Regulation of the Gap Junction Interplay in the Rat Epididymis

Regulation of the Gap Junction Interplay in the Rat Epididymis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Regulation of the Gap Junction Interplay in the Rat Epididymis Daniel Cyr, Cécile Adam, Julie Dufresne, Mary Gregory This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4731767/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract During postnatal development of the epididymis, a change in the expression of gap junction proteins, or connexins (Cxs), occurs, in which Gjb2 (Cx26) and Gja1 (Cx43) levels in the proximal epididymis are decreased, while Gjb1 (Cx32), Gjb4 (Cx30.3) and Gjb5 (Cx31.1) levels increase. The mechanism(s) responsible for the switch in Cx expression is unknown. The aims of this study are: 1) to identify the mechanisms responsible for the decrease in GJB2 protein levels and the increase in other Cxs during postnatal development. Results indicate that decreased Gjb2 expression does not induce changes in the expression of other Cxs in rat RCE-1 principal cells, suggesting a lack of compensatory expression. Sequence analysis of both Gjb2 and Gjb1 promoters identified common multiple response elements to steroid hormones. Using RCE-1 cells, we showed that glucocorticoids increased Gjb2 expression, while estradiol had no effect. Orchidectomy in rats resulted in a significant increase in GJB2 and decreased GJB1 in the caput and corpus epididymidis. Changes in Cxs protein levels were prevented by administering testosterone in orchidectomized rats. Similar results were observed in the prostate, another androgen-receptive organ. LNCaP cells, which are androgen-responsive, showed that exogenous dihydrotestosterone (DHT) exposure resulted in a decrease in Gjb2 mRNA levels concomitant with increased Gjb1 levels. Using a GJB1 promoter construct we showed that DHT could induce transactivation of the luciferase transgene, while transactivation using two GJB2 promoters were not altered. Together, our results suggest that androgens and glucocorticoids regulate the expression of Cxs in the epididymis. General Cell Biology & Physiology Connexins gap junctions androgen epididymis development gene expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The differentiation of epithelial cells can be associated with changes in the expression of connexins (Cxs) and intercellular gap junctional communication (GJIC) in a variety of tissues, including the testis and epididymis (Cyr, 2011 , Kidder and Cyr, 2016 ). In the epidermis, keratinocyte differentiation is synchronized with the decrease in the expression of gap junction protein B2 (GJB2; connexin26) and gap junction protein A1 (GJA1; connexin43) and a concomitant increase in gap junction protein B3 (GJB3; connexin31) and gap junction protein B4 (GJB4; connexin30.3)(Brissette, et al., 1994 ). This phenomenon is also observed during the differentiation of mammary gland cells, in which GJB2 levels increase during pregnancy while GJB1 (connexin32) is only expressed during lactation (Locke, et al., 2000 ). Previous studies from our laboratory have shown that in the rat epididymis, both GJA1 and GJB2 levels decrease while GJB1, GJB4 and GJB5 (connexin31.1) levels increase during postnatal development (Dufresne, et al., 2003 ). Changes in expression of different Cxs may be the result of varying factors, such as hormones, that regulate the expression of different Cxs; however, there are also examples suggesting compensatory regulation of different Cxs. In the mammary gland, inhibition of GJB2 also decreases GJB6 levels (Stewart, et al., 2014 ). A similar correlation between GJB2 and GJB6 was also observed in the cochlea, where removal of GJB2 induced a developmental delay in GJB6 expression (Crispino, et al., 2011 ). Transgenic mice lacking G jb 1 in the pancreas (Chanson, et al., 1998 ) and liver (Nelles, et al., 1996 ) displayed diminished GJB2 protein levels.. In the epididymis, no information is available on the effect of GJB2 decrease on other Cxs during development. Steroid hormones influence the establishment and maintenance of epididymal functions. Estrogen receptor 1 (ESR1) and ESR2 are present throughout the epididymis and have been shown to play an important role in epididymal development and function (Hess, 2003 , Hess, et al., 2021 ). The glucocorticoid receptor (NR3C1) is also present in multiple epididymal cell types, including principal, basal, narrow, and apical cells (Gladstones, et al., 2012 , Silva, et al., 2010 ). Glucocorticoid deprivation has been reported to increase the expression of the androgen receptor in the cauda epididymidis (Silva, Queiroz, Honda and Avellar, 2010 ). Epididymal development is also known to be highly regulated by androgens, which play a role in the differentiation of the Wolffian duct into formation of the epididymis and in its postnatal development (Joseph, et al., 2009 , Ribeiro, et al., 2017 , Robaire and Viger, 1995 , Wilbourne, et al., 2023 ). Several studies have shown the importance of androgens on the development and functions of the epididymis. Indeed, the removal of androgens leads to weight loss of the epididymis, a decrease in the diameter of the tubules, apoptosis of epithelial cells and morphological alterations in epididymal principal cells(Fan and Robaire, 1998 , Robaire and Hamzeh, 2011 , Robaire, et al., 2015 ). Phosphorylation, expression levels and localization of GJA1 have been shown to be androgen-dependent in the initial segment in rats (Cyr, et al., 1996 ). GJA1 is localized between principal and basal cells in the epididymis, while in orchidectomized animals GJA1 is also present between principal cells. This effect is inhibited by the administration of testosterone to orchidectomized rats. In wild boar, administration of flutamide, an anti-androgen, during gestation, decreases Gja1 mRNA levels in the cauda epididymidis (Hejmej and Bilinska, 2018 , Lydka, et al., 2011 ). This change is maintained in adult wild boars and is associated with decreased AR levels. Other studies have reported that Gjb3 mRNA levels are decreased in orchidectomized rats and are restored with DHT implants(Chauvin and Griswold, 2004 , Hamzeh and Robaire, 2010 ). Furthermore, in the human epididymis, the phosphorylation of GJA1 is also regulated by epidermal growth factor (EGF)(Dube, et al., 2012 ). There is no information on the mechanisms that regulate the switch from Gjb2 expression during development of the epididymis, during which tall columnar cells develop into principal cells expressing GJB1, GJB4 and GJB5(Dufresne, Finnson, Gregory and Cyr, 2003 , Dufresne, et al., 2022 ). Identifying the mechanisms regulating Cxs during differentiation opens the door to multiple factors and signaling pathways. Indeed, the epididymis is a regionalized and complex organ, with gene and protein expression profiles specific to the epididymal segment and age of animals being studied (Avram, et al., 2004 , Dufresne, Finnson, Gregory and Cyr, 2003 , Henderson, et al., 2006 , Hsia and Cornwall, 2004 , Kirchhoff, 1999 ). The hypothesis of the present study is that steroid hormones are implicated in a common mechanism of regulation of the expression of different Cxs in the epididymis. The present objectives are to identify the mechanisms responsible for the decrease in Gjb2 and the increase in other Cxs during postnatal development of the rat epididymis. Materials and Methods Cell culture and treatment RCE-1 cells Rat epididymal principal cells (RCE-1) (Dufresne et al. 2005) grown in Dulbecco-modified Eagle medium/Ham nutrient mixture F12 (DMEM/HAM F12, Sigma-Aldrich, Oakville, ON, Canada) supplemented with 5% fetal calf serum (FBS), 2 mM L-glutamine, 10 µg/ml insulin, 10 µg/ml transferrin, 80 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml cAMP, 50 U/ml penicillin, 50 µg/ml streptomycin and 5 nM testosterone at 32°C in an incubator containing 5% CO 2 . The cells were grown in 6-well plates coated with collagen IV (BD Biosciences, Mississauga, ON). Treatment with estradiol A stock solution of 17β-estradiol (Sigma-Aldrich) with a concentration of 100 µM was prepared in ethanol. RCE-1 cells were seeded in 24-well collagen-coated plates (5 µg/cm 2 ; BD Biosciences). After 24 h, the cells were rinsed with PBS, and the medium replaced with complete medium without phenol red and containing charcoal-stripped serum (Wisent, St-Bruno, QC, Canada) for 48 h. The cells were then exposed to ethanol (control condition), phenol red or estradiol (10 and 100 nM) for 48 h. The medium was changed at 24 hours. Dexamethasone treatment Stock solutions of dexamethasone (Sigma-Aldrich) and hydrocortisone (Sigma-Aldrich) of 100 µM were prepared in ethanol. The cells were seeded on 24-well plates coated with collagen IV (5 µg/cm 2 ; BD Biosciences). After 24 h, the cells were rinsed in PBS and the medium was replaced with complete medium without hydrocortisone and containing stripped serum (Wisent) for 48 h. The cells were then exposed to ethanol (control), hydrocortisone (80 ng/ml) or dexamethasone (10 and 100 nM) for 48 h. The medium was changed at 24 h. LNCaP cells Human LNCaP prostate carcinoma cells (Horoszewicz, et al., 1980 ) were cultured in RPMI medium (R7509, Sigma-Aldrich) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES and 10U/ml Penicillin-Streptomycin (Sigma-Aldrich). The cells were cultured in flasks of 75 cm 2 at 37 ° C with a change of medium every two days. A 100 nM stock solution of 5α-dihydrotestosterone (DHT; Sigma-Aldrich) was prepared in ethanol. For DHT exposure, cells were seeded in 12-well plates in complete medium. After 24 h, the medium was changed to complete medium containing 5% stripped serum (Wisent). After 24 h, the cells were exposed to ethanol (control condition) or 10 nM DHT dissolved in ethanol for 24 h. RNA interference Small interfering RNAs (siRNAs) against Gjb2 (15 nM; Qiagen, Toronto, ON, Canada) and a control nonsense RNA (scramble, 15 nM; Qiagen) were transfected into RCE-1 cells using the Hiperfect transfection agent and following the manufacturer's instructions (Qiagen). The cells were seeded on 24-well plates and transfected 24 hours later with siRNAs. The cells were incubated for 48 h with siRNAs. The cells were then lysed and total RNA was extracted using a NucleoSpin RNA extraction kit (Macherey-Nagel, Bethlehem, PA) according to the manufacturer's instructions. An aliquot of 500 ng of RNA was denatured at 65°C for 10 min and cooled on ice for 5 min. The RNA was subsequently reversed-transcribed into cDNA using oligo dT primers (R&D System Inc., Minneapolis, MN) and MMLV reverse transcriptase (Sigma-Aldrich) for 1 h at 42°C. Levels of Gjb1, Gjb2 , Gja1, Gjb4, Gjb5 and Gapdh were quantified by RT-PCR using the primers listed in Supplementary Table 2. The products were then analyzed on an agarose gel (GelDoc Imaging system, Bio-Rad Laboratories), excised, purified with the ZymoClean Gel DNA recovery kit (Zymo Research, Irvine, CA) and sequenced (Genome Québec Innovation Center, McGill University, Montreal, QC, Canada). RT-qPCR At the end of treatment, cells were lysed and the total RNA was extracted using the NucleoSpin RNA extraction kit (Macherey-Nagel) according to the manufacturer's instructions. A 400ng aliquot of total RNA was transcribed into cDNA using the qScript cDNA superMix kit (Quanta Biosciences, Gaithersburg, MD). The levels of G jb 2, G jb 1, Nfkbia , Hspb8 , and Gapdh in RCE1 cells were determined by qPCR using gene specific primers (Supplemental Table 2). Levels of human G jb 2, G jb 1, Yy1 , Igf1 and Gapdh in LnCap cells were also determined using qPCR (Supplemental Table 3). qPCR analyses were done using a 2 µ1 aliquot of cDNA in 15 µl of PerfeCTa SYBER Green Supermix (Quanta Biosciences) and 0.3 µM of each forward and reverse primer. The DNA was amplified by denaturation at 94°C for 5 min followed by 40 cycles at 94°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The products were analyzed on an agarose gel, excised, purified (ZymoClean Gel DNA recovery kit; Zymo Research) and sequenced (Genome Québec). Data were normalized to Gapdh and relative delta-delta Ct used to express relative transcript levels. Animals Adult male Sprague Dawley rats (350-400g) were obtained from Charles River Laboratories (St-Constant, QC, Canada). The animals were kept under a photoperiod of 12 hours of light and 12 hours of darkness with food and water ad libitum . Orchidectomy experiments were performed as previously described (Turmel, et al., 2011 ). The rats were anaesthetized with intraperitoneal injection of ketamine/xylazine/acepromazine (50/5/2.5 mg/kg) and received a subcutaneous injection of an analgesic (buprenorphine; 0.3 mg/kg). Polydimethylsiloxane implants were prepared with testosterone as previously described (Stratton, et al., 1973 ) and whose diffusion properties are known (Brawer, et al., 1983 ). Subcutaneous implants containing no testosterone or 3 implants of 6.2 cm of testosterone which mimic normal epididymal testosterone levels were surgically placed on the back of the animals. Sham-operated animals (n = 4) were used as controls. Seven days following surgery the animals were euthanized with CO 2 and cervical dislocation. The epididymides were removed, divided into three segments (head, body and tail), frozen in liquid nitrogen and stored at -80°C. All animal protocols used in this study were approved by the INRS Institutional Committee for Animal Care. Western blot Total proteins from the epididymides and ventral prostates of orchidectomized and control rats were extracted as previously described (Turmel et al. (Turmel, Dufresne, Hermo, Smith, Penuela, Laird and Cyr, 2011 ). Briefly, the tissues were ground in liquid nitrogen and homogenized in 3 ml/g of cold RIPA lysis buffer (1X PBS; 1% Igepal CA-630; 0.5% sodium deoxycholate; 0.1% SDS; 10 mg/ml phenylmethylsulfonyl fluoride (PMSF); 100 mM sodium orthovanadate) supplemented with a cocktail of protease inhibitors (Sigma–Aldrich). The samples were placed on ice for 30 min and centrifuged at 10000 x g at 4°C for 10 min to dispose of cellular debris. The supernatants were collected and protein concentrations determined using the Pierce BCA Protein Assay kit (ThermoFisher, Ottawa, ON). Samples were stored at -80° until used for Western blots. Proteins (50 µg) were thawed on ice, diluted in Laemmli buffer, and heated for 5 min at 94°C. The proteins were then separated on a 12% polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane using a Transblot apparatus (Bio-Rad Laboratories, Mississauga, ON). The membranes were blocked for 1 h with 5% non-fat milk dissolved in TBS (Tris-buffered saline) containing 0.1% Tween-20 (TBST) at room temperature. The membranes were then incubated at 4°C overnight (18 hours) with gentle rocking with either an anti-GJB2 mouse antibody (see Supplemental Table 1), a rabbit anti-GJB1 antibody (see Supplemental Table 1), an anti-GJB4 rabbit antibody (see Supplemental Table 1) or an anti-GJB5 mouse monoclonal antibody (see Supplemental Table 1). All primary antibodies were appropriately diluted in the blocking solution. Overnight incubation with primary antibodies was followed by a series of washes in TBST. The membranes were then incubated for 1 hr at room temperature with a secondary antibody conjugated to horseradish peroxidase (HRP). Depending on the primary antibody, a goat anti-rabbit antibody (see Supplemental Table 1) or a goat anti-mouse antibody (see Supplemental Table 1) was used. Protein bands for each connexin were revealed by the addition of Clarity Western ECL (Bio-Rad Laboratories) substrate and analyzed using the ChemiDoc MP (Bio-Rad Laboratories) imaging system. The membranes were stripped twice for 10 min in stripping solution (0.1 M glycine, 20 mM magnesium acetate, 50 mM KCl, pH 2.2), rinsed with TBST, blocked as described above and subsequently incubated with a rabbit anti-Tubulin antibody (Supplemental Table 1) for 1 hr at room temperature. Tubulin was used to normalize protein levels on the membrane. A secondary goat anti-rabbit antibody was used (Supplemental Table 1) to reveal the tubulin band as described above. Immunofluorescence of Epididymal Sections Epididymides from sham-operated control and orchidectomized rats were frozen in OCT compound (Fisher Scientific, Ottawa, ON) on dry ice and stored at -86˚C until sectioning. Frozen sections (10µm) were fixed in ice-cold methanol for 15 min at -20˚C. Sections were rehydrated and washed 3 times in cold Tris-buffered saline with TBS-T, pH 7.4, plus glycine (0.3M) at room temperature for 10 min each wash. Sections were then incubated with blocking solution (TBS-T + glycine (0.3 M) plus 5% goat serum and 1% bovine serum albumin (BSA) for 60 min at room temperature in a humidified chamber. After rinsing in TBS-T + glycine solution, sections were incubated with either a rabbit polyclonal anti-GJB1 or GJB2 antibody (Supplemental Table 1) diluted in blocking solution at room temperature for 2 hr. Sections were washed three times with TBS-T and subsequently incubated with a goat anti-rabbit IgG-Alexa Fluor 488 (Supplemental Table 1) conjugated secondary antibody (2µg/mL) at room temperature for 45 min in blocking buffer containing a Hoechst blue dye (1µg/mL, Biotium, Hayward, CA). Finally, sections were washed twice with TBS-T and once with TBS and mounted with Fluoromount (Southern Biotech, Birmingham, AL). Immunofluorescence was examined under a Zeiss LSM 780 confocal microscope. Images were processed using the Zen software (Oberkochen, Germany). Rapid amplification of cDNA Ends (RACE) The 5' region of Gjb1 transcript was amplified as described in Adam et al . (Adam and Cyr, 2016 ) using the primers shown in Supplemental Table 4. Briefly, total RNA was extracted from 56-day-old rat epididymis using the Illustra RNAspin Mini commercial kit according to the manufacturer's instructions (GE Healthcare, Montréal, QC). The 5' region of the Gjb1 transcript was amplified using the FirstChoice RLM-RACE kit (RNA Ligase Mediated Rapid Amplification of cDNA Ends, Ambion, Austin, TX), following the manufacturer's instructions. PCR amplifications were performed using 94°C for 5 min, 35 cycles at 94°C for 30 sec, melting temperature (Tm) for 30 sec, and 72°C for 30 sec cycles. The products were analyzed on a 2% agarose gel containing ethidium bromide. The bands were excised, purified (ZymoClean Gel DNA recovery kit) and sequenced (Genome Québec). Cloning of Gjb1 and Gjb2 promoters Total genomic DNA used to clone the promoters was extracted from livers of adult Sprague Dawley rats using the GenElute Mammalian Genomic DNA Purification (Sigma-Aldrich) kit and following the manufacturer's instructions. Primers used to amplify Gjb1 and Gjb2 promoters are shown in Supplementary Table 4. Gjb1 promoter A 1650 bp fragment from the 5' region of the P1 promoter and a 1173 bp fragment of the P2 promoter were amplified by PCR (5 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at TA and 3 min at 72°C). PCR products were visualized on a 0.7% agarose gel containing ethidium bromide and bands of interest were excised and purified (ZymoClean Gel DNA recovery kit). The fragments of the P1 and P2 promoters were inserted directionally into the pGL3-Basic vector (Promega, Madison WI) upstream of the luciferase gene. The restriction sites SacI-NheI and XhoI-NheI were used for P1 and P2, respectively. After transfection of TOP10 (Invitrogen) chemically competent bacteria, plasmids were purified (Plasmid Midi kit, Qiagen), and sequenced (Genome Québec). Cloning of the 5162 bp of the Gjb1 P1 promoter was performed in two stages. First a 2916 bp fragment of the Gjb1 promoter (-2052/+64 bp start of exon 1) was amplified using the primers indicated in Supplementary Table 1 (P2 cloning (3 kb) F and R). The resulting amplicon was separated on a 0.7% agarose gel and purified as described above. The DNA was incubated with T4 DNA polymerase and nucleotides to generate blunt ends and digested with the KpnI (New England Biolabs) restriction enzyme. The remaining 2759 bp fragment was inserted into the pGL3-Basic plasmid previously digested with KpnI and SmaI using ligase (New England Biolabs). The resulting − 2761/+64 construct was used to transform TOP10 competent bacteria. The resulting clones were analyzed and verified by enzymatic digestion. The − 2761/+64 construct was purified as described above and its identity confirmed by sequencing. A second PCR amplification yielded a 2510 bp fragment of the Gjb1 promoter located from − 5162 to -2652 bp relative to the start of exon 1. This amplification was carried using primers described in Supplemental Table 1 (P2 cloning (5 kb) F and R) and purified as described above. The − 2761/+64 construct was digested by KpnI (New England Biolabs) purified and ligated to the 2510 bp fragment to generate a -5076/+64 bp construct. This final construct was used to transform TOP10 bacteria as described above. The clones were analyzed by digestion and sequenced (Génome Québec). The expression of the transgene was confirmed using both hepatic MHC1 and RCE-1 cells (Supplemental Fig. 1). Gjb2 promoter A fragment of 3036 bp (-4407 to -1371) relative to the transcription initiation site of the 5' region of the Gjb2 promoter was amplified by PCR (5 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at annealing temperature (Tm, Table 4), and 3 min at 72°C). Primers used (Promoter (5 kb) F and R) are shown in Supplementary Table 4. The PCR product was analyzed on a 0.7% agarose gel containing ethidium bromide. The DNA band of interest was excised and purified using the ZymoClean Gel DNA recovery kit (Zymo Research). The Gjb2 promoter (position − 1564/+133 relative to the translational start site) was ligated into MluI and Bmg1 sites of the pGL3-Basic plasmid upstream of the luciferase gene. The construct was used to transform TOP10 bacteria as described above. The clones obtained were analyzed by digestion and sequenced (Génome Québec). Statistical analysis All statistical tests were performed using GraphPad Prism (GraphPad Software, San Diego, CA). One-factor analysis of variance (ANOVA) tests followed by the Newman-Keuls test or Student tests (T-test) were used to analyze the data. A value of p < 0.05 was considered significant. Results Effects of the decrease in Gjb2 on the expression of other Cxs Our first objective was to assess whether the previously observed decrease in Gjb2 during postnatal development (Dufresne, Finnson, Gregory and Cyr, 2003 ) could explain the variation in the expression of other Cxs in the epididymis. Using RCE-1 cells and an siRNA directed against Gjb2 (Fig. 1 A) we demonstrated that Gjb2 mRNA levels were decreased by approximately 60% (P < 0.0001) in comparison to a scrambled siRNA sequence (scramble, Fig. 1 B). mRNA expression levels for Gjb1, Gjb4, Gjb5 and Gja1 were not affected by the decrease in Gjb2 (Fig. 1 A, C). Similar results were observed 6 days after transfection with siRNA against Gjb2 . Identification of common and regulatory regions of Cxs promoters In order to evaluate the presence of common mechanism(s)/transcription factors regulating the expression of epididymal Cxs during postnatal development, we analyzed and compared the promoter sequences of the different Cxs ( Gjb1, Gjb2, Gjb4, Gjb5 and Gja1) expressed in the epididymis using BLAST Software to identify common response elements. No conserved regions were identified between the different Cxs. The promoter sequences of Gjb2 and Gja1 were also compared to examine whether or not a homologous sequence could explain the decrease in their expression after day 28 in rats (Dufresne, Finnson, Gregory and Cyr, 2003 ). This same rationale was applied to the promoter sequences of Gjb1 , Gjb4 and Gjb5 , which were compared to identify if there was any homologous sequence which would explain the increase in the expression of these Cxs during differentiation. No homologous regions were observed between the different Cxs. Analysis of the 5kb promoter region of the Gjb2 promoter and Gjb1 (Supplemental Figs. 2 and 3) revealed several response elements of the androgen, glucocorticoid and estrogen (ESR1) receptors, suggesting a role of these steroid hormones on the expression of these Cxs. Note that these response elements were also identified on the promoter region of Gjb4 , Gjb5 and Gja1 using sequence analysis software. Role of glucocorticoids and estradiol on Gjb2 expression Our analysis of the Gjb2 and Gjb1 promoter sequences allowed us to identify glucocorticoid and estrogen response elements on the DNA sequences of both Cxs. Our third objective was to assess whether glucocorticoids and/or estradiol were involved in the variation of Cxs expression during postnatal epididymis development. To do this, we first determined whether these hormones were involved in the decrease in Gjb2 expression observed during development (Dufresne, Finnson, Gregory and Cyr, 2003 ). RCE-1 cells were exposed for 48 hrs to hydrocortisone (80 ng/ml) and dexamethasone (10 and 100 nM) Gjb 2 mRNA levels were increased with exposure to hydrocortisone and dexamethasone (Fig. 2 A). The response of RCE-1 cells to glucocorticoids was assessed by measuring levels of Nfkbia , a glucocorticoid-dependent gene (Silva et al. (Silva, Queiroz, Honda and Avellar, 2010 ). A small but significant increase in Nfkbia mRNA levels was observed in RCE-1 cells exposed to either hydrocortisone or dexamethasone (Fig. 2 B). To assess the role of estrogens in regulating Gjb2 expression in the epididymis, RCE-1 cells were treated with estradiol (10 and 100 nM) for 48 hrs. There was a slight but not significant increase in the expression of Gjb2 mRNA levels at the two doses (Fig. 2 C). The estrogenic activity of phenol red (Berthois, et al., 1986 ) was also evaluated and no differences in the expression of Gjb2 were noted. The response of RCE-1 cells to estradiol was assessed by measuring the gene Hspb8 (Yang, et al., 2006 ) which displays a dose-dependent response to estradiol (Fig. 3 D). Role of androgens on Cxs expression in the epididymis To assess the role of androgens on the expression of Cxs in the epididymis, an in vivo approach was used, in which rats were orchidectomized or orchidectomized and given testosterone implants to mimic endogenous epididymal levels of testosterone. Controls were sham-operated. Western blot analysis of protein isolated from the caput, corpus and cauda epididymidis indicated that GJB2 protein levels in each region of the epididymis were significantly increased in the caput and corpus of orchidectomized animals, while testosterone maintained levels of GJB2 comparable to those of control animals. A similar pattern was observed in the cauda, but levels were not significantly different (Fig. 3 ). In contrast, GJB1, and GJB4 protein levels were decreased as a result of orchidectomy but were increased in rats orchidectomized and with testosterone implants. GJB5 levels in the caput and corpus epididymidis were not altered by orchidectomy alone but were increased in orchidectomized rats with testosterone implants. In all cases there were no significant effects in the cauda epididymidis. Immunolocalization of GJB2 revealed that GJB2 was localized primarily at the base of the epithelium between basal cells (Fig. 4 A). In orchidectomized rats, immunostaining intensity was increased in the caput, corpus and cauda epididymidis. Some staining was also observed over the sperm in the lumen of control rats, although this appears to be non-specific. Unlike GJB2, which was absent in controls, GJB1 was present along the lateral plasma membrane of principal cells. Punctate staining was evident in all regions of the epididymis and could be seen in stacked images (Fig. 4 B). In orchidectomized rats, GJB2 staining was largely absent or weakly expressed. There was very little staining observed between principal cells, although some weak staining could be observed at the base of the epithelium. Stacked images of the caput epididymis further demonstrate the lack of GJB2 staining in the epithelium. Role of androgens on Cxs expression in the ventral prostate To determine if the effects of androgens on Cxs in the epididymis were epididymis-specific or represent a generalized tissue response, the regulation of each Cx was assessed in the ventral prostate, which, like the epididymis, is known to be an androgen-dependent tissue. Our results indicated that GJB2 protein levels increased in the