Ultrastructural Pathology, 2015; 39(2): 121–130 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2015.1009222
ORIGINAL ARTICLE
Age-Dependent Sex Hormone-Binding Globulin Expression in Male Rat Yaqing Li, MD1, Xiaoli Li, MD1, Huijie Fan, MD, PhD1, Xiaoran Li, MSc2,3, Yali Zhong, MD1, Jing Cao, MD1,4, Dandan Yu, MD1, Mingzhi Zhang, MD, PhD1, Jian-Guo Wen, MD, PhD5, Li Geng, MD, PhD1, and Zhenhe Suo, MD1,2,3 1
Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Henan, Zhengzhou, China, Department of Pathology, The Norwegian Radium Hospital, Oslo, Norway, 3Department of Pathology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway, 4Departments of Pathology, The Third Affiliated Hospital of Zhengzhou University, Henan, Zhengzhou, China, and 5The Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
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ABSTRACT Sex hormone binding globulin (SHBG) is known as a carrier protein, classically thought to be mainly synthesized in the liver and then secreted into the circulating system, where it binds to sex steroids with a high affinity and modulates the bioavailability of these hormones. In humans, the organs other than the liver known to produce SHBG include the brain, uterus, testis, prostate, breast and ovary, and the locally expressed SHBG is considered to play an important role in various physiological and pathological processes. A few studies of SHBG in rats were reported, but systemic SHBG studies in consideration of different organs and aging are currently missing. So we examined the SHBG expression in the brain, liver, prostate, and serum in 40 Sprague–Dawley male rats in four different groups (newborn, 2, 6, and 12 months old, respectively) with 10 in each group by immunohistochemistry, immunofluorescience microscopy, qRT-PCR, ELISA, western blotting, and laser confocal microscopy. We discovered that SHBG was increasingly expressed in all the three tissues along with age, and the SHBG protein expression was observed in the cytoplasm and membrane of neurons, hepatocyte, and prostate epithelial cells. The ELISA assay of the sera also supported an increasing SHBG level along with age. It is concluded that the locally synthesized SHBG in the liver, brain, and prostate and the circulating SHBG of male SD rats are positively associated with age, further indicating a potential role of SHBG in aging. Keywords: Age-dependent, laser confocal microscopy, male rat, prostate, SHBG
by controlling the availability of androgens and estrogens. In plasma, SHBG controls the metabolic clearance rate of sex steroids. Except for the sertoli cells in testis, it was reported that the SHBG gene was also expressed in other tissues in rats. It has been reported that the SHBG gene expression was discovered in fetal rat liver and adult brain [3], while others identified the protein in the rat hypothalamo-neurohypophyseal system, colocalized with oxytocin [4,5]. The intrinsic expression
Sex hormone-binding globulin (SHBG) or androgenbinding globulin (ABG) is an extracellular androgen and estrogen carrier. It interacts with its ligands, 5adihydrotestosterone (5a-DHT), and testosterone, with high affinity [1]. In rat, it has been verified that the sertoli cells produce the SHBG which influences androgen actions in the seminiferous tubules and epididymis [2]. There is strong evidence that the binding protein does act to modulate the gene regulatory actions of nuclear sex steroid receptors
Received 31 December 2014; Accepted 14 January 2015; Published online 13 April 2015 Correspondence: Dr. Zhenhe Suo, Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Montebello, Ullernchausseen 70, N-0310, Oslo, Norway. E-mail: [email protected] Dr. Li Geng, Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China. E-mail: [email protected]
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122 Y. Li et al. of SHBG in magnocellular hypothalamic neurons, in part co-localized with either vasopressin or oxytocin, was also described recently. With immunoelectronmicroscopic double labeling, they found co-localization of oxytocin and SHBG in a portion of the large dense-core vesicles in paraventricular and supraoptic perikarya and in axonal varicosities in the median eminence and in the posterior lobe. SHBG was found throughout the brain, with highest levels in the hippocampus and cortex and lowest levels in the diencephalon and cerebellum [6]. In human, it is reported that SHBG levels increased with increasing age in the circulating system [7], and the role of circulating SHBG in aging and ageassociated physiological changes such as osteoporosis and obesity has been verified. SHBG is an active participant in many of these age-associated physiological changes [8]. Jirikowski et al. [9] have recently demonstrated that SHBG is co-localized in prostate and benign prostate hyperplasia with oxytocin. The colocalization of these two important endocrine elements in the prostate may suggest an interaction in their function in prostate cells. Hoskin et al. [10] have found significantly decreased circulating SHBG levels in women with Alzheimer’s disease and suggested that SHBG may play more important role in brain than in liver. In addition, results from the Study of Women Across the Nation (SWAN) indicate an important relationship between SHBG and metabolic syndrome components and SHBG has a stronger relationship with obesity and metabolic disease endpoints than estrogen or testosterone [11,12]. Schock et al. [13] demonstrated increased SHBG in cardiomyocytes of men with dilated cardiomyopathy, another possible indication of SHBG in the process of heart aging. In the female rats, it was found that infusions of SHBG into the medial preoptic area-anterior hypothalamus could facilitate female sexual receptivity, suggesting that SHBG may be a marker of sexual activity and reproductive fitness and it may also mean that SHBG is the ultimate aging hormone [14]. SHBG is colocalized in vesicles with the reproductive hormone oxyctin, which is also verified to be released into the blood during sexual activity in a number of species [15]. But in rats, it is still not fully understood whether SHBG plays a role in aging process and age-associated physiological changes. Therefore, the purposes of our present study were to characterize the age-association of SHBG expression in the serum, liver, brain, and prostate of different age groups of male Sprague– Dawley (SD) rats, and to further explore the SHBG cellular localization in these tissues. The reason why we chose only male rats in this study was due to our project in a prostate cancer mouse model study, which is currently undergoing in our lab. It was discovered that SHBG distributed in the cytoplasm and membranes in neurons of brain, hepatocytes and prostate epithelial cells, and its expression level generally
increased with age. Furthermore, age-dependent increasing levels of SHBG were verified in the sera of the rats. Collectively, our results support an agedependent SHBG expression both in the liver, brain and prostate tissues and in the serum of SD rat, indicating a role of SHBG in aging.
MATERIAL AND METHODS Ethics statement Animals used in the present study were maintained and euthanized in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The animal study was approved by the Institutional Animal Care and Use Committees of the China Medical Association (Permit Number: SCXK 2013-0001).
The animals Forty Sprague–Dawley (SD) male rats, of clean grade and male gender (divided into newborn, 2-, 6-, and 12-month old groups and each age group with 10 rats), were obtained from The Experimental Animal Center of Shandong anti-lu pharmaceutical co., LTD, Shandong, China. About 10% chloral hydrate was used for anesthesia by intraperitoneal injection, and the dosage was .4 ml/100 g body weight. Then the animals were dissected and the left frontal area of the cerebrum of the brain, liver, and dorsal prostate were removed for further immunohistochemistry, immunofluorescence microscopy, quantitative RT-PCR, Western blotting, and laser confocal microscopy assays. Serum specimens were collected from all the rats. The sera were centrifuged at 4000 rpm for 15 min, and then the supernatant was immediately removed and the sediments were kept frozen at 80 C until use.
