Systems Biology in Reproductive Medicine
http://informahealthcare.com/aan ISSN: 1939-6368 (print), 1939-6376 (electronic)
informa
Syst Biol Reprod M ed, 2014; 60(5): 257 -2 6 2 © 2014 Inform a Healthcare USA, Inc. DOI: 10.3109/19396368.2014.921738
healthcare
RESEARCH C O M M U N IC ATIO N
Connexin 43 expression in Sprague-Dawley rat seminiferous epithelium after in utero exposure to flutamide Chunhong Cai1,2, Dan Zhao2, Cao Ma2, Yongbo Zhang2, Xing Wu2, Guanghui Wei1,2, and Dawei He1,2* 'Department of Pediatric Urology, Children's Hospital of Chongqing Medical University, Chongqing, China and 2Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China Abstract
Keywords
This study explored the expression of connexin 43 (Cx43) in the testes of prepubertal SpragueDawley (SD) rats following in utero flutamide (Flu) exposure. Connexins constitute the major protein type in gap junctions. Connexin 43, the most prominent connexin family member expressed by testes, is localized at the base o f seminiferous tubules in humans and rodents, and may be involved in fertility. Flutamide was injected subcutaneously into pregnant SD rats on gestational days 12-21 (25 mg/kg/day). Immunohistochemistry, Western blotting, and real-time PCR was used to investigate the distribution and the expression o f Cx43 protein and mRNA in the testis on postnatal day 20 (PD20). Following Flu-exposure, Cx43 was observed between Sertoli cells in the seminiferous tubules. On PD20, no Cx43 protein was expressed by the spermatogonial cell layer o f the seminiferous tubules in the controls, but was observed in the Flu-exposed group. Western blotting showed that Cx43 was expressed at significantly lower levels in Flu-exposed testes than controls on PD20 (p
C
u. re
* & 2-e c ro
***
o “ O
Figure 2. Expression o f connexin 43(Cx43) protein in flutamide (FIu)exposed Sprague-Dawley rat testes (n= 3). Western blots performed on testis extracts (50 pg protein) with the anti-Cx43 and the GAPDH antibody found the expected molecular size (upper panel). A significant decrease in Cx43 protein expression in the undescended testicle (Flu-C) and descended testicle (Flu-nC) groups of postnatal day (PD) 20 rats exposed to flutamide compared to control groups (lower panel). ***p< 0.001. Each band was quantified by densitometry using the Quantity One 21.0.0 software and expressed as arbitrary units (m ean ±S D ). The graphic shows pooled data from three independent experiments.
PD20 are shown in Figure 2. Connexin 43 protein expression was lower in undescended testes in the Flu-exposed group compared to controls on PD20 (p< 0.001), but no difference in Cx43 expression was found between undescended and descended testes in the Flu-exposed group at this time point. In the mammalian testis, immunohistochemistry and hybridization histochemical studies have demonstrated that Cx43 participates in the formation of homocellular gap junctions between adjacent Sertoli cells, and the heterocellular gap junction between Sertoli cells and spermatogonia or spermatocytes [Li et al. 2010; Palmiero et al. 2003; Tan et al. 1996]. With regard to our results, immunostaining for Cx43 showed that the distribution and expression of the Cx43 protein were different in the seminiferous tubule
259
(Figure 1A-C). The distribution and expression of Cx43 protein in the testicular seminiferous tubule showed that it moved from the basal compartment to the adluminal compartment in the control group. However, its expression remained abnormally distributed in the spermatogonium layer of the seminiferous tubule basilar membrane in the Flu-exposed group. This finding suggested that in utero Flu-exposure induces an abnormal expression pattern of Cx43 that could influence the function of gap junctions and the migration of sperm cells across the seminiferous epithelium, as has been described previously [Durlej et al. 2011; Kopera et al. 2011]. During the progression of spermiogenesis, and when the spermatocyte moves into the lumen, Cx43 associ ates with plakophilin-2 to regulate the dynamic opening closing mechanism of the blood-testis barrier [Li et al. 2009], Therefore, the abnormal expression pattern of Cx43 could hinder the progression of spermatogenesis. Some studies have indicated that the increased expression of Cx43 could inhibit the proliferation of Sertoli cells, stimulating them to differentiate and mature. The loss of Cx43 could lead to the failure of spermatogenesis [Bozec et al. 2004; Cyr 2011; Lampe and Lau 2004; Salian et al. 2009]. In this current study, a significant decrease in the level of Cx43 protein expression was observed in the testes of Flu-exposed rats compared with controls on PD20 (/? < 0.01; Figure 1A-C, Table 1). Furthermore, a