Compositional analysis of chondroitin/dermatan sulfate in rhesus monkeys (Macaca mulatta).

Glycosaminoglycans (GAGs) are found in various tissues and are involved in many physiological functions. Since the rhesus monkey (Macaca mulatta) is the most widely used nonhuman primate in biomedical research, an understanding of the compositions of GAGs in their tissues is important. The aim of this study was to determine the content and sulfation pattern of disaccharides contained in several tissues of the rhesus monkey. The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from several tissues of female and male rhesus monkeys. Compositional analysis was performed after digestion with chondroitinases ABC and ACI to reveal the sulfation pattern of the CS/DS hybrid chain. This study revealed that the major CS/DS disaccharide units present in the tissues were A and C types. The E and iE types were specifically distributed not only in the tracheal tissue but also in gastrointestinal tissues.

Compositional analysis of the glycosaminoglycan family in velvet antlers of Sika deer (Cervus nippon) at different growing stages.

Glycosaminoglycans (GAG) from the velvet antlers of Sika deer (Cervus nippon) at the different growing stages (Fukurozuno, Anshi, and Santajo) of bred and wild deer were isolated and their concentrations and sulfation patterns were analyzed. GAG were digested with chondroitinase ABC, ACI, heparinase-I and -III, and keratanase-II into the corresponding repeating disaccharides of chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan, heparan sulfate (HS), and keratan sulfate. Cartilaginous tissues contained CS-DS at high concentrations with an almost equal ratio of 4- and 6-sulfates, while 4-sulfate-type CS-DS predominantly occupied ossified tissues, but at low concentrations. High O- and N-sulfation degrees of HS correspond to high ossification. Dynamic quantitative changes in CS-DS and compositional changes in CS-DS and HS were closely associated with the mineralization of deer antlers.

Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C2C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.

Expression of anti-Müllerian hormone and its type II receptor in germ cells of maturing rat testis.

This work aimed to clarify the expression and roles of anti-Müllerian hormone (AMH) and its type 2 receptor (AMHR2) in seminiferous tubules of maturing rat testes. By quantitative RT-PCR, we determined the relative expressions of Amh, Amhr2, Scp1, Rsbn1, Ngfr, and Rhox5 in rat testes aged 5-49 days (d), and in germ cells and Sertoli cells isolated from 21d testes. Smad 1,5 and 8 expressions were also determined in 21d testes and isolated germ cells. Moreover, we performed in situ hybridization (ISH) of Amh and Amhr2 in 21d testes, and immunohistochemical staining (IHCS) in 10, 15 and 21d testes using antibodies of AMH and AMHR2. In 21d testes, expression of the spermatocyte specific gene, Scp1, increased but that of the round spermatid specific gene, Rsbn1, was faint. By ISH and IHCS, expressions of AMH and AMHR2 were strongly observed in spermatocytes of 21d testes, but not in spermatogonia. In 21d testes, expressions of immature Sertoli cell specific gene, Ngfr, and mature Sertoli cell specific gene, Rhox5, were observed. IHCS confirmed the presence of AMH and AMHR2 in Sertoli cells. Smad 1, 5 and 8 were highly expressed in 21d testes and isolated germ cells. These results indicate that not only immature Sertoli cells but also spermatocytes express AMH and AMHR2 in maturing testes. In this study, we first clarified that spermatocytes coexpressed AMH and AMHR2 in rats. We speculated that AMH produced by spermatocytes and Sertoli cells binds AMHR2 of spermatocytes and acts through SMADs.

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

Sex differences in COL1A1 Expression and Collagen Content in Skeletal Muscle of Mature and Juvenile Shamo Chickens.

Collagen content is an important parameter affecting meat consistency. Sex differences in collagen were therefore studied in mature and juvenile Shamo chickens. The pectoral (PT), lateral iliotibial (ITL), medial part of puboischiofemoral (PIF), and lateral part of gastrocnemius (GCL) muscles were weighed, and their COL1A1 expression levels and total collagen content were analyzed. Body and muscle weights were significantly higher in males than in females of all ages. Muscle/body weight ratios were also higher in mature males than in females, but this difference was not observed in juveniles. In mature chickens, COL1A1 expression was higher in the PIF and GCL muscles; this was not the case in juvenile chicken muscles. Sex differences in collagen content were observed only in the ITLs of mature chickens. A positive correlation between muscle weight and intramuscular collagen content was found for PT and GCL, but not for ITL and PIF, muscles. These results suggest that the sex difference in intramuscular collagen content only occurs in specific muscles and that COL1A1 expression is not necessarily related to collagen content in mature chickens. Factors that determine the intramuscular collagen content likely differ by muscle type.

