Tetsuo Ogawa, PhD, Department of Anatomy, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Email: email@example.com
A longevity gene product, Sir2 (silent information regulator 2) is a NAD-dependent histone deacetylase involved in longevity in yeasts, worms and flies. The mammalian homolog of Sir2, SIRT1(sirtuin 1), has been shown to play important roles related to anti-aging effects (regulating apoptosis, stress tolerance, insulin resistance, and fat metabolism). Recently, SIRT1 expression has been demonstrated to occur at as early as embryonic day 10.5 in mice. SIRT1 during developing period may be involved in the mechanism of developmental origins of adult diseases, such as diabetes and cardiovascular disease. To investigate the contribution of SIRT1, it is important to reveal the distribution of this protein during development. In the present study, we demonstrated the distribution of immunoreactivity of SIRT1 in mouse organs during prenatal and neonatal development by staining a wide variety of serial sections. The SIRT1 immunoreactivity was strongly observed in the neuroepithelial layer, dorsal root ganglion, trigeminal ganglion, eyes, roots of whiskers, and internal organs, including the testis, liver, heart, kidney, and lung during the fetal period. Neurons which had finished migrating still showed relatively strong immunoreactivity. The immunoreactivity was completely absorbed by the blocking peptide in an absorption test. During the postnatal period, the immunoreactivities in most of these organs, except the heart and testis weakened, with the liver most dramatically affected. As SIRT1 expression was demonstrated in a wide variety of developing organs, further study to investigate prenatal factors which affect SIRT1 expression and its activity is important.
Intrauterine conditions, such as undernutrition have been associated with a higher incidence of lifestyle-associated diseases, such as type 2 diabetes and cardiovascular diseases in adulthood (McMillen and Robinson 2005). Understanding what occurs in the developing fetal organs under such conditions is important to preventing lifestyle-associated diseases.
Recently, SIRT1 expression has been demonstrated to occur at as early as embryonic day (ED) 10.5 (Sakamoto et al. 2004). The function of SIRT1 strongly depends on the availability of nutrients (Cohen et al. 2004; Nemoto et al. 2004; Chen et al. 2005). Therefore, the contribution of SIRT1 to the mechanism(s) by which fetal malnutrition affects lifestyle-associated diseases should be focused on.
Strong immunoreactivity for SIRT1 was demonstrated in the heart, spinal cord, dorsal root ganglion and neuroepithelium of the brain (Sakamoto et al. 2004). Information from the previous in vivo study seemed to be based on sections around the median plane. More information is needed for investigating roles of SIRT1 in the developmental origins of adult diseases. In the present study, we examined SIRT1 immunoreactivity during prenatal and neonatal development based on serial sections, which makes it possible to observe a wide variety of the fetal tissues. The results of the immunohistochemistry were partly supported by an immunoblot analysis.
MATERIALS AND METHODS
ICR and C57BL/6J mice were purchased from Japan SLC (Hamamatsu, Japan). The animals were housed at the Animal Institution at Showa University. They were maintained in cages in a ventilated animal room with controlled temperature and relative humidity with a 12-h light : 12-h dark schedule and had access to chow and tap water ad libitum. All animal experiments were started after a period of acclimation of at least one week. Pregnant animals were obtained by housing females with males (1–3 females/male). The day a vaginal plug was observed was designated as ED0. Birth day was designated postnatal day 0 (PD0). All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Showa University.