PCR and sequencing assay About 200 mg of fresh brain, liver, and prostate from each rat was placed in a reaction tube with 1 ml Trizol (Invitrogen, Carlsbad, CA) and homogenized mechanically (Ultraturrax T3, Biosafter, Beijing, China) followed by sonication. Homogenized samples were incubated for 5 min at RT. Then .6 ml chloroform was added and the tubes were shaken by hand for 15 s. After an incubation of 3 min at RT, samples were centrifuged at 12,000 g for 15 min at 4 C. The aqueous phase, containing the isolated RNA, was placed into a new tube. For RNA precipitation samples were mixed with 1 ml of 100% isopropanol, incubated for 10 min at RT and then again centrifuged for 10 min at 12,000 g at Ultrastructural Pathology
Sex hormone binding globulin in rat 4 C. Supernatant was removed from the tube. The pellet was resuspended by briefly vortexing in 3 ml of 75% ethanol. After centrifugation (7500g, 5 min, 4 C), the supernatant was discarded. The air-dried pellet was resuspended in 50 ml of RNase-free water. After that the genomic DNA elimination reaction was conducted. Up to 1 mg of total RNA, 1.0 ml of gDNA eraser were added to a total of 10.0 ml reaction for 2 min at 42 C. Then the total RNA of the tissues was extracted, and each RNA sample (3 mg) was subjected to cDNA synthesis by means of RevertAid First Strand cDNA Synthesis (Thermo Scientific, West Palm Beach, FL). Quantitative real-time PCR was performed using the Quantitative SYBR Green PCR Kit (Thermo Scientific, West Palm Beach, FL) on the ABI PRISM 7500 Fast system (Applied Biosystems, Life Technologies, Foster City, CA). All experiments were performed as specified in the protocols of the manufacturer. The primers used were as follows: SHBG forward primer, CTTCTTGGCTCACCCTT CAC; SHBG reverse primer, ACCTCCATCTTTGG TCCTTG; GAPDH forward primer: ACAGCAA CAGGGTGGTGGAC; GAPDH reverse primer: TTTGAGGGTGCAGCGAACTT. All samples were analyzed for three parallel samples and repeated three times at least, and the relative expression of the target gene was calculated with 2 DD Ct values with the Application Relative Quantification Study Program (Applied Biosystems, Life Technologies, Foster City, CA). About 3% agarose gel electrophoresis and Sequencing (Sangon Biotech, Shanghai, China) of the product were conducted to confirm the specificity of the PCR products.
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density of the target protein compared with GAPDH was quantified with the Bio-Rad Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA).
Immunohistochemistry All the liver, brain, and prostate tissues of each animal were fixed with 4% buffered paraformaldehyde (PFA, Hangzhou, China) and embedded in paraffin blocks before 4 mm sections were prepared for immunohistochemical examination. The sections were deparaffinized in xylene and then microwaved in 10 mM citrate buffer (pH 6.0) in order to unmask the epitopes. Endogenous peroxidase activity was blocked by incubation with .03% hydrogen peroxide in methanol for 20 min. Sections were incubated with the polyclonal rabbit anti-human SHBG antibody (1:300, Cat. no. 251544, Abbiotec, San Diego, CA) for 30 min at room temperature. After gently rinsed three times with washing buffer, the sections were then incubated with the peroxidase-labeled polymer conjugated to goat anti-rabbit IgG (Boaos, Beijing, China) for 30 min before stained for 2 min with 3‘3-diaminobenzidine tetrahydrochloride (DAB) (ZSGB-Bio, Beijing, China), counterstained by hematoxylin, dehydrated, and mounted in Diatex. Non-immune rabbit IgG in exactly the same concentration as the primary antibody was applied as a negative control, and known SHBG positive human liver tissue sections were used as positive controls.
Immunofluorescence microscopy Western blotting The tissues were dissolved with lysis buffer containing RIPA buffer (Thermo Scientific, West Palm Beach, FL), 1% PMSF, 1% aprotinin, 1% leupeptin, 1% pepstatin, and .5% vanadate by pipetting gently up and down, put on ice before spun down at 14,000 rpm for 15 min at 4 C to release total protein. About 50 mg proteins from each sample in SDS-loading buffer was boiled for 5 min and subjected to 10% SDS-PAGE electrophoresis and then electro-transferred to highquality polyvinylidene difluoride (PVDF) membrane (Boster, Wuhan, China). The membrane was blocked with 5% fat-free milk for 1 h at room temperature and incubated overnight at 4 C with a goat anti-human SHBG antibody (1:300, Cat. no. 251544, Abbiotec, San Diego, CA). After washing with PBS-tween .05% (PBST), the blot was incubated with rabbit anti-goat IgG HRP antibody for 45 min at room temperature (1:500, Boster, Wuhan, China). After three washes with PBST, the blot was visualized using an enhanced chemiluminescence detection kit (ECL, Amersham, UK) by following the manual guide. The relative band !
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SHBG immunofluorescence microscopy was performed on 4 mm sections. The sections were deparaffinized in xylene and then microwaved in 10 mM citrate buffer (pH 6.0) to unmask the epitopes. Endogenous peroxidase activity was blocked by incubation with .03% hydrogen peroxide in methanol for 20 min. Sections were incubated with the polyclonal rabbit antihuman SHBG antibody (1:300, Cat. no. 251544, Abbiotec, San Diego, CA) diluted in 1:150 for 30 min at room temperature. After gently rinsing three times with washing buffer, the sections were incubated with the Cy3-labeled polymer conjugated to goat anti-rabbit IgG (1:100, Boaos, Beijing, China), and the nuclear was counterstained by DAPI (Beyotime, China), mounted in liquid contained antifluorescent and then evaluated under a 58 nm fluorescence microscope. To further explore the exact location of SHBG in neurons or the neurogliocyte, the direct immunofluorescent double staining method was used for the brain tissues. The anti-Nestin antibody (1:200, ab82375, Abcam, England, UK) was used for specific labeling the stem-like neurons as shown by Codega et al. [16].
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Laser confocal microscopy Laser confocal microscopy was used to capture fluorescently labeled cells in slices. All the methods are the same as the method for the immunofluorescience microscopy. All images were obtained using the Olympus FluoView 1000 confocal microscope (Olympus, Hamburg, Germany) at a 1024 1024 pixel resolution with 63 objective.
Serum SHBG assay The sera of the rat SHBG were determined by ELISA assay kit (Sangon Biotech, Shanghai, China). The samples were assayed following the manufacturer’s protocol in determining the serum SHBG concentrations. The sensitivity and the assay range of the ELISA test is 12.5 nmol/L–200 nmol. The SHBG levels in the culture medium were determined using a standard curve of SHBG. The serum SHBG concentrations are expressed in ng/ml.
Statistical analyses All the experiments were performed at least three times. All data were processed by SPSS 17.0 software (SPSS Inc., Chicago, IL). The experimental data were expressed as mean ± standard deviation (X ± SD). The expression level between different age groups was analyzed by AVONA, and the difference between every two groups was analyzed by the Bonferroni method. Mean values were considered significantly different when p .05.