ventral prostate of orchidectomized rats (not shown), and this increase was abrogated by androgens. Levels of GJB1, GJB4 and GJB5 also displayed lower protein levels in orchidectomized rats compared to controls, while testosterone implants in orchidectomized rats increased levels of each Cx, suggesting a comparable response to that observed in the epididymis. Role of androgens on Gjb1 and Gjb2 expression in LNCaP cells Androgen regulation of Cxs was further assessed in vitro using a well-characterized androgen-responsive human prostate cancer cell line, LNCaP cells (Horoszewicz, et al., 1983 ). LNCAP cells were cultured in the presence of 10 nM DHT. Gjb2 mRNA levels were decreased by about 35% in cells exposed to DHT (Fig. 5 A). A previous study showed that the Igf1 gene is a sensitive marker of androgen response (Wu, et al., 2007 ). Our data showed that Igf1 mRNA levels were increased in LNCaP cells exposed to DHT in each set of experiments to assess the effects on androgens on LNCaP cells (Fig. 5 A, B, C). No differences were observed in Gapdh mRNA levels with androgen treatment. Gjb1 levels were also significantly increased in LNCaP cells exposed to DHT (Fig. 5 B). The transcription factor YY1, which has been described as an activator of Gjb1 (Field, et al., 2003 , Piechocki, et al., 2000 ) and which can interact with the androgen receptor to regulate its transcriptional activity (Deng, et al., 2009 ) was not affected by DHT treatment (Fig. 5 C). Effects on androgens on the transactivation of the Gjb2 and Gjb1 promoters Activity of the Gjb2 promoter Analysis of the Gjb2 promoter (Supplemental Fig. 2) identified several androgen response elements. In order to determine if androgens regulate the transcription of the Gjb2 gene, a construct containing 1.5 kb of the Gjb2 rat promoter (Adam and Cyr, 2016 ) was transfected into LNCaP cells in the presence or absence of DHT (Fig. 6 ). The construction shows luciferase activity 9 times higher than that of the empty plasmid pGL3, indicating that the cells were able to transcribe the transgene. No differences in luciferase activity were observed in cells exposed to DHT as compared to control (Fig. 6 A). Several androgen response sites have been identified upstream of 1.5 kb of the Gjb2 promoter. To assess the role of these regions, a 5 kb fragment of the Gjb2 promoter was amplified and cloned. These constructs were then transfected into LNCaP cells with or without exposure to DHT (Fig. 6 B). Activity of Gjb1 promoter P1 under the influence of androgens In order to evaluate the role of androgens in the transcription of the Gjb1 promoter, a 5 kb fragment of the Gjb1 promoter was amplified and cloned into the PGL3 plasmid. The efficacy of the construct was determined using rat hepatic MH1C1 cells (not shown). The 5 kb construct containing the Gjb1 promoter was transfected into LNCaP cells with and without DHT (Fig. 6 C). Our data indicate that the activity of the Gjb1 promoter was markedly increased with exposure to DHT. Discussion The mechanisms regulating the expression of Cxs during epididymal development remain poorly understood. It has been shown that Gjb2 and Gja1 mRNA levels are decreased concomitant with increased Gjb1 , Gjb4 and Gjb5 mRNA levels (Dufresne, Finnson, Gregory and Cyr, 2003 ) during postnatal development. While mRNA levels of Gja1 do not change as dramatically as those of Gjb2 , the expression of Gja1 becomes associated primarily with basal cells during development (Dufresne, Finnson, Gregory and Cyr, 2003 ). In the present study, we showed that the decrease in Gjb2 alone does not appear to be responsible for the variation in expression of other Cxs in the epididymis. Studies in the liver, pancreas, epidermis, and mammary gland have shown that decreased expression of specific Cxs can result in changes in the expression of other Cxs (Chanson, Fanjul, Bosco, Nelles, Suter, Willecke and Meda, 1998, Langlois, et al., 2007 , Nelles, Butzler, Jung, Temme, Gabriel, Dahl, Traub, Stumpel, Jungermann, Zielasek, Toyka, Dermietzel and Willecke, 1996, Stewart, Plante, Bechberger, Naus and Laird, 2014 ). In addition, specific removal of Gjb2 in the mammary gland and in the cochlea caused a developmental delay in Gjb6 (Cx30) expression (Crispino, Di Pasquale, Scimemi, Rodriguez, Galindo Ramirez, De Siati, Santarelli, Arslan, Bortolozzi, Chiorini and Mammano, 2011, Stewart, et al., 2015 ). However, although we observed a 60% decrease in Gjb2 mRNA levels using siRNA, we observed no difference in the expression of Gjb1, Gjb4 , Gjb5 and Gja1 in RCE-1 cells, suggesting tissue-specific differences in either the regulation or interplay of multiple connexins. Analysis of the Gjb2 promoter revealed several response elements to estradiol, glucocorticoids and androgens. We evaluated the role of each of these hormones to determine if these were involved in the repression of Gjb2 expression. In RCE-1 cells treated with either hydrocortisone or dexamethasone, an increase in GJB2 levels was noted. Glucocorticoids have been implicated in the development of the male reproductive tract by allowing the establishment and maintenance of spermatogenesis (Saxena and Paul, 1987 , Weber, et al., 2000 ). The glucocorticoid receptor (GR) has been shown to be present in principal cells of the epididymis (Gladstones, Burton, Mark, Waddell and Roberts, 2012 , Silva, Queiroz, Honda and Avellar, 2010 ). Its expression is more intense in early epididymal development and during differentiation (Gladstones, Burton, Mark, Waddell and Roberts, 2012 ). We have previously shown that the transcription factors SP1 and TFAP2A bind to the Gjb2 promoter to activate its transcription during the early stages of epididymal differentiation (Adam and Cyr, 2016 ). Given that we have observed an increase in Gjb2 with glucocorticoids, it is possible that they may act indirectly to increase Gjb2 expression by influencing SP1 and TFAP2A binding during the early stages of development (Silva, Queiroz, Honda and Avellar, 2010 ). Our data indicate that estradiol had no significant effects on Gjb2 mRNA levels in vitro . Studies have reported that injection of estradiol benzoate in rats at seven days of age decreases Gjb2 expression in the corpus epididymidis at high doses (Lee, 2015b ). In addition, the effects of estradiol benzoate on Cxs expression differ between the initial segment and the body of the epididymis (Lee, 2014 , Lee, 2015a , Lee, 2015b ). This suggests that the effects of estrogen on Cxs are modulated by dose and epididymal region. Our results support the notion that estrogen is not directly involved in the decreased Gjb2 expression observed during postnatal epididymal development. Androgens are major regulators of the development and maintenance of epididymal functions. Removal of androgens alters the structure of the tubule and causes a wave of apoptosis (Fan and Robaire, 1998 ). The present results support the notion that androgens modulate the expression of Cxs in the epididymis. We observed that GJB2 levels were significantly increased in the caput and corpus epididymidis following orchidectomy and decreased in orchidectomized animals treated with testosterone. In addition, we observed a decrease in protein levels of GJB1 and GJB4 in the caput and corpus epididymidis of orchidectomized rats, an effect that was mitigated by testosterone. These results are consistent with the previously reported variations in Cxs expression in the epididymis during development, when testosterone levels begin to increase during postnatal development (Dufresne, Finnson, Gregory and Cyr, 2003 ). Furthermore, the lack of effect observed in the cauda epididymidis is consistent with previous data that showed that the regulation of Cxs in the cauda was different from that in the proximal regions of the epididymis (Dufresne, Finnson, Gregory and Cyr, 2003 ). It has also been shown that the levels, localization and phosphorylation of GJA1 are androgen-dependent in the initial segment of the epididymis (Cyr, Hermo and Laird, 1996 ). Similarly, in wild boar, neonatal exposure to flutamide decreased GJA1 protein levels in the tail of the epididymis but not in other regions (Lydka, Kopera-Sobota, Kotula-Balak, Chojnacka, Zak and Bilinska, 2011 ). In the rat, Pannexin 1 (Panx1), a protein related to connexins and involved in the formation of transmembrane channels, was increased following orchidectomy in the caput and corpus epididymidis, but not in the cauda (Turmel, Dufresne, Hermo, Smith, Penuela, Laird and Cyr, 2011 ). Immunofluorescence of GJB2 and GJB1in rat epididymis tissue sections supported the data observed by western blot, in which GJB2 appeared to slightly increase in orchidectomized rats, while GJB1 immunostaining was decreased in orchidectomized rats relative to controls. The data support the effects of androgens as exerting an inhibition of GJB2 gene expression in the epididymis while promoting the expression of GJB1. The data also support previous reports that changes in the expression of different connexins during development are regulated by testicular factors, including androgens (Dufresne, Finnson, Gregory and Cyr, 2003 ). Several Cxs, including GJA1, GJB1 and GJB2 (Czyz, et al., 2012 , Huynh, et al., 2001 , Li, et al., 2015 , Meda, et al., 1993 , Mehta, et al., 1996 , Tate, et al., 2006 ) are expressed in the rat prostate, an organ known to be highly regulated by androgens (Cleutjens, et al., 1996 , Heyns, 1990 , Huynh, Alpert, Laird, Batist, Chalifour and Alaoui-Jamali, 2001 , Robaire, 1979 , Yamashita, 2004 ). As no data regarding on the presence of GJB4 and GJB5 in the prostate were available in the literature, western blot experiments were performed using the prostates of rats from the different in vivo experimental groups. We identified the presence of GJB4 and GJB5 in the prostate of control (sham-operated) rats. For all Cxs with the exception of GJB5, we observed a similar regulation in the prostate to that observed in the epididymis following orchidectomy plus androgen maintenance. A previous study reported an increase in mRNA and GJA1 protein levels in the prostate after orchidectomy, and these effects were inhibited with the maintenance of androgen levels (Huynh, Alpert, Laird, Batist, Chalifour and Alaoui-Jamali, 2001 ). However, in that study, no changes were observed on Gjb1 mRNA levels. Another team investigating the effect of flutamide, an anti-androgen, showed that flutamide injection during development decreased androgen receptor levels and GJA1 expression in the prostate (Hejmej, et al., 2013 ). The androgen receptor has also been reported to be involved in decreasing GJA1 expression in prostate cancer cells (Chen, et al., 2015 ). It is interesting to note that while GJB5 is regulated by androgens in the prostate, it does not appear to be similarly regulated in the epididymis. It has been shown that GJB1, GJB3, GJB4 and GJB5 are also expressed by epididymal basal cells (Mandon, et al., 2015 ). It is possible that in the epididymis, basal cell-specific factors maintain GJB5 expression after orchidectomy. Our results, as well as those of previous studies, suggest that androgens regulate the expression of some Cxs in both the epididymis and prostate, and suggest a common regulatory mechanism for some, but not all Cx genes. To understand the mechanisms that are involved in the action of androgens on Cxs expression, we used the LNCaP cell line, a widely used cellular model to study the effect of androgens (Horoszewicz, Leong, Kawinski, Karr, Rosenthal, Chu, Mirand and Murphy, 1983) et al. 1983). Our results support in vivo observations in both the epididymis and ventral prostate, in which Gjb2 mRNA levels were decreased while Gjb1 mRNA levels were increased. These results occurred independent of changes in the YY1 transcription factor, which stimulates the expression of Gjb1 in hepatocytes (Field, Tate, Chipman and Minchin, 2003 ). YY1 can act as a repressor or activator, depending on which cofactors it recruits (Shi, et al., 1997 ). Our results suggest that YY1 is not involved in the androgenic response of LNCap cells and the regulation of GJb2 and GJb1 . Using both a 1.6 and 5kb sequence of the Gjb2 promoter, our data suggest that DHT does not directly regulate the transcription of GJB2. We previously reported that both SP1 and TFAP2 transcription factors regulate minimal promoter activity of Gjb2 in the epididymis (Adam and Cyr, 2016 ). Our data suggests that androgen-dependent factors may be implicated in the down-regulation of Gjb2 and that androgens are involved in decreasing Gjb2 expression. We have recently shown that factors SP1 and TFAP2A bind to the Gjb2 promoter to activate its transcription during the early stages of epididymal differentiation (Adam and Cyr, 2016 ). Knowing that SP1 and AR factors are able to form a complex to regulate the expression of the NRIP gene in the prostate (Chen, et al., 2008 ), AR may interact with SP1 to decrease Gjb2 expression. Thus, by its binding to SP1, AR is able to bind to DNA via an SP1 site and not an androgen-response element (ARE) site in prostate cells (Chen, Tsao, Wang and Chen, 2008 , Eisermann, et al., 2013 ). In addition, TFAP2A has been shown to be the major co-regulator of the AR and facilitates the binding of AR to chromatin in the epididymis, suggesting a central role for TFAP2A in the response to androgens in this tissue (Pihlajamaa, et al., 2014 ). These studies, in conjunction with our results, suggest that AR may interact with SP1 and TFAP2A factors and/or their binding sites located on the Gjb2 promoter to decrease its expression. The Gjb1 gene can be transcribed from two tissue-dependent promoters (Neuhaus, et al., 1996 , Neuhaus, et al., 1995 ). The promoter P1 is used in the pancreas and liver (Neuhaus, Bone, Wang, Ionasescu and Werner, 1996 , Neuhaus, Dahl and Werner, 1995 ) and the P2 promoter is active in nerve fibers, allowing transcription of shorter mRNA(Neuhaus, Bone, Wang, Ionasescu and Werner, 1996 ). Our data reveal that the promoter P1 is used for the transcription of Gjb1 in the rat epididymis. The results of the luciferase assays show an increase in the activity of the Gjb1 promoter in cells exposed to DHT. These data are the result of preliminary experiments and have yet to be confirmed. They do suggest, however, that at least some of the elements regulating androgen responses are present within the 5 kb of the Gjb1 promoter. In conclusion, we have provided evidence from both in vivo and in vitro studies which suggest that the switch in expression of Cxs during epididymal development is regulated by androgens, which exerts opposing regulatory effects on the regulation of GJB2 and other Cxs. We have also provided preliminary data indicating that the activity of the P1 promoter in G jb1 expression is influenced, if not regulated in part, by androgens, and that this may also be the case for the P2 promoter. Knowing that androgens can activate and influence a multitude of signaling pathways and induce various cellular responses, further studies are needed to elucidate the role(s) and mode(s) of action of androgens in regulating Cxs expression in the epididymis. Declarations Data Availability : Data are available from the corresponding author. Summary Statement : The expression of connexins during epididymal development is regulated by androgens which inhibit the expression of Gjb2 and Gja1 in principal cells and stimulate the expression of Gjb1 , Gjb4 and Gjb5 . References Adam C, Cyr DG (2016) Role of specificity protein-1 and activating protein-2 transcription factors in the regulation of the gap junction protein beta-2 gene in the epididymis of the rat. 