Identification of some calcium binding proteins and neural cell markers in rat testis and epididymis during postnatal development.

Calcium-binding proteins (CaBPs) have an essential role in male reproduction by modulating calcium ion metabolism. Although the brain and testis are structurally and functionally different, they show a high degree of transcriptomic and proteomic similarities. The purpose of the present study was to explore some CaBPs (Iba-1, Calbindin, Calretinin and Parvalbumin) and neural cell markers (CNPase, NG2 and Drebrin) expression in rat testis and epididymis during postnatal periods by using immunohistochemistry. Iba-1, calbindin, calretinin, parvalbumin, CNPase, NG2 and drebrin were expressed in the epididymal epithelium in each postnatal period. Iba-1 and calbindin expression showed stage-dependent expression in spermatids. CaBPs and neural cell markers showed a positive reaction in Leydig cells in the postpubertal and mature periods. Sertoli cells, gonocytes, spermatogonium, and peritubular myoid cells showed heterogeneity in the expression of CaBPs and nerve markers throughout postnatal development. Interestingly, CNPase, NG2 and drebrin were positive in spermatocytes, spermatids, and sperm. Expression dynamics of calcium-binding proteins and nerve markers showed differences in germ cells and somatic cells during postnatal development. The expression of these proteins in the testis and epididymis supports that they may have important roles in reproductive physiology.

Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development.

Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5-19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.

Expression patterns of natriuretic peptides in pre-hibernating and hibernating Anatolian ground squirrel (Spermophilus xanthoprymnus) kidney.

Hibernation is characterized by marked suppression of renal function. Natriuretic peptides (NPs) are involved in the regulation of renal function. However, the role of NPs in the renal function during hibernation remains unclear. We aimed to investigate the distribution patterns of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) in Anatolian ground squirrel (Spermophilus xanthoprymnus) kidneys during pre-hibernation and hibernation periods. Cortical proximal tubules showed weak ANP immunoreactivity, with moderate staining on the brush border during the pre-hibernation period. In the hibernation period, moderate ANP immunoreactivity was seen in cortical proximal tubules, with very weak reaction in hibernating cortical distal tubules, medullary proximal and collecting tubules. Cortical proximal and distal tubules of both periods had strong and weak BNP immunoreactivity, respectively. Medullary proximal, distal and Henle's loop segments showed very weak BNP immunoreactivity during pre-hibernation. Medullary distal, proximal and collecting tubules and Henle's loop segments had moderate staining during hibernation. In both periods, cortical proximal tubules displayed strong immunoreactivity to CNP. Distal tubules had moderate CNP staining during pre-hibernation, albeit weak staining during hibernation. Medullary proximal tubules exhibited moderate to strong immunoreactivity during pre-hibernation. Medullary distal and proximal tubules had weak and moderate CNP staining, respectively, during pre-hibernation. In both periods, Henle's loop segments displayed moderate CNP immunoreactivity. Glomeruli had similar weak ANP, BNP and CNP staining in both periods. These results suggest that heterothermic conditions differently affected the expression of NPs in the squirrel kidney. This different expression of NPs may contribute to the renal adaptation during hibernation.

Spatiotemporal expression patterns of natriuretic peptides in rat testis and epididymis during postnatal development.

Natriuretic peptide (NP) family is composed of atrial, brain and C-type NP (NPPA, NPPB and NPPC). Here, we aimed to investigate NP expression in testis and epididymis during postnatal development. NPPA expression was observed in gonocytes at prepubertal period but in only spermatocytes in pachytene and leptotene/zygotene stage at pubertal period. In prepubertal and pubertal periods, we detected NPPB expression in only Leydig cells. However, NPPC expression was detected in all of the gonocytes and Sertoli cells, spermatocytes and some interstitial cells in prepubertal and pubertal periods. In postpubertal and mature periods, NPPA and NPPB staining were detected in Leydig cells, elongated and round spermatids but not in spermatogonia and spermatocytes. However, we observed NPPC expression in all cells of the seminiferous tubules and Leydig cells in the postpubertal and mature periods. Epididymal epithelium showed intense NPPC expression during postnatal period but weak NPPA and NPPB expression in prepubertal and pubertal periods. The expression of three NPs in the testis significantly increased after puberty. In conclusion, puberty had a significant effect on NP expression in testis. Unlike NPPA and NPPB, expression of NPPC in all cells of the seminiferous tubule suggests that NPPC is effective in each step of spermatogenesis.

Expression patterns of natriuretic peptides in pre-hibernating and hibernating anatolian ground squirrel (Spermophilus xanthoprymnus) lung.

