Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2005; 7 (3):295-301 |
|
| กก | |
This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - Original Article - Localization and potential function of androgen receptor in rat salivary gland Hong-Jun Li1, Bing Yao1, Wei Liang2, Yong-Mei Wang4, Qi-Lu Xu3, Yu-Feng Huang1 1Department of Reproduction, Nanjing Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing 210002, China Abstract Aim: To investigate the localization and quantity of androgen receptor (AR) in the salivary glands of rats with further analysis on the effect of castration. Methods: Sixty male Wistar rats, aged 30-60 days, were randomly divided into three groups (castrated, sham-operated and normal controls) with 20 rats in each group. The rats in the castrated group were castrated and the submaxillary glands were removed after 1 week. The salivary glands of the rats in the sham-operated and the normal control groups were also removed. Parts of the salivary glands were fixed for immunohistochemistry and in situ hybridization assays. Other parts were used for Western blot. Results: AR immunoreactivity in the three groups was localized in the glandular epithelial cells of the serous acinus and the glandular duct of the salivary gland, mainly in the nuclei. AR mRNA hybridization signals in the salivary glands of the castrated group were mainly distributed in the epithelial cells of the convoluted and secretary ducts; AR mRNA in the sham-operated and the normal control groups were found in the epithelial cells of the convoluted, the secretary and the excretory ducts. The quantity of AR in the salivary glands was decreased significantly in the castrated rats compared with the sham-operated and the normal controls. Moreover, epidermal growth factor (EGF) secreted by the salivary glands was also decreased in the castrated rats. Conclusion: Castration appears to affect the production of AR in the salivary gland and the distribution of the AR mRNA and could further affect the function of the salivary gland. The changes of AR and the distribution of AR mRNA may play an important role in the interactions between the testes and the salivary gland. (Asian J Androl 2005 Sep; 7: 295-301) Keywords: androgen receptor; immunohistochemistry; in situ hybridization; Western blot; salivary gland Correspondence to: Dr Bing Yao, Department of Reproduction, Nanjing Jinling Hospital, Clinical School of Medical College, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, China. Tel: +86-25-8086-0174, Fax: +86-25-8481-5730 1 Introduction The androgen receptor (AR), a member of the steroid/nuclear receptors superfamily, is a ligand-dependent transcription factor that increases or decreases the expression of its target genes and is involved in the regulation of development, differentiation and maintenance of male reproductive functions as well as in the generation of sexually dimorphic characteristics in nongenital tissues. Upon androgen binding, the AR acquires a new conformational state that renders it to be capable of interacting with the specific steroid response element. Liganded AR is also able to interact with other transcriptional factors and coactivators. The coactivators further recruit complexes with chromatin remodeling activities [1]. AR is reported to colocalize with epidermal growth factor receptor (EGF-R) at the membrane of the prostate cancer cell line, LNCaP. Androgen has an effect on epidermal growth factor (EGF)-mediated signaling related to invasion in LNCaP [2]. Some genes are involved in the effect of androgen on the modulating secretion of EGF in this cell line, such as prostin-1, urokinasetype plasminogen activator and neutral endopeptidase [2]. Recently, AR has been shown to be associated with c-Src kinase family membranes and caveolin-1 in LNCaP. Both src and caveolin-1 are also capable of interacting with EGFR and are involved in EGF-stimulated signaling [3]. Studies found that the mature dysfunction of the sperm, ultrastructural changes of the Leydig cells and a decrease in plasma luteinizing hormone (LH) level happened after the salivary gland of the rat was removed [4]. These changes can be reversed by administration of EGF that is produced mainly in the salivary gland [5]. Furthermore, testosterone could increase the EGF expression [6] and change the amylase activity in the salivary gland. The EGF mRNA levels could fall after male mice were castrated [7]. So it is hypothesized that an axis might exist between the testis and the salivary gland [8]. Our previous studies demonstrated that gonadotropin-releasing hormone (GnRH) could stimulate the secretion of EGF from the salivary gland [9]. GnRH receptor and AR were co-localized in the salivary gland [10]. AR mRNA was distributed in the glandular epithelial cells of the serous acinus and the epithelial cells in all gland ducts [11]. These data suggested that: 1) AR could be produced by the salivary gland; 2) in the salivary gland, AR is localized in the epithelium from which the EGF is secreted; 3) androgen might bind to AR to modulate the secretion of EGF as well as GnRH. In addition, because androgen often regulates the expression of its own receptor [12], castration may also be expected to affect the AR in the salivary gland. The present experiment employed immunohistochemical and in situ hybridization methods to investigate the localization of AR and the distribution of AR mRNA in the salivary gland following castration and on the level of EGF in the salivary gland.