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List of antibodies used in the study. supplFig1b.jpg Transactivation of the luciferase reporter gene using the GJB1 promoter sequence in rat epididymal RCE1 principal cell line. The P1 promoter (1.6kb) and P2 promoter (5kb) constructs were used to transfect RCE1 cells using lipofectamine 2000 as described in the Material and Methods. Co-transfection with a phRL-TK plasmid containing a Renilla reporter gene was used to assess transfection efficiency. Each experiment was done in triplicate. Significance was assessed by ANOVA (** P< 0.05). SupplFig2b.jpg Gjb2 promoter sequence analysis. The 5kb region upstream of the Gjb2 promoter's initiation site was analyzed using Transfac-TESS, Alibaba2 and TF-search software. Several elements of response to glucocorticoids (yellow), estrogen (blue) and androgens (bold underlined) have been identified. The beginning of exon 1 is indicated in purple. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4731767","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326282880,"identity":"e91163d2-39f5-407b-b4a4-4cd4064f21ac","order_by":0,"name":"Daniel Cyr","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIie2PoQ7CMBRFX7Jkqmy2M4xPeAuWj+kHDAIOteAwJNjxF/sARJcKTGF2CWYJCWpiBINA0G5oOhwJPam44p3cWwCL5Rfh+s11cjj/QkGdXNZfgU4h2M/wDsecNwghnuVNLPYJ+GtDWSBnLE8Rouw0zUR6FUAl+6wgj1GoSQzlIBOE63+ZlKJG8WwVUiklAfQrg1KqFugUUIoDSA0tQVljvkEa7aSrFnJBaGlo8Yp43DyWk9CTzuWuhg39raHlDYUR7xLpdd8SrvrfWiwWy5/xAoa0SH3tIUj+AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6566-783X","institution":"INRS-Centre Armand-Frappier Santé Biotechnologie","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Cyr","suffix":""},{"id":326282881,"identity":"efc5edca-095b-45af-8342-7ba33de61f5f","order_by":1,"name":"Cécile Adam","email":"","orcid":"","institution":"INRS-Centre Armand-Frappier Santé Biotechnologie","correspondingAuthor":false,"prefix":"","firstName":"Cécile","middleName":"","lastName":"Adam","suffix":""},{"id":326282882,"identity":"efdbfedd-044f-46bb-be95-ae69a4948816","order_by":2,"name":"Julie Dufresne","email":"","orcid":"","institution":"INRS-Centre Armand-Frappier Santé Biotechnologie","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Dufresne","suffix":""},{"id":326282883,"identity":"d4709d38-b097-4761-9f57-9366baf089a7","order_by":3,"name":"Mary Gregory","email":"","orcid":"","institution":"INRS-Centre Armand-Frappier Santé Biotechnologie","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"","lastName":"Gregory","suffix":""}],"badges":[],"createdAt":"2024-07-12 17:04:49","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4731767/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4731767/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60416680,"identity":"72ee6008-3e58-48c7-a399-167230ea6afb","added_by":"auto","created_at":"2024-07-16 13:59:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":767743,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of decreased expression of \u003cem\u003eGjb2\u003c/em\u003e by siRNA on mRNA levels of other epididymal Cxs.\u0026nbsp; RCE-1 cells were transfected with an siRNA directed against \u003cem\u003eGjb2\u003c/em\u003e for 48 hrs (n=3). The mRNA levels of the different Cxs were evaluated by PCR and normalized by \u003cem\u003eGapdh\u003c/em\u003e. A) Agarose gel showing the mRNA levels of different Cxs expressed in RCE-1. PCR in absence of cDNA was used as a negative control (H\u003csub\u003e2\u003c/sub\u003eO). B) Quantification of \u003cem\u003eGjb2\u003c/em\u003e mRNA levels 48h after treatment with siRNA (n=3). C) Quantification of the mRNA levels for Gjb1, Gjb4, \u003cem\u003eGjb5\u003c/em\u003e and \u003cem\u003eGja1\u003c/em\u003e in RCE-1 cells 48 hrs after treatment with \u003cem\u003eGjb2\u003c/em\u003e siRNA. Data are expressed as the mean ± SEM; each experiment was done in triplicate. Statistical differences were determined by ANOVA. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/950f8e1360170117a8c40af9.png"},{"id":60416676,"identity":"f8982bac-4c94-45cd-ba11-a1300b20d972","added_by":"auto","created_at":"2024-07-16 13:59:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227358,"visible":true,"origin":"","legend":"\u003cp\u003eRole of glucocorticoids and estradiol on \u003cem\u003eGjb2\u003c/em\u003e mRNA levels in RCE-1 cells.\u0026nbsp; RCE-1 cells were exposed to hydrocortisone (80 ng/ml) or dexamethasone (10 and 100 nM) for 48 hrs (n=3) and \u003cem\u003eGjb2\u003c/em\u003e(A) mRNA levels were measured. The glucocorticoid-dependent gene \u003cem\u003eNkfbia\u003c/em\u003e was used as a positive control (B). RCE-1 cells were exposed to phenol red or estradiol (10 and 100 nM) for 48 hours and \u003cem\u003eGjb2\u003c/em\u003e(C) mRNA levels measured (n=3). The estradiol-dependent gene \u003cem\u003eHspb8\u003c/em\u003e was used as a positive control (D). CTRL: control; Hydrocort: hydrocortisone; DEX: dexamethasone; E\u003csub\u003e2\u003c/sub\u003e: estradiol. The data are expressed as the mean ± SEM. Each experiment was done in triplicate. Statistical differences were determined by ANOVA. *P \u0026lt; 0.05; **P \u0026lt; 0.005; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0005.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/61d3e80246668f60fea129e4.png"},{"id":60416675,"identity":"cdeb9d34-047f-44fa-8dc5-c8e6331f60f1","added_by":"auto","created_at":"2024-07-16 13:59:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1125567,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of GJB2, GJB1, GJB4, and GJB5 levels in the rat epididymis. Western blot analysis of different epididymal Cx levels in sham-operated control (CTRL), orchidectomized (ORCH) and orchidectomized rats with testosterone implants (ORCH + T) (n=4 rats per group). Total proteins were isolated in each of the 3 regions of the epididymis (caput, corpus, cauda) 7 days following surgery. Protein levels were normalized using tubulin; data are presented as the mean ±SEM. Statistical differences were determined by ANOVA. *P \u0026lt; 0.05; **P \u0026lt; 0.005; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0005.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/9d6ce737b55b7bc3f2c1da6f.png"},{"id":60416683,"identity":"ea31a5c5-af6e-4f4b-872d-4bf67d8a38e7","added_by":"auto","created_at":"2024-07-16 13:59:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2674699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunolocalization of GJB2 and GJB1 in the epididymis of control and orchidectomized rats\u003c/strong\u003e. Confocal immunofluorescent micrographs of GJB2 (green) and GJB1 (green) in caput, corpus and cauda epididymidis of sham-operated control and orchidectomized rats sampled 7 days after surgery. White arrows indicate immunostaining. GJB1 was localized primarily in the base of the epithelium while GJB2 was present both basally and along the lateral plasma membranes of principal cells. Nuclei are stained blue. P-Principal cells; IT=interstitium; Lu=lumen.\u003c/p\u003e","description":"","filename":"Fig4b.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/715738ef338f94ac0c523f57.jpg"},{"id":60416679,"identity":"7a3fbfa5-b3d7-4b94-81d1-61cd70ff2523","added_by":"auto","created_at":"2024-07-16 13:59:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1059359,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DHT on \u003cem\u003eGjb1,\u003c/em\u003e \u003cem\u003eGjb2\u003c/em\u003e, \u003cem\u003eIgf1\u003c/em\u003e and \u003cem\u003eYy1\u003c/em\u003e mRNA levels in LNCaP cells. LNCaP cells were treated with 10 nM DHT for 24 hrs and total RNA was isolated. Levels of Gjb2 (A; n=6), \u003cem\u003eGjb1\u003c/em\u003e (B; n=4), and \u003cem\u003eYy1\u003c/em\u003e (C; n=3) were quantified by RT-qPCR. \u003cem\u003eIgf1\u003c/em\u003e was quantified by RT-qPCR as a positive control (A-C). The data are expressed as the mean ±SEM; Statistical differences were determined by ANOVA***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005.\u003c/p\u003e","description":"","filename":"Fig5b.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/e414710ae2217cd92578fdcb.jpg"},{"id":60417561,"identity":"b003f968-d645-46cc-8baa-e96e2d3299fd","added_by":"auto","created_at":"2024-07-16 14:07:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1290228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGjb1 and Gjb2\u003c/em\u003e promoter activities in DHT-treated LNCaP cells. \u0026nbsp;LNCaP cells were transfected with a construct containing 1.5 kb (A) or 5 kb (B) of the rat \u003cem\u003eGjb2 \u003c/em\u003epromoter upstream of a luciferase reporter gene. The cells were then exposed with or without to DHT for 24 hrs. Luciferase activity was then measured and the data expressed as the mean ± SEM; (C) LNCaP cells were transfected with a construct containing 5 kb of the \u003cem\u003eGjb1 \u003c/em\u003erat promoter upstream of a luciferase reporter gene. The cells were then exposed to DHT for 24 hrs and luciferase activity was measured. Data are expressed as the mean ± SEM. Each analysis was performed in triplicate.\u003c/p\u003e","description":"","filename":"Fig6b.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/6eafb5c637dfa2ec9a641de6.jpg"},{"id":60418166,"identity":"58d2d9d9-16fc-44de-ac96-d2fc1eec8786","added_by":"auto","created_at":"2024-07-16 14:15:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7336958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/a1c0cfb1-2524-4151-abf2-2e5309b23a44.pdf"},{"id":60416681,"identity":"c379efc0-6202-47d0-a75d-0d55b3a83ce9","added_by":"auto","created_at":"2024-07-16 13:59:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15704,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. List of antibodies used in the study.\u003c/p\u003e","description":"","filename":"TableS1Antibodies.docx","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/546b01bb602fbd27e2abbbc8.docx"},{"id":60416678,"identity":"44f8c4b8-ddfe-4120-91c6-eafbc8214bd4","added_by":"auto","created_at":"2024-07-16 13:59:45","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":769293,"visible":true,"origin":"","legend":"\u003cp\u003eTransactivation of the luciferase reporter gene using the GJB1 promoter sequence in rat epididymal RCE1 principal cell line. The P1 promoter (1.6kb) and P2 promoter (5kb) constructs were used to transfect RCE1 cells using lipofectamine 2000 as described in the Material and Methods. Co-transfection with a phRL-TK plasmid containing a Renilla reporter gene was used to assess transfection efficiency. Each experiment was done in triplicate. Significance was assessed by ANOVA (** P\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"supplFig1b.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/a6b5d1cf2c42f51cc44213ae.jpg"},{"id":60416682,"identity":"7eaeb3c1-00c2-4181-be49-e125456594d5","added_by":"auto","created_at":"2024-07-16 13:59:46","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6537546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGjb2\u003c/em\u003epromoter sequence analysis. The 5kb region upstream of the \u003cem\u003eGjb2\u003c/em\u003e promoter's initiation site was analyzed using Transfac-TESS, Alibaba2 and TF-search software. Several elements of response to glucocorticoids (yellow), estrogen (blue) and androgens (bold underlined) have been identified. The beginning of exon 1 is indicated in purple.\u003c/p\u003e","description":"","filename":"SupplFig2b.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4731767/v1/3fa9f9c52c281299dd730bb6.jpg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eRegulation of the Gap Junction Interplay in the Rat Epididymis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe differentiation of epithelial cells can be associated with changes in the expression of connexins (Cxs) and intercellular gap junctional communication (GJIC) in a variety of tissues, including the testis and epididymis (Cyr, \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e, Kidder and Cyr, \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the epidermis, keratinocyte differentiation is synchronized with the decrease in the expression of gap junction protein B2 (GJB2; connexin26) and gap junction protein A1 (GJA1; connexin43) and a concomitant increase in gap junction protein B3 (GJB3; connexin31) and gap junction protein B4 (GJB4; connexin30.3)(Brissette, et al., \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e). This phenomenon is also observed during the differentiation of mammary gland cells, in which GJB2 levels increase during pregnancy while GJB1 (connexin32) is only expressed during lactation (Locke, et al., \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e). Previous studies from our laboratory have shown that in the rat epididymis, both GJA1 and GJB2 levels decrease while GJB1, GJB4 and GJB5 (connexin31.1) levels increase during postnatal development (Dufresne, et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eChanges in expression of different Cxs may be the result of varying factors, such as hormones, that regulate the expression of different Cxs; however, there are also examples suggesting compensatory regulation of different Cxs. In the mammary gland, inhibition of GJB2 also decreases GJB6 levels (Stewart, et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). A similar correlation between GJB2 and GJB6 was also observed in the cochlea, where removal of GJB2 induced a developmental delay in GJB6 expression (Crispino, et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Transgenic mice lacking G\u003cem\u003ejb\u003c/em\u003e1 in the pancreas (Chanson, et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e) and liver (Nelles, et al., \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e) displayed diminished GJB2 protein levels.. In the epididymis, no information is available on the effect of GJB2 decrease on other Cxs during development.