Preparation of the mouse tissues was performed as described in our previous report (Ogawa et al. 2005). The fetal bodies were placed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (4% paraformaldehyde [PFA]) and held at 4°C for 2 days. To collect neonatal tissues, pups were deeply anesthetized using sodium pentobarbital and transcardially perfused with 4% PFA. Tissues were removed and postfixed for 2 days. Following fixation, the specimens were embedded in 10% gelatin and sections were cut at a thickness of 45 µm by a vibratome. Every fourth section was collected in four bottles. A set of fourth sections was stained for SIRT1. The sections were incubated in a free-floating manner at each of the following steps. After inactivation of endogenous peroxidase in phosphate-buffered saline (PBS) (10 mM phosphate buffer containing 0.9% NaCl) containing 3% H2O2 for 15 min and washing in PBS (5 min × 3 washes), the sections were transferred to a blocking solution (PBS containing 5% normal horse serum and 0.3% Triton X-100) and incubated for 1 h. We used polyclonal rabbit anti-Sir2α[Millipore, Temecula, CA, USA]. (To raise this antibody, His-tagged fusion protein corresponding to amino acids 1–131 of mouse Sir2α was used). The sections were incubated in the blocking solution containing the primary antibody (1:1000 dilution) at 4°C overnight. After washing out the primary antibodies in PBS, sections were incubated with secondary antibody (biotinylated anti-rabbit IgG, Vector laboratories, Burlingame, CA, USA) diluted in the blocking solution (1:200) for 90 min. After washing out the secondary antibody in PBS, the sections were subjected to an avidin/biotin-immunoperoxidase reaction using a Vectastain ABC kit (Vector Laboratories) with visualization of the antigen using diaminobenzidine as a substrate. To facilitate comparisons among each age and tissue, some sections from each internal organ (liver, kidney, lung, heart, and testis), and from each age (ED18, PD0, PD3, PD11, and PD16) were incubated in the same bottle and processed for immunostaining simultaneously. To confirm the specificity of the immunoreactivity, we used another antibody to SIRT1 (polyclonal goat anti-SIRT1, sc-19857 [Santa Cruz Biotechnology, Santa Cruz, CA, USA] ) for which a blocking peptide (sc-19857P [Santa Cruz Biotechnology] ) is available (we could not obtain a blocking peptide for the polyclonal rabbit anti-Sir2α antibody from Millipore). The absorption test was carried out by incubating the first antibody (1 µg/mL) with the blocking peptide (20 µg/mL) for 3 h at room temperature. After the incubation, the staining process was as described above. The sections from ED14 C57BL/6J fetuses were used for the absorption test.
Samples for immunoblotting were prepared using an AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Valencia, CA, USA). The frozen tissues were homogenized with an ultrasonic homogenizer in ice-cold lysis buffer (1:24 wt/vol for the liver and 1:7 wt/vol for the heart). Equal amounts of protein (10 µg) were separated on a 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gel, and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). Blots were blocked for 30 min at room temperature with 1% blocking solution (Block Ace Powder; Yukijirushi, Sapporo, Japan). After blocking treatment, the solution was replaced with polyclonal rabbit anti-Sir2α antibody (1:1000) at 4°C overnight. The blots were then incubated with the conjugated anti-rabbit IgG-horseradish peroxidase (1:3000; Amersham Bioscience, Little Chalfont Buckinghamshire, UK). As a protein loading control, mouse monoclonal antibody against β-actin (Sigma, Saint Louis, MO, USA) was used. Immunoreactive proteins were visualized using chemiluminescent substrate (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, Rockford, IL, USA).
Figure 1 shows SIRT1 immunoreactivity in the fetal tissues at ED11. Strong immunoreactivity was observed in the neuroepithelium, olfactory epithelium, spinal cord, dorsal root ganglion, optic cup and lens, and otic vesicle. Immunoreactivity was observed in the heart, too.
At ED14, several major organs under active development were recognizable. The SIRT1 immunohistochemistry in these organs is represented in Figure 2 (part of the head) and Figure 3 (part of the trunk). In the head, strong immunoreactivity was observed in the roots of the whiskers, the lens, the retina, the epithelium of the nasal cavity, the neuroepithelium of the forebrain, the cortical plate of the telencephalon, the trigeminal ganglion, the external germinal layer of the cerebellum, and large neurons in the pontomedullary area, including the prepositus glossal nucleus (Fig. 2). In the trunk, the spermatogonium had very strong immunoreactivity (Fig. 3). Relatively strong reactions were also observed in the developing lung buds, heart muscles, liver, dorsal root ganglion cells, metanephric vesicles and tubules. The gut mucosa, including the stomach also showed immunoreactivity.
