Angiogenic and anti-angiogenic factors during the post-hatching growth of the quail (Coturnix coturnix japonica) spleen.

Vascular endothelial growth factor (VEGF) family members are responsible for endothelial cells' growth, proliferation, migration, angiogenesis, vascular permeability, and differentiation and proliferation of non-endothelial cell types. VEGF and its receptors are found in mammalian lymphoid organs. The present study was conceived to determine (a) the presence and localization of angiogenic VEGF and its receptors (Fms-like tyrosine kinase 1 [Flt1/fms], fetal liver kinase 1 [Flk1]/kinase insert domain receptor [KDR], Fms-like tyrosine kinase 4 [Flt4]) and vascular endothelial growth inhibitor (VEGI) in the quail spleen; and (b) whether their expressions in the spleen components change during the post-hatching growth of the organ, using immunohistochemistry. Immunohistochemical stainings showed that VEGI, VEGF, and VEGF receptors were expressed in many components, including the vascular endothelial and smooth muscle cells, ellipsoid-associated cells (EACs), and immune cells, of quail spleen and that VEGF and its receptors' immunostaining intensity scores (ISs) varied depending on the post-hatching growth period, while VEGI-IS did not change. In addition, ISs of VEGI, VEGF, Flt1/fms, and Flt4 in EACs were weak to moderate, while flk1/KDR-IS in EACs adjacent to the capsule of Schweigger-Seidel sheaths (ellipsoids) was higher than other proteins, supports a more important and specific role of Flk1/KDR in the EAC function. These specific expressions of VEGI, VEGF, flt1/fms, flk1/KDR, and flt4 proteins in splenic cell types suggest their particular roles, in the functional development of splenic components and thus, are critical to post-hatching maturation of quail spleen. These findings indicate that the expression levels of VEGF, Flt1/fms, and Flt4, except Flk1/KDR, are low in the quail spleen, and only a few components of the spleen express VEGF, Flt1/fms, and Flt4 under normal conditions.

Heat Shock Proteins Are Differentially Expressed in the Domestic Cat (Felis catus) Testis, Epididymis, and Vas Deferens.

Heat shock proteins (HSPs) play key roles in controlling the morphological transformation of germ cells during spermatogenesis and posttesticular maturation of sperm. This study aims to reveal the presence and localization patterns of large adenosine triphosphate (ATP)-dependent HSPs family members in adult domestic cat testis and excurrent ducts utilizing Western blot, immunohistochemistry, and immunofluorescence techniques. The results indicated that the relative amounts of heat shock protein D1 (HSPD1)/HSP60, heat shock protein C (HSPC)/HSP90, and heat shock protein H (HSPH)/HSP105/110 were highest in the testis, while heat shock protein A (HSPA)/HSP70 was highest in the corpus epididymis. HSPs exhibited spermatogenic stage-dependent localization patterns in germ cells. Sertoli and Leydig cells were positive for other HSPs except for HSPC/HSP90. The tubules rectus and rete testis epithelia showed only HSPD1/HSP60 and HSPA/HSP70 immunoreactivity, while the ciliated cells of efferent ductules were positive for all HSPs. In the epididymis and vas deferens, HSPs localizations were cell and region specific. HSPD1/HSP60 was localized in the midpiece of the immature spermatozoa tail, while HSPA/HSP70 and HSP90 were found only in the proximal cytoplasmic droplet (CD). HSPH/HSP105 was observed in CD and the principal piece but not the midpiece. Overall, the different expression of HSPs throughout the domestic cat testis and excurrent ducts indicates their critical roles in maintaining reproductive functions under physiological conditions.

The immunolocalization of cadherins and beta-catenin in the cervix and vagina of cycling cows.