\u003c/p\u003e\n\u003cp\u003eSteroid hormones influence the establishment and maintenance of epididymal functions. Estrogen receptor 1 (ESR1) and ESR2 are present throughout the epididymis and have been shown to play an important role in epididymal development and function (Hess, \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e, Hess, et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The glucocorticoid receptor (NR3C1) is also present in multiple epididymal cell types, including principal, basal, narrow, and apical cells (Gladstones, et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e, Silva, et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Glucocorticoid deprivation has been reported to increase the expression of the androgen receptor in the cauda epididymidis (Silva, Queiroz, Honda and Avellar, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Epididymal development is also known to be highly regulated by androgens, which play a role in the differentiation of the Wolffian duct into formation of the epididymis and in its postnatal development (Joseph, et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e, Ribeiro, et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e, Robaire and Viger, \u003cspan class=\"CitationRef\"\u003e1995\u003c/span\u003e, Wilbourne, et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Several studies have shown the importance of androgens on the development and functions of the epididymis. Indeed, the removal of androgens leads to weight loss of the epididymis, a decrease in the diameter of the tubules, apoptosis of epithelial cells and morphological alterations in epididymal principal cells(Fan and Robaire, \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e, Robaire and Hamzeh, \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e, Robaire, et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Phosphorylation, expression levels and localization of GJA1 have been shown to be androgen-dependent in the initial segment in rats (Cyr, et al., \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e). GJA1 is localized between principal and basal cells in the epididymis, while in orchidectomized animals GJA1 is also present between principal cells. This effect is inhibited by the administration of testosterone to orchidectomized rats. In wild boar, administration of flutamide, an anti-androgen, during gestation, decreases \u003cem\u003eGja1\u003c/em\u003e mRNA levels in the cauda epididymidis (Hejmej and Bilinska, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e, Lydka, et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). This change is maintained in adult wild boars and is associated with decreased AR levels. Other studies have reported that \u003cem\u003eGjb3\u003c/em\u003e mRNA levels are decreased in orchidectomized rats and are restored with DHT implants(Chauvin and Griswold, \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e, Hamzeh and Robaire, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Furthermore, in the human epididymis, the phosphorylation of GJA1 is also regulated by epidermal growth factor (EGF)(Dube, et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThere is no information on the mechanisms that regulate the switch from \u003cem\u003eGjb2\u003c/em\u003e expression during development of the epididymis, during which tall columnar cells develop into principal cells expressing GJB1, GJB4 and GJB5(Dufresne, Finnson, Gregory and Cyr, \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e, Dufresne, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Identifying the mechanisms regulating Cxs during differentiation opens the door to multiple factors and signaling pathways. Indeed, the epididymis is a regionalized and complex organ, with gene and protein expression profiles specific to the epididymal segment and age of animals being studied (Avram, et al., \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e, Dufresne, Finnson, Gregory and Cyr, \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e, Henderson, et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e, Hsia and Cornwall, \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e, Kirchhoff, \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e). The hypothesis of the present study is that steroid hormones are implicated in a common mechanism of regulation of the expression of different Cxs in the epididymis. The present objectives are to identify the mechanisms responsible for the decrease in \u003cem\u003eGjb2\u003c/em\u003e and the increase in other Cxs during postnatal development of the rat epididymis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eRCE-1 cells\u003c/h2\u003e \u003cp\u003eRat epididymal principal cells (RCE-1) (Dufresne et al. 2005) grown in Dulbecco-modified Eagle medium/Ham nutrient mixture F12 (DMEM/HAM F12, Sigma-Aldrich, Oakville, ON, Canada) supplemented with 5% fetal calf serum (FBS), 2 mM L-glutamine, 10 \u0026micro;g/ml insulin, 10 \u0026micro;g/ml transferrin, 80 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml cAMP, 50 U/ml penicillin, 50 \u0026micro;g/ml streptomycin and 5 nM testosterone at 32\u0026deg;C in an incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were grown in 6-well plates coated with collagen IV (BD Biosciences, Mississauga, ON).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTreatment with estradiol\u003c/h2\u003e \u003cp\u003eA stock solution of 17β-estradiol (Sigma-Aldrich) with a concentration of 100 \u0026micro;M was prepared in ethanol. RCE-1 cells were seeded in 24-well collagen-coated plates (5 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e; BD Biosciences). After 24 h, the cells were rinsed with PBS, and the medium replaced with complete medium without phenol red and containing charcoal-stripped serum (Wisent, St-Bruno, QC, Canada) for 48 h. The cells were then exposed to ethanol (control condition), phenol red or estradiol (10 and 100 nM) for 48 h. The medium was changed at 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDexamethasone treatment\u003c/h2\u003e \u003cp\u003eStock solutions of dexamethasone (Sigma-Aldrich) and hydrocortisone (Sigma-Aldrich) of 100 \u0026micro;M were prepared in ethanol. The cells were seeded on 24-well plates coated with collagen IV (5 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e; BD Biosciences). After 24 h, the cells were rinsed in PBS and the medium was replaced with complete medium without hydrocortisone and containing stripped serum (Wisent) for 48 h. The cells were then exposed to ethanol (control), hydrocortisone (80 ng/ml) or dexamethasone (10 and 100 nM) for 48 h. The medium was changed at 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLNCaP cells\u003c/h2\u003e \u003cp\u003eHuman LNCaP prostate carcinoma cells (Horoszewicz, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) were cultured in RPMI medium (R7509, Sigma-Aldrich) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES and 10U/ml Penicillin-Streptomycin (Sigma-Aldrich). The cells were cultured in flasks of 75 cm\u003csup\u003e2\u003c/sup\u003e at 37 \u0026deg; C with a change of medium every two days. A 100 nM stock solution of 5α-dihydrotestosterone (DHT; Sigma-Aldrich) was prepared in ethanol. For DHT exposure, cells were seeded in 12-well plates in complete medium. After 24 h, the medium was changed to complete medium containing 5% stripped serum (Wisent). After 24 h, the cells were exposed to ethanol (control condition) or 10 nM DHT dissolved in ethanol for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA interference\u003c/h2\u003e \u003cp\u003eSmall interfering RNAs (siRNAs) against \u003cem\u003eGjb2\u003c/em\u003e (15 nM; Qiagen, Toronto, ON, Canada) and a control nonsense RNA (scramble, 15 nM; Qiagen) were transfected into RCE-1 cells using the Hiperfect transfection agent and following the manufacturer's instructions (Qiagen). The cells were seeded on 24-well plates and transfected 24 hours later with siRNAs. The cells were incubated for 48 h with siRNAs. The cells were then lysed and total RNA was extracted using a NucleoSpin RNA extraction kit (Macherey-Nagel, Bethlehem, PA) according to the manufacturer's instructions. An aliquot of 500 ng of RNA was denatured at 65\u0026deg;C for 10 min and cooled on ice for 5 min. The RNA was subsequently reversed-transcribed into cDNA using oligo dT primers (R\u0026amp;D System Inc., Minneapolis, MN) and MMLV reverse transcriptase (Sigma-Aldrich) for 1 h at 42\u0026deg;C. Levels of \u003cem\u003eGjb1, Gjb2\u003c/em\u003e, \u003cem\u003eGja1, Gjb4, Gjb5\u003c/em\u003e and \u003cem\u003eGapdh\u003c/em\u003e were quantified by RT-PCR using the primers listed in Supplementary Table\u0026nbsp;2. The products were then analyzed on an agarose gel (GelDoc Imaging system, Bio-Rad Laboratories), excised, purified with the ZymoClean Gel DNA recovery kit (Zymo Research, Irvine, CA) and sequenced (Genome Qu\u0026eacute;bec Innovation Center, McGill University, Montreal, QC, Canada).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR\u003c/h2\u003e \u003cp\u003eAt the end of treatment, cells were lysed and the total RNA was extracted using the NucleoSpin RNA extraction kit (Macherey-Nagel) according to the manufacturer's instructions. A 400ng aliquot of total RNA was transcribed into cDNA using the qScript cDNA superMix kit (Quanta Biosciences, Gaithersburg, MD). The levels of G\u003cem\u003ejb\u003c/em\u003e2, G\u003cem\u003ejb\u003c/em\u003e1, \u003cem\u003eNfkbia\u003c/em\u003e, \u003cem\u003eHspb8\u003c/em\u003e, and \u003cem\u003eGapdh\u003c/em\u003e in RCE1 cells were determined by qPCR using gene specific primers (Supplemental Table\u0026nbsp;2). Levels of human G\u003cem\u003ejb\u003c/em\u003e2, G\u003cem\u003ejb\u003c/em\u003e1, \u003cem\u003eYy1\u003c/em\u003e, \u003cem\u003eIgf1\u003c/em\u003e and \u003cem\u003eGapdh\u003c/em\u003e in LnCap cells were also determined using qPCR (Supplemental Table\u0026nbsp;3). qPCR analyses were done using a 2 \u0026micro;1 aliquot of cDNA in 15 \u0026micro;l of PerfeCTa SYBER Green Supermix (Quanta Biosciences) and 0.3 \u0026micro;M of each forward and reverse primer. The DNA was amplified by denaturation at 94\u0026deg;C for 5 min followed by 40 cycles at 94\u0026deg;C for 15 sec, 60\u0026deg;C for 30 sec and 72\u0026deg;C for 30 sec. The products were analyzed on an agarose gel, excised, purified (ZymoClean Gel DNA recovery kit; Zymo Research) and sequenced (Genome Qu\u0026eacute;bec). Data were normalized to \u003cem\u003eGapdh\u003c/em\u003e and relative delta-delta Ct used to express relative transcript levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult male Sprague Dawley rats (350-400g) were obtained from Charles River Laboratories (St-Constant, QC, Canada). The animals were kept under a photoperiod of 12 hours of light and 12 hours of darkness with food and water \u003cem\u003ead libitum\u003c/em\u003e. Orchidectomy experiments were performed as previously described (Turmel, et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The rats were anaesthetized with intraperitoneal injection of ketamine/xylazine/acepromazine (50/5/2.5 mg/kg) and received a subcutaneous injection of an analgesic (buprenorphine; 0.3 mg/kg). Polydimethylsiloxane implants were prepared with testosterone as previously described (Stratton, et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) and whose diffusion properties are known (Brawer, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Subcutaneous implants containing no testosterone or 3 implants of 6.2 cm of testosterone which mimic normal epididymal testosterone levels were surgically placed on the back of the animals. Sham-operated animals (n\u0026thinsp;=\u0026thinsp;4) were used as controls. Seven days following surgery the animals were euthanized with CO\u003csub\u003e2\u003c/sub\u003e and cervical dislocation. The epididymides were removed, divided into three segments (head, body and tail), frozen in liquid nitrogen and stored at -80\u0026deg;C. All animal protocols used in this study were approved by the INRS Institutional Committee for Animal Care.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eTotal proteins from the epididymides and ventral prostates of orchidectomized and control rats were extracted as previously described (Turmel et al. (Turmel, Dufresne, Hermo, Smith, Penuela, Laird and Cyr, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Briefly, the tissues were ground in liquid nitrogen and homogenized in 3 ml/g of cold RIPA lysis buffer (1X PBS; 1% Igepal CA-630; 0.5% sodium deoxycholate; 0.1% SDS; 10 mg/ml phenylmethylsulfonyl fluoride (PMSF); 100 mM sodium orthovanadate) supplemented with a cocktail of protease inhibitors (Sigma\u0026ndash;Aldrich). The samples were placed on ice for 30 min and centrifuged at 10000 x g at 4\u0026deg;C for 10 min to dispose of cellular debris. The supernatants were collected and protein concentrations determined using the Pierce BCA Protein Assay kit (ThermoFisher, Ottawa, ON). Samples were stored at -80\u0026deg; until used for Western blots.