They are composed of intercellular pores that allow the passage of small molecules (<1 kDa). These pores are composed of hexomeric connexins from each cell, which are themselves formed by the oligomerization of connexins. Connexins are a family of approximately 20 proteins. Some connexins are widely distributed among different tissues, whereas the distribution of others is more specific [4, 5]. Connexin 43 (Cx43), for example, is present in many tissues, including the testis [6–8] and epididymis [9].

In the testis, Cx43 is localized between adjacent Sertoli cells, Sertoli cells and germ cells, and between Leydig cells [10]. Cyr et al. [9] reported that Cx43 co-localizes with the tight junction protein occludin at the base of the seminiferous epithelium in the area of the blood-testis barrier. Other connexins have also been identified in the testis. These include Cx26 and Cx32, which are present between Sertoli cells in the apical region of the seminiferous tubules [11]. Cx33 is also present between Sertoli cells but is localized toward the basal region of the seminiferous tubules and co-localizes with Cx43 [10], whereas the expression of Cx50 is limited to germ cells [11]. Transcripts for Cx31, Cx37, Cx40, and Cx45 have also been identified in the testis [11], but their localization remains to be established.

In the epididymis, Cx43 is localized along the plasma membrane between principal and basal cells, and its localization in the initial segment of the epididymis is androgen-dependent [9]. It has recently been shown that other connexins are also present in the epididymis. Using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction enzyme mapping, the Cx26, Cx30.3, Cx31.1, and Cx32 have been identified. The expression of these connexins is segment-specific and age-dependent, and their localization varies [12].

An important role of gap junctions is their implication in the regulation of cell growth and differentiation by controlling the passage of small molecules, including secondary messengers, between adjacent cells [4]. Thyroid hormones also regulate cellular differentiation, but few studies have focused on the effects of thyroid hormones on intercellular junctions. In rat liver epithelial cells, thyroid hormones can modulate Cx43 levels in a dose-dependent manner [13]. This regulation appears to be via a direct action of the thyroid hormone receptor on the Cx43 promoter, which contains a thyroid response element [14]. There are no studies on the regulation of gap junctions in the testis and epididymis by thyroid hormones.

Thyroid hormones have been implicated in the differentiation and maturation of a variety of fetal tissues including the testis [15, 16]. Cooke et al. [17] reported that neonatal hypothyroidism alters testicular weight. A lack of thyroid hormones during neonatal development causes a 40% increase in the testicular weight of adult rats and increases daily sperm production [17]. This increase in testis size is caused by a delay in differentiation of Sertoli cells, resulting in a greater number of Sertoli cells, as well as a greater number of Leydig cells [18, 19]. Studies have shown that hypothyroidism in adult rats causes morphological changes in the caput and corpus epididymidis and is associated with a decrease in the number of epithelial cells [20]. However, neonatal hypothyroidism causes an increase in adult epididymal weight [17]. Thyroid hormone receptors are present in the rat testis throughout postnatal development [21–23]. Furthermore, both type 1 and type 2 iodothyronine deiodinases, which are responsible for the conversion of L-thyroxine to its active metabolite, 3,5,3'-triiodo-L-thyronine, are present in the testis, but their activity is higher in 12- and 22-day-old rats compared with that of adults [24]. In the epididymis, Del Rio et al. [25] reported high-affinity, low-capacity binding sites of T3 in the nuclei of the epididymis, suggesting that thyroid hormone can act directly on the epididymis.

The objectives of the present study were to investigate the effects of propylthiouracil (PTU)-induced neonatal hypothyroidism on Cx43 mRNA levels and immunolocalization of Cx43 protein in the testis and epididymis of rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Timed-gestation Sprague-Dawley female rats were purchased 1 wk prior to parturition from Charles River Canada, Inc. (St-Constant, QC). Pups were removed from their mothers within 12 h of birth, sexed, and randomly reassigned to a lactating dam. There were 11 male pups per dam. Rats were maintained on a 12L:12D photoperiod and both food and water were available ad libitum. To determine the presence of thyroid hormone receptors in the epididymis, 26- and 91-day-old male Sprague-Dawley rats were purchased and housed under the same conditions. All procedures were approved by the University Animal Care Committee.