The adherens junctions (AJs) maintain the epithelial cell layers' structural integrity and barrier function. AJs also play a vital role in various biological and pathological processes. AJs perform these functions through the cadherin-catenin adhesion complex. This study investigated the presence, cell-specific localization, and temporal distribution of AJ components such as classical type I cadherins and beta-catenin in the cow cervix and vagina during the estrous cycle. Immunohistochemistry and Western blot analysis results demonstrated that beta-catenin and epithelial (E)-, neural (N)-, and placental (P)-cadherins are expressed in the cow cervix and vagina during the estrous cycle. These adhesion molecules were localized in the membrane and cytoplasm of the ciliated and non-ciliated cervical cells and the stratified vaginal epithelial cells. Positive immunostaining for P-, N-cadherin, and beta-catenin was also observed in the vascular endothelial cells of the cervical and vaginal stroma. Quantitative immunohistochemistry examinations revealed that in the cervical and vaginal epithelia, P-cadherin's optical density values (ODv) were the highest; in contrast, the N-cadherin ODv were the lowest. The ODv of P-cadherin and beta-catenin in the cervical epithelium and E-cadherin in the vagina were significantly higher in the luteal phase versus the follicular phase of the estrous cycle. Furthermore, the ODv of P-cadherin, N-cadherin, and beta-catenin in the cervix's central and peripheral epithelial regions were different during the estrous cycle. These findings indicate that classical cadherins and beta-catenin in the cervix and vagina exhibit cell- and tissue-specific expression patterns under the influence of estrogen and progesterone hormones during the estrous cycle.

The abundance and localization of claudin-1 and -5 in the adult tomcats (Felis catus) testis, tubules rectus, rete testis, efferent ductules, and epididymis.

Tight junctions (TJ) are the anatomical component of blood-testis (BTB) and blood-epididymis (BEB) barriers and contain many proteins, including claudins. The presence of claudins in domestic cat testis and epididymis has not been previously described. This study aimed to determine whether claudin-1 and claudin-5 participate in the structure of BTB and BEB and whether their amounts differ between the testis and epididymal segments of adult cats, using Western blotting (WB) and immunohistochemistry. WB results demonstrated that claudin-1 was significantly lower in the testis than in all epididymal segments and higher in the corpus epididymis than in the cauda, while claudin-5 in the testis was significantly lower than in the caput and corpus. Claudin-1 was absent at the Sertoli-Sertoli junctions, while claudin-5 was detected at the level of the BTB during stages I and VIII. Both claudins were observed in the pachytene spermatocytes and the developing acrosome of the round and elongating spermatids. Claudin-5 was also detected in the cytoplasm of some spermatogonia, Sertoli cells, and late spermatid acrosome. In the epididymal segments, both claudins were localized to the area of the tight junctions and along the entire length of the lateral plasma membranes of adjacent principal cells and between principal and basal cells. These results may indicate that in the domestic cat, claudin-1 and -5 participate as both tight junction proteins and adhesion molecules in the BEB's structure, claudin 5 is a component of the BTB, and both proteins may be involved in postmeiotic germ cell development, especially acrosome development.

Testicular Expression of Antioxidant Enzymes and Changes in Response to a Slow-Release Deslorelin Implant (Suprelorin® 4.7 mg) in the Dog.

Spermatogenesis takes place in a hypoxic environment, and antioxidant enzymes protect germ and somatic cells from free radical-mediated damage. Expression of the antioxidant enzyme system in the canine testis has not yet been investigated. We hypothesized that the slow-release GnRH superagonist deslorelin 4.7 mg implant, which induces temporary reversible suppression of endocrine and germinative testicular function, would affect the testicular expression of antioxidant enzymes compared to untreated adult and prepubertal dogs. The goal of this study was to investigate and compare gene (by qPCR, in whole-tissue homogenates) and protein expression (by immunohistochemistry) of superoxide dismutase (SOD1, SOD2), catalase (CAT), glutathione peroxidase (GPx1), and glutathione disulfide reductase (GSR) in the testes of untreated adult (CON, n = 7), prepubertal (PRE, n = 8), and deslorelin-treated (DES, n = 5, 16 weeks after implantation) dogs. We found that in DES dogs, the gene expression of SOD1 was significantly (p < 0.05) lower and GPx1 was higher than in CON, and SOD2 was higher than in PRE. Expression of all, except for the SOD2 mRNA, differed between the CON and PRE dogs. Immunohistochemistry showed distinct cell-specific localization and expression patterns for the antioxidant enzymes in each experimental group. Additionally, in the CON animals, cell-specific SOD1, CAT, and GSR expression was dependent on the stage of the seminiferous epithelium cycle. These findings confirm that members of the antioxidant enzyme system are present in normal adult and prepubertal testis as well as in the deslorelin-treated downregulated adult canine testis, and that this local antioxidant system protects developing germ cells and somatic cells from oxidative damage. Different expression patterns of antioxidant enzymes in various germ cell populations and stages of the seminiferous epithelium cycle may indicate differences in their susceptibility to oxidative stress depending on their developmental and maturation stage. The continued presence of the antioxidant enzymes in the testis of DES dogs offers protection to spermatogonia as well as Sertoli and Leydig cells from oxidative stress during temporary infertility, potentially contributing to ensure the reversibility of suppression and the return of normal spermatogenesis and steroidogenesis after the end of deslorelin treatment.