\u003c/p\u003e \u003cp\u003eProteins (50 \u0026micro;g) were thawed on ice, diluted in Laemmli buffer, and heated for 5 min at 94\u0026deg;C. The proteins were then separated on a 12% polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane using a Transblot apparatus (Bio-Rad Laboratories, Mississauga, ON). The membranes were blocked for 1 h with 5% non-fat milk dissolved in TBS (Tris-buffered saline) containing 0.1% Tween-20 (TBST) at room temperature. The membranes were then incubated at 4\u0026deg;C overnight (18 hours) with gentle rocking with either an anti-GJB2 mouse antibody (see Supplemental Table\u0026nbsp;1), a rabbit anti-GJB1 antibody (see Supplemental Table\u0026nbsp;1), an anti-GJB4 rabbit antibody (see Supplemental Table\u0026nbsp;1) or an anti-GJB5 mouse monoclonal antibody (see Supplemental Table\u0026nbsp;1). All primary antibodies were appropriately diluted in the blocking solution. Overnight incubation with primary antibodies was followed by a series of washes in TBST. The membranes were then incubated for 1 hr at room temperature with a secondary antibody conjugated to horseradish peroxidase (HRP). Depending on the primary antibody, a goat anti-rabbit antibody (see Supplemental Table\u0026nbsp;1) or a goat anti-mouse antibody (see Supplemental Table\u0026nbsp;1) was used. Protein bands for each connexin were revealed by the addition of Clarity Western ECL (Bio-Rad Laboratories) substrate and analyzed using the ChemiDoc MP (Bio-Rad Laboratories) imaging system. The membranes were stripped twice for 10 min in stripping solution (0.1 M glycine, 20 mM magnesium acetate, 50 mM KCl, pH 2.2), rinsed with TBST, blocked as described above and subsequently incubated with a rabbit anti-Tubulin antibody (Supplemental Table\u0026nbsp;1) for 1 hr at room temperature. Tubulin was used to normalize protein levels on the membrane. A secondary goat anti-rabbit antibody was used (Supplemental Table\u0026nbsp;1) to reveal the tubulin band as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence of Epididymal Sections\u003c/h2\u003e \u003cp\u003eEpididymides from sham-operated control and orchidectomized rats were frozen in OCT compound (Fisher Scientific, Ottawa, ON) on dry ice and stored at -86˚C until sectioning. Frozen sections (10\u0026micro;m) were fixed in ice-cold methanol for 15 min at -20˚C. Sections were rehydrated and washed 3 times in cold Tris-buffered saline with TBS-T, pH 7.4, plus glycine (0.3M) at room temperature for 10 min each wash. Sections were then incubated with blocking solution (TBS-T\u0026thinsp;+\u0026thinsp;glycine (0.3 M) plus 5% goat serum and 1% bovine serum albumin (BSA) for 60 min at room temperature in a humidified chamber. After rinsing in TBS-T\u0026thinsp;+\u0026thinsp;glycine solution, sections were incubated with either a rabbit polyclonal anti-GJB1 or GJB2 antibody (Supplemental Table\u0026nbsp;1) diluted in blocking solution at room temperature for 2 hr. Sections were washed three times with TBS-T and subsequently incubated with a goat anti-rabbit IgG-Alexa Fluor 488 (Supplemental Table\u0026nbsp;1) conjugated secondary antibody (2\u0026micro;g/mL) at room temperature for 45 min in blocking buffer containing a Hoechst blue dye (1\u0026micro;g/mL, Biotium, Hayward, CA). Finally, sections were washed twice with TBS-T and once with TBS and mounted with Fluoromount (Southern Biotech, Birmingham, AL). Immunofluorescence was examined under a Zeiss LSM 780 confocal microscope. Images were processed using the Zen software (Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRapid amplification of cDNA Ends (RACE)\u003c/h2\u003e \u003cp\u003eThe 5' region of \u003cem\u003eGjb1\u003c/em\u003e transcript was amplified as described in Adam \u003cem\u003eet al\u003c/em\u003e. (Adam and Cyr, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) using the primers shown in Supplemental Table\u0026nbsp;4. Briefly, total RNA was extracted from 56-day-old rat epididymis using the Illustra RNAspin Mini commercial kit according to the manufacturer's instructions (GE Healthcare, Montr\u0026eacute;al, QC). The 5' region of the \u003cem\u003eGjb1\u003c/em\u003e transcript was amplified using the FirstChoice RLM-RACE kit (RNA Ligase Mediated Rapid Amplification of cDNA Ends, Ambion, Austin, TX), following the manufacturer's instructions. PCR amplifications were performed using 94\u0026deg;C for 5 min, 35 cycles at 94\u0026deg;C for 30 sec, melting temperature (Tm) for 30 sec, and 72\u0026deg;C for 30 sec cycles. The products were analyzed on a 2% agarose gel containing ethidium bromide. The bands were excised, purified (ZymoClean Gel DNA recovery kit) and sequenced (Genome Qu\u0026eacute;bec).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning of\u003c/b\u003e \u003cb\u003eGjb1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eGjb2\u003c/b\u003e \u003cb\u003epromoters\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal genomic DNA used to clone the promoters was extracted from livers of adult Sprague Dawley rats using the GenElute Mammalian Genomic DNA Purification (Sigma-Aldrich) kit and following the manufacturer's instructions. Primers used to amplify \u003cem\u003eGjb1\u003c/em\u003e and \u003cem\u003eGjb2\u003c/em\u003e promoters are shown in Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGjb1 promoter\u003c/h2\u003e \u003cp\u003eA 1650 bp fragment from the 5' region of the P1 promoter and a 1173 bp fragment of the P2 promoter were amplified by PCR (5 min at 94\u0026deg;C, 35 cycles of 30 sec at 94\u0026deg;C, 30 sec at TA and 3 min at 72\u0026deg;C). PCR products were visualized on a 0.7% agarose gel containing ethidium bromide and bands of interest were excised and purified (ZymoClean Gel DNA recovery kit). The fragments of the P1 and P2 promoters were inserted directionally into the pGL3-Basic vector (Promega, Madison WI) upstream of the luciferase gene. The restriction sites SacI-NheI and XhoI-NheI were used for P1 and P2, respectively. After transfection of TOP10 (Invitrogen) chemically competent bacteria, plasmids were purified (Plasmid Midi kit, Qiagen), and sequenced (Genome Qu\u0026eacute;bec).\u003c/p\u003e \u003cp\u003eCloning of the 5162 bp of the \u003cem\u003eGjb1\u003c/em\u003e P1 promoter was performed in two stages. First a 2916 bp fragment of the \u003cem\u003eGjb1\u003c/em\u003e promoter (-2052/+64 bp start of exon 1) was amplified using the primers indicated in Supplementary Table\u0026nbsp;1 (P2 cloning (3 kb) F and R). The resulting amplicon was separated on a 0.7% agarose gel and purified as described above. The DNA was incubated with T4 DNA polymerase and nucleotides to generate blunt ends and digested with the KpnI (New England Biolabs) restriction enzyme. The remaining 2759 bp fragment was inserted into the pGL3-Basic plasmid previously digested with KpnI and SmaI using ligase (New England Biolabs). The resulting \u0026minus;\u0026thinsp;2761/+64 construct was used to transform TOP10 competent bacteria. The resulting clones were analyzed and verified by enzymatic digestion. The \u0026minus;\u0026thinsp;2761/+64 construct was purified as described above and its identity confirmed by sequencing. A second PCR amplification yielded a 2510 bp fragment of the \u003cem\u003eGjb1\u003c/em\u003e promoter located from \u0026minus;\u0026thinsp;5162 to -2652 bp relative to the start of exon 1. This amplification was carried using primers described in Supplemental Table\u0026nbsp;1 (P2 cloning (5 kb) F and R) and purified as described above. The \u0026minus;\u0026thinsp;2761/+64 construct was digested by KpnI (New England Biolabs) purified and ligated to the 2510 bp fragment to generate a -5076/+64 bp construct. This final construct was used to transform TOP10 bacteria as described above. The clones were analyzed by digestion and sequenced (G\u0026eacute;nome Qu\u0026eacute;bec). The expression of the transgene was confirmed using both hepatic MHC1 and RCE-1 cells (Supplemental Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGjb2 promoter\u003c/h2\u003e \u003cp\u003eA fragment of 3036 bp (-4407 to -1371) relative to the transcription initiation site of the 5' region of \u003cem\u003ethe Gjb2\u003c/em\u003e promoter was amplified by PCR (5 min at 94\u0026deg;C, 35 cycles of 30 sec at 94\u0026deg;C, 30 sec at annealing temperature (Tm, Table\u0026nbsp;4), and 3 min at 72\u0026deg;C). Primers used (Promoter (5 kb) F and R) are shown in Supplementary Table\u0026nbsp;4. The PCR product was analyzed on a 0.7% agarose gel containing ethidium bromide. The DNA band of interest was excised and purified using the ZymoClean Gel DNA recovery kit (Zymo Research). The \u003cem\u003eGjb2\u003c/em\u003e promoter (position \u0026minus;\u0026thinsp;1564/+133 relative to the translational start site) was ligated into MluI and Bmg1 sites of the pGL3-Basic plasmid upstream of the luciferase gene. The construct was used to transform TOP10 bacteria as described above. The clones obtained were analyzed by digestion and sequenced (G\u0026eacute;nome Qu\u0026eacute;bec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical tests were performed using GraphPad Prism (GraphPad Software, San Diego, CA). One-factor analysis of variance (ANOVA) tests followed by the Newman-Keuls test or Student tests (T-test) were used to analyze the data. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of the decrease in\u003c/strong\u003e \u003cstrong\u003eGjb2\u003c/strong\u003e \u003cstrong\u003eon the expression of other Cxs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur first objective was to assess whether the previously observed decrease in \u003cem\u003eGjb2\u003c/em\u003e during postnatal development (Dufresne, Finnson, Gregory and Cyr, \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e) could explain the variation in the expression of other Cxs in the epididymis. Using RCE-1 cells and an siRNA directed against \u003cem\u003eGjb2\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) we demonstrated that \u003cem\u003eGjb2\u003c/em\u003e mRNA levels were decreased by approximately 60% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in comparison to a scrambled siRNA sequence (scramble, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). mRNA expression levels for \u003cem\u003eGjb1, Gjb4, Gjb5\u003c/em\u003e and \u003cem\u003eGja1\u003c/em\u003e were not affected by the decrease in \u003cem\u003eGjb2\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, C). Similar results were observed 6 days after transfection with siRNA against \u003cem\u003eGjb2\u003c/em\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification of common and regulatory regions of Cxs promoters\u003c/h2\u003e\n \u003cp\u003eIn order to evaluate the presence of common mechanism(s)/transcription factors regulating the expression of epididymal Cxs during postnatal development, we analyzed and compared the promoter sequences of the different Cxs (\u003cem\u003eGjb1, Gjb2, Gjb4, Gjb5 and Gja1)\u003c/em\u003e expressed in the epididymis using BLAST Software to identify common response elements. No conserved regions were identified between the different Cxs. The promoter sequences of \u003cem\u003eGjb2\u003c/em\u003e and \u003cem\u003eGja1\u003c/em\u003e were also compared to examine whether or not a homologous sequence could explain the decrease in their expression after day 28 in rats (Dufresne, Finnson, Gregory and Cyr, \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). This same rationale was applied to the promoter sequences of \u003cem\u003eGjb1\u003c/em\u003e, \u003cem\u003eGjb4\u003c/em\u003e and \u003cem\u003eGjb5\u003c/em\u003e, which were compared to identify if there was any homologous sequence which would explain the increase in the expression of these Cxs during differentiation. No homologous regions were observed between the different Cxs.\u003c/p\u003e\n \u003cp\u003eAnalysis of the 5kb promoter region of the \u003cem\u003eGjb2\u003c/em\u003e promoter and \u003cem\u003eGjb1\u003c/em\u003e (Supplemental Figs. 2 and 3) revealed several response elements of the androgen, glucocorticoid and estrogen (ESR1) receptors, suggesting a role of these steroid hormones on the expression of these Cxs. Note that these response elements were also identified on the promoter region of \u003cem\u003eGjb4\u003c/em\u003e, \u003cem\u003eGjb5\u003c/em\u003e and \u003cem\u003eGja1\u003c/em\u003e using sequence analysis software.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRole of glucocorticoids and estradiol on\u003c/strong\u003e \u003cstrong\u003eGjb2\u003c/strong\u003e \u003cstrong\u003eexpression\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eOur analysis of the \u003cem\u003eGjb2\u003c/em\u003e and \u003cem\u003eGjb1\u003c/em\u003e promoter sequences allowed us to identify glucocorticoid and estrogen response elements on the DNA sequences of both Cxs. Our third objective was to assess whether glucocorticoids and/or estradiol were involved in the variation of Cxs expression during postnatal epididymis development. To do this, we first determined whether these hormones were involved in the decrease in \u003cem\u003eGjb2\u003c/em\u003e expression observed during development (Dufresne, Finnson, Gregory and Cyr, \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). RCE-1 cells were exposed for 48 hrs to hydrocortisone (80 ng/ml) and dexamethasone (10 and 100 nM) Gjb\u003cem\u003e2\u003c/em\u003e mRNA levels were increased with exposure to hydrocortisone and dexamethasone (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The response of RCE-1 cells to glucocorticoids was assessed by measuring levels of \u003cem\u003eNfkbia\u003c/em\u003e, a glucocorticoid-dependent gene (Silva et al. (Silva, Queiroz, Honda and Avellar, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). A small but significant increase in \u003cem\u003eNfkbia\u003c/em\u003e mRNA levels was observed in RCE-1 cells exposed to either hydrocortisone or dexamethasone (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eTo assess the role of estrogens in regulating \u003cem\u003eGjb2\u003c/em\u003e expression in the epididymis, RCE-1 cells were treated with estradiol (10 and 100 nM) for 48 hrs. There was a slight but not significant increase in the expression of \u003cem\u003eGjb2\u003c/em\u003e mRNA levels at the two doses (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). The estrogenic activity of phenol red (Berthois, et al., \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e) was also evaluated and no differences in the expression of \u003cem\u003eGjb2\u003c/em\u003e were noted. The response of RCE-1 cells to estradiol was assessed by measuring the gene \u003cem\u003eHspb8\u003c/em\u003e (Yang, et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e) which displays a dose-dependent response to estradiol (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eRole of androgens on Cxs expression in the epididymis\u003c/h2\u003e\n \u003cp\u003eTo assess the role of androgens on the expression of Cxs in the epididymis, an \u003cem\u003ein vivo\u003c/em\u003e approach was used, in which rats were orchidectomized or orchidectomized and given testosterone implants to mimic endogenous epididymal levels of testosterone. Controls were sham-operated. Western blot analysis of protein isolated from the caput, corpus and cauda epididymidis indicated that GJB2 protein levels in each region of the epididymis were significantly increased in the caput and corpus of orchidectomized animals, while testosterone maintained levels of GJB2 comparable to those of control animals. A similar pattern was observed in the cauda, but levels were not significantly different (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, GJB1, and GJB4 protein levels were decreased as a result of orchidectomy but were increased in rats orchidectomized and with testosterone implants. GJB5 levels in the caput and corpus epididymidis were not altered by orchidectomy alone but were increased in orchidectomized rats with testosterone implants. In all cases there were no significant effects in the cauda epididymidis.