2 Materials and methods 2.1 Reagents Nitro-blue-tetrazolium (NBT) and 5-bromo-4-chloro-3-indilyl-phosphate (BCIP) were supplied by the Department of Histology and Embryology of the Second Military Medical University. The ABC kit was a Vector product (Burligame, CA,USA). The in situ hybridization kit was purchased from the Boster Company in Wuhan, China. The Digoxigenin-labeled AR oligonucleotide probe was produced from TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China). The sequence of the probe was, 5'-ctggttgtca ctacggagct ctcacttgtg cagctgcaa-3', which was used for AR mRNA in situ hybridization. The polyclonal AR antiserum (PG-21) was from UpState Biotech (Lake Placid, NY, USA). Antiserum produced against EGF(3124) in rabbit was purchased from Wakunaga Co. (Hiroshima, Japan). The anti-rabbit IgG-peroxidase and BM blue POD substrate were from Roche (Indianapolis, IN, USA). The densitometric analysis was performed with Image Gauge software (Fuji, Tokyo, Japan). 2.2 Animals and sample collection Sixty male Sprague-Dawley rats (200 g-250 g, postnatal 30-60 days) bred in our hospital were used for the present experiments. Rats were maintained in light (5:00-19:00) and temperature (23 ºC ± 3 ºC) controlled rooms and allowed free access to food and tap water. All procedures employed in the present study were done following the guidelines for animal treatment of Nanjing Jinling Hospital, which is in accordance with the principles and procedure of the NIH guide for the care and use of laboratory animals. The animals were randomized into three groups of 20 rats each. One group was used as unoperated controls, another group was sham-operated, and the third group was surgically castrated under anesthesia as previously described [13]. After one week, all rats were sacrificed at the same time of the day by decapitation. The salivary gland was quickly removed and divided into two parts. One part was immediately fixed in Bouin's solution for 12 h, embedded in paraffin, then 5-mm sections were prepared. The other part (approximately 300 mg) was suspended in 1 mL 0.01 mol/L PBS, pH 7.4, and homogenized (3 × 10 s bursts on ice) with a Polytron homogeniser. Then this suspension was centrifuged at 10000 × g for 20 min at 4 ºC. The supernatant was taken and quantified. The protein was then kept at -20 ºC. 2.3 AR immunohistochemical procedure The paraffin sections were dewaxed, hydrated and incubated in methanol-H2O2 for 20 min to remove endogenous peroxidase. They were then stained according to the immunohistochemical ABC method. Tissue sections were incubated at 4 ºC for 24 h in the primary antibodies of PG-21 (1:1000 dilution). The secondary antibody, biotin-labeled goat anti-rabbit IgG (1:200 dilution), was incubated at room temperature for 1 h, and ABC complex (1:100 dilution) was incubated at room temperature for 30 min. The negative control tissue sections were subjected to the same procedures except that the diluted rabbit serum replaced the first antibody. 2.4 Hybridization procedure The in situ hybridization of AR mRNA was carried out on the paraffin sections. The sections were dewaxed, hydrated, and then rinsed in distilled water three times for 5 min each time, and in proteinase K (1 µg/mL) at 37 ºC for 20 min. The sections were post-fixed in 4 % paraformaldehyde for 5 min, and rinsed in distilled water three times for 5 min each time. The hybridization buffer containing the AR oligonucleotide probe (2.0 µg/mL) was applied to the appropriate sections and hybridization was conducted in a sealed humid box at 43 ºC for 12-18 h. The sections were washed in 2 × saline sodium citrate (SSC) at 37 ºC twice for 5 