The Efficacy of a 3ß-Hydroxysteroid Dehydrogenase Inhibitor for the Termination of Mid-Term Pregnancies in Dogs.

Progesterone (P4) is the only hormone needed to maintain pregnancy in dogs. Therefore, a competitive inhibitor of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) could be a safe and effective option to terminate pregnancy by inhibiting P4 synthesis. To address this hypothesis, we investigated the efficacy of trilostane (TRL), a competitive inhibitor of 3ß-HSD, in terminating pregnancy in dogs. Twenty-one dogs between days 30 and 38 of pregnancy were randomly assigned to one of two treatment groups (trilostane (TRL) and aglepristone (AGL)) and an untreated control (CON) group (n = 7 dogs each). Fetal heart rates (FHRs) (measured at 12 h intervals) and serum P4 concentrations (measured at 6 h intervals) were evaluated. The pregnancy termination rates were 0% and 100% in the TRL and AGL groups, respectively. The decrease in the FHR in the TRL and AGL groups was significantly lower than that observed in the CON group. There was a marked decrease in P4 concentrations in the TRL group 6, 54, and 102 h after the initiation of treatment. The luteal expression of StAR appeared to be weaker in the AGL group than the CON group. In conclusion, although a treatment-induced decrease was observed in plasma P4 concentrations, a seven-day TRL treatment alone was not effective in terminating pregnancies. Further studies are needed on the effects of the prolonged administration of TRL with varying doses and frequencies for the termination of mid-term pregnancy in dogs.

Expression of cadherins and some connective tissue components in cow uterus and placenta during pregnancy.

The implantation and placental development processes are regulated with cell adhesion molecules and remodeling of the maternal endometrium's extracellular matrices (ECM) and fetal chorion. This study aimed to investigate the distribution and localization of some classical cadherins (E-, N-, and P-cadherins) and extracellular matrix components collagen type 5α1, fibronectin, and laminin in the cow placentomes during pregnancy using immunohistochemical and Western blotting analyses. The study results confirmed the expression of E- and P-cadherins, collagen type Vα1 (COLVα1), fibronectin, and laminin in the cow placentomes, but not N-cadherin. Throughout the pregnancy, E- and P- cadherins, COLVα1, and laminin were localized in the luminal and glandular epithelium of the inter-caruncular endometrium, caruncular epithelium, and the uninucleate (UNCs) and binucleate trophoblast giant cells (BNCs/TGCs). E- cadherin immunoreactivity in the first pregnancy period was strong in the UNCs while moderate in the BNCs/TGCs. However, it was weak in both trophoblast in the second and third pregnancy periods. In the fetal trophoblasts, P- cadherin and laminin immunostainings were more intense in the BNCs/TGCs than UNCs. The fetal and maternal stromal cells were also positive for P- cadherin, COLVα1, fibronectin, and laminin. The immunostaining intensity of COLVα1 and fibronectin in the stromal extracellular matrix of the placentomes decreased as the pregnancy progressed. The endothelia of fetal and maternal vessels were positive for all proteins. The presence and distinct localization of cadherins and ECM proteins in the cow placentome components support the role of these molecules in regulating placental cell growth, migration, and matrix production during pregnancy.

Heat shock proteins exhibit distinct spatiotemporal expression patterns in the domestic cat (Felis catus) ovary during the oestrous cycle.