\u003c/p\u003e\n \u003cp\u003eImmunolocalization of GJB2 revealed that GJB2 was localized primarily at the base of the epithelium between basal cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). In orchidectomized rats, immunostaining intensity was increased in the caput, corpus and cauda epididymidis. Some staining was also observed over the sperm in the lumen of control rats, although this appears to be non-specific. Unlike GJB2, which was absent in controls, GJB1 was present along the lateral plasma membrane of principal cells. Punctate staining was evident in all regions of the epididymis and could be seen in stacked images (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). In orchidectomized rats, GJB2 staining was largely absent or weakly expressed. There was very little staining observed between principal cells, although some weak staining could be observed at the base of the epithelium. Stacked images of the caput epididymis further demonstrate the lack of GJB2 staining in the epithelium.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eRole of androgens on Cxs expression in the ventral prostate\u003c/h2\u003e\n \u003cp\u003eTo determine if the effects of androgens on Cxs in the epididymis were epididymis-specific or represent a generalized tissue response, the regulation of each Cx was assessed in the ventral prostate, which, like the epididymis, is known to be an androgen-dependent tissue. Our results indicated that GJB2 protein levels increased in the ventral prostate of orchidectomized rats (not shown), and this increase was abrogated by androgens. Levels of GJB1, GJB4 and GJB5 also displayed lower protein levels in orchidectomized rats compared to controls, while testosterone implants in orchidectomized rats increased levels of each Cx, suggesting a comparable response to that observed in the epididymis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eRole of androgens on Gjb1 and Gjb2 expression in LNCaP cells\u003c/h2\u003e\n \u003cp\u003eAndrogen regulation of Cxs was further assessed \u003cem\u003ein vitro\u003c/em\u003e using a well-characterized androgen-responsive human prostate cancer cell line, LNCaP cells (Horoszewicz, et al., \u003cspan class=\"CitationRef\"\u003e1983\u003c/span\u003e). LNCAP cells were cultured in the presence of 10 nM DHT. \u003cem\u003eGjb2\u003c/em\u003e mRNA levels were decreased by about 35% in cells exposed to DHT (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). A previous study showed that the \u003cem\u003eIgf1\u003c/em\u003e gene is a sensitive marker of androgen response (Wu, et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). Our data showed that \u003cem\u003eIgf1\u003c/em\u003e mRNA levels were increased in LNCaP cells exposed to DHT in each set of experiments to assess the effects on androgens on LNCaP cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, C). No differences were observed in \u003cem\u003eGapdh\u003c/em\u003e mRNA levels with androgen treatment. \u003cem\u003eGjb1\u003c/em\u003e levels were also significantly increased in LNCaP cells exposed to DHT (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eThe transcription factor YY1, which has been described as an activator of \u003cem\u003eGjb1\u003c/em\u003e (Field, et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e, Piechocki, et al., \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e) and which can interact with the androgen receptor to regulate its transcriptional activity (Deng, et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e) was not affected by DHT treatment (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEffects on androgens on the transactivation of the\u003c/strong\u003e \u003cstrong\u003eGjb2\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eGjb1\u003c/strong\u003e \u003cstrong\u003epromoters\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eActivity of the Gjb2 promoter\u003c/h2\u003e\n \u003cp\u003eAnalysis of the \u003cem\u003eGjb2\u003c/em\u003e promoter (Supplemental Fig. 2) identified several androgen response elements. In order to determine if androgens regulate the transcription of the \u003cem\u003eGjb2\u003c/em\u003e gene, a construct containing 1.5 kb of the \u003cem\u003eGjb2\u003c/em\u003e rat promoter (Adam and Cyr, \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) was transfected into LNCaP cells in the presence or absence of DHT (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The construction shows luciferase activity 9 times higher than that of the empty plasmid pGL3, indicating that the cells were able to transcribe the transgene. No differences in luciferase activity were observed in cells exposed to DHT as compared to control (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Several androgen response sites have been identified upstream of 1.5 kb of the \u003cem\u003eGjb2\u003c/em\u003e promoter. To assess the role of these regions, a 5 kb fragment of the \u003cem\u003eGjb2\u003c/em\u003e promoter was amplified and cloned. These constructs were then transfected into LNCaP cells with or without exposure to DHT (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eActivity of Gjb1 promoter P1 under the influence of androgens\u003c/h2\u003e\n \u003cp\u003eIn order to evaluate the role of androgens in the transcription of the \u003cem\u003eGjb1\u003c/em\u003e promoter, a 5 kb fragment of the \u003cem\u003eGjb1\u003c/em\u003e promoter was amplified and cloned into the PGL3 plasmid. The efficacy of the construct was determined using rat hepatic MH1C1 cells (not shown). The 5 kb construct containing the \u003cem\u003eGjb1\u003c/em\u003e promoter was transfected into LNCaP cells with and without DHT (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Our data indicate that the activity of the \u003cem\u003eGjb1\u003c/em\u003e promoter was markedly increased with exposure to DHT.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe mechanisms regulating the expression of Cxs during epididymal development remain poorly understood. It has been shown that \u003cem\u003eGjb2 and Gja1\u003c/em\u003e mRNA levels are decreased concomitant with increased \u003cem\u003eGjb1\u003c/em\u003e, \u003cem\u003eGjb4\u003c/em\u003e and \u003cem\u003eGjb5\u003c/em\u003e mRNA levels (Dufresne, Finnson, Gregory and Cyr, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) during postnatal development. While mRNA levels of \u003cem\u003eGja1\u003c/em\u003e do not change as dramatically as those of \u003cem\u003eGjb2\u003c/em\u003e, the expression of \u003cem\u003eGja1\u003c/em\u003e becomes associated primarily with basal cells during development (Dufresne, Finnson, Gregory and Cyr, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we showed that the decrease in \u003cem\u003eGjb2\u003c/em\u003e alone does not appear to be responsible for the variation in expression of other Cxs in the epididymis. Studies in the liver, pancreas, epidermis, and mammary gland have shown that decreased expression of specific Cxs can result in changes in the expression of other Cxs (Chanson, Fanjul, Bosco, Nelles, Suter, Willecke and Meda, 1998, Langlois, et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Nelles, Butzler, Jung, Temme, Gabriel, Dahl, Traub, Stumpel, Jungermann, Zielasek, Toyka, Dermietzel and Willecke, 1996, Stewart, Plante, Bechberger, Naus and Laird, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, specific removal of \u003cem\u003eGjb2\u003c/em\u003e in the mammary gland and in the cochlea caused a developmental delay in \u003cem\u003eGjb6\u003c/em\u003e (Cx30) expression (Crispino, Di Pasquale, Scimemi, Rodriguez, Galindo Ramirez, De Siati, Santarelli, Arslan, Bortolozzi, Chiorini and Mammano, 2011, Stewart, et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, although we observed a 60% decrease in \u003cem\u003eGjb2\u003c/em\u003e mRNA levels using siRNA, we observed no difference in the expression of \u003cem\u003eGjb1, Gjb4\u003c/em\u003e, \u003cem\u003eGjb5\u003c/em\u003e and \u003cem\u003eGja1\u003c/em\u003e in RCE-1 cells, suggesting tissue-specific differences in either the regulation or interplay of multiple connexins.\u003c/p\u003e \u003cp\u003eAnalysis of the \u003cem\u003eGjb2\u003c/em\u003e promoter revealed several response elements to estradiol, glucocorticoids and androgens. We evaluated the role of each of these hormones to determine if these were involved in the repression of \u003cem\u003eGjb2\u003c/em\u003e expression. In RCE-1 cells treated with either hydrocortisone or dexamethasone, an increase in GJB2 levels was noted. Glucocorticoids have been implicated in the development of the male reproductive tract by allowing the establishment and maintenance of spermatogenesis (Saxena and Paul, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Weber, et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The glucocorticoid receptor (GR) has been shown to be present in principal cells of the epididymis (Gladstones, Burton, Mark, Waddell and Roberts, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Silva, Queiroz, Honda and Avellar, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Its expression is more intense in early epididymal development and during differentiation (Gladstones, Burton, Mark, Waddell and Roberts, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). We have previously shown that the transcription factors SP1 and TFAP2A bind to the \u003cem\u003eGjb2\u003c/em\u003e promoter to activate its transcription during the early stages of epididymal differentiation (Adam and Cyr, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Given that we have observed an increase in \u003cem\u003eGjb2\u003c/em\u003e with glucocorticoids, it is possible that they may act indirectly to increase \u003cem\u003eGjb2\u003c/em\u003e expression by influencing SP1 and TFAP2A binding during the early stages of development (Silva, Queiroz, Honda and Avellar, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur data indicate that estradiol had no significant effects on \u003cem\u003eGjb2\u003c/em\u003e mRNA levels \u003cem\u003ein vitro\u003c/em\u003e. Studies have reported that injection of estradiol benzoate in rats at seven days of age decreases \u003cem\u003eGjb2\u003c/em\u003e expression in the corpus epididymidis at high doses (Lee, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). In addition, the effects of estradiol benzoate on Cxs expression differ between the initial segment and the body of the epididymis (Lee, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Lee, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e, Lee, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). This suggests that the effects of estrogen on Cxs are modulated by dose and epididymal region. Our results support the notion that estrogen is not directly involved in the decreased \u003cem\u003eGjb2\u003c/em\u003e expression observed during postnatal epididymal development.