Emel Alan

Immunohistochemical localization of beta defensins in the endometrium of rat uterus during the postpartum involution period

Abstractβ-Defensins are small cationic molecules that have antimicrobial actions against bacteria, fungi and viruses and contribute to mucosal immune responses at epithelial sites. The female reproductive tract is an important site of defensin production. This study was conducted to determine the possible changes in proportions and localization of β-defensin 1-4 in the rat uterus at the 1st, 3th, 5th, 10th and 15th days of postpartum and at the period of diestrus using immunohistochemical techniques. In the present study, it was determined that β-defensin 1-4 were generally found in all structural components of the endometrium (luminal and glandular epithelium, stromal cells and blood vessels) in both the nucleus and the cytoplasm of cells during the involution period and diestrus. Suprisingly, immunoreaction of β-defensin 2 was also observed in the lateral membrane of the luminal and glandular epithelial cells on the 10th day of involution and immunostaining of β-defensin 4 was also localized in the apical membrane of the luminal and glandular epithelial cells. The current study demonstrated β-defensin 1-4 immunoreactivities in the endothelium of blood vessels were stronger throughout the involution period. Although β-defensins 2 and 3 were localized in both the nuclei and the cytoplasm of endothelial cells, β-defensins 1 and 4 were present in only cytoplasm. These results show that the most component of rat endometrium expresses human β-defensin 1-4 in a involution-dependent manner. Therefore it may be asserted that these molecules constitute a organised protection to prevent uterus from probable infections during the involution process.

The differences between the localizations of MUC1, MUC5AC, MUC6 and osteopontin in quail proventriculus and gizzard may be a reflection of functional differences of stomach parts

Mucins are high molecular weight glycoproteins which constitute the major component of the mucus layer and are produce by many epithelial tissues in vertebrates. Osteopontin (OPN) is an adhesive phosphorylated glycoprotein that is expressed by a broad range of tissues and cells. Although gastric mucins MUC1, MUC5AC, MUC6 and OPN have been widely used in histological studies and in diagnostic pathology in order to diagnose gastric carcinomas, their localizations in the stomach of quail have not yet been studied. In this study, the localizations of MUC1, MUC5AC, MUC6 and OPN in the proventriculus and gizzard of Japanese quail during the post‐hatching period were compared at light microscope levels by applying immunohistochemical methods. In all ages studied, the immunoreactivity of MUC5AC was present in the lining epithelium of both folds and superficial proventricular glands in the proventriculus, whereas MUC1, MUC6 and OPN reactivity was found in the oxynticopeptic cells of profound proventricular glands. In addition, some cells in the fold epithelium of the proventriculus showed a positive reaction to OPN. The immunoreactivity of MUC1 in gizzard was different from that of MUC5AC. Although MUC5AC was expressed in the cells of both the surface epithelium and profound glands of the gizzard, MUC1 was only localized in the profound glands of the gizzard. However, MUC6 and OPN immunoreactivity was absent in the gizzard. The results indicated that the differences between the localizations of MUC1, MUC5AC, MUC6 and OPN in quail proventriculus and gizzard may be a reflection of functional differences of stomach parts. Although the biological significances of the expressions of MUC1, MUC5AC, MUC6 and OPN in the quail stomach remains unknown, these notable glycoproteins may be associated with barrier function, host defence, and/or secretion.

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. Microsc. Res. Tech. 79:192–208, 2016.

Distribution, density and histochemical profiles of the lung mast cells during the post-hatching period of Japanese quails (Coturnix coturnix japonica).

The distribution, density and histochemical characteristics of mast cells in the lungs of the Japanese quail were investigated during the post-hatching period. In the period starting from the first to the 60th day post-hatching, based on proteoglycan content, three types of mast cells, which were alcian blue-positive, safranin O-positive and alcian blue/safranin O-positive, were found to exist in the lungs. The application of staining with berberine sulphate demonstrated that, similar to the distribution of safranin O-positive cells, the heparin-containing cells were located in the periphery of large blood vessels. The percentages of mast cells in different localization sites of the lungs were found to vary with age in the post-hatching period with toluidine blue staining. The lack of any statistically significant increase/decrease in the number of mast cells per unit area of the right and left lung lobes is partially in favour of the proposal that the mast cell number increases with the growth of the lung volume in the post-hatching period.

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. Microsc. Res. Tech. 79:285–297, 2016.

The profile of the epidermal growth factor system in rat endometrium during postpartum involution period.

The epidermal growth factor (EGF) plays a crucial role in the control of uterine cell proliferation, growth and differentiation. This study was designed to investigate the spatiotemporal expression pattern and localization of the EGF receptor/ligand system during the process of uterine involution using immunohistochemistry. Our results indicated that the expression of the ErbB/HER receptors and their ligands varied with structural changes in the uterus at different days of involution. Supranuclear punctate ErbB1 immunostaining was observed in the luminal and glandular epithelial cells and endometrial fibroblasts. Moderate ErbB2/HER2 immunoreactivity was observed in the lateral membrane and cytoplasm of the epithelial cells on the 1st, 3rd and 5th days and was decreased on the other days of involution. The amount of nuclear and cytoplasmic ErbB3/HER3 and ErbB4/HER4 immunostaining remained constant throughout the postpartum period. The EGF immunoreaction was weak in the luminal and glandular epithelium throughout the involution period. Although the cytoplasmic AREG immunoreactivity in the glandular epithelium was stronger on the 1st and 3rd days compared with the other days of involution, NRG1 immunostaining was weak on the 1st and 3rd days and was moderate in the apical cytoplasm on the 10th and 15th days of involution. The macrophages displayed strong cytoplasmic immunoreactivity for ErbB3/HER3, ErbB4/HER4, EGF, AREG and NRG. Strong, moderate and weak immunostaining for ErbB2/HER2, ErbB4/HER4 and other proteins (ErbB1, ErbB3, AREG and NRG), respectively, was present in the myometrial smooth muscle cells. These findings support the hypothesis that the EGFsystem plays a role in the development of various physiological changes associated with uterine involution.