Experimental Protocol

The experimental protocol used in this study was based on previous experiments in which PTU in the drinking water of lactating dams induced neonatal hypothyroidism in rat pups. Pups recovered normal circulating concentrations of thyroid hormones shortly after PTU treatment was ceased at weaning [26]. Two experimental groups were used: controls and those treated with PTU. In the PTU-treated group, lactating dams were given drinking water containing 0.02% PTU (Sigma Chemicals, Mississauga, ON) dissolved in 1% cherry-flavored Kool-Aid (Kraft, Montreal, QC, Canada). Control dams were given 1% Kool-Aid alone in their drinking water. Kool-Aid was added to the water to increase palatability and to encourage PTU drinking (R. Hess, personal communication). PTU treatment started at birth and was stopped on Day 24; thereafter, clean water was provided. Pups were weaned on Day 28. Animals were weighed and killed by asphyxiation with CO2. Rats were sampled at 14, 18, 22, 26, 30, and 91 days of age. These time points were chosen in relation to the formation of the blood-testis barrier, which occurs between Days 15 and 19 [27], and the blood-epididymal barrier, which is formed between Days 18 and 21 [28]. Time points were selected before, during, and after these barriers are formed. Day 26 was chosen because it was shortly after the PTU treatment ceased, and Day 30 was selected because it was shortly after weaning. Rats were considered adults at 91 days of age. At the time of sampling, the testes were removed and rapidly frozen in liquid nitrogen. Epididymides were divided into the caput-corpus (initial segment, caput, and corpus epididymidis) and cauda epididymidis prior to being frozen in liquid nitrogen. Testes and epididymal segments were subsequently stored at -86°C. Some of the testes and epididymides were fixed for histological examination by immersion in Bouins fixative.

Western Blot Analysis

Frozen epididymides of 26- and 91-day-old rats were homogenized in buffer (0.23 M sucrose, 10 mM Tris-HCl pH 7.6, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 100 µg/ml phenylmethyl-sulfonyl fluoride (PMSF), 1 µg/ml pepstatin, 2 µg/ml antipain [Sigma Chemicals]). The cellular debris was removed by centrifugation for 10 min at 10 000 x g. A 100-µg aliquot of supernatant was diluted in loading buffer (Laemmli buffer), boiled for 10 min, and cooled on ice. The samples were loaded onto a 10% polyacrylamide gel with a 4% stacking gel [29]. Electrophoresis was performed at 120 V for 1.5 h until the dye front reached the end of the gel. The proteins on the gel were then transferred onto a nitrocellulose membrane using a Bio-Rad Transblot apparatus (BioRad Laboratories, Inc., Mississauga, ON) at 120 V for 1 h at 4°C. The transfer of colored molecular weight markers was used to assess the efficiency of transfer. The presence of the thyroid hormone receptor was determined using the TR{alpha}1 antibody, which recognizes both the TR{alpha}1 and TRß1 thyroid hormone receptor subunits (Santa Cruz Biotechnology Laboratories, Santa Cruz, CA). The presence of the thyroid hormone receptor was detected with a streptavidin-alkaline phosphatase-conjugated secondary antibody (Blotting Detection Kit, BioRad). Adult rat testis was used as a positive control.

Northern Blot Analyses

Total cellular RNA from four distinct pools of frozen tissues from two individuals were used for Northern blot analyses. Total cellular RNA from testis, caput-corpus and cauda epididymidis was isolated using the guanidinium isothiocyanate method [30]. A 10-µg aliquot of total RNA from each sample was then separated by electrophoresis in a 1.2% agarose-formaldehyde gel and transferred onto a charged nylon membrane (GeneScreen Plus, Dupont Chemicals, Mississauga, ON).