Quantitative, compositional, and immunohistochemical analyses of chondroitin sulfate, dermatan sulfate, and hyaluronan in internal organs of deer (Cervus nippon centralis and C. n. yesoensis) and cattle (Bos taurus).

Chondroitin sulfate (CS) + dermatan sulfate (DS) and hyaluronan (HA) concentrations and the sulfation patterns of CS-DS in the cartilaginous tissues and alimentary canals of Honshu Sika deer, Hokkaido Sika deer, and cattle were investigated in the present study. CS + DS concentrations were high in cartilaginous tissues, namely, the trachea and scapular cartilage region (5- 12 g*), and low in the alimentary canal (~0.3 g*). HA concentrations were low in cartilaginous tissues and the alimentary canal (~0.2 g*). All tissues mainly contained A-type [HexAGalNAc(4-sulfate)] and C-type [HexAGalNAc(6-sulfate)] CS + DS. The ratios of A-type/C-type CS + DS were 1.2- 3.1 and 0.9- 16.4 in cartilaginous tissues and the alimentary canal, respectively. CS + DS predominantly comprised ß-D-GlcA and α-L-IdoA in cartilaginous tissues and the alimentary canal, respectively. The alimentary canal characteristically contained up to 14 % highly sulfated E-type [HexAGalNAc(4,6-disulfate)] and D-type [HexA(2-sulfate)GalNAc(6-sulfate)] CS + DS. The specific distributions of CS and DS were immunohistochemically confirmed using CS + DS-specific antibodies. Although the omasum of cattle is more likely to have higher concentrations of CS + DS and HA, no significant species differences were observed in the concentrations or sulfation patterns of CS + DS among species for Honshu Sika deer, Hokkaido Sika deer, and cattle. (*per 100 g of defatted dry tissue).

Expression and localization of the neuropeptide Y-Y4 receptor in the chick spleen: mRNA upregulation by high ambient temperature.

High ambient temperatures (HT) can increase diencephalic neuropeptide Y (NPY) expression, and central injection of NPY attenuates heat stress responses while inducing an antioxidative state in the chick spleen. However, there is a lack of knowledge about NPY receptor expression, and its regulation by HT, in the chick spleen. In the current study, male chicks were used to measure the expression of NPY receptors in the spleen and other immune organs under acute (30 vs. 40 ± 1°C for 3 h) or chronic (30 vs. 40 ± 1°C for 3 h/day for 3 days) exposure to HT and in response to central injection of NPY (47 pmol, 188 pmol, or 1 nmol). We found that NPY-Y4 receptor mRNA was expressed in the spleen, but not in other immune organs studied. Immunofluorescence staining revealed that NPY-Y4 receptors were localized in the splenic pulp. Furthermore, NPY-Y4 receptor mRNA increased in the chick spleen under both acute and chronic exposure to HT. Central NPY at two dose levels (47 and 188 pmol) and a higher dose (1 nmol) did not increase splenic NPY-Y4 receptor mRNA expression or splenic epinephrine under HT (35 ± 1°C), and significantly increased 3-methoxy-4-hydroxyphenylglycol (MHPG) concentrations under HT (40 ± 1°C). In conclusion, increased expression of NPY-Y4 receptor mRNA in the spleen under HT suggest that Y4 receptor may play physiological roles in response to HT in male chicks.

The fibrous tapetum of the horse eye.