RESULTS Age-dependent SHBG expression in the brain For the brain tissues, all the samples were collected from the left frontal area of the cerebrum of the brain. SHBG protein expression was observed in the cytoplasm and membrane of neurons in all the tissues of different age groups both immunohistochemically and immunofluorescence microscopically (Figures 1 and 2A). Western blotting assay of the brain samples demonstrated an immunoreactive band of 44 kDa SHBG expression (Figure 3A). Quantitative analysis of the blots revealed an age-associated increasing SHBG expression and the difference between every two groups was significant except the newborn group (Figure 3B). QRT-PCR of SHBG showed that the mRNA levels of SHBG were markedly increasing as the age increased. Except for no significant difference was observed between the 2-month group and the
6-month group, SHBG expression difference in other every two groups was all significant (p .05) (Figure 4A). The SHBG RT-PCR of the brain and sequencing results showed that the PCR products were SHBG specific (Supplementary materials).
Age-dependent SHBG expression in the liver In contrast to the previous reports, the SHBG expression in the liver also increased as the rats got older. The newborn rat livers did express SHBG, but the expression of this gene/protein was the lowest, following increasing expression as age increasing. The immunopositivity of the SHBG antibody was also cytoplasmic and membranous verified by both immunohistochemistry (Figure 1) and immunofluorescience microscopy (Figure 2B). Western blot assay always revealed the highest SHBG expression in the 12-month old rats and lowest in the newborn rats (Figure 3A). Quantitative analysis of the Western blots confirmed that this difference was statistically significant between the 12-month old rats and other groups (Figure 3B). QRT-PCR examinations verified the highest SHBG mRNA in the 12-month old group. The differences were significant between the newborn and 6-month groups, between the newborn and 12month groups and between the 2-month and 12-month groups (all with 5 .001, Figure 4B). The SHBG RT-PCR of the liver and sequencing results showed that the PCR products were SHBG specific (Supplementary materials).
Age-dependent SHBG expression in the dorsal prostate Since the prostate tissues of the newborn rats were too limited to isolate protein, Western blotting of the newborn rat prostate tissues was not possible. Immunohistochemically (Figure 1) and immunofluorescience microscopically (Figure 2C), SHBG protein expression was clearly revealed in the cytoplasm and cell membrane and the immunoreactivity was confined to the prostate epithelial cells. To further quantify the SHBG expression, both Western blotting and qRT-PCR were performed in these prostate samples. It was repeatedly shown by Western blotting that significantly higher levels of SHBG were expressed in the samples of 12-month old rats compared with the SHBG expression in the 2-month old rats (p5.001). In addition, there were significantly higher levels of SHBG protein expression in the 12-month old rats than those in 6-month rat groups as well (p = .004) (Figure 3). However, QRT-PCR of these samples showed variable expression of SHBG in different groups. Except for no significant difference was observed between the 2-month and 6-month Ultrastructural Pathology
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FIGURE 1. SHBG expression by immunohistochemistry in tissues (200). (A) Cytoplasmic and membranous SHBG positivity of the neurons of brain, the hepatocytes and the prostate epithelial cells in newborn, 2-month, 6-month, and 12-month old rats. (B) Positive SHBG expression (left) and negative control (right) in normal human liver sections.
groups (p = .075), there was a significant SHBG RNA expression difference in all the other two groups (p .05) (Figure 4C). Again, the SHBG RT-PCR of the prostate and sequencing results showed specific SHBG (Supplementary materials).
(25.967 ± 3.176 nmol/L), and 12-month rats (25.581 ± 4.997 nmol/L). The significant circulating SHBG concentration difference was observed between every two groups except for that between the 6-month and 12-month groups (Figure 6).
Laser confocal microscopy DISCUSSION To further explore the cellular location of the SHBG protein, laser confocal microscopy was performed in the 12-month old rats. This assay verified the cellular and membrane location of the SHBG protein in the neurons of brain, hepatocytes and prostate epithelial cells (Figure 5).
The SHBG levels in sera Circulating SHBG was significantly lower in the newborn rats (13.280 ± 2.294 nmol/L) compared with that in the 2-month (19.594 ± 6.492 nmol/L), 6–month !
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In our study, we demonstrated an age-dependent SHBG expression in the circulation and the organs including liver, brain, and prostate both at mRNA and protein levels using qRT-PCR, immunohistochemistry, immunofluorescience microscopy, western blotting, and laser confocal microscopy. Meanwhile, we also revealed the changes of the serum SHBG protein in consideration of age in male rats. The group difference in SHBG mRNA level was not always significant, which may be explained as rather complicated post-transcription level regulation in the
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FIGURE 2. (A) Results of immunofluorescience microscopy of the brain (200). From left to right: SHBG, Nestin, SHBG + Nestin, SHBG + Nestin + DAPI. (B) Results of immunofluorescience microscopy of the liver ( 200). From left to right: SHBG, DAPI, SHBG +DAPI. (C) Results of immunofluorescience microscopy of the prostate (200). From left to right: SHBG, DAPI, SHBG, and +DAPI.
prostate as earlier reported in 1991 [4]. To verify the specificity of products and the accuracy of the results of the real-time quantitative PCR, we designed the primers of SHBG, which span the sixth and seventh exons and contain an intron of 311 bp. The results of the agarose gel electrophoresis also supported the specificity and accuracy of the PCR results. Furthermore, sequencing of the SHBG RT-PCR products of all the tissues verified the SHBG sequence. In human, it is known that circulating SHBG plays a crucial role in aging and age-dependent pathologies, although the exact mechanism remains unclear. But in the rats, SHBG expression in the circulation and the specific organs are not fully elucidated, although there are some reports showing that SHBG expressed in the brain and fetal liver [1,3]. To our best knowledge, this is the first report characterizing SHBG expression in the brain, liver, prostate, and the circulation in male
rats at both mRNA and protein levels throughout different age groups. It is verified in our present study that SHBG expression in both mRNA and protein levels is positively associated with aging in these three different tissues, and the SHBG protein expression is confined to the cell cytoplasm and membrane confirmed by the assays of immunohistochemistry, immunofluorescience microscopy, and laser confocal microscopy. The ELISA assay also demonstrated that SHBG exists in the serum and its level was elevating as the age increased. For the brain tissues, we only examined the left front cerebrum of the rats. In this study, we verified the earlier SHBG expression in this region of rats [3]. In addition, we also discovered an age-dependently positive SHBG expression in the same region, which was not explored ever before. We first examined these samples with immunohistochemistry and discovered Ultrastructural Pathology
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FIGURE 3. Western blotting assay of SHBG expression. (A) Some representative blots where SHBG expression is highest in the 12-month old rats and lowest in the newborn rats in all the three tissues. (B) Histogram of the Western blotting results in the tissues. **p .01 and ***p .001.
FIGURE 4. Quantitative RT-PCR results of SHBG expression in the samples of brain, liver, and prostate of rats are shown in (A), (B), and (C), respectively. *p .05; **p .01, and ***p .001.
an immunoreactivity difference, which encouraged us to apply other technologies to confirm such a finding. The later immunofluorescience microscopy, qRT-PCR, and Western blotting all were in line with the notion that the SHBG expression in the brain is significantly associated with aging in the rats. There were reports that the SHBG protein or mRNA was undetectable in adult rat liver [1,17]. We are currently not aware why its expression was not detected in the earlier studies. With the same assays for SHBG expression in the brain, we could also confirm its expression in the liver and prostate of !