The Cx43 cDNA probe was a gift from Dr. E. Beyer (University of Chicago, Chicago, IL) [31]. The epithelial-cadherin (E-Cad) cDNA was amplified by RT-PCR using the following specific primers: forward, 5'-TGCCCCAGTATCGTCCCCGT-3'; reverse, 5'-CGGTTGCCCATTCGTTCAGATAA-3' [32]. Total RNA (500 ng) was reverse transcribed using an oligo(dT)16 primer. The cDNA was amplified using 30 cycles of denaturation at 94°C (60 sec), annealing at 55.6°C (60 sec), and elongation at 72°C (60 sec). The PCR product was separated on an agarose gel and the 230-base pair (bp) product was isolated from the gel using the Qiaex II extraction kit (Qiagen, Valencia, CA) and cloned into the T/A cloning site of the pCR II plasmid (Invitrogen, Palo Alto, CA). The plasmid was then used to transform competent bacteria, and positive clones were selected by color. The insert was isolated by restriction enzyme digest and agarose gel electrophoresis, and was purified using the Qiaex II extraction kit. Rat E-Cad was sequenced and its identity was confirmed by sequence homology using the basic local alignment search tool (BLAST) (GenBank, National Center for Biotechnology Information, Washington, D.C.).

Both the Cx43 and E-Cad cDNA probes were labeled by random priming with [32P]-dCTP (Oligonucleotide Labeling kit, Amersham-Pharmacia Biotech, Baie d'Urfe, QC). The membranes were standardized for RNA loading by hybridization with an end-labeled 18S rRNA oligonucleotide probe [33]. The resulting unsaturated exposed phosphorus screens were scanned using a PhosphorImage analyzer (Molecular Dynamics, Sunnyvale, CA) and quantified using ImageQuant software (Molecular Dynamics). The integrated area under the curve for the cDNA probe was standardized against the signal for the 18S rRNA to determine the relative levels of Cx43 and E-Cad mRNA.

Immunocytochemistry

Bouin-fixed tissues were dehydrated and embedded in paraffin. Sections (5 µm) were subsequently treated as previously described [33]. For occludin immunostaining, prior to blocking free aldehyde with glycine, antigen retrieval was performed by boiling the sections in a citrate solution (0.02 M citric acid, 0.01 M sodium citrate) for 10 min in a microwave oven.

Immunocytochemical localization of Cx43 and occludin in the testis and epididymidis were performed using affinity-purified polyclonal rabbit antisera (Cx43, 100 µg/ml, Santa Cruz Biotechnology; occludin, 10 µg/ml, Zymed Laboratories, South San Francisco, CA). Antibody binding to Cx43 and occludin were detected using the horseradish peroxidase method as previously described [33]. Slides incubated with normal rabbit antiserum were used as negative controls because immunoabsorption was not possible due to a lack of Cx43 and occludin antigens.

Statistical Analyses

One-way ANOVA was conducted on the data from Northern blots for statistical analyses. Results from control and PTU-treated groups were compared for each age. Levels at each sampling time were also compared one to another within each group. All analyses were performed using SigmaStat computer software (Jandel Scientific Software, San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body And Tissue Weights

Body weights of the treated animals were lower than those of the control rats during PTU administration. At the end of the treatment, the PTU-treated rats weighed approximately 59% less than controls. Once the treatment was stopped, the growth rate of PTU-treated pups increased. However, the weights of the treated adult rats (Day 91) were still significantly lower than those of control rats. The mean adult testis weight in the treated group was 35% greater than that of controls, despite the lower body weights. Epididymal weights of adult rats were not significantly altered by PTU treatment.

Presence of the Thyroid Hormone Receptor in the Epididymis

Western blot analysis using total protein was performed to determine the presence of the thyroid hormone receptor (TR) in the epididymis. Results indicate a single band of 52.5 kDa in the epididymis of both the 21- and 91-day-old rats. This corresponds to the molecular weight of the TR (Santa Cruz Biotechnology). A slightly higher molecular weight protein of 53 kDa was present in the testis (Fig. 1). This corresponds to the testicular thyroid hormone receptor that has been reported to have a slightly higher molecular weight in the testis [19].