The tapetum lucidum is a light-reflective tissue in the eyes of many animals. Many ungulates have a fibrous tapetum. The horse has one of the largest eyes of any living animal and also has excellent vision in low-light environments. This study aimed to clarify the macroscopic tapetal shape, relationship between the tapetal thickness and the degree of pigmentation of the retinal pigment epithelium (RPE), spatial relationship between the visual streak and the tapetum, and wavelength of the light reflected from the tapetum in the horse. Macroscopically, weak light revealed the tapetum as a horizontal band located dorsal to and away from the optic disc. The tapetum expanded dorsally as the illumination increased. The tapetal tissue consisted of lamellae of collagen fibrils running parallel to the retinal surface; these spread over almost the entire ocular fundus and were thicker in the horizontal band dorsal to the disc. Only the horizontal band of the tapetum was covered by unpigmented RPE, suggesting that this band reflects light and is responsible for mesopic and scotopic vision. The visual streak was located in the ventral part of the horizontal band, ventral to the thickest part of the tapetum. The wavelength of the light reflected from the horizontal band of the tapetum was estimated from the diameter and interfibrous distance of the collagen fibrils to be approximately 468 nm. Therefore, the light reflected from the tapetum should be more effectively absorbed by rods than by cones, and should not interfere with photopic vision.

Tumor suppressor candidate TUSC3 expression during rat testis maturation.

Analysis of microarray data obtained by comparing gene expression between 2-week-old infant and 7-week-old mature SD rat testes revealed novel targets involved in tumor suppression. Reverse-transcription polymerase chain reaction and Northern blotting indicated that Tusc3 gene expression was upregulated in the normal maturing testis and prostate and other organs such as the cerebrum and ovary. Tumor suppressor candidate 3 protein expression was detected in these same organs at a size of about 40 kDa, in accord with the predicted molecular size. In situ hybridization and immunohistochemistry showed that mRNA and protein localization were prevalent in the testis spermatocytes and interstitial cells such as the Leydig cells, as well as prostate epithelial cells. These data suggest that TUSC3 is deeply involved in spermatogenesis in the testis, inducing sperm differentiation and maturation, and plays a role in normal prostate development and tumor suppression.

Diamond squid (Thysanoteuthis rhombus)-derived chondroitin sulfate stimulates bone healing within a rat calvarial defect.

Chondroitin sulfate (CS) has been suggested to be involved in bone formation and mineralization processes. A previous study showed that squid-derived CS (sqCS) has osteoblastogenesis ability in cooperation with bone morphogenetic protein (BMP)-4 in vitro. However, in vivo, osteogenic potential has not been verified. In this study, we created a critical-sized bone defect in the rat calvaria and implanted sqCS-loaded gelatin hydrogel sponges (Gel) into the defect with or without BMP-4 (CS/BMP/Gel and CS/Gel, respectively). At 15 weeks, bone repair rate of CS/Gel-treated defects and CS/BMP/Gel-treated defects were 47.2% and 51.1%, respectively, whereas empty defects and defects with untreated sponges showed significantly less bone ingrowth. The intensity of von Kossa staining of the regenerated bone was less than that of the original one. Mineral apposition rates at 9 to 10 weeks were not significantly different between all treatment groups. Although bone repair was not completed, sqCS stimulated bone regeneration without BMP-4 and without external mesenchymal cells or preosteoblasts. Therefore, sqCS is a promising substance for promotion of osteogenesis.

Collagen Content and Collagen Fiber Architecture in the Skin of Shamo Chicken, a Japanese Game Fowl.

Collagen content and collagen fiber architecture in the skin of Shamo chickens were compared between sexes and body parts. Cervical, thoracic, dorsal, femoral, and crural skin samples were collected and their collagen content was analyzed. Collagen fiber specimens were prepared for scanning electron microscopy using the cell maceration method with a NaOH solution. Sex differences in collagen content were only observed in the femoral skin of mature chickens, but not in 10-week-old chicks. The difference in collagen content between body parts was obvious; femoral and crural skin had higher collagen content than those of other parts in both sexes. Scanning electron microscopy indicated that the collagen fiber architecture was quite different between the superficial and deep layers in the dermis, with the former consisting of loosely tangled band-like collagen fibers, and the latter composed of thick and dense layers of collagen bundles in a parallel arrangement. The width of collagen fibers in the superficial layer of the dermis differed between sexes in the dorsal, femoral, and crural skin. From these results, it is likely that the difference in collagen content in the femoral skin is not due to sex hormones but other factors, such as mechanical stimulation in daily activity. Additionally, collagen fiber width in the superficial layer is likely related to the difference in collagen content between sexes and between body parts.