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newborn and 12-month old rats. Most importantly, the SHBG expression in these organs is similar to the expression in the brain, i.e. its expression is positively associated with age, both at the mRNA level and in the protein level, and the expression is confined to the cytoplasm and cell membrane as well. The above findings are interesting, since the exact role of SHBG produced in the organs other than liver is largely unknown, although circulating SHBG has already been correlated to aging in human. At the same time, in our study, we also found that the circulating SHBG level was also increasing in the
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FIGURE 6. Results of serum SHBG examinations. **p .01, ***p .001.
FIGURE 5. Results of laser confocal microscopy of SHBG in the tissues of a 12-month old rat. SHBG positivity is dominantly positioned in the membrane and cytoplasm in the neurons of the brain (A), hepatocytes of the liver (B), and the prostate epithelial cells (C).
elder rats compared with the newborn and the 2-month groups. In human, Muller et al. [18] have tested 400 men and found increasing SHBG with an average of 1.1% every year in circulation as they get older. While Dechaud et al. [19] also found a similar primary increase in SHBG levels with aged people. There are several examples of changes in blood SHBG levels associated with pathologies appearing in the elderly. The phenomenon that SHBG is increasing with age has been postulated that SHBG is a passive steroid carrier, which is to say that the change in SHBG levels with age is a response to the decrease in gonadal steroid production. In addition, in some pathological states, for example, the benign prostatic hypertrophy, breast cancer, obesity and lipid metabolism, Alzheimer’ s disease, osteoporosis and
cardiovascular disease, it has also been verified that the level of circulating SHBG is also variable [20]. While it is true that SHBG production in both the liver [21] and the brain [22] is controlled by steroid levels, it is far from definitive that a drop in gonadal production of steroids is the primary cause of an increase in SHBG as people get older. Decreased SHBG and elevated testosterone are associated with metabolic syndrome and glucose intolerance in women, and SHBG, but not testosterone, is independently associated with metabolic syndrome in overweight women with polycystic ovary syndrome (PCOS) and is associated with insulin resistance and PCOS diagnostic criteria [23]. Whats more, it was also reported that lower levels circulating SHBG independently associated with metabolic syndrome in preelderly and elderly men in China, probably an independent predictor of metabolic syndrome [24]. A case–control study included 125 incident hepatocellular carcinoma (HCC) cases and 247 individually matched controls conducted by Lukanova et al. have shown that the observed associations of HCC with prediagnostic SHBG, free testosterone, and IGF-I concentrations are in directions opposite to that expected under the etiological hypotheses. Circulating SHBG has a potential to be a prediagnostic risk marker for HCC [25]. In older men, SHBG, but not T and E2, is positively and independently associated with endothelium-dependent vasodilation (EDV) in resistance arteries. In both sexes, SHBG was positively and independently associated with endothelium-independent vasodilation (EIDV), indicating that SHBG has certain relationships with endothelial function in older subjects [26]. However, all these studies focused only on circulating SHBG. It is currently not clear whether the SHBG produced in the organs other than liver is also secretory and its exact function except for minoring steroid level. Nevertheless, our present study merits additional Ultrastructural Pathology
Sex hormone binding globulin in rat studies on such a possibility, in order to better understand not only the aging process but also aging-associated health problems such as cancer. It was also reported that SHBG could be internalized into some cells. The non-tumorigenic 3T3 fibroblast cells, which do not have either androgen receptors or estradiol receptor-a (ERa), still are stimulated to become tumorigenic by steroids. It was verified that the 3T3 cells internalize SHBG, so it may be a novel mechanism or steroid-induced tumorigenesis [27]. Caldwell et al. have demonstrated that infusions of SHBG into the hypothalami of rats increase their female sexual receptivity except when SHBG is coupled to dihydrotestosterone (DHT) suggesting that SHBG has an active function in behavioral neuroendocrinology [14]. Caldwell et al. have also revealed that both human neuronal and non-neuronal cells in brain take up SHBG in vitro and in vivo. Analysis of the uptake of (125 I-SHBG) radiolabeled SHBG in HT22 hippocampal cells stably transfected with cDNA for ERb (HT22-ERb) and the fluor (Alexa-555TM) labeled SHBG infused into the lateral cerebroventricles of ovariectomized rats showed that SHBG uptake was seen in specific parts of the choroid plexus and periventricular cells as well as into cells in the paraventricular nucleus, the medial forebrain bundle, and the habenula, a suggestion of SHBG internalization by brain cells, which may be affected by the presence of ERb. The gonadal steroids have numerous effects in brain and the discovery that the steroid-binding protein SHBG is taken up into neurons and brain cells may demand a change in thinking about how steroids are delivered to brain cells to affect neurophysiology [28]. Results in a recent study of SHBG in the rat olfactory system by immunocytochemistry and RT-PCR suggest that SHBG may be of functional importance for rapid effects of olfactory steroids on limbic functions including the control of reproductive behaviors through pheromones [29]. In our previous study [30], we verified a role of SHBG in upregulating the stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples was significantly associated with poor clinicopathological features. In a later study, we have also demonstrated that ovarian cancer cells produced SHBG and higher SHBG expression in ovarian carcinoma was associated with unfavorable clinicopathological features [31]. Both studies indicate a role of SHBG in cancer progression. Gebhart et al. [32] found that neuron-like differentiated PC 12, one of the best characterized neuron-like cell lines derived from the pheochromocytoma cells in rat, were induced to greatly increase their SHBG production by long term treatment with estradiol. The authors reasoned that this effect was apparently not mediated by the classical estradiol receptors since tamoxifen had no effect on this !
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cell line, a finding similar to our prostate cancer study [30].
CONCLUSION In summary, our current study confirms the SHBG expression in the liver, brain, and prostate of male rats, and its expression is positively associated with aging, indicating that the locally expressed SHBG may have a much broader yet unknown function in organs other than liver. While in human the SHBG level in circulation has been associated with aging and agedependent pathologies, the potential role of SHBG in rats should be elaborated further, in special consideration of aging and aging related diseases including cancer. In order to elucidate the exact role of this gene and protein, SHBG gene knock out animal models will be necessary.
DECLARATION OF INTEREST The authors report that they have no conflicts of interests. The article was supported by the National Natural Science Foundation of China (81272824).