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  		FIG. 1. Western blot of thyroid hormone receptor {alpha} in the epididymis. Total protein extract from epididymides of rats at 26 and 91 days of age and testes at 91 days were separated on SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was incubated with thyroid hormone antibody (TR1), and the protein was detected using a streptavidin-alkaline phophatase-conjugated antibody. Adult testis was use as a positive control. E, Epididymis; TE, testis; MW, molecular weights

Effects of PTU On Cx43 in the Testis

The developmental pattern of Cx43 mRNA levels in control rat testis indicate that levels were low until 22 days of age, increased 1.5-fold at Day 26, and remained constant thereafter. However, the differences between the ages were not statistically significant. The pattern is the same for PTU-treated rats. There were no significant differences in Cx43 mRNA levels between controls and PTU-treated rat testis at any of the ages tested (Fig. 2).



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  		FIG. 2. Northern blot analyses of Cx43 levels in rat testis during postnatal development. Total cellular RNA was isolated from testes of rats at age 14, 18, 22, 26, 30, and 91 days. A single band of approximately 3.0 kilobase was detected. The intensity of the bands at different ages was determined by densitometry. The blots were then reprobed with an 18S rRNA probe to standardize for RNA loading. Data were normalized relative to 14-day-old control rats (n = 4). Closed bars represent the mean values for control rats and open bars represent those of PTU-treated rats. Data are expressed as means ± SEM from four separate pools of tissue

Morphologically, the seminiferous tubules of PTU-treated rats (Fig. 3B, D, and F) were smaller than controls (Fig. 3A, C, and E) from Day 14 until Day 30. In the seminiferous tubules of PTU-treated rats, there was no lumen in 18-, 22-, and 26-day-old rats, and few tubules had a clearly defined lumen by 30 days of age (Fig. 3B, D, and F). This is in sharp contrast to control rats, which displayed a well-defined lumen by 18 days of age.



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  		FIG. 3. Immunolocalization of Cx43 in testis of control (A, C, E) and PTU-treated rats (B, D, F) of 22 (A–D) and 30 (E, F) days of age. Sections were immunostained with anti-Cx43 antibody. At both ages, the seminiferous tubules of PTU-treated rats are smaller than those of control animals. In PTU-treated rats, the lumen of the seminiferous tubule is absent in testes of 22-day-old rats and begins to form only in 30-day-old rats. By contrast, in control rats the lumen of the seminiferous tubule is already formed by 22 days of age. In the testis of control (A, C) and PTU-treated 22-day-old rats (B, D), Cx43 immunostaining was present along the lateral margins of plasma membranes of Sertoli cells (arrows). In PTU-treated rats, however, a more cytoplasmic immunoreaction was observed (arrowheads). In 30-day-old control rats, Cx43 was localized at the base of the seminiferous tubule between Sertoli cells and forms discontinuous wavy reactive bands (arrows). Punctate staining is also present more apically between Sertoli and germ cells (E). In PTU-treated rats, Cx43 was also present between Sertoli cells but a strong cytoplasmic reaction remained (arrowheads) (F). L, Leydig cells; Lu, lumen; G, germ cells; rs, round spermatids; g, spermatogonia; S, Sertoli cells. Magnification x400 in (A, B) x640 in (C–F)

Testes of 14, 18, 22, and 30 days were used for immunolocalization of Cx43. In young animals (Days 14–18), Cx43 immunostaining was localized at the apical and lateral margins of Sertoli cells. However, the staining was still diffuse throughout the seminiferous tubules and there were no differences between controls and PTU-treated rats (data not shown). In testes from 22-day-old rats, Cx43 immunostaining was still mainly localized along the lateral plasma membrane of Sertoli cells, but there was a more intense reaction in the basal region of seminiferous tubules (Fig. 3A and C). In testes from PTU-treated rats, Cx43 immunostaining along the plasma membrane was much weaker than in control rats (Fig. 3B and D). In 30-day-old rats, the difference in Cx43 localization between control and PTU-treated rats was more pronounced. In controls, Cx43 was localized mainly at the base of the tubules between Sertoli cells, as discrete ribbon-like strands (Fig. 3E). In testes from PTU-treated rats, there was an intense cytoplasmic immunostaining for Cx43 with little immunostaining along the lateral plasma membrane of Sertoli cells (Fig. 3F). In testes from 22- and 30-day-old rats, some tubules were stained more intensely than others. The levels of Cx43 are known to be related to the stage of the seminiferous tubules. In all cases, no staining was observed in sections incubated with normal rabbit serum, which was used as a negative control (data not shown).