Effects of different coating materials on the morphological characteristics of chicken adenohypophyseal folliculo-stellate cells in vitro.

Chicken adenohypophyseal cells were cultured in plates coated with different materials, and their morphologies were examined to confirm the characteristics of chicken folliculo-stellate (FS) cells in vitro. The adenohypophyseal cells were dispersed with a collagenase/trypsin mixture in media and seeded in plates coated in either poly L-lysine (PLL), collagen, or laminin. After 7 days of culture, the cells were fixed and immunocytochemistry was performed. 5-Bromo-2'-deoxyuridine incorporation test indicated that the proliferation activity of the culture cells was different based on the coating materials, and it was higher in the collagen-coated plate than two other coating materials. Fluorescence immunocytochemistry was also performed using mixed antibodies against growth hormone, prolactin, luteinizing hormone ß-subunit, basic cytokeratin (bCK), and S100B. The culture cells on the PLL- and laminin-coated surfaces were round or oval in shape, and bCK-immunopositive FS cells were morphologically indistinguishable from endocrine cells. In the collagen-coated plate, many endocrine cells were round or oval in shape, but FS cells displayed a larger and flattened morphology. S100B-immunoreactions were localized in the nuclei of bCK-immunopositive FS cells. These results suggest that culturing the chicken adenohypophyseal cells in the collagen-coated plate enables the distinction of FS cells from endocrine cells.

Qualitative and quantitative analyses in sulfated glycosaminoglycans, chondroitin sulfate/dermatan sulfate, during 3 T3-L1 adipocytes differentiation.

Chondroitin sulfate/dermatan sulfate (CS/DS) is a member of glycosaminoglycans (GAGs) found in animal tissues. Major CS/DS subclasses, O, A, C, D, and E units, exist based on the sulfation pattern in d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine repeating units. DS is formed when GlcA is epimerized into l-iduronic acid. Our study aimed to analyze the CS/DS profile in 3 T3-L1 cells before and after adipogenic induction. CS/DS contents, molecular weight (Mw), and sulfation pattern were analyzed by using high-performance liquid chromatography. CS/DS synthesis- and sulfotransferase-related genes were analyzed by reverse transcription real-time PCR. CS/DS amount was significantly decreased in the differentiated (DI) group compared to the non-differentiated (ND) group, along with a lower expression of CS biosynthesis-related genes, chondroitin sulfate N-acetylgalactosaminyltransferase 1 and 2, as well as chondroitin polymerizing factor. GAGs in the DI group also showed lower Mw than those of ND. Furthermore, the A unit was the major CS/DS in both groups, with a proportionally higher CS-A in the DI group. This was consistent with the expression of carbohydrate sulfotransferase 12 that encodes chondroitin 4-O-sulfotransferase, for CS-A formation. These qualitative and quantitative changes in CS/DS and CS/DS-synthases before and after adipocyte differentiation reveal valuable insights into adipocyte development.

Housekeeping gene expression variability in differentiating and non-differentiating 3T3-L1 cells.

Normalization is a crucial step in gene expression analysis to avoid misinterpretation. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of 10 candidate housekeeping genes in non-differentiated (ND) and differentiated (DI) 3T3-L1 cells on days 5 and 10. We used geNorm, NormFinder, BestKeeper, RefFinder, and the ∆Ct method to evaluate expression stability. The findings revealed that (1) the expression levels of the reference genes changed over time, even in non-differentiating cells, and (2) peptidylprolyl isomerase A (Ppia) and TATA box-binding protein (Tbp) were stable reference genes for 10 days in both undifferentiated and differentiated 3T3-L1 cells. Notably, the expression of known reference genes in non-differentiating cells was altered throughout the experiment.

The complete control of murine pregnancy from embryo implantation to parturition.