min each time, then in 0.1 × SSC at 37 ºC for 10 min, incubated in mouse anti-Dig antibody at 37 ºC for 2 h, rinsed in 0.5 mol/L TBS three times for 2 min each time, incubated in biotin labeled goat anti-mouse IgG at 37 ºC for 1 h, rinsed in 0.5 mol/L TBS three times for 2 min each time, incubated in SABC-AP at 37 ºC for 30 min, and rinsed in 0.5 mol/L TBS four times for 5 min each time. The alkaline phosphatase reaction was conducted by incubation with the complex solution of NBT and BCIP for 30-50 min. The sections were washed in water to stop the reaction, then dehydrated in alcohol, cleared in xylene, and mounted on Canada balsam. The negative controls were processed as described above but in the absence of the labeled probe and were incubated with normal mouse serum instead of mouse anti-Dig antibody. An additional control group of in situ hybridization was carried out as previously described in which the sections were treated with labeled probe mixture in the presence of a 100-fold excess concentration of unlabeled probe mixture [14]. 2.5 Western blot Following the above procedure, 30 µg of each sample was separated with 12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and then transferred onto immun-blotTM PVDF membrane (Hercules, CA, USA). The membrane was then blocked and incubated with PG-21 (1:1000 diluted with blocking solution) or anti-EGF (3124) antibody (1:1000 diluted with blocking solution) at room temperature for 1 h. After three 5-min washes, the membrane was incubated with anti rabbit IgG-Peroxidase antibody (1:1000 diluted with blocking solution) at room temperature for 1 h. After a further three 10-min washes, the BM blue POD substrate(Indianapolis, IN, USA) was added and the blue band appeared. A picture was taken and Image Gauge software was used to analyze the density of each band. 2.6 Statistical analysis The results were evaluated statistically using Student's t-test and P < 0.05 was considered significant.
3 Results 3.1 Localization of AR in salivary gland AR is dark brown in color with a light yellow, non-staining background in AR immunoreactive positive cells (Figure 1). The glandular epithelial cells of the serous acinus and the epithelial cells of the secretory duct, the excretory duct and the granular convoluted tubule showed AR immunoreactivity and the immunoreactive materials were mainly distributed in the nuclei. The distribution of AR in salivary glands is similar in castrated, sham-operated and normal groups (Figure 1). 3.2 Distribution of AR mRNA in rat salivary gland The AR hybridization signal of dark blue color can be easily identified (Figure 2). AR mRNA hybridization signals were detected in the epithelium of the salivary gland serous acinus, but not in the mucous acinus. Epithelial cells in the intercalated duct and the secretory duct in castrated rats exhibited AR mRNA hybridization signals, which were present in cytoplasm but not in nuclei, but the signals of AR mRNA in the sham-operated and the normal control groups were distributed in the epithelial cells of intercalated duct, secretory duct and excretory duct. The patterns were the same as that we have reported previously [11] (data not shown). 3.3 Effects of castration on the expression of AR and EGF in salivary gland The expression of AR and EGF in the salivary gland was analyzed by Western blot. As shown in Figure 3, castration resulted in a significant decrease in AR compared with that in the sham operation and the normal control groups (P < 0.05). EGF was also decreased significantly in the castrated rats compared with those in the sham-operated and the normal control rats (P < 0.05) (Figure 4).