Heat shock proteins (HSP) are significant regulators of cell proliferation, differentiation and apoptosis. HSP participate in ovarian physiology through proliferative and apoptotic mechanisms and the modulation of sex steroid receptor functions. We investigated whether the expression and localisation patterns of HSP in the domestic cat ovary vary with the oestrous cycle stage. Immunohistochemical analysis revealed cell type-specific localisation patterns of HSPD1/HSP60, HSPA/HSP70, HSPC/HSP90 and HSPH/HSP105 in several ovarian cells of the domestic cat, including oocytes, follicular (granulosa and theca cells) and luteal cells, stromal and thecal interstitial cells, stromal cells, and vascular endothelial and smooth muscle cells during the anoestrous, follicular and luteal phases of the oestrous cycle. Western blot results showed that the expression of three HSP (HSPD1/HSP60, HSPA/HSP70 and HSPH/HSP105) varied with the oestrous cycle stage. While the maximal expression of HSPD1/HSP60 and HSPH/HSP105 occurred during the luteal phase, the expression of HSPA/HSP70 was minimal. The expressions of HSPA/HSP70 and HSPH/HSP105 were low during the follicular phase compared to the anoestrous phase. In conclusion, the alterations that occur in the expression of HSP in the domestic cat ovary during the different stages of the oestrous cycle imply that these proteins participate in the regulation of ovarian function under different physiological conditions.

The distribution and immunolocalization of fibroblast growth factors (FGFs) in the rat oviduct during early pregnancy and the post-partum period.

The mammalian oviduct provides a favourable environment for several reproductive processes, including ovum transport, sperm capacitation, fertilization and pre-implantation embryonic development. This environment is regulated by cyclic ovarian steroids, that is oestrogen, and growth factors. Fibroblast growth factors (FGFs) regulate the differentiation and growth of various cell types in the female genital tract. This study aimed to determine the localization of FGF1, FGF2, FGF receptor 1 (FGFR1) and 2 (FGFR2) in the rat oviduct, by immunohistochemistry, on day 5 of pregnancy and post-partum days 1, 3 and 5, and to demonstrate the possible functions of these proteins during early pregnancy and the post-partum period. On all examination days, cytoplasmic and nuclear FGF1 immunoreactivity was detected in the epithelium lining the infundibulum, ampulla and isthmus of the oviduct. Immunoreactivity was much stronger in the basal bodies of the cilia on the epithelium lining the infundibulum and ampulla. FGF1 immunoreactivity was also detected in stromal cells, myocytes and endothelial cells. Cytoplasmic FGF2 immunoreactivity was observed in the tunica muscularis, vascular myocytes and endothelial cells. While strong cytoplasmic FGF2 immunoreactions were observed in the stromal cells of the lamina propria, the luminal epithelium, some stromal cells and smooth muscle cells displayed a rather weak FGFR1 and FGFR2 immunoreactivity. Immunoreaction intensity did not differ between the periods examined. This study shows that FGF1, FGF2, FGFR1 and FGFR2 are produced by rat oviduct cells during pregnancy and the post-partum period, and reproductive physiology is regulated not only by hormonal mechanisms, but also by growth factors.

Abundances and localizations of Claudin-1 and Claudin-5 in the domestic cat (Felis catus) ovary during the estrous cycle.

Claudins (CLDNs) are major Ca2+-independent cell adhesion molecules functioning at tight junctions (TJ). The presence and localization of cell adhesion molecules are important for understanding the mechanisms associated with follicular and luteal development in the ovary. In this study, there was an examination of whether CLDN-1 and CLDN-5 are present in a cell- and stage-specific manner during follicular and luteal development in the domestic cat ovary using immunohistochemistry and Western blot analysis. While results from immunoblot analyses revealed there were relatively similar abundances of CLDN-5 protein in three phases of the ovarian cycle, the abundance of CLDN-1 in the luteal phase was greater than those measured in the follicular and anestrous phases (P < 0.01). Results with immunohistochemistry indicate CLDN-1 and -5 are mainly localized in the cell nuclei and cytoplasm of all tissues of the cat ovary. In follicles, throughout the development from primordial to large antral follicles, CLDN-1 and -5 were present in oocytes, and the granulosa and theca cell layers. In follicles at all stages of atresia, there were cell-type and stage-specific protein distributions with immunostaining present in granulosa, thecal interstitial, and fibroblast-like cells. In corpora lutea, both small and large luteal cells stained positively for both claudins. In conclusion, the specific presence and localization patterns of CLDN-1 and -5 in the cat ovary is suggestive that these TJ proteins could have local functions in the regulation of most ovarian functions such as follicle development and atresia, ovulation, and corpus luteum formation and regression.

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.

Expression and localisation of epidermal growth factor receptors and their ligands in the lower genital tract of cycling cows.