comparison of the expression of Cx43 mRNA between Flu-exposed rats with undescended testes and those with descended testes suggested that embryonic Flu-exposure could lead to cryptorchidism and the failure of germ cell development in cryptorchid testicles via the aberrant expression of Cx43. It has been previously demonstrated that Flu induces germ cell sloughing, which might result from the downregulation of Cx43 and the alteration of cell junction proteins [Sobarzo et al. 2009]. Therefore, the downregulation of Cx43 could inhibit the differentiation and maturation of Sertoli cells, leading to the failure of spermatogenesis. Although the exposure to Flu was only during pregnancy, the effects of androgenic interference are noted postnatally, as it influences spermatocyte maturity and spermatogenesis [Durlej et al. 2011], Rats treated with Flu on embryonic days 16-17 resulted in deranged gubernacular migration during the postnatal inguinoscrotal period, resulting in cryptorchidism in most rodents [Husmann and McPhaul 1992; Shono et al. 2004; Shono et al. 1994]. In the seminiferous tubules of human and rodent testes, this protein is distributed mainly in the basal compartment [Batias et al. 1999; Risley et al. 1992; Steger et al. 1999]. In our study, we found that rats had undescended testes in the Flu-exposed group on PD20, but there was no difference in the level of Cx43 protein expression between rats with undescended and descended testes. In the horse, there was a clear reduction of Cx43 expression in those with undescended testes [Hejmej and Bilinska 2008], It remains possible that Flu can alter the expression and distribution of Cx43, as the level of Cx43 expression was normal in the control group with normally descended testes. Further investigation will be needed to explore the Cx43 between the descended testes with undescended testes exposed to flutamide.
260
Syst Biol Reprod Med, 2014; 60(5): 257-262
C. Cai et al.
Our current studies confirmed that embryonic exposure to the anti-androgen, flutamide, interfered with normal Cx43 expression by the testicular spermatogenic epithelium. This could lead to disorders in the migration and development of male germ cells. However, the specific upstream and downstream regulatory mechanisms related to the expression of the Cx43 gene, and the expression changes of possible downstream genes, remain to be elucidated. M a t e r ia ls a n d M e th o d s A n im als a n d e x p e rim e n ta l design
The Ethics Committee of ChongQing Medical University provided approval (reference number: 20100333) for the study. Outbred SD rats were used in this experiment. The animals were kept at a constant temperature of 18-21°C, with approximately 50% humidity and a fixed 12-h controlled light/dark cycle, with free access to a standard diet and water. Fertile female rats were placed with two male rats until the commencement of pregnancy. The animals were examined each morning for vaginal plugs; the day of the appearance of plugs was taken as day 1 of gestation. Flutamide (F9397, Sigma-Aldrich Co., St. Louis, MO, USA) was dissolved in 5% ethanol and com oil and injected subcutaneously into the pregnant rats (25 mg/kg/day) on gestational days 12-21, as described previously [McIntyre et al. 2001]. The Flu-exposed groups of experimental animals were exposed prenatally on gestational days 12-21. The control animals were given vehicle alone. On PD20, in the control group, 10 immature male rats were selected at random; in the Flu-exposed group, respectively, 10 immature male rats with undescended testicles and 10 with normally descended testicles were selected at random. The testes were removed from each group of rats.
presence of a rabbit polyclonal anti-Cx43 antibody (1:2,000, abll370; Abeam, USA; http://www.abcam.com). As a nega tive control, the primary antibody was omitted in the treatment of some slides. Subsequently, sections were incubated for 20 min at 37°C in a humidified chamber with a horseradish peroxidase-secondary antibody conjugated polymer, goat anti-rabbit IgG (PV-6001, Zhongshan Goldenbridge Biotechnology Co. Ltd., China; http:// www.zsbio.com). Bound antibody was visualized by 3,3'diaminobenzidine (DAB, ZLI-9018; Zhongshan Goldenbridge Biotechnology). At the end of the reaction, all the sections were counterstained with hematoxylin. The sections were examined with an Olympus microscope (BX-51, Olympus, Japan, http://www.olympusmicro.com) and photographs were taken. W e s te rn b lo ttin g analysis
Each testis was cut into two small cubes, of which one was fixed in Bouin’s fixative, dehydrated, and embedded in paraffin. The second cube was immediately snap-frozen in liquid nitrogen for RNA and protein isolation.