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medial preoptic area, medial hypothalamus and pituitary. Neuroendocrinology 2003;78: 61–71. Moran LJ, Teede HJ, Noakes M, et al. Sex hormone binding globulin, but not testosterone, is associated with the metabolic syndrome in overweight and obese women with polycystic ovary syndrome. J Endocrinol Invest 2013; 36: 1004–10. Pang XN, Hu Y, Yuan Y, et al. Lower levels sex hormonebinding globulin independently associated with metabolic syndrome in pre-elderly and elderly men in China. J Geriatr Cardiol 2013;10: 28–33. Lukanova A, Becker S, Husing A, et al. Prediagnostic plasma testosterone, sex hormone-binding globulin, IGF-I and hepatocellular carcinoma: Etiological factors or risk markers?. Int J Cancer 2014;134: 164–73. Maggio M, Cattabiani C, Lauretani F, et al. SHBG and endothelial function in older subjects. Int J Cardiol 2013;168: 2825–30. Caldwell JD, Suleman F, Jirikowski GF. Internalization of sex hormone binding globulin into fibroblast 3T3 cells. Horm Metab Res 2007;39: 620–2. Caldwell JD, Shapiro RA, Jirikowski GF, Suleman F. Internalization of sex hormone-binding globulin into neurons and brain cells in vitro and in vivo. Neuroendocrinology 2007;86: 84–93. Ploss V, Gebhart VM, Dolz W, Jirikowski GF. Sex hormone binding globulin in the rat olfactory system. J Chem Neuroanat 2014;57–58C: 10–14. Ma Y, Liang D, Liu J, et al. SHBG is an important factor in stemness induction of cells by DHT in vitro and associated with poor clinical features of prostate carcinomas. PloS One 2013;8: e70558. Huang R, Ma Y, Holm R, et al. Sex hormone-binding globulin (SHBG) expression in ovarian carcinomas and its clinicopathological associations. PloS One 2013;8: e83238. Gebhart VM, Jirikowski GF. Estrogen dependent expression of sex hormone binding globulin in PC 12 cells. Steroids 2014;81: 26–30.
Supplementary material available online Supplementary Figure 1
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ORIGINAL ARTICLE
Age-Dependent Sex Hormone-Binding Globulin Expression in Male Rat Yaqing Li, MD1, Xiaoli Li, MD1, Huijie Fan, MD, PhD1, Xiaoran Li, MSc2,3, Yali Zhong, MD1, Jing Cao, MD1,4, Dandan Yu, MD1, Mingzhi Zhang, MD, PhD1, Jian-Guo Wen, MD, PhD5, Li Geng, MD, PhD1, and Zhenhe Suo, MD1,2,3 1
Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Henan, Zhengzhou, China, Department of Pathology, The Norwegian Radium Hospital, Oslo, Norway, 3Department of Pathology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway, 4Departments of Pathology, The Third Affiliated Hospital of Zhengzhou University, Henan, Zhengzhou, China, and 5The Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
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ABSTRACT Sex hormone binding globulin (SHBG) is known as a carrier protein, classically thought to be mainly synthesized in the liver and then secreted into the circulating system, where it binds to sex steroids with a high affinity and modulates the bioavailability of these hormones. In humans, the organs other than the liver known to produce SHBG include the brain, uterus, testis, prostate, breast and ovary, and the locally expressed SHBG is considered to play an important role in various physiological and pathological processes. A few studies of SHBG in rats were reported, but systemic SHBG studies in consideration of different organs and aging are currently missing. So we examined the SHBG expression in the brain, liver, prostate, and serum in 40 Sprague–Dawley male rats in four different groups (newborn, 2, 6, and 12 months old, respectively) with 10 in each group by immunohistochemistry, immunofluorescience microscopy, qRT-PCR, ELISA, western blotting, and laser confocal microscopy. We discovered that SHBG was increasingly expressed in all the three tissues along with age, and the SHBG protein expression was observed in the cytoplasm and membrane of neurons, hepatocyte, and prostate epithelial cells. The ELISA assay of the sera also supported an increasing SHBG level along with age. It is concluded that the locally synthesized SHBG in the liver, brain, and prostate and the circulating SHBG of male SD rats are positively associated with age, further indicating a potential role of SHBG in aging. Keywords: Age-dependent, laser confocal microscopy, male rat, prostate, SHBG
by controlling the availability of androgens and estrogens. In plasma, SHBG controls the metabolic clearance rate of sex steroids. Except for the sertoli cells in testis, it was reported that the SHBG gene was also expressed in other tissues in rats. It has been reported that the SHBG gene expression was discovered in fetal rat liver and adult brain [3], while others identified the protein in the rat hypothalamo-neurohypophyseal system, colocalized with oxytocin [4,5]. The intrinsic expression
Sex hormone-binding globulin (SHBG) or androgenbinding globulin (ABG) is an extracellular androgen and estrogen carrier. It interacts with its ligands, 5adihydrotestosterone (5a-DHT), and testosterone, with high affinity [1]. In rat, it has been verified that the sertoli cells produce the SHBG which influences androgen actions in the seminiferous tubules and epididymis [2]. There is strong evidence that the binding protein does act to modulate the gene regulatory actions of nuclear sex steroid receptors
Received 31 December 2014; Accepted 14 January 2015; Published online 13 April 2015 Correspondence: Dr. Zhenhe Suo, Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Montebello, Ullernchausseen 70, N-0310, Oslo, Norway. E-mail: [email protected] Dr. Li Geng, Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China. E-mail: [email protected]
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122 Y. Li et al. of SHBG in magnocellular hypothalamic neurons, in part co-localized with either vasopressin or oxytocin, was also described recently. With immunoelectronmicroscopic double labeling, they found co-localization of oxytocin and SHBG in a portion of the large dense-core vesicles in paraventricular and supraoptic perikarya and in axonal varicosities in the median eminence and in the posterior lobe. SHBG was found throughout the brain, with highest levels in the hippocampus and cortex and lowest levels in the diencephalon and cerebellum [6]. In human, it is reported that SHBG levels increased with increasing age in the circulating system [7], and the role of circulating SHBG in aging and ageassociated physiological changes such as osteoporosis and obesity has been verified. SHBG is an active participant in many of these age-associated physiological changes [8]. Jirikowski et al. [9] have recently demonstrated that SHBG is co-localized in prostate and benign prostate hyperplasia with oxytocin. The colocalization of these two important endocrine elements in the prostate may suggest an interaction in their function in prostate cells. Hoskin et al. [10] have found significantly decreased circulating SHBG levels in women with Alzheimer’s disease and suggested that SHBG may play more important role in brain than in liver. In addition, results from the Study of Women Across the Nation (SWAN) indicate an important relationship between SHBG and metabolic syndrome components and SHBG has a stronger relationship with obesity and metabolic disease endpoints than estrogen or testosterone [11,12]. Schock et al. [13] demonstrated increased SHBG in cardiomyocytes of men with dilated cardiomyopathy, another possible indication of SHBG in the process of heart aging. In the female rats, it was found that infusions of SHBG into the medial preoptic area-anterior hypothalamus could facilitate female sexual receptivity, suggesting that SHBG may be a marker of sexual activity and reproductive fitness and it may also mean that SHBG is the ultimate aging hormone [14]. SHBG is colocalized in vesicles with the reproductive hormone oxyctin, which is also verified to be released into the blood during sexual activity in a number of species [15]. But in rats, it is still not fully understood whether SHBG plays a role in aging process and age-associated physiological changes. Therefore, the purposes of our present study were to characterize the age-association of SHBG expression in the serum, liver, brain, and prostate of different age groups of male Sprague– Dawley (SD) rats, and to further explore the SHBG cellular localization in these tissues. The reason why we chose only male rats in this study was due to our project in a prostate cancer mouse model study, which is currently undergoing in our lab. It was discovered that SHBG distributed in the cytoplasm and membranes in neurons of brain, hepatocytes and prostate epithelial cells, and its expression level generally
increased with age. Furthermore, age-dependent increasing levels of SHBG were verified in the sera of the rats. Collectively, our results support an agedependent SHBG expression both in the liver, brain and prostate tissues and in the serum of SD rat, indicating a role of SHBG in aging.