The epidermal growth factor receptor (ErbB) family and its ligands are essential for the regulation of multiple cellular processes required for mammalian reproduction. The objectives of this study were to investigate the expression and localisation of ErbB subtypes (ErbB1-4) and selected ligands, namely epidermal growth factor (EGF), amphiregulin (AREG) and neuregulin (NRG), in the cervix and vagina of cycling cows and to determine possible steroid hormone-dependence of their expression using immunohistochemistry. All four ErbBs and EGF, AREG and NRG proteins were found to be localised in the nucleus and cytoplasm of different cells in the cervix and vagina, and their expression differed during the oestrous cycle. During the follicular phase, in both the cervix and vagina, ErbB1, ErbB2, ErbB3, ErbB4 and EGF expression was higher in the luminal epithelium (LE) than in stromal and smooth muscle (SM) cells (P<0.05). During the luteal phase, the expression of ErbB1, ErbB3 and EGF in the LE was significantly different from that in stromal and SM cells in the cervix, whereas the expression of EGF and AREG differed in the vagina compared to the cervix (P<0.05). Throughout the oestrous cycle, in both the cervix and vagina, although ErbB2/human epidermal growth factor receptor 2 expression in the LE and SM cells was significantly higher than in the stromal cells (P<0.05), NRG expression was similar in the LE, stromal and SM cells (P>0.05). Overall, these results suggest that all four ErbBs and the EGF, AREG and NRG proteins may collectively contribute to several cellular processes in the bovine cervix and vagina during the oestrous cycle.

The expression and localization of Toll-like receptors 2, 4, 5 and 9 in the epididymis and vas deferens of a adult tom cats.

Toll-like receptors (TLRs) are important molecules, which provide protection against infections of the reproductive tract. This study demonstrates for the first time the expression and localization patterns of TLRs in the caput, corpus and cauda segments of the epididymal duct (ED) and the vas deferens (VD) of adult domestic cats using immunohistochemistry and western blotting. While immunoblot analyses revealed relatively similar protein levels for TLRs 2, 4, 5, and 9 in three segments of the ED, the protein levels of TLR2 and TLR4 in the VD were found to be significantly higher than those measured in the ED segments (P < 0.05). On the other hand, immunostaining showed that TLRs exhibited regional- and cell-specific localization patterns. TLR2 and TLR5 were immunolocalized to the nucleus and cytoplasm of the principal cells in all ducts. TLR4 was restricted to the stereocilia, and TLR9 was located in the cytoplasm of the principal cells. Narrow cells displayed positive immunoreactions for TLR4 and TLR5. The basal cells of the different ED segments were positive for all four TLRs. TLR2, TLR5 and TLR9 were detected in the cytoplasmic droplets of the spermatozoa. TLR4 and TLR9 were detected along the entire length of the sperm tail, whilst TLR2 and TLR5 were absent in the midpiece. TLR2 and TLR5 were also detected in the equatorial segment of the sperm head. These results suggest that TLR2, TLR4, TLR5 and TLR9 are important not only for the protection of the ED, VD and spermatozoa but also for the maturation and storage of spermatozoa in the ED and VD, respectively.

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.

Tissue distribution of some immune cells in bovine reproductive tract during follicular and luteal phase.

More recent studies indicate that immune cells which secrete their secretory products or cytokines play an important role in reproductive system. In our study, immune cell populations (CD8+ T lymphocytes, CD68+ macrophages, plasma cells, siderophages, eosinophils) and expression of major histocompatibility complex (MHC) class I and class II were examined in female reproductive tract during follicular (n = 13) and luteal phase (n = 10). Plasma cells and eosinophil granulocytes are present in few numbers in luminal epithelium, but abundant in longitudinal muscle layer of uterus, whereas siderophages are the dominant cell type in stroma. Moreover, MHC-I and -II+ cells are expressed by individual cells in organ layers, while CD8+ T cells and CD68+ macrophages are dominant in epithelium and muscle layer, respectively. In conclusion, we did not found significant changes in immune cells according to follicular and luteal phases, but localization and numbers in each organ have changed according to both organ and layers. These results indicate that these factors may play a crucial role not only to generate an immune response but also to have a role in regulation of physiological functions in female reproductive organs.

Toll-like receptor expression patterns in the rat uterus during post partum involution.