In adult rats, occludin has been shown to localize to tight junctions between Sertoli cells that form the blood-testis barrier. Previous studies in our laboratory have shown that Cx43 co-localizes with occludin in the seminiferous tubules of adult mice [3]. Given that Cx43 localization was disrupted in PTU-treated rats, and because the seminiferous tubules of PTU-treated rats have no lumen by 22 days of age, we wanted to know whether or not occludin localization was also altered by PTU treatment, and whether this could explain why Cx43 does not localize to the basal region of the seminiferous tubules between adjacent Sertoli cells. In testes from 22-day-old control rats, occludin was located mainly at the base of the tubules between Sertoli cells and also between some germ cells (Fig. 4A). In PTU-treated rats, occludin immunostaining was more diffuse throughout Sertoli cells (Fig. 4B). In 30-day-old control rats, occludin was localized to the base of the seminiferous tubules between adjacent Sertoli cells (Fig. 4C), but in testes from PTU-treated rats, the staining in the majority of the tubules was still localized throughout the tubules, although some tubules showed a similar pattern of occludin immunostaining as in control rats (Fig. 4D). Sections treated with normal rabbit serum were unreactive (data not shown).



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  		FIG. 4. Immunolocalization of occludin in the testis of 22- (A, B) and 30-day-old (C, D) rats. In control rats of both ages, occludin immunostaining (arrows) appears at the base of the seminiferous tubules where tight junctions form the blood-testis barrier (A, C). In 22-day-old PTU-treated rats, the lumen of the seminiferous tubule is absent and occludin immunostaining (arrowheads) is present throughout the tubules (B). By 30 days of age, the lumen has formed and occludin is localized at the base of some tubules (not shown), however, in most tubules, the staining is still present throughout the seminiferous tubule (arrowheads) (D). L, Leydig cells; Lu, lumen; IT, intertubular space; S, Sertoli cells. Magnification x640

Effects of PTU on Cx43 in the Epididymis

Cx43 mRNA levels were measured in the caput-corpus and cauda regions of the epididymis from control and PTU-treated rats. In the caput-corpus epididymidis of control rats, Cx43 mRNA levels were already high at Day 14 and they increased to peak levels by 22 days of age. Cx43 mRNA levels subsequently decreased from Days 30 to 91 by almost 70% (Fig. 5A). Levels at Day 91 were significantly different from those at all other time points in both control and PTU-treated rats. In the cauda epididymidis, Cx43 mRNA levels were constant throughout the postnatal development period and peaked at adulthood (91 days) (Fig. 5B) [12]. In the caput-corpus epididymidis, Northern blot analyses indicated a significant decrease in the levels of Cx43 mRNA in PTU-treated rats compared with controls at 14, 18, and 22 days of age. Although Cx43 mRNA levels were also lower at 26, 30, and 91 days of age, these differences were not statistically significant (Fig. 5A). There were no significant differences between control and PTU-treated Cx43 mRNA levels in the cauda epididymidis at any of the ages sampled (Fig. 5B).



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  		FIG. 5. Northern blot analyses of Cx43 mRNA levels in rat epididymides during postnatal development. Total cellular RNA was isolated from either the proximal (A) or distal (B) segments. A single 3.0-kilobase band was obtained. The blots were standardized using an 18S rRNA probe. The intensity of the bands was determined by densitometry. Data were normalized to data from the 14-day-old control rat. Closed bars represent the mean values for control rats and open bars those of PTU-treated rats. Data are expressed as means ± SEM (n = 4). Asterisks indicate a significant difference between control and PTU-treated rats (P < 0.005)

Immunolocalization of Cx43 was conducted on epididymides of control and PTU-treated rats at 14, 18, 22, and 30 days of age. There was no Cx43 immunostaining in epididymidis at 14 and 18 days of age. In 22-day-old rats, a Cx43 immunoperoxidase reaction was localized in the basal region of the epididymal lumen between principal and basal cells in all segments of the epididymis (Fig. 6A, C, E, and G). In epididymides of PTU-treated rats, Cx43 was either absent or present at very low levels in the initial segment (Fig. 6B), caput (Fig. 6D), and corpus epididymidis (Fig. 6F). In contrast, Cx43 immunostaining in the cauda epididymidis was similar to control rats, as Cx43 was localized between basal and principal cells (Fig. 6H).