MATERIAL AND METHODS Ethics statement Animals used in the present study were maintained and euthanized in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The animal study was approved by the Institutional Animal Care and Use Committees of the China Medical Association (Permit Number: SCXK 2013-0001).
The animals Forty Sprague–Dawley (SD) male rats, of clean grade and male gender (divided into newborn, 2-, 6-, and 12-month old groups and each age group with 10 rats), were obtained from The Experimental Animal Center of Shandong anti-lu pharmaceutical co., LTD, Shandong, China. About 10% chloral hydrate was used for anesthesia by intraperitoneal injection, and the dosage was .4 ml/100 g body weight. Then the animals were dissected and the left frontal area of the cerebrum of the brain, liver, and dorsal prostate were removed for further immunohistochemistry, immunofluorescence microscopy, quantitative RT-PCR, Western blotting, and laser confocal microscopy assays. Serum specimens were collected from all the rats. The sera were centrifuged at 4000 rpm for 15 min, and then the supernatant was immediately removed and the sediments were kept frozen at 80 C until use.
PCR and sequencing assay About 200 mg of fresh brain, liver, and prostate from each rat was placed in a reaction tube with 1 ml Trizol (Invitrogen, Carlsbad, CA) and homogenized mechanically (Ultraturrax T3, Biosafter, Beijing, China) followed by sonication. Homogenized samples were incubated for 5 min at RT. Then .6 ml chloroform was added and the tubes were shaken by hand for 15 s. After an incubation of 3 min at RT, samples were centrifuged at 12,000 g for 15 min at 4 C. The aqueous phase, containing the isolated RNA, was placed into a new tube. For RNA precipitation samples were mixed with 1 ml of 100% isopropanol, incubated for 10 min at RT and then again centrifuged for 10 min at 12,000 g at Ultrastructural Pathology
Sex hormone binding globulin in rat 4 C. Supernatant was removed from the tube. The pellet was resuspended by briefly vortexing in 3 ml of 75% ethanol. After centrifugation (7500g, 5 min, 4 C), the supernatant was discarded. The air-dried pellet was resuspended in 50 ml of RNase-free water. After that the genomic DNA elimination reaction was conducted. Up to 1 mg of total RNA, 1.0 ml of gDNA eraser were added to a total of 10.0 ml reaction for 2 min at 42 C. Then the total RNA of the tissues was extracted, and each RNA sample (3 mg) was subjected to cDNA synthesis by means of RevertAid First Strand cDNA Synthesis (Thermo Scientific, West Palm Beach, FL). Quantitative real-time PCR was performed using the Quantitative SYBR Green PCR Kit (Thermo Scientific, West Palm Beach, FL) on the ABI PRISM 7500 Fast system (Applied Biosystems, Life Technologies, Foster City, CA). All experiments were performed as specified in the protocols of the manufacturer. The primers used were as follows: SHBG forward primer, CTTCTTGGCTCACCCTT CAC; SHBG reverse primer, ACCTCCATCTTTGG TCCTTG; GAPDH forward primer: ACAGCAA CAGGGTGGTGGAC; GAPDH reverse primer: TTTGAGGGTGCAGCGAACTT. All samples were analyzed for three parallel samples and repeated three times at least, and the relative expression of the target gene was calculated with 2 DD Ct values with the Application Relative Quantification Study Program (Applied Biosystems, Life Technologies, Foster City, CA). About 3% agarose gel electrophoresis and Sequencing (Sangon Biotech, Shanghai, China) of the product were conducted to confirm the specificity of the PCR products.
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density of the target protein compared with GAPDH was quantified with the Bio-Rad Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA).
Immunohistochemistry All the liver, brain, and prostate tissues of each animal were fixed with 4% buffered paraformaldehyde (PFA, Hangzhou, China) and embedded in paraffin blocks before 4 mm sections were prepared for immunohistochemical examination. The sections were deparaffinized in xylene and then microwaved in 10 mM citrate buffer (pH 6.0) in order to unmask the epitopes. Endogenous peroxidase activity was blocked by incubation with .03% hydrogen peroxide in methanol for 20 min. Sections were incubated with the polyclonal rabbit anti-human SHBG antibody (1:300, Cat. no. 251544, Abbiotec, San Diego, CA) for 30 min at room temperature. After gently rinsed three times with washing buffer, the sections were then incubated with the peroxidase-labeled polymer conjugated to goat anti-rabbit IgG (Boaos, Beijing, China) for 30 min before stained for 2 min with 3‘3-diaminobenzidine tetrahydrochloride (DAB) (ZSGB-Bio, Beijing, China), counterstained by hematoxylin, dehydrated, and mounted in Diatex. Non-immune rabbit IgG in exactly the same concentration as the primary antibody was applied as a negative control, and known SHBG positive human liver tissue sections were used as positive controls.
Immunofluorescence microscopy Western blotting The tissues were dissolved with lysis buffer containing RIPA buffer (Thermo Scientific, West Palm Beach, FL), 1% PMSF, 1% aprotinin, 1% leupeptin, 1% pepstatin, and .5% vanadate by pipetting gently up and down, put on ice before spun down at 14,000 rpm for 15 min at 4 C to release total protein. About 50 mg proteins from each sample in SDS-loading buffer was boiled for 5 min and subjected to 10% SDS-PAGE electrophoresis and then electro-transferred to highquality polyvinylidene difluoride (PVDF) membrane (Boster, Wuhan, China). The membrane was blocked with 5% fat-free milk for 1 h at room temperature and incubated overnight at 4 C with a goat anti-human SHBG antibody (1:300, Cat. no. 251544, Abbiotec, San Diego, CA). After washing with PBS-tween .05% (PBST), the blot was incubated with rabbit anti-goat IgG HRP antibody for 45 min at room temperature (1:500, Boster, Wuhan, China). After three washes with PBST, the blot was visualized using an enhanced chemiluminescence detection kit (ECL, Amersham, UK) by following the manual guide. The relative band !
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SHBG immunofluorescence microscopy was performed on 4 mm sections. The sections were deparaffinized in xylene and then microwaved in 10 mM citrate buffer (pH 6.0) to unmask the epitopes. Endogenous peroxidase activity was blocked by incubation with .03% hydrogen peroxide in methanol for 20 min. Sections were incubated with the polyclonal rabbit antihuman SHBG antibody (1:300, Cat. no. 251544, Abbiotec, San Diego, CA) diluted in 1:150 for 30 min at room temperature. After gently rinsing three times with washing buffer, the sections were incubated with the Cy3-labeled polymer conjugated to goat anti-rabbit IgG (1:100, Boaos, Beijing, China), and the nuclear was counterstained by DAPI (Beyotime, China), mounted in liquid contained antifluorescent and then evaluated under a 58 nm fluorescence microscope. To further explore the exact location of SHBG in neurons or the neurogliocyte, the direct immunofluorescent double staining method was used for the brain tissues. The anti-Nestin antibody (1:200, ab82375, Abcam, England, UK) was used for specific labeling the stem-like neurons as shown by Codega et al. [16].
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Laser confocal microscopy Laser confocal microscopy was used to capture fluorescently labeled cells in slices. All the methods are the same as the method for the immunofluorescience microscopy. All images were obtained using the Olympus FluoView 1000 confocal microscope (Olympus, Hamburg, Germany) at a 1024 1024 pixel resolution with 63 objective.