Toll-like receptors (TLRs) belong to a family of pathogen recognition receptors and play critical roles in detecting and responding to invading pathogens. TLR expression could be significant because, in the uterus, the reproductive tract is an important site of exposure to and infection by pathogens during the post partum involution period. To clarify the expression and localisation patterns of TLRs in the rat uterus on Days 1, 3, 5 and 10 post partum (PP1, PP3, PP5 and PP10 respectively), immunohistochemistry and western blotting were used to analyse TLR1-7, TLR9 and TLR10. The immunohistochemistry results indicated that TLR1-7, TLR9 and TLR10 were localised in both the cytoplasm and nuclei of luminal and glandular epithelium, stromal fibroblasts and myometrial cells in the rat uterus. In the luminal epithelium, TLR4-7 were also found in lateral membranes, whereas TLR10 was present in apical membranes. Western blot analysis revealed that the expression of TLR proteins increased with the number of days post partum, reaching a maximum on PP10, although levels did not differ significantly from those on PP1 (P>0.05). These findings confirm that TLR1-7, TLR9 and TLR10 are constitutively expressed in uterine cells and that localisation pattern of TLRs in the endometrium varies with structural changes in the uterus on different days of involution. These results suggest that TLRs may play a role in uterine repair and remodelling during physiological involution.

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.

Adult-onset hyperthyroidism impairs spatial learning: possible involvement of mitogen-activated protein kinase signaling pathways.

Given evidence that mitogen-activated protein kinase (MAPK) activation is part of the nongenomic actions of thyroid hormones, we investigated the possible consequences of hyperthyroidism for the cognitive functioning of adult rats. Young adult rats were treated with L-thyroxine or saline. Twenty rats in each group were exposed to Morris water maze testing, measuring their performance in a hidden-platform spatial task. In a separate set of rats not exposed to Morris water maze testing (untrained rats), the expression and phosphorylated levels of p38-MAPK and of its two downstream effectors, Elk-1 and cAMP response element-binding protein, were evaluated using quantitative reverse transcriptase-PCR and western blotting. Rats with hyperthyroidism showed delayed acquisition of learning compared with their wild-type counterparts, as shown by increased escape latencies and distance moved on the last two trials of daily training in the water maze. The hyperthyroid rats, however, showed no difference during probe trials. Western blot analyses of the hippocampus showed that hyperthyroidism increased phosphorylated p38-MAPK levels in untrained rats. Although our study is correlative in nature and does not exclude the contribution of other molecular targets, our findings suggest that the observed impairments in acquisition during actual learning in rats with hyperthyroidism may result from the increased phosphorylation of p38-MAPK.

Region-specific localization of NOS isoforms and NADPH-diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus).

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage-dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)-NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH-d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH-d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell-specific localization in the efferent ductules and region- and cell-specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation.

Involution dependent changes in distribution and localization of bax, survivin, caspase-3, and calpain-1 in the rat endometrium.

The endometrial layer of the uterus is characterized by continuous cycle of cell growth and apoptosis in response to hormonal changes. Apoptosis is regulated by several apoptotic regulators, but their significance in involuting uterus has not been well understood. For that reason, aim of this study was to investigate possible role of apoptosis-related proteins (bax and survivin) and enzymes (caspase-3 and calpain-1) in the involuting uterus of the rat, using immunohistochemistry. Our results indicated cytoplasmic and nuclear immunostaining for bax, caspase-3, calpain-1 and survivin proteins were found in the endometrial epithelium and stromal cells such as fibroblasts, mast cells and macrophages, and blood vessels; however, calpain-1 immunoreactivity in the endometrial fibroblast was quite weak or absent. Supranuclear punctate bax immunolabelling was also observed in the endometrial fibroblasts and luminal and glandular epithelial cells from days 1st and 3rd following parturition, respectively. Although survivin was localized in the apical cytoplasm underneath the apical membrane of the luminal epithelium on the 1st and 3rd days, it was also localized in the apicolateral membrane and basal cytoplasm on the 10th and 15th days of involution. Immunostainigs demonstrated that expression patterns of all examined proteins varied with structural changes in the luminal epithelium, and number of immunopositive fibroblasts for bax, caspase-3 and survivin increased with advance of postpartum days and reached a maximum on postpartum days 10 and 15. These results suggest that the process of postpartum involution of endometrium may be regulated by apoptotic and non-apoptotic activity of bax, caspase-3, calpain-1, and survivin.