Figures 4–9 show SIRT1 immunohistochemistry from ED14 to PD16 or adult hood in the liver, kidney, heart, lung, testis and cerebral cortex.
In the liver, ED14 tissue exhibited relatively strong immunoreactivity. Thereafter it became very weak, and faint until PD16 (Fig. 4). This organ exhibited the most dramatic decrease in immunoreactivity during development.
In the kidney, strong immunoreactivity was observed in the metanephric vesicle, and then gradually decreased in the Bowman capsule, and the collecting tubule, but not the glomerulus, depending on age (Fig. 5). At PD16, it became weak, but still remained, especially in the uriniferous tubule and collecting duct (Figs 5–6, inserted Fig).
The cardiac muscle showed relatively strong immunoreactivity throughout the experiment (Fig. 6).
In the lung, the lung bud exhibited strong immunoreactivity, which remained in the structures surrounding the alveolus (Fig. 7). There tended to be a decrease in reactivity between PD11 and 16 (Fig. 7).
The spermatogonium in the testis had very strong SIRT1 immunoreactivity at ED14 (Fig. 8). The immunoreactivity remained strong until the adult stage, and distributed in most cells in the seminiferous tubules at all stages of spermatogenesis (Fig. 8-6, inserted Fig).
In the cerebral cortex, SIRT1 immunoreactivity was detected, not only in the neuroepithelial layer (the stem cells and progenitor cells), but also in the neurons located in the cortical plates where immature cells that have ceased migrating accumulate (Fig. 9). The immunoreactivity remained, but with a tendency of decrease until PD18 (Fig. 9).
The absorption test demonstrated that the immunoreactivity represented the presence of SIRT1 protein (Fig. 10). The distribution of immunoreactivity presented by the goat polyclonal antibody for which a blocking peptide is available was the same as that by the rabbit polyclonal antibody demonstrated in Figures 1–9. Figure 10 demonstrated that the immunoreactivity was completely blocked by the blocking peptide in the roots of whiskers, cortical plate of the forebrain, eyes, dorsal root ganglion, liver, kidney, heart, and lung.
The result of the immunoblot analysis is shown in Figure 11. In the liver, SIRT1 protein expression decreased gradually from ED14 to PD16, while not in the heart, confirming the results of immunohistological observation (Fig. 11).
In the present study, we demonstrated the distribution of SIRT1 in diverse developing mouse organs. Except the heart and testis, all other organs examined showed an attenuated expression of SIRT1 until PD16, suggesting active roles for SIRT1 during development.
Sakamoto et al. (2004) demonstrated high levels of SIRT1 expression in the brain, spinal cord, dorsal root ganglion and heart in mouse fetuses, and theorized a role for SIRT1 in neurogenesis and cardiogenesis. The same authors showed a predominant expression of SIRT1 in the heart, and analyzed, extensively, the alterations in its expression (mRNA) level. While their data was based on the gene expression, we focused our analysis at the level of the protein. SIRT1 is a very well-known protein, known to play an important role in the calorie restriction-induced longevity effect. This protein is up-regulated during calorie restriction or after food deprivation (Cohen et al. 2004; Rodgers et al. 2005); these authors have demonstrated changes at the level of the SIRT1 protein, but not at the level of gene expression. Keeping in mind these findings, we have focused on the protein itself, rather than on the gene. Sakamoto et al. (2004) carried out analysis of gene expressions from ED12.5, and detected a rapid decrease in the mRNA level from ED12.2 to ED14.5. We carried out immunoblot analysis only after ED14, and therefore, failed to detect the higher expression of SIRT1 at ED12.5. We just detected SIRT1 immunoreactivity in the ED11 heart. Protein levels in the heart from ED14 to and after birth were examined, but there appear to be no dramatic changes, considering previous (Sakamoto et al. 2004) and our results.