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  		FIG. 6. Immunolocalization of Cx43 in the epididymis of control and PTU treated rats. Cx43 was immunolocalized in the initial segment (A, B), caput (C, D), corpus (E, F), and cauda (G, H) epididymidis of control (A, C, E, G) and PTU-treated (B, D, F, H) rats at 22 days of ages. In control rats, Cx43 was localized at the basal region of the epididymal epithelium between principal and basal cells in all regions of the epididymis (arrows). In PTU-treated rats, Cx43 was nondetectable in the initial segment, caput, and corpus epididymidis (B, D, and F). In the cauda epididymidis (H), however, Cx43 was localized at the base of the epithelium between basal and principal cells, and the intensity of the staining was similar to that of control rats (G). b, basal cells; p, principal cells; C, clear cell; IT, interstitial space; Lu, lumen. Magnification x640

Effects of PTU On E-Cad in the Epididymis

Unlike in the testis, Cx43 in the epididymis is not localized to epididymal tight junctions present in the apical region of the epididymal tubules between adjacent principal cells. Previous studies have suggested that in certain epithelial cells, adherens junctions can regulate the formation of gap junctions [34]. We therefore wanted to establish whether E-Cad, a protein associated with adhering junctions, was also regulated by PTU. Northern blot analyses of E-Cad mRNA levels in the epididymis indicated that PTU treatment had no effect on E-Cad mRNA levels at all ages tested, except at 14 days of age in both proximal and distal segments of the epididymis. However, the effects of PTU on E-Cad mRNA levels in the caput-corpus and cauda epididymidis are markedly different. In the caput-corpus epididymidis, PTU treatment resulted in a significant decrease in E-Cad mRNA levels (Fig. 7A), whereas in the cauda epididymidis, E-Cad mRNA levels were significantly increased (Fig. 7B). There were no significant differences found within a group when comparing levels for each age group. Immunolocalization of E-Cad during epididymal development indicated that E-Cad was not regulated by PTU treatment (data not shown), suggesting that PTU-induced lower Cx43 mRNA levels are not related to alterations in E-Cad.



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  		FIG. 7. Northern blot analyses of E-cad mRNA levels in rat proximal (A) and distal (B) epididymides. A single band of 4.7 kilobase was obtained. RNA loading was standardized using an 18S rRNA probe. Densitometry was used to determine intensity of the bands. Closed bars represent the mean values for control rats and open bars those of PTU-treated rats. Data are expressed as a percentage of 14-day-old control rats ± SEM (n = 4). Asterisks indicate a significant difference between control and PTU-treated rats (P < 0.005)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Testicular intercellular communication mediated by Cx43 gap junctions is believed to represent an essential process for spermatogenesis [2]. The relatively large number of different connexins and their localization in the testis is suggestive of the importance of intercellular gap junctional communication in testicular functions [6, 7, 9, 10]. Limited information is available on the factors that regulate Cx43 and other connexins in the testis. The proliferation of Sertoli cells is regulated in part by thyroid hormones, which appear to regulate the differentiation of Sertoli cells from immature to mature cells during the first 3 wk of postnatal life. This differentiation of cells within a complex syncitium such as the seminiferous tubule is likely to involve the formation of new cellular interactions and communication processes between cells. Our results indicate that PTU-induced neonatal hypothyroidism appears to modulate the cellular localization of Cx43 in the developing seminiferous tubule. Although PTU treatment does not alter testicular Cx43 mRNA levels, the cellular targeting of Cx43 to the lateral plasma membranes of Sertoli cells appears to be altered by neonatal hypothyroidism. In control rats we observed a pronounced Cx43 cytoplasmic immunostaining in both 14- and 18-day-old rats. This cytoplasmic immunostaining disappeared in older rats, as Cx43 appeared to localize exclusively to the lateral plasma membranes of Sertoli cells. In 30-day-old PTU-treated rats the Cx43 cytoplasmic immunostaining was still present in the testis. The intracellular targeting of Cx43 occurs primarily through a Golgi-mediated pathway in which connexins oligomerize into connexins and are subsequently targeted to the plasma membrane [4, 35, 36].