4 Discussion Salivary proteins come in a variety of different forms with diverse functions. In some cases, a salivary protein's function has yielded to biochemical studies. For example, salivary amylase digests dietary starches, glycoproteins lubricate hard and soft tissues, and some peptides retard the growth of bacteria and fungi in the oral cavity. For other salivary proteins, however, the function has remained elusive. AR and androgen binding protein (ABP) are the two of those. Verhoeven and Wilson [15] found an androgen-binding complex to exist from birth and throughout the life of normal mice that could be precipitated at 40 % saturation with ammonium sulfate. Studies demonstrated that ABP is produced de novo in the submaxillary gland and to be composed of two different disulfide-bridge subunits, alpha (ca. 12 000 daltons) and beta (ca. 8800 daltons). ABP binds with the male sex steroid hormone in mouse salivary glands [16], but does not precipitate out of saliva before 70 % ammonium sulfate saturation, indicating that the AR and mouse salivary ABP are not the same protein. Sawada and Noumura [17] using anti-AR antibodies found that the specific immunoreactivity to AR appeared only in the cell nuclei of the acini, the intercalated ducts, the granular convoluted tubules (GCT) and the excre tory striated ducts in the salivary glands of mice of both sexes. This result is consistent with our previous findings in the salivary gland of the male rat using PG-21 [10]. This distribution suggested that AR might have the biologic function in salivary gland. It is reported that androgen often regulates the expression of its own receptors [12], indicating a biologic relationship exists, but, the effect of castration on AR in extra-testis organs such as the prostate or salivary gland remains obscure. Kumar et al. [18] reported that the levels of AR mRNA in the prostate increased following castration. The maximum level was achieved in 5 days, and a decline in the level of AR mRNA was observed after 5 days. However, Husmann et al. [19] found AR in the prostate decreased 24 h after castration. Nagai et al. [20] found AR mRNA increased in the castrated male mice, while the administration of testosterone reduced the AR mRNA in female mice, and suggested that the amount of AR mRNA in mouse salivary gland can be reduced by androgen. In our present study, we used 1-week-old castrated rats and found AR immunoreactivity decreased. The localization of AR after castration is similar to that in our two controls. This result is consistent with Sawada and Noumura's [17] findings that 1-week castration did not cause any changes in the intracellular distribution of androgen receptor and the percentage of immunoreactive cells in each region of the adult salivary gland. The reasons our results are different from other reports [21] might be: 1) the method for tissue preparing is different (we utilized Bouin's solution, which can protect the protein from being lost from the tissue); and 2) the antibody is different (PG-21 is a polyclonal antibody raised against a synthetic peptide corresponding to the first 21 N-terminal amino acids of the androgen receptor). The exact reason should be further clarified by comparing the distribution of AR in different tissues using this fixed method and PG-21 to those of Sar et al. [21]. This work is currently being done in our laboratory. The hybridization signals of AR mRNA were detected in glandular epithelial cells of serous acinus and epithelial cells but not in epithelial cells of the excretory duct in the castrated rats. This different distribution compared to AR immunoreactivity may reflect a modulation of the transcript of AR mRNA in the salivary gland. We, hence, suggest that the decrease of AR in castrated rats may be caused by the change in distribution of AR mRNA. Our laboratory is planning to investigate the detailed mecha nism by organ culture and to further study the quantity of AR mRNA using real-time reverse transcriptase PCR (RT-PCR) to clarify the mechanism of the decrease of AR after castration. EGF is a potent regulator of cell growth and differentiation in many tissues, and its level is regulated by several hormones in a tissue-specific fashion. Testosterone can increase EGF levels in the salivary glands. Treating female mice with testosterone first raises the level of prepro-EGF messenger RNA in the salivary gland 3 days after the treatment, and the levels approach those in the male after approximately 1-2 weeks [6]. The castration of adult male mice can cause EGF mRNA levels in the salivary gland to begin to fall within 24 h [6]. In the present study, we found a decrease of EGF in the salivary gland 1 week after castration. Because this decrease is accompanied by the decrease of AR, we hypothesized that the decrease in the expression of EGF in the salivary gland might be related to that of AR. More and more evidence indicates that the salivary gland exerts not only an exocrine function but also an endocrine function. For the exocrine function, the salivary gland secretes many enzymes in the oral cavity to exert many digestive functions; for the endocrine function, it can synthesize EGF and nerve growth factor (NGF). Recent studies revealed that the salivary gland is closely related to the testis. EGF secreted mainly from the salivary can modulate the spermatogenesis as an autocrine and/or paracrine factor. Aida et al. [22] reported that EGF played a role in male germ cell development and suggested that EGF might be involved in the paracrine control of spermatogenesis in vivo. Removing the salivary gland can also cause a decrease in testosterone secreted from the testis [23]. On the other hand, testosterone can increase the expression of EGF from the cultured salivary gland [6]. The secretion of amylase from the salivary gland and the production of EGF mRNA in salivary glands were decreased by castration [7]. If the functional relationship between the salivary gland and the testis can be clarified, it will help us to understand the detailed function of the testis and to further modulate the function of the testis involved in reproduction. The detailed mechanism of the modulation in androgen, AR and the EGF mRNA in the salivary gland after castration needs to be further studied.