Serum SHBG assay The sera of the rat SHBG were determined by ELISA assay kit (Sangon Biotech, Shanghai, China). The samples were assayed following the manufacturer’s protocol in determining the serum SHBG concentrations. The sensitivity and the assay range of the ELISA test is 12.5 nmol/L–200 nmol. The SHBG levels in the culture medium were determined using a standard curve of SHBG. The serum SHBG concentrations are expressed in ng/ml.
Statistical analyses All the experiments were performed at least three times. All data were processed by SPSS 17.0 software (SPSS Inc., Chicago, IL). The experimental data were expressed as mean ± standard deviation (X ± SD). The expression level between different age groups was analyzed by AVONA, and the difference between every two groups was analyzed by the Bonferroni method. Mean values were considered significantly different when p .05.
RESULTS Age-dependent SHBG expression in the brain For the brain tissues, all the samples were collected from the left frontal area of the cerebrum of the brain. SHBG protein expression was observed in the cytoplasm and membrane of neurons in all the tissues of different age groups both immunohistochemically and immunofluorescence microscopically (Figures 1 and 2A). Western blotting assay of the brain samples demonstrated an immunoreactive band of 44 kDa SHBG expression (Figure 3A). Quantitative analysis of the blots revealed an age-associated increasing SHBG expression and the difference between every two groups was significant except the newborn group (Figure 3B). QRT-PCR of SHBG showed that the mRNA levels of SHBG were markedly increasing as the age increased. Except for no significant difference was observed between the 2-month group and the
6-month group, SHBG expression difference in other every two groups was all significant (p .05) (Figure 4A). The SHBG RT-PCR of the brain and sequencing results showed that the PCR products were SHBG specific (Supplementary materials).
Age-dependent SHBG expression in the liver In contrast to the previous reports, the SHBG expression in the liver also increased as the rats got older. The newborn rat livers did express SHBG, but the expression of this gene/protein was the lowest, following increasing expression as age increasing. The immunopositivity of the SHBG antibody was also cytoplasmic and membranous verified by both immunohistochemistry (Figure 1) and immunofluorescience microscopy (Figure 2B). Western blot assay always revealed the highest SHBG expression in the 12-month old rats and lowest in the newborn rats (Figure 3A). Quantitative analysis of the Western blots confirmed that this difference was statistically significant between the 12-month old rats and other groups (Figure 3B). QRT-PCR examinations verified the highest SHBG mRNA in the 12-month old group. The differences were significant between the newborn and 6-month groups, between the newborn and 12month groups and between the 2-month and 12-month groups (all with 5 .001, Figure 4B). The SHBG RT-PCR of the liver and sequencing results showed that the PCR products were SHBG specific (Supplementary materials).
Age-dependent SHBG expression in the dorsal prostate Since the prostate tissues of the newborn rats were too limited to isolate protein, Western blotting of the newborn rat prostate tissues was not possible. Immunohistochemically (Figure 1) and immunofluorescience microscopically (Figure 2C), SHBG protein expression was clearly revealed in the cytoplasm and cell membrane and the immunoreactivity was confined to the prostate epithelial cells. To further quantify the SHBG expression, both Western blotting and qRT-PCR were performed in these prostate samples. It was repeatedly shown by Western blotting that significantly higher levels of SHBG were expressed in the samples of 12-month old rats compared with the SHBG expression in the 2-month old rats (p5.001). In addition, there were significantly higher levels of SHBG protein expression in the 12-month old rats than those in 6-month rat groups as well (p = .004) (Figure 3). However, QRT-PCR of these samples showed variable expression of SHBG in different groups. Except for no significant difference was observed between the 2-month and 6-month Ultrastructural Pathology
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FIGURE 1. SHBG expression by immunohistochemistry in tissues (200). (A) Cytoplasmic and membranous SHBG positivity of the neurons of brain, the hepatocytes and the prostate epithelial cells in newborn, 2-month, 6-month, and 12-month old rats. (B) Positive SHBG expression (left) and negative control (right) in normal human liver sections.
groups (p = .075), there was a significant SHBG RNA expression difference in all the other two groups (p .05) (Figure 4C). Again, the SHBG RT-PCR of the prostate and sequencing results showed specific SHBG (Supplementary materials).
(25.967 ± 3.176 nmol/L), and 12-month rats (25.581 ± 4.997 nmol/L). The significant circulating SHBG concentration difference was observed between every two groups except for that between the 6-month and 12-month groups (Figure 6).
Laser confocal microscopy DISCUSSION To further explore the cellular location of the SHBG protein, laser confocal microscopy was performed in the 12-month old rats. This assay verified the cellular and membrane location of the SHBG protein in the neurons of brain, hepatocytes and prostate epithelial cells (Figure 5).
The SHBG levels in sera Circulating SHBG was significantly lower in the newborn rats (13.280 ± 2.294 nmol/L) compared with that in the 2-month (19.594 ± 6.492 nmol/L), 6–month !
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In our study, we demonstrated an age-dependent SHBG expression in the circulation and the organs including liver, brain, and prostate both at mRNA and protein levels using qRT-PCR, immunohistochemistry, immunofluorescience microscopy, western blotting, and laser confocal microscopy. Meanwhile, we also revealed the changes of the serum SHBG protein in consideration of age in male rats. The group difference in SHBG mRNA level was not always significant, which may be explained as rather complicated post-transcription level regulation in the
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FIGURE 2. (A) Results of immunofluorescience microscopy of the brain (200). From left to right: SHBG, Nestin, SHBG + Nestin, SHBG + Nestin + DAPI. (B) Results of immunofluorescience microscopy of the liver ( 200). From left to right: SHBG, DAPI, SHBG +DAPI. (C) Results of immunofluorescience microscopy of the prostate (200). From left to right: SHBG, DAPI, SHBG, and +DAPI.
prostate as earlier reported in 1991 [4]. To verify the specificity of products and the accuracy of the results of the real-time quantitative PCR, we designed the primers of SHBG, which span the sixth and seventh exons and contain an intron of 311 bp. The results of the agarose gel electrophoresis also supported the specificity and accuracy of the PCR results. Furthermore, sequencing of the SHBG RT-PCR products of all the tissues verified the SHBG sequence. In human, it is known that circulating SHBG plays a crucial role in aging and age-dependent pathologies, although the exact mechanism remains unclear. But in the rats, SHBG expression in the circulation and the specific organs are not fully elucidated, although there are some reports showing that SHBG expressed in the brain and fetal liver [1,3]. To our best knowledge, this is the first report characterizing SHBG expression in the brain, liver, prostate, and the circulation in male
rats at both mRNA and protein levels throughout different age groups. It is verified in our present study that SHBG expression in both mRNA and protein levels is positively associated with aging in these three different tissues, and the SHBG protein expression is confined to the cell cytoplasm and membrane confirmed by the assays of immunohistochemistry, immunofluorescience microscopy, and laser confocal microscopy. The ELISA assay also demonstrated that SHBG exists in the serum and its level was elevating as the age increased. For the brain tissues, we only examined the left front cerebrum of the rats. In this study, we verified the earlier SHBG expression in this region of rats [3]. In addition, we also discovered an age-dependently positive SHBG expression in the same region, which was not explored ever before. We first examined these samples with immunohistochemistry and discovered Ultrastructural Pathology
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FIGURE 3. Western blotting assay of SHBG expression. (A) Some representative blots where SHBG expression is highest in the 12-month old rats and lowest in the newborn rats in all the three tissues. (B) Histogram of the Western blotting results in the tissues. **p .01 and ***p .001.