Tissues were homogenized on ice with a cold Tris/EDTA buffer (50 mM Tris, 2mM EDTA, pH 7.5), sonicated, and centrifuged at 20,000 g for 30 min at 4°C. The protein concentration for each sample was estimated using the Bradford dye-binding procedure with BSA as a standard. Homogenates containing 50 pg of protein were solubilized in a sample buffer and heated at 99.9°C for 5 min. Equal amounts of protein and a prestained dual color protein molecular weight marker (Beyotime) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Then, blots were blocked overnight at 4°C with shaking in a solution of non-fat dry milk (5%) in phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST), followed by incubation with rabbit polyclonal antibody against Cx43 (1:5,000; Abeam) and mouse polyclonal antibody against GAPDH (1:5,000; Beyotime) for 4h at 37°C. The membranes were washed and incubated with a goat anti-rabbit IgG and anti-mouse IgG linked to horseradish-peroxidase (1:2,000; Zhongshan Goldenbridge Biotechnology) for 2 h at 37°C and blots were developed with an enhanced chemiluminescence system. Autoradiographic band intensities were quantified by Quantity One 21.0.0 software. For each sample, Western blots were performed at least three times.
Im m u n o h is to c h e m is try
R e a l-tim e q u a n tita tiv e PCR
Embedded testes were sectioned into 5-pm sections, deparaffinized, and rehydrated using standard techniques. Endogenous peroxidase activity was blocked by incubation in a solution of hydrogen peroxide (0.3%) in distilled water. To optimize immunohistochemical staining, sections were immersed in citrate buffer (0.01 M, pH 6.0) and placed in a pressure cooker (full pressure reached after 10 min) for high temperature antigen retrieval. Afterwards, sections were incubated for 3h at 37° C in a humidified chamber in the
Expression levels of Cx43 were quantified using real-time PCR on the StepOne™ Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). 01igo7 nucleotide sequences of the primers used are described in Table 2. Total cellular RNA from testes was isolated using RNA pure total RNA fast isolation kit (RP1202, Bioteke Co., Beijing, China); Nanodrop-1000 software was used to check the RNA concentration and agarose gel electrophoresis was used to check RNA quality. Then, 1 pg of RNA from each sample was
Tissue p re p a ra tio n
Table 2. Oligonucleotide sequences used in the quantitative PCR measurements. Gene name
GenBank accession no.
Connexin43
NM_012567.2
P-actin
NM_031144.2
Oligonucleotide sequence F:5'-CCGACGACAACCAGAATGCC-3' R: 5'-CCAACTCCACGGGAACGAA-3' F:5'- GGAGATTACTGCCCTGGCTCCTA-3' R: 5'-GACTCATCGTACTCCTGCTTGCTG-3'
Product size 233 bp 150 bp
DOI: 10.3109/19396368.2014.921738
reverse-transcribed to cDNA using a PrimeScript™ RT reagent kit (DRR037S, TaKaRa Biotechnology Co. Ltd., Dalian, China) according to the manufacturer’s instructions. For real-time PCR reactions, a master mix of the following reaction components was prepared to the indicated endconcentration: 8.8 pi RNase free water, 0.1 pi forward primer (20 pM), 0.1 pi reverse primer (20 pM), 9.0 pi 2.5 x Real Master Mix/20 x SYBR solution (FP202, Qiagen, Germany; http:// www.qiagen.com). PCR conditions were as follows: denaturation program (95°C for 3 min), amplification pro gram repeated 40 times (95°C for 20 s, 60°C for 15 s, 60°C for 30 s with a single fluorescence measurement), melting curve program (60-95°C with a heating rate of 0.3°C per second and a continuous fluorescence measurement), and finally a cooling step to 4°C. Samples were run in triplicate, and the average Ct (threshold cycle) values for Cx43 was normalized to the level of the housekeeping gene, P-actin. Statistical analysis
Data were expressed as the m ean±SD and the normal distribution of results were analyzed. Statistical analysis was carried out by ANOVA followed by the LSD test for multiple comparisons, or by the Student’s t-test to compare the differences between two sets of data, p-values
http://informahealthcare.com/aan ISSN: 1939-6368 (print), 1939-6376 (electronic)
informa
Syst Biol Reprod M ed, 2014; 60(5): 257 -2 6 2 © 2014 Inform a Healthcare USA, Inc. DOI: 10.3109/19396368.2014.921738
healthcare
RESEARCH C O M M U N IC ATIO N
Connexin 43 expression in Sprague-Dawley rat seminiferous epithelium after in utero exposure to flutamide Chunhong Cai1,2, Dan Zhao2, Cao Ma2, Yongbo Zhang2, Xing Wu2, Guanghui Wei1,2, and Dawei He1,2* 'Department of Pediatric Urology, Children's Hospital of Chongqing Medical University, Chongqing, China and 2Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China Abstract