A role for SIRT1 in the differentiation of neuroprogenitor cells into neurons and oligodendrocytes has been reported (Hisahara et al. 2008). In the central nervous system, we have demonstrated that SIRT1 was expressed, not only in the neuroepithelial layer (the stem cells and progenitor cells), but also in the neurons which had finished migrating. The results suggested other roles for SIRT1, such as in neuroprotection, through the regulation of transcription factors that have been recently reported (Luo et al. 2001; Hasegawa and Yoshikawa 2008).
The expression of SIRT1 weakened in many organs where a contribution of SIRT1 was reported in adults. Accumulating evidence shows that in adulthood, SIRT1 seems to be induced and activated with physiological conditions, such as nutrition and stress change (Cohen et al. 2004; Nemoto et al. 2004; Rodgers et al. 2005; Wu et al. 2006; Suwa et al. 2008; Canto et al. 2009). The expression of SIRT1 in the cortical neurons was induced after oxygen and glucose deprivation (Wang et al. 2009). Thus, after the developmental period, the expression of SIRT1 seems to enter dormancy and be regulated by a fine system depending on changes in physiological conditions. The liver showed the most dramatic decrease in immunoreactivity during development, which may be due to the change in its function from transient hematopoiesis to actual hepatic functions.
On PD16, the SIRT1 immunoreactivity still remained in the heart and the uriniferous tubule and collecting duct in the kidney. These tissues in adults were reported to have SIRT1, whose function was demonstrated to be protection from cell death induced by oxidative stress (Alcendor et al. 2004; 2007; Kume et al. 2006; Hasegawa et al. 2008; Hsu et al. 2008). This function of SIRT1 may operate from early in organogenesis.
During the fetal period, we detected very strong immunoreactivity in the testis. To determine if SIRT1 plays a role in mammalian gene silencing, two strains of mice carrying a null allele of SIRT1 were created (McBurney et al. 2003; Coussens et al. 2008). The mice were smaller and most died during the early postnatal period. Some of the mice that survived to adulthood showed sterility in both sexes. Analysis of spermatozoa from the cauda epididymis of the SIRT1 null mice showed dramatically decreased numbers of mature sperm and the sperm had an abnormal structure with no motility (McBurney et al. 2003). These results and the present results strongly suggest that SIRT1 plays an important role in the development and maintenance of germ cells from the very early embryonic stage.
A product of the longevity gene Sir2 has been demonstrated to have a histone deacetylase activity in yeasts (Imai et al. 2000). SIRT1 has also been reported to show deacetylase activity in mammalian tissues and cells (Vaziri et al. 2001; Brunet et al. 2004; Daitoku et al. 2004; van der Horst et al. 2004; Yeung et al. 2004; Jeong et al. 2007; Li et al. 2008). SIRT1 not only catalyzes histone, but also deacetylates lysine residues on various proteins (Zeng et al. 2009). Covalent histone modifications regulate gene expression (Strahl and Allis 2000). Acetylation of lysine on histone proteins neutralizes the positive charge of histone and decreases affinity between histone and DNA (negatively charged), which renders DNA more accessible to transcription factors and increases the chance for modifications, such as methylation. Considering that SIRT1 has histone deacetylase activity, SIRT1 definitely plays a role in modulating gene expression in each organ under development. Intrauterine conditions, such as undernutrition have been associated with a higher incidence of lifestyle-associated diseases in adulthood (McMillen and Robinson 2005). In mice, altered levels of DNA methylation and expression of some genes have been reported in the liver of offspring of mothers suffering from undernutrition during pregnancy (Gluckman et al. 2007). It is important to investigate the contribution of SIRT1 to lifestyle-associated diseases because SIRT1 activity strongly depends on NAD, which should be increased under conditions of undernutrition.