Testicular tight junctions are composed of several different transmembrane proteins, including occludin and several claudins [37–40]. It has been reported that Cx43 and occludin can co-localize within intercellular junctional complexes, including the testis [37, 41–43]. This co-localization is related to the binding of both of these proteins to cytosolic tight junctional scaffolding proteins such as zona occludens-1 (ZO-1) [41, 42]. The interaction between Cx43, occludin, and ZO-1 suggest that the effects of thyroid hormones on Cx43 localization may be indirect and result from alterations in the formation of tight junctions between adjacent Sertoli cells. The formation of the lumen in the seminiferous tubule of PTU-treated rats was delayed. Because lumen formation requires the presence of tight junctions and the resulting vectorial transport across the epithelium toward the lumen, we hypothesized that the increased cytoplasmic Cx43 immunostaining may be the result of a delay in the formation of tight junctions between adjacent Sertoli cells. In rats, tight junctions in Sertoli cells are formed at the base of the seminiferous tubule at approximately 14 days of age, which results in the formation of the blood-testis barrier [27]. The resulting lumen of seminiferous tubules is first apparent by approximately 16 days of age in rats [44]. In the present experiment, the lumen of the seminiferous tubules of control rats is apparent by 18 days of age. In contrast, seminiferous tubules of PTU-treated rats became evident only in 30-day-old rats. This suggests that thyroid hormones may be necessary for the formation of tight junctions between Sertoli cells. Previous studies by Van Haaster et al. [44] reported that T3 administration can accelerate testicular lumen formation in rats.

The pattern of occludin immunostaining was similar to that previously reported [37, 39]. Our results indicate that at 22 and 30 days of age in PTU-treated rats, occludin is not yet localized to the base of the tubule between adjacent Sertoli cells, as was observed in control rats (Fig 4A and C). Alterations in the localization of occludin between Sertoli cells in PTU-treated rats suggest that hypothyroidism delays the formation of tight junctions between Sertoli cells. In fact, the absence of specific occludin localization in the area of Sertoli-Sertoli tight junctions is similar to the organization of seminiferous tubules present in younger animals prior to the formation of the blood-testis barrier [37, 43]. Although thyroid hormones are known to act directly on Cx43 to regulate its expression [24, 25, 45, 46], the results obtained in this study suggest that the effects observed on the localization of this protein, and of occludin, as well as the effects observed on lumen formation, may be related to a general delay in testicular development. This observation is in agreement with previous reports of altered testicular development in PTU-treated rats [47].

In both control and PTU-treated rats, some tubules displayed a more intense Cx43 immunoreaction than others. Previous studies have shown that Cx43 immunostaining is dependent on the stage of the tubule [6, 7, 10]. It is interesting that this differential expression of Cx43 between different tubules also occurs in immature animals and is not regulated by thyroid hormones.

Northern blot analyses indicate that no differences exist in Cx43 mRNA levels between control and PTU-treated rats, suggesting that thyroid hormones do not affect the levels of Cx43, but rather, they affect the intercellular targeting of Cx43 in the testis.

In the epididymis, our results indicate that neonatal hypothyroidism results in a reduction in Cx43 mRNA levels in the caput-corpus region of the epididymis during PTU treatment, but that this effect does not occur in the cauda epididymidis. Thyroid hormones are known to affect the morphology of the epididymis [20]. However, little is known regarding the regulation of cellular interactions by thyroid hormones. Some studies have shown that thyroid hormones enhance gap junctional communication and Cx43 expression in vitro in rat hepatocytes [13]. Autoimmune thyroid disease causing hypothyroidism is associated with a reduction in cell-cell communication and the formation of Cx43 gap junctions in thyroid tissues [45, 46]. Stock and Sies [14] recently reported that the thyroid hormone receptor binds to a response element in the Cx43 promoter, which stimulates Cx43 transcription in rat hepatocytes, thereby increasing gap junctional communication [14]. These studies suggest that the decrease in Cx43 mRNA and protein levels in the epididymis of PTU-treated rats results from a lack of thyroid hormones.