Anatolian ground squirrel (Spermophilus xanthoprymnus) is a true hibernator. This animal transiently reduces pulmonary function during hibernation. Continuance of pulmonary function is very important to survive ground squirrels during the hibernation. Natriuretic peptides may be key players in the modulation of pulmonary hemostasis. However, NPs' role in pulmonary function during hibernation remains unclear. We aimed to investigate the localization and distribution of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) in squirrel lungs during pre-hibernation and hibernation periods using immunohistochemistry. Our immunohistochemical data indicate that ANP, BNP, and CNP were produced by the mucosal epithelium of terminal and respiratory bronchioles, smooth muscle cells in the lamina propria of terminal bronchioles and vascular smooth muscle cells, alveolar type II cells, and macrophages. ANP immunoreactivity was weaker than BNP and CNP immunoreactivities in these cells. The results also demonstrate that the number of ANP, BNP and CNP positive alveolar type II cells tended to increase, although statistically non-significant, during the hibernation period, but the expression of NPs in other pulmonary cells is unaffected by hibernation. This study firstly investigates ANP, BNP and CNP distribution in the Anatolian ground squirrel lung. However, further studies are required to dissect their functional roles during the hibernation.

Localization profiles of natriuretic peptides in hearts of pre-hibernating and hibernating Anatolian ground squirrels (Spermophilus xanthoprymnus).

The Anatolian ground squirrel (Spermophilus xanthoprymnus) is a typical example of true mammalian hibernators. In order to adapt to extreme external and internal environments during hibernation, they lower their body temperatures, heart rates and oxygen consumption; however, pathological events such as ischemia and ventricular fibrillation do not occur in their cardiovascular systems. During the hibernation, maintenance of cardiac function is very important for survival of ground squirrels. Natriuretic peptides (NPs) are key factors in the regulation of cardiovascular hemostasis. Since NPs' role on the protection of heart during hibernation are less clear, the aim of this study was to investigate dynamic changes in NPs content in the cardiac chambers and to reveal the possible role of NPs on establishing cardiac function in ground squirrel during hibernation using immunohistochemistry. The immunohistochemical results indicate that cardiac NP expressions in atrial and ventricular cardiomyocytes were different from each other and were sex-independent. ANP and BNP were expressed in a chamber-dependent manner in female and male squirrel hearts. Furthermore, cardiac NPs expression levels in hibernation period were lower than those at the pre-hibernation period. During prehibernation period, ANP, BNP and CNP were expressed in the white and beige adipocytes of epicardial adipose tissue (EAT); while during hibernation period, the brown adipocytes of EAT were positive for BNP and CNP. These data suggest that the hibernation-dependent reduction in levels of NPs, particularly ANP, in cardiac chambers and EAT may be associated with low heart rate and oxygen consumption during hibernation. However, further studies are needed to better delineate the roles of NPs during the hibernation.

Expression pattern of galectin-1 and galectin-3 in rat testes and epididymis during postnatal development.

Galectins are a family of lectins-binding beta-galactosides involved in a variety of extracellular and intracellular processes, thereby contributing to homeostasis, cell adhesion, cellular turnover, and immunity. This study aimed to determine the localization and expression of galectin-1 (Gal-1) and galectin-3 (Gal-3) in the testis and epididymis of rats at postnatal [(prepubertal (day 5), pubertal (day 20), postpubertal (day 50) and mature (day 70)] periods by using immunohistochemistry and Western blotting. Gal-1 and Gal-3 were differentially expressed in different types of cells in the testis and epididymis during postnatal development. While we detected Gal-1 expression in some spermatogenic cells and Leydig cells in the testis, not in the epididymal epithelium, Gal-3 was expressed in Sertoli cells, peritubular myoid cells, Leydig cells, smooth muscles and interstitial CD68-positive macrophages. Epithelial cells of the corpus and cauda epididymis showed an intense Gal-3 expression. Gal-1 expression was higher in the testis than in the epididymis on days 50 and 70. The expression of Gal-3 in the testis increased from the prepubertal to mature period. While the expression difference of Gal-3 was not statistically significant in the testis and epididymis until puberty, Gal-3 expression in the postpubertal and mature periods was higher in the epididymis. The expression of Gal-3 in the corpus and cauda epididymis was higher than that in the caput epididymis. In conclusion, our findings suggest that puberty has potential regulatory effect on the expression of galectins in testis and epididymis of rats. Gal-1 and 3 may play a role in the development of the reproductive system and the preservation of the immune-privileged environment in the testis, due to their pro-apoptotic and anti-apoptotic functions. The presence of intense expression of Gal-3 in the corpus and cauda epididymis may contribute to the maturation and storage of spermatozoa.