The Process of Apoptosis in a Holocrine Gland as Shown by the Avian Uropygial Gland

This study was designed to elucidate the presence of apoptosis and the localization of apoptosis‐related Bax and survivin proteins and proliferating cell nuclear antigen (PCNA) within the chicken uropygial gland, a specialized holocrine secretory gland. In day‐old chicks, survivin and Bax immunoreactivities were observed in the cell cytoplasm of the germinative and secretory layers of the luminal epithelium and tubules. During this period, the TUNEL reaction, an indication of apoptosis, was only sporadically positive in the tubules. From the 7th day to the 150th day of posthatching, survivin was detected in the cytoplasm of cells in the germinative layer and in the nuclei of some cells in the secretory layers of the gland. The germinative layer cells showed weak homogeneous cytoplasmic staining for Bax, whereas the cells of the secretory and intermediate layers of luminal epithelium and tubules exhibited granular cytoplasmic staining. After day 7, TUNEL‐positive cells were observed in the secretory and degenerative layers of the luminal epithelium and central tubules. After day 12, some TUNEL‐positive cells were also seen in the peripheral tubules. At all posthatch ages, the cytoplasm and nucleus of the germinative layers of luminal epithelium and tubules reacted with PCNA, whereas only a small number of cell nuclei in the secretory layers were immunopositive. These results support the theory that specific PCNA/Bax/survivin expression patterns could reflect particular cell differentiation states in the uropygial gland and that holocrine secretion in the gland is realized mainly by way of apoptosis. Anat Rec, 296:504–520, 2012.

Expression of vascular endothelial growth factor receptors and their ligands in rat uterus during the postpartum involution period

Abstract Vascular endothelial growth factor (VEGF) and its specific receptors, FLt1/fms, Flk1/KDR and FLt4, play important roles in vasculogenesis, and physiological and pathological angiogenesis. Whether angiogenic growth factors are involved in regulating angiogenic processes during the postpartum involution period (PP) of the rat uterus is unknown. We used immunohistochemistry to analyze the expression levels of VEGF, the fms-like tyrosine kinase 1 (FLt1/fms), the kinase insert domain-containing region 1 (Flk1/KDR), Fms-related tyrosine kinase 4 (FLt4) and vascular endothelial growth inhibitor (VEGI) in the rat uterus during the days 1, 3, 5, 10 and 15 of the PP to determine the temporal and spatial expressions of VEGF and its receptors during the PP. Throughout the PP, cytoplasmic and membrane staining of VEGI, VEGF and their receptors were observed in the lumens, crypts and glandular epithelial cells as well as in connective tissue and vascular endothelial and smooth muscle cells in the endometrium. We found that the intensity of the immunoreactions in the endometrium varied with the morphological changes that occurred during involution. Immunoreactions for VEGI, VEGF and their receptor, Flk1/KDR, in the luminal epithelial cells were stronger than those in the glandular epithelial and stromal cells, particularly during PP 1, 3 and 5, which suggests that these peptides may contribute to re-epithelialization of the endometrium. On the other hand, Flt1/fms immunoreactivity was strong mainly in the stromal cells during the PP. The presence of VEGF and its receptors (FLt1/fms, Flk1/KDR, FLt4) in the stromal cells and blood vessels during the PP suggests that they may contribute to regulating stromal repair and angiogenesis in the involuting uterus of the rat.

Immunohistochemical Demonstration of S-100 Protein in the Chicken Uropygial Gland During the Post-Hatching Period

S-100 proteins are calcium-activated signaling proteins that interact with other proteins to modulate biological functions such as cell migration, growth, proliferation, differentiation, apoptosis, contraction, and the inflammatory response. Although S-100 proteins are expressed in neuronal and non-neuronal tissues, their localization in the uropygial gland was not known. This study concerns the immunohistochemical detection of localization of S-100&agr;, S-100&bgr;, and wbS-100 in the chicken uropygial gland during development from days 1–150 days post-hatching. In 1-day-old chicks, the nuclei and cytoplasm of cells in the luminal epithelium and the tubules of the gland stained positively for S-100&agr;, S-100&bgr;, and wbS-100. Seven days after hatching, immunoreactivity for S-100&agr;, S-100&bgr;, and wbS-100 was detected in the nucleus and cytoplasm of cells in the germinative and intermediate layers of the central zone, but was found only in the germinative layer of the peripheral zone. In addition, in cells of the degenerative and secretory layers of both the central and peripheral zones, S-100&agr;, S-100&bgr;, and wbS-100 were present in the plasma membrane. In uropygial glands studied from days 7–150 post-hatching, the number of immunopositive cells in the central zone increased with the advance of age and glandular growth. The results indicated that the chicken uropygial gland contains both &agr;&bgr; and &bgr;&bgr; dimers. Although the biological significance of the expression of wbS-100 and its subunits in the uropygial gland remains unknown, these notable calcium-binding proteins may be associated with the processes of gland-cell differentiation and apoptosis, and the antimicrobial properties of preen wax or lipids.

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.