\u003c/p\u003e \u003cp\u003eAndrogens are major regulators of the development and maintenance of epididymal functions. Removal of androgens alters the structure of the tubule and causes a wave of apoptosis (Fan and Robaire, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The present results support the notion that androgens modulate the expression of Cxs in the epididymis. We observed that GJB2 levels were significantly increased in the caput and corpus epididymidis following orchidectomy and decreased in orchidectomized animals treated with testosterone. In addition, we observed a decrease in protein levels of GJB1 and GJB4 in the caput and corpus epididymidis of orchidectomized rats, an effect that was mitigated by testosterone. These results are consistent with the previously reported variations in Cxs expression in the epididymis during development, when testosterone levels begin to increase during postnatal development (Dufresne, Finnson, Gregory and Cyr, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Furthermore, the lack of effect observed in the cauda epididymidis is consistent with previous data that showed that the regulation of Cxs in the cauda was different from that in the proximal regions of the epididymis (Dufresne, Finnson, Gregory and Cyr, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). It has also been shown that the levels, localization and phosphorylation of GJA1 are androgen-dependent in the initial segment of the epididymis (Cyr, Hermo and Laird, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Similarly, in wild boar, neonatal exposure to flutamide decreased GJA1 protein levels in the tail of the epididymis but not in other regions (Lydka, Kopera-Sobota, Kotula-Balak, Chojnacka, Zak and Bilinska, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the rat, Pannexin 1 (Panx1), a protein related to connexins and involved in the formation of transmembrane channels, was increased following orchidectomy in the caput and corpus epididymidis, but not in the cauda (Turmel, Dufresne, Hermo, Smith, Penuela, Laird and Cyr, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmunofluorescence of GJB2 and GJB1in rat epididymis tissue sections supported the data observed by western blot, in which GJB2 appeared to slightly increase in orchidectomized rats, while GJB1 immunostaining was decreased in orchidectomized rats relative to controls. The data support the effects of androgens as exerting an inhibition of GJB2 gene expression in the epididymis while promoting the expression of GJB1. The data also support previous reports that changes in the expression of different connexins during development are regulated by testicular factors, including androgens (Dufresne, Finnson, Gregory and Cyr, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral Cxs, including GJA1, GJB1 and GJB2 (Czyz, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Huynh, et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Li, et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Meda, et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Mehta, et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Tate, et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) are expressed in the rat prostate, an organ known to be highly regulated by androgens (Cleutjens, et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Heyns, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Huynh, Alpert, Laird, Batist, Chalifour and Alaoui-Jamali, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Robaire, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Yamashita, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). As no data regarding on the presence of GJB4 and GJB5 in the prostate were available in the literature, western blot experiments were performed using the prostates of rats from the different \u003cem\u003ein vivo\u003c/em\u003e experimental groups. We identified the presence of GJB4 and GJB5 in the prostate of control (sham-operated) rats. For all Cxs with the exception of GJB5, we observed a similar regulation in the prostate to that observed in the epididymis following orchidectomy plus androgen maintenance. A previous study reported an increase in mRNA and GJA1 protein levels in the prostate after orchidectomy, and these effects were inhibited with the maintenance of androgen levels (Huynh, Alpert, Laird, Batist, Chalifour and Alaoui-Jamali, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, in that study, no changes were observed on \u003cem\u003eGjb1\u003c/em\u003e mRNA levels. Another team investigating the effect of flutamide, an anti-androgen, showed that flutamide injection during development decreased androgen receptor levels and GJA1 expression in the prostate (Hejmej, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The androgen receptor has also been reported to be involved in decreasing GJA1 expression in prostate cancer cells (Chen, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It is interesting to note that while GJB5 is regulated by androgens in the prostate, it does not appear to be similarly regulated in the epididymis. It has been shown that GJB1, GJB3, GJB4 and GJB5 are also expressed by epididymal basal cells (Mandon, et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It is possible that in the epididymis, basal cell-specific factors maintain GJB5 expression after orchidectomy. Our results, as well as those of previous studies, suggest that androgens regulate the expression of some Cxs in both the epididymis and prostate, and suggest a common regulatory mechanism for some, but not all Cx genes.\u003c/p\u003e \u003cp\u003eTo understand the mechanisms that are involved in the action of androgens on Cxs expression, we used the LNCaP cell line, a widely used cellular model to study the effect of androgens (Horoszewicz, Leong, Kawinski, Karr, Rosenthal, Chu, Mirand and Murphy, 1983) et al. 1983). Our results support \u003cem\u003ein vivo\u003c/em\u003e observations in both the epididymis and ventral prostate, in which \u003cem\u003eGjb2\u003c/em\u003e mRNA levels were decreased while \u003cem\u003eGjb1\u003c/em\u003e mRNA levels were increased. These results occurred independent of changes in the YY1 transcription factor, which stimulates the expression of \u003cem\u003eGjb1\u003c/em\u003e in hepatocytes (Field, Tate, Chipman and Minchin, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). YY1 can act as a repressor or activator, depending on which cofactors it recruits (Shi, et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Our results suggest that YY1 is not involved in the androgenic response of LNCap cells and the regulation of \u003cem\u003eGJb2\u003c/em\u003e and \u003cem\u003eGJb1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eUsing both a 1.6 and 5kb sequence of the \u003cem\u003eGjb2\u003c/em\u003e promoter, our data suggest that DHT does not directly regulate the transcription of GJB2. We previously reported that both SP1 and TFAP2 transcription factors regulate minimal promoter activity of \u003cem\u003eGjb2\u003c/em\u003e in the epididymis (Adam and Cyr, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our data suggests that androgen-dependent factors may be implicated in the down-regulation of \u003cem\u003eGjb2\u003c/em\u003e and that androgens are involved in decreasing \u003cem\u003eGjb2\u003c/em\u003e expression. We have recently shown that factors SP1 and TFAP2A bind to the \u003cem\u003eGjb2\u003c/em\u003e promoter to activate its transcription during the early stages of epididymal differentiation (Adam and Cyr, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Knowing that SP1 and AR factors are able to form a complex to regulate the expression of the NRIP gene in the prostate (Chen, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), AR may interact with SP1 to decrease \u003cem\u003eGjb2\u003c/em\u003e expression. Thus, by its binding to SP1, AR is able to bind to DNA via an SP1 site and not an androgen-response element (ARE) site in prostate cells (Chen, Tsao, Wang and Chen, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Eisermann, et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition, TFAP2A has been shown to be the major co-regulator of the AR and facilitates the binding of AR to chromatin in the epididymis, suggesting a central role for TFAP2A in the response to androgens in this tissue (Pihlajamaa, et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These studies, in conjunction with our results, suggest that AR may interact with SP1 and TFAP2A factors and/or their binding sites located on the \u003cem\u003eGjb2\u003c/em\u003e promoter to decrease its expression.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eGjb1\u003c/em\u003e gene can be transcribed from two tissue-dependent promoters (Neuhaus, et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Neuhaus, et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The promoter P1 is used in the pancreas and liver (Neuhaus, Bone, Wang, Ionasescu and Werner, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Neuhaus, Dahl and Werner, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and the P2 promoter is active in nerve fibers, allowing transcription of shorter mRNA(Neuhaus, Bone, Wang, Ionasescu and Werner, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Our data reveal that the promoter P1 is used for the transcription of \u003cem\u003eGjb1\u003c/em\u003ein the rat epididymis. The results of the luciferase assays show an increase in the activity of the \u003cem\u003eGjb1\u003c/em\u003e promoter in cells exposed to DHT. These data are the result of preliminary experiments and have yet to be confirmed. They do suggest, however, that at least some of the elements regulating androgen responses are present within the 5 kb of the \u003cem\u003eGjb1\u003c/em\u003e promoter.\u003c/p\u003e \u003cp\u003eIn conclusion, we have provided evidence from both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies which suggest that the switch in expression of Cxs during epididymal development is regulated by androgens, which exerts opposing regulatory effects on the regulation of GJB2 and other Cxs. We have also provided preliminary data indicating that the activity of the P1 promoter in G\u003cem\u003ejb1\u003c/em\u003e expression is influenced, if not regulated in part, by androgens, and that this may also be the case for the P2 promoter. Knowing that androgens can activate and influence a multitude of signaling pathways and induce various cellular responses, further studies are needed to elucidate the role(s) and mode(s) of action of androgens in regulating Cxs expression in the epididymis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e: \u0026nbsp;Data are available from the corresponding author. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSummary Statement\u003c/strong\u003e: The expression of connexins during epididymal development is regulated by androgens which inhibit the expression of \u003cem\u003eGjb2\u003c/em\u003e and \u003cem\u003eGja1\u003c/em\u003e in principal cells and stimulate the expression of \u003cem\u003eGjb1\u003c/em\u003e, \u003cem\u003eGjb4\u003c/em\u003e and \u003cem\u003eGjb5\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdam C, Cyr DG (2016) Role of specificity protein-1 and activating protein-2 transcription factors in the regulation of the gap junction protein beta-2 gene in the epididymis of the rat. 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Endocrinology 165\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, Zhao W, Zhao J, Pan J, Wu Q, Zhang Y, Bauman WA, Cardozo CP (2007) Identification of androgen response elements in the insulin-like growth factor I upstream promoter. Endocrinology 148:2984\u0026ndash;2993\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamashita S (2004) Localization of estrogen and androgen receptors in male reproductive tissues of mice and rats. Anat Rec Discov Mol Cell Evol Biol 279:768\u0026ndash;778\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang C, Trent S, Ionescu-Tiba V, Lan L, Shioda T, Sgroi D, Schmidt EV (2006) Identification of cyclin D1- and estrogen-regulated genes contributing to breast carcinogenesis and progression. Cancer Res 66:11649\u0026ndash;11658\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Materials","content":"\u003cp\u003eSupplementary Figure 3 and Supplementary Tables 2-4 are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"64c1909d-653f-4e8e-9c92-3f981eb5e661","identifier":"10.13039/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","awardNumber":"2023-05026","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Connexins, gap junctions, androgen, epididymis, development, gene expression","lastPublishedDoi":"10.21203/rs.3.rs-4731767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4731767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring postnatal development of the epididymis, a change in the expression of gap junction proteins, or connexins (Cxs), occurs, in which \u003cem\u003eGjb2\u003c/em\u003e (Cx26) and \u003cem\u003eGja1\u003c/em\u003e (Cx43) levels in the proximal epididymis are decreased, while \u003cem\u003eGjb1\u003c/em\u003e(Cx32), \u003cem\u003eGjb4\u003c/em\u003e (Cx30.3) and \u003cem\u003eGjb5 \u003c/em\u003e(Cx31.1) levels increase. The mechanism(s) responsible for the switch in Cx expression is unknown. The aims of this study are: 1) to identify the mechanisms responsible for the decrease in GJB2 protein levels\u003cem\u003e \u003c/em\u003eand the increase in other Cxs during postnatal development. Results indicate that decreased \u003cem\u003eGjb2\u003c/em\u003e expression does not induce changes in the expression of other Cxs in rat RCE-1 principal cells, suggesting a lack of compensatory expression. Sequence analysis of both \u003cem\u003eGjb2\u003c/em\u003e and \u003cem\u003eGjb1\u003c/em\u003e promoters identified common multiple response elements to steroid hormones. Using RCE-1 cells, we showed that glucocorticoids increased \u003cem\u003eGjb2\u003c/em\u003e expression, while estradiol had no effect. Orchidectomy in rats resulted in a significant increase in GJB2 and decreased GJB1 in the caput and corpus epididymidis. Changes in Cxs protein levels were prevented by administering testosterone in orchidectomized rats. Similar results were observed in the prostate, another androgen-receptive organ. LNCaP cells, which are androgen-responsive, showed that exogenous dihydrotestosterone (DHT) exposure resulted in a decrease in \u003cem\u003eGjb2\u003c/em\u003emRNA levels concomitant with increased \u003cem\u003eGjb1\u003c/em\u003e levels. Using a GJB1 promoter construct we showed that DHT could induce transactivation of the luciferase transgene, while transactivation using two GJB2 promoters were not altered. Together, our results suggest that androgens and glucocorticoids regulate the expression of Cxs in the epididymis.\u003c/p\u003e","manuscriptTitle":"Regulation of the Gap Junction Interplay in the Rat Epididymis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-16 13:59:41","doi":"10.21203/rs.3.rs-4731767/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"884e51b0-069e-485f-a1cc-415b3c7dc504","owner":[],"postedDate":"July 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34529623,"name":"General Cell Biology \u0026 Physiology"}],"tags":[],"updatedAt":"2024-07-16T13:59:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-16 13:59:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4731767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4731767","identity":"rs-4731767","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}