References 1 Rosinski-Chupin I, Huaulme JF, Rougeot C, Rougeon F. The transcriptional response to androgens of the rat VCSA1 gene is amplified by both binary and graded mechanisms. Endocrinology 2001; 142: 4550-9. 2 Bonaccorsi L, Carloni V, Muratori M, Formigli L, Zecchi S, Forti G, et al. EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen-sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR). Int J Cancer 2004; 112: 78-86. 3 Mariotti A, Kedeshian PA, Dans M, Curatola AM, Gagnoux-Palacios L, Giancotti FG. EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion. J Cell Biol 2001; 155: 447-58. 4 Boyer R, Jame F, Arancibia S. A non-exocrine function of the salivary gland. Ann Endocrinol 1991; 52: 307-22. 5 Wahab-Wahlgren A, Martinelle N, Holst M, Jahnukainen K, Parvinen M, Soder O. EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro. Mol Cell Endocrinol 2003; 201: 39-46. 6 Sheflin LG, Brooks EM, Keegan BP, Spaulding SW. Increased epidermal growth factor expression produced by testosterone in the salivary gland of female mice is accompanied by changes in poly-A tail length and periodicity. Endocrinology 1996; 137: 2085-92 7 Shear M, Barbakow F, King G. Histochemically demonstrable changes in amylase activity in submaxillary glands of puberal male mice following castration. S Afr J Med Sci 1975; 40: 133-44. 8 Yan YC, Sun YP, Zhang ML. Testis epidermal growth factor and spermatogenesis. Arch Androl 1998; 40: 133-46. 9 Yao B, Huang W, Huang Y, Chui Y, Wang Y, Li H, et al. A study on the localization and distribution of GnRH and its receptor in rat salivary glands by immunohistochemical, in situ hybridization and RT-PCR. Life Sci 2003; 72: 2895-904. 10 Yao B, Fan J, Wang YM, Cui YX, Huang YF. The distribution of androgen receptor and its coexistence with gonadotropin releasing hormone receptor in rat salivary gland. Zhonghua Nan Ke Xue 2002; 8: 32-4. 11 Xu QL. The localization and potential function of androgen receptor mRNA in rat salivary gland. Zhonghua Nan Ke Xue 2003; 9: 681-3. 12 Lu SF, McKenna SE, Cologer-Clifford A, Nau EA, Simon NG. Androgen receptor in mouse brain: sex differences and similarities in autoregulation. Endocrinology 1998 ; 139(4): 1594-601. 13 Colombel MC, Buttyan R. Hormonal control of apoptosis: the rat prostate gland as a model system. Methods Cell Biol 1995; 46: 369-85. 14 Bauer-Dantoin AC, Jameson JL. Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle. Endocrinology 1995; 136: 4432-8. 15 Verhoeven G, Wilson JD. Cytosol androgen binding in submandibular gland and kidney of the normal mouse and the mouse with testicular feminization. Endocrinology 1976; 99:79-92. 16 Dlouhy SR, Taylor BA, Karn RC. The genes for mouse salivary androgen-binding protein (ABP) subunits alpha and gamma are located on chromosome 7. Genetics 1987; 115: 535-43. 17 Sawada K, Noumura T. Developmental pattern of androgen receptor immunoreactivity in the mouse submandibular gland. Zool Sci 1995; 12: 243-8. 18 Kumar VL, Majumder PK, Kumar V. In vivo modulation of androgen receptor by androgens. Asian J Androl 2002; 4: 229-31. 19 Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD. Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to nuclei of target cells in the rat and human prostate. Endocrinology 1990; 126: 2359-68. 20 Nagai M, Nemoto T, Masuda T, Ota M. Reduction of androgen receptor mRNA concentration by testosterone in mouse submandibular gland. Endocr J 1995; 42: 31-8. 21 Sar M, Lubahn DB, French FS, Wilson EM. Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 1990; 127: 3180-6. 22 Wahab-Wahlgren A, Martinelle N, Holst M, Jahnukainen K, Parvinen M, Soder O. EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro. Mol Cell Endocrinol 2003 ; 201(1-2): 39-46. 23 Pollegrini A, Conte G, Ricciardi MP, Ferdeghini M. Effects of the ablation of the submaxillary gland on the rat testis. Z Mikrosk Anat Forsch 1988; 102: 760-8. |
| กก | |