Keywords
This study explored the expression of connexin 43 (Cx43) in the testes of prepubertal SpragueDawley (SD) rats following in utero flutamide (Flu) exposure. Connexins constitute the major protein type in gap junctions. Connexin 43, the most prominent connexin family member expressed by testes, is localized at the base o f seminiferous tubules in humans and rodents, and may be involved in fertility. Flutamide was injected subcutaneously into pregnant SD rats on gestational days 12-21 (25 mg/kg/day). Immunohistochemistry, Western blotting, and real-time PCR was used to investigate the distribution and the expression o f Cx43 protein and mRNA in the testis on postnatal day 20 (PD20). Following Flu-exposure, Cx43 was observed between Sertoli cells in the seminiferous tubules. On PD20, no Cx43 protein was expressed by the spermatogonial cell layer o f the seminiferous tubules in the controls, but was observed in the Flu-exposed group. Western blotting showed that Cx43 was expressed at significantly lower levels in Flu-exposed testes than controls on PD20 (p
C
u. re
* & 2-e c ro
***
o “ O
Figure 2. Expression o f connexin 43(Cx43) protein in flutamide (FIu)exposed Sprague-Dawley rat testes (n= 3). Western blots performed on testis extracts (50 pg protein) with the anti-Cx43 and the GAPDH antibody found the expected molecular size (upper panel). A significant decrease in Cx43 protein expression in the undescended testicle (Flu-C) and descended testicle (Flu-nC) groups of postnatal day (PD) 20 rats exposed to flutamide compared to control groups (lower panel). ***p< 0.001. Each band was quantified by densitometry using the Quantity One 21.0.0 software and expressed as arbitrary units (m ean ±S D ). The graphic shows pooled data from three independent experiments.
PD20 are shown in Figure 2. Connexin 43 protein expression was lower in undescended testes in the Flu-exposed group compared to controls on PD20 (p< 0.001), but no difference in Cx43 expression was found between undescended and descended testes in the Flu-exposed group at this time point. In the mammalian testis, immunohistochemistry and hybridization histochemical studies have demonstrated that Cx43 participates in the formation of homocellular gap junctions between adjacent Sertoli cells, and the heterocellular gap junction between Sertoli cells and spermatogonia or spermatocytes [Li et al. 2010; Palmiero et al. 2003; Tan et al. 1996]. With regard to our results, immunostaining for Cx43 showed that the distribution and expression of the Cx43 protein were different in the seminiferous tubule
259
(Figure 1A-C). The distribution and expression of Cx43 protein in the testicular seminiferous tubule showed that it moved from the basal compartment to the adluminal compartment in the control group. However, its expression remained abnormally distributed in the spermatogonium layer of the seminiferous tubule basilar membrane in the Flu-exposed group. This finding suggested that in utero Flu-exposure induces an abnormal expression pattern of Cx43 that could influence the function of gap junctions and the migration of sperm cells across the seminiferous epithelium, as has been described previously [Durlej et al. 2011; Kopera et al. 2011]. During the progression of spermiogenesis, and when the spermatocyte moves into the lumen, Cx43 associ ates with plakophilin-2 to regulate the dynamic opening closing mechanism of the blood-testis barrier [Li et al. 2009], Therefore, the abnormal expression pattern of Cx43 could hinder the progression of spermatogenesis. Some studies have indicated that the increased expression of Cx43 could inhibit the proliferation of Sertoli cells, stimulating them to differentiate and mature. The loss of Cx43 could lead to the failure of spermatogenesis [Bozec et al. 2004; Cyr 2011; Lampe and Lau 2004; Salian et al. 2009]. In this current study, a significant decrease in the level of Cx43 protein expression was observed in the testes of Flu-exposed rats compared with controls on PD20 (/? < 0.01; Figure 1A-C, Table 1). Furthermore, a comparison of the expression of Cx43 mRNA between Flu-exposed rats with undescended testes and those with descended testes suggested that embryonic Flu-exposure could lead to cryptorchidism and the failure of germ cell development in cryptorchid testicles