Expression profile of some neuronal and glial cell markers in the ovine ileal enteric nervous system during prenatal development.

The enteric nervous system (ENS) is a network of neurons and glia found in the gut wall and governs this gastrointestinal function independently from the central nervous system (CNS). ENS comprises the myenteric plexus (MP) and the submucous plexus (SP). In this study, we examined the expression profile of neurofilament heavy chain (NF-H), neuron-specific enolase (NSE), calcyclin (S100A6), vimentin and glial fibril acidic protein (GFAP) in ovine ileal enteric neurons and enteric glia cells (EGCs) during prenatal development using an immunohistochemical method. The material of the study consisted of 15 different fetal ileum tissues obtained between days 60 and 150 of pregnancy. NF-H was observed in the majority of ganglion cells in SP and MP throughout the fetal period. It was determined that there was no NF-H reaction in some ganglion cells in Peyer's patches of internal submucosal plexus (ISPF). In the early stage of pregnancy (60-90 days), there was no expression of NSE and S1006 in ileum. After this period, NSE and S1006 were expressed in the ganglion cells of the plexus, indicating an increase in the amount of expression towards the end of pregnancy. In the early period, vimentin expression was only detected in intramuscular interstitial cells (ICs) (60-90 days), but later (90-150 days) it was also seen in the cells around the ganglion cells in the plexus. On days 60-90 of gestation, GFAP expression only occurred in MP, but in later stages, staining was also detected in SP. In the plexus, an immunoreactivity was present in EGCs forming a network around the ganglion cell. During the last period of gestation (120-150 days), the number of GFAP-positive plexus increased, with the majority of these stained cells being observed in MP. Interestingly, weak staining or reaction did not occur in ISPF, unlike other plexuses. In conclusion, this is the first study that demonstrated the expression of NF-H, vimentin, S100A6, NSE and glial fibril acidic protein (GFAP) in ovine ileal ENS in the prenatal period. In the last period of gestation (120-150 days), the expression profile of ENS was similar to that of adult animals. The expression of the used markers increased toward the end of pregnancy. Our results suggest that neurons and EGCs show heterogeneity, and GFAP and NF-H cannot be used as panenteric glial or panneuronal markers, respectively. We also demonstrated, for the first time, the prenatal expression of S100A6 in enteric neurons and the possibility of using this protein for the identification of enteric neurons.

Prenatal development and histochemical characteristics of gastrointestinal mucins in sheep fetuses.

The object of this study was to describe the prenatal development and histochemical properties of mucins in the sheep gastrointestinal tract. To determine changes in the mucin profile, the sections were stained with specific histochemical stains for carbohydrates. While neutral and mixed mucins were observed in the superficial epithelial cells of the abomasal pyloric region, acidic mucins were detected in the secretory ducts and corpus of the glands. Acidic mucins consisted predominantly of sialomucins. In the duodenal villi, the number of goblet cells containing neutral mucins increased toward the end of gestation, whereas Brunner's glands contained acidic mucins until the 95th day of gestation and both acidic and neutral mucins thereafter. The jejunal goblet cells contained either acidic, neutral, or mixed mucins. Goblet cells containing acidic mucins, which were mainly localized to the ileal crypts and villi, mostly contained sulfated mucins. While villi were observed in the proximal colon until the 115th day of gestation, later the typical crypt structure emerged. During the period in which the villi were found in the proximal colon, the goblet cells containing sulphomucins were predominant, whereas the goblet cells containing sialomucins were predominant after the typical crypt structure was formed. In conclusion, gastrointestinal mucins may be involved in the formation of meconium during the prenatal period, and acidic mucins may contribute to the strength of the intestinal barrier against pathogens and digestive enzymes, as the barrier is not fully functional after birth.

Intestinal macrophages in Peyer's patches, sacculus rotundus and appendix of Angora rabbit.