In the present experiment, PTU treatment was stopped on Day 24 and the mRNA levels for Cx43 returned to control levels by 26 days of age, while protein levels, as estimated immunocytochemically, returned to control levels by 30 days of age. The transient effect and rapid return to normal can be explained by the short half-life of Cx43 (approximately 1.3 h) [36]. It has also been shown by Kirby et al. [47] that thyroid hormone levels return to normal within 15 days after the end of treatment. It is interesting that Cx43 levels are not regulated by thyroid hormones in the cauda epididymidis. This suggests that thyroid hormones are not the only factors regulating Cx43 and that other factors are likely involved in its regulation. This is not the first demonstration of a differential regulation of mRNA or protein levels along the epididymis. Cyr et al. [9] have shown that the localization of Cx43 in the epididymis was regulated by androgens only in the initial segment of the organ [9]. Gregory et al. [48] have also demonstrated that androgens regulate claudin-1 expression exclusively in the initial segment. Other genes such as CRES, 5{alpha}-reductase, and GGT are also regulated by testicular factors in the initial segment only [49]. Likewise, SGP-2 mRNA levels are androgen-repressed only in the distal corpus and cauda epididymidis [50]. Whether or not thyroid hormones preferentially regulate specific regions of the epididymis remains to be established.

Unlike in the testis, Cx43 does not co-localize with tight junctional proteins in the epididymis (Cyr, unpublished observations). The cell adhesion protein E-Cad, which is present in the epididymis, is also known to modulate the formation of gap junctions in certain epithelial cells [34, 51–53]. Results from the present study indicate that PTU treatment did not significantly alter E-Cad mRNA levels or E-Cad immunolocalization compared with that of control rats. It is therefore unlikely that the regulation of Cx43 by PTU involves alterations in E-Cad-mediated cell adhesion. The differential regulation of E-Cad in the proximal and distal regions of the epididymis is unknown but is unlikely due to a direct regulation by thyroid hormones, because the effect occurred only in 14-day-old rats and was not maintained throughout the treatment period.

Whereas the observed results are likely due to PTU-induced hypothyroidism, we cannot rule out the possibility that PTU exerts a toxic effects on cellular junctions in the testis and epididymis. However, Cooke and Meisami [17] reported that neonatal rats treated with PTU from birth to Day 25 via lactation, and also given T4 and T3 replacement during that period, showed normal testis size, indicating that the PTU-induced alterations in testis size was in fact related to the thyroid status of the animals and not due to a toxic effect of PTU.

In summary, our results have shown that the cellular targeting of testicular Cx43 is in part regulated by thyroid hormones in developing rats. Whether or not this effect is the result or the cause for a delay in the differentiation of Sertoli cells remains to be established. Unlike those of testes, Cx43 mRNA levels in the proximal regions of the epididymis appear to be thyroid hormone-dependent. These results suggest that thyroid hormones may play an important role in the development of intercellular communication in both the testis and epididymis.


   ACKNOWLEDGMENTS
 
We thank Dr. Rex Hess (University of Illinois) for his helpful suggestions and Dr. E. Beyer (University of Chicago) for his generous gift of Cx43 cDNA. We thank S. DeBellefeuille, L. Falcone, M. Gregory, and Dr. K. Finnson for their assistance.


   FOOTNOTES
 
1 This study was supported by the Toxic Substances Research Initiative (Health Canada) and the Natural Sciences and Engineering Research Council of Canada via grants to D.G.C. Back

2 Correspondence: Daniel G. Cyr, INRS-Institut Armand-Frappier, Université du Québec, 245 boul Hymus, Montreal (Pointe-Claire), QC, Canada H9R 1G6. FAX: 514 630 8850; daniel.cyr{at}inrs-sante.uquebec.ca Back

3 Present address: U.S. Environmental Protection Agency, Raleigh, NC Back

Received: 20 August 2002.

First decision: 15 September 2002.

Accepted: 18 October 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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