FIGURE 4. Quantitative RT-PCR results of SHBG expression in the samples of brain, liver, and prostate of rats are shown in (A), (B), and (C), respectively. *p .05; **p .01, and ***p .001.
an immunoreactivity difference, which encouraged us to apply other technologies to confirm such a finding. The later immunofluorescience microscopy, qRT-PCR, and Western blotting all were in line with the notion that the SHBG expression in the brain is significantly associated with aging in the rats. There were reports that the SHBG protein or mRNA was undetectable in adult rat liver [1,17]. We are currently not aware why its expression was not detected in the earlier studies. With the same assays for SHBG expression in the brain, we could also confirm its expression in the liver and prostate of !
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newborn and 12-month old rats. Most importantly, the SHBG expression in these organs is similar to the expression in the brain, i.e. its expression is positively associated with age, both at the mRNA level and in the protein level, and the expression is confined to the cytoplasm and cell membrane as well. The above findings are interesting, since the exact role of SHBG produced in the organs other than liver is largely unknown, although circulating SHBG has already been correlated to aging in human. At the same time, in our study, we also found that the circulating SHBG level was also increasing in the
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FIGURE 6. Results of serum SHBG examinations. **p .01, ***p .001.
FIGURE 5. Results of laser confocal microscopy of SHBG in the tissues of a 12-month old rat. SHBG positivity is dominantly positioned in the membrane and cytoplasm in the neurons of the brain (A), hepatocytes of the liver (B), and the prostate epithelial cells (C).
elder rats compared with the newborn and the 2-month groups. In human, Muller et al. [18] have tested 400 men and found increasing SHBG with an average of 1.1% every year in circulation as they get older. While Dechaud et al. [19] also found a similar primary increase in SHBG levels with aged people. There are several examples of changes in blood SHBG levels associated with pathologies appearing in the elderly. The phenomenon that SHBG is increasing with age has been postulated that SHBG is a passive steroid carrier, which is to say that the change in SHBG levels with age is a response to the decrease in gonadal steroid production. In addition, in some pathological states, for example, the benign prostatic hypertrophy, breast cancer, obesity and lipid metabolism, Alzheimer’ s disease, osteoporosis and
cardiovascular disease, it has also been verified that the level of circulating SHBG is also variable [20]. While it is true that SHBG production in both the liver [21] and the brain [22] is controlled by steroid levels, it is far from definitive that a drop in gonadal production of steroids is the primary cause of an increase in SHBG as people get older. Decreased SHBG and elevated testosterone are associated with metabolic syndrome and glucose intolerance in women, and SHBG, but not testosterone, is independently associated with metabolic syndrome in overweight women with polycystic ovary syndrome (PCOS) and is associated with insulin resistance and PCOS diagnostic criteria [23]. Whats more, it was also reported that lower levels circulating SHBG independently associated with metabolic syndrome in preelderly and elderly men in China, probably an independent predictor of metabolic syndrome [24]. A case–control study included 125 incident hepatocellular carcinoma (HCC) cases and 247 individually matched controls conducted by Lukanova et al. have shown that the observed associations of HCC with prediagnostic SHBG, free testosterone, and IGF-I concentrations are in directions opposite to that expected under the etiological hypotheses. Circulating SHBG has a potential to be a prediagnostic risk marker for HCC [25]. In older men, SHBG, but not T and E2, is positively and independently associated with endothelium-dependent vasodilation (EDV) in resistance arteries. In both sexes, SHBG was positively and independently associated with endothelium-independent vasodilation (EIDV), indicating that SHBG has certain relationships with endothelial function in older subjects [26]. However, all these studies focused only on circulating SHBG. It is currently not clear whether the SHBG produced in the organs other than liver is also secretory and its exact function except for minoring steroid level. Nevertheless, our present study merits additional Ultrastructural Pathology
Sex hormone binding globulin in rat studies on such a possibility, in order to better understand not only the aging process but also aging-associated health problems such as cancer. It was also reported that SHBG could be internalized into some cells. The non-tumorigenic 3T3 fibroblast cells, which do not have either androgen receptors or estradiol receptor-a (ERa), still are stimulated to become tumorigenic by steroids. It was verified that the 3T3 cells internalize SHBG, so it may be a novel mechanism or steroid-induced tumorigenesis [27]. Caldwell et al. have demonstrated that infusions of SHBG into the hypothalami of rats increase their female sexual receptivity except when SHBG is coupled to dihydrotestosterone (DHT) suggesting that SHBG has an active function in behavioral neuroendocrinology [14]. Caldwell et al. have also revealed that both human neuronal and non-neuronal cells in brain take up SHBG in vitro and in vivo. Analysis of the uptake of (125 I-SHBG) radiolabeled SHBG in HT22 hippocampal cells stably transfected with cDNA for ERb (HT22-ERb) and the fluor (Alexa-555TM) labeled SHBG infused into the lateral cerebroventricles of ovariectomized rats showed that SHBG uptake was seen in specific parts of the choroid plexus and periventricular cells as well as into cells in the paraventricular nucleus, the medial forebrain bundle, and the habenula, a suggestion of SHBG internalization by brain cells, which may be affected by the presence of ERb. The gonadal steroids have numerous effects in brain and the discovery that the steroid-binding protein SHBG is taken up into neurons and brain cells may demand a change in thinking about how steroids are delivered to brain cells to affect neurophysiology [28]. Results in a recent study of SHBG in the rat olfactory system by immunocytochemistry and RT-PCR suggest that SHBG may be of functional importance for rapid effects of olfactory steroids on limbic functions including the control of reproductive behaviors through pheromones [29]. In our previous study [30], we verified a role of SHBG in upregulating the stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples was significantly associated with poor clinicopathological features. In a later study, we have also demonstrated that ovarian cancer cells produced SHBG and higher SHBG expression in ovarian carcinoma was associated with unfavorable clinicopathological features [31]. Both studies indicate a role of SHBG in cancer progression. Gebhart et al. [32] found that neuron-like differentiated PC 12, one of the best characterized neuron-like cell lines derived from the pheochromocytoma cells in rat, were induced to greatly increase their SHBG production by long term treatment with estradiol. The authors reasoned that this effect was apparently not mediated by the classical estradiol receptors since tamoxifen had no effect on this !
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cell line, a finding similar to our prostate cancer study [30].
CONCLUSION In summary, our current study confirms the SHBG expression in the liver, brain, and prostate of male rats, and its expression is positively associated with aging, indicating that the locally expressed SHBG may have a much broader yet unknown function in organs other than liver. While in human the SHBG level in circulation has been associated with aging and agedependent pathologies, the potential role of SHBG in rats should be elaborated further, in special consideration of aging and aging related diseases including cancer. In order to elucidate the exact role of this gene and protein, SHBG gene knock out animal models will be necessary.
DECLARATION OF INTEREST The authors report that they have no conflicts of interests. The article was supported by the National Natural Science Foundation of China (81272824).
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