via the aberrant expression of Cx43. It has been previously demonstrated that Flu induces germ cell sloughing, which might result from the downregulation of Cx43 and the alteration of cell junction proteins [Sobarzo et al. 2009]. Therefore, the downregulation of Cx43 could inhibit the differentiation and maturation of Sertoli cells, leading to the failure of spermatogenesis. Although the exposure to Flu was only during pregnancy, the effects of androgenic interference are noted postnatally, as it influences spermatocyte maturity and spermatogenesis [Durlej et al. 2011], Rats treated with Flu on embryonic days 16-17 resulted in deranged gubernacular migration during the postnatal inguinoscrotal period, resulting in cryptorchidism in most rodents [Husmann and McPhaul 1992; Shono et al. 2004; Shono et al. 1994]. In the seminiferous tubules of human and rodent testes, this protein is distributed mainly in the basal compartment [Batias et al. 1999; Risley et al. 1992; Steger et al. 1999]. In our study, we found that rats had undescended testes in the Flu-exposed group on PD20, but there was no difference in the level of Cx43 protein expression between rats with undescended and descended testes. In the horse, there was a clear reduction of Cx43 expression in those with undescended testes [Hejmej and Bilinska 2008], It remains possible that Flu can alter the expression and distribution of Cx43, as the level of Cx43 expression was normal in the control group with normally descended testes. Further investigation will be needed to explore the Cx43 between the descended testes with undescended testes exposed to flutamide.
260
Syst Biol Reprod Med, 2014; 60(5): 257-262
C. Cai et al.
Our current studies confirmed that embryonic exposure to the anti-androgen, flutamide, interfered with normal Cx43 expression by the testicular spermatogenic epithelium. This could lead to disorders in the migration and development of male germ cells. However, the specific upstream and downstream regulatory mechanisms related to the expression of the Cx43 gene, and the expression changes of possible downstream genes, remain to be elucidated. M a t e r ia ls a n d M e th o d s A n im als a n d e x p e rim e n ta l design
The Ethics Committee of ChongQing Medical University provided approval (reference number: 20100333) for the study. Outbred SD rats were used in this experiment. The animals were kept at a constant temperature of 18-21°C, with approximately 50% humidity and a fixed 12-h controlled light/dark cycle, with free access to a standard diet and water. Fertile female rats were placed with two male rats until the commencement of pregnancy. The animals were examined each morning for vaginal plugs; the day of the appearance of plugs was taken as day 1 of gestation. Flutamide (F9397, Sigma-Aldrich Co., St. Louis, MO, USA) was dissolved in 5% ethanol and com oil and injected subcutaneously into the pregnant rats (25 mg/kg/day) on gestational days 12-21, as described previously [McIntyre et al. 2001]. The Flu-exposed groups of experimental animals were exposed prenatally on gestational days 12-21. The control animals were given vehicle alone. On PD20, in the control group, 10 immature male rats were selected at random; in the Flu-exposed group, respectively, 10 immature male rats with undescended testicles and 10 with normally descended testicles were selected at random. The testes were removed from each group of rats.
presence of a rabbit polyclonal anti-Cx43 antibody (1:2,000, abll370; Abeam, USA; http://www.abcam.com). As a nega tive control, the primary antibody was omitted in the treatment of some slides. Subsequently, sections were incubated for 20 min at 37°C in a humidified chamber with a horseradish peroxidase-secondary antibody conjugated polymer, goat anti-rabbit IgG (PV-6001, Zhongshan Goldenbridge Biotechnology Co. Ltd., China; http:// www.zsbio.com). Bound antibody was visualized by 3,3'diaminobenzidine (DAB, ZLI-9018; Zhongshan Goldenbridge Biotechnology). At the end of the reaction, all the sections were counterstained with hematoxylin. The sections were examined with an Olympus microscope (BX-51, Olympus, Japan, http://www.olympusmicro.com) and photographs were taken. W e s te rn b lo ttin g analysis
Each testis was cut into two small cubes, of which one was fixed in Bouin’s fixative, dehydrated, and embedded in paraffin. The second cube was immediately snap-frozen in liquid nitrogen for RNA and protein isolation.