The largest pool of macrophages in the body is harboured by the intestinal mucosa. As the principal phagocytic component of the immune system, macrophages are essential for maintaining mucosal homeostasis as they prevent commensal bacteria from adhering to mucosal epithelial cells. This study provides a RAM11 immunohistochemical and electron microscopic investigation of the existence, localization and distribution of intestinal macrophages in organized gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), the sacculus rotundus (SR) and the appendix, in the Angora rabbit. Although rabbit intestinal macrophages did not express the tissue macrophage marker macrosialin (CD68), they expressed RAM11. RAM11-positive intestinal macrophages were mostly localized to the subepithelial dome region, interfollicular area and germinal centres (GCs) of the GALT and the lamina propria or submucosa of the ileum and jejunum devoid of PPs and were also observed in the follicle-associated epithelium of PPs, but not in that of the SR and appendix. RAM11-positive macrophages containing engulfed apoptotic bodies were present in the GCs of the lymphoid follicles in the GALT. Electron microscopy further revealed multiple macrophages containing apoptotic bodies within the GCs of the follicles in the GALT. Some macrophage aggregations were observed in the GC and between the GC and the corona region of the follicles in the SR and appendix. Rabbit intestinal macrophages thus undertake both potent phagocytic activity and the efficient scavenging of apoptotic cells. Immunohistochemical data suggest that RAM11 can be reliably used for the determination of intestinal macrophages in the GALT of rabbits.

Identification of intestinal M cells in isolated lymphoid follicles and Peyer's patches of the Angora rabbit.

The presence, distribution, and localization of M cells in isolated lymphoid follicles (ILF) and in follicle-associated epithelia (FAE) covering Peyer's patches (PP) in Angora rabbits were investigated by immunohistochemistry and electron microscopy. Although PP could macroscopically be identified along the length of the mucosal and serosal surfaces of jejunum and ileum, the presence of ILF could only be located microscopically. Typical M cells in FAE were detected within the periphery of the dome regions of the PP, and immature columnar M cells in the FAE resided in the vicinity of the crypts. M cells in the FAE of both ILF and PP showed vimentin-positive reaction. M cells in the FAE of ILF were morphologically similar to the immature M cells found in the FAE of PP. Typical mature M cells were also observed in the FAE of a few ILF. In contrast to FAE of PP, numerous goblet cells were observed in the FAE of many ILF. Moreover, among intestinal villi, we noticed villi-like solitary lymphoid structures that showed abundant lymphocytes in their lamina propria and that were surrounded with vimentin-positive cells and goblet cells. Thus, the occurrence of copious immature M cells and goblet cells, in addition to the detection of villi-like solitary lymphoid structures full of lymphocytes in the FAE of many ILF, indicate that ILF do not complete their immunological maturation in contrast to PP. Various antigenic stimulations conceivably induce the formation and maturation of ILF along the length of the small intestine. The morphological resemblance between ILF M cells and PP M cells suggests that these two types of cells perform similar or the same immunological functions.

The effects of experimental aflatoxicosis on the exocrine pancreas in quails (Coturnix coturnix japonica).

The present investigation was undertaken to assess the effects of aflatoxin (AF) on the exocrine pancreas in quails by means of light and electron microscopy. A total of 30 quails were divided into three groups, each composed of ten animals. Total AF was incorporated into the diet of these groups, at doses of 0, 2.5, and 5.0 mg of AF/kg feed, ppm, respectively. The quails were raised in cages with electrical heating and 24-h lighting for a period of 3 weeks. Ad libitum access was provided to feed and drinking water. Pancreas samples were taken for light and electron microscopic examination from animals that were killed by means of cervical dislocation at the end of the study. Light microscopic examination demonstrated mild mononuclear cell infiltration of exocrine tissue and vacuolisation of acinar cells in the group fed on AF at 2.5 ppm. On the other hand, electron microscopic examination demonstrated degranulation of the rough endoplasmic reticulum (rER) of acinar cells, decrease in the number of zymogen granules and free ribosomes and polisomes, and dilatation of capillaries in the group fed on AF at a dose of 2.5 ppm. Numerous degenerative acinar cells were determined in the group fed a diet containing 5.0 ppm AF, in addition to the findings common with the other group exposed to the toxin.

Simultaneous determination of chlorophyll a and chlorophyll b by derivative spectrophotometry.

Chlorophyll a (Chl a) and chlorophyll b (Chl b) plant pigments, which are important in the food industry and are beneficial as environmental pollution indicators, have been extracted with a novel solvent mixture (1:1 v/v acetone-propanol) not containing chloroform and simultaneously determined by first-derivative spectrophotometry. The results were statistically compared to those obtained by the ordinary absorption spectrophotometric reference utilizing the principle of additivity of absorbances. The testing of the developed method in synthetic mixtures of Chl a and Chl b and in real plant material samples (grass, spinach, chard, purslane, black cabbage, crisp lettuce, rocket, dill and seaweed) proved successful in that the developed extractive derivative spectrophotometric method was both rapid and precise, and was not dependent on the Chl a/b ratio in contrast to the reference method which was adversely affected by the latter parameter.