The ovary is the main secretory source of progestin and estrogen and is indispensable to the maintenance of all events of pregnancy in mice. The purpose of this study was to control all processes of pregnancy in mice, from embryo implantation to parturition, without ovaries. The ovaries were removed before embryo implantation, and a single injection of medroxyprogesterone acetate (MPA) was given. Embryo implantation was induced by leukemia inhibitory factor, which can substitute 17ß-estradiol (E(2)). Continuous exposure to E(2) was necessary at mid-pregnancy, when placentation was completed. All mice sustained pregnancy without ovaries before parturition, which was initiated by the removal of E(2) and MPA. Murine pregnancy is a complicated process involving embryo implantation, placentation, and parturition. Complete control of pregnancy was achieved with the simple treatment of MPA and E(2) after induction of embryo implantation. Here, time-dependent events in the uterus during pregnancy could be realized without the ovaries, because the initiation of each event could be stringently controlled by hormonal treatments.

Expression of a novel sphingosine 1-phosphate receptor motif protein gene in maturing rat testes.

We screened a novel sphingosine 1-phosphate receptor type 5 motif-containing gene, LOC290876, from maturing rat testes by differential display. Gene expression was testis-specific, increased at week 7, and continued for 15 weeks. PCR analysis clarified two gene transcript isoforms, which were expressed at the same level in all samples detected in Northern blot. The deduced amino acid sequences of the two isoforms revealed differences in carboxyl terminal sequences. Gene and protein expression in the testes was dominant in the spermatocytes, and protein expression was localized to the nucleus. Taken together, these findings suggest that the LOC290876-encoded gene product is not involved in sphingosine signaling, but has distinct roles in the nucleus during the processes of spermatocyte maturation and meiosis producing spermatids.

Rat stem-cell leukemia gene expression increased during testis maturation.

We found that stem-cell leukemia (SCL), also known as T cell acute-lymphocytic leukemia (Tal-1) gene expression, was upregulated in the maturing rat testis. Strong expression of Tal-1 was detected in the normal maturing rat testis by Northern blotting. Western blotting revealed the protein size to be about 34 kDa. Protein expression was wide-spread in spermatocytes, spermtids and spermatogonia in accordance with the seminiferous epithelium cycle, as determined by an analysis of immunohistochemistry. Gene expression of Tal-1 regulatory gene, NKX3.1, was negatively correlated with Tal-1 expression. Human Tal-1 expression in the maturing testis as well as in bone marrow was observed, which suggests that the gene product is a novel cancer-testis antigen candidate. Taken together, TAL-1 may be involved in cell division, morphological changes, and the development of spermatogenic cells in the normal rat testis.

Preliminary study of the gene expression of sulfation and degradation enzymes for chondroitin sulfate in glycerol-treated C2C12 myoblast cells.

In this study, we induced chemical damage of C2C12 myoblasts that had differentiated into myotubes with glycerol, and four sulfation enzymes for chondroitin sulfate (CS) [carbohydrate sulfotransferase (Chst) 12, Chst15 and Chst3 and uronyl 2-O-sulfotransferase (UST)] and two CS degradation enzymes [hyaluronidase (Hyal) 1 and Hyal2] were examined for changes in gene expression. Treatment of myoblasts with 5% glycerol significantly increased the expression levels of the sulfation enzymes Chst12 and Chst15 and the degradation enzymes Hyal1 and Hyal2. However, the expression levels of the other two genes (Chst3 and Ust) showed no change. Differences in the expression levels of these enzymes may help to understand the difference in responsiveness of myoblasts to glycerol after muscle injury in vivo or in vitro.

Chondroitin sulfate E downregulates N-cadherin and suppresses myotube formation.

Myogenesis, the formation of muscle fibers, is affected by certain glycoproteins, including chondroitin sulfate (CS), which are involved in various cellular processes. We aimed to investigate the mechanism underlying CS-E-induced suppression of myotube formation using the myoblast cell line C2C12. Differentiated cells treated with 0.1 mg/ml CS-E for nine days showed multinucleated and rounded myotubes with myosin heavy chain positivity. No difference was found between the CS-E-treated group with rounded myotubes and CS (-) controls with elongated myotubes in the levels of phospho-cofilin, a protein involved in the dynamics of actin cytoskeleton. Interestingly, N-cadherin, which is involved in the gene expression of myoblast fusion factors (myomaker and myomixer), was significantly downregulated at both the mRNA and protein levels following CS-E treatment. These results suggest that N-cadherin downregulation is one of the mechanisms underlying the CS-E-induced suppression of myotube formation.