Tissues were homogenized on ice with a cold Tris/EDTA buffer (50 mM Tris, 2mM EDTA, pH 7.5), sonicated, and centrifuged at 20,000 g for 30 min at 4°C. The protein concentration for each sample was estimated using the Bradford dye-binding procedure with BSA as a standard. Homogenates containing 50 pg of protein were solubilized in a sample buffer and heated at 99.9°C for 5 min. Equal amounts of protein and a prestained dual color protein molecular weight marker (Beyotime) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Then, blots were blocked overnight at 4°C with shaking in a solution of non-fat dry milk (5%) in phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST), followed by incubation with rabbit polyclonal antibody against Cx43 (1:5,000; Abeam) and mouse polyclonal antibody against GAPDH (1:5,000; Beyotime) for 4h at 37°C. The membranes were washed and incubated with a goat anti-rabbit IgG and anti-mouse IgG linked to horseradish-peroxidase (1:2,000; Zhongshan Goldenbridge Biotechnology) for 2 h at 37°C and blots were developed with an enhanced chemiluminescence system. Autoradiographic band intensities were quantified by Quantity One 21.0.0 software. For each sample, Western blots were performed at least three times.
Im m u n o h is to c h e m is try
R e a l-tim e q u a n tita tiv e PCR
Embedded testes were sectioned into 5-pm sections, deparaffinized, and rehydrated using standard techniques. Endogenous peroxidase activity was blocked by incubation in a solution of hydrogen peroxide (0.3%) in distilled water. To optimize immunohistochemical staining, sections were immersed in citrate buffer (0.01 M, pH 6.0) and placed in a pressure cooker (full pressure reached after 10 min) for high temperature antigen retrieval. Afterwards, sections were incubated for 3h at 37° C in a humidified chamber in the
Expression levels of Cx43 were quantified using real-time PCR on the StepOne™ Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). 01igo7 nucleotide sequences of the primers used are described in Table 2. Total cellular RNA from testes was isolated using RNA pure total RNA fast isolation kit (RP1202, Bioteke Co., Beijing, China); Nanodrop-1000 software was used to check the RNA concentration and agarose gel electrophoresis was used to check RNA quality. Then, 1 pg of RNA from each sample was
Tissue p re p a ra tio n
Table 2. Oligonucleotide sequences used in the quantitative PCR measurements. Gene name
GenBank accession no.
Connexin43
NM_012567.2
P-actin
NM_031144.2
Oligonucleotide sequence F:5'-CCGACGACAACCAGAATGCC-3' R: 5'-CCAACTCCACGGGAACGAA-3' F:5'- GGAGATTACTGCCCTGGCTCCTA-3' R: 5'-GACTCATCGTACTCCTGCTTGCTG-3'
Product size 233 bp 150 bp
DOI: 10.3109/19396368.2014.921738
reverse-transcribed to cDNA using a PrimeScript™ RT reagent kit (DRR037S, TaKaRa Biotechnology Co. Ltd., Dalian, China) according to the manufacturer’s instructions. For real-time PCR reactions, a master mix of the following reaction components was prepared to the indicated endconcentration: 8.8 pi RNase free water, 0.1 pi forward primer (20 pM), 0.1 pi reverse primer (20 pM), 9.0 pi 2.5 x Real Master Mix/20 x SYBR solution (FP202, Qiagen, Germany; http:// www.qiagen.com). PCR conditions were as follows: denaturation program (95°C for 3 min), amplification pro gram repeated 40 times (95°C for 20 s, 60°C for 15 s, 60°C for 30 s with a single fluorescence measurement), melting curve program (60-95°C with a heating rate of 0.3°C per second and a continuous fluorescence measurement), and finally a cooling step to 4°C. Samples were run in triplicate, and the average Ct (threshold cycle) values for Cx43 was normalized to the level of the housekeeping gene, P-actin. Statistical analysis
Data were expressed as the m ean±SD and the normal distribution of results were analyzed. Statistical analysis was carried out by ANOVA followed by the LSD test for multiple comparisons, or by the Student’s t-test to compare the differences between two sets of data, p-values
